NATIONAL EYE INSTITUTE Cover Photograph A Y-79 human retinoblastoma cell treated with pigment epithelium-derived factor (PEDF). Photograph courtesy of Dr. Gerald Chader, Chief, Laboratory of Retinal Cell and Molecular Biology, NEI. Dr. Chader's report begins on page 255. NATIONAL INSTITUTES OF NIH LIBRARY AUG 2 2 1995 National Eye iNSTiTore BLDG 10, 10 CENTER DR. BETHESDA, MD 20892-1150 Annual Report Fiscal Year 1994 U.S. Department of Health and Human Services Public Health Service National Institutes of Health ^t 1 Table of Contents Statement of the Institute Director i Carl Kupfer, M.D. Extramural Research 7 Report of the Associate Director 9 Jack A. McLaughlin, Ph.D. Division of Basic Vision Research 9 Peter Dudley, Ph.D. Retinitis Pigmentosa 9 Glaucoma 10 Keratoconus of the Cornea 11 Acquired Immunodeficiency Syndrome 12 Retinal Neuroscience — Molecular Basis of Signaling 12 Corneal Angiogenesis 14 Lens Development 14 Neural Processing of Visual Information 15 Development 15 Personal Guidance System for the Visually Impaired 16 Division of Collaborative Clinical Research 17 Richard Mowery, Ph.D. Retinal Diseases 17 Glaucoma 19 Corneal Diseases 20 Strabismus, Amblyopia, and Visual Processing 20 Division of Biometry and Epidemiology 23 Report of the Acting Director 25 Roy C. Milton, Ph.D. Research Highlights 25 Research Activities 26 Publications 29 FY 1994 NEl Annual Report Office of International Program Activities 31 Report of the Acting Assistant Director 33 Terrence Gillen, M.A., M.B.A. Highlights of Recent Scientific Advances Resulting From International Activities 33 Summary of International Programs and Activities 34 Activities With International and Multinational Organizations 37 Extramural Programs 37 Intramural Programs and Activities 37 Office of Science Policy and Legislation 39 Report of the Associate Director 41 Michael P. Davis, M.S. Policy, Legislation, Planning, and Evaluation Branch 41 Carmen P. Moten, Ph.D. Management Information Systems Branch 43 David Scheim, Ph.D. Office of Health, Education, and Communication 47 Report of the Director 49 Judith A. Stein, M.A. National Eye Health Education Program 49 25th Anniversary Program 50 PubUc Inquiries Program 50 Scientific Reporting 51 Office of the Scientific Director 53 Office of the Scientific Director Francisco M. de Monasterio, M.D., D.Sc. Anatomical Studies of the Primate Visual System 55 Physiological Studies of the Primate Visual System 57 Helen H. Hess, M.D. Biochemistry of Retina and Pigmented Epithelium in Health and Disease .... 59 Laboratory of Immunology 63 Report of the Chief 65 Robert B. Nussenblatt, M.D. Table of Contents Section on Experimental Immunology Charles E. Egwuagu, Ph.D., M.P.H. Transgenic Rat and Mouse Models for the Study of Intraocular Effects of IFN-y and Autoimmunity 71 Analysis of T Lymphocytes and Cytokines Involved in Experimental Autoimmune Uveoretinitis 74 Igal Gery, Ph.D. Immune Responses to Ocular Antigens 77 Section on Clinical Immunology Frangois G. Roherge, M.D. Inhibition of EAU in Monkey With Humanized Anti IL-2 Receptor 82 Study of the Effect of NPC 15669, an Inhibitor of Neutrophil Recruitment in Uveitis 85 Study of the Role of Nitric Oxide in Uveitis 87 Study of Immunosuppressants for the Treatment of Uveitis in Animal Models 90 Section of Ocular Gene Therapy Karl G. Csaky, M.D., Ph.D. Ocular Gene Transfer 91 Section on Immunopathology Scott M. Whitcup, M.D. The Diagnosis and Treatment of Human Uveitis and AIDS-Related Ocular Disease 94 Chi-Chao Chan, M.D. Immunopathology in Eyes With Experimental and Clinical Ocular Diseases ... 99 Scott M. Whitcup, M.D. Immunologic Mechanisms of Ocular Disease 105 Ocular Toxicity of 2',3'-Dideox)dnosine(ddI) 109 Chi-Chao Chan, M.D. Cytokines and Ocular Antigens in the Eye 112 Section on Immunoregulation Robert B. Nussenblatt, M.D. Cyclosporine Therapy in Uveitis 113 Oral Administration of Antigen and the Ocular Immime Response 116 Rachel R. Caspi, Ph.D. Cellular and Immunogenetic Mecharusms in Uveitis 119 Marc D. de Smet, M.D. Characterization of Immune Responses to Retinal Specific Antigens 125 Surgical Management of Uveitis 128 Ocular Manifestations of the Acquired Immune Deficiency Syndrome 129 Section on Immunology and Virology John J. Hooks, Ph.D. Interferon System in Cellular Function and Disease 132 Studies on the Bioregulatory Aspects of the Retinal Pigment EpitheHal Cell ... 135 Virus Infections in the Eye 138 Chandrasekharam N. Nagineni, Ph.D. Role of Retinal Pigment Epithelium in Retinal Disorders 143 John J. Hooks, Ph.D. Toxoplasmosis Infections in the Eye 147 ¥Y 1994 NEI Annual Report Section of Genetics and Molecular Immunology Moncef Jendoubi, Ph.D. Gene Targeting of Invariant Chain Gene: A Tool To Study Immunoregulation in Autoimmune Diseases 149 Retinal Survival in Transgenic Mice Expressing Human Ornithine 6-Aminotransferase 152 Enzymatic Correction of OAT Deficiency: Progress Toward Gene Therapy to Ocular Genetic Disease 155 Isolation and Characterization of the Mouse OAT Gene for Gene Targeting ... 160 Gene Therapy for Ocular Genetic Disease 163 Immunopathology of Ocular Diseases in Humans 164 Laboratory of Mechanisms of Ocular Diseases i65 Report of the Chief 167 /. Samuel Zigler, Jr., Ph.D. Section on Cataracts /. Samuel Zigler, Jr., Ph.D. Structure and Composition of Lens Crystallins With Respect to Cataractogenesis 169 Donita L. Garland, Ph.D. Oxidation of Proteins in Cataractogenesis 173 Studies on Human Lens Proteins 176 Deborah Carper, Ph.D. Structure and Expression of Polyol Pathway Enzymes 179 Paul Russell, Ph.D. Characterization of the Lens 183 Lenticular Expression of the HTV Protease 186 Autoantibodies to Lens Crystallins 188 James Fielding Hejtmancik, M.D., Ph.D. Inherited Ocular Diseases 190 Section on Pathophysiology W. Gerald Robison, Jr., Ph.D. Ultrastructure and Function of the Cells and Tissues of the Eye 195 Laboratory of Molecular and Developmental Biology 199 Report of the Chief 201 Joram Piatigorsky, Ph.D. Section on Cellular Differentiation Peggy S. Zelenka, Ph.D. Proto-oncogene Expression During Lens Differentiation and Development .... 204 Section on Molecular Genetics Joram Piatigorsky, Ph.D. CrystaUin Genes: Structure, Organization, Expression, and Evolution 209 Molecular Biology of the Cornea 217 Table of Contents Section on Molecular Structure and Function Graeme J. Wistow, Ph.D. Moleciilar Biology and Functions of the Lens Proteins 220 Section on Regulation of Gene Expression Ana B. Chepelinsky, Ph.D. Genetically Engineering the Eye With the aA-CrystaUin Promoter 224 Regulation of Expression of Lens Fiber Membrane Genes 228 Section on Transgenic Animal and Genome Manipulation Eric Wawrousek, Ph.D. NEl Cential Transgenic Animal Production Fadlity 231 a-CrystaUin Gene Disruption in the Mouse 234 Transgenic Animal Models 237 Laboratory of Ocular Therapeutics 241 Report of the Chief 243 Peter F. Kador, Ph.D. Peter F. Kador, Ph.D. Pharmacology of Ocular Complications 244 Sanai Sato, M.D., Ph.D. NADPH Reductases and Polyol Pathway in Ocular Complications 250 Laboratory of Retinal Cell and Molecuur Biology 253 Report of the Chief 255 Gerald J. Chader, Ph.D. Section on Biochemistry Barbara YJiggert, Ph.D. Vitamin A and Ocular Tissues 258 Section on Gene Regulation Susan Gentleman, Ph.D. Microtubule Stability as a Factor in Retinal Degenerations 263 Diane E. Borst, Ph.D. Molecular Genetics of the Eye and Ocular Diseases 266 Gerald J. Chader, Ph.D. Molecular Biology of the Retina and Pigment Epithelium 270 Visual Control Mechanisms and Hereditary Degeneration 273 T. Michael Redmond, Ph.D. Molecular Biology of Outer Retina-Specific Proteins 277 Section on Molecular Biology Toshimichi Shinohara, Ph.D. Molecular Biology of Experimental Autoimmune Uveitis 280 Molecular Biology of Phototransduction 283 Laboratory of Sensorimotor Research 287 Report of the Chief 289 Robert H. Wurtz, Ph.D. FY 1994 NEI Annual Report Section on Visual Behavior David Lee Robinson, Ph.D. Visuomotor Properties of Neurons in the Thalamus 293 Section on Neuro-Ophthalmologic Mechanisms Michael E. Goldberg, M.D. Cerebral Cortical Mechaiusms for Eye Movements and Visual Attention 297 Section on Oculomotor Control Frederick A. Miles, D.Phil. Visual Motion and the Stabilization of Gaze 301 Section on Visuomotor Integration Robert H. Wurtz, Ph.D. Visuomotor Processing in the Primate Brain 305 Section on Neural Modeling Lance M. Optican, Ph.D. Ii\formation Processing by Visual System Neurons 310 Ophthalmic Genetics and Clinical Services Branch 315 Report of the Chief 317 Muriel L Kaiser-Kupfer, M.D. Section on Cataract and Corneal Diseases Manual B. Datiles, M.D. The Effects of Corneal Contact Lenses on the Cornea 321 Documentation and Monitoring of Opacities in the Human Lens 322 Use of Human Lens Material for Determining Possible Causes of Cataracts 325 Muriel I. Kaiser-Kupfer, M.D. Addendum to Use of Human Lens Material for Determining Possible Causes of Cataracts 328 Carl Kupfer, M.D. Anterior Chamber Anomalies Associated With Glaucoma or Ocular Hjrpertension 330 Section on Ophthalmic Genetics Muriel L Kaiser-Kupfer, M.D. Pigment Dispersion With and Without Glaucoma 332 Visual Function and Ocular Pigmentation in Albinism 335 Gyrate Atrophy of the Choroid and Retina and Other Retinal Degenerations . . 338 NTH Interinstitute Genetics Program: The Genetics Clinic 342 Usher Syndrome — Clinical and Molecular Studies 345 A Double-Masked Controlled Randomized CUnical Trial of Topical Cysteamine [II] 347 A Double-Masked Controlled Randomized Clinical Trial of Topical Cysteamine [I] 348 Mark H. Scott, M.D. Characteristics of Macular Scotomas in Patients With Primary Monofixation Syndrome 350 Table of Contents Section on Eye Services Rafael Caruso, M.D. Clinical Psychophysics of the Visual System 351 Clinical Electrophysiology of the Visual System 353 Visual Function Diagnosis Service 355 Index 357 Statement of the Institute Director statement of the Institute Director Carl Kupfer, M.D. In this first year of our new research plan Vision Research — A National Plan: 1994- 1998, vision researchers have made sig- nificant progress in achieving the stated goals and objectives that are considered so impor- tant to improving the visual health of the American people. During this fiscal year, 1,210 research grants were funded for nearly $235 million, and an additional 19 research and development contracts were funded for approximately $7.8 million. Another $30 mil- lion was expended in support of the intramu- ral research program. As this year's annual report demonstrates, the investment by Ameri- can taxpayers in vision research has led to several important breakthroughs by our intra- mural and extramural laboratories and clinical scientists and has continued to improve our understanding of the pathological processes involved in diseases of the eye and disorders of vision. As an example, the National Eye Institute (NEI)-supported intiamural scientists continue to provide new and important data on the role and function of the visual motor system. A study investigating the control of movement by visual input and the systems in the brain that perform this vital function is under way. Recentiy, these scientists tested the hypothesis that the spread of activity in the superior coUi- culus, a brain structure critical to rapid eye movement, controls the amplitude of such movement. Minute injections of a neurotrans- mitter inhibitor into the superior colliculus changed the direction of the eye movement. This suggests that not only the end-point but also the path taken by the eye is influenced by the superior colliculus; this information is pro- foundly important not only for understanding how the brain controls eye movement but also how it controls movements in general. This remarkable advance in understanding the sen- sory-motor system creates opportunities to examine new mechanisms of controlling movement and could lead to a better under- standing of how to control disease-induced disorders of eye movement control. Intramural researchers have also contin- ued their important studies on the role of aldose reductase (AR) in inhibiting ocular compUcations associated with diabetes. They have developed an animal model (galactose- fed dogs) that develops both the chnical and histological lesions associated with all stages of diabetic retinopathy. Using this model, investigators have conducted studies to image noninvasive cataract formation and to mea- sure the levels of AR in the lens in vivo. With this technique, they have also demonstrated a direct correlation between inhibition of AR in the lens and the prevention of the retinal peri- cyte degeneration, which leads to diabetic retinopathy. Intramural scientists are continuing their basic and clinical studies on the natural histo- ry and the role of the ornithine aminotiansfer- ase gene in the development of gyrate atrophy (GA), an inherited degenerative retinal disor- der. The research is currentiy directed at ge- netically altering the defective gene and cul- turing skin tissues that will be used as a tiansfer vehicle for inserting the altered gene in GA patients. This research may lead not only to an effective therapy for GA but may be extended to genetic interventions for other blinding genetic disorders. FY 1994 NEI Annual Report Scientists in NEI's intramural laboratories have discovered several genes that may be implicated in macular degeneration, a leading cause of bUndness in older Americans. In addition, a new gene has been cloned that could be the defective gene in Bardet-Biedl's syndrome, a hereditary disease of blindness and mental retardation. These investigators also have discovered a protein that promotes the survival of neurons within the central ner- vous system. This protein (PEDF) promotes maturation of photoreceptor-Uke cells derived from the retina. It also greatly lengthens the Ufe-span of brain neurons in tissue culture experiments. With these characteristics, future researchers will explore whether PEDF can be used in transplantation procedures that could be helpful in conditions such as Parkinson's disease. NEI intramural scientists are examining the role of ceU adhesion molecules and cyto- kines in the development of ocular inflamma- tory disease. CeU adhesion molecules are surface proteins that are important for antigen sensitization and leukocytes' migration to inflammation sites. Researchers are currentiy investigating compounds that block cell adhe- sion molecules as a treatment for uveitis. They have been able to inhibit significantiy the development of endotoxin-induced uveitis in mice with a single injection of anti-Mac-1 antibody. Research is now under way to de- velop and test topically administered sub- stances that can block critical cell adhesion molecules. If animal studies are successful, scientists plan to test therapies based on block- ing critical cell adhesion molecules and cyto- kines in the cUnical trials of uveitis patients. Scientists in NEI's intramural program have been studying the trabecular meshwork, a tissue in the eye that has been implicated in the development of open-angle glaucoma. In a series of experiments, investigators have analyzed the proteins present in the four quadrants of the normal trabecular meshwork and are now examining the differences be- tween normal tissue and glaucomatous trabec- ular meshwork and the changes that precede glaucoma. Such results would offer important clues on the causes of open-angle glaucoma and may provide the foundation for the devel- opment of effective therapies. The most common causes of blindness in the United States are associated with the aging process. NEI epidemiologists have initiated a natural history study to assess the cUnical course, prognosis and risk factors of age-re- lated macular degeneration (AMD), and cata- ract. Further investigation of the risk factors in the development and progression as well as their pathophysiologies may lead to methods for preventing these debilitating ocular disor- ders. Additionally, the effects of pharmaco- logic doses of antioxidants — ^vitamins C and E and beta-carotene — and zinc on the incidence and progression of AMD and antioxidants on the incidence and progression of lens opacities wiU be assessed as part of a randomized clini- cal trial, using the cohort of 4,600 patients developed for the natural history study. NEI-supported extramural scientists have reported the loss of neuronal cells in the brains and retinas of acqviired immunodefi- ciency syndrome (AIDS) patients, which is thought to contribute to the neurologic and retinal dysfunction often associated with hu- man immunodefiency virus (HIV)-1 infection. Retinal ganglion cells involved in the trans- mission of information from the retina to the brain appear to undergo physical changes in these patients. These scientists have shown that a complex web of interactions between the cells of the immune system and the neu- rons is involved, involving a protein known as gpl20. This protein is found on the surface of the HIV virus. They have found that adding gpl20 to retinal ganglion cells growing under laboratory conditions causes injury to the cells. They have also found a receptor antagonist that may give protection from gpl20 neurotox- icity, which may one day lead to the develop- ment of the means to prevent the neurological manifestations of AIDS. An abnormal proliferation and leakage of retinal blood vessels leading to loss of vision are characteristic of a number of conditions in which the oxygen supply to the retina is ob- Statement of the Institute Director structed. These conditions include severe diabetic retinopathy, retinal vascular occlu- sions, and retinopathy of prematurity. Recent studies supported by the NEI have demon- stiated that a protein called vascular endothe- lial growth factor (VEGF) is present in greatly increased amounts in the ocular fluid of pa- tients with these conditions. Production of VEGF is also enhanced by experimental proce- dures that decrease the oxygen supply to the retina. As VEGF is well known to be capable of stimulating blood vessel growth in the eye, these new studies indicate that VEGF may play a major role in diabetic retinopathy and other ischemic eye conditions. Investigators will expand the search for ways to either block the production of VEGF or to block its ability to stimulate the growth of new blood vessels. Because several inherited macular degen- erative diseases share significant similarities to AMD, NEI-supported scientists are searching for defective genes in affected farrdlies. Inves- tigators have localized gene mutations to spe- cific chromosomes for three forms of inherited macular degeneration. These investigators have shown that a mutation in one of these genes causes macular degeneration, while a different mutation in the same gene causes retinitis pigmentosa (RP). The information gained from these studies should help in understanding the cause of the more prevalent AMD. During this last year, investigators from the NEI-supported Prospective Evaluation of Radial Keratotomy (RK) reported the results of their 10-year foUowup study. The data contin- ue to indicate that RK remains a reasonably safe and effective method for correcting myo- pia (nearsightedness). However, more than 40 percent of RK-operated eyes continue to have a gradual shift toward farsightedness. This finding suggests that some people who have RK may need glasses at an earlier age for poor close-up vision, a common problem after age 40, than if they had chosen not to have the surgery. Data on quality of life, symptoms related to glare and fluctuating vision, and subjective visual function remain to be analyzed. Last year NEI-supported researchers re- ported the localization of the gene for juvenile onset glaucoma, a form of the disease charac- terized by early adulthood onset and elevated intraocular pressure, to chromosome 1. These researchers have now mapped the gene to a region of chromosome 1 extensive enough to contain approximately 20 genes. Once the exact location of this gene is determined, the gene can be cloned and sequenced. The hope is that the deoxyribonucleic acid (DNA) se- quence of the juvenile glaucoma gene will reveal clues about the fundamental cause of glaucoma. A new technique, which uses microlaser stimulation and microelecfrodes, allowed researchers supported by the NEI to stimulate selected nerve cells in individual circuits in this dense meshwork of cells. Results from this approach show how individual nerve cells function together. Previously, these results were very difficult to obtain because of the inaccessibility of these functional uruts. Fur- ther refinement of this approach could lead to the development of a powerful tool for isolat- ing discrete networks of nerve cells and for learning how they function and organize in- formation. During fiscal year (FY) 1994, results from a NEI-supported study the Orinda Longitudi- nal Study of Myopia (OLSM) were published suggesting a possible genetic link to myopia. Measurements were made in more than 700 school children ages six to 14 on each child's refractive error, corneal curvature, crystaUine lens power, and axial ocular dimensions. Parents reported their own vision status. The investigators found that school children whose parents are nearsighted have differently shaped eyeballs than children whose parents are not nearsighted. Children of myopic parents had longer eyes even before the onset of myopia. These results suggest that the premyopic eye in children with a family history of myopia already resembles the elongated eye present in niyopia. The OLSM will continue to provide a wealth of informa- tion on ocular and refractive development in children in the years ahead. FY 1994 NEI Annual Report These are highlights of a few of the re- reports and project descriptions contained in search accompUshments of the intramural and this annual report. It is a pleasure to provide extramural laboratory and clinical scientists this recognition of the researchers' efforts in supported by the NEI during FY 1994. More addressing the visual health needs of our detail on these and other accompUshments in Nation, the field of vision research can be found in the Carl Kupfer, M.D. Director National Eye Institute Extramural Research Report of the Associate Director for Extramural Research) Jack A. McLaughlin, Ph.D. Research activities supported by the extramural Vision Research program address the leading causes of blind- ness and unpaired vision in the United States, including retinal diseases, corneal diseases, cataract, glaucoma, strabismus, and ambly- opia. The program seeks to increase under- standing of the normal development and function of the visual system; to understand the causes of and to better diagnose, prevent, and treat sight-threatening conditions; and, to enhance the rehabilitation, training, and quaU- ty of life of individuals who are partially sighted or bUnd. In working to this end, the Vision Re- search program supports vision research through grants, cooperative agreements, and research and development contracts; encourag- es high quality cHnical research, including dinical trials and other epidemiologic studies; encourages research training and career devel- opment in the sciences related to vision; sponsors scientific workshops in high-priority research areas to encourage exchange of information among scientists; and carries out a construction, alteration, and instrumentation program of grants for public and private nonprofit vision research facilities. For FY 1994, an estimated total of $248,425,000 was expended for NEl extramural grants, cooperative agreements, and research and development contracts in the following categories and amounts: Research Grants $233,541,000 Research Training Awards $7,294,000 Research and Development Contract $7,590,000 Total Extramural Support $248,425,000 The following sections highlight some of the recent accomplishments of NEl-supported investigators. Division of Basic Vision Research Peter Dudley, Ph.D., Director Retinitis Pigmentosa The major diseases of the retina affect primari- ly the photoreceptor ceUs and the neighboring tissue called the pigmented epithelium. The photoreceptors are fragile and easily damaged in the face of hereditary defects, aging, toxic agents, overexposure to Ught, and dietary deficiencies. They are targets for such dis- eases as RP and AMD, which are leading causes of blindness. The basis for understand- ing these diseases Ues in gaining fundamental knowledge of the molecular machinery and cellular organization of these specialized ceUs. Structural information, combined with de- tailed biochemical and physiological knowl- edge, sets the stage for a molecular dissection of genetic diseases. Recent identification of defects in photoreceptor-specific genes in some inherited retinal diseases support this concept. FY 1994 NEI Annual Report RP is one of the most common human inherited eye disorders, causing bUndness from degeneration of the rod and cone photo- receptors in the retina. Patients with RP develop night bUndness and loss of midperi- pheral vision early in the disease. As the disease progresses, a shrinking island of central vision remains resulting in "tunnel vision." Unfortunately, many patients are bUnd by middle age. In the United States, RP affects 50,000 to 100,000 people of all races. RP exhibits heterogeneity, meaning that differ- ent mutations in a person's genetic material or DNA cause similar cUnical symptoms. There are two forms: "allelic heteogeneity," refers to different mutations within a single gene (rho- dopsin for example), and "locus heteroge- neity," which refers to gene defects at different chromosome locations. RP can be transmitted as an autosomal dominant, autosomal reces- sive, or X-Unked trait. The understanding of RP, currentiy an incurable retinal degeneration, has progressed recentiy with the discovery of a number of ge- netic mutations in important photoreceptor proteins. In about one-quarter of the cases of RP, mutations have been identified in the genes for rhodopsin, peripherin/RDS, rod phosphodiesterase-p subunit, and the cyclic guanosine monophosphate (cGMP)-gated channel. Although most cases are considered to be monogenic, i.e., in one particular family only one chromosome locus is thought to be defective. Dr. Dryja, from the Harvard Medi- cal School, has demonstrated an unusual in- heritance pattern in three famUies with RP called "digenic inheritance." Mutations in two genes, the unlinked photoreceptor-specific genes-ROMl and peripherin/RDS, were found to be responsible for this particular form of RP. The interesting feature of this unusual form of inheritance is that although variability in disease severity was observed in patients with other mutations {e.g., rhodopsin), the complete lack of clinical symptoms in some persons with a peripherin/RDS gene muta- tion, had not been observed. The explanation for this was revealed when a mutation in ROMl was discovered in patients who also had the peripherin/RDS mutation resulting in disease expression. Norrie's disease (ND) is a rare X-Unked hereditary eye disease characterized by con- genital bUndness. Investigations so far show the disease-causing gene to be located on the X chromosome. Genetic analysis has pinpoint- ed a chromosome site that may be the actual gene. Four separate mutations have been identified that cause deletions of part of the chromosome housing the apparent ND gene. But formal conclusive evidence that the ND gene is involved in the disease awaits identifi- cation of more gene mutations in ND patients. Dr. Wong, from Duke Uruversity, has discovered a new mutation in the ND gene of a male infant that results in loss of a small part of the ND protein. The normal ND protein, which appears to function in retinal and brain development, contains 11 cysteine amino acids. The point mutation discovered by Dr. Wong results in a loss of two cysteine residues. The discovery of another separate point mutation increases the Ust of mutations in the ND protein and appears to confirm the importance of the fuU complement of 11 cysteines in its function. This discovery may result in a useful diagnostic test to confirm an initial clinical detennination of ND. Glaucoma In the United States, about two milUon people have glaucoma, but, because of the insidious nature of the disease, many are unaware of its presence. AdditionaUy, about five milUon Americans, some of whom wUl develop glau- coma, have elevated intraocular pressure (lOP). Primary open-angle glaucoma (POAG) is the most severe form of the disease and is most common in people older than age 60. Approximately 80,000 people with this form of the disease will become bUnd. African Ameri- cans are affected disproportionately from POAG with a risk factor five times that of the white population for people older than age 40. The rate for blindness due to POAG in African Americans is six times higher than that of the 10 Extramural Research rest of the population, reflecting a more severe disease. The mechanism by which the optic nerve is damaged by glaucoma is not known. The relative influence of genetic and environmen- tal factors is also unclear. However, there is some hope for understanding the cause of the disease because juvenile onset glaucoma, a form of the disease characterized by early adulthood onset and elevated lOP, displays an autosomal dominant pattern of inheritance. This mode of inheritance makes a genetic approach ideal for the study of this disease. Dr. Stone, from the University of Iowa, Dr. Richards, from the University of Michigan, and Dr. Wiggs, from the Tufts New England Medical Center, have identified a number of families with sufficient numbers of individuals with glaucoma so that it is now possible to perform genetic linkage studies. To date med- ical histories have been collected fiom families in Michigan, New England, and Iowa. The ultimate goal is to identify a "glaucoma gene." Recentiy, one disease-associated gene has been mapped by Unkage analysis to chromosome 1. Corroborating data from difterent laboratories using different families have confirmed this location. Linkage analysis has placed the gene to within approximately a 20 to 80 gene re- gion on the chromosome. A possible association between juvenile onset glaucoma and POAG may Ue in identi- fying a causal factor for elevated lOP, be it a block in the aqueous humor outflow pathway or some other as yet unidentified factor. Keratoconus of the Cornea Keratoconus is a progressive condition charac- terized by norvinflammatory thinning and protrusion of the cornea, leading to its cone- shaped appearance. Keratoconus has an inci- dence of about five in 10,000 in the general population, and approximately 100,000 kerato- conus patients require eye care in the United States annually. Most of these patients need multiple contact lens fittings during their hfe and 10 to 20 percent ultimately require a pro- cedure called a penetrating keratoplasty to correct their condition. Recent retrospective studies suggest that keratoconus is the leading cause for cornea transplantation in the United States. Dr. Rabinowitz, from the Cedar-Sinai Medical Center, has been examining the genetic basis for keratoconus. He is using a techruque called videokeratography to obtain an accurate assessment as early as possibleof the chances for developing this condition. PUot studies on patients with advanced dis- ease indicated three distinguishing features: central corneal steepness, nonsymmetric steep- ness when comparing the superior and inferi- or corneal regions, and large central refractive differences between the right and left corneas. These features were quantitatively indexed and appUed to broader family studies. This work suggests an autosomal dominant mode of heredity with variable expressivity. Dr. Rabinowdtz's group is exploring three independent approaches to identifying a kera- toconus gene(s); cytogenetic studies have been initiated. These have not yet been informa- tive, although there is a report in the Uterature that Angehnans' syndrome patients with kera- toconus as one of their ocular findings carry a deletion on the long arm of chromosome 15. Family pedigree studies have been initiated, using videokeratographic data. To date 200 normal subjects, 110 keratoconic patients, and 210 family members have been analyzed and have donated blood for future genetic studies. Enrollment is approximately 60 percent com- plete, and, so far, more than 70 family pedi- grees have been constructed. These include several large families with multiple aftected members in at least four generations. Analy- sis will be performed within the year to deter- mine the genetic mode of inheritance. Longi- tudinal candidate gene studies are under way in one of the large multigenerational families. Some specific genes involved in the synthesis of collagen have been excluded. Other colla- gen genes and the genes for enzymes involved in coUagen metaboUsm are being explored. 11 FY 1994 NEI Annual Report Acquired Immunodeficiency Syndrome As many as one-third of adults with AIDS eventually develop neurological symptoms, including problems with memory, coordinated movement, and sensation. These problems can occur in the face of almost complete ab- sence of direct infection of nerve ceUs or neu- rons by the HIV-1. Recently, loss of neuronal cells in the brains and retinas of AIDS patients has been observed and is thought to contrib- ute to the neurologic and retinal dysfunction. Retinal gangUon cells involved in the trans- mission of information from the retina to the brain appear to undergo physical changes in these patients. What are the mechanisms that cause the observed changes in retinal gangUon cells in AIDS patients? Dr. Lipton, from Children's Hospital in Boston, has been studjdng this problem. There appears to be growing scien- titic evidence that for infections like HIV, which lead to the injury of neurons, a complex web of interactions between ceUs of the im- mune system and neurons is involved. Dr. Lipton has been studjdng one particular pro- tein involved in this system called gpl20. Gpl20 is a protein present on the outer surface of the HTV virus and is associated with increased intracellular concentrations of the ion calcium (Ca"^*). When gpl20 is added to retinal ganglion ceUs growing under labora- tory conditions, the cells are injured. How does this happen? In general, when cells take up Ca"^"^ in an uncontrolled fashion, they die. Dr. Lipton has shown that there is a certain kind of receptor for the neurotransmitter glutamate that is apparentiy involved in this process. Glutamate is actively taken back into ceUs after release as a neurotransmitter so that it can be reused. Apparentiy gpl20 can hin- der this "reuptake." Recentiy, Dr. Lipton has discovered a novel antagonist to the glutamate receptor that may give protection from gpl20 neurotoxicity. In addition to its affect on glutamate, gpl20 can also cause macro- phages — ceUs that can swallow up and de- stroy bacteria and foreign materials — ^to re- lease substances toxic to neuronal cells. Retinal Neuroscience— Molecular Basis of Signaling Vision begins when cells in the retina called rods and cones capture Ught and initiate a series of biochemical events that send an elec- trical message to the brain. Rod cells respond to dim light. The red-, green-, and blue-sensi- tive cones are sensitive to bright Ught and give us color vision. The orchestrated interplay of molecules in photoreceptors is called signaling or visual phototransduction and describes the way Ught is converted to an electrical signal destined for the brain. Light, which is focused by the cornea and the lens, enters the retina as energy particles caUed photons, which are absorbed by rho- dopsin. Attached to rhodopsin is a smaU molecule, vitamin A, which changes its molec- ular configuration sUghtiy when struck by a photon. This change in configuration or isom- erization is the initial event in a process caUed the transduction cascade, which now becomes rapidly amplified as rhodopsin coUides with many molecules of transducin. Transducin also has a smaller molecule attached to it. In this case, it is not a vitamin but a nucleotide called guanosine triphosphate (GTP). The binding of GTP to transducin is a consequence of the initial contact with rhodopsin. Trans- ducin then dissociates into separate parts, one of which binds to yet another molecule caUed phosphodiesterase (PDE), activating it. PDE, an enzyme, actuates the triggering event in phototransduction — ^the conversion of cGMP into an altered form caUed simply GMP to denote the fact that it is no longer a circular molecule. This hydrolysis or spUtting of cGMP closes a molecular gate in the mem- brane of the rod ceU resulting in a decreased flow of smaU ions Uke sodium into the ceU. This change in membrane potential of the ceU generates an electrical signal to the neighbor- ing retinal ceUs and then to the brain for sig- nal decoding. Current research has now turned to estab- Ushing the complete molecular basis of signal- ing in visual ceUs, signal termination, and adaptation. Knowledge of the structure of 12 Extramural Research each of the components of the phototransduc- tion cascade is necessary to understand the mechanisms behind the dynamics of, and potential sites for, regulation of signaling in visual cells. The electrical events that initiate vision begin with the capture of light that leads to the closing of cGMP-controUed membrane ion charmels. These ion channels directly control the flow of ions across the outer cell mem- brane. Rods and cones use similar but not identical molecvdar components to transduce Hght into neural signals to the brain, having their own distinct visual pigments as well as enzymes necessary for phototransduction. In general, although cones appear to have a similar phototransduction scheme to rods, the exact mechanisms underlying their low sensi- tivity and quick response to Ught remain un- clear. To study the basis for these differences Dr. Molday in Vancouver, British Columbia, has cloned the cGMP-gated channels from rod and cone photoreceptors of the chicken retina and examined their molecular and electrical properties. Using a special technique to engi- neer the cells to express specific molecules, he demonstrated that chicken rod and cone cells each synthesize different forms of cGMP-gated channels. The characterization of the rod channel protein is of great scientific interest because of its central involvement in phototransduction. Dr. Yau, from The Johns Hopkins School of Medicine, has cloned a protein from human retina that has about one-third similarity in structure to a similar protein from bovine retina. By itself, the human-derived protein does not self-associate to form a functional working channel. However, when combined with the channel protein isolated from bovine retina it functions characteristically like the native channel. What does this mean? It implies that the newly discovered human protein is but one of a group of different proteins that when assembled together form the native charmel protein. It is a clue that the channel protein may be made up of different subunits, a characteristic shared by other channel proteins that are controlled by small molecules like cGMP. At the end of a signaling event, the mole- cules involved must return to their original state. A key feature in the recovery and reac- tivation of phototransduction is the involve- ment of the ion calcium in Ca**-sensitive regu- lation of guanylyl cyclase (GC), an enzyme that synthesizes cGMP. During visual trans- duction, closure of the cGMP-gated channels reduces Ca** influx, while efflux by Na^ K^ and Ca*"^ continues, causing a decrease in Ca** within the cell. This fall in Ca^* stimulates GC and allows recovery of the sensitivity to Ught. Dr. Palczewski, from the University of Wash- ington, has shown that the regulation of GC is mediated by a novel photoreceptor-specific protein. The cloning and characterization of this molecule have shown there are three areas on the molecule that can bind Ca**, which suggests it has features in common with a large family of molecules called calci- um-binding proteins. In addition. Dr. Hurley, from the University of Washington, has shown that human photoreceptors use similar physio- logically relevant proteins in responding to and recovering from Ught. Human color vision involves three sepa- rate Ught-capturing molecules, caUed the red, green, and blue photopigments, to designate the wavelength of Ught to which they are most receptive. Although the sequence of amino acids for the red and green pigments are identical, except for 15 of their 365 amino acids, their Ught absorption characteristics differ sigruficantly. In an extensive study using a technique caUed mutagenesis to selec- tively and individuaUy alter specific amino adds. Dr. Oprian, from Brandeis University, has determined that there are only seven ami- no acids responsible for the Ught absorption differences between the red and green pig- ments. The mechanisms by which these ami- no acids determine the Ught absorbing proper- ties of the photopigment are unknown. How- ever, they do indicate how sensitive biological molecules can be to small changes in amino add composition. These changes can have health impUcations when the change or "muta- 13 FY 1994 NEI Annual Report tion" leads to disease. A good example is RP, where certain mutations in the gene coding for rhodopsin result in disease. On the other hand, some mutations in the gene coding for rhodopsin can manifest themselves only in an altered sensitivity to wavelengths of light with no impact on quality of life. The physical structure of the biologically active subunit of rod cell transducin was de- scribed recentiy by Dr. Hamm, from the Uni- versity of Illinois. Her research suggests how "nucleotide exchange" might occur in photo- transduction so as to induce changes in the surfaces of proteins to activate them and ex- plain a mechanism for GTPase activity not evident from previous studies. Dr. McKay, from Stanford University, has determined the crystal structure of recoverin, a recentiy dis- covered member protein that serves as a calci- um sensor. Ca*^ plays a critical role in the recovery phase of visual excitation and in adaptation to background Ught. The light- induced lowering of Ca"^* in retinal rod outer segments appears to function in system recov- ery after a Ught response. With the recent discovery that calcium-bound recoverin pro- longs the photoresponse, most likely blocking the phosphorylation of photoexdted rhodop- sin, the information obtained on the crystal structure will lead to further insights about recovery and adaptation in vision. Corneal Angiogenesis The growth of new capillary blood vessels, neovascularization or angiogenesis, occurs during many normal ocular processes such as wound healing and embryonic development. However, angiogenesis can also be a compo- nent of serious eye pathologies such as diabet- ic retinopathy, corneal clouding following infection or traumatic injury, and neovascular glaucoma. The presence of blood vessels in the cornea makes it much more difficult for a transplanted cornea to survive and remain clear. Current treatment of corneal neovasculari- zation relies on the use of topical steroids. Unfortunately, these agents have significant side effects, including immunosuppression, osteoporosis, stomach ulcers, and diabetes. Dr. Proia, from Duke Uruversity School of Medicine, and Dr. Schwartzman, from the New York Medical College, are developing new categories of angiostatic drugs to inhibit new vessel growth with a more favorable therapeutic profile. Recent advances include the discovery of a novel class of angiostatic steroids with fewer side effects. An indepen- dent family of angiostatic substances, Uke the cytochrome oxidase P450 inhibitors flurbipro- fen and clotrimazole, has also been recentiy described. These drugs decrease corneal inflammation and neovascularization by affecting the levels of prostaglandins synthe- sized during the inflammatory process. Lens Development Lens formation begins with lens epithelial ceU division and proceeds with differentiation into fiber cells. Fiber cells are terminally differenti- ated cells that are essentially "sacs" of proteins devoid of cellular structures. Continuous differentiation of epithelial cells into fiber cells is required to maintain lens tiansparency. Any disruption of the internal controls that balance growth and differentiation has impli- cations for cataract formation. Determining the signals that control epi- thelial cell division and differentiation into fiber cells has been an area of active investiga- tion. Using model systems. Dr. Paul Over- beek, from the Baylor College of Medicine, has shown the importance of growth factors in this process. Growth factors are a diverse group of small molecules that can trigger cell division. A challenge to lens biologists is to determine which of the many known growth factors are responsible for cell differentiation. Scientists have identified a number of poten- tially important growth factors including insu- Un-Uke growth factor I (IGF-I), fibroblast growth factor (FGF), and a novel factor found in the vitreous — ^lentropin. Dr. Overbeek has been stud5dng these growth factors by putting the genes for their expression into a mouse and creating what is called a transgenic mouse so that one can assign a functional role to 14 Extramural Research various growth factors. Overexpression of IGF-I and FGF in the lens of transgenic mice leads to congenital cataracts, substantiating the importance of these molecules in lens develop- ment and providing scientists with a model for cataract formation. Dr. DePinho, from the Albert Einstein Medical School, and Dr. Creep, from the University of Wisconsin, are interested in how lens development is controlled. Because fiber cell formation is essential for maintenance of lens transparency, preserving the balance between cell division and cell differentiation is critical. Recentiy, the retinoblastoma (Rb) and protein(p)53 tumor suppressor genes have been shown to play a role in eye development. Transgenic mice in which the normal function- ing Rb gene is knocked out demonstrate a condition caUed microphthalmia (small eyes). In these mice, lens cell DNA s5Tithesis pro- ceeds unimpeded while cell elongation and differentiation is inhibited. Continuous epi- thelial cell division is incompatible with nor- mal lens functioning. To avoid this problem it appears that p53 acts as a backup regulatory gene. Thus, in the absence of Rb activity, p53 is expressed causing apoptosis or programmed cell death and resulting in the microphthalmic condition. Neural Processing of Visual Information Understanding visual processing and its disor- ders requires knowledge of the human ner- vous system, including molecular, genetic, chemical, cellular, and integrative processes that underlie perception and the control of eye movements. As this knowledge increases it will help research aimed at preventing or treating disorders such as strabismus (mis- alignment of the eyes), amblyopia (commonly known as "lazy eye"), myopia, and neuro- ophthalmological disorders. Although disorders of visual processing may not always cause total blindness, they may seriously diminish the quaUty of life of those they afflict. Because these conditions affect more than 10 percent of the population, they constitute serious public health problems. Continued advancement of clinical investiga- tion in this field rests upon an improved un- derstanding of basic visual mechanisms. Development Using a new technique to trigger activity in selected nerve cells in a circuit. Dr. Katz, fiom Duke University, has shown that developing visual circuits are quite different than anatom- ical studies alone would indicate. Katz and his colleagues are using an approach he devel- oped called "laser photostimulation" or "caged glutamate" to show how the wiring in the mammaUan visual system changes during development. The nervous system is a dense network of many individual nerve cells that communicate with each other at speciaUzed gaps called synapses by chemical messengers called trans- mitters. The sending cell releases the trans- mitter at the synapse and the receiving cell responds if it has an appropriate receptor for the transmitter. Currently, Dr. Katz is explor- ing the visual circuits in the brain that use one common transmitter, glutamate. Thin slices of the nerve tissue are bathed in glutamate, which is "caged" — each molecule is trapped within a surrounding molecule that renders it inactive. A tiny electrode is inserted inside a single nerve cell and the surrounding tissue is bombarded with minute spots fiom an ulfiavi- olet laser. This bombardment releases the glutamate from its chemical cage causing that single nerve cell to become active. This gives researchers information on how the surround- ing neurons functionally interact. Initial results indicate that the visual system does not produce large numbers of neurons that are subsequently pruned during development. Instead, there is more of a redistribution of synapses in a circuit rather than an eHmination during development. Of greater significance for the field of neurosci- ence, however, is that this method promises to become a powerful tool to trace how networks of brain ceUs function and organize informa- tion. 15 FY 1994 NEI Annual Report Dr. McConnell, from Stanford University, is investigating how the highly organized cellular patterns and connections among nerve cells in the adult visual cortex arise during development. When these nerve cells are "bom," they receive instructions to leave the site of their origin and migrate long distances through a complex environment to other sites in the brain where they acquire a new identi- ty. Dr. McConnell wants to determine if an individual nerve cell "knows" who it is during its development. This is done by transplant- ing young nerve cells (whose normal fate she can predict) from one brain to another and then allowing the behavior of the transplanted nerve cell to reveal its "commitment" to its normal identity. What has been found is that by the time they are bom, nerve cells seem to "know" exactly who they are. In fact the pro- genitor cells, or mothers of the young cells, actually make decisions about the fates of the progeny, and they do so in a way that is ex- quisitely sensitive to the local environment in which the young nerve cells are generated. Dr. McConnell has been tr5dng to find out how the progerutor cells generate different types of nerve cells at specific times in devel- opment. It appears that the progenitors can- not make the right choice without information from neighboring cells. The progenitor cells are either sending signals to one another or receiving them from other nerve cells, which provide instructions that are essential for nor- mal fate determination. Currently, studies are aimed at trying to identify the molecules and genes that are involved in this process. This research will provide us with insights into how the complex connections of the brain are formed and how inappropriate connections are established when this process is flawed. Personal Guidance System for the Visually Impaired Drs. Loomis and Klatzky, researchers from the University of California, Santa Barbara, are developing a "personal guidance system" that "shows" the environment to blind users. The users wear stereo earphones mounted on glasses through which they receive informa- tion that tells them how to navigate through an unfamiliar environment. The system, housed in a backpack, picks up signals from the global positioning system (GPS) that was developed for the military and is transmitted from satellites. The system integrates this positional information with a geographic information system (CIS) in the form of a computerized map to create a "virtu- al acoustic display" that the user perceives as a talking map where preprogrammed objects and landmarks announce themselves as words through the earphones at the appropriate time and volume to cue the user to their precise location as he or she navigates. This provides the user with information on distance and direction for successful navigation through an unfamiliar environment. The device relies on triangulation of signals from GPS satellites to determine precise location. The information is transmitted to the system's on-board computer that contains a digitized map. The user wears an electroiuc compass that tells the system exactiy where he or she is. The system then generates speech that the user perceives as coming from the location of the object or land- mark in the real world. This allows the user to preview a trip permitting a rehearsal of a planned walk. Other workers are developing similar systems that use the GPS, but this is the first one to use a virtual acoustic display. Dr. Loomis and his colleagues plan to minia- turize the system further to make it less bulky and easier to carry. A cane or a guide should still be used with this system to inform the user of any obstacles that are not on the com- puter map. When the personal navigation system is fuUy implemented, it promises to expand the mobility of bUnd persons for navi- gating through unfamiliar areas and signifi- cantiy improve their ability to carry out every day tasks. 16 Extramural Research Division of Collaborative Clinical Research Richard Mowery, Ph.D., Director The Division of Collaborative Clinical Re- search plans and directs a program of grant, cooperative agreement, and contract support for appUed cUnical vision research, including clinical trials, natural history studies, surveys, cohort studies, and case-control studies. Cur- rently, the division manages 19 clinical trials, 17 epidemiology projects, three eye health education demonstration projects, and 10 analysis and planning projects with an annual budget of $38.6 million. Following are highlights of some of the major findings from these research projects. Retinal Diseases Macular Degeneration AMD is the leading cause of legal blindness in the United States for persons older than age 60. Despite the major public health signifi- cance of AMD, the cause of the disease is not understood and major risk factors, other than age, have not been determined. For instance, a case-control study of risk factors for AMD was conducted on Long Island, New York, among individuals ages 50 to 79. Patients with "wet" (exudative) AMD and patients with "dry" (nonexudative) AMD were compared with individuals without the disease. An increased risk for both AMD types were asso- ciated with current smoking, light eye-color, family history of AMD, poorly controlled hypertension, and certain nutritional factors. This study indicates that many causative fac- tors may contribute to the disease. Beneficial treatment effects of laser photo- coagulation for some of the people with the neovascular form of the disease have been reported for the Macular Photocoagulation Study. However, laser photocoagulation does not restore severe vision loss due to the dis- ease, and some people lose vision despite treatment. Arumal research and preUminary epidenni- ologic studies suggest a protective role for certain micronutrients in the development of both cataract and macular degeneration. In particular, vitamins and certain trace minerals with antioxidant capabilities are being studied for their role in age-related vision loss. Cur- rently, published studies are intriguing but are not conclusive in establishing a role for antiox- idant nutrients in the development or worsen- ing of AMD or cataract. The Age Related Eye Diseases Study (AREDS) has as one of its goals to evaluate the effect of high-dose antioxidant vitamins and zinc on the progression of macular dis- ease and lens changes. Patients with minor to severe drusen pathology and minimal lens opacities are being enrolled in the AREDS, randomized to a high-dose dietary supple- ment or placebo, and followed for a minimum of seven years to assess the progression of both eye disorders. Because patients enrolled will not have optically significant lens opaci- ties, this study will also provide information regarding the risk factors for cataract forma- tion. The prospective nature of this study, its focus on the progression of drusen pathology, and its evaluation of the effects of high-dose supplements make this a unique and much needed study. Eleven clinical centers, a coor- dinating center, a photographic reading center, a drug distribution center, a central laboratory, and the provider of the supplements — Ameri- can Cyanamid/Storz-Lederle — participate in the study. Patient enrollment and randomiza- tion are ongoing. Diabetic Retinopatliy A well-coordinated pubUc health approach to diabetic retinopathy requires accurate data regarding the incidence and associated risk factors for the disease. Diabetes is a major cause of visual impairment and blindness in the United States. Results from a 10-year population-based cohort study conducted in southern Wisconsin recentiy reported the 10- year incidence rate of blindness (visual acuity of 20/200 or worse) in diabetic persons was 1.8 percent, 4.0 percent, and 4.8 percent in FY 1994 NEI Annual Report younger-onset, older-onset taking insulin, and older-onset not taking insulin, respectively. This rate of blindness increased with age and with duration of diabetes. Respective 10-year rates of visual impairment (visual acuity of 20/40 or worse) were 9.4 percent, 37.2 percent, and 23.9 percent. For all three diabetic groups, macular edema or more severe reti- nopathy was associated with greater visual loss. Also, visual loss was associated with smoking (younger-onset group) and increased systoHc blood pressure (older onset group taking insuUn). These data indicate visual loss is common in diabetic populations and identi- fy several modifiable risk factors for visual loss due to diabetes. The data also assist in the estimation of costs for diabetic care. Retinopathy of Prematurity Retinopathy of prematurity (ROP) is a serious pubUc health problem among low-birth-weight infants. The NEI supported a multicenter trial of Cryotherapy for Retinopathy of Prematur- ity, which demonstrated that cryotherapy reduces by approximately one-half the risk of unfavorable retinal and functional outcome from threshold ROP. Cryotherapy, although helpful, is expensive, ablates a significant portion of the retina, is not always successful, and its long-term sequelae are unknown. Despite the highly significant advances in the treatment of ROP by crvotherapy, ROP continues to affect an increasing number of very low-birth- weight survivors. The NEI is currently supporting the Supplemental Thera- peutic Oxygen for Prethreshold Retinopathy of Prematurity (STOP-ROP) Study. The STOP- ROP is a multicenter, controlled cUnical trial initiated to determine if supplemental thera- peutic oxygen wiU reduce the proportion of infants with prethreshold retinopathy of pre- maturity who advance to threshold ROP. In addition to ophthalmic outcomes, this study wdll obtain neonatal outcome information, including data on growth, ventilatory stability, chronic lung disease, neurological maturation, and length of hospital stay. Recruitment for this study began in early 1994 and is being conducted at 20 clinical centers in the United States. In addition to the support being pro- vided by the NEI, the Institute has arranged collaboration and support for this study from the National Institute for Quid Health and Human Development (NICHD) and the Na- tional Institute for Nursing Research. Cytomegalovirus Retinitis C5^omegalovirus (CMV) retinitis is by far the most crucial ocular problem for people with AIDS who Uve more than one year. A poten- tially bUnding disease of the retina, CMV retinitis affects between 25 and 30 percent of people with AIDS. Through the Studies of the Ocular CompUcation of AIDS (SOCA), a net- work of investigators with expertise in AIDS clirucal research, retinal disease, and clinical trial methodology, the NEI has expedited the testing of tieatments for CMV retinitis. These investigators demonstiated the safety and efficacy of foscamet for the treatment of CMV retinitis. Foscamet was also shown to extend the life expectancy of people with AIDS by approximately four months compared with patients who took ganciclovir. AU patients v\dth CMV retinitis relapse when anti-CMV therapy is discontinued. Therefore, continu- ous maintenance therapy is required. Even on continuous maintenance therapy, all patients with CMV retinitis eventually relapse. Although relapse can often be controlled, with each relapse, additional retina is des- troyed. Therefore, treatment strategies de- signed to prolong the time to relapse are needed. The SOCA investigators are currentiy comparing the use of combination therapy using foscarnet and ganciclovir with standard therapy to see if it will extend the time to relapse and disease progression. Other stud- ies that are being designed wiU use newly developed oral compounds and implant devices to look at safety and efficacy issues, extension of relapse time, plus the improve- ment in quality of Ufe. 18 Extramural Research Glaucoma Epidemiology Glaucoma is a major cause of visual impair- ment in the African-American populations. African Americans have an earUer age of onset of the disease compared with Caucasians, suffer bUndness at a greater rate, and may be more resistant to treatment. Unfortunately, few well-designed epidemiologic studies have examined the prevalence rate of glaucoma in African-American populations and Umited information exists on risk factors for the disease. The Barbados Eye Study is a population based-prevalence survey that was recently conducted among 4,709 residents of Barbados ages 40 to 84. The prevalence of glaucoma was found to be seven percent. Male gender and a family history of glaucoma were associ- ated with the disease. Open-angle glaucoma was diagnosed by strict definition, including both visual field and optic disc criteria. This miivimum, conservative prevalence estimate of glaucoma was high, particularly among those older than age 50. If study results are extrap- olated to the population of Barbados, the prevalence of glaucoma is one in 11 among persons older than age 50, one in rune in per- sons older than age 60, and increases to one in six in persons older than age 70. Comparison of this study with that conducted in 2,395 urban African Americans and 2,913 urban Caucasian residents of Baltimore, indicates the age-adjusted prevalence of glaucoma in Barba- dos is 1.5 times higher than that in the Balti- more African- American population and 7.1 times higher than in the Baltimore Caucasian population. As such, a higher prevalence of glaucoma in African-Caribbean populations compared with African- American popvdations is found and these differences raise the ques- tion of possible genetic factors. The contribution of genetic factors to glaucoma are planned for investigation in a study designed to address familial aggregation of the disease. The surviving parents, sibUngs, spouses, and children (older than age 40) of 100 Barbados participants with glaucoma will be examined for eye disorders, and risk factor information wiU be obtained. This informa- tion wiU increase our understanding of the inheritance of glaucoma and have direct clini- cal implications in identifying those family members at high risk for developing the dis- A report of family history from the Balti- more Eye Survey, a population-based preva- lence survey conducted among 5,308 African- American and Caucasian residents of east Baltimore as a risk factor for glaucomas, has been published. Glaucoma was diagnosed in 161 participants in this survey. Participants in this survey were interviewed to determine if first-degree relatives (parents, siblings, and children) had glaucoma. Results indicated a higher risk of glaucoma in siblings than in parents. However, these estimates may be biased because glaucoma was self-reported, not diagnosed, and prior knowledge of glau- coma diagnosis among participants influenced their recall of affected individuals. Treatment Currentiy, the most common treatment for glaucoma is the use of medications to lower pressure in the eye. Although the medications have been demonstrated to be very effective at lowering pressure, the impact of lowering pressure on visual field loss is not completely understood. The economic effect of glaucoma treatment with medications is substantial, accounting for millions of doUars in yearly expenditures in the United States alone. Fur- thermore, these pressure-lowering medications can often have significant side effects and require the patient to adhere to a strict admin- istration schedule, thus potentially having a profound effect on a patient's quality of Ufe. The NEl is currentiy supporting three new cUnical trials designed to evaluate treatment strategies for ocular hypertension and glauco- ma. The Ocular Hypertension Treatment Study is designed to determine whether medi- cal reduction of intraocular pressure prevents or delays the onset of visual field loss and /or 19 FY 1994 NEI Annual Report optic disc damage. Recruitment of patients began in early 1994 at 21 clinical sites throughout the United States. Fifteen hun- dred ocular hypertensive subjects, at least 500 of whom will be African Americans, are being randomly assigned to either close observation or to a stepped medical regimen. Participants will be followed for a minimum of five years. The Early Manifest Glaucoma Trial is a randomized, controlled clinical trial designed to determine whether and to what extent reduction of intraocular pressure (lOP) influ- ences the course of chronic open-angle glauco- ma. Investigators at the University of Lund in Makno, Sweden, collaborating with investi- gators from the State University of New York at Stony Brook, are studying 300 patients vsdth newly diagnosed glaucoma. Participants are being randomized to receive pressure lower- ing treatment or observation with no or de- layed treatment. Both groups are followed closely with computerized perimetry and fundus photography. Recruitment of patients began in 1993 and will continue for an esti- mated two to three years. FoUowup of pa- tients will be conducted for approximately four years. Filtration surgery can also be effective in controlling lOP. When successful, filtration surgery may provide the most effective long- term, consistent control of lOP with the least Ukehhood of requiring supplemental medica- tions. The Collaborative Initial Glaucoma Treatment Study is designed to compare the efficacy of a medical regimen with filtration surgery as the initial treatment for newly diagnosed glaucoma. This clinical trial will compare the two treatment strategies in terms of controUing TOP and will specifically investi- gate the impact of these treatment strategies on the participants quality of Ufe. Recruit- ment of the 600 patients began in November 1993. The study is being conducted at 11 clinical centers located throughout the Uruted States. Corneal Diseases Prospective Evaluation of Radial Keratotomy Study Approximately 25 percent of adults in the western world have myopia. Some of these people may be candidates for radial keratoto- my (RK), a procedure aimed at correcting or reducing myopia through surgical incisions made in the cornea. The NEI has been sup- porting a multicenter controlled clinical trial designed to evaluate the short- and long-term safety and efficacy of a single standardized technique of RK. The five-year followoip results from the Prospective Evaluation of Radial Keratotomy (PERK) Study indicated that RK was safe and that few very serious complications resulted from the procedure. However, it was difficult to predict the outcome for an individual eye. In addition, the refractive error continued to change in some patients over time. In 1994, the PERK investigators completed 10-year foUowup examinations of patients who were enrolled in the PERK Study. Data from these examinations will soon be available and wiU be one of the few sources of informa- tion on the long-term stability of RK. Strabismus, Ambiyopia, and Visual Processing Optic Neuritis and Multiple Sclerosis More than one-half of aU people with first- time optic neuritis, a vision-impairing inflam- mation of the optic nerve that affects more than 25,000 Americans each year, will eventu- ally develop multiple sclerosis. Multiple scle- rosis is a debOitating disease of the central nervous system that affects as many as 500,000 Americans. Based on data from two years of foUowup of patients enrolled in the Optic Neuritis Treatment Trial, researchers found that treating first-time optic neuritis patients with a combination of intravenous and oral 20 Extramural Research corticosteroids lowers their risk of developing miiltiple sclerosis. The results from this re- search, pubUshed in Tlie New England Journal of Medicine, offer the first scientific evidence that intravenous corticosteroids help to delay the progression of multiple sclerosis. It also suggests that this treatment may provide simi- lar benefits for people with not only optic neuritis but other early symptoms of multiple sclerosis. Myopia Results from an NEI-supported study pub- lished in the Journal of the American Medical Association suggest a genetic Unk to myopia. Researchers from the University of California, Berkeley, School of Optometry report on 716 school children ages six to 14 who are enrolled in the OLSM. Measurements were made on each child's refractive error, corneal curvature, crystalline lens power, and axial ocular dimen- sions. Parents reported their own vision status. The investigators found that school children whose parents are myopic have different shaped eyeballs than children whose parents are not nearsighted. Children of myopic parents had longer eyes even before the onset of myopia. These results suggest that the premyopic eye in children with a family history of myopia already resembles the elongated eye present in myopia. The OLSM wiU provide a wealth of infor- mation on ocular and refractive development in children in the years ahead. Future results may provide the eye care provider or pediatri- cian with the answer to the question frequent- ly asked by myopic parents — "What are the chances that my child will develop myopia?" Ocular Injuries Ocular trauma is a major cause of monocular blindness in the United States and, in 1986, was responsible for estimated hospital costs of up to $200 mUlion. Self-reports of lifetime ocular injuries were obtained in the popula- tion-based Baltimore Eye Survey of adults older than age 40. Ocular injuries were re- ported by 14.4 percent of participants (n=5308); men reported a greater number of ocular injuries than women. The number of injuries were similar among both African- American and Caucasian men, however, visu- al consequences of injuries were more severe among African- American men. 21 Division of Biometry and Epidemiology Report of the Acting Director, Division of Biometry and Epidemiology Roy C. Milton, Ph.D. The Division of Biometry and Epidemi- ology (DBE) is made up of a Clinical Trials Branch, an Epidemiology Branch, and a Biometry Section. Dr. Roy Milton is the Acting Director for the division. Drs. Frederick Ferris HI and Robert Sperduto serve as chiefs of the two branches, respective- ly; Dr. Roy Milton is the head of biometry. The DBE has three main functions: re- search, education, and consultation. Research is the dominant function. It is the division's mission to plan, develop, and conduct human population studies concerned with the cause, prevention, and treatment of eye disease and vision disorders, with emphasis on the major causes of blindness. This includes studies of incidence and prevalence in defined popula- tions, prospective and retrospective studies of risk factors, natural history studies, clinical trials, genetic studies, and studies to evaluate diagnostic procedures. The DBE carries out a program of educa- tion in biometric and epidemiologic principles and methods for the vision research communi- ty. This program consists of courses, work- shops, a fellowship program for ophthalmolo- gists, publications, and consultation and collaboration on research. The DBE provides biometric and epidemi- ologic assistance to the NEI intramural and extramural staff and to vision research work- ers elsewhere. The assistance ranges from consultation to collaboration as coinvestigator. Research Highlights The Eye Disease Case-Control Study One of the diseases studied in the Eye Disease Case-Control Study was idiopathic macular holes, a disease by which women are more affected than men. Results from this study showed that higher fibrinogen levels and a history of glaucoma were associated with an increased risk of the condition. The use of exogenous estrogens was associated with a decreased risk. The fibrinogen finding was unexpected, and it is not clear whether this is a chance finding or whether higher levels of fibrinogen can increase susceptibility to the forces of vitreous traction, perhaps by compro- mising the macular blood supply or by some yet unexplained mechanism. Within this large case-control study, a sub- study was conducted to evaluate the relation- ships between dietary intake of carotenoids and vitamins A, C, and E and the risk of neo- vascular macular degeneration, the leading cause of irreversible blindness among adults. A higher intake of dietary carotenoids was associated with a lower risk. Among the specific carotenoids, lutein and zeaxanthin were most strongly associated with a reduced risk of this form of macular degeneration. Several food items rich in carotenoids were inversely associated with macular degenera- tion. In particular, a higher frequency of in- take of spinach or collard greens was associat- ed with a substantially lower risk for AMD. 25 FY 1994 NEI Annual Report The Framingham Offspring Eye Study Eye examination data from 1,086 parents ex- amined in the Framingham Eye Study and 896 offspring examined in the Framingham Off- spring Eye Study (FOES) were used to study familial associations for nuclear, cortical, and posterior subcapsular lens opacities. For any pair of siblings, if one sibUng had a nuclear opacity the odds of the other sibling having such an opacity were estimated to be more than triple. Similar findings were noted for posterior subcapsular opacity. The strong associations between siblings for nuclear and posterior subcapsular opacities suggest there is clustering of lens opacities within families. The clustering may be due to genetic or envi- ronmental factors. The Italian-American Natural History Study of Age-Related Cataract The ItaUan- American Natural History Study of Age-Related Cataract has estimated the inci- dence and progression of cortical, nuclear, and posterior subcapsular opacities in a large fol- lowup study. The three-year cumulative inci- dence for persons ages 65 to 74 years (the largest group studied) was 18 percent, six percent, and six percent for cortical, nuclear, and posterior subcapsular opacities. Progres- sion was much higher than incidence for each type of opacity. The study suggested that patient age, baseUne lens status, cataract grading system, definition of change, and analytic methodology may have important effects on estimates of cataract incidence and progression. The Early Treatment Diabetic Retinopathy Study Recent publications from the Early Treatment Diabetic Retinopathy Study (ETDRS) multicen- ter, randomized clinical trial include an evalu- ation of the effect of aspirin versus placebo on mortality and morbidity from all causes and specifically from cardiovascular disease. This study is one of two primary prevention stud- ies of the effect of aspirin on cardiovascular disease and is the only large study to include women. The results are similar to the studies on males without diabetes and demonstrate that aspirin use reduces the risk of cardiovas- cular disease. Previous ETDRS publications had not reported any increase in the occurrence of vit- reous /preretinal hemorrhages in study pa- tients assigned to aspirin. With new data suggesting the importance of aspirin use in persons with diabetes who are at risk for cardiovascular disease, it was important to investigate this possible risk of aspirin use in more depth. Data published recentiy {ETDRS Report No. 20) demonstrate that the severity and duration of these hemorrhages were not significantly affected by the use of aspirin and that there were no ocular contiaindications to its use in persons with diabetes who require it for tieatment of cardiovascxilar disease or for other medical indications. Research Activities Clinical Trials The Early Treatment in Diabetic Retinopathy Study The ETDRS was designed to determine when to use photocoagulation for diabetic retinopa- thy. Patients with macular edema, preproli- ferative retinopathy, and mild or moderate proliferative retinopathy were studied. Vari- ous treatment strategies of focal and scatter (panretinal) photocoagulation were compared with no photocoagulation. In addition, the study evaluated the placebo-contioUed effects of daily administration of aspirin on the inci- dence of microvascular and macrovascular complications. The study also investigated factors associated with the progression of disease. Recruitment was completed in March 1985 with the enrollment of 3,711 patients. In De- cember 1985, the study reported that focal photocoagulation of clinically significant dia- betic macular edema substantially reduces the risk of visual loss. It was further reported that focal treatment increases the chances of 26 Division of Biometry and Epidemiology visual improvement, decreases the frequency of persistent macular edema, and causes only minor visual field losses. Subsequent reports indicated that whether treated early with scatter photocoagulation or followed closely and treated as soon as they reached the high- risk stage, all eyes had low rates of severe visual loss. Scatter photocoagulation is not recommended for eyes with mUd or moderate nonproliferative diabetic retinopathy, provided careful followup can be maintained. When retinopathy is more severe, scatter photocoag- ulation should be considered and usually should not be delayed if the eye has reached the high-risk proliferative stage. Sixteen ETDRS reports have been pub- lished and additional manuscripts are being prepared. Drs. Lloyd Aiello and Frederick L. Ferris, HI serve as cochairmen. Dr. Richard L. Mowery is project officer, and Dr. Emily Y. Chew serves as a member of the analysis planning group. The results of effects of aspi- rin on vitreous hemorrhage in patients with diabetes mellitus have been accepted for pub- lication. A report of accommodation in the ETDRS population has been submitted for publication. Analyses in progress include the association of serum Upids and retinal hard exudates, risk factors for severe visual loss, risk factors for development of high-risk pro- liferative diabetic retinopathy, fibrovascular proliferation associated with macular edema, and causes of severe visual loss. In collabora- tion with Dr. Thomas Gardner, from Hershey Medical Center, results of analyses of digoxin and retinopathy have also been submitted for publication. In addition, patients with mild to prolifer- ative retinopathy are being followed with ex- tensive psychophysical testing in the NEI Clinical Center, to determine the mechanisms for loss of visual acuity in diabetic retinopa- thy. An additional study of long-term foUow- up of diabetic retinopathy following laser photocoagulation is under way at the NEI CUnical Center. The Krypton-Argon Regression of Neovascularization Study The CUrucal Trials Branch began the Krypton- Argon Regression of Neovascularization Study (KARNS) in three pUot clinics in December 1983. The major objective of this randomized cUnical trial is to compare red krypton laser with blue-green argon laser panretinal photo- coagulation for treating neovascularization on the optic nerve head caused by diabetic reti- nopathy. Twenty-nine new cUrucs were en- rolled in KARNS starting in August 1984. At the termination of the study in June 1990, a total of 1,063 patients had been randomized. This study is unique for the NEI because the functions for both the coordinating center and the fundus photography reading center are being handled by staff of the Clinical Trials Branch. Another feature of this multicenter trial is that the participating clinics receive no financial reimbursement from the NEI for their participation. Drs. Ferris and Chew direct this study along with Dr. Lawrence Singerman. KARNS Report No. 1 {Ophthalmology, 1993) showed the two treatments were equally effective in arresting neovascularization of the disc; additional analyses are under way. The Linxian Eye Study The NEI joined an ongoing NCI-supported clinical trial of nutrition and cancer in north central China in 1991 to determine whether the vitamin /mineral dietary supplements administered in the Linxian Cancer Trials for the preceding five years have affected the risk of age-related cataract and AMD. Eye exami- nations were conducted in 1991 on 5,390 members of the Linxian Study cohort. Dr. Sperduto is project officer, and the project team includes Drs. Milton and Chew from DBF and Dr. Tian-Sheng Hu, an ophthalmolo- gist from Beijing. Findings for cataract have been pubUshed. Analyses are being conduct- ed for the macular degeneration component. 27 FY 1994 NEI Annual Report The Italian-American Trial of Age-Related Cataract A new collaborative Italian-American cUnical trial is being planned to study the effect of a broad spectrum vitamin /mineral supplement on the risk of age-related cataract. Twelve hundred people will be randomized to either the supplement or a matching placebo and followed for five years. Recruitment into the study will begin in early 1995. The study wlU complement the ongoing AREDS, which is being conducted in the United States. Dr. Sperduto is the project officer and Dr. Milton is a member of the project team. Intramural Program Clinical Trials Drs. Ferris and Chew and Ms. Remaley are collaborating with Dr. Nussenblatt on four additional randomized cUrucal trials in the NEI Intramural Program of the Clinical Cen- ter: (a) a trial of a sustained-release intraocu- lar drug deUvery system for ganciclovir thera- py of cytomegalovirus retinitis in patients with AIDS; (b) a trial to evaluate the efficacy of a heparin-surface modified intraocular lens in reducing the incidence and severity of post- operative inflammatory episodes following extracapsular surgery in uveitis patients with cataracts; (c) a trial of anti-inflammin, a pep- tide, in the treatment of anterior uveitis; and (d) a trial of oral S-antigen or retinal extract versus placebo in patients with uveitis. Other Dr. Ferris now represents the NEI on the Data Monitoring Committee of the United Kingdom Prospective Diabetes Study, a clinical trial of alternative treatment regimens in the manage- ment of patients with diabetes. FoUowup is scheduled to continue in this study until 1997. Epidemiology The Age-Related Eye Disease Study The AREDS is designed to coUect natural history data of 4,600 patients of ages 55 to 80 years with bilateral drusen of different types or with unilateral advanced AMD. This study will evaluate the rates of development and progression of AMD, the rates of visual loss due to retinal lesions of AMD, and the risk factors associated with the development and progression of AMD. Evaluation of lens change over the 10-year AREDS study period will provide an opportunity to evaluate factors associated with the development of cataracts. In addition, a cUnical trial wiU be performed to determine whether antioxidants (vitamin C, E, and beta-carotene) and zinc can prevent the development or retard the progression of AMD and cataract. There are 11 cUnical cen- ters, a photographic reading center, a central laboratory, and a coordinating center. Identi- fication of study participants began in Septem- ber 1990. In November 1992, participants were evaluated with qualifying visits, and participants were randomly assigned to the study medications begirming in February 1993. Dr. Ferris, chairman; Dr. Sperduto, director of lens project; and Dr. Chew are directing the scientific aspects of the AREDS; Dr. Natalie Kurinij is project officer. The Eye Disease Case-Control Study The Eye Disease Case-Control Study is de- signed to identify risk factors for neovascular macular degeneration, idiopathic branch reti- nal vein occlusion, idiopathic central retinal vein occlusion, rhegmatogenous retinal de- tachment, and idiopathic macular hole. Dr. Sperduto is the study chairman, Ms. Rita Hil- ler is director of data analysis, and Dr. Chew is a member of the project team. Two papers were published this year: risk factors for idio- pathic macular hole and the effect of dietary carotenoids and vitamins A, C, and E on neo- vascular age-related macular degeneration. The Diabetes in Early Pregnancy Study Dr. Chew and Ms. Remaley, in collaboration with Dr. James Mills of the NICHD, examined the effects of pregnancy on diabetic retinopa- thy in the Diabetes in Early Pregnancy Study. Data collection terminated in 1985, and a man- uscript has been submitted. Further analyses on the effects of pregnancy in proliferative retinopathy are planned. 28 Division of Biometry and Epidemiology The Italian-American Natural History Study of Age- Related Cataract The Italian- American Natural History Study of Age-Related Cataract was designed to esti- mate the rates of development and progres- sion of the different types of lens opacities and the associated risk factors. Dr. Sperduto is the project officer; Dr. Milton and Ms. Remaley are members of the project team. Three pa- pers were pubUshed this year, including one that provides estimates of the rates of cataract development and progression. Analyses of risk factors for cataract development and pro- gression are under way. The Framingham Offspring Eye Study Dr. Sperduto is the project officer. Dr. Milton is the alternate project officer, and Drs. Podgor and Freidlin and Ms. HUler are members of the project team for FOES. This study is de- signed to examine famihal relationships for age-related cataract and AMD among parents examined in the Framingham Eye Study (1973 to 1975) and their children examined 1989 to 1991. Dr. Podgor has used generalized esti- mating equation methodology in the analyses of these data. A manuscript describing the study's findings for cataract has been accepted for publication. A manuscript describing the association of opacities between and within eyes of individuals has been submitted. Anal- yses of macular degeneration are planned. Other A manuscript has been accepted for publica- tion on risk factors for strabismus, using data from the NICHD Collaborative Study and in collaboration with Dr. Mark Klebanoff, from the NICHD. The DBF project team includes Drs. Chew, Tamboli, Zhao, and Podgor and Ms. Remaley. Esotropia developed in three percent and exotropia in 1.2 percent of the children followed for seven years. Esotropia was more common in Caucasians than in African Americans. The occurrence of exotro- pia was similar in the two races. Maternal cigarette smoking during pregnancy and low birth weight were independent and important risk factors for both esotropia and exotropia. Analyses are under way on sibling association in strabismus and on risk factors for congeni- tal cataract. Drs. Valerie Freidlin and Marvin Podgor continued to provide consultations with NEI CUrucal Center investigators, especially for various studies of measurement of lens opaci- ties. Dr. Freidhn is collaborating with Dr. EUwein in management and analysis of Medi- care data on ophthalmologist services. Statistical Methods Dr. Podgor and Dr. Joseph Gastwirth, from the George Washington University, collaborat- ed in an investigation of the use of scores for stratified data. A paper has been accepted for publication. Drs. Podgor, Gastwirth, and Cyrus Mehta, from Cytel Corporation and Harvard University, have proposed methodol- ogy for efficiency robust tests for ordered 2xK contingency tables. A paper has been submit- ted. Ongoing Activities Members of DBE are active in consultations and educational and professional activities, including referees for professional journals, associate editors or members of editorial boards, members of data and safety monitor- ing committees for clinical trials, training of staff fellows, invited and contributed presenta- tions at professional society and other meet- ings, advisory committees for grant-supported cooperative agreements, and technical advi- sors to the World Health Organization (WHO). Publications Chew EY, Klein ML, Murphy RP, Remaley NA, Ferris FL and The Early Treatment Dia- betic Retinopathy Study Research Group: Effects of aspirin on vitreous hemorrhage in patients with diabetes meUitus. ETDRS Re- port No. 20, Arch Ophthalmol, in press. 19 FY 1994 NEI Annual Report Chew E, Remaley NA, Tamboli A, Zhao J, Podgor MJ, BGebanoff M: Risk factors for esotropia ar\d exotropia. Arch Ophthalmol, in press. Ferris FL: How effective are treatments for diabetic retinopathy? Commentary. JAMA 269(10):1290-1291, 1993. Ferris HI FL, Freidlin V, Kassof A, Green SB, Milton RC: Relative letter and position diffi- culty on visual acuity charts from the Early Treatment of Diabetic Retinopathy Study. Am J Ophthalmol 116:735-740, 1993. Javitt JC, AieUo LP, Chiang Y, Ferris FL, Can- ner JK, Greenfield S: Preventive eye care in people with diabetes is cost-saving to the Federal Government. Implications for health- care reform. Diabetes Care 17(8):909-917, 1994. Magno BV, Freidlin V, DatHes MB: Reproduci- bility of the NEI Scheimpflug cataract imaging system. Invest Ophthalmol Vis Sci 35:3078-3084, 1994. Magno BV, Freidlin V, Lasa MSM, Datiles MB: Comparison of linear, multilinear and mask microdensitometric analysis of Scheimpflug images of the lens nucleus. Invest Ophthalmol Vis Sci, in press. Maraini G, Rosmini F, Graziosi P, Tomba MC, Bonacini M, Cotichini R, Pasquini P, Sperduto RD, and the Italian American Cataract Study Group: Influence of type and severity of pure forms of age-related cataract on visual acuity and contrast sensitivity. Invest Ophthalmol Vis Sci 35:262-267, 1994. Nussenblatt RB, De Smet M, Podgor M, Lane C, Polls M, Pizzo P, Perry C, Beifort Jr R: The use of the flarephotometry in the detection of cytomegalic virus retinitis in AIDS patients. AIDS 8:135-136 [letter], 1994. Podgor MJ: Review of Gibbons, JD (1993) Nonparametric Measures of Association. J Am Stat Assoc 89:719 [book review], 1994. Podgor MJ, Gastwirth JL: On nonparametric and generalized tests for the two-sample prob- lem with location and scale change alterna- tives. Stat Med 13:747-758, 1994. Podgor MJ, Gastwirth JL: A cautionary note on appl5dng scores in stratified data. Biomet- rics, in press. Rosmini F, Stazi MA, Milton RC, Sperduto RD, Pasquini P, Maraiiu G, and the Italian- American Cataract Study Group: A dose-re- sponse effect between a sunlight index and age-related cataract. Ann Epidemiol 4:266-270, 1994. Sastry SM, Sperduto RD, Waring GO, Remaley NA, Lynn MJ, Blanco PE, Miller DN: Radial keratotomy does not affect intraocular pres- sure. Refractive and Corneal Surgery 9:459-464, 1993. Seddon JM, Ajani UA, Sperduto RD, HiUer R, Blair N, Burton TC, Farber MD, Gragoudas ES, Haller J, Miller DT, Yannuzzi LA, Willett W: Dietary carotenoids, vitamins A, C, and E and advanced age-related macular degenera- tion — a multicenter study. JAMA, in press. Sperduto RD: Age-related cataracts — scope of problem and prospects for prevention. Prev Med, in press. The Eye Disease Case-Control Study Group: Risk factors for idiopathic macular holes. Am J Ophthalmol, in press. The Framingham Offspring Eye Study Group: Familial aggregation of lens opacities: the Framingham Eye Study and the Framingham Offspring Eye Study. Am J Epidemiol, in press. The Italian-American Cataract Study Group: Incidence and progression of cortical, nuclear and posterior subcapsular cataracts. Am J Ophthalmol, in press. 30 Office of International Program Activities Report of the Acting Assistant Director for International Program Activities Terrence Gillen, M.A., M.B.A. The mission of the NEI is to reduce the prevalence of blindness, visual impair- ment, and eye disease worldwide through basic and applied research and train- ing. Although excellent ophthalmic proce- dures and eye-care delivery systems are acces- sible in the developed world, adequate health care is not readily available in aU parts of the developing world. This widening gap in visual health between developed and develop- ing nations threatens to have ominous conse- quences. If present trends continue, the num- ber of blind people — estimated at 24 mil- lion — ^wUl more than quadruple during the next 40 years. As many as 90 percent of these blind people wiU Uve in developing countries. This large-scale disablement caused by bHndness is not only a costly obstacle to economic development, it is also a catastroph- ic loss of human potential in the areas of the world most desperately in need of a healthy w^orkforce. In addition, because more than 80 percent of aU cases of blindness can be consid- ered avoidable — that is, they could have been prevented or could be cured using available and locally appropriate technology — such deprivation is a truly needless denial of a basic human right for mUhons of people. Therefore, the NEI undertakes international activities to facUitate the development and application of effective prevention and inter- vention programs. These efforts are coordi- nated by the Institute's Office of International Program Activities (OIPA), which was created in February 1989. The OIPA enhances NEI's international programs by: • Evaluating available health technologies, promoting the most cost-effective inter- vention and prevention programs, and encouraging their availability for affected populations, especially in developing countries. • Conducting collaborative applied research studies to develop preventive methods for tieating specific eye diseases. • Conducting contioUed dinical evaluations of promising research findings. • Exchanging information on recent scientif- ic advances and their appropriate applica- tion to visual problems. The NEI supports international research on six blinding diseases that have a major worldwide effect: cataract, onchocerciasis, ocular toxoplasmosis, glaucoma, diabetic retinopathy, and vitamin A deficiency. Highlights of Recent Scientific Advances Resulting From International Activities Because cataract is responsible for about one- half of the developing world's curable bHnd- ness and is a major problem for the United States as well, the NEI has developed a collab- orative research program that includes projects to prevent blindness from cataract with collab- orating groups in Italy, India, and Latin Amer- ica. In addition, health services research expertise from the NEI is made available to 33 FY 1994 NEI Annual Report selected collaborating partners through train- ing activities and the conduct of joint research projects. For example, intramural scientists from NEl's Laboratory of Mechanisms of Ocular Diseases (LMOD) in collaboration with col- leagues at the Centre for CeUular and Molecu- lar Biology in Hyderabad, India, are studying aging-related modifications to lens crystalUns. These scientists have demonstrated that chem- icals present in smoke, either from tobacco products or from wood fires, can directly damage lenses in organ culture studies. In addition, molecular geneticists in the NEI's Section on Cataract have initiated gene linkage studies with scientists at Osmarua University and the L.V. Prasad Eye Institute in Hyderabad on selected families with heredi- tary cataract. The collaborative Italian-American Study of the Natural History of Age-Related Cataract has completed a four-year foUowup study of cataract. Objectives of the natural history study were to estimate the rates of develop- ment and progression of the various tj^es of lens opacities, identify risk factors associated with the development and progression of cataracts, and evaluate cataract classification schemes. A manuscript describing study results has been accepted for publication in the American Journal of Ophthalmology. Summary of International Programs and Activities Country-to-Country Activities Barbados Open-angle glaucoma is the leading cause of blindness in African Americans and is a major cause of visual impairment and disability. The incidence of glaucoma has not been mea- sured precisely in any population, and the risk factors related to its development are largely unknown. Since 1988, the Barbados Eye Study has examined more than 4,200 persons ages 40 to 86 years as part of a population-based study to determine the prevalence and risk factors for glaucoma and other eye disorders such as diabetic retinopathy, AMD, cataract, and visual impairment. In 1992, the Barbados Incidence Study was initiated to estimate the incidence of glaucoma and other ocular disor- ders in individuals in the Barbados prevalence survey who were free of disease. In addition, risk factor analysis wiU be conducted to iden- tify associations with development of glauco- ma and to characterize those who have pro- gressive eye disease. (See "The Barbados Eye Study: Prevalence of Open-Angle Glaucoma" in the June 1994 issue of Archives of Ophthal- mology, 112 (6): 821-829.) Brazil In collaboration with the U.S. National Insti- tute of AUergy and Infectious Diseases (NIAID), NIH and three Brazilian scientific organizations in Sao Paulo — Escola PauUsta de Medicina, CUnica Erexim, and Laboratory Fleury — ^the NEI developed a research pro- gram on the immunology, basic mechanisms, and epidemiology of toxoplasmosis in south- em Brazil. The prevalence of ocular toxoplas- mosis in this population was found to be more that 30 times higher than previous esti- mates for the same condition elsewhere. In this population, ocular toxoplasmosis appears to be a sequela of postnatal rather than con- genital infection. Studies performed in 1993 on postnatal blood from newborns in southern Brazil have shown a low percentage of immu- noglobtdin M positivity, further suggesting that the disease in southern Brazil is acquired. India The NEI and the Indian Council of Medical Research (ICMR) have developed a collabora- tive blindness research program under the 1983 Indo-U.S. Science and Technology Initia- tive. This program includes projects to reduce blindness from vitamin A deficiency, cataract, and Eales' disease in India. Indian govern- ment funds for the work come through the ICMR, and U.S. Government funds are pro- vided through the National Science Founda- 34 International Program Activities tion and the NEI. In addition, the NEI collab- orates with Indian scientists under the U.S.- Indo Subcommission program. The NEI director, deputy director, and special advisor to the director participated as consultants to the World Bank to develop a proposal by the Government of India for an initiative in cataract blindness control. Techni- cal meetings have been held in New Delhi and Madurai to provide the knowledge base on which training and surgical guidelines can be developed for a twofold expansion of cataract surgery, with expUcit attention to the quaUty and extent of vision restoration. Intramural scientists from the NEI, LMOD are collaborating with colleagues at the Centre for Cellular and Molecular Biology on studies on aging-related modifications to lens crystal- Uns. Cataracts typically occur at an earHer age and are more heavily pigmented in the Indian population than in the U.S. population. In an attempt to elucidate the molecular mechan- isms underlying this difference in color, the scientists are comparing over a wide range of ages the fluorescence spectra for normal intact lenses from the Indian population with age- matched Eye Bank lenses from the U.S. popu- lation. The Indian population lenses have signiftcantiy greater amounts of pigmented fluorescent compounds than do the U.S. popu- lation lenses. These compounds may play a direct role in cataractogenesis through their ability to function as photosensitizers. In organ culture studies, collaborating investigators have demonstrated that chemi- cals present in smoke, either from tobacco products or from wood fires, can directly damage lenses. A primary site of damage appears to be the cell membrane. Epidemi- ological studies have indicated that smoke- derived compounds are probable risk factors for cataract. These studies will continue in an effort to identify the pathological mechanisms involved. A paper describing the studies to date has been submitted for publication. Molectdar geneticists in the NEI, Section on Cataract have initiated gene linkage studies with scientists at Osmania University and the L.V. Prasad Eye Institute in Hyderabad on selected families with hereditary cataract. The prevalence of consanguineous marriages in this region of India greatly increases the hkeU- hood of recessive cataract phenotypes. Blood samples from individuals in suitable pedigrees are being shipped from India to the NEI for linkage analysis. A geneticist from Osmania University, who was trained at the NEI in relevant techniques, has returned to Hyder- abad to estabUsh a laboratory so that much of the work can be performed in India. In one family the cataract trait has been Unked to a particular chromosome and a potential candi- date gene has been identified. In September, Dr. Prem Prakash, chief of the Dr. Rajendra Prasad Centre for Ophthal- mic Sciences, a component of the All India Institute of Medical Sciences, visited the NEI to discuss possible future research collabora- tion. Italy The Collaborative Italian-American Study of the Natural History of Age-Related Cataract has completed a four-year foUowup study of cataract. Objectives of the natural history study were to estimate the rates of develop- ment and progression of the various types of lens opacities, identify risk factors associated with the development and progression of cataracts, and evaluate cataract classification schemes. A total of 1,297 patients participated in the foUowup study. Data collection lasted from April 1989 to April 1994. Organizations participating in the study included the Insti- tute of Ophthalmology at the University of Parma, the Laboratory for Epidemiology and Biostatistics at the Istituto Superiore di Sanita in Rome, and the NEI in the United States. A manuscript describing study results has been accepted for publication in the American Jour- nal of Ophthalmology. Investigators at the University of Parma and NEI are also collaborating in a study to determine whether the complete or partial deletion of the glutathione-S-fransferase I gene 35 FY 1994 NEI Annual Report is an important risk factor in the development of senile cataract. Blood has been drawn from approximately 300 cataract patients and is now being analyzed to determine complete or partial gene deletion at the University of Parma and the NEFs LMOD. A new collaborative Italian-American study is being planned to evaluate the effect of multivitamin supplements on the risk of cataract development and progression. Ap- proximately 1,200 subjects will be randomized to either a multivitamin /mineral supplement or matching placebo and followed for five years. Professor Giovanni Mararni, from the University of Parma, and Dr. Robert Sperduto, from the NEI, will be the study's principal investigators. The Laboratory for Epidemiolo- gy and Biostatistics, Istituto Superiore di Sanita, Roma, and the DBE at the NEI serve as the study's Coordinating Centers. The seven- year study was scheduled to start in late 1994. Mexico An international collaboration has been estab- lished by scientists in the NEI, LMOD, Section on Cataract, to investigate the relationship between enzyme deficiency diseases and cata- ract. For example, a candidate gene study was initiated to determine whether a deficien- cy in sorbitol dehydrogenase in a family where several members have congenital cata- racts is due to changes in SDH gene structure or expression. This study is possible through the cooperation of the Unidad de Investiga- don Biomedica y Hospital de Pediatria, Insti- tuto Mexicano del Soguro Social, Guadalajara, Mexico. Sweden Many eye diseases, especially retinal degener- ations, could be successfully treated if human retinal transplantation were possible. In animal models, visual cells that have been transplanted do not develop and function normally. However, a new differentiating factor has been discovered and is being ex- pressed using molecular biology techniques at the NEI. This factor, which is a protein that causes neuronal-Uke differentiation, is being tested in vitro by NEI collaborators in Sweden at the University of Gothenburg and the University of Lund to determine if it wiU cause retinal cell differentiation. The ultimate purpose of these investigations is to develop cells that could be transplanted into the hu- man eye in vivo and function normally. In collaboration with protein biochemists at the Karolinska Institute in Stockholm, NEI cataract researchers are investigating the evolutionary relationships of ^-crystaUin, an enzyme /crystaUin of certain species, with other oxido-reductases. Establishing such relationships with enzymes of known function should help to identify the physiological roles of ^-crystalUn in the lens and in other tissues where it is present at low levels. A paper reporting these analyses is being prepared. The Early Marufest Glaucoma Trial is a randomized, controlled clinical trial to deter- mine whether and to what extent reduction of lOP influences the course of chronic open- angle glaucoma. Investigators at the Universi- ty of Lund are collaborating with investigators at the State University of New York at Stony Brook and will study an estimated 300 pa- tients with newly diagnosed disease. Partici- pants will be randomized either to pressure- lowering treatment or to observation without treatment. Both groups will be followed close- ly with computerized perimetry and fundus photography. Recruitment of patients began in 1993 and will continue for an estimated two years. FoUowup of patients will be conducted for four years. United Kingdom The UK Prospective Diabetes Study is a pro- spective randomized study of different thera- pies to determine whether improved blood glucose control or improved blood pressure control of noninsuUn-dependent diabetes will reduce morbidity and mortality. The study began in 1977 and has recruited more than 5,100 newly diagnosed diabetic patients. Patients who fail to respond to diet therapy are randomized to diet therapy or active 36 International Program Activities therapy with sulfonylurea, insulin, or metformin. As part of the study, hypertensive diabetic patients have been randomized to tight blood pressure control (with either an angiotensin-converting enzyme inhibitor or p- blocker) or to less tight control. The develop- ment and progression of diabetic retinopathy in these patients are being assessed by retinal photography. The study is completing 11 years of patient followup. Activities With International and Multinational Organizations During the past year, the NEI has supported investigations of bUnding eye diseases that have a worldwide effect. These studies are implemented through bilateral agreements between foreign countries and the United States; other types of country-to-country programs such as those supported by U.S. Agency for International Development; and collaborative activities with the WHO, the Pan American Health Organization, foundations, and private and voluntary organizations such as the Lions Clubs International. The NEI is continuing to provide technical advice to Lions Clubs International in the development of its $100 million SightFirst initiative, a global sight conservation program aimed at substantially reducing the prevalence and incidence of preventable and curable vision loss. In FY 1994, the ISTEI continued its activities as a WHO Collaborating Center for the Pre- vention of Blindness. The NEI director contin- ues to serve on the WHO's Special Advisory Panel in the Prevention of Blindness. Other NEI staff members have, on request, consulted to the WHO program. The NEI is working closely with nongovern- mental organizations in designing service and research programs to reduce the prevalence of bUndness, regardless of its etiology, through- out the world. A special emphasis last year and in the next few years will be an evalua- tion of program performance in selected coun- tries. Extramural Programs In FY 1994, NEI granted 14 awards to foreign institutions in six countries. Research and training projects were supported in lens and cataract, glaucoma, visual system develop- ment, photoreceptors, phototransduction, visual cortex, visual abnormahties, Leber's disease, nutrition of the eye, ocular compUca- tions of diabetes, and the prevention of blind- ness. Awards covered both basic and cUnical research projects. Intramural Programs and Activities The NEI continues to serve as an international center for research and training on eye dis- ease. In FY 1994, 25 visiting fellows, 21 visit- ing associates, 17 visiting scientists, 19 special volunteers, and three guest researchers from more than 20 countries conducted research at the NEI facilities in Bethesda, Maryland. Their work included basic laboratory investi- gations on the molecular structure and devel- opment of the visual system, sensory and motor disorders of vision, and the biochemical bases of retinal and corneal diseases and cata- ract development. In addition, visiting scien- tists collaborated with NEI investigators in clinical studies to define, treat, and prevent vision disorders, such as genetic and develop- mental defects, ocular inflammatory disease, and ocular compUcations due to systemic conditions such as diabetes. 37 Office of Science Policy and Legislation Report of the Associate Director for Science Policy and Legislation Michael P. Davis, M.S. The Office of Science Policy and Legis- lation is responsible for program plan- ning, analysis, and evaluation activi- ties; development and maintenance of a com- puterized management information system; and legislative and other program coordina- tion activities. In addition to the activities listed below, the office had one organizational change during the year. The Scientific Report- ing Branch was made an office within the Office of the Director and is now called the Office of Health, Education, and Communica- tion (OHEC). Policy, Legislation, Planning, and Evaluation Branch Carmen P. Moten, Ph.D., Chief During FY 1994, the Policy, Legislation, Plan- ning, and Evaluation Branch provided numer- ous reports concerning research-related activi- ties of the NEL Specific activities included the preparation of recurring and ad hoc program analyses in response to requests from the NIH, The U.S. PubUc Health Service (PHS), and the U.S. Department of Health and Hu- man Services (DHHS); serving as the focal point on program planning, analysis, evalua- tion, and legislation; and planning, coordinat- ing, carrying out, and monitoring NEI pro- gram evaluations. Principal activities for this branch are specified below: Preparation of NEI Scientific Advances- 1995 Congressional Justification. -FY Preparation and submission for the Annual Report of Aging-Related Eye Disease Research. Eye diseases and disorders, such as AMD, cataract, glaucoma, and diabetic retinopa- thy, are causes of bHndness and visual impairment among older Americans. NEI submission for the Survey on Nursing Research and Related Activities Supported by NIH, FYs 1989 to 1992. Response to the Fogarty International Center (FIC) concerning the NIH report to the Senate on Biodiversity. NEI submission on Gene Therapy and He- reditary Rare Diseases. Much of the molec- ular genetics research that is conducted by NEI researchers is instrumental in devel- oping the basic understanding necessary to pursue gene therapy treatment strate- gies for rare ocular and visual system hereditary disorders. NEI submission for the top scientific ac- complishments in basic research, applied research, and clinical trials. Review of various PHS FY 1996 Legisla- tive Proposals from the Health Resources and Services Administration and the Food and Drug Administration. The NEI felt compelled to respond to the legislative proposal Regulation of Human Tissue for Therapeutic Use. The proposal intends to provide a regulatory program for banked human tissues that addresses the funda- mental differences between human tissues used for medical products. Fees wiU also 41 FY 1994 NEI Annual Report be charged for registration, operating permits, and inspections to support the cost of the program. The NEI believes that such fees could threaten human eye tissue transplantation. Almost all, if not all, eye banks of the Eye Bank Association of American are 501 (c)3 organizations. They depend heavily on philanthropic contributions in providing eye tissue for research or for patient care. Most rural and small eye banks would have serious difficulty with the imposition of fees, giv- en the difficulties of meeting current oper- ation budgets. NEI submission for supported projects that are evaluating cigarette smoking as a potential risk factor for eye dis- eases — Healthy People 2000 Tobacco Prior- ity Area. NEI submission for the 1994 NIH Disease Prevention Annual Report. The report highlighted basic research, applied re- search and cUnical investigation, interven- tion studies, and professional and public education. NEI submission report to the Office of the Director (OD), Office of Disease Preven- tion for the Fiscal Years 1992 and 1993 Prevention Outlays and Full Time Equivalent Positions. Report to the OD, Office of Science PoUcy and Technology for the 2994 Minority Health Legislation. The NEI supports three demonstration projects aimed at develop- ing, implementing, and evaluating com- prehensive culture-specific and communi- ty-based education programs for the pre- vention of diabetic retinopathy. NEI Planning Activities Report, in response to the OD, Office of Strategic Planning and Evaluation request. NEI submission for the NIH Legislative Implementation Plan — NIH Revitalization Act of 1993. • NEI submission for the FY 1996 NIH Plan for HIV-Related Research. Analyses of draft materials from the PHS Office of Disease Prevention and Health Promotion for editorial review of the Guide to Clinical Preventive Services report of the U.S. Preventive Services Task Force. The branch also has been involved in researching, writing, and editing various reports requested by the NIH, PHS, DHHS, Congress, and nongovernmental organizations and individuals, including the following: NEI submission for the NIH Intramural Research Program to the Subcommittee on Appropriations Health and Education, Congressmen Louis Stokes. • Report to the Schepens Eye Research Insti- tute on the NEI extramural funding for various eye diseases and disorders. • NEI submission for the 1995 White House Conference on Aging. The mandate of such conferences is to produce recommenda- tions for aging policy to span the next decade. • Report of the NEI director for the 1993- 1994 Biennial Report of the NIH. The report described research accomplishments, outlined future opportunities, and as- sessed important poUcy issues. • NEI submission for the Diabetes Mellitus Interagency Coordinating Committee (DMICC) Annual Report. The report in- cluded recent activities of the structure and function of polyol pathway enzjTnes, epidemiology of diabetic retinopathy, and advances in retinal cell biology. • NEI submission on Research Activities Related to Space. The report highlighted two areas of research, ultraviolet radiation on the deUcate tissues of the eye, and the structure and function of the vestibular- ocular reflex. 42 Science Policy and Legislation NEI submission for the Annual Legislative Weekend of the Congressional Black Caucus Health Braintrust. The report highlighted epidemiologic studies, clinical trials. Na- tional Eye Health Education Program, demonstration projects, and support for minority scientists, institutions, and stu- dents. The following information was submitted to the NEI Financial Management Branch: • NEI report of FY 1993 Actual Outlays for Trans-NIH Research Areas. The report areas included: accidents and injuries, aging and age-related diseases, Alzheimer's disease, arthritis and muscu- loskeletal disorders, breast cancer, cystic fibrosis, diabetes, diagnostic imaging /dia- gnostic radiology, drug development, funding for children (0 to 21), gene map- ping for both the Human Genome Project and in nonhumans, gene therapy research, health and behavior research, immunology research, infant mortality /low birth weight, kidney diseases, medical rehabil- itation research, minority aids, minority health and assistance, neurofibromatosis, neuroscience, nutrition, orphan drugs, prevention, sexually transmitted diseases, sickle cell diseases, smoking and health, space medicine, stroke, vaccine develop- ment, vaccine-related, and women's health. • Total NEI Basic /Applied/Development research by mechanism, in a tabular for- mat, for the Office of Financial Manage- ment and Budget Exhibit 44 A. • Written responses to questions submitted by Appropriations Subcommittee mem- bers. The branch contributed support for the Office of Health Education and Communi- cation (OHEC) concerrung the NEI grant port- folio. This portfolio included various types of eye disease-related research conducted by specific investigators such as: Retinal cell transplantation studies, RP and related diseases, cataract, dry eye, glaucoma, and tissue plasminogen activa- tor. The branch provided detailed information for various NIH offices, including: A description of NEI-ouflays for disease prevention research for the Office of Dis- ease Prevention. An analysis of all nutrition-related re- search supported by the NEI for inclusion in the Human Nutrition Research Information Management Re-port. NEI submission on Skin Diseases Activi- ties/Congressional Report. The report in- cluded a table of all skin disease-related research funded by NEI and research activites of the corneal angiogenesis, ge- netic studies of keratoconus, cloning of a human corneal desmosomal protein, and collaborative activities. • NEI table submission of all arthritis and musculoskeletal diseases-related research for the Arthritis and Musculoskeletal Diseases Interagency Coordinating Committee (AMDICC) Annual Report. The branch also provided editorial review of a variety of letters, reports, and other narra- tive materials for other offices within the NEI. Management Information Systems Branch David Scheim, Ph.D., Chief During the past fiscal year, the Manage- ment Information Systems Branch (MISB) has upgraded four of its network servers in Build- ing 31 and EPS to Windows NT advanced server, providing improved speed, rehability, and connectivity. MISB initiated and super- vised a contract for the extension of its local area network to a Windows NT server and 10 workstations distributed among the NEI 43 FY 1994 NEI Annual Report intramural laboratories, allowing access to administrative data and computer support services. This has enabled MISB, for example, to grant intramural offices access to the Status of Funds program, which MISB also provided to additional users in the administrative and executive offices of Building 31. The integrat- ed architecture provided by the migration to the Windows NT network operating system allows all current and any additional users to run this program from one file server location. The MISB managed the installation of the TAIMS timekeeping system on approximately 20 workstations within the NEI. Bringing this system to fuU production entailed assistance and problemsolving for users, particularly in the transmission of aggregated data. The MISB also provided similar assistance in the installation of the ATRAIN system for generat- ing training requests and installed an official airlines guide flight scheduling program for the administrative office. The NEI upgraded several users from Word Perfect 5.1 to 6.0 and arranged traiiung for NEI staff to make this transition. The MISB upgraded five printers to the Hewlett Packard LaserJet 4 series. Eight modems were upgraded from 2,400 to 14,400 bits per second, allowing much faster commu- nications speeds for remote usage of the NEI LAN by NEI staff on tiavel or working after hours at home. MISB installed two CD ROM drives, primarily for use in software installa- tions and installed a magneto-optical drive for more reliable daily and weekly backups of all network data. The MISB, in addition, has con- tinued to provide support and maintenance for all 95 workstations in Building 31 and EPS, aU software used, and the network infrastruc- ture with no contractor support. The MISB planned and implemented a comprehensive documentation contiact for the NEI's microcomputer LAN, associated hard- ware and software, and aU custom-developed systems at a cost of $23,500. This project, when completed in FY 1995, wiU provide extensive textual documentation of NEI micro- computer systems; cabling diagrams; and online documentation of equipment, processes, and problem resolution procedures accessible to MISB and all NEI staff. Database tables and access systems for the online components of this effort were developed by MISB staff. The MISB began a joint effort with the NIAID for the development of a cUent-server personnel tracking system to maintain infor- mation on aU permanent and temporary em- ployees. Development will be performed by an NIAID contractor, but MISB input and design assistance will be provided in exchange for consideration of particular NEI require- ments. If successful, such collaborative efforts will be used for the subsequent development of a procurement tracking system using state- of-the-art software compatible with the NEI's current database platforms. The MISB evaluated automated grants awards systems in use at NIH and arranged a $14,000 contract for the customization of one such system for use by the NEI Extramural and Collaborative Program. This contract specifies capabilities provided to the NEI on par with those provided to another ICD through a contract costing about $1 million. Most of the work on this contact was complet- ed in FY 1994. MISB provided extensive enhancements to its grants information systems during FY 1994. Microsoft SQL Server, the database server for all systems, was upgraded to version 4.2 to allow enhanced functionality. JAM and JAM/DBI, the cUent tools for these systems, were also upgraded to allow additional func- tionality. The existing NEI snapshot, coundl letter, and grants coding systems were up- graded for this new environment. MISB reprogrammed the grants master on- line update and SCORE coding routines using the JAM cUent-server front end in which the NEI's other grant modules are developed. It also converted aU historical grants and SCORE data into cUent-server tables, using custom- developed data conversion and checking rou- tines. The NEI's council letter generation program was also modernized into a more 44 Science Policy and Legislation streamlined client-server process using R&R report writer, which, after evaluation of sever- al such tools, was selected as a future report- ing platform. These conversions have allowed the older and less reliable Paradox database applications to be completely phased out. Automated security, login, and usage tracking functions integrated with LAN login were developed by MISB staff. These func- tions provide automatic login to grants infor- mation systems for NEI staff and also auto- matic tracking of system usage. Between January and September of 1994, a total of 4,198 logins to grants information systems were recorded, not including system testing by MISB staff. Also, daily check and backup procedures for aU active NEI databases, which run automatically each night, were implement- ed through custom programming by MISB. The NEI's weekly grants update batch procedures, comprising 24 procedures and 100,000 lines of code previously written in Paradox 3.0, were completely reprogrammed by MISB staff in Microsoft SQL Server Trans- act SQL language, extensively tested, and implemented. The new batch update proce- dures run more quickly and reUably and contain extensive built-in system checks to automatically halt processing if an error is detected. The reprogramming was performed to consolidate the NEI's grants system by eliminating an obsolete platform, allowing for easier maintenance and future development in the more reliable, state-of-the art, cUent-server architecture. The new update procedures have run on weekends virtually error-free since mid-1994. The MISB has continued to provide cus- tom information reports to NEI staff for inter- nal use and pubUc distribution, with 70 new mainframe requests and an increasing volume of microcomputer-based production reports logged for FY 1994, with rapid turnaround achieved in every case. Weekly and monthly reports, as well, continue to be provided. In addition to its own programming efforts, the MISB has continued to support NEI staff in the use of information resources provided by DRG, the NLM, and other sources, including the DRG information system, CRISP, FOCUS, WYLBUR, MEDLINE, Grateful Med, Legislate, the electronic NIH library catalog. Gopher, and other specialized systems. The MISB has continued to handle a number of IRM functions for the NEI, includ- ing its environment and resources report, strategic plan, tactical plan, budget report, and security functions. MISB staff has continued to represent the NEI on a number of NIH- wide committees, including the Office Techni- cal Coordinators and its network subcommit- tee, the ADP Extramural Programs Coordi- nating Committee and its steering committee, the Database Technology Task Force, the NIH lead users group, the Campus Users Research Exchange, and the Technical LAN Coordina- tors Committee. 45 Office of Health, Education, and Communication Report of the Director of the Office of Health Education and Communication Judith A. Stein, M.A. In a reorganization, the NEI has estab- Ushed the OHEC within the NEI, OD. This new office replaces the Scientific Reporting Branch previously part of the NEI's Office of Science PoUcy and Legislation. The activities of this office include the National Eye Health Education Program (NEHEP); special activities such as the traveling science museum exhibit; dissemination of research results; publications; response to inquiries from pubUc, health professionals, and the media; and advising NEI staff on aU aspects of NEI and NTH scientific reporting, knowledge transfer, health education, and press relations. National Eye Health Education Program The NEHEP began the development of a diabetic eye disease education program for Hispanics/ Latinos with diabetes. Eight focus groups were conducted across the country to learn more about the knowledge, attitudes, and practices of this target audience as related to diabetes and eye health. Groups were con- ducted with Central Americans in Washing- ton, D.C.; Puerto Ricans in New York City; Mexican Americans in Los Angeles; and Cu- ban Americans in Miami. An ad hoc working group on Hispanic outreach met to provide recommendations to the NEHEP staff on the development of an education program. It is anticipated this program wiU be launched in spring 1995. A television pubUc service announcement (PSA) on glaucoma was produced and distrib- uted nationally in September 1994. The PSA stiesses the importance of eye examinations for people at risk for glaucoma especially African Americans older than age 40 and everyone older than age 60. In addition, radio and print PSA's on glaucoma and diabetic eye disease were distributed to media outlets reaching target audiences at risk for these diseases. The Third National Eye Health Education Conference was held in December 1993. The purpose of this conference was to provide an opportunity for the members of 51 organiza- tions in the NEHEP Partnership to share their program and interests and to develop collabo- rative eye health education programs at the community level. One idea that originated at the conference was to conduct an awareness campaign on diabetic eye disease during National Diabetes Month in November. In February, the Ameri- can Diabetes Association (ADA) and the NEI joined forces to coordinate this event. Nine other Partnership organizations offered sup- port to increase awareness among people with diabetes about the importance of an annual dilated eye examination. These organizations will coordinate local activities. A special bro- chure was developed for November, adapted from the existing NEHEP Don't Lose Sight of Diabetic Eye Disease brochure. The ADA wiU use its 1-800-DIABETES number as a place to call for a referral to an eye care professional and more information on diabetes and diabetic eye disease. The referral program is coordi- nated with the American Academy of Oph- thalmology and the American Optometric 45 FY 1994 NEI Annual Report Association. An extensive media campaign will also be conducted to complement local efforts. This includes a video news release, press release, print advertisement campaign, and a radio program targeted to African- American radio stations. 25th Anniversary Program The NEI celebrated its 25th anniversary with a nationwide public education program to promote the benefits of vision research. The centerpiece of the celebration was the travel- ing exhibit VISION, which was developed to highlight the sight-saving results of vision research funded through American tax dollars. VISION premiered in San Francisco, CaHforiua at the Exploratorium in October 1993. During 1994, it has been displayed at the Museum of Science and Industry in Chica- go, Illinois; the Museum of Discovery and Science in Fort Lauderdale, Florida; Union Station in Washington, D.C.; and at the Louisi- ana Nature & Science Center in New Orleans. An estimated 125,000 people have visited the exhibit to date. It will travel to approximately 13 more cities, including Boston, Massachu- setts; Jacksonville, Florida; Houston, Texas; Los Angeles, California; Portland, Oregon; and Seattie, Washington, during the next few years. In each location where the exhibit is dis- played, NEI grantees and local chapters of the voluntary and/ or professional organizations plan a series of regional events designed to increase the public's awareness of vision re- search. These events include public lectures, vision screenings, press conferences, science writers seminars, and educational programs for school age children. The NEI also is developing a school curric- ulum program for children in grades four through eight. The program consists of three lesson plans, interactive classroom activities, and previsit and postvisit exercises. Topics covered include the anatomy and physiology of the eye and visual system, common eye diseases and disorders, and eye safety. The program is designed as a supplement to any science or health curriculum and can be used by either a guest speaker (vision researcher or eye care professional), or the classroom teacher. The curriculum will be pilot tested this fall in several communities throughout the coun- try and will be available in spring 1995. The Association for Research in Vision and Oph- thalmology will print and distribute the pro- gram to its 11,000 members this November and will highlight it during the association's annual meeting in May. A marketing and promotion plan wiU be developed to target science and health educators for children in grades four through eight nationwide. Public Inquiries Program The OHEC staff responded to more than 16,000 inqviiries from the general public, pa- tients and their farrdlies, students, health pro- fessionals, legislators, and the media in FY 1994, including 14 pieces of controlled corre- spondence representing five congressional inquiries and a Presidential proclamation. This reflects an approximate six percent in- crease in inquiries over last year. To handle the increase in pubUc inquiries more efficientiy, the OHEC staff developed standard information packets on commonly requested eye disorders and diseases, includ- ing sarcoidosis, blepharitis, RK, RP, and cata- ract surgery, and more. To assist health pro- fessionals find additional materials on specific eye diseases and disorders, staff members prepared listings of the materials found in the Eye Health Education subfile in the Combined Health Information Database. Two new bro- chures, written in an easy-to-read format, were developed for people at risk for cataract and AMD. 50 Office of Health, Education, and Communication The OHEC staff also handled 38 Freedom of Information requests. Scientific Reporting The NET publication Clinical Trials Supported by the National Eye Institute was developed and printed in faU 1993. This publication provides information on 22 extramural and intramural clinical trials supported by the NEl. Each dinical trial description includes the purpose and design of study, patient eligibility criteria, patient recruitment status, results to date, and participating cUnical centers. 51 Office of the Scientific Director DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00122-14 OSD PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Anatomical Studies of the Primate Visual System PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Francisco M. de Monasterio M.D., D.Sc. Medical Officer OSD, NEI COOPERATING UNITS (if any) LAB/BRANCH Office of the Scientific Director INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.40 PROFESSIONAL: 0.40 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project involves the study of the anatomical properties and organization of cells in the visual system of primates, with emphasis on the retina and the visual cortex. The studies include the pattern distribution of selectively stained cones in the retina of macaques. The results have provided information on the probable retinal circuitry of the blue-sensitive cone pathway of primate retinal cells. 55 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives To study the anatomical properties and neural organization of the primate visual system. Methods Retinal histological processing, intravitreal injection of dyes, computer modeling, and spatial statistical analyses of point and area patterns are used. Major Findings The anatomical studies of this project were affected once again by further underground construction work in a building just across the street, in front of the laboratory; in addition, the building housing the laboratory under- went structural renovation during most of this period. Such construction affected both mi- crotomy and microphotography, limiting work to the evening or night hours. The point pattern of blue-sensitive cones selectively stained with tissue-reactive nonfluorescent dyes (Procion black) was examined at various eccentricities of the macaque retina in distortion-free whole mounts. The point pattern was analyzed in terms of its angular structure and disorder using spatial statistical techniques described in earlier work. Comparison of the parafoveal, extrafoveal central retina, and peripheral retina point patterns indicates that the degree of disorder of the pattern increases slowly with eccentricity, from about 18 percent to about 30 percent. This is equivalent to dis- turbing each point of a lattice of unit area by 0.18 to 0.30 units along a randomly selected azimuth. Despite this disorder, the pattern maintains its regularity with eccentricity, and side-by-side blue-sensitive cones are very rarely seen. These results indicate that the blue-cone pattern of macaque retina does not foUow a lattice distribution and support a previous model for the development of this point pattern, based on an exclusionary hard core surrounded by a probabilistic soft shell. Significance to Biomedical Research and the Program of the Institute Information on the anatomical properties of blue-sensitive cones is important not only to the functional properties of these cones inves- tigated in different basic disciplines but also to the clinical research and diagnosis of acquired retinal disease. The data obtained from the eye of diabetic human donors are particularly promising in this respect. Proposed Course These studies will be continued. NEI Research Program Retinal Diseases — Retinal Neuroscience DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00065-17 OSD PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Physiological Studies of the Primate Visual System PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Francisco M. de Monasterio M.D., D.Sc. Medical Officer OSD, NEI COOPERATING UNITS (if any) LAB/BRANCH Office of the Scientific Director INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.60 PROFESSIONAL: 0.60 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project involves the study of the physiological organization of neurons of the visual system of nonhuman primates that may serve as a model for the human visual system. Emphasis is given to the spectral and spatial properties and central projections of retinal ganglion cells and cells from the lateral geniculate body and the visual cortex of macaques. Recordings from color-opponent retinal ganglion cells and parvocellular geniculate cells show essentially identical spectral response bandwidths under similar conditions of stimulation. On the basis of these bandwidth data, both types of cells can be grouped in subtypes which show a direct correspondence with specific color-opponent varieties. The bandwidth data essentially show no difference between geniculate and retinal color-opponent cells subserving similar areas of the visual field. 57 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives To study the neural organization underlying the processing of visual information at differ- ent levels of the primate visual system. Methods Intracellular and extracellular recordings from single neurons, extracellular recordings of mass responses, computer video stimulation, tangent screen chromatic and spatial stimula- tion. Major Findings The single-ceU studies of this project were affected once again by further underground construction work in a building just across the stieet, in front of the laboratory; this work resulted in often violent shaking that defeated vibration-isolation measures. In addition, the buUding housing the laboratory underwent stiuctural renovations during most of this period. The response bandwidth of color-oppo- nent ganglion cells and parvocellular genicu- late cells was examined with spectral lights in conditions of neutral chromatic adaptation. The same stimulating conditions were used for both cells, which were typically recorded from the same anesthetized animals. As noted in previous work, spectral response band- widths have specific "signatures" when plot- ting bandwidth against the wavelength of the peak sensitivity. The average half-bandwidth at half-maximum sensitivity was 24 nano- meters (run) for retinal gangUon cells and 22 nm for parvocellular geniculate cells. Aver- aged spectral bandwidths of these cells fall in various distinct signature subgroups matching subgroups based on color-opponent response varieties. No significant differences, either in aver- age half-bandwidth or spectral signature, were found between color-opponent, center-sur- round geniculate, and ganglion cells. In fact, for neurons subserving the same or similar areas of the central visual field, the geniculate and retinal bandwidth data could not be distinguished from one another. These results provide further support for an essentially one- to-one relationship between these neurons. Significance to Biomedical Researcli and the Program of the Institute Numerous behavioral, psychophysical, and electrophysiological studies show that the visual performance and characteristics of macaques and humans are extremely similar to one another, so that an understanding of nonhuman primate physiology provides a useful animal model for human visual func- tion. Proposed Course These studies will be continued. NEI Research Program Stiabismus, Amblyopia, and Visual Process- ing — Structure and Function of Central Visual Pathways. Publications de Monasterio FM: Operating system errors in DOS 5. J PC Tech 4:30-37, 1993. de Monasterio FM: Direct access to interrupt handlers in the system kernel. / PC Tech, in press. DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00135-22 OSD PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Biochemistry of Retina and Pig men ted Epithelium in Health and Disease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Helen H. Hess M.D. Medical Officer (Research) OSD, NEI COOPERATING UNITS (if any) Laboratory of Chemoprevention, National Cancer Institute (M. Anzano, Ph.D.) LAB/BRANCH Office of the Scientific Director INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 1.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects Q (b) Human tissues [x] (c) Neither □ (a1) IVIinors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Effects of nutrition, oxidation, and other environmental factors (light intensity or darkness) on incidence and progress of posterior subcapsular opacities (PSO) associated with genetically influenced retinal degeneration are being studied in pink-eyed Royal College of Surgeons (RCS) rats, in which rod photoreceptor outer segment debris accumulates secondary to a phagocytic defect in retinal pigmented epithelium (RPE). Peroxidation in polyunsaturated fatty acids in debris lead to water-soluble toxic aldehydes, detectable in the vitreous and toxic to lens cells and membranes. Dystrophic rats fed a natural ingredient diet (NIH-07) were highly sensitive to retina light damage, beginning at an intensity of 10 to 40 lux, and 27 percent of the rats developed mature cataracts by 5 to 12 mondis. Rhodopsin bleaching is essential for retina light damage and PSO. In vitro, free retinaldehyde has been shown to be a photosensitizer to generate singlet oxygen, an extremely damaging oxidant for both lipids and proteins, and this also may occur in vivo. In RCS rats reared at 10 to 40 lux, a purified diet (AIN-76A) fortified with antioxidants (0.4 percent p-carotene + 0.01 percent BHT) prevented PSO and mature cataracts. A diet containing additional antioxidants (1,000 mg/Kg diet of vitamin C and 150 mg/Kg vitamin E) retarded retinal degeneration during the time the cataracts would have had their onset (23 to 53 postnatal days) if NIH-07 had been fed. Higher concentrations of vitamin E did not show additional retardation of retinal degeneration. Effects of increasing environmental lighting in incidence of bilateral mature cataracts were studied in pink-eyed RCS rats fed the NIH- 07. Incidence of bilateral mature cataracts (BMC) was 5 percent in rats reared in 10 to 40 lux of cyclic light; but was 25 percent in rats reared in 110 lux of constant light; 70 percent in 270 lux of constant light; and 100 percent in 65-day-old rats given 48 hours of high intensity light (7500 lux). After lengthy or intense illumination, occurrence of disturbed meridional rows of lens epithelial cells and posterior nucleated (Wedl) cells pointed to proliferation of germinative zone epithelial lens cells from deoxyribonucleic acid (DNA) damage. At low illumination, damage can be repaired (stationary cataracts and rare BMC). The results are consistent with the hypothesis of PSC causation by DNA damage to lens epithelial cells. Agents that can have this effect include products of peroxidation of polyunsaturated fatty acids, short wavelength radiation (UV, X-rays, P and y rays) and numerous chemical mutagens such as N-nitroso-N-methylurea (NMU). Normal albino rats, injected with NMU at a concentration and dosage sufficient to cause breast cancer, developed BMC by 5 months of age. These cataracts were PSC of a more severe nature than ever seen in RCS rats (exposed to excessive light), with abnormally large cells (some binucleate) not only at the posterior pole but encircling the lens. The retinal showed advanced degeneration, ^g PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Additional Personnel J. Samuel Zigler, Ph.D. Chief, LMOD, NEI Jr. Joseph J. Knapka FhD. Nutrition Consultant, Veterinary Research Program (VRP), Nation- al Center for Research Resources (NCRR) Dennis Bernard M.S. Nutritionist, VRP, NCRR Maria Anzano Ph.D. Expert, Laboratory of Chemoprevention, NCI Objectives This project is designed to study the biochemi- cal and bionutritional relationships among lens, retinal photoreceptors, retina, retinal pigment epitheUum (RPE), and biological fluids in health and disease. It also involves exploring the possibilities for slowing the rate of retinal degeneration and preventing lens opacities and mature cataracts, which are often associated with retinal degeneration in rats and humans. The cataract that develops in the Royal College of Surgeons (RCS) rat is a posterior subcapsular cataract (PSC), a tj^e that makes up about 10 percent of cases of age-related cataract and occurs in 40 percent of cases of retinitis pigmentosa. PSC also is seen in other hereditary retinal diseases and in radiation damage to the lens. It is characterized by genetically damaged lens epitheUal cells, which fail to undergo normal differentiation into lens fibers and instead multiply and migrate to the posterior subcapsular region to form an opacity. PSC and retinal degeneration are pro- duced in rats by the chemical mutagen N- nitrosoN-methylurea (NMU). This agent has been used to produce breast and prostate tumors as well as gUal tumors of the brain in rats. Chemopreventive agents have been found to reduce the incidence of breast and prostate cancers in rats using dual agents, one to reduce or halt multiplication of cells, the other to cause differentiation of the cells. An objective will be to test whether these princi- ples can be used to reduce incidence of PSC and retinal degeneration in rats given NMU. The rat C-6 gUal cell hne, used for the past 30 years, was derived from rat brain glial cells transformed by NMU in vivo; several clones were obtained, C-6 being one of the more differentiated. Transformation of lens epitheU- al cells in vivo by NMU potentially could yield a similarly useful ceU Une. Methods The RCS rat is being studied as an animal model of hereditary retinal degeneration that results from a defect in the RPE as well as a type of cataract that is secondary to retinal degeneration. Bionutrition has been used as a tool to combat Upid peroxidation in the RCS rat retina and to prevent water-soluble toxic aldehyde byproducts from reaching and damaging the lens. The RCS rat cataract is not genetic because the mutant gene is ex- pressed not in the lens but in the RPE; it is instead an outcome of environmental risk factors of both internal and external origin. Thus, the RCS rat is a living laboratory, and the cataracts are susceptible to orchestration by varjdng risk factors and preventive mea- sures. Defined diets were prepared and fed to congenic affected and unaffected RCS rats in controlled experiments. The diets were fed to young breeding pairs before they produced their first offspring and to their offspring after weaning so that the experimental animals received their diets from conception to date of observation. Clinical findings were recorded after indirect ophthalmoscopic and biomicro- scopic sHt-lamp examination. Postmortem examination of the eye included dissecting microscopy and light microscopy of stained specimens. At appropriate times, photogra- phy was used to record in vitro or in vivo data. Analytical methods included standard bio- chemical, fluorometric, and separation proce- dures. Special environmental lighting condi- tions were used to determine histopathological effects on the lens and retina. 60 Ojfice of the Scientific Director In collaborative studies with the National Cancer Institute (NCI), the direct acting de- oxyribonucleic acid (DNA) alkylating agent NMU was injected intiavenously in a dosage sufficient to produce cancer of the breast in female rats. Experimental rats were given chemopreventative agents. "Contiol" rats we studied were normals injected with NMU or saline; lenses and retinas were examined histopathologically. Eyes were fixed in 10 percent neutral formalin, embedded in plastic, sectioned at 2 /xm, and stained with haema- toxyUn and eosin. Major Findings (1) Bilateral mature cataracts in RCS rats. Dystiophic RCS rats fed a natural ingredient diet and reared from birth at a low-hght intensity have a 27 percent incidence of ma- ture cataracts (MC) in one year, most of which are unilateral. In a study of incidence of bilateral MC (BMC) in the RCS rat as a func- tion of environmental light intensity, we found that at low-Ught intensity (10 to 40 lux) only 5 percent of rats had BMC in one year, while the remaining eyes had stationary cataracts (clear lens fibers between the opacity and capsule). In previous studies, when reared from birth in 100 lux of constant Ught, 78 percent of rats has MC, 28 percent of which were BMC. Rats exposed to higher intensities of Ught starting at 22 to 28 postnatal days (when debris containing rhodopsin and poly- unsaturated fatty acids is maximal) has a higher incidence of BMC: at 270 lux of con- stant Ught, 70 percent of rats had MC, 70 percent of which were BMC; and at 7500 lux for 48 hours, 100 percent of rats had MC, 100 percent BMC. We concluded that rats reared in 10 to 40 lux cycUc Ught had DNA damage that was repaired in the majority of lenses, and BMC were rare. In constant Ught of higher intensity, repair did not occur and most or aU rats had BMC. The results support the hypothesis of cataractogenesis by toxic aldehydes from peroxidized retina Upids, damaged by singlet oxygen generated by the sensitizer retinaldhyde. Such aldehydes (detected in the vitreous) can damage pro- teins, Upids, and nucleic adds. Occurrence of disturbed meridional rows of lens epitheUal ceUs and posterior nucleated (Wedl) cells pointed to proliferation of germinative zone epitheUal lens ceUs, possibly from DNA dam- age. These results are consistent with a hypoth- esis of PSC causation by DNA damage to lens epitheUal ceUs in the germinative zone. Agents able to have this effect include prod- ucts or peroxidation of polyunsaturated fatty acids (abundant in retina rod outer segments and synapses), short wavelength radiation (ultraviolet, x-ray, P, and y rays), and numer- ous chemical mutagenic agents such as NMU. (2) BMC in NMU-injected normal albino rats. Examination of the "control" NMU-inject- ed normal rats, as compared with saline- injected rats, revealed BMC at five months of age in the NMU-injected rats. This histopath- ological appearance of the cataracts was that of posterior subcapsular cataracts of a severe nature with abnormaUy large ceUs of Wendl not only at the posterior pole but encircling the lens; some ceUs were binucleate. The retina showed advanced generate changes. (3) Antioxidant diets in RCS rats. Antioxi- dant diets that prevent the cataracts in pink- eyed RCS dystrophic rats have the effect of retarding retinal degeneration. None of the diets we have tried stops the degeneration, but, when a certain degree of retardation is achieved, the lens is protected. Last year we increased the concentiation of vitamin E in the AIN-76 purified diet that already had been supplemented with 0.4 percent p-carotene plus 0.01 percent of BHT (to prevent oxidation of carotene in the cage hopper) as well as 1000 mg/Kg vitamin C and 150 mg/Kg vitamin E. We did not succeed in showing any additional retardation of the retinal degeneration (beyond 55 days), perhaps because apoptosis occurs in this rat strain. Proposed Course As pink-eyed, tan-hooded dystrophic breeders become available from the National Institutes of Health (NIH) Foundation Colony, lens 61 FY 1994 NEI Annual Report epithelial cell whole mounts will be prepared to compare the numbers of cells in lenses with and without cataract. Collaborative studies of the mutagen NMU as a cataractogenic agent will continue. Principles learned from chemoprevention in rats with breast and prostate cancer would involve using dual agents that can (1) halt or slow multiplication of lens epitheUal cells and (2) cause differentiation into lens fibers. Collaboration to find the mutant autoso- mal recessive rdy gene on chromosome 3 of the RCS rat has been investigated. Until now, this study has not been feasible because the rat genome had not been well studied. How- ever, knowledge of the rat genome has been proceeding apace. A molecular biologist in NCRR, VRP, where "fingerprinting" of some of the strains of rats in the Foundation Colonies (including RCS rats) is being pursued, may be interested in this gene, which appears to be involved in RPE phagocytosis. NEI Research Program Lens and Cataract — Pathogenesis of Cataract Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Hess HH: Incidence of bilateral mature cata- racts in Royal College of Surgeons (RCS) rats and environmental Ught stress. Invest Ophthal- mol Vis Sci 35(suppl):2137, 1994. 62 Laboratory of Immunology Report of the Chief Laboratory of Immunology Robert B. Nussenblatt, M.D. The Laboratory of Immunology (LI) has finished its eighth year. The sections of the laboratory include: the Immu- nology and Virology Section headed by Dr. John J. Hooks; the Section on Experimental Immunology headed by Dr. Igal Gery, who is also the deputy head of the Laboratory; the Section of Immunoregvdation headed by Dr. Rachel Caspi; the Section on Experimental Immunopathology headed by Dr. Chi-Chao Chan; the Section on Clinical Immunology whose acting head is Dr. Marc de Smet; and the Section on Molecular Biology headed by myself as acting head of an interdisciplinary group. Section on Clinical Immunology The Section on Clinical Immunology has continued to focus on major areas of clinical relevance, including new interventional stud- ies. The section has continued its study of patients with acquired immunodeficiency sjmdrome (AIDS) in collaboration with the National Institute of AUergy and Infectious Diseases (NIAID). A randomized study to evaluate a slow-release implant filled with ganciclovir was tested in AIDS patients with cytomegalovirus (CMV) retinitis. These im- plants are placed directly into the eye through the pars plana. They have been calibrated to release therapeutically effective doses of ganciclovir over an eight-month period. Recruitment for this study has come to an end, and the results wiU be tabulated and reported in the near future. This randomized study may yield important information about a new alternative to systemic anti-CMV thera- py for patients who cannot tolerate intrave- nous therapy or those who do not wish to be treated with systemic therapy. The potential for a marked improvement in quality of life in these patients is a serious consideration. Pediatric AIDS patients continue to be seen and evaluated for the incidence of ocular infection. This study is done in conjunction with Dr. Phihp Pizzo and the National Cancer Institute (NCI). The group has been extremely active in the development of new ways in which the ocular immune response can be interfered with. This has included the study of the effect of NPC- 15669, an inhibitor of neutrophil recruitment in uveitis. It was found that the injection of this compound into rats wiU significantly reduce the severity of endotoxin- induced uveitis (EIU), a disease that is mediat- ed mainly by pol5anorphonuclear cells as well as macrophages. Of great interest as well was that treatment of rats late in the process of experimental autoimmune uveitis (EAU), a disease thought to be mainly mediated by T-ceUs, could also have a marked inhibition of the evolution of their disease. Of great poten- tial interest as a therapeutic mode in the near future is the effectiveness of humanized anti-interleukin (IL)-2 receptor antibodies in the treatment of autoimmune uveoretinitis in monkeys. This work has been performed in collaboration with Dr. Thomas Waldmann of the NCI as well as Drs. James Raber and 65 FY 1994 NEI Annual Report Martin Kriete, from the Veterinary Research and Resources Section, National Eye Institute (NEI). The humanized antitact antibody is an anti-IL-2 receptor antibody originally pro- duced in mice but has been modified by replacing all but its binding region with hu- man immunoglobulin elements. Cynomolgus monkeys induced with EAU were treated with this antibody; the disease was markedly limited in the group treated with the antitact humanized antibody. These findings are in marked contradistinction to the inflammation that was seen in control groups. This work has great significance because of the implica- tions for the potential for human therapeutic studies to occur in the not-too-distant future. From a basic scientific point of view, these studies also implicate the potential role of IL- 15 m this ongoing process. Section on Molecular Biology This new interdisciplinary group, the Section on Molecular Biology, has been in existence now for more than two years. The attempt is to better understand approaches to gene therapy, whether they be local or systemic in nature. The group has had a long-term inter- est in focusing on the regulation of the omithine-6-aminotransferase (OAT) gene. Work continued in the development of a knockout gene model for this disorder. Addi- tionally, interactions with a variety of individ- uals, including those from Johns Hopkins University and the State University of New Yf "k at Stony Brook, have begun to develop re ±ods by which therapy for gyrate atrophy could be introduced through skin cells. Work by this group has also emphasized studying direct ocular gene transfer using ElA deficient adenovirus constructs. These have been used to transfer genes into isolated retinal pigment epithelial cells (RPE) followed then by trans- plantation of these RPE cells. A mutant of transforming growth factor beta (TGF-P) has been placed into the adenovirus construct. It appears from early experiments that the RPE cells are capable of sustaining replication of ElA deficient adenovirus. Section on Immunoregulation The Section on Immunoregulation has main- tained its great interest in the development and study of animal diseases of experimental auto-ocular autoimmune disease. One aspect of the work has been to characterize further murine EAU because the mouse model offers quite important differences from other rodent models of uveitis. The section found a possi- ble correlation between the pathogenicity of autoimmune T-ceUs and their lymphokine production expression of functional adhesion molecules as well as the expression of some surface antigens in the examination of the Lewis rat model for uveitis. Antigen-specific Lewis rat T-ceU lines and sublines have been developed; one is specific for the major patho- genic epitope on the human retinal soluble S-antigen (S-Ag) and three are specific to the major pathogenic epitopes of bovine inter- photoreceptor retinoid-bin ding protein (IRBP). Though these lines have different degrees of uveitogenicity, the four T-ceU lines produce roughly equivalent amounts of interferon gamma (IFN-y) tumor necrosis factor, IL-3, IL- 6, and TGF-p. Of interest, IL-4 cannot be detected. Similarly, there are essentially equal amounts of functional adhesion molecules being ex- pressed; however, the nonpathogenic subline was the poorest responder to antigen stimula- tion with respect to proliferation in IL-2 pro- duction. The nonpathogenic subline wiU show almost no expression of CD-4. These results would support the contention that class 2 restricted recognition of autoantigens within the neuroretina by uveitogenic T-lymphocytes must occur as an initial step in the induction of experimental uveitis. Therefore, a defect in this step wiU preclude marked uveitogenicity of these cells. C5^okine genes within the eye in murine experimental uveitis have been studied. T-cells that have been specifically grown in the presence of the retinal protein IRBP or to its peptides can induce uveitis with adoptive transfer. These cell lines show an unrestricted cytokine profile in vitro. Looking at messenger ribonucleic add (mRNA) pro- 66 Laboratoiy of Immunolog]/ duction, a TH-1 type cytokine profile (IL-2, IFN-v, and tumor necrosis factor alpha [TNFa], was present in the eyes of mice that had EAU induced with the transfer of these cells Unes. These results suggest that these lymphokines are important for the induction of uveitis and that a lack of IL-4 mRNA in the eyes of mice that had experimental uveitis would argue that predominately TH-1 type cells were present. Additionally, the group has identified a major pathogenic epitope in the IRBP molecule that is recognized by mice of the H2R haplotype. Another approach of suppressing ocular autoimmunity was through the induction of oral tolerance. EAU susceptible B-IO.A mice were fed IRBP or a control solution. Results indicated that three feedings of 0.2 mL of IRBP every other day before immunization did not protect mice against this form of uveitis, whereas a similar regimen of five doses was in fact protective. However, of interest was supplementing the nonprotective three times regimen with one intraperitoneal administration of recombinant human (rHum) IL-2 resulted in disease suppression that was equal to that of the protective feeding regi- men. Analysis of the Peyer's patch cells of fed mice showed a large increase in the produc- tion of TGF-P, IL-4, and IL-10 in those animals that were fed IRBP and received IL-2, as compared with those animals that only re- ceived IRBP feedings. The group would propose that IL-2 treatment enhances the protection from experimental uveitis by stimu- lating regulatory cells that ultimately produce cytokines such as TGF-P, IL-4, and IL-10. This interest in immunosuppression oral tolerance has also resulted in an ongoing randomized masked study to look at the effectiveness of oral tolerization. This study continues to be in progress and will evaluate the usefulness of S-Ag oral adnvinistiation in the induction of tolerance in uveitis patients. It is hoped that this study will be completed in the next year. Section on Experimental Immunology The Section on Experimental Immunology has continued its long-term investigation of the pathogenesis of inflammatory eye diseases. In this ongoing interest, they reported the uveito- gerucity of recoverin this year. Recombinant recoverin was found to be highly uveitogenic in Lewis rats, inducing a severe experimental uveitis at doses as low as 10 /xg per rat. The clinical and histopathologic changes induced by recoverin were very similar to those that one sees with disease induced by the retinal S-Ag. Of great clinical interest is that recover- in has been suggested by many to be the antigen to which antibodies are developed in carcinoma-associated retinopathy syndrome. Work by the group has continued to look at the uveitogenicity and antigenicity of rHums- Ag in primates. Monkeys of three species have been immunized with recombinant S-Ag molecules. The ocular changes observed closely resemble those seen in previous studies of monkeys immunized with bovine S-Ag. Additionally, lymphocyte proliferative re- sponses were quite similar to those previously seen. Transgeruc mice were also evaluated by this group during the year. Transgenic mice that expressed foreign antigens in their lens were developed by collaborators at the Labo- ratory of Molecular and Developmental Biolo- gy (LMDB), NEI. These foreign antigens were expressed under control of the aA-crystalline promoter. Development of immunotolerance in transgenic mice was examined by measur- ing their capacity to mount specific immune responses following immunization with a corresponding antigen, which was emulsified in agevin. The main finding showed that the expression of chloramphenicol acetyltiansfer- ase (CAT) in the lens had littie effect on the 67 FY 1994 NEl Annual Report capacity of the transgenic nnice to respond against this antigen. Of interest was that in contrast to the findings with CAT, expression of human fibroblasts (HEL) in the lens pro- duced a state of complete tolerance to this antigen. HEL transgenic mice failed to devel- op any detectable antibody or ceUular immune response against this antigen. The group has also explored the phenome- non of oral tolerance by looking at the oral administration of the bacterial product N-acetyl-D-glucosaminyl-|3 (l-4)-N-acetyl-L- muranyl-L-alanyl-D-isoglutamine (GMDP) . When GMDP was given along with the uveit- ogenic peptide, it significantiy enhanced the level of tolerance induced by the peptide. The role of CD-8 positive lymphocytes in the process that produces oral tolerance was also examined by testing whether mice deficient of these cells were capable of developing oral tolerance. The CD-8 deficient animals used were p-2 m-mice. These mice then have very low expression of major histocompatibility complex (MHC) class 1 molecules and a severe deficiency of CD-8 positive cells. Feeding these mice with ovalbumin produced remarkable levels of immunotolerance that closely resembled those observed in the simi- larly treated control animals. This finding thus indicated that CD-8 positive cells are not essential for the induction of oral tolerance, at least when induced by the procedure used in this study. Further work by this group looking at transgenic models has evaluated the effects of IFN-Y on the physiology of the eye and the role of elevated MHC class 2 in the eye. Both transgenic rat and mice strains were generated by microinjection of deoxyribonucleic acid firagment containing a murine aA-crystalline promoter, which was then fused to the coding sequence of murine IFN-y gene. In both the rat and mouse models, ectopic expression of IFN-Y in the lens affecting the growth of the w^hole eye resulted in cataract thickening of the anterior lens capsule, rupture of posterior capsule, impairment of the lens fiber forma- tion as well as microphthalmia and micropha- kia. The group has an ongoing interest in the role of the T-ceU receptor and how it relates to autoimmune intraocular inflammatory disease. The goal is to develop anti-T-ceU receptor therapies for the treatment of uveitis. In doing this, athymic as well as euthymic rats were injected with cells from antigen-stimulat- ed T-ceU lines specific for a major pathogenic epitope for bovine IRBP. The findings suggest that the time of appearance of cytokine-pro- ducing T-cells in the retina is influenced by the immunologic status of the rat, the differ- ence in circulating cytokines in athymic rats might affect parameters such as vascular permeability and could facilitate penetration of T-ceUs into the eye. Additionally, the stiong TH-1 Uke cytokine profile was detected in R-16 rats, but this was not detected in AK-16 rats. This might suggest that the cytokine profile observed in the refina is influenced by the identity of the pathogenic epitope. The Clinical Branch, in collaboration with the Experimental Immunology Section, has looked at a variety of immunologic questions as they relate to ocular disease. A continuing interest in ceU adhesion molecules has 5delded new information. The effective treatment with the monoclonal antibody directed against lymphocyte function-associated antigen (LFA- 1) and very late activation antigen (VLA)-4 demonstrates that the anti-LFAl antibodies sigruficantiy inhibited the development of EIU. This was in contrast to the anti-VLA-4 anti- body that had no effect on the development of intraocular inflammation. They have also seen that systemic treatment with anti-IL-12 mono- clonal antibodies exacerbates the development of EIU. These findings were similar to previ- ous studies with anti-IFN-y antibody. They have also noted that topical heparin signifi- cantiy inhibited the development of allergic conjunctivitis in mice. This group has also been very active in the evaluation of uveitis in patients. The group has been involved in a number of studies designed to improve the treatment of uveitis. They have recentiy completed a prospective, double-masked randomized study 68 Laboratory of Immunology of diamox for uveitis cystoid macular edema. They have also completed a pUot study exam- ining the efficacy and toxicity of a chemother- apy regimen for therapy of patients with central nervous system (CNS) lymphoma involving the brain or eye. This has been done in collaboration with the Medicine Branch of NCI. A clinical trial is also under way evaluating heparin surface-modified intraocular lens in patients with uveitis. Section on Experimental immunopathology The Section on Experimental Immunopatholo- gy has continued to study the immunopathol- ogy of various inflammatory cells and ocular resident cells in a variety of experimental models of uveitis. This group has developed a particular expertise in in\munohistochemis- try and in situ hybridization techniques that have provided the whole laboratory with the ability to identify and topographically localize immunocompetent cells. Additionally, it analyzes the alteration of surface markers on ocular resident cells and the production of cytokines in experimental uveitic models as well as in tissue obtained from patients undergoing surgery. They have demonstrated that higher levels of S-Ag and its mRNA are expressed in nonretinal ocular cells such as the lens, the ciliary body, and the trabecular meshwork of EAU rats. This was particularly so in animals that received long-term steroid therapy. The group has also evaluated several new experimental models for uveitis. Experimental melanin induced uveitis (EMIU) can be induced with immuni- zation using bovine choroidal and RPE mela- run protein. EMIU is characterized by a bilateral recurrent iris, scleritis, and choroidi- tis. The main infiltrating cells in this model are T-cells of CD-4 origin seen in the early stage of the disease and CD-8 positive cells that infiltrate at later stages. There is an abundant expression of adhe- sion molecules and MHC class 2 antigens on the ocular resident cells one to two days before ocular inflammation is noted. Of interest in this disorder is the fact that recur- rences occur one month after the first attack becomes quiescent. The group has continued its interest in evaluating an animal model for acquired ocular toxoplasmosis. This is done by the infection of a virulent strain of Toxo- plasma gondii (ME49) into mice. Focal ocular inflammation as well as RPE involvement are seen about two weeks after the infection. About one month after infection, ocular in- flammation becomes stable and only occasion- al cysts can be seen. The group evaluated 12 cases of intraocular lymphoma diagnosed at the NEI between 1984 and 1992. These were aU non-Hodgkin's large B-ceU lymphomas of the CNS. The prompt appropriate handling of specimens and the review by an experienced cytopathologist have been shown to be excep- tionally critical to the diagnosis of intraocular lymphoma. They have also evaluated three affected members of a Chinese-American family with Bietti's crystalline retinopathy. Crystalline lysosomal materials are observed in lymphoc5^es and skin fibroblasts of these patients. Section on Immunology and Virology The Section on Immunology and Virology has continued to emphasize its interest in the study of the RPE cells. This section has devel- oped a new method using RPE choroidal explants to initiate cell growth. By monitoring the clusters of cells growing around the ex- plants, they were able to select purely epitheU- al cells and discard the nonepitheUal cells at the primary culture stage. Using this tech- nique, they have established primary cell lines of human RPE from cells of elderly patients. The cell culture origin was confirmed by immunochemical staining for cytokeratin with monoclonal antibodies. Human RPE cultures secrete significant quantities of IL-6 and inter- cellular adhesion molecule 1 (ICAM-1) but no IL-1 in response to stimulation by inflammato- ry mediators. 69 FY 1994 NEI Annual Report These observations are important in un- derstanding posterior uveitis that may be caused by infections or an autoimmune pro- cess. Lymphocytes and macrophages infiltrate into the retina and secrete cytokines such as IL-1, TNFa, IFN-Y, and IL-2 that would initi- ate immune reactions. In response to these cytokines, the retinal resident cells could locally produce lL-6 as well as ICAM-1 to amplify the rmmunopathologic process. The group has continued its interest in the field of ocular toxoplasmosis. They were able to demonstrate in this model that 100 percent of mice develop cysts in the brain, but retinal cysts could be found late in the course of the disease. This section has also developed polymerase chain reaction techniques for the detection of a variety of viruses in the eye. This ability will be applied in the future for the evaluation of intraocular samples for the presence of a variety of viruses that are thought to be path- ogenic in the eye. 70 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00280-03 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) Transgenic Rat and Mouse Models for the Study of Intraocular Effects of IFN-y and Autoimmunity PRINCIPAL INVESTIGATOR (List other professiortal personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: Charles E. Egwuagu Ph.D., M.P.H. Senior Scientist LI, NEI Otheis: Robert B. Nussenblatt Chi-Chao Chan Ana B. Chepelinsky Jorge Sztein Rashid Mahdi M.D. Scientific Director NEI M.D. Head, Section on Immi nology U, NEI Ph.D. Head, Section on Regulation of Gene Expression LMDB, NEI D.V.M., Ph.D. Visiting Associate U, NEI B.S. Biologist U, NEI COOPERATING UNITS (if any) Laboratory of Immunology SECTION Section on Experimental Immunology INSTITUTE AND LOCATION NEI, Nm, Bethesda, MP 20892 TOTAL STAFF YEARS: PROFESSIONAL: 1.2 1.2 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues \x\ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) To Study the possible role of interferon (IF^^)-Y in ocular pathogenesis and, specifically, the linkage between its induction of aberrant major histocompatibility complex (MHC) class II expression and predisposition to ocular autoimmune diseases, we generated transgenic mice with constitutive expression of IFN-7 in the eye. Although the mouse has. up to this point proven to be a useful model, the rat is the preferred strain for the study of experimental autoimmune uveoretinitis (EAU). EAU is an animal disease that shares essential features with several human uveitic diseases such as sympathetic ophthalmia, birdshot retinochoriodopathy, Behget's disease, and Vogt-Koyanagi-Harada (VKH) syndrome. Consequently, we have generated a IFN-7 transgenic Sprague Dawley rat strain. In FY 1993-1994 we focused on characterizing both the rat and mouse IFN-7 transgenic models with the goal of establishing a comprehensive and complementary transgenic animal system that would be useful for studying the in vivo effects of IFN-y in the eye. 71 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The objectives of this project include the study of the possible role of interferon gamma (IFN- Y) in the eye. Aberrant expression of the major histocompatibihty complex (MHC) class n molecules is an early event in a number of human autoimmune diseases, and IFN-y induces high levels of MHC class II protein biosynthesis. Therefore, we generated trans- genic animals with selective secretion of IFN-7 in their eye tissues. These transgenic mice and rats are ideally suited for studying the effects of IFN-7 on the physiology of the eye and the role of elevated MHC class II predis- position to intraocular autoimmune diseases. Methods Transgenic rat and mouse strains were gener- ated by microinjection of a deoxyribonucleic add fragment containing the murine aA-crys- taUin promoter (aACry) fused to the coding sequence of murine IFN-y gene. Polymerase chain reaction (PCR) and reversed transcrip- tion-PCR (RT/PCR) were used to screen for the presence of the transgene and conduct messenger ribonucleic acid (mRNA) analyses, respectively. Methacrylate-embedded eye sections were analyzed for morphology and cryosections for immunoperoxidase antibody staining. Major Findings In both rat and mouse models, ectopic expres- sion of IFN-y in the lens affected the growth of the whole eye, resulting in cataract, thicken- ing of anterior lens capsule, rupture of posteri- or capsule, impairment of lens fiber formation, microphthalmia, and microphakia. Additional effects in the mouse include blepharophimosis, arrest of retinal differentiation, serous retinal detachment with presence of macrophages in the subretinal space, persistent hyperplastic primary vitreous, and corneal vascularization. Unlike the mouse, the rat anterior chamber is well formed, and the retina is intact with focal retinal serous detachment. MHC class 11 mRNA levels were significantly increased in the transgenic mouse eyes, and MHC class 11 proteins were expressed in their corneas, irises, cihary bodies, choroids, lens, and retinal pigment epthelia. At the molecular level, the pattern of lens gene expression was perturbed, and expression of gene coding for adhesion molecules and IFN-y-inducible transcription factor was up-regulated in transgenic eyes. Significance to Biomedical Researcti and ttie Program of ttie Institute The aACry /IFN-y transgeruc rat is the first transgenic rat strain generated for vision research. Constitutive expression of IFN-y, and its induction of MHC class II molecules in the eye, provides a useful model to address: (1) the linkage between aberrant MHC class 11 expression and predisposition to autoimmuni- ty, (2) the role of IFN-y in the treatment of inflammatory eye diseases and in ACAID, and (3) understanding cytokine signaling during embryonic eye development. The rat and mouse models complement each other for elucidation of the in vivo effects of IFN-y in the eye. Proposed Course We intend to continue studying the molecular basis of IFN-y actions in the eye with particu- lar emphasis on the rat. A major focus will be to estabUsh primary and long-term cultures of IFN-y-expressing epithelial lens cells as these cell lines would be valuable in studies aimed at understanding the mechanism of transcrip- tional activation in the aACry-IFN-y animals. NEI Research Program Retinal Diseases — ^Inflammatory Diseases Publications Egwuagu CE, Sztein J, Reid W, Chan C-C, Mahdi R, Nussenblatt RB, Chepehnsky AB: Gamma interferon expression disrupts lens 72 Laboratory of Immunology and retinal differentiation in bransgenic mice. Dev Biol, in press. Egwuagu CE, Sztein J, Reid W, Chan C-C, Mahdi R, Nussenblatt RB, Chepelinsky AB: Transgenic rat and mouse models for stii dying the role of gamma interferon and MHC Class n in intiaocular diseases and autoimmunity, in Nussenblatt RB, Gery 1 (eds). Sixth Interna- tional Symposium of the Immunology and Immu- nopathology of the Eye. Amsterdam, Nether- lands, Elsevier Press, in press. Egwuagu CE, Sztein J, Reid W, Chan C-C, Mahdi R, Nussenblatt RB, Chepelinsky AB: Ectopic expression of gamma interferon in the eyes of transgenic mice induces ocular pathol- ogy and MHC class II gene expression. Invest Opthalmol Vis Sci 35:332-341, 1994. Egwuagu CE, Sztein J, Reid W, Chan C-C, Mahdi R, Nussenblatt RB, Chepehnsky AB: Transgenic rat and mouse models for the study of intraocular effects of IFN-y and autoimmunity. Invest Opthalmol Vis Sci 35/4 (suppl):3391, 1994. 73 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00262-05 LI 1 PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Analysis of T Lymphocytes and Cytokines Involved in Experimental Autoimmune Uveoretinitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Charles E. Egwuagu Others: Igal Gery Robert B. Nussenblatt Rachel Caspi Rashid Mahdi Alexanda T. Kozhich Phyllis B. Silver Ph.D., M.P.H. Senior Research Scientist Ph.D. Head, Section on Experimental Immunology M.D. Scientific Director Ph.D. Visiting Associate B.S. Biologist Ph.D. Visiting Fellow B.S. Biolog ist LI, NEI LI, NEI NEI LI, NEI LI, NEI LI, NEI LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Section on Experimental Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.7 PROFESSIONAL: 0.7 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated autoimmune disease that serves as a model of human intra-ocular inflammatory diseases (uveitis). It is initiated in susceptible animals by immunization with retinal antigens such as interphotoreceptor retinoid-binding protein (IRBP) or S-antigen (S-Ag) or by adoptive transfer of activated S-Ag- or IRBP-specific uveitogenic T lymphocytes. We had previously demonstrated that VpS-expressing T cells accumulate in the retina during EAU. In FY 1993-1994, we sought to define the T-cell subsets and cytokines present in the retina of athymic and euthymic Lewis rats after adoptive transfer of uveitogenic or nonuveitogenic T lymphocytes. Our results indicate that (1) the temporal appearance of cytokine-producing T cells in the retina is influenced by the immunological status of the rat and might affect parameters such as vascular permeability that influence the penetration of lymphocytes into the eye. (2) Cytokine messenger ribonucleic acid (mRNA) transcripts were detected in the retinas of animals immunized with uveitogenic T lymphocytes, as well as in the retinas of rats injected with Con A-specific T cells. However, rats injected with Con A-specific T cells neither developed EAU nor was there detection of VpS* T cells in their retinas. (3) Detection of interferon (IFN)-y transcripts was temporally correlated with the appearance of VpS"^ T cells in the retina and the onset of disease. Taken together, our data suggest that infiltration of the retina by activated T-cells is not sufficient for disease induction; ocular-antigen specific Thl- like VpS^ lymphocytes appear to be necessary for EAU induction. 74 PHS 6040 (Rev. 5/92) Laboratory of Immunology Project Description Objectives This project is aimed at determining the clo- nality of the T lymphocytes that mediate intraocular autoimmune diseases. Identifica- tion of the pathogenic T-cell subset in experi- mental autoimmune uveoretinitis (EAU) is relevant to our goal of developing anti-T-ceU receptor (TCR) therapies for the treatment of uveitis. Our effort during fiscal year 1993-1994 focused on analyses of T cells pres- ent at the autoimmune site and the lympho- kines that they produce. Methods Athymic and euthymic rats were injected with cells from an antigen (Ag)-stimulated T-ceU line specific to the major pathogenic epitope of bovine interphotoreceptor retinoid-binding protein (IRBP) (peptide R16: amino acids 1177-1191; R16 rats) or with concanavalin A (ConA)-stimulated splenocytes (ConA rats). In a separate experiment, euthymic rats were injected with an Ag-stimulated T-cell line specific to the pathogenic epitope of rat IRBP (peptide AK16: amino acids 273-283; AK16 rats) or with purified proton derivative (PPD)-stimulated primed lymph node cells (PPD rats). Retinas were sampled every 12 hours after uveitogervic challenge and reversed transcription polymerase chain reaction (RT/PCR) was used to analyze messenger ribonucleic acid (mRNA) levels for TCR VpS, interferon gamma (IFN-y), tumor necrosis factor alpha (TNF-a), interleukin (IL)-2, IL-6, CD-4, and CD-8. Major Findings (1) Analyses of cytokine mRNAs in retinas of athymic and euthymic ConA and R16 rats showed that in athymic rats IFN-y, TNF-a, IL- 2, IL-6, CD-4, and CD-8 mRNAs were detected after 12 hours, whereas in the euthymic rats they were only detected after 24-84 hours. Retinas of ConA rats showed essentially the same cytokine profile as retinas of R16 rats. Cytokine mRNAs were not detected in retinas of animals that did not receive cells. (2) Analyses of cytokine mRNAs in euthymic AK16 and PPD rats showed that TNFa, IL-6, CD-4, and CD-8 mRNAs were detected after 24 hours, but IFN-y and IL-2 transcripts were not detectable even after 120 hours. In contrast to ConA rats, cytokine mRNA expression was not detected in retinas of PPD rats. (3) In aU experiments, the ap- pearance of Vp8* T cells in the retina coincid- ed with the temporal expression of IFN-y in the retinas of rats with experimental autoim- mune uveitis (EAU). A similar correlation was not observed for any of the other cyto- kines. Significance to Biomedical Research and the Program of the institute (1) The time of appearance of cytokine-pro- ducing T cells in the retina appears to be influenced by the immunological status of the rat. Differences in levels of circulating cyto- kines in athymic rats might affect parameters such as vascular permeability and could facilitate penetration of T cells into the eye. (2) A Th-1-like cytokine profile was detected in R16 rats, whereas it was not de- tected in AK16 rats. This might suggest that the cytokine profile observed in the retina is influenced by the identity of the pathogenic epitope. Proposed Course Analyses of uveitogervic T-ceU clonotypes and the lymphokines they produce during EAU will be continued to identify the relevant autoaggressive T cells involved. Our study will be expanded to include analyses of speci- mens obtained from patients with ocular sarcoidosis and anterior and posterior uveitis. NEI Research Program Retinal Diseases — ^Inflammatory Disorders 75 FY 1994 NEI Annual Report Publications Egwuagu CE, Bahmanyar S, Mahdi R, Nussenblatt RB, Gery I, Caspi R: Predominant usage of Vp8.3 T cell receptor in a T ceU line that induces experimental autoimmune uveo- retinitis. Clin Immunol & Immunopathol 65:152, 1992. Egwuagu CE, Caspi R, Mahdi R, Gery I, Nussenblatt BR: Evidence for selective accu- mulation of Vp8+ T lymphocytes in experi- mental autoimmune uveoretinitis induced by two different retinal antigens / Immunol 151:1627, 1993. Kozhich AT, Kawano Y, Egwuagu CE, Caspi RR, Maturi RK, Berzofsky JA, Gery I: A pathogenic autoimmune process targeted at a surrogate epitope. / Exp Med, in press Mahdi RM, Caspi RR, Kozhich AT, Kozhich OA, Silver PB, Nussenblatt RB, Egwuagu CE: C3^okine mRNA expression following adop- tive transfer of uveitogenic T cells into athymic and euthjonic Lewis rats. Invest Opthalmol Vis Sci 35/4 (suppl):1432, 1994. 76 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00069-17 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Immune Responses to Ocular Antigens PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Igal Gery Alexander Kozhich Eddy Anglade Scott M. Whilcup Chi-Chao Chan Robert B. Nussenblatt Barbara Vistjca Nathan Felix James Lai Eric Wawrousek Christina M. Sax Ph.D. Ph.D. M.D. M.D. M.D. M.D. B.A. B.A. B.A. Ph.D. Ph.D. Head, Section on Experimental Immunology Visiting Fellow Senior Staff Fellow Associate Clinical Director Head, Section of Immunopathology Scientific Director Microbiologist Special Volunteer Guest Researcher Head, Section on Transgenic Animals and Genome Manipulation Senior Staff Fellow U, NEI LI, NEI U, NEI CB, NEI LI, NEI LI, NEI LI, NEI LI, NEI LI, NEI LMDS, NEI LMDB, NEI COOPERATING UNITS (if any) Biotechnology Unit, Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases (Joseph Shiloach, Ph.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Experimental Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892-1858 TOTAL STAFF YEARS: 2.8 PROFESSIONAL: 2.4 0.4 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ {a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Targeted at learning about inflammatory eye diseases grouped under the term "uveitis," this project continued to focus mainly on learning about ocular antigens capable of inducing experimental autoimmune uveoretinitis (EAU), an animal model for uveitis in humans, and procedures that modulate this disease. The major achievements of this project in FY 1994 include: (1) We discovered that recoverin, a retinal calcium-binding protein, is highly uveitogenic in rats, producing severe inflammatory changes in eyes of rats of all four strains tested. In addition to identifying a new uveitogenic retinal molecule, this observation provides evidence to support the assumption that recoverin is the target for an autoimmune process that causes a condition termed cancer-associated retinopathy (CAR). (2) Recombinant human S-antigen, which has become available by recombinant deoxyribonucleic acid (DNA) technologies, was found to be uveitogenic in primates, inducing severe inflammatory ocular changes that closely resemble those induced by bovine S-antigen. Lymphocytes from the immunized monkeys were used to identify the peptides that are selected as the immunodominant determinants, i.e., the ones that are the target for the immune response in animals immunized with the whole protein. Monkeys of different species responded against different peptides, whereas four monkeys of the same species (cynomolgus) remarkably responded against the same selected peptides. (3) Blood lymphocytes collected at different time points from a human donor exhibited consistency in their responding strongly to the same selected peptides of human S-antigen. Yet, marked changes were observed in the capacity of individual dominant peptides to stimulate lymphocytes collected at different time points. (4) To examine the effect of sequestration on the immunogenicity of lens proteins, transgenic (TG) mice were developed in which foreign antigens are selectively expressed in the lens. Two different types of response were observed: mice expressing chloramphenicol aminotransferase (CAT) responded to this antigen similarly to their wildtype controls, while TG mice expressing hen egg lysozyme (HEL) failed to respond against this antigen, due to a state of complete immunotolerance. (5) Oral tolerance, a procedure used to inhibit pathogenic autoimmune processes, was found to be enhanced by treating the fed animals with certain bacterial products, with the best effect achieved with glucosaminyl muramyl dipeptide (GMDP). (6) The role of CDS lymphocytes in the process of oral tolerance induction was examined by testing the capacity of mice deficient in these cells to develop oral tolerance. CDS deficient mice resembled their controls in developing tolerance, thus showing that CDS cells are not always essential for induction of oral tolerance. 77 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives Studies conducted during fiscal year (FY) 1994 were aimed at the following: (1) to examine the uveitogenicity of recoverin, a calcium- binding protein specific to the retina, that was reported to be the target for autoantibodies detected in the majority of cases with cancer- associated retinopathy (CAR); (2) to test the uveitogenicity of recombinant human S-anti- gen (rHumS-Ag) in primates and to identify the peptide determinants that are selected as the immunodominant epitopes by the immune system of the immunized monkeys; (3) to monitor at different timepoints the changes that may occur among the subpopulations of human blood Ij^nphocytes that respond against different epitopes on HumS-Ag; (4) to investigate the level of sequestration of pro- teins within the lens by testing the develop- ment of immunotolerance toward foreign antigens expressed in the lens of transgenic mice (expression of foreign proteins in other organs usually produces tolerance); (5) to test the capacity of bacterial products to enhance oral tolerance, a procedure that is extensively used to inhibit pathogenic autoimmune pro- cesses, including uveitis in man. In the sys- tem used here, feeding with S-Ag is used to inhibit the development of experimental autoimmune uveoretinitis (EAU) in rats; and (6) to investigate the role of CD-8 lymphocytes in the process that brings about oral tolerance by testing the capacity of CD-8 deficient mice to develop oral tolerance. Methods Recombinant recoverin was kindly provided by Dr. L. Stryer, from Stanford University; rHumS-Ag was prepared by Dr. Joseph Shiloach, from the National Institute of Diabe- tes and Digestive and Kidney Diseases (NIDDK), as described in our Annual Report, FY 1993; peptides were sjnithesized and puri- fied by Applied Biosystem, Foster City, Cali- fornia. Monkeys of different species (rhesus, artcoides, and cjmomolgus) were provided by the National Institutes of Health (NIH) animal facility, and R2 m -/- mice (CD-8 deficient) were provided by the NCI Twinbrook Facility. Blood samples were collected at different timepoints from a single donor, and the mononuclear leukocytes were separated on Isolymph gradients. Published conventional methods were used to immunize animals and measure their immune response as weU as development of EAU. Adoptive fransfer of EAU was carried out as described by Mochizuki et al. {Invest Ophthalmol Vis Sci 26:1, 1985). Levels of chloramphenicol acetyltrans- ferase (CAT) were measured by a biphase CAT assay, and those of human fibroblasts (HEL) were determined by the particle con- cenfration fluorescence immunoassay. Major Findings Uveitogenicity of recoverin. Recombinant re- coverin was found to be highly uveitogenic in Lewis rats, inducing severe EAU at doses as low as 10 fig per rat. The clinical and histo- pathological changes induced by recoverin closely resemble those elicited in rats by S-Ag, reaching in many cases the maximum grade of severity of 4+. Recoverin is also found in the pineal gland, and most rats with EAU also developed pineal inflammation. In addition to Lewis rats, recoverin was found to induce EAU in three other rat sfrains — ^BN, WF, and ACL Similarly to observations with other uveitogenic antigens, however, the severity of changes in these strains was lower than in Lewis rats. EAU induced by recoverin was found to be ceU-mediated; it is readily and adoptively fransferred to naive recipients by IjTnph node or spleen cells from immunized donors. Moreover, antibodies to recoverin seem to play a minor role, if any, in the patho- genic process because recipients developed disease even in the absence of antibodies, as seen in rats injected with recoverin-sensitized spleen cells that were stimulated in culture with Concanavalin A. Uveitogenicity and antigenicity ofrHum S-A8 in primates. Monkeys of three different species (artcoides, rhesus, cynomolgus) developed uveitis when immunized with rHumS-Ag. 78 Laboratory of Immunology The ocular changes closely resembled those observed in previously published studies in eyes of monkeys immunized with bovine S-Ag (Nussenblatt RB, et al.. Arch Ophthalmol 99:1090, 1981). Peripheral blood lymphocytes from the immunized monkeys responded well against the rHumS-Ag molecule as well as against bovine S-Ag. The monkey lymphocytes were also tested for their proliferative response against 40 synthetic overlapping peptides that span the entire sequence of HumS-Ag. Lymphocytes from monkeys of the three tested species recognized and responded well against differ- ent peptides; a finding that indicates that different determinants of HumS-Ag serve as the "immunodominant epitopes" for the im- mune system of monkeys of different genetic makeups. On the other hand, all four cyno- molgus monkeys remarkably recognized the same immunodominant regions of HumS-Ag at sequences 21-61, 71-90, 121-140, 171-200, and 281-310. Response of human lymphocytes against HumS-AS peptide determinants: Specificity pattern is conserved. Peripheral blood lympho- cytes collected from a single donor at different timepoints were used to examine the possible changes in the repertoire of responsiveness toward HumS-Ag and its 40 overlapping peptides. Six blood samples, collected at timepoints spanning over six months, were tested in the present study. Significant re- sponses were found at all timepoints against four regions, localized at sequences 71-90, 121-140, 171-200, and 341-360. Marked varia- tions were noted, however, among the six blood samples in the level of response to each of these four peptides, thus producing differ- ences in the hierarchy of their stimulating capacity. Immune responses in transgenic mice express- ing foreign antigens in their lens. Transgenic mice that express foreign antigens in their lens were developed by collaborators at the LMDB, NEl. Mice expressing CAT were developed by Dr. Chris Sax, but mouse Unes expressing HEL were established by Dr. Eric Wawrousek. Both foreign antigens were expressed under the control of the aA-crystaUin promoter. Sensitive immunological tests (see Methods section) showed high concentrations of CAT or HEL in the lens, but neither antigen could be detected in any other organ or in the blood of the transgenic mice. Development of im- munotolerance in the transgenic mice was examined by measuring their capacity to mount specific immune responses following immunization with the corresponding antigen, emulsified in complete Freund's adjuvant. The main findings include: (1) Expression of CAT in the lens had Uttie effect on the capaci- ty of the transgenic mice to respond against this antigen. Transgenic mice immunized with CAT produced antibodies to CAT with levels similar to those of their wild-type con- trols. The transgenic mice also developed cellular immune response to CAT, albeit with levels sUghtiy lower than those monitored in the wild-type controls. (2) In contrast to the findings with CAT, expression of HEL in the lens produced a state of complete tolerance to this antigen; HEL transgenic mice failed to develop any detectable antibody or cellular immunity against HEL following immuniza- tion with this antigen. Enhancement of oral tolerance by bacterial products. Oral administration of the bacterial product N-acetyl-D-glucosaminyl-P (l-4)-N- acetyl-L-muramyl-L-alanyl-D-isoglutamine (GMDP) along with a uveitogenic peptide significantly enhanced the level of tolerance induced by the peptide. The study was car- ried out in rats in which EAU is induced by peptide 1181-1191 of bovine interphotorecep- tor retinoid-binding protein (IRBP). Feeding with peptide 1181-1191 reduces the disease development, and the level of inhibition was further eivhanced by cofeeding with GMDP. A sUght enhancing effect was also induced by bacterial Upopolysaccharide (LPS), but Salmo- nella typhimurium mitogen (STM) and the des- (N-acetyl-D-glucosaminyl) analog of GMDP had no detectable effect in this system. The capacity of GMDP to enhance oral tolerance was further demonstrated by the finding that the cellular immunity to peptide 1181-1191 was iivhibited remarkably more in rats fed 79 FY 1994 NEI Annual Report with the combination of this peptide and GMDP than in those fed with the peptide alone. Induction of oral tolerance in CD-8 cell-defi- cient mice. The role of OD-S lymphocytes in the process that produces oral tolerance was examined by testing whether mice deficient of these cells are capable of developing oral tolerance. The QD-S-deficient animals used here were p2m -/- mice, i.e., animals in which the disruption of the p2-microglobulin gene causes very low expression of MHC class I molecules and severe deficiency of CD-S"^ ceUs. Feeding these mice with ovalbumin (three or five times, 1 mg per mouse) produced remark- able levels of immunotolerance that closely resembled those observed in the similarly treated control animals. This finding indicates that the CD-8 cells are not essential for the induction of oral tolerance, at least when induced by the procedure used in this study. Significance to Biomedicai Research and the Program of the Institute (1) The finding that recoverin is highly uveitogenic underscores the unique character- istic of the retina, i.e., its content of mvdtiple molecules with the capacity of initiating path- ogenic autoimmune processes; previous stud- ies have identified four other uveitogenic proteins in the retina. In addition, the capaci- ty of recoverin to initiate a pathogenic autoim- mune process supports the notion that this protein is the target for the putative autoim- mune process that brings about the retinal damage in CAR. (2) The present study is the first to show that HumS-Ag is highly uveitogeruc in pri- mates. Moreover, this observation supports the notion that autologous S-Ag plays a major role in the immunopathogenic process of uveitis in man. This study also provides, for the first time, information concerning the peptide determinants of HumS-Ag that are immunodominant in monkeys in which im- munization with HumS-Ag produced uveitis. As expected, monkeys of different species varied in their selection of the dominant peptides, but the similarity among the four cynomolgus monkeys was quite surprising because these animals are outbred. The latter observation may suggest that uveitic patients with partial identity of histocompatibility anti- gens may also exhibit similarity in their selec- tion of immunodominant epitopes. (Such an observation was made with multiple sclerosis patients). (3) The study of responsiveness to HumS- Ag peptides of a human donor at different timepoints has provided information concern- ing the fluctuations among lymphocyte clones with specificity toward autologous peptides. The finding that responses to the same few epitopes were observed in aU blood samples thus indicates that only small and quantitative fluctuations occur among the clones of Ijmi- phocj^es that recognize the dominant epitope of an autologous antigen. (4) The experiments with the transgenic mice have yielded new information on the development of immunotolerance against antigens expressed inside the encapsulated lens. The findings so far, of two patterns of response against CAT or HEL suggest that lens proteins can be treated in different ways by the immune system. (5) The observation that the efficacy of oral tolerance can be enhanced by cotreating the animals with GMDP provides a new strategy for the inhibition of pathogenic im- munemediated processes such as uveitis. The potential to erihance oral tolerance is of great importance because the feeding procedure usually produces only partial inhibition of autoimmune diseases. (6) The finding that CD-8-deficient mice develop oral tolerance shows that this subpop- ulation of lymphocytes is not essential for the induction of oral tolerance in the system used in the present study. CD-8 ceUs were shown in other studies to play a major role in the induction of oral tolerance (Weiner H, et al., Annu Rev Immunol 12:809, 1994), and thus, our data provide direct evidence to the notion that 80 Labomtoiy of Immunology at least two different mechanisms participate in this process. Proposed Course Our future efforts will focus on the following issues: (1) Other retinal proteins, mainly those related to recoverin, will be tested for uveito- genicity; (2) the response to HumS-Ag of primates and human subjects will be further analyzed, mainly by attempts to establish and analyze cell Unes with specificity toward this molecule and its peptide detemunants; (3) the development of tolerance against foreign antigens expressed in the lens will be further analyzed, mainly with regard to the mecha- nisms that bring about tolerance and the difference between the responses to CAT and HEL; and (4) more effort wiU be focused on studies aimed at enhancing oral tolerance, the mechanisms involved in this phenomenon, and its usage for suppression of pathogenic autoimmune processes. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications Chan C-C, Hikita N, Dastgheib K, Whitcup SM, Gery I, Nussenblatt RB: Experimental melanin-protein induced uveitis in the Lewis rat: Immunopathological processes. Ophthalmology 101:1275-1280, 1994. Gery I, Chanaud NP III, Anglade E: Recoverin is highly uveitogenic in Lewis rats. Invest Ophthalmol Vis Sci 35:3342-3345, 1994. Gery I, StreUein JW: Autoimmunity in the eye and its regulation. Cuir Opinion Immunol, in press. Kasner L, Chan C-C, Whitcup SM, Gery I: The paradoxical effect of tumor necrosis factor alpha (TNF-a) in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 34:2911-2917, 1993. Kozhich AT, Kawano YI, Egwuagu GE, Gaspi RR, Maturi RK, Berzofsky JA, Gery I: A pathogenic autoimmune process targeted at a surrogate epitope. / Exp Med 180:133-140, 1994. Sasamoto Y, Kawano YI, Wiggert B, Chader GJ, Gery I: Induction of unresponsiveness in adult rats by immunodominant and nondomi- nant peptides. Cell Immunol 152:286-292, 1993. 81 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00298-01 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Inhibition of EAU in Monkey With Humanized Anti IL-2 Receptor PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Francois G. Roberge Others: Yan Guex-Crosier M.D. Igal Gery Ph.D. Chi-Chao Chan M.D. Robert B. Nussenblatt M.D. Visiting Scientist LI, NEI Special Volunteer LI, NEI Deputy Laboratory Chief LI, NEI Head, Section on LI, NEI Immunopathology M.D. Scientific Director LI, NEI COOPERATING UNITS (if any) VRRS, NEI (James Raber, Ph.D.); VRRS, NEI (Martin Kriete, Ph.D.); NCI (Thomas Waldmann, M.D.); Hoffmaim LaRoche, Nutley, NJ (John Hakimi) LAB/BRANCH Laboratory of Immunology SECTION Clinical Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.7 PROFESSIONAL: 0.7 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Experimental autoimmune uveoretinitis (EAU) was induced in Cynomolgus monkey by immunization with recombinant human retinal S-antigen. At the onset of ocular disease, animals were treated for 28 days with intravenous injection of humanized anti-Tac, an anti-interleukin (IL)-2 receptor antibody originally produced in mouse but modified by replacing all but its binding region with human immunoglobulin elements. Controls were treated with vehicle alone. The animals were examined twice a week during the treatment period. The progression of the disease was markedly limited in the group treated with anti-Tac-H, while the severity of the inflammation continued to increase in the control group. The in vivo results were correlated with a significant inhibition of monkey lymphocyte proliferation stimulated by IL-2 when anti-Tac-H was added to the culture medium. 82 PHS 6040 (Rev. 5/92) Laboratory of Immunology Project Description Clinical Protocol Number 93255 Objectives General Goal of the Study. Evaluate the effec- tiveness of humanized anti-interleukin (IL)-2 receptor antibodies for the therapy of autoim- mune diseases. Specific Objectives (1) Evaluate the effect of two humanized anti-IL-2 receptor antibodies, anti-Tac-H, and Mik-bl-H, respectively directed at the a and p chain of the IL-2R, in the treatment of active uveoretinitis in monkey. (2) Evaluate the possibUity of a beneficial therapeutic effect in using anti-Tac-H and Mik-bl-H in combination or in the form of recombinant hybrid molecules. (3) Correlate the therapeutic response with in vitro study of inhibition of prolifer- ation of monkey lymphocytes by anti-Tac-H and Mik-bl-H. (4) Measure the response of treated mon- keys against the antibodies used for treatment. Methods Animal Immunization and Examination. Cyno- molgus morJceys, between 2.0 and 3.0 kg, are immuriized with recombinant human retinal S-antigen at 40 /ig/kg emulsified in Hunter's TiterMax adjuvant by subcutaneous injection at the nape of the neck. Twice a week, starting 14 days after im- munization, the animals are sedated with ketamine, the pupils dilated with topical atropine, and the eyes examined by indirect ophthalmoscopy for signs of uveoretinal inflammation. Treatment Protocol. The animals are ran- domized in two groups of five individuals. Treatment consists of intravenous injection of anti-Tac-H or Mik-bl-H at 2.0 mg/kg, or control vehicle. The treatment is instituted on the day of presentation of the first sign of experimental autoimmune uveitis. Antibody injections are done following a schedule of alternating three- and four-day periods, for a total of eight injections, covering a total treat- ment period of 28 days. In addition, the pupils are kept dilated with the application of atiopine ophthalmic ointment 0.5 percent. Tests Performed Preimmunization • Blood drawn for complete blood count (CBC) and serum for baseline measure- ments. At initiation of therapy • Funduscopy and fundus photography. • Fundus fluorescein angiogram. • Measurement of intraocular pressure with a Tonopen tonometer. • Blood drawn for CBC, serum creatinine, slL-2R, and serum for baseline measure- ments. Once a week • Fundus photography. • Measurement of intraocular pressure with a Tonopen tonometer. • Blood collection for anti-idiotypic antibody and sIL-2R, anti-S-Ag Ab, and level of anti-Tac-H or Mik-bl-H. At termination of experiment • Funduscopy and fundus photography. • Fundus fluorescein angiogram. • Measurement of intraocular pressure with a Tonopen tonometer. • Blood drawn for CBC, serum creatinine, anti-idiotypic antibody, sIL-2R, anti-S-Ag Ab, level of anti-Tac-H or Mik-bl-H. • Eyes are collected for histopathological examination. 83 FY 1994 NEI Annual Report Major Findings On average, the ocular inflammation was stabilized in the animals treated with anti-Tac-H. There was a marked progression of the disease in the control group. Given the caveat of the small sample number, the statis- tical analysis of the present data by the Mann- Whitney test on the averaged variation per animal showed significance at a value of p < 0.01. We also observed a marked decrease of the intraocular pressure (-50 percent) in the inflamed eyes. There was no significant difference between the two treatment groups. We have also tested the activity of various anti-IL-2 receptor antibodies in the inhibition of IL-2 driven proliferation of monkey periph- eral blood lymphocytes. The results con- firmed the inhibitory effect of anti-Tac-H and a hybrid of anti-Tac-H and Mik-bl-H that had been observed with human lymphocytes. Mik-bl-H alone was not inhibitory but pro- duced an increased inhibition when used in combination with anti-Tac-H. We were also surprised to find that the antibody 7G7/B6, which does not block the IL-2 signal on hu- man lymphocytes, was a strong uihibitor of monkey lymphocyte proliferation. In conclusion, the results of this first experiment indicate that the humanized anti-Tac antibody could be useful in the thera- py of some autoimmune diseases. We are now conducting a similar experiment to evalu- ate Mik-bl-H. In light of the results of that experiment, we should decide in what direc- tion to develop the work. Already we feel that the next experiment should include a group to confirm the results obtained with anti-Tac-H. We are also trying to obtain IL-15 to pursue the in vitro evaluation of the activity of the monoclonal antibodies on the monkey lymphocytes. Significance to Biomedical Research and the Program of the Institute The effectiveness of humanized anti-Tac in monkey suggests that this therapeutic ap- proach could be useful in the treatment of autoimmune diseases in human. Because the molecule is mostly of human origin, it appears that it would be better tolerated in patients, thereby avoiding the production of neutraliz- ing antibodies. Proposed Course We will conduct a similar experiment to evaluate Mik-bl-H. In light of the results of that experiment, we will decide in what direc- tion to develop the work, possibly using a combination of antiTac-H with Mik-bl-H. A forthcoming experiment shovdd include a group to confirm the results obtained with anti-Tac-H. We are also trying to obtain IL-15 to pursue the in vitro evaluation of the activity of the monoclonal antibodies on the monkey lymphocytes. NEI Research Program Retinal Diseases — Inflammatory Diseases 84 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00300-01 LI PERIOD COVERED October 1, 1993 to September 30 , 1994 TITLE OF PROJECT (80 characters or less. Title must fit or} orte line between the borders.) Study of the Effect of NPC 15669, an Inhibitor of Neutroph il Recruitment iiLUyeitis^ PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: Frangois G. Roberge M.D. Visiting Scientist LI, NEI Others: Margaret Cheung Kourosh Dastgheib Seiji Hayashi Chi-Chao Chan M.D. M.D. M.D. M.D. Ph.D. Senior Staff Fellow Visiting Fellow Volunteer Fellow Head, Section on Immunopathology LI, NEI LI, NEI LI, NEI LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Clinical Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.9 PROFESSIONAL 0.9 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Polymorphonuclear neutrophils (PMN) participate in the inflammatory infihrate of uveitis. At present, the importance of the role played by these cells is largely unknown. We have studied the influence that an inhibitor of PMN recruitment could have on two types of uveitis: (1) endotoxin-induced uveitis (EIU), a disease mediated mainly by PMN and macrophages, and (2) experimental autoimmune uveoretinitis (EAU), a T-cell-mediated disease in which the early infiltrate is composed mainly of PMN. We have found that injection of NPC 15669 in rats significantly reduced the severity of EIU. More surprisingly, treatment of rats late in the process of EAU induction also inhibited the evolution of this disease. 85 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives (1) Evaluate the effect of NPC 15669 in the inhibition of protein exudation and cellular infiltration in the anterior chamber of the eye after subcutaneous injection of endotoxin. (2) Evaluate the effect that the inhibition of polymorphonuclear neutrophils recruitment could have on the evolution of experimental autoimmune uveitis (EAU) in the rat. Methods Uveitis was induced in Lewis rats with a subcutaneous injection of Upopolysaccharride (LPS). Treahnent with NPC 15669 started three hours after injection of endotoxin. NPC at doses of 12, six, or three mg/kg or vehicle alone were given by intraperitoneal (i.p.) injections repeated six times at two-hour intervals. After 24 hours, one eye was collect- ed for histology, and the aqueous humor was aspirated from the other eye for the measure- ment of protein and cells in the exudate. For EAU, Lewis rats were immuruzed with S-antigen (S-Ag) in Hunter's adjuvant. NPC was given three times a day at 30 mg/kg i.p. from day 10 to day 16 after immunization. EAU was evaluated by histopathology on the eye collected at the end of the treatment period. Major Findings Treatment with NPC produced a dose-depen- dent inhibition of endotoxin-induced uveitis (EIU). At the dosages of 12, six, and three mg/kg, there was a dose-dependent reduction in the leukocyte count in the aqueous humor, accompanied by a parallel decrease in the protein concentration. Histological examina- tion also showed a reduction in the inflamma- tory infiltrate of the iris and ciliary body. NPC was also effective in preventing the induction of EAU. In a representative experiment, four out of 14 NPC-treated rats developed mild EAU with an average severity grading of 1.37, whereas 10 out of 14 vehicle-treated rats developed EAU at a disease severity of 3.5. Significance to Biomedical Researcii and tlie Program of ttie Institute We conclude that the inhibition of neutrophil recruitment by NPC 15669 is effective in preventing intraocular inflammation. The treatment is effective even when instituted very late in a T-ceU mediated disease such as EAU. This finding suggests that the recruit- ment of PMN leukocytes plays a determining role in the dynamic of intraocular inflamma- tion. Proposed Course The mechanism of action of NPC 15669 in uveitis will be studied. The method wiU consist of measuring the expression of CD-18 adhesion molecule on the surface of inflamma- tory cells, stimulated in vitro and in vivo in the presence or absence of the drug. NEI Research Program Retinal Diseases — ^Inflammatory Diseases Publications Cheung MK, Dastgheib K, Chan C-C, Roberge EG: Inhibition of PMN recruitment by NPC 15669 prevents endotoxin induced uveitis. Invest Ophthalmol Vis Sci 35(4):1684, 1994. DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00299-01 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one Ime between the borders.) Study of the Role of Nitric Oxide in Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Frangois G. Roberge M.D. Others: Seiji Hayashi M.D. Chi-Chao Chan M.D. David Parks M.D. Margaret Cheung M.D., NamTram Pham M.D. Kourosh Dasthgieb Ph.D. Visiting Scientist Volunteer Fellow Head, Section on Immunopathology Senior Staff Fellow Senior Staff Fellow Volunteer Fellow Visiting Fellow LI, NEI LI, NEI LI, NEI LI, NEI LI, NEI LI, NEI LLNEI COOPERATING UNITS (if any) National Institute of Allergy and Infectious Diseases, Laboratory of Parasitic Diseases (Ricardo Grazzinelli, Ph.D.) LAB/BRANCH Laboratory of Immunology SECTION Clinical Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.9 PROFESSIONAL: 1.9 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) We have studied the role of nitric oxide (NO) in two types of uveitis: (1) Anterior uveitis represented by the model of endotoxin-induced uveitis (EIU) in the rat and (2) toxoplasma retinochoroiditis using a mouse model. In EIU, we have found NO levels that peak in the anterior chamber of the eye 2 hours before cellular infiltration and a sharp rise in protein exudation. Inhibition of NO production with a structural analog of L-arginine prevented the induction of EIU. In contrast, inhibiting NO synthesis during infection with Toxoplasma gondii caused an exacerbation of the disease. Spleen cell cultures from infected mice produced a large amount of NO. NO production was associated with a reduced viability and proliferation of the lymphocytes to toxoplasma antigen. The lymphocyte proliferative response was restored by blocking NO production. In conclusion, it appears that NO has diverse, even opposing roles in uveitis, depending on the type and mechanism of the inflammatory process involved. 87 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives (1) Measure the production of nitric oxide (NO) in the eye in the course of uveitis in- duced by endotoxin. (2) Evaluate the effect of inhibiting the production of NO on the evolution of endo- toxin-induced uveitis (EIU). (3) Examine the effect of inhibiting NO production on the evolution of Toxoplasma gondii (T. gondii) infection in mice. (4) Evaluate the role of NO in the im- mune response against T. gondii. Methods EIU. EIU was induced in Lewis rats with a subcutaneous injection of Upopolysaccharide (LPS) at 300 fig/ kg. Aqueous humor was collected from groups of five rats every two hours for the first 12 hours, at 16 hours, and 24 hours. NO levels in the aqueous humor were measured by a colorimetric assay based on the Griess reaction. Protein levels and the number of leukocytes were also determined. In some experiments, rats were treated by intraperitoneal injections with N'"-nitro-L- arginine methyl ester (L-NAME), an L-argin- ine analogue acting as a specific inhibitor of NO synthesis. The aqueous humor of one eye was collected after 24 hours, and the contralat- eral eye was examined by histopathology. Toxoplasmosis. Infection w^as induced in C57bl/B6 mice by i.p. injection of 10-20 cysts of T. gondii strain ME49. Aminoguanidine (AMG), an inhibitor of NO sjmthase, or vehi- cle control w^as administered i.p. every eight hours at 35 mg/kg. At the end of the second week, the eyes and brain were collected for histopathological examination. In other exper- iments, the spleens from infected and normal mice were collected tw^o, four, and six weeks after infection and the cells stimulated with toxoplasma antigen. The proliferative res- ponse was measured by ^H-thymidine incor- poration in the presence or absence AMG at 1 mM. In parallel cultures, the level of NO produced was measured by a colorimetric diazotization assay of nitrite accumulated in the supernatant. Interferon gamma (IFN-y) and prostaglandin E2 (PGE2) were also mea- sured by specific enzyme-Unked immuno- sorbent assay. Major Findings In EIU, the analysis of the aqueous humor after LPS injection showed a sharp peak of NO at eight hours, followed two hours later by a rise in protein and cell entry into the eye. Treatment of rats with L-NAME markedly reduced the level of NO and inhibited the induction of ocular inflammation by LPS. The aqueous humor protein exudate decreased by 73 to 82 percent, and the cellular infiltration was abrogated. The histopathological exami- nation of the eyes also showed a similar inhibition of iris and ciliary body tissue infil- tration in the treated rats. The inhibition of NO production with AMG in mice infected with T. gondii resulted in the exacerbation of disease. On histopatho- logical examination, the eyes of the infected mice treated with AMG showed increased inflammation in the retina, the choroid, and the vitreous compared with the infected con- trols. The brain showed an increased number of toxoplasma tachizoites infiltrating the tissue accompanied by a marked inflammation in AMG-treated animals. In addition, there was an accelerated evolution toward intraceUvdar cysts formation. When spleen ceUs from infected mice were cultured in the presence of toxoplasma antigen, there was a negative eftect on the survival and proliferation of the lymphocytes. This effect was correlated with high levels of IFN-y, NO, and PGE2 produc- tion. The presence of AMG in the culture medium reestablished the lymphocyte prolifer- ative response. Preventing PGE2 secretion with indomethacin also increased this re- sponse in proportion to an effect on NO production. Laboratoiy of Immunology Significance to Biomedical Research and the Program of the Institute In conclusion, it appears that NO has diverse opposing effect in uveitis depending on the type and mechanism of the inflammatory process involved. In anterior uveitis, the use of NO inhibitors could lead to improved therapy. In toxoplasmosis, however, it would be indicated to avoid drugs that inhibit NO synthesis. An important implication of these observations concerns the management of toxoplasma uveitis. It is a common practice to add corticosteroids to the antibiotic regimen in severe sight-threatening toxoplasmosis. In view of the negative regulation of NO produc- tion by corticosteroids, the use of antiinflam- matory drugs devoid of effect on the NO synthase should be considered. Proposed Course Research wiU be directed at identifying ocular cells that may produce NO. Finding such cells in the anterior segment of the eye could lead to a new therapeutic approach in the treatment of uveitis. the levels expressed in sensitive and resistant strains with toxoplasmosis. We will also study the possible effect of NO inhibition on triggering reactivation of chronic latent toxo- plasmosis. NEI Research Program Retinal Diseases— Inflammatory Diseases Publications Hayashi S, GazzineUi R, Chan C-C, Pham N, Roberge FG: In vivo inhibition of nitric oxide enhances ocular and CNS inflammation in murine toxoplasmosis. Invest Ophthalmol Vis Sci 35(4):1685, 1994. Parks DJ, Cheung MK, Chan C-C, Roberge FG: The role of nitric oxide in uveitis. Arch Ophthalmol 112:544, 1994. Roberge FG, Hayashi S: Nitric oxide is re- sponsible for the inhibition of lymphocyte proliferation in Toxoplasma gondii infection. Invest Ophthalmol Vis Sci 35(4):1685, 1994. We will also study the kinetics of NO pro- duction in vivo in mice infected with T. gondii. In particular, we will try to identify cells expressing NO S3rnthase in the eye and the brain during infection. Later we wiU compare 89 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00279-03 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Study of Immunosup pressants for the Treatment of Uveitis in Animal Models PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: Frangois G. Roberge Others: Chi-Chao Chan Marc D. de Smet Robert B. Nussenblatt Dan Martin Margaret Cheung David Parks M.D. Visiting Scientist LI, NEI M.D. Head, Section on Immunopathology LI, NEI M.D. Visiting Scientist LI, ISfEI M.D. Scientific Director NEI M.D. Senior Staff Fellow LI, NEI M.D., Ph.D. Senior Staff Fellow LI, NEI M.D. Senior Staff Fellow LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Clinical Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: PROFESSIONAL: 0.0 CHECK APPROPRIATE BOX(ES) (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) THIS PROJECT HAS BEEN TERMINATED. 90 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00301-01 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Ocular Gene Transfer PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Karl G. Csaky M.D., Ph.D. Medical Officer LI, NEI Others: Daniel Sullivan Ph.D. IRTA Fellow LI, NEI Eddy Anglade M.D. Staff Fellow LI, NEI Csaba Salaman M.D. Visiting Scientist LI, NEI Robert Interrante M.S. Special Volunteer LI, NEI COOPERATING UNITS (if any) S.U.N.Y. at Stony Brook, Stony Brook, ^fY (L. Taichman, M.D., Ph.D.); Department of Pediatrics, The Johns Hopkins University, BaUimore, MD (D. Valle, M.D.); Human Genome Center, NTH, Bethesda, MD (M. Blaese, M.D.); Massachusetts Institute of Technology, Department of Chemical Engineering, Boston, MA (D. Mooney, Ph.D.) LAB/BRANCH Laboratory of Immunology SECTION Section of Ocular Gene Therapy INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 2.75 PROFESSIONAL: 2.25 0.5 CHECK APPROPRIATE BOX(ES) [xj (a) Human subjects [x] (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) An ocular gene therapy section is being developed that emphasizes the study of direct ocular gene transfer using ElA-deficient adenovirus constructs, the transfer of genes ex vivo into isolated retinal pigment epithelial cells prior to their transplantation, and the use of ornithine aminotransferase (OAT) transfected keratinocytes to treat patients with gyrate atrophy. A mutant of transforming growth factor beta-1 (TGF-P), which is secreted in an active form, has been placed into an adenovirus construct. Studies involving transduction of retinal pigment epithelial cells have shown that these cells can be efficiently transducted at a low multiplicity of infection and are capable of producing high levels of a biologically active TGF-(3. Adenovirus-TGF-P has also been injected into the anterior chamber of rats. Immunohistochemical analysis of these eyes has suggested the presence of secreted TGF-p. Human retinal pigment epithelial cells (RPE) have been transfected with plasmids containing beta-galactosidase (P-gal). Studies are underway to examine the optimal modes of transfection. Parallel studies are being done with isolated rat RPE cells. Both cell lines are capable of growth on thin synthetic basement membrane polymers. These polymers are transplanted, as a single cell layer, into the subretinal space of rats. OAT has been successfully transfected into OAT-deficient fibroblasts (CHO cells) and into keratinocytes, isolated from patients with gyrate atrophy. Studies are presently underway to examine ways to increase OAT levels and activity in these cells. 91 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives An ocular gene therapy section is being estab- lished, the objective of which is to apply molecular biology techniques in the treatment of human ocular disease. Major areas of interest include direct ocular gene transfer using El A-deficient adenoviruses, transfection of retinal pigment epithelial cells ex vivo before their transplantation and the transfection of omithine-6-aminotransferase (OAT) into keratinocytes from patients with gyrate atro- phy (GA). Optimization of expression of OAT in cultured keratinocytes from patients with GA will allow us to determine if transfected keratinocytes can be used as a sufficient source of OAT to treat this disorder. Methods Using standard cloning techniques, adenovirus constructs are being synthesized. Using polymerase chain reaction. Northern, Western, Southern, and enzyme-linked immunosorbent assay (ELISA) methods, sequence and expres- sion data from various adenovirus constructs such as transforming growth factor (TGF)-p and OAT are being generated. Site-directed mutagenesis of the OAT gene and alteration in the promoters and their proximity to translational start sites are being performed. This will aUow us to identify OAT constructs that, when transfected into kera- tinocytes, will most efficiently degrade circu- lating ornithine. Standard tissue-culture techniques and the study of S5mthetic base- ment membrane polymers are used to study the transplantation of retinal pigment epitheli- um (RPE) into rodent eyes. This approach win allow the determination of the usefulness of these techniques in the treatment of animals with ocular neovascularization and diabetic macular edema. Major Findings Human RPE cells appear to be efficiently transduced by El A-deficient adenovirus. By using a lac-Z/ElA-adenovirus construct, human RPE were shown to express P-galacto- sidase activity in 100 percent of transduced cells. El A-deficient adenovirus constructs with TGF-P have been produced. As a result of site-directed mutagenesis, the TGF-|3 construct is secreted in an active form as a 24 kDa protein. This is unlike endogenous TGF-pi, which is secreted as a 90 to 110 kDa protein and requires activation by a latent factor to become biologically active. We have been able to show that the efficient transduction of human RPE can be readily achieved with the use of ElA-deficient adenovirus-TGF-p. Even at low multiplicity of infection, studies have shown that expression of active TFG-P can be achieved to pharmacological levels. TFG-P can be quantified by an ELISA assay, has been shown to be biologically active, and correlates with ribonucleic acid expression. The virus also has been injected into the anterior cham- ber of rats. At one to seven days following injection, immunohistochemical staining has demonstrated the presence of TGF in the corneal and iris stroma. Injection with nuU ElA-deficient adenovirus lacked TGF-p ex- pression, suggesting specificity of the expres- Human and transformed rat RPE have been isolated and grown in culture. Transfec- tion of cytomegalovirus (CMV)-lacZ constructs into these cell Unes, with Upofectant or calci- um phosphate, have jdelded transfection efficiencies of 20 to 30 percent. Transfection efficiency, into human RPE, was increased by contransfecting attenuated adenovirus with lipofectamine. This achieved a transfection efficiency of 90 percent. Both cell types have been grown, as a monolayer, on a 20 fim thick polylactic/polyglycoUc poljoner. Transplanta- tion of these sheets into the subretinal space of rats is now being performed. 92 Laboratory of Immunology Keratinocytes from patients with GA have been isolated and grown in culture. They exhibit low levels of endogenous OAT activi- ty. Various OAT constructs have been gener- ated. Expression of full-length OAT inserts, under a CMV or long terminal repeat (LTR) promoter, have been tested in CHO ceUs. Under LTR control, this construct yielded a twentyfold higher OAT activity than mock transfected ceUs. However, when transfected into the isolated keratinocytes, the increase was only threefold. Several OAT constructs were made with varying lengths of untranslat- ed regions and portions of the mitochondrial leader to the protein removed. Studies are now underway to determine which of these constructs is most efficient at expressing biologically active OAT. Significance to Biomedical Research and the Program of the Institute Direct ocular gene transfer has the potential for treatment in hereditary ocular diseases. In diseases such as GA where a deficiency of OAT is responsible for the clinical findings, gene therapy offers the possibility of cure. From a biological perspective, direct gene transfer into ocular tissues allows the study of the biology of overexpression of certain pro- teins into the eye. When animal models of disease are available, the use of direct gene transfer v^th adenovirus or transplantation of transfected RPE cells can identify critical proteins in the amelioration of these diseases. For example, TGF-P has been proposed as a downregulator of the immune response. By overexpressing TGF-P in animal models of uveitis, direct examination of this hypothesis can be obtained. In the event that keratinocytes from pa- tients with GA transfected with a construct of OAT exhibit sufficient OAT activity to de- grade significant serum ornithine, these cells wdll then be replaced onto the patient's skin. If serum ornithine levels fall, this will offer the first gene therapy for an ocular disease. Proposed Course The biology of overexpression of TGF-P in rodent models of uveitis will be further exam- ined. Various other inflammatory cytokines and inhibitors such as interleukin (IL)-IO and IL-1 receptor antagonist will also be investi- gated in this fashion. Further investigation of the effect of transduction by adenovirus on RPE cells will be performed. Subretinal injec- tions of adenovirus constructs will result in transduced RPE cells overexpressing selected proteins in vivo. The biologic significance of this overexpression in the subretinal space can then be examined. We will examine RPE ceUs, transplanted in a monolayer in the subretinal space, to deter- mine if these cells are able to function normal- ly. The kinetics of gene expression fiom these cells, transfected ex vivo, will also be deter- mined. The development of other mutants of OAT will continue. Once assayed for biological activity in degrading ornithine, these con- structs will be examined for expression and activity in cultured keratinocytes from GA patients. If sufficient OAT is produced that is capable of degrading adequate amounts of serum ornithine, further steps wdll be taken to replace these keratinocytes in the GA patients. Adenovirus constructs containing OAT will be created, and the study of their expression following ocular injections in mouse OAT knockout models of GA will be accomplished. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders 93 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00268-04 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) The Diagnosis and Treatment of Human Uveitis and AIDS-Related Ocular Disease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Scott M. Whitcup M.D. Staff Medical Officer CB, NEI Others: Robert B. Nussenblatt Marc D. de Smet Chi-Chao Chan M.D. Scientific Director NEI M.D. Visiting Scientist LI, NEI M.D. Medical Officer LI, NEI COOPERATING UNITS (if any) Department of Medicine, The Johns Hopkins University, Baltimore, MD (David R. MoUer, M.D.) LAB/BRANCH Clinical Branch/Laboratory of Immunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 1.0 0.0 CHECK APPROPRIATE BOX(ES) fxl (a) Human subjects n (a1) Minors |~1 (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The title of this project has been changed from "The Diagnosis and Treatment of Human Uveitis" to "The Diagnosis and Treatment of Human Uveitis and AIDS-Related Ocular Disease," to reflect the expanded scope of the project to include the study of ocular disease in patients with AIDS. In addition, this project was previously listed in the Laboratory of Immunology but is now listed in the Clinical Branch. The goal of this project is to develop improved methods for diagnosing and treating human uveitis and AIDS-related ocular disease. This project encompasses clinical trials evaluating new diagnostic and therapeutic approaches and immunologic and histologic studies on blood and tissue specimens obtained from patients. A number of studies have focused on improving diagnostic tests for uveitis. Examination of lacrimal gland and conjunctival biopsies from patients with sarcoidosis has demonstrated lymphocytic infiltration and specific T-cell receptor repertoires despite a lack of granuloma formation, and these results should help improve the diagnostic yield of biopsies for the diagnosis of sarcoidosis. We have also shown that lumbar puncture can miss malignant involvement of the leptomeninges in patients with intraocular lymphoma and that ventricular taps are needed. The Clinical Branch is involved in a number of studies designed to improve the treatment of uveitis and ocular malignancy. A prospective, double-masked, randomized study of acetazolamide for uveitis-associated cystoid macular edema has just completed enrollment, and data will be analyzed this year. In addition, we completed a pilot study examining the efficacy and toxicity of a chemotherapy regimen for therapy of patients with central nervous system lymphoma involving the brain or eye. A complete response was obtained in seven of eight patients; the other patient had a partial response. A clinical trial evaluating a heparin surface-modified intraocular lens in patients with uveitis is under way, and a natural history study of patients with uveitis and good vision is in progress. Finally, the Clinical Branch is involved in studies with ocular complications related to AIDS. We are retrospectively reviewing the ophthalmologic examinations of 550 AIDS patients to improve diagnosis of ocular complications such as infection. In addition, we are participating in an open-label study of cidofovir for the treatment of resistant cytomegalovims (CMV) retinitis in patients with AIDS. 94 PHS 6040 (Rev. 5/92) Laboratory of Immunology Project Description Additional Personnel Emily Chew M.D. Visiting Scientist BEP, NEI Frederick Ferris, MD. Chief, Clinical Trials m Branch BEP, NEI George P. MX). Medical Officer Chrousos NICHD/DEB George M.D. Visiting Scientist Mastorakos NICHD/DEB Igal Gery PhD. Deputy Chief LI, NEI Susan Mellow R.N. Nurse Specialist CB, NEI R. Christopher MD. Senior Staff Fellow Walton LI, NEI Eddy Anglade M.D. Senior Staff Fellow LI, NEI Clinical Protocol Numbers 87-EI-0104 91-EI-0030 91-EI-0139 92-EI-0070 92-EI-0108 92-EI-0138 92-EI-0157 94-EI-0189 Objectives The title of this project has been changed from "The Diagnosis and Treatment of Human Uveitis" to "The Diagnosis and Treatment of Human Uveitis and AIDS-Related Ocular Disease," to reflect the expanded scope of the project to include the study of ocular disease in patients with acquired immunodeficiency syndrome (AIDS). The goal of this study is to develop better methods for the diagnosis and treatment of human uveitis and ocular disease associated with AIDS. We are also interested in defining the pathophysiology of inflamma- tory eye diseases by performing immunologic and histologic studies on tissue specimens and blood samples obtained from patients. Methods Diagnosis of Uveitis and Malignancy (1) To improve the diagnostic yield of con- junctival and lacrimal gland biopsies for sarcoidosis, tissue specimens are examined using immunohistochemical staining and polymerase chain reaction (PCR). Conjuncti- val and lacrimal gland biopsies are performed and snap frozen in OCT from patients with known sarcoidosis. Immunohistochemical staining is per- formed using primary monoclonal antibodies against T-ceU markers, T-ceU receptors, Kveim antigen, and various interleukins. The ceU receptor repertoire is also studied using PCR techniques. Results will be compared with biopsies from patients with other uveitic conditions such as Behcet's disease to deter- mine the specificity of these results. (2) Intraocular lymphoma often masquer- ades as an idiopathic uveitis that delays the start of appropriate therapy. We continue to prospectively coUect data on patients with intraocular lymphoma to improve diagnosis in these patients. (3) Samples of aqueous humor, iris, and vitreous are obtained from patients undergo- ing ocular surgery for uveitis. Samples wiU be assayed for both cytokines and ceU-adhesion molecules and compared with samples ob- tained from patients undergoing routine cataract surgery in an attempt to identify immunologic markers for ocular inflammation. (4) Animals with experimental autoim- mune uveitis develop an associated pineaUtis. It is not known whether patients with human uveitis also have associated inflammation of the pineal gland. We are examining patients with uveitis for the presence of pinealitis using magnetic resonance imaging (MRI). Treatment of Uveitis of IVIalignancy (1) The efficacy of acetazolamide for the treatment of uveitis-associated macvdar edema 95 FY 1994 NEI Annual Report is being evaluated in a masked crossover study comparing acetazolamide with placebo. Visual acuity and the height of the macular edema measured on fluorescein angiography wiU be used as primary endpoints. (2) We are investigating the tieatment of patients with non-Hodgkin's lymphoma involving the brain or eye. The NEI is partici- pating in a pilot study to evaluate the efficacy and toxicity of a chemotherapy regimen for primary therapy of patients presenting with central nervous system lymphoma involving the brain or eye. High-dose methotrexate and leucovorin rescue is used in combination with thiotepa, vincristine, dexamethasone, and intrathecal cytarabine, and G-colony stimulat- ing factor (G-CFS) when needed. Radiation therapy is deferred until patients show evi- dence of disease progression. Patients are followed by cUnical ophthalmologic examina- tions as well as MRI and nuclear magnetic resonance spectroscopy. Laboratory correlates, including immunohistochemistry and viral sequence detection, are performed on available tissue. The data obtained from this study wUl be used to design a more inclusive study of chemotherapy in ocular and central nervous system lymphoma. (3) The purpose of this project is to evalu- ate the ability of a heparin surface-modified intraocular lens to reduce the incidence and severity of postoperative inflammation in patients with uveitis undergoing cataract surgery. Patients with a history of uveitis and catar ts, scheduled for cataract extraction with ^_^iacement of an intraocular lens, will be randomized to receive either a heparin sur- face-modified intraocular lens or a nonmodi- fied lens. The primary endpoint of the study is inflammatory cell deposits in the intraocular lens. Secondary endpoints will include other measures of ocular inflammation and visual acuity. Eighty patients will be recruited for the study. (4) The clinical course of patients with Behcet's disease who were treated wdth cyclo- sporine alone was compared retrospectively with patients who were treated with com- bined cyclosporine and prediusone. AIDS (1) We are retrospectively reviewing the ophthaLmologic examinations of 550 patients with AIDS in an attempt to improve the diagnosis of cytomegalovirus (CMV) retinitis. Information, including patient symptoms, visual acuity, and ocular examination data and CD-4 T-lymphocyte counts, wiU be ana- lyzed to establish criteria for screening pa- tients for ophthalmologic disease. We are also using laser interferometry to measure anterior chamber flare to determine whether this device can provide a sensitive and specific test for CMV retinitis in patients with AIDS. (2) The NEI is participating in an open- label, randomized, multicenter study of the safety and efficacy of cidofovir for the treat- ment of relapsing CMV retinitis in patients with AIDS. Patients with evidence of CMV retinitis progression while receiving ganci- clovir and /or foscamet therapy wiU be ran- domly assigned to one of two dose levels of cidofovir. Patients will undergo ophthalmo- logic examination at baseUne and at regular intervals thereafter. The primary endpoints are safety and time to progression of CMV retinitis. (3) Eyes obtained at autopsy are studied in an attempt to understand the pathophysiol- ogy of AIDS-related ocular disease. In addi- tion, the cUnical course of aU patients with AIDS and ocular disease are prospectively studied. Major Findings Diagnosis of Uveitis and IMalignancy (1) We have recruited nine patients with biopsy-proven sarcoidosis into the protocol examining conjunctival and lacrimal gland biopsies. None of the biopsies had gramiloma formation diagnostic for sarcoidosis. Never- theless, substantial areas of lymphocytic infil- tration were present on most biopsies, and we 96 Laboraton/ of Immunology are assessing the T-cell receptor repertoire in these specimens. (2) Evaluation of the diagnostic data from patients with intraocular lymphoma has shown that despite the absence of malignant cells in the cerebral spinal fluid obtained by lumbar puncture, malignant cells were demon- strated in a sample simultaneously obtained from an Omaya reservoir placed in the ventri- cles of five patients. The data suggest that malignant leptomeningeal involvement may be missed by lumbar puncture alone. (3) Studies on the levels of soluble inter- cellular adhesion molecule 1 in the serum, iris specimens, and aqueous of patients with uveitis are in progress. (4) We continue to recruit patients in the protocol using MRl to evaluate the pineal gland in patients with uveitis. Treatment of Uveitis and Malignancy (1) We have completed enrollment of patients in the crossover study of acetazolamide for the treatment of uveitic cystoid macular edema. The last patient is completing the study course; the randomization will be unmasked, and the data will be analyzed during the next year. Previous treatment with corticosteroids alone failed to control the uveitis in aU patients. Ten patients were given cyclosporine therapy alone (mean dosage, 8.6 mg/kg of body weight per day), and nine patients were given lower dosages of cyclosporine (mean dosage, 6.2 mg/kg of body weight per day) in combi- nation with prednisone (mean dosage, 29.4 mg per day). The mean foUowup on therapy was 51 months. After three months of therapy, a trend toward greater improvement in visual acuity was noted in patients treated with combined cyclosporine and prednisone com- pared with those receiving cyclosporine alone (17.8 letters vs. 10.2 letters, p = .24); after one year, little difference was observed in the improvement between the two groups (5.8 letters vs. 3.3 letters, p = .80). However, a trend toward greater renal toxicity was seen in patients treated with cyclosporine alone after both three months and one year of therapy. Because of either a suboptimal therapeutic response or adverse effects, all patients treated with cyclosporine alone at baseline had pred- nisone added to their regimen after a mean time of 23.5 months. Overall, visual acuity remained stable or improved in 28 of 37 eyes (75.7 percent) over the course of therapy. The data suggest that combined cyclosporine and prednisone therapy is an effective treatment for Behget's uveitis and may be less toxic than therapy with cyclosporine alone. (2) Eight patients were eru-oUed in this pilot study and recruitment was completed in November 1993. Seven of eight patients had complete remission on this protocol; one patient had a partial response. Defiiute grade rV toxicity occurred in or\ly one patient. The phase 11 trial examining chemotherapy for the treatment of central nervous system lympho- ma has just started recruiting patients. (3) Seven patients have been enrolled in the protocol examining the heparin surface- modified intraocular lens in patients with uveitis. (4) We reviewed 19 patients with severe ocular Behget's disease treated with combined cyclosporine and corticosteroid therapy. AIDS (1) Laser interferometry has showm increased flare in the anterior chambers of patients with CMV retinitis and may be a useful screening test of ocular inflammatory disease in patients with AIDS. (2) Recruitment into the trial of ddofovir for CMV retinitis has started. (3) We have found an increased severity of CMV retinitis in children with AIDS as compared with adults. This increased severity appears to be related to a delay in diagnosis because chUdren are less apt to complain of visual symptoms and seek ophthalmologic evaluation than adults. Increased vigilance in FY 1994 NEI Annual Report screening children with ADDS for the develop- ment of ocular compUcations appears warrant- ed. We also described atypical findings of mild peripheral retinopathy in a patient with acute retinal necrosis following chicken pox, which may have been related to early acy- clovir therapy. Significance to Biomedical Research and the Program of the Institute Uveitis accounts for about 10 percent of the visual impairment in the United States. A major goal of the NEI is to improve the meth- ods for diagnosing and treating uveitis in an attempt to preserve useful vision in patients with inflammatory eye disease. In addition, many patients with AIDS develop severe, sight-threatening ocular disease. CMV retini- tis is the most common cause of impaired visual acuity in patients with AIDS, occurring in 10 to 30 percent of AIDS patients. An important goal of the NEI is to improve our methods of diagnosing AIDS-related eye disease and to develop and test new therapies for these disorders. Proposed Course We will continue to recruit patients with intraocular lymphoma, uveitis, and AIDS- related ocular disease into the clinical trials detailed earUer. A new protocol that looks at novel therapeutic approaches for CMV retini- tis will be started during the next year. We have completed the enrollment of patients into the crossover study of acetazolamide for uveitic cystoid macular edema and will start the analysis of the data. In addition, we have completed our collection of ophthalmologic data from the 550 patients with AIDS. Our plan is to analyze the compiled data and issue recommendations for the screerving of patients with AIDS for ocular disease. NEI Research Program Retinal Diseases — Inflammatory Diseases, Cancer Publications Callanan DG, Cheung MK, Martina DF, de Smet MD, Whitcup SM, Nussenblatt RB: Outcome of uveitis patients treated with long- term cyclosporine. Invest Ophthalmol Vis Sci 35(suppl):2094, 1994. Friedman SM, Mames RN, Whitcup SM: Acute retinal necrosis after chickenpox in a patient with acquired immunodeficiency syndrome. Arch Ophthalmol 111:1607-1608, 1993. Whitcup SM, Salvo EC Jr, Nussenblatt RB: Combined cyclosporine and corticosteroid therapy for sight-threatening uveitis in Behcet's disease. Am J Ophthalmol 118:39-45, 1994. Whitcup SM, Nussenblatt RB: Treatment of autoimmune uveitis. Ann N Y Acad Sci 696:307-318, 1993. Whitcup SM, de Smet MD, Rubin BI, Palestine AG, Martin DF, Bumier M Jr, Chan C-C, Nussenblatt RB: Intraocular lymphoma: Clinical and histopathologic diagnosis. Oph- thalmology 100:1399-1406, 1993. 98 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00222-09 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) Immunopathology in Eyes With Experi m ental and C linical Ocular Diseases^ PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PL- Chi-Chao Chan M.D. Head, Section on LL NEI Immunopathology Others: Qian Li M.D. Visiting Associate LI, NEI Kourosh Dastgheib M.D. IRTA Fellow LI, NEI Bo Peng M.D. Visiting Fellow LI, NEI Deborah Luyo Technician LI, NEI Scott M. Whitcup M.D. Associate Director CB, NEI Charles E. Egwuagu Ph.D. Medical Officer LI, NEI Franjois G. Roberge M.D. Visiting Scientist LI, NEI Rachel R. Caspi Ph.D. Visiting Scientist LI, NEI Igal Gery Ph.D. Deputy Chief LI, NEI Robert B. Nussenblatt M.D. Scientific Director NEI COOPERATING UNITS (if any) Regulation of Gene Expression Section, LMDB, NEI (Ana B. Chepelinsky, Ph.D.); Ophthalmic Genetics and Services Branch, NEI (Muriel Kaiser- Kupfer, M.D.); Department of Ophthalmology, University of New South Wales, Sydney, Australia (Denis Wakefield, M.D.); Department of Ophthalmology, Kurume University, Kurume, Japan (Manabu Mochizuki, M.D.) Laboratory of Immunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 5.0 PROFESSIONAL: 4.0 1.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The identity and topographic localization of immunocompetent cells and the alteration of surface markers on ocular resident cells in animals with various experimental uveitis were analyzed by immunohistochemical studies and in situ hybridization. Previously, we have demonstrated that T lymphocytes were the predominantly infiltrating cells in experimental autoimmune uveoretinitis (EAU), yet both macrophages and polymorphonuclear neutrophils were the predominantly infiltrating cells in endotoxin-induced uveitis (EIU). Cytokines (e.g., interferon gamma), inflammatory mediators (e.g., nitric oxide), and some retinal proteins (e.g., S-antigen (S-Ag)) play an important role in the immunopathogenesis of EAU and EIU. Agents that modulate them can alter the immunopathology of the experimental model. For example, prolonged corticosteroid therapies enhance S-Ag expression in nonretinal ocular tissues of rats with EAU. Several new animal models, including experimental melanin-protein-induced uveitis (EMIU), experimental blepharitis, murine allergic conjunctivitis, and murine toxoplasmosis, have been developed and studied. They represented different entities of uveitis in humans. EMIU closely resembles Vogt-Kayanagi-Harada syndrome and sympathetic ophthalmia; experimental blepharitis induced by immunization of monoclonal antibody of Id 16/6 resembles idiopathic blepharitis; murine allergic conjunctivitis induced by compound 48/80 resembles allergic conjunctivitis; murine toxoplasmosis infected with T. gondii resembles acquired ocular toxoplasmosis. Specimens from human ocular tissues with various diseases, including uveitis, retinal and corneal diseases, tumors, and metabolic genetic disorders, are studied using immunohistochemical and in situ hybridization techniques as well as light and electron microscopic examinations. In uveitis, immunocompetent cells and lymphokines besides the stimulators (e.g., infectious organisms) are valuable adjuncts to the clinical diagnosis and the understanding of pathogenesis of the diseases. In nonuveitic conditions, alteration of cellular membrane surface markers and intracytoplasmic organelles of the ocular resident cells (e.g., crystalline inclusions in Beitti's crystalline dystrophy; B-cell marker in intraocular lymphoma) may reflect cellular damage and abnormalities in these diseases. Elucidating the immunopathological role of the relationships between infiltrating inflammatory or malignant cells and other resident cells in the clinical behavior of various diseases will increase our understanding of human ocular disorders and provide better treatment. 99 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives This program is designed to evaluate the clinical manifestation, histopathology, and immunopathology of the ocular tissue when experimental uveitis (experimental autoim- mune uveitis [EAU], endotoxin-induced uve- itis [EIU], experimental melanin-induced uveitis [EMIU], murine toxoplasmosis, etc.) are induced and /or modulated by different im- munosuppressive agents in various animal species. Ocular tissues obtained from patients with various diseases, including inflammatory and noninflammatory disorders, are all includ- ed. The infiltrating inflammatory cells, ocular resident cells and their products, cytokines, and metaboUtes are examined. These findings will help us understand ocular inflammation and the pathogenesis of each disease exam- ined in humans. Methods CUnical examinations include flashlight, sUt- lamp and fundus examination, under the dissecting microscopes of experimental ani- mals and patients. Pathological examinations include routine histologic techniques for Ught and electron microscopy, immunofluorescence and avidin-biotin-peroxidase complex method, and in situ hybridization techniques. Enzyme- linked immunosorbent assay technique is also performed for cytokines or protein analysis of the serum and intraocular fluids. Major Findings We have continued to study the immunopa- thology of various inflammatory cells and ocular resident cells in different experimental models of uveitis. We have demonstrated that higher levels of S-antigen (S-Ag) and its mes- senger ribonucleic acid (mRNA) are expressed in nonretinal ocular cells (the lens, the ciUary body, and the trabecular meshwork) of rats with EAU, in particular those that also re- ceived long-term corticosteroid treatment. This experimental result supports our previ- ous finding of S-Ag and its mRNA detected in irises of some uveitic patients who received long-term steroids. Several new experimental models for uveitis in humans have been investigated by us. EMIU is induced by immuruzation with bovine choroidal and retinal pigment epitheli- um (RPE) melanin protein and is characterized by bilateral, recurrent iridocycUtis and choroi- ditis. In EMIU, Uke EAU, the main infiltrating cells are T lymphocytes of QD-4 cells seen in the early stage and CD-8 cells seen in the late stage of the disease. Expressions of adhesion molecules and major histocompatibility (MHC) class II antigens are observed on ocular resi- dent cells one to two days before ocular in- flammation (eight to 10 days postimmuniza- tion). However, unlike EAU, recurrence occurs one month later. Acquired ocular toxoplasmosis is devel- oped by the infection of an avirulent strain of Toxoplasma gondii (T. gondii) (ME49) in C57BL / 6 mice. Focal ocular inflammation and RPE involvement are observed after 15 days of infection. Four weeks after infection, ocular inflammation becomes stable and rare cysts can be found. Treatment of mice with anti- bodies (CD-4 plus CD-8) or cj^okines (interfer- on gamma [IFN-7] or tumor necrosis factor alpha [TNF-a]) results in a marked increase of ocular inflanrmiation associated with the pres- ence of the parasite. In the study of transgenic mice v^th constitutive expression of IFN-y in the eye, we have reported that the growth of the eye is affected, resulting in microphthalmia, cata- racts, arrest of retinal differentiation, and enhancement of the expression of MHC class n. In a study of the role of nitric oxide, we have demonstrated that competitive blocking of NO formation with the L-arginine analogue is sufficient to inhibit the induction of EIU. Using immunopathological techniques, we examine ocular tissues obtained from patients with various octilar diseases to help visualize the pathology and the kinetics of the specific disease process. The findings provide useful 100 Laboratory of Immunology information for understanding the pathologi- cal mechanisms of the disease, determining the diagnosis, and guiding the subsequent management of the patient. Iris biopsies from patients with uveitis and cataracts were stud- ied. All except one specimen from uveitic patients had T lymphocyte and macrophage infiltration. The consistent spatial correlation between the presence of T cells and IFN-y as well as between the presence of macrophages and defensin are observed. Retinal necrosis, neovascularization, marked chorioretinal inflammation, particular- ly of T lymphocytic infiltration, and the ab- sences of bradyzoites are the characteristic findings in fetal eyes infected with T. gondii. Polymerase chain reaction (PCR), a sensitive and specific technique to identify infectious agents, and immunohistochemistry are helpful for the diagnosis of ocular toxoplasmosis. Twelve cases of intraocular lymphoma diagnosed at the NEI between 1984 and 1992 were retrospectively reviewed. AU were non- Hodgkin's large B cell lymphoma of the cen- tral nervous system. The prompt, appropriate handling of specimens and review by an experienced cytopathologist are critical to the diagnosis of intraocular lymphoma. Malig- nant cells often are present in the vitreous before and /or in the cerebral spinal fluid. Multiple vitrectomies and lumbar punctures may be necessary before the correct diagnosis is made. Didanosine (DDI), used in the treatment of acquired immunodeficiency syndrome, is associated with toxic retinopathy. We have studied the first pathological report and dem- onstrated numerous membranous lamellar inclusions and cytoplasmic bodies in the affected RPE cells. These data show that the DDI retinopathy resiilts from the pathology of RPE cells. We evaluated three affected members of a Chinese-American family with Bietti's crystal- line retinopathy. Crystalline lysosomal mate- rials are observed in Ijmiphocytes and skin fibroblasts of these patients. The advanced panchorioretinal atrophy with crystals and complex Upid inclusions in the choroidal fibroblast was first documented in this study. Significance to Biomedical Research and the Program of the Institute Immunopathological findings on experimental uveitides have provided information on vari- ous inflammatory cells and ocular resident cells during the process of ocular inflamma- tion. This information helps us to better understand the mechanisms of uveitis and select or evaluate novel pharmacological agents as well as provide suitable therapeutic intervention of uveitis in humans. Studies of ocular tissues obtained from patients with various disorders have enabled us to gain information on the pathogenesis, diagnosis, and management of these ocular diseases. The finding that corticosteroids enhance S- Ag expression in nonretinal ocular tissues of rats with EAU may have clinical implication. The alteration of protein (such as S-Ag) ex- pression on the lens, the ciliary body, and the trabecular meshwork may contribute to the ocular side effects induced by prolonged corticosteroid therapy in patients. Melanin protein is capable of inducing autoimmune uveitis, resembHng noninfectious recurrent iridocyclitis and choroiditis in hu- mans. EMIU is another useful model for the study of uveitis such as Vogt-Kayanaki- Harada Sjmdrome and the evaluation of immunomodulating agents. Murine toxoplasmosis is a practical model for us to understand the respective roles of T. gondii proliferation and immune mechanisms involved in the pathogenesis of acquired ocular toxoplasmosis. T lymphocytes as well as TNF-a and IFN-y are crucial elements in controlling parasite growth. In immunocom- promised hosts such as AIDS patients, ocular lesions can be more severe and result from parasite proliferation rather than from an autoimmune process. 101 FY 1994 NEI Annual Report Early indicators of ocular toxoplasmosis in the fetus are infiltrating T cells and the ab- sence of tachyzoite antigens in the lesions. PCR for the diagnosis of ocular toxoplasmosis is helpful. The study demonstrates that T cells and macrophages are part of the inflammatory response in uveitis and that they secrete IFN-y and defensrn, respectively. These findings may have implications for the treatment of ocular uveitis. Antibody or specific immuno- modulatory therapy that abrogates the effects of cytokines such as IFN-y or defensins may decrease the extent and severity of ocular inflammation. Proposed Course AU experimental models, including EAU, EIU, EMIU, allergic conjunctivitis, experimental blepharitis, and murine toxoplasmosis will be studied clinically, histopathologicaUy, and immunopathologically in different strains and species. Various pharmacological agents and the role of cytokines, enzymes, metaboUtes, and cellular markers will be evaluated in these models. Also, we propose continuation of the analysis of human specimens to understand their immunopathogenesis. NEI Research Program Retinal Diseases — ^Inflammatory Diseases Publications Anderson W, Chan C-C, Nussenblatt KB, Whitcup SM: Topical heparin inhibits com- pound 48/80 induced allergic conjunctivitis. Invest Ophthalmol Vis Sci 35(4):1291, 1994. Brezin AP, Kasner L, Thulliez P, Li Q, Daffos F, Nussenblatt KB, Chan C-C: Ocular toxo- plasmosis in the fetus: Immunohistochemistry and DNA ampUcation. Retina 14:19-26, 1994. Caspi RR, Chan C-C, Grubbs B, Silver PB, Wiggert B, Parsa CF, Bahmanyar S, BiUiau A, Heremans H: Endogeneous systemic interfer- on-gamma has a protective role against ocular autoimmunity in mice. / Immunol 152:890-899, 1994. Chan C-C, Gery 1, Nussenblatt RB, Mozes E, Singer DS: Periocular inflammation in mice with experimental systemic lupus erythemato- sus (SLE): A new experimental eye disease and its modulation. Invest Ophthalmol Vis Sci 35(4):1988, 1994. Chan C-C, Hikita N, Dastgheib K, Whitcup SM, Gery 1, Nussenblatt RB: Experimental melariin-protein induced uveitis in the Lewis rat: Immuno-pathological processes. Ophthal- mology 101:1275-1280, 1994. Chan C-C, Palestine AG, Li Q, Nussenblatt RB: The diagnosis of ocular toxoplasmosis by the use of immunocytology and the polymer- ase chain reaction. Am J Ophthalmol 117:803- 805, 1994. ChepeUnsky AB, Robinson ML, Overbeek PA, Parker DM, Chan C-C, Jamieson S, Dickson C: FGF-3 ectopic expression induces differentia- tion of central lens epitheUa and appearance of secretory epitheUa in the eyes of tiansgeruc mice. Invest Ophthalmol Vis Sci 35(4):1998, 1994. Cheung MK, Martin DF, Chan C-C, Callanan DG, Cowan CL, Nussenblatt RB: Reactive lymphoid hyperplasia: Diagnosis by chorio- retinal biopsy. Am J Ophthalmol, in press. Cheung MK, Dastgheib K, Chan C-C, Roberge RG: Inhibition of PMN leukocyte recruitment by NPC 15669 prevents endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 35(4):1684, 1994. Dastgheib K, Hikita N, Sredni B, Albeck M, Sredni D, Nussenblatt RB, Chan C-C: Ocular inflammation stimulated by the immunomo- dulator AS 101 (ammonium trichloro (dioxy- ethelene-o-o') teUurate). Curr Eye Res, in press. Dastgheib K, Hikita N, Walton RC, Hayashi S, Chan C-C: Experimental melanin-protein induced uveitis (EMIU): Susceptibility and 102 Laboratory of Immunology recurrence. Invest 35(4):1541, 1994. Ophthalmol Vis Sci DeBarge LR, Chan C-C, Greenberg SC, McLean IW, Yannuzzi LA, Nussenblatt RB: Chorioretinal, iris and ciliary body infiltration by juvenile xanthogranuloma masquerading as uveitis. Surv Ophthalmol 39:65-71, 1994. Egwuagu CE, Sztein J, Chan C-C, Mahdi R, Nussenblatt RB, Chepelinsky AB: Gamma interferon expression disrupts lens and retinal differentiation in transgenic mice. Dev Biol, in press. Egwuagu CE, Sztein J, Chan C-C, Reid W, Mahdi R, Nussenblatt RB, Chepelinsky AB: Ectopic expression of gamma interferon in the eyes of transgenic mice induces ocular pathol- ogy and MHC class 11 gene expression. Invest Ophthalmol Vis Sci 35(4):332-341, 1994. Egwuagu CE, Sztein J, Chan C-C, Mahdi R, Nussenblatt RB, ChepeUnsky AB: Transgenic rat and mouse models for the study of intraoc- iilar effects of gINF and autoimmunity. Invest Ophthalmol Vis Sci 35(4):1987, 1994. GazzineUi RT, Brezin A, Li Q, Nussenblatt RB, Chan C-C: Toxoplasma gondii: Acquired ocular toxoplasmosis in the murine model, protective role of TNF-alpha and IFN-gamma. Exp Parasitol 78:217-229, 1994. Hayashi S, GazzineUi R, Chan C-C, Pham N, Roberge FG: In vivo inhibition of nitric oxide enhances ocular and CNS inflammation in murine toxoplasmosis. Invest Ophthalmol Vis Sci 35(4):1685, 1994. Hikita N, Dastgheib K, Mochizuki M, Nussenblatt RB, Chan C-C: Effect of topical FK506 on experimental melanin-protein in- duced uveitis (EMIU) in rats. Invest Ophthal- mol Vis Sci 35(4):1540, 1994. HoUand EJ, Olsen TW, Chan C-C, Bergstrom L, Palestine AG, Nussenblatt RB: Kinetics of corneal transplant rejection in the rat penetrat- ing keratoplasty model. Cornea 13:317-323, 1994. Jang S, Li Q, Whitcup SM, Peng B, Nussenblatt RB, Chan C-C: Susceptibility to endotoxin induced uveitis varies in different murine strains. Invest Ophthalmol Vis Sci 35(4):1685, 1994. Kaiser-Kupfer MI, Chan C-C, MarkeUo TC, Crawford MA, Caruso RC, Csaky KG, Guo J, Gahl WA: Bietti's crystalline dystrophy: Natural history, biochemical and clinical pathologic correlations. Am J Ophthalmol, in press. Lai JC, Wawrousek EF, Sipe JD, Whitcup SM, Chan C-C, Igal G: Ocular and systemic im- munological profile of interleukin-lb (IL-1) transgenic mice. Invest Ophthalmol Vis Sci 35(4):1988, 1994. Li Q, Dastgheib K, Hibita N, Egwaugu C, Nussenblatt RB, Chan C-C: TGF-pi mRNA expression in experimental melanin-protein induced uveitis (EMIU) and in experimental autoimmune uveitis (EAU). Invest Ophthalmol Vis Sci 35(4):1807, 1994. Li Q, Abe T, Kikuchi T, Nussenblatt RB, Shinohara T, Chan C-C: Corticosteroids enhance S-antigen in non-retinal ocular tissues of rats with experimental autoimmune uveitis. Exp Mol Pathol 60:27-38, 1994. MacCumber MW, Dastgheib K, Bressler NM, Chan C-C, Harris M, Fine S, Green WR: CHnicopathologic correlation of the multiple recurrent serosanguineous retinal pigment epithelial detachments syndrome. Retina 14:143-152, 1994. MiUer-Rivero NE, Rizzo LV, Chan C-C, Wiggert B, Nussenblatt RB, Caspi RR: Sup- pression of IRBP-induced EAU in mice by feeding IRBP and its potentiation by interleukin-2. Invest Ophthalmol Vis Sci 35(4):1865, 1994. Murali S, Hardten DR, DeMartelaere S, Olevsky OM, Mindrup EA, Hecht ML, Chan C-C, Holland EJ: Effect of topical adminis- tered platelet-derived growth factor on corneal wound strength. Curr Eye Res, in press. 103 FY 1994 NEI Annual Report Parks DJ, Cheung MK, Chan C-C, Roberge FG: The role of rutric oxide in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 35(4):1685, 1994. Parks DJ, Cheung MK, Chan C-C, Roberge FG: The role of nitric oxide in uveitis. Arch Ophthalmol 112:544-546, 1994. Peng B, Li Q, Roberge F, Whitcup SM, Luyo D, Chan C-C: Topical rapamycin inhibits allergic conjunctivitis in a murine model. Invest Ophthalmol Vis Sci 35(4):1292, 1994. Rizzo LV, Silver PB, GazzineUi RT, Chan C-C, Wiggert B, Caspi RR: Expression of cytokine genes within the eye in murine EAU. Invest Ophthalmol Vis Sci 35(4):1862, 1994. Sartani G, Silver PB, Strassmann G, Chan C-C, Caspi RR: Effect of suramin treatment on induction of EAU. Invest Ophthalmol Vis Sci 35(4):1862, 1994. Silver PB, Rizzo LV, Chan C-C, Donoso LA, Wiggert B, Caspi RR: Identification of a major pathogenic epitope in the IRBP molecule recognized by mice of the H-2' haplotype. Invest Ophthalmol Vis Sci 35(4):2061, 1994. Suh EDW, Vistica BP, Chan C-C, Raber JM, Gery I, Nussenblatt RB: Splenectomy abro- gates the induction of oral tolerance in experi- mental autoimmune uveoretinitis. Curr Eye Res 12:833-839, 1993. Walton RC, Lai JC, Chanaud NP HI, Chan C- C, Gery I, Whitcup SM: Inhibition of experi- mental autoimmune uveitis by MDL 28,842. Invest Ophthalmol Vis Sci 35(4):1865, 1994. Wawrousek EF, Chan C-C, Lai JC, Gery I: Progressive inflammatory disease and neovascularization in the eyes of interleukin- Ib transgenic mice. Invest Ophthalmol Vis Sci 35(4):1988, 1994. Whitcup SM, Hayashi S, Rizzo L, Lai JC, GazzineUi R, Nussenblatt RB, Chan C-C: Systemic anti-IL-12 antibody exacerbates endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 35(4):1481, 1994. Whitcup SM, Dastgheib K, Nussenblatt RB, Walton RC, Pizzo PA, Chan C-C: A clinico- pathologic report of the retinal lesions associ- ated with Didanosine. Arch Ophthalmol, in press. Whitcup SM, Hikita N, Shirao M, Miyasaka M, Mochizuki M, Nussenblatt RB, Chan C-C: Monoclonal antibodies against CD54 and CDlla prevent and inhibit endotoxin-induced uveitis. Exp Eye Res, in press. Zierhut M, Chan C-C, Duijvestijn A, Nussenblatt RB, Whitcup SM: High endotheli- al venules in IRBP-induced experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 35(4):1807, 1994. Wakefield D, Li Q, McCluskey P, Nussenblatt RB, Chan C-C: Immunohistochemical localiza- tion of T lymphocytes and macrophages and expression of interferon gamma and defensin in uveitis. Ocul Immunol Inflam, in press. 104 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00270-04 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Immunologic Mechanisms of Ocular Disease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Scott M. Whitcup M.D. Staff Medical Officer CB, NEI Others: Chi-Chao Chan M.D. Robert B. Nussenblatt M.D. Sei-Ichi Ishimoto M.D. Head, Section on Immunopathology Scientific Director Visiting Associate LI, NEI NEI U NH (CRADA) COOPERATING UNITS (if any) LAB/BRANCH Clinical Branch/Laboratory of Immunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.4 PROFESSIONAL: 0.4 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The title of this project has been changed from "cell adhesion molecules in ocular inflammation" to "immunologic mechanisms of ocular disease" to reflect the broadening scope of experiments to encompass the role of cell adhesion molecules and other immunologic factors, including cytokines, in the pathogenesis of ocular diseases, particularly uveitis, ocular malignancy, and ocular disease related to AIDS. In addition, this project was previously listed in the Laboratory of Immunology but is now listed in the Clinical Branch. The goal of this project is to study the immunologic mechanisms involved in the pathogenesis of ocular inflammation and ocular malignancy and to develop and test therapies based on these data. Recently, we have concentrated on the role of cell adhesion molecules and cytokines in the development of ocular inflammatory disease. Cell adhesion molecules (CAMs) are surface proteins important for antigen sensitization and the migration of leukocytes to sites of inflammation. We are currently investigating compounds that block CAMs as a treatment for uveitis and other ocular inflammatory diseases. During the past year, we showed that a single injection with a monoclonal antibody against the CAMs, lymphocyte function-associated antigen-1 (LFA-1) and Mac-1, can inhibh the development of uveitis by more than 66 percent. In contrast, treatment with a monoclonal antibody against a third CAM, vascular adhesion molecule-1 (VCAM-1) failed to inhibit in vivo disease. Antibodies against these cell adhesion molecules also inhibited lymphocyte proliferation in vitro by up to 70 percent. We also studied the role of interleukin-12 on the development of acute intraocular inflammation and showed that systemically administered antibody against IL-12 exacerbates the endotoxin-induced uveitis. Similarly, intraocular IL-12 inhibited disease. These data suggest that endotoxin-induced uveitis may be a Th-2-dependent disease. Finally, we investigated the effect of a number of therapeutic agents in animal models of ocular inflammation. We demonstrated that MDL 28,842, an inhibitor of S-adenosyl-L-homocysteine hydrolase, decreases the ocular inflammation in animals with experimental autoimmune uveitis (EAU). In other experiments, we showed that topically administered heparin inhibits the development of allergic conjunctivitis induced by mast cell degranulation. 105 PHS 6040 (Rev. 5/92) FY 1994 NEl Annual Report Project Description Additional Personnel Rachel Caspi Ph.D. Visiting Associate LI, NEI Igal Gery Ph.D. Deputy Chief LI, NEI Qian Li MD. Visiting FeUow LI, NEI R. Christopher MX). Senior Staff Fellow Walton LI, NEI Objectives The goal of this project is to study the immu- nological mechanisms involved in the patho- genesis of ocular inflammation and malignan- cy and to develop and test new therapies for these disorders. The project has focused on investigating the role of ceU-adhesion mole- cules and cytokines in ocular inflammation and testing new therapeutic agents using in vitro and in vivo models of ocular inflammato- ry disease. Methods Animal Models of Ocular Inflammation. Endo- toxin-induced uveitis (EIU) is induced by injecting 100 jig of Salmonella typhimurium endotoxin into one footpad of Lewis rats or 200 /Ag into one footpad of C3H-Hen mice. Experimental autoimmune uveitis (EAU) in mice is induced by immunizing BIO.A mice with 50 /Lig of interphotoreceptor retinoid binding protein in complete Freund's adju- vant, with pertussis toxin injected intraperitoneaUy. Finally, allergic conjunctivi- tis was induced in mice using topical adminis- tration of compound 48/80, a mast cell degranulating agent. Histology and Immunohistockemistry of Ocular Inflammation. Enucleated arumal eyes and human ocular tissue are immediately snap frozen and embedded in OCT. The expression of ceU-adhesion molecules and presence of cytokines is then assessed with immunohistochemical staining using an avi- din-biotin-peroxidase complex method on frozen sections of ocular tissue. Eyes are also embedded in methyl methacrylate, and four micron sections are examined for histologic evidence of inflammation. In the model of compound 48/80 induced allergic conjunctivi- tis, tarsal and bulbar conjunctiva were re- moved and processed for routine histology or frozen for immunohistochemical staining as described earUer. In vitro lymphocyte proliferation assays are performed as previously detailed {Cell Immunol 122:251, 1989). Major Findings (1) In a study examining the effect of treat- ment with a monoclonal antibody against lymphocyte function-associated antigen (LFA)- 1 and very late activation antigen (VLA)-4, we demonstrated that anti-LFA-1 antibody signifi- cantly inhibited the development of EIU. In contrast, anti-VLA-4 antibody had no effect on the development of intraocular inflammation. The data suggest that compounds blocking LFA-1 but not VLA-4 should be effective for the treatment of acute ocular inflammation. Studies in vitro showed that antibodies against LFA-1 and Mac-1 inhibited lymphocyte prolif- eration but that anti-VLA-4 antibody had less effect. These data suggest that anti-LFA-1 and anti-Mac-1 antibody may be useful in the treatment of lymphocyte-mediated ocular inflammatory disease. Finally, increased expression of ceU-adhesion molecules was noted in corneal specimens with allograft failure, suggesting that antibodies against adhesion molectiles may prevent rejection. (2) We found that systemic treatment with anti-interleukin (IL)-12 monoclonal anti- body exacerbates the development of EIU. The mean number of inflammatory cells ± S.E.M. was 41.6 ± 9.3 for mice treated with anti-IL-12 antibody, 17.6 ± 3.5 for mice treated with IFN- Y IL-12, and 17.7 ± 21 for control mice. Addi- tional studies showed that intraocular admin- istration of IL-12 inhibits the development of EIU. Similar to previous studies with anti- interferon gamma (IFN-y) antibody, systemic 106 Laboratory of Immunology anti-IFN-y antibody exacerbates intraocular inflammation. This may be due to decreased generation of Th-1 cells and increased genera- tion of Th-2 ceUs. (3) MDL 28,842, a potent irreversible inhibitor of S-adenosyl-L-homocysteine hydro- lase was shown to inhibit the development of EAU in mice. Animals treated with MDL 28,842 at 2.5 and 5.0 mg/kg/day had signifi- cantiy less disease when compared with contiols (p < 0.009). Importantiy, there was no significant weight loss in the treated ani- mals. These data suggest that MDL 28,842 may be a useful therapeutic agent in the treatment of uveitis in humans. (4) Topical heparin significantiy inhibited the development of allergic conjunctivitis in mice. Preliminary studies revealed no ocular toxicity. These findings suggest that topical heparin may provide a well-tolerated and effective treatment for allergic conjunctivitis. Significance to Biomedical Research and ttie Program of the Institute One major mission of the NEl is to under- stand the mechanisms of sight- threatening eye disease so that new and effective therapies can be developed. The expression of cell- adhesion molecules appears to be a funda- mental mechanism in the development of intraocular inflammation. Cytokines are also important in the pathogenesis of ocular in- flammation, and certain cytokines such as IL- 12 and IL-IG appear to have a regulatory role on uveitis. With this understanding, we hope to develop new anti-inflammatory therapy for ocular inflammation. The testing of these therapeutic agents in models of ocular inflam- mation allows the development of new thera- pies for patients with ocular inflammatory disease, which accounts for approximately 10 percent of the visual impairment in the United States. Proposed Course We plan to continue our experiments investi- gating the role of ceU-adhesion molecules and cytokines in uveitis. In addition to using monoclonal antibodies against adhesion mole- cules, we are testing small molecules to block the cell adhesion molecules because these agents may be administered topically. We have started experiments studying the role of CAMs and cytokines in other inflammatory diseases, including secondary glaucoma, corneal allograft rejection, and intraocular malignancies. We are also continuing our experiments with MDL 28,842. NEl Research Program Retinal Diseases — Inflammatory Diseases Publications Anderson W, Chan C-C, Nussenblatt RB, Whitcup SM: Topical heparin inhibits com- pound 48/80 induced allergic conjunctivitis. Invest Ophthalmol Vis Sci 35(suppl):1291, 1994. Jang S, Li Q, Whitcup SM, Peng B, Nussenblatt RB, Chan C-C: Susceptibility to endotoxin induced uveitis varies in different murine strains. Invest Ophthalmol Vis Sci 35(suppl):1685, 1994. Walton RC, Lai JC, Chanaud NP, Chan C-C, Gery I, Whitcup SM: Inhibition of experimen- tal autoimmune uveitis by MDL 28,842. Invest Ophthalmol Vis Sci 35(suppl):1865, 1994. Whitcup SM, Hayashi S, Rizzo, Lai JC, Gazzinelli R, Nussenblatt RB, Chan C-C: Systemic anti-IL-12 antibody exacerbates endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 35(suppl):1481, 1994. Whitcup SM, Nussenblatt RB, Price FW Jr, Chan C-C: Expression of cell adhesion mole- cules in corneal graft failure. Cornea 12:475-80, 1993. Whitcup SM: The role of cell adhesion mole- cules in endotoxin-induced uveitis. Regional Immunol, in press. Whitcup SM, Hikita N, Shirao M, Miyasaka M, Tamatani T, Mochizuki M, Nussenblatt RB, 107 FY 2994 NEI Annual Report Chan C-C: Monoclonal antibodies against Patents CD54 (ICAM-1) and CDlla (LFA-1) prevent and inhibit endotoxin-induced uveitis. Exp U.S. patent (applied for) Eye Res, in press. Contract/CRADA Reports Zierhut M, Chan C-C, Duijvestijn A, Nussenblatt RB, Whitcup SM: High endotheU- Allergan CRADA al venules in IRBP-induced experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 35(suppl):1807, 1994. 108 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00269-04 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title mus( fit on one line between the borders.) Ocular Toxicity of 2',3'-Dideoxvinosine (ddl) PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Scott M. Whitcup M.D. Staff Medical Officer CB, NEI Others: Robert B. Nussenblatt M.D. Scientific Director NEI Rafael Caruso M.D. Visiting Scientist OGCS, NEI COOPERATING UNITS (if any) Pediatric Branch, National Cancer Institute (Philip A. Pizzo, M.D.); Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases (Clifford H. Lane, M.D.); Clinical Oncology Program, National Cancer Institute (Robert Yarchoan, M.D.) Clinical Branch/Laboratory of Immunology Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.4 PROFESSIONAL: 0.4 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (b) Human tissues □ (c) Neither |~1 (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project, "Ocular toxicity of 2',3'-Dideoxyinosine (ddl)", was previously listed in the Laboratory of Immunology but is now a project in the Clinical Branch, ddl is a purine analog with antiretroviral activity currently used to treat patients with the acquired immunodeficiency syndrome (AIDS), both adults and children, in clinical protocols at the NIH. The purpose of this study is to prospectively follow patients treated with ddl for the development of ocular complications secondary to drug toxicity. Ninety-five children with symptomatic (CDC class P-2) HIV infection were enrolled in a Phase I-II study to assess the safety and antiretroviral activity of ddl. More than 100 children treated with ddl have been examined, and 6 children have developed peripheral atrophy of the retinal pigment epithelium during ddl therapy. Eyes with ddl- associated retinal lesions have now been examined histologically. Microscopic examination of these lesions revealed multiple areas of retinal pigment epithelial (RPE) loss, some surrounded by areas of hypertrophy or hypopigmentation of the RPE. Partial loss of the choriocapillaris and neurosensory retina were also noted in areas of diseased RPE. Transmission electron microsocopy showed numerous membranous lamellar inclusions and cytoplasmic bodies in the RPE cells. These data show that didanosine primarily affects the RPE and that the choriocapillaris and overlying neurosensory retina are also dystrophic in areas of RPE loss. We also continue to follow a group of 75 adults treated with ddl with periodic fundus examinations and electro- oculograms. One aduh previously developed retinal lesions while treated with ddl, but no additional adult patients have developed retinal lesions. 209 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Additional Personnel R. Christopher MD. Senior Staff Fellow Walton LI, NEI Objectives The goal of this study is to monitor patients treated with dideoxyinosine (ddl) for the development of ocular complications. Methods (1) Patients treated with ddl are given com- plete eye examinations every three to four months, including dilated ophthalmoscopy and fundus photography of any abnormal retinal findings. Patients treated with the higher dosages of ddl also receive periodic electro-oculograms to assess the electrophy- siologic function of the retinal pigment epithe- Hum (RPE). (2) Eyes from one child with ddl-associat- ed retinal lesions were studied histologically. Eyes were fixed in 10 percent buffered forma- lin for routine histology and electron micros- copy. Sections were stained with hematoxylin and eosin, periodic acid Schiff, and Gomori's methenamine silver. Small fragments of chorioretinal tissue in the area of the lesions were embedded in an epoxy resin, and ultra- thin sections were stained with uranyl acetate and lead citrate for transmission electron microscopy. Major Findings (1) Six children have now developed periph- eral atrophy of the RPE during ddl therapy. The lesions are scalloped areas of RPE atrophy with hyperpigmented borders and occur predominantly in the midperiphery of the fundus in both eyes. These retinal lesions slowly progress if ddl therapy is continued, but central visual acuity remains unaffected. During the past year, one child developed a few retinal lesions while on ddl. (2) No additional adults developed retinal lesions. (3) Gross examination of the eyes re- vealed multiple 1 to 2 mm round lesions of RPE loss, many with areas of RPE hyperpig- mentation at their periphery or centrally located. The lesions spanned from 3 mm posterior of the ora serrata to the midperi- phery and extended circumferentially in each eye. Microscopic examination revealed a normal cornea, iris, cihary body, lens, and optic nerve in both eyes. There were multiple areas of RPE loss, and some of these areas of RPE hypertrophy and/ or RPE hypopigmenta- tion were located at their margins. Chorioreti- nal adhesion and partial loss of the choriocapi- llaris and outer retina to the level of the inner nuclear layer were also noted in areas of RPE loss. Transmission electron microscopy re- vealed enlarged RPE cells with large, spherical melanin granules (RPE hypertrophy) and other RPE cells with a reduced number of pigment granules (RPE hypopigmentation). Degenerating RPE and neural cells were also seen. Bruch's membrane was intact, except in the areas of chorioretinal adhesion. A large number of membranous lamellar inclusions up to 20 nm in thickness and concentric membra- nous cytoplasmic bodies measuring up to 2 /xm in diameter were clustered between the melanin granules of some RPE cells. Significance to Biomedical Research and the Program of the Institute It has been determined that ddl is a drug with in vitro and in vivo activity against human immunodeficiency virus infection. One of the missions of the NEI is to monitor patients for the development of ocular toxicity and assess the effect such toxicity has on vision. Proposed Course The detailed study of the retinal lesions associ- ated with ddl has been completed. Although aU HIV patients followed on NIH protocols will continue to be followed for the develop- ment of ocular complications, this specific project was completed on September 30, 1994. 110 Lahoratory of Immunology NEI Research Program Retinal Diseases — Photoreceptors and Pigment Epithelium Publications Nguyen B-Y, Shay LE, Wyvill KM, Pluda JM, Brawley O, Cohen RB, Whitcup SM, Venzon DJ, Broder S, Yarchoan R: A pUot study of sequential therapy with Zidovudine plus acyclovir, didanosine, and dideoxycytidine in patients with severe human immunodeficiency virus infection. / Infect Dis 168:810-817, 1993. Whitcup SM, Dastgheib K, Nussenblatt RB, Walton RC, Pizzo PA, Chan C-C: A clinico- pathologic report of the retinal lesions associ- ated with didanosine. Arch Ophthalmol, in press. Ill DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00264-04 LI PERIOD COVERED October 1, 1992 to Septemb e r 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Cytokines and Ocular Antig e ns in the Eye PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Chi-Chao Chan M.D. Head, Section on LI, NEI Immunopathology Others: Robert B. Nussenblatt Igal Gery Qian Li Louis Kasner M.D. Scientific Director LI, NEI Ph.D. Head, Section on LI, NEI Experimental Immunology M.D. Visiting Fellow LI, NEI M.D. Fellow LLNEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL: 0.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x| (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project has been terminated and combined with project number ZOl EY 00222-08 LI. 112 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00115-16 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Cvclosporine Therapy in Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Robert B. Nussenblatt M.D. Scientific Director NEI Others: Marc D. de Smet M.D. Scott Whitcup M.D. Chi-Chao Chan M.D. Visiting Scientist Senior Staff Fellow Medical Officer LI, NEI LI, NEI LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS; 0.5 PROFESSIONAL: 0.5 0.0 CHECK APPROPRIATE BOX(ES) fxl (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Cyclosporine (Cs), an endecapeptide fungal product with specific anti-T-cell characteristics, is being administered to patients with sight-threatening ocular inflammatory disease of noninfectious origin who have failed on either corticosteroid or cytotoxic agent therapy to test its efficacy in the treatment of uveitis. Within the context of these ongoing studies, the combined use of CsA and ketaconazole has been tested in a randomized masked study of a small group of patients whose uveitis was well controlled with Cs. The combination allowed a significant reduction in the dose of Cs needed to control the disease. In some instances, the dose could be reduced by as much as 90 percent. No significant increase in side effects was noted. A phase I/II randomized trial using CsA and CsG has ended. There is a definite trend showing that combined use of a Cs and low to moderate steroid doses is efficacious in preventing the progression of uveitis. An effective dose of Cs appears to be around 5 mg/kg. At this dosage, toxicity has been reduced for up to 12 months of followup. CsG was more effective than CsA in treating cystoid macular edema. Patients who remain on Cs long term continue to be followed to gain further information about renal (or other) toxicity. 113 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Biologist LI, NEI Project Description Additional Personnei Barry Grubbs Clinical Protocol Number 81-EI-33 Objectives Cyclosporine (Cs), an endecapeptide obtained from fungi, has been shown to have specific anti-T-cell activity {Transplant Proc 12:234, 1980). We have reported cyclosporine' s excep- tional effectiveness in preventing the induction of S-antigen autoimmune uveitis in rats as w^eU as inhibiting the disease once immuniza- tion has occurred (J Clin Invest 67:1228, 1981). The goal of this study is to test CsA versus CsG to test their efficacy in treating patients with bilateral sight-threatening posterior uveitis of an autoimmune nature. Methods Patients 18 years of age or older, of either sex (females not pregnant), who have not done weU on more conventional medical therapy were admitted to this study. All patients must have a bilateral sight-threaterung uveitis of noninfectious etiology that was not satisfac- torily controlled by either corticosteroid or cytotoxic agent therapy. Lymphocyte cultures are prepared, and the immune cells are tested against various crude ocular extracts as well as purified human S-antigen to assess evi- dence of cellular immune memory, which is considered to be the in vitro equivalent of the anamnestic response in vivo. Patients chosen are treated with CsA or a new analog called CsG in a phase I/II trial to evaluate the safety and activity of CsG versus CsA. During this period, the patients' cHnical and immunologic courses are closely monitored. Specific atten- tion is given to renal function changes, a frequent side effect. Patients who need to continue CsA because of their ocular disease for more than one year may be asked to undergo renal biopsy to evaluate the revers- ible and irreversible components to CsA renal toxicity. Some patients who have been en- tered on previous CsA studies and have continued to be followed in the eye clinic wiU continue to be monitored for their renal func- tion and to determine how and when cyclo- sporine can safely be tapered. Major Findings CsA has been effective in the treatment of some cases of posterior uveitis. An improve- ment in the inflammatory activity and visual acuity was seen in most patients treated to date. The particular responsiveness of pa- tients with the ocular manifestations of Behcet's disease to this agent has been corrob- orated by a masked, randomized trial per- formed in Japan. The improvement in the clinical condition was supported by a concom- itant improvement in electrophysiologic test- ing, particularly contrast sensitivity. Patients treated with CsA had no abnor- malities of natural killer cell activity before the initiation of therapy, nor was any noted after- ward. CsA significantiy decreased skin test responsiveness but did not alter lymphocj^e proliferation or antibody production in pa- tients. Renal toxicity has been noted in some patients on long-term therapy, necessitating the addition of systemic corticosteroids and a decrease in CsA dosage. At three months, approximately 78 percent of the patients entering this open study were considered therapeutic successes, but 62 percent were considered successes at one year. Seventeen patients treated long term with CsA underwent renal biopsy. These biopsy specimens were read in a masked fashion by a group of renal disease specialists who com- pared these biopsies with those from age-mat- ched controls. An irreversible component of CsA toxicity covdd be identified in the main being renal tubular afrophy accompanied by interstitial fibrosis. The majority of the indivi- duals' biopsies had normal serum creatinine values, but a correlation could be made 114 Laboratory of Immunology between the alterations noted and previous serum creatinine elevations for some period of time. The Cs A/G trial has shown that CsG and CsA have overall equal value in treating uveitis. However, CsG was more effective in reducing cystoid macular edema than was CsA, particularly at lower dosages. Significance to Biomedical Research and tiie Program of ttie Institute Uveitis is one of the most frustrating problems in all of ophthalmology. Present modes of therapy for patients with severe ocular inflam- matory disease are inadequate and nonspecif- ic. CsA appears effective in treating posterior uveitis of noninfectious etiology. This is the first new agent in decades to be found useful in treating the severe form of this condition; therefore, it is important that the optimum therapeutic schedule be developed. Newer therapeutic strategies have already begun. Proposed Course Newer studies to look at various Cs combina- tions will continue. NEI Research Program Retinal and Diseases — Inflammatory Diseases Publications de Smet MD, Nussenblatt, RB: Clinical use of cyclosporine in ocular disease. Int Ophthalmol Clin 33(4):31-45, 1993. Nussenblatt RB, deSmet MD, Rubin B, Freidlin V, Whitcup SM, Davis J, et al: A masked randomized, dose response study between cyclosporine A and G in the treatment of sight-threatening uveitis of noivinfectious etiology. Am J Ophthalmol 115:583-591, 1993. 215 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00278-03 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Oral Administration of Antigen and the Ocular Immune Response PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and instittJte affiliation) PI: Robert B. Nussenblatt M.D. Scientific Director NEI Others: Igal Gery Susan Whitcher Marc D. de Smet Ph.D. M.S. M.D. Head, Section on LI, NEI Experimental Immunology Clinical Protocol Assistant LI, NEI Visiting Scientist LI, NEI COOPERATING UNITS (if any) Laboratory of Immunology Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.8 PROFESSIONAL 0.3 0.5 CHECK APPROPRIATE BOX(ES) [xj (a) Human subjects |~| (a1) Minors |~| (a2) Interviews □ (b) Human tissues □ (c) Neitiier SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The effect of the oral administration of various antigens on the ocular immune response has been tested in the animal model for severe intraocular inflammatory disease, experimental autoimmune uveioretinitis, which is induced by both retinal S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). Oral tolerance could be induced by repeatedly feeding rats with S-Ag. A putative suppresser cell that was CDS positive could be isolated from the spleen of such animals and transferred to other animals to induce a similar toleragenic effect. In addition, the role of the spleen was confirmed in ongoing animal experiments. A randomized masked trial to evaluate the usefulness of S-Ag feeding in patients with intraocular inflammatory diseases continues. A pilot study was performed in two patients that showed the induction of such tolerance, and these patients continue to be followed. 116 PHS 6040 (Rev. 5/92) Laboratory of Immunology Project Description Objectives Exploring means at imniunon^odulation has been a major role of this laboratory. Although extensive experimentation has used various immunosuppressive agents, there has also been a major thrust in an attempt to use other modes of immunosuppression. The goal of this series of experiments, in animals as well as in humans, is to test the efficacy of oral tolerance with uveitogenic antigens in the treatment of animals induced with experimen- tal autoimmune uveitis and in patients with bilateral sight-threatening posterior and inter- mediate uveitis of an autoimmune nature. Methods Six- to 10- week-old Lewis rats of either sex are used in these experiments. Animals are fed various antigens before and after the induction of experimental uveioretinitis. The feeding regimen includes whole molecules such as the retinal S-antigen (S-Ag) and interphotorecep- tor retinoid-binding proteins as well as their fragments. In a subset of experiments, some animals will also undergo splenectomy before the initiation of these experiments, but others will receive sham procedures. We are at- tempting to evaluate the clinical course of their disease and corroborate the cUnical observations with histopathology at various points after the initiation of the experiments. The goal is to evaluate the role of the spleen as well as the role of various fragments in the ability to induce this tolerogenic state. In the studies to be performed with pa- tients, those individuals who have bilateral uveitis of a noninfectious cause and who are 18 years or older of either sex wiU be consid- ered for the study. Additionally, their lym- phocytes must demonstrate an in vitro preUf- orative response to the retinal S-Ag. The patients also need to be on systemic immuno- suppressive therapy, whether it be corticos- teroids, cytotoxic agents, or cyclosporine. The goal of this study will be to assess, in individ- uals who need high amounts of immunosup- pressive therapy to control their disease, whether the addition of oral feeding of retinal antigens wiU induce a toleragenic state. This study will be performed in a random- ized, double-masked fashion in which some patients wiU receive S-Ag, other patients wiU receive a retinal mixture containing several antigens, and still others wiU receive placebo. The intent is to reduce the amount of immu- nosuppressive therapy they are taking with the hope that a toleragenic state can be in- duced by the feeding of these antigens. Major Findings In the animal work, the spleen appears to play an important role in the induction of oral tolerance of S-Ag. In addition, the spleen is essential for adoptive transfer of tolerance by splenocytes from S-Ag fed donors. Thus, it would be logical to assume that the spleen acts as a sight for induction and /or ampUfica- tion of ceUs with suppressive activity. The pilot study has demonstrated that, at least in two patients, a toleragenic state could be induced with the feeding of antigen at the dosages that are planned for this study. One patient with par planitis and the other v^dth Behcet's disease have been able to discontinue their medication completely or reduce it to exceptionally low dosages. Significance to Biomedical Researcf) and the Program of the Institute Uveitis is one of the most frustrating problems in all of ophthalmology. The present modes of therapy for patients with severe ocular inflammatory disease all have limitations, particularly because of their secondary side effects. In identifying patients with an im- mune response to the retinal S-Ag, we will have been able to induce an immunosuppres- sive state without the use of pharmacologic agents. Furthermore, the induction of this tolerance would be antigen specific. ii: FY 1994 NEI Annual Report Proposed Course Publications The randomized study will begin shortly. Nussenblatt RB, de Smet MD, Weiner HL, Gery I: The treatment of the ocular complica- NEI Research Program tions of Behcet's disease with oral tolerization, in Wechsler B, Godeau P (eds): Sixth Interna- Retinal Diseases — Inflammatory Diseases tional Conference on Behget's Disease. New York, Excerpta Medica, 1993. 118 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00184-12 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit or) one line between the borders.) Cellular and Immunogenetic Mechanisms in Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Rachel R. Caspi Ph.D. Visiting Scientist LI, NEI Others: Phyllis Silver B.S. Biologist LI, NEI Luiz Rizzo M.D. Visiting Associate LI, NEI Gil Sartani M.D. Visiting Fellow LI, NEI Xu Hui Ph.D. Visiting Fellow LI, NEI Sun Bing Ph.D. Visiting Fellow LI, NEI Chi-Chao Chan M.D. Medical Officer LI, NEI COOPERATING UNITS (if any) National Institute of Child Health and Development (Lawrence M. Nelson, M.D.); Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases (Ronald L. Wilder, M.D., Ph.D.); Bone Marrow Transplantation Unit, National Cancer Institute (Frances Hakim, Ph.D.); Research and Development, Wills Eye Hospital, Philadelphia, PA (Larry A. Donoso, M.D., Ph.D.) LAB/BRANCH Laboratory of Immunology Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 5.1 PROFESSIONAL: 5.1 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Cellular mechanisms in ocular immunologically mediated disease are being studied in animal models of experimental autoimmune uveoretinitis (EAU). Rats and mice are immunized with retina-derived antigens or synthetic peptides, representing fragments of these antigens, to induce EAU. Susceptibility to disease induction is being evaluated in various strains of known genetic makeup, in the hope of delineating the hereditary mechanisms that predispose to uveitis. In vivo functional long-term T-cell lines and clones are developed from lymphoid organs of rats and mice immunized with uveitogenic ocular proteins. The functional properties and antigen receptors of these cells are being studied to develop strategies for in vivo targeting of the autoiiimiune cells. EAU in rats and mice serves as a template for the evaluation of new drugs and compounds as well as for the study and characterization of the participating cells and their factors. The goals of these studies are to identify the immunogenetic factors predisposing to uveitic disease, learn about the pathogenic mechanisms involved, characterize the immunoreactive cells and their mediators, and finally to utilize this knowledge for designing rational approaches to immunotherapy. 119 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Additional Personnel Robert B. M.D. Scientific Director, NFEI Nussenblatt Chief, LI, NEI Charles E. PhD. Staff FeUow, NEI Egwuagu Rashid Mahdi Biologist, LI, NEI Igal Gery Ph.D. Head, Section on Experimental Immunology, NEI Objectives The development and study of animal models of experimental ocular autoimmune disease permits the study of cellular and genetic factors that may be generally involved in ocular autoimmunity. Experimental autoim- mune uveitis (EAU) in rats and mice serves as a template for the evaluation of new drugs and compounds as well as for the study and characterization of the participating cells and their factors. Long-term maintenance of T cells in vitro permits the investigators to sepa- rate and selectively grow various T-cell sub- sets. The goals are to use the EAU model in rats and mice for the study of cellular mecha- nisms in ocular autoimmunity. Specifically, we are trying to delineate discrete stages in the immunopathogenic process and to devise strategies to specifically disrupt them at criti- cal points. One means to this end is to establish, characterize, and use retinal antigen-specific T- cell Unes and clones. These lines and clones permit us to learn about cells capable of ocular immunomodulation in the positive and negative sense, to earn about migration and localization of autcmmune lymphocytes, and to study their interactions with other lym- phoid and nonlymphoid cells in eliciting effector mechanisms. Finally, we use the EAU model for the study of hereditary mechanisms controlling genetic susceptibility and resis- tance to ocular autoimmune disease. The study and understanding of these parameters will help not only in the development of new therapies but also possibly in the prevention of ocular disease. Methods Rats and mice of various strains are immu- nized with purified S-antigen (S-Ag) or with interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant or with various pathogenic peptides derived from these proteins that are synthesized in the laboratory. After the development of disease, eyes are processed for histopathology and examined for disease, and lymphoid cells from the blood, lymph nodes, or eyes are taken. Cells thus obtained are placed in culture either with mitogen or with the retinal antigen with which the donor aiumal was immunized. Responses of the immune cells are studied. Cells are also expanded in culture and used to transfer EAU to nonimmune aiumals to study the cell population responsible for disease induction. Similar methods are used to study cells responsible for disease suppres- sion. Long-term ceU lines are developed and in some cases are cloned. These cell Unes and clones are then tested for functional character- istics such as the ability to induce or suppress ocular disease, production of soluble media- tors, expression of various cell-surface mole- cules, response to therapeutic agents, and interactions with other cells in culture. Major Findings The possible correlation between the pathoge- nicity of autoimmune T cells and their lym- phokine production, expression of functional adhesion molecules and expression of some surface antigens, was examined in the Lewis rat EAU model. We used four retinal antigen- specific Lewis rat T-cell lines and sublines: one specific to the major pathogenic epitope of the human retinal soluble antigen (S-Ag; residues 337-356), and three specific to the major patho- genic epitope of the bovine IRBP (residues 1177-1191). The lines have different degrees of uveitogenicity, from highly pathogenic to nonpathogenic. AU four T-cell Unes produced roughly equivalent amounts of interferon 120 Laboratory of Immunology gamma (IFN-y), l5nTiphotoxin/ tumor necrosis factor (TNFa/p), interleukin (IL)-3, IL-6, and transforming growth factor beta (TGF-P). IL-4 activity could not be detected. The lines also expressed similar levels of functional adhesion molecules, as measured by binding to cultured rat aorta endothelial cells. The nonpathogenic subline, however, was the lowest responder to antigenic stimulation with respect to proUfera- tion and IL-2 production. Examination of ceU- surface antigens showed that in contiast with the other lines, the majority of cells in the nonpathogenic subline lacked detectable expression of QD-4. No difference was found in the level of expression of the IL-2 receptor and T-ceU antigen receptor between the four lines. Because CD-4 is the restricting element in these lines, reduced QD-4 expression in the nonpathogenic subUne may at least partially explain its poor response in vitro to antigenic stimulation. All three attributes could be coimected to lack of pathogenicity of this line in vivo. These results support the contention that class H-restricted recognition of autoantigen within the neuroretina by uveitogenic T lymphocytes must occur as an initial step in the pathogene- sis of EAU. A defect in this step wiU preclude pathogenesis, regardless of some other func- tional attributes possessed by effector T cells such as production of inflammatory lympho- krnes and expression of adhesion molecules. We have also studied the expression of cytokine genes within the eye in murine EAU. We have previously described several T-ceU Unes specific to the retinal protein IRBP or to its peptides that can induce EAU on adoptive transfer into naive mice. We have also shown that such cell Unes elaborate an unrestricted cytokine profile in vitro. The purpose of this study was to investi- gate what set of cytokines was being ex- pressed by these cells in vivo within the target organ. C57BL/6 athymic mice and BIO. A euthymic mice were injected intravenously with histocompatible uveitogenic T cells. Control animals were immunized with an established uveitogenic regimen of IRBP. The eyes were harvested 11 to 21 days later after perfusing the animals with 10 mL of PBS /heparin (to ensure that the cytokines detected did not originate from passenger lymphocytes passing through the retinal vessels). One eye was sent for histological examination, and the other eye was processed for ribonucleic acid (RNA) extiaction. The RNA was reverse-transcribed, and the result- ing complementary deoxyribonucleic acid was ampUfied using primers for several cytokines of interest. The results showed that although the uveitogenic T-ceU Unes and clones had an unrestricted cytokine profile in vitro, predomi- nantiy Th-1-type cytokine messenger ribonu- cleic acid (mRNA) (IL-2, IFN-y, and TNF-a) were present in eyes of mice that had EAU induced with those cells. Eyes of actively immuruzed animals showed a less restricted lymphokine profile, with most eyes having detectable mRNA for IL-2, IFN-y, and IL-4. Eyes of control animals that had neither been immunized nor adoptively transferred with T cells were negative for all cytokines tested. These results suggest that IL-2, IFN-y, and TNF-a are involved in murine EAU. Taken together with a lack of IL-4 mRNA in eyes of mice that had EAU induced by adoptive transfer, the data argue that predominantiy Th-1 tjrpe ceUs were present in the uveitic eyes. Because adoptive EAU was induced with T-ceU lines having an unrestricted cyto- kine profile (representing a Th-0 or a mixed Th-l+Th-2 population), this might imply that either a differentiation or a selection event occurred during development of EAU. In the mouse model of EAU, we have identified a major pathogenic epitope in the IRBP molecule that is recognized by mice of the H-2r haplotype. Overlapping 20-amino add peptides, spanning the entire human IRBP molecule, were synthesized and used to immunize C57BL/10 (H-2b), BIO.BR (H-2k), and BlO.Rlll (H-2r) mice. Bovine IRBP was used as a positive control. EAU was examined by histopathology 28 days after immunization. In vivo and in vitro immunological responses were assessed by delayed type hypersensitivity (DTH) and lymphocyte proliferation, respectively. 121 FY 1994 NEI Annual Report Peptide 161-180, spanning the sequence SGIPYVISYLHPGSTVSHVD, was found to be highly pathogenic for BIO.RIII mice but not for the other strains. EAU occurred in 20/20 BIO.RIII mice after immunization with 50 yitg of peptide. The average disease score was 2.7 as compared with 3.2 for IRBP-immunized mice. A dose-response curve showed that peptide 161-180 remained maximally patho- genic at 25 /ig, but incidence and scores were reduced at 10 fxg. The truncated 14-mer 165- 178 was also highly pathogenic (100-200 /ig/ mouse), suggesting that it contained the pathogenic epitope. Mice immunized with the peptide, or with whole IRBP, had positive DTH and lymphocj^e responses in vitro to the immunizing as weU as to the reciprocal anti- gen. These results indicate that peptide 161- 180 appears to contain an epitope that is pathogenic to mice of the H-2r, but not H-2b or the H-2k haplotjq^es. High incidence and high severity scores as well as immunological crossrecognition between the peptide and IRBP in vivo and in vitro suggest that this peptide contains a major pathogenic epitope. In another study, the compound suramin was evaluated for its efficacy to prevent EAU. Suramin has been in clirucal use for treatment of parasitic diseases and some types of cancer and is known to have immunosuppressive properties. EAU was induced in BIO.A mice by immunization with the whole IRBP protein and in Lewis rats by immunization with peptide 35 of S-Ag or by adoptive transfer of a T-ceU Une specific to this peptide (SP-35 Une). Actively immunized animals were treated with suramin (30-100 mg/kg, l.P.) to cover either the afferent or the efferent stage of EAU (days and seven or days seven and 14, respectively). Adoptively transferred animals (considered to represent efferent-stage disease) were treated on day minus one. Control animals were injected with phosphate-buffered saline at the corresponding times. EAU was assessed by cHrucal evaluation and by histopa- thology performed approximately one week after onset. Immunological responses were assessed by DTH and lymphocyte prolifera- tion to the immunizing antigen. The results revealed that treatment of BIO.A mice with 100 mg/kg of suramin completely prevented disease when given during the afferent stage and ameliorated disease when given during the efferent stage of EAU. The same dose and regimen were somewhat less effective in preventing EAU in Lewis rats: Afferent treatment lowered the incidence and ameUo- rated disease scores by approximately 50 percent, whereas efferent treatment (of either immunized or adoptively transferred rats) had Uttie or no effect on disease. Effect on DTH responses and lymphocyte proliferation rough- ly paralleled the effect on EAU. Thus, afferent treatment with suramin suppressed EAU and immunological responses, whereas treatment during the efferent stage was less effective, suggesting interference with antigen priming. The response to treatment may in part be dependent on the species in that mice re- sponded to the same treatment better than rats. To our knowledge, this is the first report of the successful use of suramin for treatment of autoimmunity. Another approach to suppressing ocular autoimmunity was through induction of oral tolerance. It was previously shown that EAU in rats can be suppressed by feeding of S-Ag. We wished to: (1) test whether a similar phenomenon exists in mice and (2) evaluate the feasibility of potentiating it by tmmuno- manipulation. For this purpose, EAU-suscep- tible BIO.A mice were fed IRBP or control solution using various regimens and were subsequently challenged with a uveitogenic regimen of IRBP. EAU was assessed by histopathology 21 days after immuruzation. Immunological responses measured included DTH, l5miphocyte proliferation, and cytokine production. The results indicated that three feedings of 0.2 mg IRBP every other day before immuni- zation did not protect mice against EAU, whereas a similar regimen of five feedings of 0.2 mg IRBP every other day was protective. However, supplementing the nonprotective 3x regimen with one intraperitoneal administra- tion of 400 units of recombinant human IL-2 122 Laboratory of Immunology on the day of immunization resulted in dis- ease suppression that was equal to that of the protective 5x regimen (p <. 0.02 compared with unfed controls). Analysis of cytokines pro- duced by Peyer's Patch ceUs of fed mice showed a large increase in production of TGFb, IL-4, and IL-10 in the 3x-fed and IL-2- treated animals compared with animals given the nonprotective 3x (no IL-2) feeding regimen and animals given the protective 5x feeding regimen. We propose that the IL-2 treatment enhances protection from EAU by stimulating regulatory cells that produce TGF-p, IL-4, and IL-10. Furthermore, the differences in lym- phokine production patterns among the exper- imental groups suggest that the mechanism of protection induced by the 3x + IL-2 regimen may differ from that induced by the 5x regi- men. It is conceivable that in the former case, protection from EAU was achieved by active suppression of the uveitogenic effector cells, whereas a mechanism of deletion or anergy might predominate in the latter. Significance to Biomedical Research and the Program of the Institute It has become increasingly clear that the cellular mechanisms and possibly the genetic mechanisms observed in animal models of uveitis reflect the mechanisms that operate in ocular immune-mediated disease in humans. The identification and characterization of the ceUs involved in ocular autoimmunity, and of their functions, will provide new understand- ing of inflammatory ocular diseases. Success- ful immunomodulation of EAU in animal models has thus far usually served as a good predictor of the clinical success of a given therapeutic modality. The continued study of basic mechanisms involved in the immuno- pathogenesis of uveitis in animal models will aid in the development of novel immunother- apeutic approaches for the control of uveitis in humans. Proposed Course This project will continue so that more infor- mation about the basic mechanisms in experi- mental uveitis may be obtained. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications Caspi RR, Chan C-C, Grubbs BG, Silver PB, Wiggert B, Heremans H: Interferon-gamma at the systemic level protects mice against experi- mental autoimmune uveoretinitis. Reg Im- munol, in press. Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover S, Silver PB, Hansen CT, WUder RL: Recruitment of naive T cells plays a pivotal role in experimental autoimmune uveoretinitis (EAU). Reg Immunol, in press. Caspi RR, Nussenblatt RB: Natural and thera- peutic control of ocular autoimmunity — rodent and man, in Goutinho A, Kazatchkine M (eds): Autoimmunity: Physiology and Disease. Wiley-Liss, Inc., New York, 1994, pp 377-405. Caspi RR, Parsa C, Chan C-C, Grubbs BG, Bahmanyar S, Heremans H, BUliau A, Wiggert B: Endogenous systemic interferon-gamma has a protective role against ocular autoimmu- nity in mice. / Immunol 152:890-899, 1994. Caspi RR, Silver PB, Chan C-C, Wiggert B, Redmond TM, Donoso LA: Immunogenetics of experimental autoimmune uveoretinitis (EAU). Reg Immunol, in press. Caspi RR: Experimental autoimmune uveoretinitis in rats and mice, in Cohen I, Miller A (eds.): Guidebook to Animal Models for Autoimmune Diseases. Academic Press, in press. Caspi RR: Thl and Th2 lymphocytes in exper- imental autoimmune uveoretinitis, in: Advanc- es in Ocular Immunology (Proceedings of the Sixth International Symposium on the Immu- nology and Immunopathology of the Eye, Bethesda, MD, 1994). Amsterdam, Elsevier Science Publishers B.V., International Congress Series, 1994, pp 55-58. 123 FY 1994 NEI Annual Report Kozhich AT, Kawano Y, Egwuagu CE, Caspi RR, Berzofsky JA, Gery I: A pathogenic autoimmune process targeted at a surrogate epitope. ; Exp Med 180:133-140, 1994. Mahdi RM, Caspi RR, Kozhich AT, Kozhich OA, Silver PB, Nussenblatt RB, Egwuagu CE: C5^okine mRNA expression following adop- tive transfer of uveitogenic T cells into athymic and euthymic Lewis rats. Invest Ophthalmol Vis Sci 35(4):1561, 1994. Malty R, Caspi RR, Nelson LM: Neonatal th5miectomy causes persistence into adulthood of the neonatal capacity for increased IL-4 production. FASEB } 8:A483, 1994. Miller-Rivero NE, Rizzo LV, Chan C-C, Wiggert B, Nussenblatt: RB, Caspi RR: Sup- pression of IRBP-induced EAU in mice by feeding IRBP, and its potentiation by interleukin-2. Invest Ophthalmol Vis Sci 35(4):1865, 1994. Prendergast RA, Coskuncan NM, Lutty GA, McLeod DS, Caspi RR: Induction of adoptive T ceU-mediated EAU: Temporal appearance of specific and control activated cells in retinal tissue. Invest Ophthalmol Vis Sci 35(4):1561, 1994. Prendergast RA, Coskuncan NM, MacLeod DS, Lutty GA, Caspi RR: T ceU tiraffic and the pathogenesis of experimental autoimmune uveoretinitis, in: Advances in Ocular Immunolo- gy (Proceedings of the Sixth International S5miposium on the Immunology and Immuno- pathology of the Eye, Bethesda, MD, 1994). Amsterdam, Elsevier Science Publishers B.V., International Congress Series, 1994, pp 59-62. Rizzo LV, Miller-Rivero NE, Chan C-C, Wiggert B, Nussenblatt RB, Caspi RR: Inter- leukin-2 treatment potentiates induction of oral tolerance. FASEB J 8:A476, 1994. Rizzo LV, MiUer-Rivero NE, Chan C-C, Wiggert B, Nussenblatt RB, Caspi RR: Effect of interleukin-2 on the induction of oral toler- ance in experimental autoimmune uveoreti- nitis, in: Advances in Ocular Immunology (Pro- ceedings of the Sixth International Sjonposium on the Immunology and Immunopathology of the Eye, Bethesda, MD, 1994). Amsterdam, Elsevier Science Publishers B.V., International Congress Series, 1994, pp 221-224. Rizzo LV, Miller-Rivero NE, Chan C-C, Wiggert B, Nussenblatt RB, Caspi RR: lnterleukin-2 treatment potentiates induction of oral tolerance in a murine model of autoim- munity. / Clin Invest 94:1668-1672, 1994. Rizzo LV, Silver PB, Gazzinelli RT, Chan C-C, Wiggert B, Caspi RR: Expression of cytokine genes wdthin the eye in murine EAU. Invest Ophthalmol Vis Sci 35(4):1862, 1994. Sartani G, Silver PB, Sh-assmann G, Chan C-C, Caspi RR: Effect of suramin treatment on induction of EAU. Invest Ophthalmol Vis Sci 35(4):1862, 1994. Savion S, Oddo S, Grover S, Caspi RR: Uveitogenic T lymphocytes in the rat: Patho- gerucity vs. lymphokine production, adhesion molecules and surface antigen expression. / Neuroimmunol, in press. Silver PB, Rizzo LV, Chan C-C, Donoso LA, Wiggert B, Caspi RR: Identification of a major pathogenic epitope in the IRBP molecule recognized by mice of the H-2r haplotype. Invest Ophthalmol Vis Sci 35(4):2061, 1994. 124 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00266-05 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on arte line between the borders.) Characterization of Immune Responses to Retinal Specific Antigens PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI Others: Igal Gery Robert B. Nussenblatt Frangois Roberge Ph.D. Head, Section on LI, NEI Experimental Immunology M.D. Scientific Director NEI M.D. Visiting Scientist LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.6 PROFESSIONAL: 0.6 0.0 CHECK APPROPRIATE BOX{ES) fxl (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) One of the characteristics of S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP), which are retinal-specific antigens, is the ability to induce an intense autoimmune inflammation in the eyes of experimental animals when injected in the presence of an adjuvant. This disease, called experimental autoimmune uveitis (EAU), is critically dependent on T cells and antigen processing by appropriate antigen- presenting cells (APCs). Antigen processing, which occurs within the endocytic vesicles of the APCs, results in the production of small polypeptide subunits. These small polypeptides must then be protected from further degradation and transported to the cell surface where the interaction with the T cell takes place. In FT 1994 we further characterized the intracellular protein first identified in FY 1993. We confirmed that the protein does belong to the heat shock family of proteins by partial amino acid sequencing of the 70kD peak. We also identified the peak at 40kD as being actin. Testing with different peptide moieties has shown that binding of immunogenic peptides by hsp70 is a selective process. Certain peptides bind well to hsp70, although other peptides have no affinity for hspVO. The process is also selective in that it requires ATP in order to release the bound peptide. We have also found increased levels of hsp antibody in the serum of patients with Behget's disease. The increase serum levels corresponded to periods of ocular inflammatory activity in the absence of any evidence of active systemic disease. 125 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Additional Personnel Sumeet Mainigi Biologist, LI, NEI Sakpana PhD. Biolgist, LRCMB, NEI Rengerajan Gerald J. Chader Ph.D. Chief, LRCMB, NEI Barbara Wiggert PhD. Head, Section on Biochemistry, LRCMB, NEI Clinical Protocol Numbers 84-EI-214 79-EI-49 Objectives In fiscal year (FY) 1994, we further character- ized the intracellular protein first identified in FY 1993. We confirmed that the protein does belong to the heat shock family of proteins by partial amino add sequencing of the 70kD peak. We also identified the peak at 40kD as being actin. Testing with different peptide moieties has shown that binding of immuno- genic peptides by heat shock proteins (HSP) 70 is a selective process. Certain peptides bind weU to HSP 70, but other peptides have no affinity for HSP 70. The process is also selective in that it requires adenosine triphos- phate to release the bound peptide. We have also found increased levels of HSP antibody in the serum of patients with Behcet's disease. The increased serum levels corresponded to periods of ocular inflammatory activity in the absence of any evidence of active systemic disease. Methods Using B-cell lysates from Epstein-Barr virus (EBV)-fransformed cells and from B-ceU iso- lates of rat spleens, the HSP 70 capable of binding to interphotoreceptor retinoid-binding progression (IRBP) fragment 1169-1191 was isolated and further characterized by amino acid sequencing. Isolation was carried out on an activated Sepharose 4b column to which an appropriate peptide was Hnked. Several experiments were carried out to determine the affinity of various peptide substitutes to infraceUular HSP 70. Human serum was tested in a standard enzyme-Unked immuno- sorbent assay using a commercial HSP 70 antigen. Major Findings We have determined that the 70 kD binding protein belongs to the heat shock family of proteins and that it is a new member of the HSP 70 family of proteins because part of its sequence has only 40 percent homology with other HSP members. The 40 kD protein is actin. The exact role of actin in antigen pre- sentation is still unknown. One simple expla- nation is that actin binds through a simple bystander phenomenon because the protein is isolated from a ceU lysate. However, mono- meric actin may weU play a more active role in antigen presentation, particularly of cyto- soUc proteins. Further studies will be neces- sary to further elucidate its role. Using cell lysates of EVB-fransformed cells, we were able to show that the binding of HSP 70 to various peptide fragments is depen- dent on the peptide sequence. Substitution of certain nonpolar amino acids by charged molecules changes the binding characteristics. Differences were also noted between patients with Behcet's disease and normal individuals in terms of binding to various residues or analogs of IRBP. Of particular interest was the finding that serum levels of HSP 70 anti- bodies varied with the level of ocular inflam- mation. Patients had levels of HSP 708 anti- bodies that were above two standard devia- tions of confrols, the level of HSP 70 antibod- ies rose significantiy in patients with ocular inflammatory episodes. This is a unique finding in ocular inflammatory disease, where it is rare to find a systemic marker of inflam- matory activity in the eye. Proposed Course In the coming year, the main emphasis will be on further elucidating the role of hsps in anfigen presentation by studying the effect of 226 Laboratoiy of Immunology cellular stress on antigen presentation in cell lines and clones. In addition, we will attempt to produce a specific polyclonal and mono- clonal antibody to the HSP 708 that was isolated in the course of these studies. Once the antibody has been generated, we will look at several more patient populations to fiarther define the role of hsp in uveitis. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications de Smet MD, Bitar G, Roberge FG, Gery I, Nussenblatt RB: Human S-antigen: Presence of multiple immunogenic and immuno- pathogenic sites in the Lewis rat. J Autoimmun 6:587-599, 1993. de Smet MD, Rengerajan K, Chader GJ, Wiggert B: Characterization of B ceU proteins binding specifically to uveitopathogenic pep- tide 1169-1191 of bovine IRBP. Invest Ophthal- mol Vis Sci 35(suppl):1865, 1994. Mainigi S, Rengarajan K, Wiggert B, Chader GJ, Nussenblati RB, de Smet MD: Elevation of serum antibody levels to heat shock protein 70 during ocular inflammatory episodes in Behcet's patients. Invest Ophthalmol Vis Sci 35(suppl):2098, 1994. Rengarajan K, de Smet MD, Chader GJ, Wiggert B: Affinity of HSP70 fiom EBV transformed human B cells for bovine and human IRBP peptides. Invest Ophthalmol Vis Sci 35(suppl):1864, 1994. Rengarajan K, de Smet MD, Chader GJ, Wiggert B: Identification of heat shock pro- tein binding to an immunodominant uveitopathogenic peptide of IRBP. Curr Eye Res 13:298-296, 1994. 127 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00276-03 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Surgical Management of Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI Others: Frangois Roberge M.D. Visiting Scientist LI, NEI Margaret Cheung M.D. Senior Staff Fellow LI, NEI David Parks M.D. Senior Staff Fellow LI, NEI COOPERATING UNITS (if any) Clinical Oncology Program, Medicine Branch, National Cancer Institute (Robert Wittes, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: PROFESSIONAL: CHECK APPROPRIATE BOX(ES) fx| (a) Human subjects r~l (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) THIS PROJECT IS INACTIVE. 128 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00218-09 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Ocular Manifestations of the Acquired Immune Deficiency Syndrome PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Marc D. de Smet M.D. Visiting Scientist LI, ^fEI Others: Robert B. Nussenblatt Scott Whitcup Margaret Cheung David Parks Frangois Roberge Chi-Chao Chan M.D. M.D. M.D. M.D. M.D. M.D. Scientific Director Assistant Clinical Director Senior Staff Fellow Senior Staff Fellow Visiting Scientist Head, Section on Immunopathology NEI CB, NEI LI, NEI LI, NEI LI, NEI LI, NEI COOPERATING UNITS (if any) Department of Critical Care Medicine, Clinical Center (Henry Masur, M.D.); Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases (H. Clifford Lane, M.D.); Pediatric Branch, National Cancer Institute (Phil A. Pizzo, M.D.) ^^^^^^^^^ LAB/BRANCH Laboratory of Immunology Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 2.0 PROFESSIONAL 2.0 0.0 CHECK APPROPRIATE BOX(ES) fxl (a) Human subjects [x] (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Patients suffering from AIDS (acquired immunodeficiency syndrome) are at risk of developing significant ocular problems, either as a result of HIV (human immune deficiency virus) itself or as a result of opportunistic infection. Some of these problems can lead to blindness if left untreated. Among the many pathogens that can lead to blindness, cytomegalovirus (CMV) is by far the most common. In FY 1994, we have evaluated the effectiveness of an intraocular delivery device in preventing the spread of CMV retinitis. We have now nearly completed recruitment for this study. Although no analysis has yet been done of the data, no significant adverse reaction has been noted, and in all cases progression of CMV retinitis has been prevented. 129 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Nurse Spedalist CB, NEI Project Description Additional Personnel Susan Mellow RN Clinical Protocol Number 90-EI-208 Objectives Patients suffering from AIDS (acquired immu- nodeficiency s5Tidrome) are at risk of develop- ing significant ocular problems, either as a result of human immunodeficiency virus itself or as a result of opportunitistic infection. Some of these problems can lead to blindness if left untreated. Among the many pathogens that can lead to blindness, cytomegalovirus (CMV) is by far the most common. In fiscal year (FY) 1994, we have evaluated the effectiveness of an intraocular delivery device in preventing the spread of CMV retinitis. We have nearly completed recruit- ment for this study. Although no data analy- sis has yet been done, no significant adverse reaction has been noted, and in all cases progression of CMV retinitis has been pre- vented. Methods This project entails the clinical evaluation, diagnosis, and treatment of retinitis in AIDS patients. It also involves the development of novel methods of therapy for the various forms of retirutis observed. Study of patho- logic tissue also is used to better understand the nature of the infectious processes. Major Findings In the past year, our major effort has centered on the treatment of CMV retinitis by using a slow release intraocular device. CMV retinitis is a major vision-threatening infection found in patients with advanced stages of AIDS. Current therapeutic modalities commit pa- tients to life-long intravenous (IV) therapy with anti-CMV drugs. These drugs require close monitoring because of their systemic toxicity. They also require the placement of a permanent IV access and hence place the patient at risk for both local and systemic infection. Recurrence of disease also tends to occur in the majority of patients. An intraocu- lar, slow-release device avoids these side effects by providing continuous antiviral therapy above the minimum inhibitory con- centration for up to eight months. Recruit- ment of patients for the study that was started in FY 1993 is now completed. This study compares patients in whom an intraocular device is placed immediately with patients in whom placement is differed until the CMV retinitis has progressed by 750 /im. Although recniitment has been completed, some patients are still in the active phase of the study. No serious side effects have been encountered, and good control of the CMV retinitis has been achieved. Full analysis of the study parameters wiU be undertaken shortly. Partic- ular attention wiU be given to the rate of bilateraHzation and to the survival of patients treated with the intraocular device. Despite treatment, CMV retinitis has a tendency to recur. With larger areas of retinal involvement, the risk of CMV detachment increases considerably, particularly in patients with peripheral involvement where vitro- retinal traction is greatest. Up to 30 percent of patients with CMV retinitis wiU develop a retinal detachment. Repair of detachments reqviires the use of a variety of vitro-retinal techniques from classical retinopexy to the use of more advanced vitreal-retinal approaches, including the use of silicone oU. The best time for intervention still remains to be determined. In FY 1994, we have begun to explore the various surgical approaches that are available for the repair of retinal detachments in pa- tients with CMV retinitis. For patients with detachments involving noninfected retina, standard scleral buckling procedures appear to be adequate. If a retinal detachment develops in involved retina, scleral buckling with sili- cone oil appears to be the most effective 130 Laboratory of Immunology approach. Many patients, however, do not recover their fuU central visual acuity. The cause of this decrease is not known and is being investigated. Significance to Biomedicai Research and the Program of the Institute The AIDS epidemic is a major pubUc health concern. CMV retinitis remains the number one cause of blindness among patients infect- ed with the AIDS virus. Early diagnosis is important because all drugs currently avail- able are only virostatic and not viroddal; thus, some progression of the lesion is seen in more than 50 percent of patients, despite anti-CMV therapy. No therapeutic modaUties that are cost effective and that reduce the incidence of progression are necessary. Management of the compUcations of CMV retinitis, particularly after recurrence and pro- gression of the disease will be an ever-increas- ing challenge as patients survive longer. Of these, the threat of retinal detachment is a major concern. The means of prophylaxis and therapy need to be developed. Proposed Course In the coming fiscal year, we are planning to evaluate the slow-release device further as well as the means of preventing second eye involvement in patients who are treated with the device. We also will study further means of treating patients with viral-related retinal detachments and means of preventing visual loss at the time of surgery. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications de Smet MD, Nussenblatt RB: Ocular mani- festations of HIV in the pediatric population, in Pizzo PA, Wilert CM (eds): Pediatric AIDS: The Challenge of HIV Infection in Infants, Chil- dren, and Adolescents. Baltimore, Williams & Wilkins, 1994 pp. 457-466. Maturi RK, Nussenblatt RB, de Smet MD: Prevalence of tear hyposecretion and vitamin A deficiency in patients with AIDS. Invest Ophthalmol Vis Sci 35(4)(suppl):1308, 1994. 131 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00232-09 LI PERIOD COVERED October 1. 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on arte Ime between the borders.) Interferon System in Cellular Function and Disease PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Caroline Percopo M.S. Biologist Chandrasekharam Nagineni Ph.D. Visiting Scientist LI, NEI LI, NEI COOPERATING UNITS (if any) Department of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.) LAB/BRANCH Laboratory of Immunology Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.5 PROFESSIONAL: 0.2 0.3 CHECK APPROPRIATE BOX(ES) l~l (a) Human subjects r~l (al) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Cytokines, such as interferon gamma (IFN-y) and interleukin (IL)-2, are a group of specialized hormone-like proteins that exert profound influences on cellular development and on a variety of cellular functions. This project has concentrated on studying the ways in which cytokines interact with cells of the immune system and with cells in the ocular microenvironment. We have shown that IFN-y and IL-2 are found within the inflamed eye in association with T-cell infiltration and major histocompatibility complex (MHC) class II antigen expression on infiltrating cells and on retinal pigment epithelial (RPE) cells. Furthermore, IFN-y- activated RPE cells can process and present antigens to helper T lymphocytes. Experimentally we demonstrated that isolated human RPE cells can be induced to produce another lymphokine, IL-6, and soluble intercellular adhesion molecule- 1 (ICAM-1) following incubation with IFN-y. IL-6 is a potent inflammatory cytokine capable of enhancing antibody production and cytotoxic T-cell activities. ICAM-1 is an adhesion molecule that mediates cell adhesion, an essential component for several immunologic functions. These studies indicate that cytokine-mediated activation of RPE cells may be a basic component of ocular immunity and an important aspect of RPE cell transplantation. These observations indicate that IFN-y induces MHC class I, class II antigen and ICAM-1 expression and IL-6 secretion by RPE cells. All of these factors may serve as a local amplification system in autoimmune and inflammatory eye disease. A better understanding of the role of cytokines in the mechanisms involved in the development of autoimmunity and inflammation may be beneficial in developing treatments for these diseases. 132 PHS 6040 (Rev. 5/92) Laboratory of Immunology Project Description Objectives This project is designed to determine the bioregulatory actions of interferon (IFN) and other cytokines and to evaluate their regulato- ry actions in the pathogenesis of disease. Methods We assayed human IFN using inhibition of vesicular stomatitis virus plaque formation in human amnion or WISH cells. IFNs were characterized by neutralization of antiviral activity with monoclonal anti-IFN immuno- globulin. Interleukin (IL)-2 biological activity was assayed by induction of proliferation of CTL cells. Soluble intercellular adhesion molecule 1 (ICAM-1) and IL-6 activity was assayed by an enzyme-Unked immunosorbent assay (ELISA) and immunoblot assays. ICAM-1 and IL-6 messenger ribonucleic acid (mRNA) were evaluated by Northern blot assays. Analytical flow cytometry was used to quantitate retinal proteins. Gene transcription techiuques such as Northern blot analysis and nuclear runoff transcription assays are being used to evaluate interferon gamma (IFN-y) modulation of retinal proteins. Major Findings Numerous studies indicate that a variety of autoimmune diseases are associated with the IFN-y-induced tissue-specific expression of major histocompatibility complex (MHC) class n molecules. During the past five years, we have identified various steps that may be involved in ocular immunopathologic mecha- nisms. In these studies of retinal degenera- tions and autoimmune diseases, we showed that a critical regulatory ceU in the retina, the retinal pigment epitheUal (RPE) cell, is capable of expressing MHC class n determinants. We can also detect IFN-7, in situ, in retinas from patients with inflammatory eye diseases as well as MHC class n positive RPE ceUs. In addition, freshly isolated human RPE ceUs can express these determinants following treat- ment with IFN-y. In animal model systems, we found that inoculation of recombinant IFN-y induces la expression of ocular cells, and treatment with anti-la antibodies can eliminate or inhibit experimental autoimmune uveitis. Recently, we showed that the RPE cell may be playing an important role in ocular immunity, acting as a resident antigen presenting cell in the retina. Also, we found that the RPE cell is capa- ble of producing the cytokine, rL-6. During the past year, we have evaluated lCAM-1 production by cytokine-activated RPE cells. RPE cell cultures were estabUshed from hu- man donor eyes. Stimulation of RPE cells with IL-la, IL-P, tumor necrosis factor alpha (TNF-a), and IFN-7 resulted in the production and release of ICAM-1. In addition, there was a concomitant increase in the RPE ceU surface expression of ICAM-1 and the production of ICAM-1 mRNA. In contrast, IL-6 and hpo- polysaccharide were not capable of inducing ICAM-1 secretion by RPE ceUs. Taken togeth- er, these studies indicate that the proinflam- matory cytokine such as IL-1, TNF, and IFN-y can activate RPE cells to release both IL-6 and soluble ICAM-1. These studies further sub- stantiate the concept that cjiiokine-mediated activation of RPE ceUs may be a basic compo- nent of ocular immunity and may have major immunological consequences for RPE ceU transplantation studies. Significance to Biomedical Research and the Program of the Institute The studies described herein highlighted the fact that the release of cytokines such as IFN-y, within the ocular microenvironment and the subsequent induction of cytokines and MHC class I and 11 antigen expression on resident and infiltrating cells may be critical elements in a cascading effect that leads to ocular cell destruction. The cell within the retina that may play a critical role in autoim- mune uveitis is the RPE cell. IFN-y induced activation of RPE cells may participate in autoimmune disease in the ocular microenvir- onment. 133 FY 1994 NEI Annual Report Cytokines produced and localized in the eye may play a critical role in normal physiol- ogy, pathogenic mechanisms, and therapeutic approaches. Because the RPE cell is a pivotal regulatory cell in the retina, an understanding of how cytokines interact with this cell will shed light on avenues for therapeutic interven- tion in pathogenic states and transplantation. Proposed Course We plan to continue our evaluation of the role of cytokines in autoimmunity and inflamma- tion. We are now developing systems in rat models to monitor directly the effects of altering c)^okine production on inflammatory eye diseases. Moreover, we will continue to characterize the antigen-presenting ability of the RPE cell to a variety of antigens and viruses. NEI Research Program Retinal Diseases — ^Inflammatory Diseases Publications Detrick B, Hooks JJ: Cytokines and effector molecules in human immunology, in Leffell MS, Bias WB, Donnenberg AD, (eds): CRC Handbook of Human Immunology. Boca Raton, Florida, CRC Press Inc., in press. Nagineni CN, Detrick B, Hooks JJ: Synergistic effects of gamma interferon on inflammatory mediators that induce interleukin-6 gene expression and secretion by human retinal pigment epithelial cells. Clin Diag Lab Im- munol 1:569-577, 1994. Nagineni CN, Detrick B, Hooks JJ: Human RPE cells secrete cytokines in response to inflammatory mediators, in Proceeding of the Sixth International Symposium on the Immunolo- gy and Immunopathology of the Eye. Amster- dam, Elsevier Science Publishers, 1994. DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00233-09 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title rrtust fit art one line between the borders.) Studies on the Bioregulato r y Aspects of the R etinal Pigment Epithelial Cell PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Chandrasekharam Nagineni Ph.D. Visiting Scientist LI, NEI Caroline Percopo M.S. Biologist LI, NEI T. Michael Redmond Ph.D. Research Biologist LRCMB, NEI R. Krishnan Kutty Ph.D. Senior Staff Fellow LRCMB, NEI David Parks M.D. Senior Staff Fellow LI, NEI Marc D. de Smet M.D. Visiting Scientist LI, NEI Robert B. Nussenblatt M.D. Scientific Director LI, NEI COOPERATING UNITS (if any) Hopital St. Louis, Paris, France (Lawrence Boumsell, M.D.); University of Nice, France (Alain Bernard, M.D.); National Institute of Dental Research (Reuben Siraganian, M.D.); The Johns Hopkins University (Stanley A. Vinores, Ph.D.; Peter Campochiaro, M.D.); Department of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Betbesda, MP 20892 TOTAL STAFF YEARS: PROFESSIONAL: 0.9 0.5 0.4 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects x (b) Human tissues (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The retinal pigment epithelial (RPE) cell plays a basic role in maintaining the structural and physiological integrity of the neural retina. We have isolated and propagated RPE cells in vitro and have developed monoclonal antibodies directed against human RPE cells. The RPE epitope is a 67 kDa protein that is closely associated with the microsomal membrane. A complementary deoxyribonucleic acid (cDNA) clone has been isolated that codes for a protein that does not match any other sequences in the databases. We are using these techniques and reagents to evaluate molecular, biochemical, and biological properties of the RPE cells. We have propagated human RPE cells in vitro and evaluated their ability to respond to cytokine activation. Transforming grov^^th factor - p (TGF-P) is a potent cytokine that modifies a variety of cellular functions. This cytokine has been identified within the retina. We found that TGF-p induces gene expression and production of Heme oxygenase (HO-1). Since HO-1 is a protective agent against oxidative damage in an O, rich environment, RPE production of this protein may protect the retina against oxidative damage. Studies are also in progress to propagate and transplant RPE cells in various animals. We have established a graft rejection model by transplanting human RPE cells into the rat retina. These studies demonstrate that both cellular and humoral aspects of the immune response are activated to reject RPE cell transplants. These studies provide the framework to evaluate cytokines and immune reactivity in RPE cell transplantation. 135 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The aim of this project is to evaluate the molecular, biochemical, and varied biologic properties of retinal pigment epitheUal (RPE) cells in normal and disease states. Moreover, we are evaluating RPE cell transplantation. Methods RPE cells are isolated and propagated in vitro. Monoclonal antibodies were generated in mice by fusing mouse spleen cells with myeloma cells. Antibodies to RPE cells are evaluated by immunoperoxidase assays and Western blot assays. The effect of drugs and cytokines are evaluated by cell viability and proliferation assays and nuclear transcription runoff assays. Major Findings (1) Cytokine Activation of RPE Cells. Trans- forming growth factor beta (TGF-P) is a potent cytokine that modifies a variety of cellular functions. This cjdiokine has been identified within the retina. We found that TGF-p induces messenger ribonucleic acid (mRNA) for heme oxygenase-1 (HO-1) and induces HO-1 protein expression in human RPE cells. Because HO-1 is a protective agent against oxidative damage in an oxygen rich environ- ment, RPE production of this protein may provide dowrvregulation of oxidative damage. (2) RPE cell transplantation. Recent studies indicate that RPE cell transplantation may be beneficial in restoration of retinal architecture in selected retinal degenerations. It is essen- tial to develop methods for large-scale prepa- rations of RPE ceU ciiltures for somatic cell genetic engineering manipulations. We are in the process of evaluating various parameters for human and rat RPE cell culture and trans- plantation. Preliminary studies show that w^e can successfully transplant human RPE cells into the rat retina. We have used this xenoge- neic RPE transplant in the rat as a graft rejec- tion model. We demonstrated that the trans- fer of cultured adult human RPE cells to the Lewis rat subretinal space eUcits a graft rejec- tion peaking at seven days posttransplanta- tion. Infiltrating cells consisted of phagocytic CD-llc/CD-18 cells as well as CD-4 and CD-8 positive cells. Systemic antibodies to the human RPE cells developed in the rats by seven to 21 days. These data indicate that both cellular and humoral aspects of the immune response are activated in this graft rejection model. Significance to Biomedical Research and the Program of the Institute The monoclonal antibodies developed in this study are the first directed solely at the RPE cell. These antibodies are potentially useful in identification of RPE cells in situ and in vitro. These antibodies, which can be used to moni- tor RPE cellular functions, may be used in providing a better understanding of the role of RPE cells in retinal degenerative disorders. Identification of the complementary deoxyri- bonucleic acid and proteins detected by the monoclonal antibodies may provide the frame- work to evaluate specific RPE cell functions. RPE cell transplantation to correct retinal degenerative processes is being actively inves- tigated in a number of laboratories. The studies reported here provide the framework to evaluate RPE ceU transplantation. Proposed Course (1) We will continue to characterize these antibodies as well as the effect of these anti- bodies on ceU function in vivo and in vitro. (2) We will isolate, propagate, and charac- terize RPE cells for transplantation studies in arumals and man. We will design effective ways to maintain the cell in culture and de- sign ways to measure and monitor ceU func- tion. NEI Research Program Retinal Diseases — Photoreceptors and Pigment Epithelium Laboratoiy of Immunology Publications Kutty RK, Kutty G, Nagineni CN, Hooks J], Chader GJ, Wiggert B: RT-PCR assay for Kutty RK, Nagineni CN, Kutty G, Hooks J], heme oxygenase-1 and henne oxygenase-2: A Chader GJ, Wiggert B: Increased expression sensitive method to estimate cellular oxidative of heme oxygenase-1 in human retinal pig- damage. Ann NY Acad of Sci, in press, ment epithelial cells by transforming growth factor-beta. / Cell Physiol 159:371-378, 1994. 137 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00240-08 LI PERIOD COVERED October 1, 1993 to September 30. 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Virus Infections in the Eye PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Caroline Percopo Yun Wang Miguel Bumier Ingeborg Kirch Yusuke Komuraski M.S. M.D. M.D. M.D. M.D. Biologist Guest Worker Visiting Scientist Guest Worker Guest Worker LI, NEI LI, NEI LI, NEI LI, NEI LI, NEI COOPERATING UNITS (if any) Department of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.); Department of Pathology, Uniformed Services University of the Health Sciences (Katherine Holmes, Ph.D.); Department of Ophthalmology, Ruprecht-Karl's University, Heidelberg, Germany (Ellen Kraus-Mackiw, M.D.); Laboratory of Biology, NCI, NIH (Charles H. Evans, M.D., Ph.D.); Department of Medicine, The Johns Hopkins Medical School, Baltimore, MD (William Burns, M.D.); Laboratory of Clinical Investigations, NIAID, NIH, (Jeffrey 1. Cohen, M.D., and Steven Strauss. M.D.) LAB/BRANCH Laboratory of Immunology Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 0.8 0.2 CHECK APPROPRIATE BOX(ES) i~| (a) Human subjects □ (a1) Minors r~| (a2) Interviews [x| (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Our studies of various virologic and immunopathologic processes that occur when viruses replicate in the ocular microenvironment comprise four areas: (1) coronavirus infection in ocular and optic nerve cells; (2) the possible roles of viruses in human diseases; (3) molecular diagnosis and pathogenesis of cytomegalovims (CMV) infections in humans, and (4) Varicella - zoster vims infections of the retina. We have established that murine coronavims can induce ocular disease and may be used as a model system for studying retinal degenerative diseases. This model has many unique features. The virus is capable of inducing an acute infection in the presence of mild retinal vascular inflammation. Initial retinal damage is followed by clearance of infectious vims and progressive retinal degeneration. In situ hybridization techniques identified that the viral RNA persists within the Muller cells and RPE cells throughout the course of the disease. Recent studies show that there are genetic and immunologic components to this disease. The retinal degenerative pathologic manifestations of the disease can be influenced by the genetics of the host. That is, some strains of mice are resistant to vims-induced retinal degenerative changes. The pathologic changes are also closely related to the development of anti-retinal and anti-RPE antibodies. These findings suggest a role for autoimmunity in the pathogenesis. This disease may be considered a model for degenerative diseases of the pigment epithelium and photoreceptors in humans. Human CMV is a herpesvims that is a major cause of blindness in children bom with congenital infections and in immunocompromised individuals. It is difficult to study CMV latency in man. Therefore cell culture models of CMV replication and latency may provide insight into a rationale for alternative treatment modalities. We identified that CMV replicates in human RPE cells. Virus replication is extremely slow and is associated with a low expression of IE viral proteins. These may be critical variables in viral persistence and viral activation within the retina. 138 PHS 6040 (Rev. 5/92) Laboratory of Immunology Project Description Objectives This project was designed to determine the various effects of virus infections on the ocular microenvironment and to study modes of antiviral therapy. Methods This study involves the propagation and quantitation of viruses such as herpes simplex virus type 1, coronaviruses, and cytomegalovi- rus (CMV) in vitro and in vivo as weU as immunocytochemical analysis of infected cells and tissues. Techniques used in the character- ization of virus infection include flow cyto- metric analysis. Western blot analysis. North- em blot analysis, in situ hybridization, and amplification of viral genes by polymerase chain reaction (PCR). Techniques used in characterization of antivirus antibodies include enzyme-Unked immunosorbent assay and neutralization assays. Major Findings (1) Coronavirus Infection in the Eye. The mu- rine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces a retinal degener- ative disease in adult Balb/c mice. In the early, acute phase one to seven days after inoculation, a niild retinal vasculitis is ob- served. The second stage is seen by day 10 and progresses for several months. This stage is characterized by a retinal degeneration in the absence of vasciiUtis or inflammation. This degenerative process is associated with a reduction of the photoreceptor layer, loss of interphotoreceptor-binding protein, abnormali- ties in the retinal pigment epitheUum (RPE) and retinal detachments. The development of the degenerative phase of the disease is con- trolled by a genetic predisposition of the host and is associated with the development of antiretinal and anti-RPE cell autoantibodies. During the past year, we have evaluated three aspects of this disease process: (a) virus persistence within the retina, (b) immunologic aspects of the disease, and (c) genetic predis- position to the disease. One of the intriguing aspects of this dis- ease process is the nature of viral clearance. The acute phase of the disease is associated with the presence of viral proteins and the detection of infectious virus within the retina. However, after day eight, infectious virus and viral proteins are not detected. Nevertheless, the retinal tissue damage characterized as retinal degeneration becomes apparent at day 10 and continues for months. The purpose of this study was to determine if the virus per- sists within the retina and other tissues during the course of this disease process. In situ hybridization was selected as a way to deter- mine if the virus persists and to identify the cellular location of this persistent infection. The presence of viral ribonucleic add (RNA) was detected by in situ hybridization with a viral complementary deoxyribonucleic add (cDNA) probe, and viral proteins were identified by immunocytochemical staining. cDN A probe representing MHV-A59 genes 4-6 was prepared by digesting the plasmid DNA (clone 2-2) with Pst 1. cDNA was labeled v^dth digoxigenin-11-dUTP and hybridized with tissue sections. The resultant hybridized probe was identified with antidigoxigenin antibody conjugated to alkaline phosphatase. When uninfected (normal) mouse eye sections were incubated with the viral cDNA probe, no reactivity was observed. In contrast, when JHM virus-infected mouse eye sections were incubated with the viral cDNA probe; positive reactivity was noted within the retina from day one to day 60 postinoculation. During the acute phase of the infection, viral RNA was found in the retina, RPE, ciliary body epitheUum, and the iris epitheUum. During the late phase of the infection, viral RNA was almost exclusively found within the retina and RPE and not in the anterior segment of the eye. Within the retina, viral RNA was detected in the ganghon ceU layer, the inner retina, the outer retina, and the RPE ceU. Pretreatment of infected eyes with RNase inhibited the reactivity and FY 1994 NEI Annual Report incubation of infected eyes with plasmid cDNA resulted in no reactivity. Immuno- cytochemical staining identified viral protein within the retina only from day one to day eight. This ocular disease is also associated with a persistent systemic infection. Both viral RNA and viral proteins were identified within the liver during the first eight days. However, only viral RNA is detected in the liver from day eight to 60. These studies show that MHV establishes an acute infection (days one to eight) where infectious virus and viral proteins are identified. This is followed by a persistent infection within the retina and Uver where only viral RNA can be detected by in situ hybridization. The coronavirus-induced retinopathy model allows us to explore some of the cellu- lar and molecular mechanisms involved in the autoimmune aspects of retinal tissue damage. During the past year, we have characterized the autoantibodies associated with the retinal degenerative process. BALB/c mice were inoculated by the intra vitreal route with 10^^ TCID50 / 5 ^tl of virus or media. At varying times after inoculation, sera and eyes were removed. The presence of antiretinal and anti-RPE cell antibodies were identified by immunocytochemical staining on normal rat eye sections and by immunoblot analysis. Sera collected from BALB/c mice from 12 to 70 days after JHM virus infection contained antiretinal autoantibodies. These antibodies are not found in the sera from normal or mock-injected mice. Antibod- ies to retinal tissue were identified as two distinct patterns of autoantibodies — retinal and RPE autoantibodies. Absorption studies were performed to characterize the autoanti- bodies. Absorption of sera with 10 percent retina, brain, or kidney tissue did not alter antiretinal or anti-RPE cell autoantibody reactivity. However, absorption of sera with lyophilized soluble fractions of rat or cow retina did inhibit both antiretinal and anti-RPE autoantibodies. Incubation of sera with lyoph- ilized soluble fractions of rat kidney or Uver did not inhibit autoantibody reactivity. The studies identify that the autoantibodies in- duced by the virus infection react with soluble proteins identified in the rat and cow retinas. This animal model of postvirus retinopathy is associated with the production of retinal- specific autoantibodies and may provide insight into the study of humoral autoimmune responses in human retinal degenerations. Because the genetic composition of the host and the virus can determine the response to infection and the resulting pathology, the third phase of our studies evaluated the effect of MHV infections on different strains of mice. We reported in the past year that the patho- logic manifestations of a virus infection in the retina can be influenced by the genetics of the host. BALB/c mice developed both the retinal vasculitis and the retinal degenerative phases of the disease, whereas CD-I mice developed only the early retinal vasculitis and were spared the degenerative disease. During the past year, we have found that the A59 virus strain as well as the JHM virus stiain both induce a biphasic retinal disease. In contrast, the inoculation of MHV-3 strain by the intravitreal route did not resvdt in a retinal degenerative disease. Within three to five days, aU of the animals died. Evalua- tion of the brain did not reveal pathologic damage. However, the liver contained patho- logic changes consistent with fvdminant acute hepatitis. These remarkably different diseases are induced by different strains of the same virus. The ability of different MHV strains to cause fatal acute hepatitis or persistent retinal disease may help to decipher the mechanisms of viral tissue tropism in this strain-specific pathogenesis. These studies demonstrate that the genetics of the virus can profoundly affect the pathology generated by a virus using the eye as a portal of entry. In summary, this model is characterized by the replication of JHM virus in the retina; producing an acute necrotizing disease of the sensory retina, resulting in only a mild inflam- matory response and a long-lasting disease (longer than 14 weeks). These studies identify that a progressive degenerative disease in the retina may be initiated by an acute virus Laboratory of Immunology infection in the absence of major inflammatory response. These studies during the past year clearly indicate that this retinal degenerative process has a persistent viral component, an immune component, and a genetic component. How these genetic and immunologic factors interact to influence the development of reti- nal degenerations are the intriguing aspects of this model. (2) Possible Role of Viruses in Human Eye Diseases. We have initiated studies to evaluate the possible involvement of viruses in the pathogenic processes of a variety of human eye diseases. We are now collecting serum samples and ocular tissue to use seroepi- demiologic approaches to detect virus and viral antigens via immunocytochemical stain- ing, in situ hybridization, and PCR assays. (3) Cytomegalovirus replication within the retina. CMV infections are frequent complica- tions in kidney and bone marrow transplant patients and human immunodeficiency virus patients. The mechanisms by which CMV is activated and repUcates within the retina is not known. We evaluated the ability of hu- man CMV to initiate replication in human RPE cells and compared this with studies in human fibroblasts (HEL) and human amnion epitheHal (WISH) cells. Human RPE ceUs were obtained from donor eyes and propagat- ed in vitro. CeUs were infected with CMV (AD169 strain) at an input multiplicity of 1. CMV replication was evaluated by immuno- fluorescence, flow cytometry. Western blot analysis, and infectivity assays. Cellular protein expression was evaluated by immuno- fluorescence and flow cytometry with mono- clonal antibodies. CMV induces a cytopathic effect, infectious virus in RPE cells, and hu- man fibroblast cells but fails to replicate in another epitheUal ceU— WISH ceUs. CMV replication in RPE cells is characterized by a prolonged period of low-virus protein expres- sion. Less than one percent of the cells con- tain viral proteins (IE, E, L) during the first 10 days. These viral proteins are not detected by flow cytometry or Western blot analysis until 15 days after inoculation. In contrast, CMV proteins are found in HEL ceUs within 24 hours. Untieated human RPE cells in vitro, express MHC class I molecules, P-2 microglob- ulin, and intracellular adhesion molecule 1 (CD-54). The CMV infection of RPE cells results in a downregulation in the expression of MHC class I molecules on the RPE cells. Cytotoxic T cells recognize the virus-infected cell only in association with MHC class I molecules on the cell surface. This study demonstrates that CMV can repUcate slowly within the RPE cell and that this repUcation can be monitored by flow cytometry. CMV replication in RPE cells is associated with the modulation of cellular protein expression, and this alteration may contribute to viral persis- tence within the retina. (4) Varicella-zoster virus (VZV) infections of the retina. VZV is a herpes virus that is fre- quenfly associated with acute retinal necrosis and other forms of retinal tissue damage. Nevertheless, there are no good in vivo or in vitro models to evaluate virus replication and latency. During the past year, we have initiat- ed studies to develop models of VZV infection of the retina and retinal ceUs. PreUminary studies indicate that human VZV can repUcate in the guinea pig retina, and this replication is associated with a chronic uveitis. In vitro studies indicate that VZV can replicate within human RPE cells. These preliminary findings suggest that we now have two approaches to evaluate VZV replication in retinal tissues. Significance to Biomedical Research and the Program of the Institute Elucidating the factors involved in viral spread and pathogenesis will yield a better understanding of diseases of viral etiology. We have established a new virus model for retinal degenerative processes in adult ani- mals. This model has many unique features. The virus is capable of inducing an acute infection in the presence of mild retinal vascu- lar inflammation. The initial retinal damage is followed by persistence of viral RNA and progressive retinal destruction, even months after infectious virus is gone. Moreover, the development of retinal degenerative process is determined by the genetics of the host and Ml FY 1994 NEI Annual Report involves the development of antiretinal auto- antibodies. This model should assist us in understanding the pathogenesis of selected human diseases of unknown etiology. We have developed new systems to evalu- ate two herpes virus infections of the retina, CMV, and VZV. We have shown that CMV repUcates within RPE cells in a slow, limited manner. The evaluation of the molecular aspects of this defect may provide critical clues in terms of the virus' ability to estabUsh persistent infections and the factors initiating viral activation within the retina. We have developed two new approaches to evaluate VZV replication within the retina. Proposed Course (1) We will continue to evaluate coronavirus infections of the eye. The role of genetic fac- tors and autoantibodies in the pathogenesis of retinal degenerations will be evaluated. The data obtained will be correlated with what is known about human retinal degenerative disorders. (2) We wiU initiate studies to determine whether certain viruses can replicate in retinal tissues and cells. Infected cells wall be evalu- ated for the release or expression of uveito- genic proteins. (3) We will continue to collect samples and initiate studies to detect the involvement of viruses in human eye diseases. (4) We will evaluate the molecular diag- nosis and pathogenesis of CMV and VZV infections in the eye. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications Bumier M, Wang Y, Detrick B, Hooks JJ: Retinal manifestations of a murine coronavirus infection: A histopathological and ultrastruc- tural study. Exp Pathol, in press. Hooks JJ: Ocular Virology, in Tabarra K (ed): Infections of the Eye. Boston, Little, Brown & Co. PubUshers, 1993. Hooks JJ, Wang Y, Komurasald Y, Percopo C, Nagineni CN, Detrick B: Molecular and immunologic mechanisms involved in corona- virus induced retinopathy, in Proceedings of the Sixth International Symposium on the Immunolo- gy and Immunopathology. Amsterdam, Excerpta Medica, Elsevier Science Publishers, 1994. Wang Y, Percopo CM, Bumier MN, Detrick B, Hooks JJ: Genetics of the virus determines retinal tissue damage induced by corona- viruses, in Proceedings of the Sixth International Symposium on the Immunology and Immunopa- thology. Amsterdam, Excerpta Medica, Elsevier Science Publishers, 1994. PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE ! NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00277-03 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between (he borders.) Role of Retinal Pigment Ep ith elium in Re tina l Dis orders PRINCIPAL INVESTIGATOR (List ottier professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Chandrasekharam N. Nagineni Ph.D. Visiting Scientist LI, NEI Others: John J. Hooks Ph.D. Head, Section on Immunology LI, NEI and Virology COOPERATING UNITS (if any) Department of Pathology, George Washington University, Washington, DC (Barbara Detrick, Ph.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 1.0 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects [x] (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The retinal pigment epithelium (RPE) plays a critical role in the regulation of retinal and choroidal function in normal and disease states. Due to limited availability of human tissues, an in vitro cell culture system is desired. Therefore, we have developed and characterized the primary cell lines of human RPE from donor eyes obtained from eye banks. Using human RPE cell cultures as a model, we conducted investigations to examine the various roles of RPE in the pathophysiology of retinal disorders. Human RPE cultures secreted significant quantities of interleukin-6 (IL-6) and intercellular adhesion molecule- 1 (ICAM-1) but not interleukin-1 (IL-1) in response to the stimulation by inflammatory mediators. Interferon gamma (IFN-7) exhibited synergistic effects in the secretion of IL-6 and ICAM-1 in the presence of suboptimal levels of tumor necrosis factor a (TNF-a) or IL-1. Cellular expression of ICAM-1, mostly localized to intercellular junctions, was observed in RPE treated with TNF-a and IFN-y. There is a close correlation between IL-6 and ICAM-1 secretion and IL-6 and ICAM-1 mRNA levels, respectively, suggesting the regulation at the gene transcription. The responses of RPE to inflammatory mediators in IL-6 and ICAM-1 secretion was rapid and sustained in the presence of stimulants but reversed to control levels quickly upon withdrawal of the stimulants, indicating the reversibility of the responses of RPE to inflammatory signals. These results show that RPE responds to inflammatory stimuli by increased cellular expression and secretion of IL-6 and ICAM-1, which may in turn perpetuate immune reactions in the pathogenesis and/or prevention of retinal and choroidal diseases. 143 PHS 6040 (Rev. 5/92) FY 1994 NEl Annual Report Project Description Additional Personnel Krishnan R. Kutty Ph.D. LRCMB, NEI Barbara Wiggert PhD. LRCMB, NEI Objectives The primary objectives of this research project are to: (1) establish primary cell lines of human retinal pigment epithelial (HRPE) cells from donor eyes, (2) develop serum-free media and other factors that can induce differ- entiation and pigmentation of HRPE, (3) investigate the role of inflammatory mediators and growth factors on cellular and molecular aspects of HRPE structure and function, and (4) evaluate the usefulness of HRPE and rat retinal pigment epithelial (RPE) cultures for transplantation studies to restore retinal func- tions in hereditary, autoimmune, and age-related disorders. Methods Primary cell cultures of HRPE are prepared by initial seeding of either freshly isolated RPE cells or RPE-choroid explants. Cells are grown in minimum essential medium supple- mented with 10 percent fetal calf serum and nonessential amino adds and antibiotics. We are developing serum-free, hormonally de- fined media and extracellular matrix factors that would induce differentiated morphology for the RPE cells. Techniques required for cell culture, im- munofluorescence, C5d:okine, and intercellular adhesion molecular 1 (ICAM-1) assays by enzyme-hnked immunosorbent assay, gel electrophoresis. Western and Northern blot- ting for proteins and ribonucleic acid (RNA), reverse tianscription polymerase chain reac- tion (RT/PCR) are developed and standard- ized in our laboratory to carry out these studies. Major Findings In the past, age of the donor was considered very critical in preparing human RPE cultures because eyes from donors older than 50 years of age did not yield fruitful cell Unes, proba- bly due to senescence-associated loss of viabil- ity. In these experiments, RPE cells were first disassociated from the eye cups by digestion with proteolytic enzymes, a treatment that might have caused initial contamination with nonepitheUal cells from which it is impossible to purify epithelial cells. Therefore, we have developed a new method using RPE-choroid explants to initiate ceU growth. By careful monitoring of clusters of cells growing around the explants, we were able (on the basis of morphology combined with experience) to select pure epithelial cells and discard nonepi- theUal cells at the primary culture stage. Using this technique, we established primary cell Unes of human RPE from eyes obtained from 81- and 87-year-old donors. The epitheUal nature of these ceU Unes was confirmed by immunochemical staining for cyiokeratin with monoclonal antibodies. All of the ceUs expressed cytokeratin at different passages. Immunoblotting analysis of cellular proteins indicated cytokeratin-18 was the predominant cytokeratin in these ceUs. Be- cause RPE is the only epitheUal ceU in the posterior segment (choroid-RPE-retina), these results estabUsh without doubt that the ceU Unes developed are, in fact, RPE. Human RPE cultures secrete significant quantities of interleukin (IL)-6 and ICAM-1, but not IL-1, in response to the stimulation by inflammatory mediators. IL-1 is the most potent stimulant of IL-6 secretion followed by tumor necrosis factor alpha (TNF-a) and Upopolysaccaride (LPS). Interferon gamma (IFN-y) had only minimal effects on IL-6 secretion. In confrast to IL-6 secretion, ICAM-1 secretion was not influenced by LPS. TNF-a, IFNy, and IL-1 had almost similar effects on ICAM-1 secretion by HRPE. The effect of IFN-Y is striking in that it can act synergjs- tically in the presence of suboptimal levels of 144 Laboratory of Immunology TNF-a or IL-1 to augment secretion of both IL-6 and ICAM-1. More than 98 percent of IL-6 produced by HRPE cells was secreted promptly into the medium. Western blot analysis of secreted IL-6 revealed multiple molecular forms suggesting that HRPE are capable of carrying out posttranslational glycosylation processes that may be important for functional activities. Cellular expression of IL-6 was not detected by immunofluorescence staining of the cells. In contrast, intense staining for ICAM-1 — that was mostly local- ized to intercellular junctions of the monolayer of epithelial cells — was observed in HRPE treated with TNF-a and /or IFN-y. Analysis of IL-6 and ICAM-1 messenger ribonucleic add (mRNA) expression by Northern blotting indicated rapid and sustained responses of HRPE to inflammatory cytokines that can be reversed quickly on withdrawal of the stimu- lus. There is a close correlation between IL-6 and ICAM-1 secretion as weU as IL-6 and ICAM-1 mRNA levels, respectively. Our results indicate that HRPE respond to LPS and inflammatory cytokines (TNF-a, IL-1, and IFN-y); and enhance IL-6 and ICAM-1 gene expression and secretion of proteins. During posterior uveitis of the eye caused by infections or autoimmune diseases, macro- phages and lymphocytes infiltrate into the retina and secrete cytokines such as TNF-a, IL-1, IFN-Y, 3rid IL-2 that would initiate im- mune reactions. These cytokines in their turn act on retinal resident cells to locally produce IL-6 and ICAM-1 to ampUfy the immunopath- ological processes. IL-6, a multipotent cyto- kine, plays a major role in the autoimmune and inflammatory disorders by its abUity to induce proliferation and differentiation of lymphocytes and production of antibodies. ICAM-1, an adhesive glycoprotein, participates in inflammatory reactions by recruiting leuko- cytes to the sites of inflammation, lymphocyte proliferation, cytotoxic T-ceU function, and T- cell mediated B-ceU activation. Several Hnes of evidence support that bacterial endotoxins and cytokines TNF-a, IL-1, IFN-Y, IL-2, and IL-6 play a critical role in uveitis and other inflammatory diseases of the eye. Intravitreal injection of IL-1, TNF-a, or IL-6 has been shown to cause uveitis in experimental animal models. Moreover, elevated levels of IL-6, IFN-y, IL-1, and TNF-a have been found in aqueous humor and vitreous aspirates of patients with uveitis, proliferative vitreoretinopathy, and other noncomplicated retinal detachments. Upregu- lation of the expression of ICAM-1 on retinal ceUs and epiretinal membranes and an in- crease in soluble ICAM-1 in vitreous of pa- tients with inflammatory retinal diseases suggest a vital role for ICAM-1 in various diseases. Our studies suggest that RPE reacts to inflammatory stimuU and secrete IL-6 and ICAM-1, thereby elevating these proteins in the local environment for the perpetuation of immunopathological processes. The synergis- tic actions of IFN-y on IL-6 and ICAM-1 secre- tion by HRPE cells in the presence of other cytokines would result in an effective amplifi- cation mechanism because several cytokines are produced simultaneously during iitflam- mation. The roles of growth factors, basic fibro- blast growth factor, transforming growth factor beta (TGF-P) and platelet-derived growth factors in RPE functions, and the regulation of secretion of these growth factors by RPE are being investigated. We found that these growth factors had no effect on the expression and secretion of IL-6, ICAM-1, and IL-1. The expression of heme oxygenase-1 (HO-1) was increased by TGF-P in HRPE ceUs by fourfold to fivefold within hours of stimu- lation. However, LPS, inflammatory cyto- kines, and other growth factors had no effect on HO-1 levels. Among ocular tissues, RPE has the highest activity of HO-1. HO-1 cata- lyzes the oxidation of heme into biliverdin and carbon monoxide. Biliverdin is converted by nonlimiting enzymatic reaction into biliru- bin, an antioxidant that offers protection of cells against heat and oxidative stress. These results suggest that TGF-P upregulates de- fense mechanisms of RPE, a phagocytic cell that is constantly subjected to chemical stress, by engulfing the shed outer segments of retinal rods and cones for protection against oxidative damage. 145 FY 1994 NEI Annual Report Significance to Biomedical Research and the Program of the Institute Primary cell lines of human RPE are an ideal in vitro model for evaluation of several func- tions of RPE and for further elucidation of the mechanisms of RPE involvement in the patho- genesis of retinal and choroidal diseases. These cells are potentially useful in cellular transplant therapy to correct hereditary and age-related macular degeneration defects in humans. Proposed Course Two of the major problems associated with human RPE cell cultures are: (1) progressive loss of pigmentation on serial passaging of cells and (2) lack of clear intercellular junc- tions and in vivo-]ike morphological appear- ance. These changes could probably be due to cytoskeletal reorganization and partial dedif- ferentiation. Development of such fully differ- entiated RPE cell lines is crucial not only to understanding cellular functions but also for cellular transplant therapy. Our goal is to examine the mechanisms by which RPE cul- tures can be induced to resume in vivo charac- teristics. Preliminary studies show that HRPE cells assume differentiated morphology on incubation in serum-free medium containing insuhn, transferrin, selenium, hydrocortisone, prostaglandins, and tri-iodothyronine. Further studies will be conducted in defining and selecting specific media composition and /or culturing on suitable extracellular matrix. The effects of inflammatory cytokines and bacterial endotoxins on HRPE wUl be evaluat- ed: (1) on the secretion and expression of IL-8 and granulocyte-macrophage colony-stimulat- ing factors; (2) on the role of anti-inflammato- ry cytokines TGF-p, IL-4, and IL-10 on ihe influences of inflammatory mediators; (3) on cellular cytoskeletal organization, intercellular junctions, and adhesion properties; and (4) characterization of growth factors and proteo- lytic enzymes secreted by RPE in response to various stimuli. These studies are likely to shed light on the role of RPE in the patho- physiology of retina and choroid, the tissues that are in close vicinity to and have direct influence on RPE. NEI Research Program Retinal Diseases — Inflammatory Diseases, Macular Degeneration, Photoreceptors and Pigment Epithelium Publications Kutty RK, Nagineni CN, Kutty G, Hooks JJ, Chader GJ, Wiggert B: Increased expression of heme oxygenase-1 in human retinal pig- ment epithelial cells by transforming growth factor-b. / Cell Physiol 159:371-378, 1994. Nagineni CN, Detrick B, Hooks JJ: Synergistic effects of gamma interferon on inflammatory mediators that induce interleukin-6 gene expression and secretion by human retinal pigment epithelial cells. Clin Diagnos Lab Immunol, (in press). Nagineni CN, Detrick B, Rhame J, Hooks JJ: Inflammatory cytokines IFN-y, TNF-a and IL-1 induce ICAM-1 secretion /shedding by human retinal pigment epithelial cells. Invest. Ophthalmol. Vis Sci 35 (4):2040, 1994. DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00287-02 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Toxoplasmosis Infections in the Eye PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: M. Cristina Martins Chandrasekharam Nagineni Miguel Bumier Robert B. Nussenblatt M.D. Guest Worker LI, NEI Ph.D. Visiting Scientist LI, NEI M.D. Visiting Scientist LI, NEI M.D. Scientific Director LI, NEI COOPERATING UNITS (if any) National Institute of Allergy and Infectious Diseases (R. Gazzinelli, M.D.) Laboratory of Immunology Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS; 0.5 PROFESSIONAL: 0.5 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects [x] (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Toxoplasma gondii infections are a major source of visual loss and blindness. Ocular toxoplasmosis may occur as a result of congenital infections, acquired infections, and as a manifestation of immunosuppression, particularly as a result of transplantation or acquired immunodeficiency syndrome (AIDS). Due to the recent resurgence of acquired ocular toxoplasmosis in Brazil and the worldwide complications of toxoplasmosis in HIV infections, we initiated studies to develop a model of acquired toxoplasmosis to evaluate the molecular mechanisms of pathogenesis and therapeutic strategies. We have developed a murine model of ocular toxoplasmosis that is characterized by retinal inflammation, chorioretinal scaring, retinal disorganization, and cyst formation. Retinal disease occurs in three different strains of mice following inoculation with toxoplasmosis by the subcutaneous or intraperitoneal routes. This model of acquired ocular toxoplasmosis is being used to evaluate the efficacy of new antiparasitic agents in controlling the development of retinal cyst formation and retinal inflammation. 147 PHS 6040 (Rev. 5/92) FY 1994 NEl Annual Report Project Description Objectives This project was designed to develop an animal model of acquired ocular toxoplasmo- sis and in vitro models of toxoplasmosis repli- cation within the retina. These models will be used to evaluate molecular mechanisms of ocular pathogenesis and to evaluate new antiparasitic drugs and cytokines. Methods This study involves the propagation and quantitation of Toxoplasmosis gondii (T. gondii) strains in vitro and in vivo, as weU as immuno- cytochemical analysis of infected ceUs and tissues. Techniques used in the characteriza- tion of T. gondii infections include histopathol- ogy, immunocytochemistry, in situ hybridiza- tion, and Western blot analysis. Techniques used in characterization of anti-T. gondii anti- bodies include enz)Tne-linked immunosorbent assays. Major Findings Adult Swiss Webster, C57BL6 and BALB/C mice were inoculated by the subcutaneous route or intraperitoneal route with 10 T. gondii cysts (S2C9 or ME49 strains) in a 1 mL vol- ume. At various times after inoculation (day seven, 14, 21, 28, and 42) the mice were sacri- ficed, and eyes and brains were removed and fixed in 10 percent buffered formalin. Fifteen hematoxylin and eosin sections of brain and eye were evaluated for the presence of T. gondii cysts. By day 14, 100 percent of the mice devel- oped cysts in the brain. Retinal inflammation was also noted in 100 percent of the animals by day 14. Chorioretinal scars were also ob- served in mice inoculated with both strains of T. gondii. Retinal cysts were found in mice 28 and 42 days after inoculation with ME49 strain and 14 and 42 days after inoculation with S2C9 strain in Swiss Webster mice. T. gondii cysts in the retina were also detected in C57BL6 mice at 14, 21, 28, and 42 days after inoculation with the S2C9 strain. This study identities an animal model of ocular toxoplas- mosis characterized by retinal inflammation, chorioretinal scaring, retinal disorganization, and cyst formation. Preliminary studies on an in vitro cell culture model for T. gondii replication in retinal tissues has revealed that T. gondii can infect and replicate in human retinal pigment epithelial cells. Initial studies also indicate that this replication can be inhibited by the addition of recombinant human interferon gamma. Significance to Biomedical Research and the Program of the Institute This is the first animal model of acquired toxoplasmosis that consists of retinal iriflam- mation, degeneration, and parasitic cyst for- mation. This model wUl allow us to evaluate the efficacy of new antiparasitic drugs in controlling the development of retinal cyst formation and retinal inflammation and scar- ring. Proposed Course We will evaluate drugs and cytokines in controlling the ocular manifestations of ac- quired T. gondii infections. NEl Research Program Retinal Diseases — ^Inflammatory Diseases 248 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00293-01 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Gene Targeting of Invariant Chain Gene: A Tool To Study Immunoregulation in Autoimmune Diseases PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Moncef Jendoubi Others: Noriko Esumi Daniel H. Lacorazza Luis J. Rivero Ph.D. M.D., Ph.D. Ph.D. Ph.D. Visiting Scientist Visiting Associate Visiting Fellow Visiting Fellow LI, NEI LI, NEI LI, NEI LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Section of Genetics and Molecular Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.9 PROFESSIONAL: 3.9 CHECK APPROPRIATE BOX(ES) n (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Class II antigens of the major histocompatibility complex (MHC) are essential in the immune response because they bind processed polypeptides for presentation to T lymphocytes. The interactions of class II MHC molecules on the antigen-presenting cell with a responding T lymphocyte are complex because they involve molecular contacts with an antigen peptide, an antigen-specific T-cell receptor, and the CD4 molecule expressed on the T lymphocytes. Most antigens must be processed in order to bind to MHC molecules and to be recognized by T lymphocytes. In this context, the majority of peptides bound by MHC class II molecules during antigen loading are not derived from ingested and processed foreign proteins but instead are primarily derived from a finite number of self-proteins. During progression outward through the exocytic pathway in the process of antigen presentation, class Il-invariant chain complexes play a critical role. Experimental autoimmune uveoretinitis (EAU) in rodents is a T-cell-mediated autoimmune response, particularly against the photoreceptors of the neural retinal cells, and can serve as a model for human uveitis. The roles of MHC and non-MHC genes have been strongly associated with EAU in rats and mice. To further study the MHC class II participation in mice animal model, we decided to generate deficient mice for the invariant chain gene (li). This glycoprotein combines with MHC class II heterodimers from the beginning of their synthesis in the endoplasmic reticulum, travels through the Golgi apparatus, and ends up in endosomal compartments where it is either proteolytically cleaved or degraded. The absence of li has been shown to affect the transport of class II molecules, resulting in a poor antigen presentation. 149 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives Targeting Vector. A replacement vector has been designed to introduce a mutation in li murine gene, where the neomycin phospho- transferase gene was inserted in the middle of the second exon. Two fragments of 0.3 Bgl II- Sac I and 9.2 Kb Sac I-EcoR I were added upstream and downstream of the neomycin gene, respectively. Furthermore, two copies of herpes simplex virus thymidine kinase (HSV- tk) that allow the negative selection were cloned at the end of the targeting vector. The targeting fragment used for the electropora- tion was excised from the backbone vector by digestion with Not I, dialyzed against TE (Tris/HCl 10 mM, pH 7.5, EDTA 1 mM), precipitated with ethanol, resuspended in an appropriate buffer at a concentration of 1 mg/ml, and analysed on agarose gel. Methods Electroporation. Embryonic stem cells (D3, from 129 /5v stiain) were cultured in standard condition on an inactivated layer of embryonal fibroblasts and fed two and one-half hours before electroporation. Cells were trypsinized, washed with DMEM, and resuspended in HBS (Hepes 25 mM, NaCl 134 mM, 5 mM KCl, Na2P04 0.7 mM, pH 7.1) at 2x10' ceUs/ml. The same volume of HBS containing 100 /ig/ml of targeting vector was added to the cell suspension and incubated 10 minutes on ice. The electroporation was carried out using a 600 BTX electroporator with the following conditions: Volts: 250; /tParadays: 300; Resis- tance: R8. Transfected cells were left at room temper- ature for 10 minutes and seeded at a concen- tration of 5x10* cells/ 10 cm Petri dish. Twen- ty-four hours later, the ceUs were selected using G418 (200 /ig active substance/ml) and ganciclovir (2 fiM). Blastocyst Injection. Blastocysts, three and one-half days old, were collected by flushing the uterus of super ovulated C57BL/6 females. Mutated cells in the U gene were injected into blastocysts before their transfer to pseudo- pregnant B6D2 FI foster mothers. Major Findings A total of 8x10' D3 cells were electroporated with the targeting vector and selected with G418 and ganciclovir for 10 days. Double- resistant clones were picked up individually and expanded in a 24-weIl plate. Deoxyribo- nucleic acid (DNA) was isolated from each clone and analyzed by polymerase chain reaction (PCR) for homologous recombination events, using a combination of primers that allow the discrimination of random integra- tions. In addition, genonuc DNA was isolated from aU double-resistant clones and analyzed by Southern blot, using both inside and out- side probes for the invariant chain gene. For further confirmation, the same studies were repeated, using various restriction enzymes. Taken together, the results of PCR and South- em blot analysis confirmed that three out of 130 double-resistant clones scored positive. These three targeted clones were further expanded and injected into blastocysts, and the embryos were later transferred into the foster mothers. Offspring carrying the mutat- ed li gene were identified by their chimeric coat color. Again, DNA was purified from all chimeric mice and analyzed by PCR as well as by Southern blot. The obtained results showed that the targeted mutation has been transmitted to the offspring. Heterozygous mice were set up for mating to generate homozygous mice mutated on both alleles of the invariant chain gene. Presentiy, we were able to create homozygous mice and to estab- lish a colony of these animals. Homozygous mice bom in the colony are normal and grow up without showing any obvious abnormality. Significance to Biomedical Researcti and ttie Program of the Institute The creation of a deficient mouse for the invariant chain gene represents a previously unavailable tool to study several important 150 Laboratory of Immunology phenomenons in the immune system, both in normal and pathological conditions. Degenerative and inflammatory diseases of the posterior pole of the eye are common causes of impaired vision and blindness throughout the world. Between 500,000 and 1,000,000 Americans suffer severe visual impairment from retinal and choroidal dis- eases. Retinal degenerative disorders consist of a diverse group of diseases frequently associated with a genetic predisposition such as major histocompatibUity complex (MHC) class II. However, in many ocular diseases the causes are unknown. In this respect, invariant chain-deficient mice wiU be very useful to study some of these ocular disorders. Thus, these mice will be used to study the implica- tion of MHC class n genes in ocular immuno- pathological diseases. Proposed Course In the future, we will focus our work on the following: (1) We will use the invariant chain gene- deficient mice to study endotoxln-induced uveitis. (2) We will study the effect of viral infec- tion such as murine coronavirus that induces an acute, long-lasting disease of the retina to clarify the degenerative and inflammatory diseases that affect the retina and the choroid in many ocular disorders (this study will be conducted in collaboration with Dr. John Hooks in the LI). (3) We wiU use these deficient mice as models to study allergic conjunctivitis, in collaboration with Dr. Chi-Chao Chan in the LI. (4) We will use these deficient mice as models to study experimental autoimmune uveitis, in collaboration with Dr. Rachel Caspi in the LI. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications Lacorazza DH, Rivero LJ, Jendoubi M: Ex- pression of the human OAT gene after retro- viral transfer into CHO deficient cell line. NIH Research Festival E05:35, 1993. Rivero LJ, Jendoubi M: Creation of invariant chain gene deficient mice as a tool to study autoimmune disease and uveitis. Sixth Inter- national Symposium on Immunology and Ophthal- mology, NIH, June 22, 1994. Rivero LJ, Jendoubi M: Gene targeting of invariant chain gene by homologous recombi- nation as a tool to study immunoregulation in autoimmune diseases. Advances in Ocular Immunology, p. 163, Amsterdam, Netherlands, Elsevier Press, 1994. Rivero LJ, Jendoubi M: Embryonic stem cell lines carrying insertional mutation. Cold Spring Harbor M Mol Gen, p 172, 1992. Rivero LJ, Jendoubi M: Mutagenesis of ESC and generation of chimeric mice. NIH Research Festival 4:23, 1992. Rivero LJ, Kozhich A, Nussenblatt RB, Jendoubi M: Retrovirus expression of orni- thine 6aminotransferase in vitro toward gene therapy. Invest Ophthalmol 34(4):807, 1993. Rivero LJ, Kozhich A, Nussenblatt RB, Jendoubi M: Retrovirus-mediated gene trans- fer and expression of human ornithine 6- aminotransferase into embryonic fibroblast. / Cell Biol 17E:251, 1993. Rivero LJ, Lacorazza DH, Kozhich A, Nussenblatt RB, Jendoubi M: Retrovirus- mediated gene transfer and expression of human ornithine delta transferase into embry- onic fibroblasts: An alternative approach to somatic gene therapy. Hum Gen Ther 5:701, 1994. 151 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00292-01 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Retinal Survival in Transgenic Mice Expressing Human Ornithine 3-Aminotransferase PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI Others: Noriko Esumi Daniel H. Lacorazza Luis J. Rivero Chi-Chao Chan M.D., Ph.D. Visiting Associate LI, NEI Ph.D. Visiting Fellow LI, NEI Ph.D. Visiting Fellow LI, NEI M.D. Head, Section on Immunology LI, NEI COOPERATING UNITS (if any) Laboratory of Immunology Section of Genetics and Molecular Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 3.9 PROFESSIONAL: 3.9 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues Jx] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Gyrate atrophy (GA) is a severe human recessive eye disease resulting in progressive loss of vision due to chorioretinal degeneration. The disorder is associated with a deficiency of the mitochondrial matrix enzyme, ornithine 5-aminotransferase (OAT), which catalyzes the interconversion of ornithine and a-ketoglutarate to A-pyrroline-5'carboxylate and glutamate. GA patients with activity have 10- to 20-fold higher levels of plasma ornithine as compared with controls. Dietery and specific hormonal administration in rats and mice have been shown to modulate the regulation of this enzyme in different tissues, suggesting OAT's important physiological role in vivo. To test this hypothesis and to find out whether ornithine 6-aminotransferase could be a common mediator for retinal cell growth, we produced two transgenic lines, expressing the human OAT gene in strains of mice normally exhibiting a progressive retinal degeneration. Here we show that transgenic mice expressing human ornithine 8-aminotransferase exhibit less severe retinal degeneration than control mice littermates. 152 PHS 6040 (Rev, 5/92) Laboratory of Immunologi/ Project Description Objectives Certain inbred strains of mice, such as FVB/N, present a progressive retinal degeneration beginning a few days after birth and becoming complete a few months later. Retinal degener- ation in these mice has been associated at least in part with a deficiency in a phosphodiester- ase activity, which leads to the accumulation of cyclic GMP in affected retina degenerative photoreceptors. We postulated that other gene products such as ornithine-6-aminotrans- ferase (OAT), which is associated with retinal degeneration in humans may be involved. Thus, persistent expression of human OAT (hOAT) in retinal degenerated mice could have a role in the development of retinal cell layers. To study the physiological relevance of OAT in vivo and to determine whether its expression could rescue the retinal cell layers from degeneration, we produced two transgenic lines (OATtg) expressing the hOAT gene in strains of mice normally exhibiting a progressive retinal degeneration. Therefore, it can be seen that transgenic mice expressing hOAT exhibit less severe retinal degeneration than control mice litter mates. Methods Production of Transgenic Mice. hOAT comple- mentary deoxjrribonucleic acid was cloned under the transcriptional element of Moloney murine leukemia retrovirus long terminal repeat (LTR-MoMuLV), and the construct was injected into zygotes of FVB/N mice strain genetic background. Biochemical Analysis of Transgenic Mice. Cells were prepared from different tissues from both transgenic mice and control litter mates; the cells were lysed in 500 mM NaCl, 50 mM Tris pH 7.5, one percent NP40. 30 /ig of protein extracts from each sample was subjected to sodium dodecyl-sulfate polyacryl- amide gel electrophoresis (SDS/PAGE) and stained with coomassie blue or transferred to nylon filters for immunoblot analysis. Histopathology. Enucleated eyes were prefixed in 4 percent phosphate-buffered glutaraldyde for one hour after being fixed in 10 percent formaldehyde overnight, dehydrat- ed, and embedded in methacrylate. Four /xm- thick sections were cut horizontally along the pupiUary-optic nerve plane of the eye and were stained with hematoxyhn-eosin. Sections were evaluated, and microphotographs were taken at a magnification of 400 X. Major Findings Biochemical Analysis. OATtg mice were bom normal. The expression of the transgene was assessed primarily by Northern blot analysis and then in several tissues using immunoblot analysis and specific antibodies raised against hOAT. When the protein extracts were sepa- rated on SDS/PAGE and stained with coomassie blue, we saw the presence of new polypeptides, and /or an enhancement of several others, at all range of molecular weights only in the OATtg tissue protein extracts. OATtg and control litter mates were analyzed for their expression of hOAT and its consequences on cell growth, particularly in the eye. The expression of the transgene was assessed primarily by Northern blot analysis and then in several tissues using immunoblot analysis. The correct size of hOAT protein in transgenic mice, as detected with specific antibodies raised against hOAT, indicated that our transgene encoded the entire protein. Histopathology. To further determine whether the expression of hOAT would have any consequence on the retinal cell layer development, we examined histopathologically OATtg and the control litter mates derived from both founders at different timepoints. At birth, no significant differences of the retinal structure, mainly the inner and outer neuro- blast layers, between wild-type and OATtg mice were observed. At one week, wild-type retina showed, as expected, an early degenera- tive development of the outer segments of the photoreceptors with a sUght reduction of the outer and inner nuclear layers (ONL and 153 FY 1994 NEI Annual Report INL). On the contrary, OATtg retina showed better preserved inner and outer segments of the photoreceptors (IS /OS) with larger num- bers of intact nuclei both in the ONL and ESTL. At two weeks, the retinal thickness be- tween the wild-type and OATtg showed remarkable differences. The ONL of wild-type retina showed no more than one row of pho- toreceptor nuclei, and the IS /OS had become a debris of degenerating membranes, owing to the defect in FVB/N with congenital retinal degeneration. In contrast, at least three rows of cells with relatively fewer pyknotic nuclei remained in the ONL of OATtg retina. In the transgenic mice, the thickness of the IS /OS was ahnost double that of the wild-type retina. The INL and the outer plexiform layer (OPL) showed better differentiation and more thickness in OATtg than in wild type. After two months, retina in the control litter mates showed complete degeneration and atrophy with total loss of all photorecep- tor cells, including the nuclei, the IS /OS, and OPL. The ONL was in direct contact with the retinal pigment epithelium (RPE). The residu- al single row of degenerated photoreceptor nuclei and the remains of IS /OS and OPL were identified in some regions. During the retinal development, the most striking differ- ence between OATtg and wild type was observed at two weeks. All three wild types showed a retinal degeneration, but the OATtg mice exhibited a retardation of the degenera- tive process. Significance to Biomedical Researcii and the Program of ttie Institute Although the physiological role of OAT and how it contributes to retinal cell growth, whether directly or indirectly, remains to be understood. The present study provides strong evidence that OAT plays a critical role in rescuing neural cell lines in the retina. In the human, many ocular diseases affect both retina and choroid and lead to blindness. This is the first time that we show a physiological role of OAT and demonstrate that it partici- pates in some extent to the survival of retinal cells. Thus, by virtue of its role as a growth- factor, OAT represents a valuable model that may aid studies of the pathogenesis and treatment of human retinal degeneration. Proposed Course In the future we will focus on the following: (1) We will breed these OATtg+ mice with OAT-deficient mice to restore the missing enzymatic activity. (2) We will study the consequences of the overexpression of the exogenous OAT on other tissues. (3) We will try to find out how the over- expression of OAT leads to the survival of retinal cells. NEI Research Program Retinal Diseases— Retinitis Pigmentosa and Other Inherited Disorders DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00294-01 LI PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Enzymatic Correction of OAT Deficiency: Progress Toward Gene Therapy to Ocular Genetic Disease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI Others: Noriko Esumi Daniel H. Lacorazza Luis J. Rivero M.D., Ph.D. Ph.D. Ph.D. Visiting Associate Visiting Fellow Visiting Fellow LLNEI LI, NEI LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology Section of Genetics and Molecular Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.9 PROFESSIONAL: CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Gyrate atrophy (GA) of the choroid and retina is an autosomal recessive chorioretinal degeneration, caused by deficiency of the mitochondrial matrix enzyme omithine-6-aminotransferase (OAT). This deficiency results in the accumulation of ornithine in the body fluids and leads to hyperomithinemia. Although the clinical phenotype is largely confined to the eye, OAT deficiency is a systemic disorder. With the final goal of applying gene therapy to this human genetic disease, we have established an in vitro model to test the correction of OAT enzymatic deficiency in mammalian cells, using OAT-recombinant retroviruses. Herein, we report the construction of several Moloney murine leukemia virus (MoMLV)-based recombinant retrovirus vectors, in which the human OAT cDNA was placed under the transcriptional control of the mouse phosphoglycerate kinase (PGK) promoter or under the enhancer-promoter regulatory element derived from MoMLV long terminal repeat (LTR). The retrovirus constructs were packaged in the PG13-GALV cell line and used to transduce C9, an OAT-deficient cell line derived from Chinese hamster ovary cells (CHO-Kl). We showed that the recombinant retrovirus transfers the hOAT gene into C9. Expression of the hOAT gene in the transduced C9-deficient cell line exceeded the level of endogenous human fibroblasts, OAT mRNA, and enzymatic activity. 155 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives Omithine-6-aminotransferase (OAT) (L-omi- thine:2-oxo-acid aminotransferase, 2.6.1.13) is a nuclear-encoded mitochondrial matrix en- zyme that catalyzes the reversible intercon- version of ornithine and a-ketoglutarate to glutamate semialdehyde and glutamate. OAT monomers are synthesized as 49-kDa precur- sors and processed to 45-kDa forms upon entry into the mitochondrial matrix where they assemble into the active homohexameric form of the enzyme. The OAT gene in hu- mans has been characterized and mapped to the site 10q26. Both rat and human comple- mentary deoxyribonucleic acid (cDNA) have been cloned. This has greatly facilitated the study of OAT's physiological role. In previ- ous studies, it has been show^n that gyrate atrophy (GA) patients have a high concen- tration of ornithine in their body fluids, up to 20 times the normal level. This hyperomithi- nemia was associated with the absence of OAT enzymatic activity. More recently, se- quencing of OAT cDNA from GA patients revealed the presence of mutations— mostly point mutations — that cause frameshift, non- sense, and missense mutations and lead to the inactivation of the OAT gene and ultimately to the absence of enzymatic activity typical of GA disease The hyperomithinemia associated with GA of the choroid and retina can be lowered to normal levels with a low-protein and low- arginine diet in all cases studied so far, and with pyridoxine hydrochloride (vitamin B6) in some cases. Because an arginine-free diet cannot be easily followed for a long period of time, this treatment is temporary and pallia- tive rather than curative. With the view of apphang a genetic tiierapy to GA patients and correcting the OAT enzymatic deficiency by supplying a functional gene, we established an in vitro model system in which we attempted to correct the enzjmnatic deficiency in an OAT- defident cell line as a first step toward a somatic gene therapy. Methods We constructed several OAT-recombinant retroviruses bearing human OAT (hOAT) cDNA under different regulatory elements, transduced them into cells — an OAT-deficient cell line — and finally studied the efficiency of OAT gene expression following retrovirus- mediated transfer. Using Southern, Northern, and Western blot analyses as well as specific OAT enzymatic assays we show that OAT recombinant retroviruses efficientiy transfer the gene to recipient cells leading to high expression of an active OAT enzyme. Major Findings Establishment and Characterization of a Recombi- nant Retrovirus Producer Cell Line. Four retro- viral vector constructs bearing the hOAT cDNA as well as the neomycin phosphotrans- ferase (neo^) gene as selectable marker were packaged into virions using the PG13-GALV cell Une. The PG13-GALV packaging ceU line was used to produce virions with Gibbon leukemia virus host range and to infect ham- ster cell Hnes, including C9. The retrovirus producer cells were screened for the presence of an unrearranged OAT recombinant retrovirus, the production of high-titer virus and the functional expres- sion of the inserted hOAT cDNA. The pattern of the Southern blot indicates that the retro- virus constructs were integrated into the packaging cell genome without rearrange- ment. Moreover, the hOAT expression was confirmed by Northern blot analysis, and the results showed the presence of the cor- responding OAT transcripts in the four trans- fected packaging cell Hnes. To determine whether the inserted hOAT recombinant retrovirus is able to produce an active enzyme, we performed an enzymatic assay on cell lysates from PG13-GALV retro- virus producer cell lines previously transfected with the constructs described above. The four different lysates showed a high expression of OAT enzyme, however, the comparison of the enzymatic activity between them demonstrate 56 Laboratory of Immunology that the PG13-GALV retrovirus producer cell line transfected by LPOSN expresses the highest OAT activity. Correction of OAT Deficiency in C9-Trans- duced Cell Line. To further assess the expres- sion of integrated hOAT sequences, we ana- lyzed the production of the OAT messenger ribonucleic acid (mRNA). We purified total cellular ribonucleic acid (RNA) from wild-type C9 cells and transduced ones with LPOSN retrovirus as well as from normal human fibroblasts. Total RNA from these cells was processed for Northern blot analysis. The results showed strong hybridization on a 5.0- kbp and a weaker hybridization on a 3.6-kbp transcript, which might suggest that the major transcript could be derived from the viral long terminal repeat and the minor transcript from the phosphoglycerate kinase (PGK) promoter. This result may suggest an interference be- tween viral and PGK promoter. The native hOAT transcript from human fibroblasts showed the corresponding size or 2.1-kbp. The hOAT transcripts in the transduced C9 cell line are in greater abundance than the endogenous hOAT mRNA in the normal human fibroblasts taking into account that the lanes contain the same amount of total RNA as confirmed by hybridization with a P-actin probe. To test if the mRNA transcript from the proviral insert was being appropriately translated and processed into a mature pro- tein, we performed a Western blot analysis using protein extracts from transduced and nontransduced C9 as well as from human fibroblasts, and an anti-hOAT antibody direct- ed against a 19-mer peptide located in the N- terminal domain of the OAT protein. No sigruficant immunological reaction was ob- served in C9 wild-type extract, while in trans- duced cells a strong reaction was seen on one polypeptide, which comigrates with an appar- ent molecular weight of 45-kDa with that detected in human fibroblasts. From these results, we infer that the OAT produced by the transduced cells has been well processed into a mature protein. To further assess whether the expressed OAT protein in the transduced cells is enzy- matically active, cell lysates were prepared from transduced and nontransduced C9 and from human fibroblasts and were analyzed for the presence of an active OAT. The deficient cell line has almost no OAT enzymatic activi- ty, but the human native fibroblast cells showed a normal activity (specific activity [SA] = 24 ± 2 nmol A^-pyrrohne-5-carboxylate [A'P5C] formed /mg of protein /h), whereas the transduced C9 cells produced at least a threefold increase of OAT activity as com- pared with human fibroblasts (SA = 65 ± 9 nmol A^P5C formed/mg of protein/h). These results show that the LPOSN provirus is capable of expressing high levels of functional hOAT enzyme that represents at least 10-fold more than the OAT residual activity in defi- cient cells. Our results demonstrate that the retro- virus-mediated transfer of hOAT results in a stable integration of the transferred gene without rearrangement into the transduced cells genome. In addition, we have shown that the gene was transcribed, translated, and processed into a protein recognized by a specific hOAT antibody and able to metabo- lize the ornithine in its natural substrate. Significance to Biomedicai Research and ttie Program of tlie Institute OAT deficiency is associated with hyperomi- thinemia and degeneration of the choroid and retina, suggesting that the accumulation of ornithine may produce a toxic effect on eye tissue. The introduction of a normal OAT gene via retrovirus transfer into somatic cells of GA patients consequentiy may turn the hyperomi- thinemia to normal level and lead to an im- provement in visual function. Retiovirus gene delivery has been extensively used in in vitro studies and in animal models as well as for somatic gene therapy in humans. Following this idea, we designed and made OAT retro- virus vectors that would provide optimal gene expression in deficient himian somatic cells FY 1994 NEI Annual Report and used this gene delivery system to evalu- ate OAT expression in vitro in an OAT-defi- dent cell line. In our previous studies, we were able to express the hOAT gene in murine embryonal fibroblasts. With the aim of achieving a higher expression of hOAT, we analyzed several recombinant retroviral vectors. Al- though all OAT retrovirus constructs present equivalent levels of functional OAT protein, the enzymatic activity was particularly higher with retrovirus, which was used to transduce an OAT-deficient cell line. Expression of the provirus was studied in the transduced C9 cells, both the 5' LTR and the internal PGK promoters were active, giving rise to two RNA transcripts. Enzymatic activity in this trans- duced deficient cell Une was at least three times higher over that of hOAT in normal human fibroblasts. However, in GA patients the OAT residual activity is about 5 percent the normal level (5 nmol A^P5C formed /mg/h); therefore, the obtained result of enzymatic activity in the transduced cells (65 nmol A^P5C formed/mg/h) would corre- spond to more than a 10-fold increase as compared with residual activity in GA pa- tients. Therefore, the level of OAT expression by the LPOSN retrovirus in the in vitro system is in the physiologic range needed for the correction of congenital OAT deficiency. In GA patients, OAT deficiency results in retinal and choroidal degeneration, indicating that OAT function is mostly needed in the eye. This raises the question about the target tissue for somatic gene delivery for gene therapy of this ocular genetic disease. Obvi- ously, the best tissue would be the retinal pigmented epithilium; however, these cells are not surgically accessible for removal, manipu- lation, and transplantation into the eye of GA patients. In addition, when these cells were isolated from rat or human eyes after biopsy, they were very difficult to maintain in culture. OAT is also highly expressed in liver, where it plays an important role in ornithine metabo- lism; therefore, hepatic cells could be consid- ered for OAT gene delivery to GA patients. These cells have been previously used success- fully by others to correct inborn genetic defi- ciency in animal models and in humans using both adenovirus and retrovirus. Although both systems have their limitations, the adeno- virus gene transfer remains an additional alternative worth pursuing for OAT gene therapy. There are currently more than 40 ap- proved human gene therapy protocols; never- theless, to date none of these trials involves ocular diseases. Our data provide the basic knowledge necessary to focus on the appropri- ate human cells as a target tissue for a future gene therapy trial in this ocular genetic dis- ease. Proposed Course Our future efforts will focus on the following issues: (1) We are correcting the enzymatic activity in cell Unes from GA patients. (2) We will assess the enzymatic activity in vivo in animal models after tissue engraft. (3) We will use the deficient animal that we are creating to assess the feasibility of gene therapy in ocular genetic disease. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Lacorazza DH, Rivero JL, Jendoubi M: Ex- pression of the human OAT gene after retro- viral transfer into CHO deficient cell Une. NIH, Research Festival, E05:35, 1993. Lacorazza DH, Jendoubi M: Correction of OAT deficiency in chinesehamster ovary cell line mediated by retrovirus gene transfer. Sixth International Symposium on Immunology and Opthalmology, NIH, June 22, 1994:25. Laboratory of Immunology Lacorazza DH, Jendoubi M: Correction of Rivero LJ, Laccorazza DH, Kozhigh A, genetic and enzymatic activity of ornithine 6- Nussenblatt RB, Jendoubi M: Retrovirus- aminotiansferase into mammalian deficient mediated gene transfer and expression of cell Unes using retrovirus mediated gene human ornithine delta tiansferase into embry- transfer. ARVO Annual Meeting, Sarasota, onic fibroblasts: An alternative approach to Florida, May 1-6, 1994: 1705. somatic gene therapy. Hum Gen flier 5:701, 1994. Lacorazza DH, Rivero LJ, Jendoubi M: Genet- ic and enzymatic correction of ornithine 6- aminotransferase into CHO deficient cell line. Gene Therapy, in press. 159 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1, 1993 to September 30, 1994 PROJECT NUMBER ZOl EY 00295-01 LI TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Isolation and Characterization of the Mouse OAT Gene for Gene Targeting PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI Others: Noriko Esumi M.D., Ph.D. Visiting Associate LI, NEI Daniel H. Lacorazza Ph.D. Visiting Fellow LI, NEI Luis J. Rivero Ph.D. Visiting Fellow LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology Section of Genetics and Molecular Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 PROFESSIONAL: 3.9 OTHER: TOTAL STAFF YEARS: 3.9 CHECK APPROPRIATE BOX(ES) D (a) Human subjects □ (b) Human tissues (xl (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Gyrate atrophy (GA) of the choroid and retina is an autosomal recessive eye disorder involving a progressive loss of vision due to chorioretinal degeneration. A variety of omithine-6-aminotransferase (OAT) gene mutations have been reported in GA patients and suspected to associate with this ocular disease. However, the precise mechanism by which the OAT deficiency and hyperomithinemia lead to the chorioretinai degeneration remains unknown. To elucidate the pathophysiological role of OAT, we are attempting to create OAT-deficient mice by gene targeting via embryonic stem (ES) cells. Toward this ultimate goal, we isolated murine OAT gene to construct OAT targeting replacement vector. 260 PHS 6040 (Rev. 5/93) Labor at oiy of Immunologic Project Description Objectives Isolation of Mouse OAT Functional Gene. The 129 mouse genomic library, OLA 129-yGEM- 12, was kindly provided by Dr. Anton Berns from the Cancer Institute, Netherlands. This library was screened with a nearly full-length rat omithine-6-aminotransferase (OAT) com- plementary deoxyribonucleic acid (cDNA) probe [4] labeled with ^^P-dCTP by random oUgonucleotide priming. Screening proce- dures were performed using standard proto- cols. Methods Polymerase Chain Reaction (PCR) Analysis. To determine the structure of positive genomic clones, PCR was performed with primers from each exon based on mouse cDNA sequence (Genebank, X64837) to ampHfy each exon and intron. Standard reaction conditions were used. Sequencing. The promising Clone 11 was subcloned into BlueScript vector (Stratagene, La Jolla, California) and designated pBSmOAT6. This plasmid was sequenced by dideoxy nucleotide chain termination method of Sanger using ^^S-dATP and the CircumVent Thermal Cycle Dideoxy DNA Sequencing Kit (New England Biolabs, Beverly, Massachu- setts). Construction of Targeting Vector. The mouse genomic fragment in pBSmOAT6 was 16 kb in length and used almost entirely to make a targeting vector. Neomycin resistance gene (Neo") was purified from pMClneo PolyA kindly provided by Dr. Mario Capecchi and inserted at Sea I site of exon 4 to disrupt the OAT gene. Herpes simplex virus thymi- dine kinase (HSV-TK) gene was purified from TGV-TK2 (Moncef Jendoubi, unpublished) originated from pIC19R/MCl-TK provided by Dr. Capecchi and added at the 3' end of the OAT fragment in targeting construct. Standard procedures were used in all subcloning and constructing processes. Major Findings Isolation and Clmracterization of Mouse OAT Gene. By genomic library screening, 16 clones were isolated from 6 x 10" phage plaques. AU clones were analyzed by PCR, and the results were compared with the PCR amplification pattern with mouse genomic DNA as a tem- plate. Among 16 clones. Clone 11 seemed to encode the functional OAT gene containing the 5' flanking region and the coding region up to exon 5. Because the OAT gene has been reported to have at least several pseudogenes and related sequences in human and rat genome [11-14], it is highly suspected that mouse genome also has at least several related se- quences. Therefore, sequencing of Clone 11 was performed to confirm that this clone encoded the functional OAT gene. Sequences of a total of 444 bases from exons 3, 4, and 5 were comparable to the sequence of the mouse OAT cDNA. Construction of Targeting Vector. Targeting vector was constructed according to a posi- tive-negative selection strategy. Using 16 kb fragment in pBSmOATG, exon 4 was disrupt- ed by insertion of Neo' gene at Sea I site for positive selection by G4I8. Two targeting vectors were made in which Neo' gene was infroduced in a forward and an opposite direction as referred to the coding sfrand of OAT gene. HSV-TK gene was added at the 3' end of the genomic fragment for negative selection by ganciclovir. The length of homolo- gous region was 14 kb upsfream from Neo"^ and 2.2 kb downstream from Neo*". This con- sfruct is designed for three favorable features: that genomic fragment used is isogenic to ES cell (129 sfrains of mice), that a positive-nega- tive selection can be used for identifying clones after elecfroporation into ES cells, and that a long homologous region is expected to yield a high frequency of homologous recom- bination events. FY 1994 NEI Annual Report We made several electroporations using different ES cells for their capacity to contrib- ute to germ-Une transmission. Cells were electroporated with OAT targeting construct replacement vector and selected with both G418 and ganciclovir, to enrich the homolo- gous recombination events. Two weeks later, resistant clones were picked up individually and grown for further analysis. Genomic DNA was purified from all clones and ana- lyzed by Southern blot using different restric- tion digests and differents probes. The results showed that several clones were recombinants on one allele. Four of them already have been injected to generate deficient mice for OAT gene. Presently we are collecting the first litters of chimeric mice for the OAT. Significance to Biomedical Research and the Program of the Institute The identification, characterization, mapping, and sequencing of the OAT gene is a very significant achievement in itself because we were able to study the functional OAT gene as well as several pseudogenes. We used 16 kb to construct two targeting vectors to mutate the functional OAT gene in ES cells. We were able to obtain 17 targeted recombinant clones, four of them have been already injected into embryos to generate deficient animals for the OAT gene. These deficient animals will be invaluable in many respects: (1) This v^ be the first animal model for ocular genetic diseases. (2) We will be able to study the physio- logical relevance of the OAT gene in vivo. (3) We wdll also find out if there is any relationship between retina degeneration and the absence of functional OAT enzyme. Proposed Course Our work in the future will focus on the following: (1) We will study the ocular physiology in the OAT deficient mice. (2) We will assess the importance of the absence of a functional OAT enzyme in vivo. (3) We wall use this animal model to assess the feasibility of gene therapy for gyrate atrophy disease. (4) We will study how to correct the enzymatic deficiency in vivo by transferring cells from normal donors to affected animals. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Esumi N, Jendoubi M: Isolation and charac- terization of the mouse ornithine 6-amino- transferase gene for gene targeting by homolo- gous recombination. Sixth International Sympo- sium of the Immunology and Immunopathology of the Eye, 1994, p 25. Esumi N, Jendoubi M: Advances in Ocular Immunology. Amsterdam, Netherlands, Elsevier Press, p. 151, 1994. Esumi N, Jendoubi M: Isolation, characteriza- tion and sequencing of the mouse ornithine 6- aminotransferase gene for gene, in prepara- tion. 162 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00288-02 LI PERIOD COVERED October 1, 1992 to September 30, 199 3 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Gene Therapy for Oc ul ar Genetic Disease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI Others: Noriko Esumi Daniel H. Lacorazza Luis J. Rivero Robert B. Nussenblatt M.D., Ph.D. Ph.D. M.D. Ph.D. Visiting Associate Visiting Fellow Visiting Fellow Scientific Director LI, NEI LI, NEI LI, NEI NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Section of Genetics and Molecular Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: PROFESSIONAL CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) THIS PROJECT HAS BEEN TERMINATED. 163 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00241-07 LI PERIOD COVERED October 1, 1 992 to Septembe r 30. 1 993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Immuno pathology of Ocular Diseases in Humans PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Others: Chi-Chao Chan Robert B. Nussenblatt Qian Li Marc D. de Smet Raymond DeBarge Scon M. Whitcup Juan Lopez Miguel Bumier Richard Fenton Dev Shah M.D. M.D. M.D. M.D. M.D. M.D. M.D. M.D. M.D. M.D. Chief, Section on Immunopathology Scientific Director Visiting Fellow Visiting Scientist Senior Staff Fellow Staff Medical Officer Visiting Associate Visiting Scientist Staff Fellow Visiting Associate LI, NEI NEI U, NEI U, ^fEI LI, NEI U, NEI LI, NEI LI, NEI LLNEI LI, NEI COOPERATING UNITS (if any) Department of Ophthalmology, Armed Forces Institute of Pathology (Ian W. McLean, M.D.); University of Minnesota, Department of Ophthalmology (Edward J. Holland, M.D.); L'Hopital de la Pitie, Paris, France (Pbuc LeHoang, M.D.) LAB/BRANCH Laboratory of Immunology Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL: 0.0 0.0 CHECK APPROPRIATE BOX(ES) (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project has been terminated and combined with Project No. ZOl EY 00222-08 LI. 164 PHS 6040 (Rev. 5/92) Laboratory of Mechanisms of Ocular Diseases Report of the Chief Laboratory of Mechanisms of Ocular Diseases J. Samuel Zigler, Jr., Ph.D. Investigators in the Laboratory of Mechanisms of Ocular Diseases (LMOD) are engaged in a broad range of studies relating to the biology of various tissues in the normal eye and the molecular mechanisms responsible for certain ocular diseases. Major emphasis has been on cataract and the various ocular complications of diabetes. The addition of Dr. Fred Bettelheim to the group as a part-time Special Volunteer and the initiation of new collaborative studies with Dr. Joseph Horwitz, from the Jules Stein Eye Institute, has broadened and strengthened our expertise in the biophysical aspects of lens opacification and in the use of human cataract samples in the laboratory. Section on Cataracts Dr. Donita Garland has developed new meth- ods for dissecting the human lens into distinct zones representing tissue produced during different stages of Ufe. Two-dimensional analysis of the proteins present in each of these regions provides a greatiy improved picture of the pattern of protein changes that occur as a function of aging and development in the lens. Marked differences are found between the nuclear and cortical protein patterns in both normal lenses and cataracts. Careful analysis of the protein content of dissected zones from intracapsular cataract lenses may provide the most definitive insight to date on the significance, with respect to cataractogenesis, of the changes that occur in both the lens crystallins and the metabolic proteins of the lens. Dr. Paul RusseU and his group are using a variety of model systems, e.g., whole animal, lens organ culture, and lens epitheUal ceU culture, to investigate the mechanisms that the lens uses to resist environmental and systemic stresses, which can lead to cataract. A great improvement in the efficiency of lens organ culture has resulted fiom the development of a simple and very effective means to identify lenses damaged during dissection. This al- lows elimination of bad lenses before experi- ments begin, thereby greatiy reducing the level of variability within experimental groups. Two new projects have been started dur- ing the past year. In one, transgenic mice that develop cataracts as the result of insertion of the human immunodeficiency virus protease gene wiU be studied to determine how expres- sion of this foreign protease in the lens leads to cataract. In the second new project, serum samples collected from patients with cataract and from normal volunteers wiU be analyzed for presence of autoantibodies in human lens proteins. By using two-dimensional elecfro- phoresis, the specific protein(s) to which antibodies have been produced can be identi- fied and correlated with cataract type and progression rate. 167 FY 1994 NEI Annual Report Dr. Fielding Hejtmancik and his group have had outstanding success in gene linkage studies on several ocular diseases. The locus for Usher's syndrome type I, which they previously Unked to chromosome llq, contin- ues to be refined using fine linkage mapping with the ultimate goal of identifying and characterizing the gene responsible for this disease. Linkage has also been obtained for two different cataract families during the past year. With the establishment of collaborative arrangements with clinical researchers in a variety of locations, including India, Italy, and Barbados, this group wiU have access to excellent material for the study of inherited cataract, retinal diseases, and glaucoma. Dr. Deborah Carper's laboratory has continued to study the role of the polyol pathway in the generation of diabetic compli- cations. Site-directed mutagenesis studies on aldose reductase (AR) have identified certain critical residues, information that wUl be invaluable in the effort to design more effec- tive inhibitors of this enzyme. Studies have also continued on sorbitol dehydrogenase, the second enzyme of the pathway, with determi- nation of the complete structure of the human gene as well as its chromosomal localization. Work is under way to identify the defect in this gene in a family that has cataracts associ- ated with sorbitol dehydrogenase deficiency. In Dr. J. Samuel Zigler's laboratory, work has concentrated on the testing of potential anticataract agents and on the analysis of the functions of lens crystallins and the role(s) they play in the normal lens and in the pro- cess of cataractogenesis. Studies done in collaboration with Dr. Joseph Horwitz are aimed at elucidating the physiological signifi- cance of the chaperone-Uke activity of a-crys- taUin. The possibility that the "enzyme/ crystallins" may be part of the lens' defenses against oxidation is being actively investigat- ed. Studies on the effect of smoke on the lens have provided further support for the view that it represents an important risk factor for cataract development. Section of Pathophysiology Dr. W. Gerald Robison and his colleagues are studying the process of diabetic retinopathy. By using advanced morphological and mor- phometric techiuques, they have analyzed the appearance of a variety of specific lesions in a rat model of this disease that are characteristic of changes seen in the human disease. They have also demonstrated that in the rat, these changes can be prevented by AR inhibitors. DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00105-15 LMOD PERIOD COVERED October 1, 1 9 93 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Structure and Composition of L e ns Crysta llin s Wit h Respect to Cataractog enesi s PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: J. Samuel Zigler, Jr. Ph.D. Research Biologist LMOD, NEI Others: Vasantha Rao Ph.D. Visiting Associate Pedro Gonzalez Ph.D. Visiting Associate Chuan Qin M.D. Visiting Fellow Frederick A. Bettelheim Ph.D. Special Volunteer LMOD, NEI LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (if any) Jules Stein Eye Institute, UCLA (J. Horwitz, Ph.D. and B. Bateman, M.D.); University of Tennessee (H.M. Jemigan, Jr., Ph.D.) National Cancer Institute (M. Krishna, Ph.D.); Centre for Cellular and Molecular Biology, Hyderabad, India (D. Balasubramanian, Ph.D. and M. Rao, Ph.D.) Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: PROFESSIONAL 4.0 4.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project, directed toward elucidation of the molecular mechanisms responsible for cataractogenesis and the development of means of prevention of this disease, places special emphasis on the structure and function of the lens crystallins and the role these proteins play in lens opacification. Until recently, crystallins were thought to be simply structural elements of the lens matrix without specific quantifiable biological functions. Two recent discoveries have provided new insights and approaches to the physiological roles of the crystallins: (1) the crystallins are either functionally active enzymes or are at least related to proteins with specific biological activities, and (2) a-crystallin is a molecular chaperone that can prevent the aggregation of denaturing proteins. We believe that crystallins have specific biological functions in addition to their structural role in forming the transparent lens tissue. The strongest example to date is the chaperone-like function of a-crystallin. This major lens protein prevents the aggregation of other proteins that are undergoing modification and denaturation. In the lens, where protein turnover is extremely limited and where the long-lived crystallins are known to undergo extensive structural modification, a-crystallin may be essential in preventing protein aggregation and precipitation. Such precipitation would destroy the optical transparency of the lens by creating light scattering centers. We have also developed evidence that the enzyme/crystallins are contributing to the antioxidative capacity of the lens, primarily by markedly increasing the pool of reduced pyridine nucleotides in the lens. We can demonstrate the utilization of the nucleotide's reducing capacity in eliminating activated species of oxygen generated by Fenton chemistry or by other mechanisms. Further support for the view that enzyme/crystallins have specific functions was obtained from studies on the expression of C- crystallin in the lenses of guinea pigs and llamas. The data clearly demonstrate that the £,-crystallin gene was recruited by the lens independently in each of the two species. This finding strongly supports a selective basis for the recruitment rather than a neutral evolution mechanism and indicates that the protein must provide significant benefit to the lens of these species. 169 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The primary objectives of this project are to: (1) elucidate processes responsible for cataract development at the molecular level, (2) inves- tigate the structures and functions of the lens crystallins, and (3) develop and use model systems for screening potential anticataract agents and test them in appropriate in vitro and animal model systems. Methods Conventional protein chemical techniques used are chromatography, electrophoresis, and isoelectrofocusing. Immunological studies of lens proteins use specific antisera. Physico- chemical analyses on the proteins are per- formed using high-pressure liquid chromatog- raphy, fluorescence, and circular dichroism techniques. Lens organ culture experiments use rat or monkey lenses and use active trans- port and membrane permeability parameters to monitor the effects of various stresses on the cultured lenses. Techniques used in analysis of nucleic adds include ribonucleic acid and deoxyribo- nucleic acid (DNA) isolation, complementary DNA and gene cloning, DNA sequencing, various electrophoretic methods, and the pol5anerase chain reaction. Major Findings (1) In collaboration with Dr. Joseph Horwitz, from the Jules Stein Eye Institute, we have further characterized the chaperone-like activi- ty of a-crystaUin and analyzed the specificity of the reaction with different target proteins. With some denaturing proteins, the presence of obligate cofactors is critical if a-crystaUin is to effectively prevent aggregation. Circular dichroism spectroscopy suggests that subtle conformational changes associated with the binding of the cofactor may be essential for interaction of a-crystallin with these proteins. Our findings suggest that a-crystaUin is spe- cifically adapted to function as a chaperone within the lens. (2) The putative promoter region previ- ously identified in the Uama ^-crystaUin gene has proved to be active by functional analysis. The studies in transfected cells also indicate that the promoter is highly lens specific, i.e., does not drive gene expression in other tis- sues. (3) The human gene for ^-crystallin has been characterized and shown to have essen- tially the same structure as found in the guinea pig and Uama. However, unlike the guinea pig and Uama genes, it lacks a second lens-specific promoter and consequentiy is not expressed at a high level in the lens. A pro- cessed pseudogene for ^-crystaUin was also found to be present in the human genome. (4) A catalytic activity that uses the re- ducing equivalents of pyridine nucleotides to eliminate free radical oxidants has been identi- fied in the lens. We beUeve that this activity, which appears to be associated with a protein, may be an important part of the lens' antioxi- dant capacity. Because NAD(P)H is renew- able via redox cycling, it is conceivable that its reducing equivalents may function in a man- ner analogous to the glutathione redox cycle. (5) Smoke has been identified as a risk factor for cataract in several epidemiological studies. In coUaboration with Dr. Ch. Mohan Rao, from the Center for CeUular and Molecu- lar Biology, Hyderabad, India, we have ex- posed organ-cultured rat lenses to a conden- sate of wood smoke. The studies indicate that components of the condensate accumulate within the lens, are metabolized, and cause damage to the ceU membranes. Histological analysis demonstrated early and severe injury to the lens epithelial ceUs. (6) Studies with Dr. Fred Bettelheim, also using rat lenses in organ culture, have estab- Ushed a calcium-induced cataract model as a system for studying the role of optical aniso- tropy fluctuations in lens opacification. Early results suggest a major role for the intermedi- 170 Laboratory of Mechanisms of Ocular Diseases ate filament protein, vimentin, in establishing and maintaining proper order within the cytoplasm of lens fibers. Loss of vimentin via calcium-induced proteolysis leads to optical anisotropy fluctuations that cause lens turbid- ity. (7) The testing of potential agents for the prevention or retardation of cataract develop- ment is being performed in the lens organ culture system on compounds with antioxida- tive capacity. Compounds that provide prom- ising results will be prepared for testing in Significance to Biomedical Research and the Program of the Institute Cataract is a major pubUc health problem worldwide. Better understanding of the biochemistry of the normal lens and of the molecular changes that occur during aging and cataract development are essential if this disease is to be controlled. Our studies are aimed primarily at elucidating the role of the lens crystallins, the primary structural ele- ments of the normally transparent lens matrix, in the processes leading to opacification. Such knowledge should contribute to the development of means of intervention that can prevent or delay the process of cataract devel- opment. Proposed Course We wiU: (1) work to establish viable model systems for testing anticataract agents and use these systems to assess the efficacy of various types of compounds, including antioxidants; (2) clarify the mechanism and the significance of the pjoidine nucleotide-dependent antioxi- dant system in the lens and investigate the possible role of enzyme /crystallins in this system; (3) continue to investigate the chaper- one-like function of a-crystallin and determine its physiological significance in the normal lens and in cataract; and (4) expand the two- dimensional analysis of proteins from human normal lenses and cataracts through the use of preliminary fractionation by affinity chroma- tography. Studies under way using blue Sepharose affinity columns suggest that this will be a very useful way to reduce the com- plexity of the protein mixture as well as to focus on specific classes of proteins. NEI Research Program Lens and Cataract— Pathogenesis of Cataract Publications Bettelheim FA, Qin C, Zigler JS Jr: Calcium cataract: A model for optical anisotropy fluctuations. Exp Eye Res, in press. Cui X-L, Qin C, Zigler JS Jr: Residual EDTA bound by lens crystallins accounts for their reported resistance to copper-catalyzed oxida- tive damage. Arch Biochem Biophys 308:207- 213, 1994. Gonzalez P, Hemandez-CalzadiUa D, Rao PV, Rodriquez IR, Zigler JS Jr, Borras T: Compar- ative analysis of the ^-crystaUin/quinone reductase gene in guinea pig and mouse. Molec Biol Evolution, 11:305-315, 1994. Gonzalez P, Rao PV, Zigler JS Jr: Organiza- tion of the human I;-crystallin/qviinone reduc- tase gene (CRYZ). Genomics 21:317-324, 1994. Heinzmaim C, Kojis TL, Gonzalez P, Rao PV, Zigler JS Jr, Polymeropoulos MH, Klisak I, Sparkes RS, Mohandas T, Bateman JB: As- signment of the ^-crystallin gene (CRYZ) to human chromosome Ip22-lp31 and identifica- tion of restriction fragment length polymorph- isms. Genomics, in press. Persson B, Zigler JS Jr, JomvaU H: A super- fanuly (MOR) of medium chain dehydrogen- ases/reductases: Sublines including t-crystal- lin, alcohol and polyol dehydrogenases, qui- none oxidoreductases, enoyl reductases, VAT- 1 and further proteins. Eur J Biochem, in press. Rao PV, Horwitz J, Zigler JS Jr: Chaperone- like activity of a-crystaUin. J Biol Chem 269:13266-13272, 1994. FY 1994 NEI Annual Report Tumminia SJ, Qin C, Zigler JS Jr, Russell P: Zigler JS Jr: Lens proteins, in Albert DM, The integrity of mammaUan lenses in organ Jakobiec F (eds): Principles and Practice of culture. Exp Eye Res 58:367-374, 1994. Ophthalmology. Basic Sciences, Philadelphia, JB Saunders Co., 1994, pp 97-113. Tumminia SJ, Rao Pv, Zigler JS Jr, Russell P: Xenobiotic induction of quinone oxidoreduc- tase activity in lens epitheUal cells. Biochim Biophys Acta, 203:251-259, 1993. 172 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00189-11 LMOD PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Oxidation of Proteins in Cataractogenesis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Donita L. Garland Ph.D. Research Chemist LMOD, NEI Others: Jose Jimenez Lx)renzo Merola Kenichi Matsuno Ph.D. M.S. Ph.D. Visiting Fellow Chemist IRTA LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.3 PROFESSIONAL: 3.3 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Oxidative processes are a major contributing factor in senile cataracts. We have demonstrated that metal catalyzed oxidation of the crystallins induces protein modifications that mimic those seen in aging, senile cataracts, and brunescent lenses. The importance of the role of metal in oxidative processes related to cataract is further supported by studies in other labs. The lens contains high levels of thiols such as glutathione that can participate in these metal-catalyzed oxidation reactions. Our lab has continued our studies on both the interaction of metals with crystallins and the mechanisms that protect the lens against deleterious oxidation reactions. A protein that protects enzymes specifically against inactivation by thiol-dependent metal-catalyzed reactions has been reported in yeast and rat tissues. (This activity is not related to catalase, glutathione peroxidase, or superoxide dismutase.) Our lab demonstrated the presence of a similar activity in lenses of bovine, guinea pig, human, pig, monkey, and rat. The antioxidant activity consistently copurifies with a subpopulation of glutathione S-transferase ju. Copper, zinc, and iron, but not calcium, induced aggregate formation in bovine lens extracts and solutions of the crystallins. Aggregation, measured by light scattering, was time dependent, occurring at metal-to-protein ratios greater than one and varying, depending on the metal and protein. Zinc induced the aggregation of P- and a- but not y-crystallin. The affinity of copper and zinc for these proteins is relatively low. The addition of EDTA, DETAPAC, L-histidine, or L-cysteine prevented zinc- and copper-induced protein aggregation and caused complete disaggregation. The treatment of a- and P-crystallin and trypsin inhibitor with diethylpyrocarbonate prevented aggregation induced by zinc but not by copper. The presence of salt decreased the metal-induced aggregation of a- and (3-crystallin. No metal-induced changes in secondary and tertiary structures of these proteins were observed by fluorescence and circular dichroism spectroscopy. 173 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The long-term goal of this project is to under- stand the role of oxidation in cataract forma- tion. The immediate objectives are to: (1) study the effect of oxidation on structure and function of lens crystallins, (2) study the interaction between crystallins and those metals that are involved in oxida- tion/reduction reactions and the effects of these interactions on crystallin solubihty and aggregate formation, and (3) characterize the enzymes that protect lens proteins against thiol-dependent metal-catalyzed oxidation. Methods Bovine, rat, and guinea pig tissues were used for these studies. We used classical methods to purify proteins. Other methods used were standard procedures for studying proteins, polyacrylamide gel electrophoresis, high- pressure Uquid chromatography, ultraviolet spectroscopy, fluorescence, circular dichroism, electron spin resonance, amino acid analysis, immunotechniques, and capillary gas Uquid chromatography. Major Findings Oxidative processes are considered to be a major contributing factor in senile cataracts. We have demonstiated that metal-catalyzed oxidation of the crystallins induces protein modifications that mimic those seen in aging, senile cataracts, and brunescent lenses. The importance of the role of metal in oxidative processes related to cataract is further sup- ported by studies in other laboratories. The lens contains high levels of thiols such as glutathione that can participate in these metal- catalyzed oxidation reactions. Our laboratory has continued studies on both the interaction of metals with crystallins and the mechanisms that protect the lens against deleterious oxida- tion reactions. (1) A protein that protects enzymes spe- cifically against inactivation by thiol-depen- dent, metal-catalyzed reactions has been reported in yeast and rat tissues. (This activity is not related to catalase, glutathione perox- idase, or superoxide dismutase.) Our labora- tory demonstrated the presence of a similar activity in the lens of bovine, guinea pig, human, pig, monkey, and rat. Following the antioxidant activity, a protein was purified from bovine lenses. Seventy percent of the protein sequence was obtained. The protein was identified as glutathione S-transferase /i, a detoxification enzyme found in most cells. This enzyme is not known to possess an antioxidant activity. The antioxidant activity consistentiy purifies with a subpopulation of glutathione S-transferase through many differ- ent purification schemes, yet we have not proved that the antioxidant activity is part of the glutathione S-transferase molecule. It is possible that this subuiut may form a hetero- dimer with glutathione S-transferase. Studies are in progress to try to resolve this issue. Other Unes of evidence indicate the pres- ence in the lens of a protein(s) more closely related to the antioxidant protein characterized from yeast and rat brain. Antibodies made against the whole protein or to an internal peptide crossreact with proteins from lens but not glutathione S-transferase. Further North- em-blot analysis of total ribonucleic acid (RNA) from cow, human, monkey, and rat lens was performed using a complementary deoxyribonucleic acid (cDNA) probe made to the rat antioxidant protein. The results clearly demonstiated the presence of specific messen- ger RNA for this particular gene. (2) Copper, zinc, and iron, but not calci- um, induced aggregate formation in bovine lens extiacts and solutions of the crystallins. Aggregation, measured by light scattering, was time dependent, occurring at metal-to- protein ratios greater than one and varying depending on the metal and protein. Zinc induced the aggregation of p- and a- but not Y-crystaUin. Copper and zinc induced the aggregation of a number of other proteins, but they had no effect on lysozyme and papain. Laboratory of Mechanisms of Ocular Diseases One explanation for the lack of effect on these two proteins is that they are basic proteins. However, copper induced the aggregation of Y-crystallin, also a basic protein. The affinity of copper and zinc for these proteins is relatively low. The addition of EDTA, DETAPAC, L-histidine, or L-cysteine prevented zinc- and copper-induced protein aggregation and caused complete disaggrega- tion. The treatment of a- and |3-crystaUin and trypsin inhibitor with diethylpyrocarbonate prevented aggregation induced by zinc but not by copper. The presence of salt decreased the metal-induced aggregation of a- and p- crystallin. No metal-induced changes in secondary and tertiary structures of these proteins were observed by fluorescence and circxilar dichroism spectroscopy. In the presence of zinc, lens crystallins were least soluble at 2.0 - 2.5 pH units higher than their isoelectric points. The effects of varying pH were fully reversible. Solubility decreased further with increasing temperature. Lowering the temperature did not induce any disaggregation, indicating that the tempera- ture effect on zinc-induced aggregation was not reversible. The mechanism of metal-induced aggrega- tion is not clear. Effects of the metals on conformation could not be demonstrated. The data obtained to date support a neutralization mechanism of aggregation more than a cross- linking mechanism, but these studies are still in progress. (3) Capillary gas chromatography (GLC) is used in this laboratory to study sugar metabolism in the lens of two animal models, rat and monkey. Standard methodologies have been used to quantitate the common sugars. Methods have been devised in this laboratory that allow the separation of sorbitol and dulcitol, the alcohol derivatives of glucose and galactose. Sugar analyses have been done on serum of animals fed various sugar diets and rat lenses incubated in vitro with a variety of sugars. The chromatogram obtained from the capillary GLC analysis of animal blood serums and lens material contains, in addition to the expected sugar derivatives, a number of unidentified metabolites present in low quan- tities. Our laboratory is in the process of identifying these. One of those metabolites present in both rat and monkey lenses, has been tentatively identified as scylloinositol. Significance to Biomedical Research and tlie Program of the Institute Oxidative processes are known to contribute to cataractogensis. Metal-catalyzed oxidation of the crystallins leads to protein modification that mimic those seen in aging, senile cata- racts, and brunescent lenses. Studies on the interaction of metals with the crystallins are highly relevant to understanding the role of these oxidation reactions in lens. Understand- ing those mechanisms present in lens for protecting against oxidative damage is impor- tant for developing interventions. Studies on glutathione S-transferase are highly relevant to cataract. Recent reports correlate deletions on glutathione S-transferase /x with certain cata- racts. Proposed Course We wiU focus our studies for fiscal year 1995 on the following: (1) pursuing the activity in lens that protects against thiol-dependent oxidation reactions, (2) assaying for glutathi- one S-transferases in epithelial layers from cataract surgeries, and (3) continuing the studies on the interaction of metals with the crystaUins. NEI Research Program Lens and Cataract — Pathogenesis of Cataract Publications Bettelheim FA, Reid MB, Garland D: Hydra- tion of gamma crystallins. Exp Eye Res 58:219- 224, 1994. DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00296-01 LMOD PERIOD COVERED October 1, 1993 to September 30, 1994^ TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Studies on Human Lens Proteins PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Donita L. Garland Ph.D. Research Chemist LMOD, NEI Others: J. Samuel Zigler, Jr. Ph.D. Senior Scientist Yvonne Duglas-Tabor B.A./B.S. Biologist LMOD, NEI OGCS, NEI COOPERATING UNITS (if any) Ophthalmic Genetics and Clinical Services Branch, NEI, NIH (Manuel B. Datiles, M.D.); Jules Stein Eye Institute, UCLA (J. Horwitz, Ph.D.) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: PROFESSIONAL: 0.7 0.7 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This laboratory has in progress a study to characterize the proteins of the human lens. The lens consists of a few very high abundance proteins called the crystallins and several hundred lesser abundance proteins. During the life of the lens, the proteins become extensively modified. Methodologies have been developed to yield good separation of the proteins using two-dimensional polyacrylamide gel electrophoresis. Due to the extensive modifications, identification of the proteins is based on immunotechniques and sequencing. Normal donor lenses varying in age from fetal to 70 years and cataracts of different etiologies have been analyzed. The regions of the normal lenses are identified by structure patterns. The cortex has been separated into three different layers, and each of the developmentally defined nuclear regions has been separated. The protein patterns in each of the cortical regions are distinguishable as cortex with the characteristic large protein spots corresponding to each of the major crystallins. The protein patterns of the nuclear regions are all similar to each other but clearly unique from those of the cortical regions. Protein spots corresponding to the major crystallins are not readily visible. Many new spots are present, including numerous low molecular weight spots that are fragments of crystallins. These results have significance in understanding the potential role of protein modification in cataractogenesis. The proteins throughout the lens are being identified, yielding a database of information on the normal human lens. The protein patterns of numerous cataracts have been determined. These data are now being analyzed with respect to the cataract etiology. 176 PHS 6040 (Rev. 5/92) Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The long-term goal of this project is to under- stand those mechanisms involved in human cataractogenesis. The immediate objectives of this project are to: (1) identify the proteins of the human lens and characterize the changes in the protein compo- sition that occur with development and aging, (2) identify the covalent modifications that the human lens proteins undergo with develop- ment and aging, and (3) characterize the proteins of human cataracts of various etiologies. Methods Human lens material was obtained from donors' eyes and from cataract surgery. Immobilized pH gradients and the Isodalt two-dimensional electrophoresis system were used to separate the lens proteins. Other techniques used include high-pressure liquid chromatography, ultraviolet /visible spectros- copy, and immunotechniques. Major Findings Normal donor lenses of ages varying from seven months to 60 years were dissected, and the lens regions were identified by suture patterns. Adult lenses were easily and clearly separated into regions corresponding to elon- gating fiber cells, outer cortex, inner cortex, adult nucleus, fetal nucleus, and embryonic nucleus. In some lenses, another layer was observed that may correspond to the juvenile nucleus. These separations are consistent with the zones of discontinuity seen microscopically The proteins of each region were separat- ed by two-dimensional electrophoresis. For the three cortex layers, the two-dimensional protein patterns are distinguishable as cortex. These patterns are characterized by large protein spots corresponding to aA-, aB-, pBl- VS-, and (3B2-crystallins as well as 100 to 200 lesser abundant spots. Comparisons of the three cortex regions yield some differences among the protein patterns. The inner cortex of the mature lens has an increase in the acidic forms of a crystallin, bBl-crystaUin spots are greatly diminished, and many lesser abundant spots are either increased or decreased. In contrast, the protein patterns of the nuclear regions of adult lenses are unique and distinguishable from those of the cortical regions. The crystaUins have undergone extensive modification. Protein spots corre- sponding to the major crystaUins are not visible. Many new spots are present, and there is a significant increase in the number of low molecular spots that are fragments of crystaUins. The protein patterns, including the low molecular weight species, are simUar in aU adult lenses analyzed. The clear distinction between the protein patterns of the cortex and those of the nucleus suggests that certain modifications of the crystaUins occur in the developmentaUy defined nuclear regions but not in the cortical regions. It is not clear if there are signals that initiate the modifications of crystaUins in the nucleus or if there are signals that prevention the modifications of proteins in the cortex. The protein patterns of the adult, fetal, and embryonic nuclear regions are simUar in the adiilt lens. The protein patterns of the nuclear regions of lenses fiom birth to about 30 years change with increasing age from a pattern closer to a cortical pattern to that of a nuclear protein pattern. For lenses older than 30 years, the protein patterns of the nuclear regions appear to remain stable. Predictable protein patterns are found in aU normal lenses in comparable regions of lenses of comparable ages. Thus, these results are consistent with the interpretation that the extensive modifications seen in the two-di- mensional protein patterns do not represent deleterious, random modifications that result from decreased lens viability or form exposure to noxious environmental agents. These 177 FY 1994 NEI Annual Report modifications are likely not related to cataract formation. Rather at appropriate developmen- tal stages, a series of reactions occur leading to the covalent modification of the crystallins. These modified forms of the crystallins may be better suited for function in the center of the lens in maintaining transparency. Numerous studies have demonstrated that with time many of the lens proteins undergo acidification. Our studies demonstrate that the enzymes such as glyceraldehyde 3-phos- phate dehydrogenase are present as multiple- charge species even in the outermost layers of the cortex. This suggests that the conversion of these enzymes to more acidic forms is not likely to be just the result of aging as has been suggested. The proteins of the cataractous regions of posterior subcapsvdar cataracts with diagnoses of serule, presenile, radiation, retinitis pigmen- tosa, and gyrate atrophy have been analyzed by two-dimensional electrophoresis. Some differences in the protein patterns exist, but, so far, no protein changes can be correlated with any disease etiology. The proteins of a large number of cortical and nuclear cataracts have been analyzed. Correlations between protein changes and cataract types have not yet been found using the present methods of classifying cataracts. The data obtained to date indicate that there are no obvious, unique crystallin modifications responsible for each type of cataract. Many of the proteins in the human lens have been identified using immunoblotting techniques. Degraded forms of aB-crystallin have been identified in both normal and cataractous lenses. Blots of human lens proteins separated by two-dimensional electrophoresis are being used to identify the targets of autoantibodies found in normal and cataract patients. Significance to Biomedical Researcti and ttie Program of tfie Institute These studies are directiy relevant to the NEI program. Characterization of the proteins of the normal human lens will provide informa- tion necessary to understand cataractogenesis. In addition, it wiU provide information on normal metabolic and developmental process- es in this unique tissue in contrast to those processes that occur as a function of aging. Proposed Course In fiscal year 1995, we will continue the char- acterization of human lens proteins. Our studies wiU include continued efforts to identi- fy the modified proteins and the modifications involved, the purification of modified crystal- lins for sequencing and mass spectroscopy, and the continued analysis of cataracts. NEI Research Program Lens and Cataract — Lens Development and Aging 178 PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00201-10 LMOD PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Structure and Exp re ssion of Polyo l Pathway Enzym es PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Deborah Carper Ph.D. Biologist LMOD, NEI Others: Susan Old Ph.D. Staff Fellow LMOD, NEI Takeshi Iwata Ph.D. Visiting Associate LMOD, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS; 3.0 PROFESSIONAL 3.0 0.0 CHECK APPROPRIATE BOX{ES) □ (a) Human subjects [x] (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) In tissues that do not require insulin for glucose uptake, the high systemic level of glucose that develops during diabetic hyperglycemia readily translates into high tissue levels of glucose. Some of this excess glucose is metabolized by the polyol pathway. Aldose reductase (AR), the first enzyme of this pathway, reduces glucose to the organic osmolyte sorbitol while sorbitol dehydrogenase (SDH), the second enzyme of the pathway, oxidizes sorbitol to fructose. The diabetes-enhanced flux of glucose through the polyol pathway has been implicated in the etiology of diabetic complications, including cataract, retinopathy, and neuropathy. Our studies have been aimed at defining the structure and regulation of AR and SDH so that new approaches to the control of this pathway may be made available in diabetic tissues. Many aldose reductase inhibitors (ARIs) have been shown to have broad substrate specificity and undesirable side effects. Emphasis on the structure/function properties of the AR enzyme will help in the refinement and design of future inhibitors. To this end, catalysis and inhibition of aldose reductase was examined following site-directed mutagenesis. In the rat, our mutagenesis studies indicated that tyrosine 48, histidine 110, and cysteine 298 are important residues in the active site and that tyrosine 48 is most likely the proton donor during substrate reduction. In addition, mutation of histidine 110, an active-site residue, decreased inhibitor effectiveness by up to 2000-fold, indicating that this residue is important in inhibitor binding. Mutations of human AR indicated that although phenylalanine 122 putatively binds ARIs, substitution of this amino acid has little affect on the inhibitor-binding constant, K,. Sorbitol dehydrogenase has been reported to be linked to cataract formation in nondiabetics. We have determined the gene structure, tissue distribution, transcriptional initiation, and chromosomal localization of human SDH. In addition we have used SSCP analysis followed by sequence comparison to analyze congenital cataract patients with SDH deficiency. Through these studies, we hope to identify the gene defect in this family. 179 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The objective of this project is to study the structure, function, and regulation of the two enzymes of the polyol pathway — aldose re- ductase (AR) and sorbitol dehydrogenase (SDH). Methods The methods used include molecular biology, protein chemistry, cell biology, and molecular genetics. Major Findings Structure-function studies of AR. X-ray crystal- lographic studies previously indicated that either Y48, HI 10, or C298 could potentiaUy function as the proton donor in the catalytic mechanism of AR. The mechanism involves binding of the substrate to the enzyme/ NADPH complex with subsequent hydride transfer from the rucotinamide ring to the carbonyl group. A proton is then donated to the carbonyl oxygen. Our studies showed that mutagenesis of rat lens tyrosine 48 to phenyl- alanine (Y48F) virtually abolished rat lens enzyme activity, even though the physical structure of Y48F appeared to be normal as evidenced by circular dichroism spectra and NADPH-binding affini*^v constants, Kd. Changes of histidine 110 to glutamine (HllOQ) and cysteine 298 to serine (C298S) resulted in mutants that were still active. Our findings would indicate that Y48 is the proton donor in the reduction reaction of rat lens AR. The potential role of the amino adds C298, HI 10, H187, and H200 in the inhibition of rat lens AR was examined by measuring the IC50 values of five different AR inhibitors. No differences between the wild tjrpe and the mutants were observed, except for HllOQ. This AR mutant was less sensitive to inhibi- tion by both the carboxylic and hydantoin classes of compounds. The greatest increase in IC50 was seen with the hydantoins. There was a three hundredfold increase for sorbinil and a two thousandfold increase for tmeristat. The carboxylic acids such as tolrestat and statiJ gave increases of approximately fivefold. These inhibitor studies indicate that HI 10 is important in inhibitor binding. The mecha- nism involved may include perturbation of the positively charged anion weU formed by Y48, HI 10, and the nicotiniamide ring with variable effects on AR inhibitors due to their inherently different structural properties. The efficacy of AR inhibitors are evaluated by their ability to lower polyol levels and prevent or retard cataract formation, retinal microangiopathies, microalbuminuria, decreased motor nerve conduction velocity, and /or deterioration of nerve ultrastructure. Because the rat is the most widely used model of diabetic complica- tions, comparative information on the catalysis and inhibition of rat and human AR has provided a baseline for designing specific inhibitors. Ovu: studies on the mutagenesis of human AR have been focused on elucidating the residues involved in inhibitor binding. X-ray crystallographic studies indicated that a num- ber of hydrophobic interactions occur between the enzyme and the inhibitor. The inhibitor was proposed to be sequestered by a hydro- phobic bridge comprising phenylalanine 122 and leucine 300. We have begun to evaluate this location by mutating phenylalanine 122 to t)n"osine and cysteine. Small changes in the affinity to various substrates. Km, and in the turnover number, Kcat, were observed. How- ever, contrary to the predicted importance of this residue, there was no significant change in the inhibitor-binding constant, Ki. Gene structure of sorbitol dehydrogenase. The complete complementary deoxyribonucleic acid (cDNA) sequence coding for human SDH and the genomic organization of this enz5nne were determined. SDH is arranged into nine exons and eight introns. The first exon con- tains 89 bp of 5' untranslated sequence, and exon nine contains 1,263 bp of 3' untranslated sequence. This considerably long stretch of 3' untranslated sequence comprises more than 60 percent of the total cDNA sequence, the im- 180 Laboratoii/ of Meclmnisms of Ocular Diseases portance of which is unknown, although it is likely to include messenger ribonucleic acid stability and translational regulation. Homol- ogous human alcohol dehydrogenase genes and the human ^-crystaUin gene are also arranged into nine exons and eight introns, but none of the spUcing points coincide with the splice points of the SDH gene. At the promoter region of human SDH, no obvious TATAA or CCAAT box was found. Interestingly, different transcription initia- tion sites were observed in Uver and lens. These different tianscription initiation sites do not affect the tianslation initiation site (ATG-codon). At the 5' flanking region of this gene, which is reported to bind Spl tianscrip- tional factor, three Spl and a CACCC box were observed. The sequential motif of the promoter region resembles that of duck lactate dehydrogenase p/e-crystaUin that is highly expressed in heart as an enzyme and in lens as a crystaUin. That SDH may also be an enzyme /crystaUin is an interesting possibUity. Fluorescence in situ hybridization localized the SDH gene to human chromosome 15q21.1. Northern blot analysis demonstrated that the highest expression of SDH was in lens. Other tissues with high expression were kidney, heart, brain, testes, retina, and the retinoblastoma ceU hne Y79. The high expres- sion of SDH in human lens is of great interest in view of previous reports showing abnormal SDH activity in red blood cells of patients who develop cataracts. A congenital cataract patient from the largest known family with red blood cell SDH enzyme deficiency was examined for muta- tions by single standard conformation poly- morphisms and DNA sequencing. In this family, four out of five brothers and their father had bilateral cataracts. Although SDH activity is reduced 18 to 78 percent of normal in all members of this family, the occurence of cataract does not appear to be correlated with the severity of the red blood cell deficiency. However, this does not rule out a correlation between cataract and lens SDH activity. In this study, a polymorphic one bp mismatch in exon four and a two bp deletion followed by a one bp mismatch in exon nine were found, but no mutation within the coding sequence was detected. Thus, the enzyme deficiency is probably not due to abnormal protein struc- ture, and other possibilities such as reduced promoter activity are now being examined. We have demonstrated high expression of SDH in human lens compared with other tissues. A previous study reported higher enzyme activity in human lens compared with other species. These data suggest that SDH may play an important role in the human lens, and dysfunction of this enzyme may lead to alterations in the polyol pathway. Significance to Biomedical Research and ttie Program of tlie Institute The polyol pathway has been implicated in ocular and other compUcations of diabetes. Regulating the enzymes of this pathway — AR and SDH — in a manner that will decrease the flux of glucose through the polyol pathway should ameliorate these compUcations. Using structure /function studies, the localization of the inhibitor site of AR wiU provide a rational basis for inhibitor drug design. By character- izing SDH, we can begin to evaluate its role in the lens and its interrelationship with AR. Proposed Course Site-directed mutagenesis studies will continue to be used to determine the inhibitor-binding site of human AR. For SDH, the functional promoter wiU be defined, and the other mem- bers of the family with lowered SDH activity and cataracts wiU be examined. NEI Research Program Lens and Cataract — Molecular Genetics Publications Bateman JB, Kojis T, Heinzmann C, Klisak I, Diep A, Carper D, Nishimura C, Mohandas T, Sparkes R: Mapping of AR gene sequences to FY 1994 NEI Annual Report human chromosomes 1, 3, 7, 9, 11, and 13. Sato S, Old S, Carper D, Kador PF: Purifica- Genomics 17:560-565, 1993. tion and Characterization of Recombinant Human Placental and Rat Lens ARs Expressed in Esche- Iwata T, Carper D: Human Sorbitol Dehydroge- richia coU. New York, Plenum Publishing nase Gene. New York, Plenum Publishing Corp., in press. Corp., in press. Old SE, Carper DA, Hohman, TC: Na,K-ATPase response to osmotic stress in primary dog lens epithelial cells. Invest Ophth- almol Vis Sci, in press. 182 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00237-09 LMOD PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on or)e line between the borders.) Characterization of the Lens PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Paul Russell Ph.D. Research Chemist LMOD, NEI Others: Carolyn Chambers Geoffrey Kidd Santa Tumminia Ph.D. Ph.D. Ph.D. Senior Staff Fellow Senior Staff Fellow Senior Staff Fellow LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (if any) Oakland University (John Reddan, Ph.D.); Purdue University (Jean Smith, Ph.D.); University of East Anglia (George Duncan, Ph.D.); National Institute of Child Health and Human Development (Jose Pichel, Ph.D. and Heiner Westphal, Ph.D.) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.6 PROFESSIONAL: 3.6 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) We are continuing our efforts in characterizing the lens and processes that may occur in cataract development. There are three aims in our research efforts: determination of the seqeuences of human crystallins, development of the in vitro lens incubation system, and examination of stress on lenses and lens crystallins. Each of these areas complements the Other and builds a base on which to study how the lens resists cataract development and the stresses that will eventually lead of opacification. The first area of work has been the determination of the sequence of human p B2-crystallin and human yS-crystallin. The human p B2-crystallin is the principal p crystallin in the lens. Two human lens complementary deoxyribonucleic acid (cDNA) libraries were made. One of the libraries was to adult lens and one was to fetal lens. The p B2-crystallin cDNA was cloned and sequenced from these libraries and the deduced amino acid sequence was determined. Work also continued on the promoter region of this crystallin in order to develop a construct that would be developmentally regulated in transgenic animals. yS-crystailin is a protein that increases in content in the lens with age. The amount of this crystallin is very often decreased with the advent of human cataract formation. The yS-crystallin sequence was determined by a combination of cDNA sequencing, electrospray ionization mass spectrometry, and fast atom bombardment mass spectrometry. In vitro lens incubation is currently one of the only ways to study the effect of environmental stresses on whole lens metabolism. Work has progressed to establish criteria for determining which of the in vitro incubated lenses has metabolic integrity and has not been injured in the dissection process. A simple test has been developed to determine the integrity of the lens in vitro in as few as 30 minutes after the start of the incubation procedure. Studies have been done in vitro to determine the response of the whole lens to oxidative insult using the content and mRNA levels of catalase, an enzyme responsible for protection against H2O2. Glutathione levels of stressed lenses have also been measured to determine the reason for the loss of this vital constituent. The effects of environmental stresses have also been studied using cell culture systems. The cell line used in these studies constitutively expresses aB-crystallin. Effects of oxidative stress and heat shock have been investigated on this crystallin that is thought to serve as a molecular chaperon. 183 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The purposes of this project are to: (1) deter- mine the components in the human lens, (2) develop model systems to examine how stresses such as oxidation can alter the tissues in the anterior segment of the eye, (3) define these model systems for use with agents that might delay cataract formation, and (4) ex- plore basic questions about the metabolism of the lens using cell and molecular biological methods. Methods Among numerous biochemical and molecular biological methods used in this research are Northern, Southern, and Western blotting of messenger ribonucleic acid, deoxyribonucleic add (DNA), and proteins. In addition, vari- ous methods for quantitation of these compo- nents such as slot-blotting are done. The polymerase chain reaction is used as is nucleic acid sequencing. Major Findings (1) Two libraries of the complementary cDNAs from human lenses have been made. One of the libraries is from adult lens, and one is from fetal lens. (2) The sequence of the human pB2-crys- tallin has been detemiined from cloned se- quences from the human lens libraries. The sequence of the human |3B2-crystallin is simi- lar to the sequence of the bovine and rat crystallins and shows the high level of homol- ogy in this crystallin. The deduced sequence of the pB2-crystallin matches exactiy the protein sequence that was published concur- rently by others. (3) The sequence of human vS-crystallin has been determined using a combination of cDNA sequencing, electrospray ionization mass spectrometry, and fast atom bombard- ment mass spectrometry. The human se- quence differs from the bovine sequence in several areas, including one peptide fragment that appears to be associated with cataract development. (4) A method for the determination of protein content in the incubation medium has been tested as a means to determine lens integrity in vitro. The method, which is rapid, can accurately predict which of the lenses in vitro has been metabolically compromised during the dissection procedure. (5) Studies on rat and monkey lenses incubated in vitro have suggested that the rapid loss of glutathione in the young rat lenses in vitro may be due to the rapid growth rate of these lenses. Older lenses and lenses from adolescent monkeys do not exhibit the rapid loss of this constituent. The loss of glutathione in the rat lens has long been an enigma. (6) aB-crystallin, a molecular chaperone, has been studied using a cultured cell line that constitutively expresses this protein. In the U373MG astroglioma ceU Hne, the accumula- tion of this crystallin has been shown to be stress dependent and phosphorylation inde- pendent. Heat shock will cause a rapid rise in the level of this protein, but within four to six hours the levels of aB-crystallin return to baseline values. Incubation of the cell with cobalt wiU also cause a rise but at a later time, and the level does not return to baseline within 72 hours. The aB-crystaUin also ap- pears to switch from water soluble to w^ater insoluble after the insult suggesting an alter- ation in the compartmentalization of this chaperone. The level of phosphorylation of the aB-crystaUin was not changed during the stress experiments, indicating phosphorylation was not an important event for the function of this protein in response to stress. Significance to Biomedical Research and the Program of the Institute The human lens needs to be characterized to determine which components are involved in the protection of this tissue from envirorunen- 184 1 Laboratory of Mechanisms of Ocular Diseases tal pressures such as oxidative stress and which components are the more susceptible to these stresses. Once the constituents in the human lens are known, the development of systems to study the whole lens is vital to understanding the process of cataract forma- tion. Obtaining a reproducible in vitro system is important to the development of anticata- ract agents. Working under definable condi- tions will allow us to formulate mechanisms and agents to ameliorate cataract develop- ment. NEI Research Program Lens and Cataract — Lens Biochemistry and Biophysics Publications Chambers C, RusseU P: Sequence of the human lens pB2 crystallin encoding cDNA. Gene 133:295-299, 1993. Kidd GL, Reddan JR, RusseU P: Differentia- tion and angiogenic growth factor message in two mammalian lens epithelial cells lines. Differentiation 6:67-74, 1994. RusseU P, Tumminia SJ, Pichel JMC: Compari- son of lens epithelial ceU Unes from transgenic animals. Invest Ophthalmol Vis Sci 35:2204, 1994. Tumminia SJ, Qin C, Zigler JS Jr, RusseU P: The integrity of mammaUan lenses in organ culture. Exp Eye Res 58:367-374, 1994. Tumminia SJ, RusseU P: aB-crystallLn expres- sion and chaperone function in human astro- gUoma ceU Une U373. Invest Ophthalmol Vis Sci 35:2212, 1994. 185 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00289-01 LMOD PERIOD COVERED Oct obe r 1, 1993 to S eptember 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Lentic ular Expressi on of the HIV Protease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute affiliation) PI: Paul Russell Ph.D. Research Chemist LMOD, NEI Others: Santa Tumminia Ph.D. Senior Staff Fellow LMOD, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.35 PROFESSIONAL: 0.35 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues Q (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Two transgenic mouse strains have recently been obtained. These strains contain the HIV protease coding sequence linked to the oA-crystallin promoter. One strain gets cataract in utero, while the other strain develops cataract at approximately 25 days. The strains are currently hemizygous. Efforts in the past several months have been directed to getting homozygous populations of these strains to determine the cause of the cataract formation and how this is related to the expression of the HIV protease in the lens. 186 PHS 6040 (Rev. 5/92) Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The purpose of this project is to determine how the expression of the human immunode- ficiency virus protease in the lens causes cataract formation. Methods We will use biochemical and molecular biolog- ical methods as well as Ught and electron microscopy to investigate cataract formation. Major Findings The homozygous mouse strains appear to get cataract formation a few days before the hemizygous animals; therefore, we are using homozygous selection. Significance to Biomedical Research and the Program of the Institute Cataract formation in the presence of a small amount of HIV protease in the lens could sug- gest that the protease is cleaving a specific protein that will start the process of cataract formation. Alternatively, the protease could be interfering v^th the process of differentia- tion in the lens. Either of these two hypothe- ses would have important imphcations either for cataract formation or for the action of the HIV virus in general. Proposed Course After developing the homozygous strains, we will investigate the mechanism involved in cataract formation in these animals. NEi Research Program Lens and Cataract — Pathogenesis of Cataract 187 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00290-01 LMOD PERIOD COVERED October 1, 1993 to Septe mb er 3 0, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Autoantibo die s to Lens Crys tallins PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Paul Russell Ph.D. Research Chemist LMOD, NEI Others: J. Samuel Zigler, Jr. Donita Garland Yvonne Duglas-Tabor Ph.D. Senior Scientist Ph.D. Research Biologist B.A./B.S. Biologist LMOD, NEI LMOD, NEI OGCSB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.05 PROFESSIONAL: 0.05 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews Jx] (b) Human tissues □ (c) Neitlier SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Experiments are under way to investigate the level of autoantibodies to lens crystallins in the serum of normal volunteers and of individuals with cataracts. The specific autoantibodies will be determined and measured using chemiluminescent Western blots of two-dimensional gels of human lens proteins. Initial efforts on some serum samples have indicated that autoantibodies are present, and these appear to react most commonly with the p-crystaUin components in the lens. 188 PHS 6040 (Rev. 5/92) Laboratoiy of Mechanisms of Ocular Diseases Project Description Objectives The purpose of this project is to determine if autoantibodies to lens are present in the serum and if the specific autoantibodies corre- late with cataract type or the rate of progres- sion of cataract. We will use chemilumines- cent Western blot analysis to investigate the increase in autoantibodies. Methods Autoantibodies will be determined using Western blots to lens proteins. The lens proteins will be separated by two-dimensional gel electrophoresis. Major Findings This project has just recently been undertaken; however, it has been determined that autoanti- bodies are present in the serum. The autoanti- bodies that have been observed are generally to proteins that are part of the p-crystallin family. Significance to Biomedical Research and the Program of the Institute This project sets out to determine if specific cataract type or the progression of cataract formation can be related to specific autoanti- bodies to lens crystallins that are present in the serum. If a correlation between these autoantibodies and progression of lens opacifi- cation can be made, it might be possible to develop a cUnical test to determine which individuals are most at risk for rapid cataract formation. Proposed Course Efforts will continue to determine the level of autoantibodies in the serum of normal indi- viduals, and then these levels will be com- pared with the ones found in the serum of patients who have cataracts. NEI Research Program Lens and Cataract — Pathogenesis of Cataract 1S9 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00272-04 LMOD PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Inherited Ocular Diseases PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: James Fielding Hejtmancik M.D., Ph.D. Medical Officer LMOD, NEI Others: John Hope Radha Ayyagari Ling Lee Masami Oguni Rita Mitra T. Padma Ph.D. Ph.D. M.S. M.D. Ph.D. Ph.D. Senior Fellow Visiting Associate Chemist Fogarty Fellow Special Volunteer Visiting Scientist LMOD, NEI LMOD, NEI LMOD, NEI LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (if any) Baylor College of Medicine (J. Towbin, M.D.); Univ. of Iowa (R. Smith, M.D.); Univ. of Texas-Houston (S. Daiger, Ph.D.); Ocular Genetics and Clinical Services Branch, NEI, NIH (M. Kaiser, M.D., R.Caruso, M.D., R. Sperduto, M.D.); Massachusetts Institute of Technology (G. Benedek, Ph.D., J. Pande, Ph.D.); Osmania Univ., Hyderabad, India (J.S. Murty, Ph.D., T. Padma, Ph.D.); L.V. Prasad F.yp Inst , Hyderabad, India (G.N. Ran, MP., S . Rasti, Ph.D.); Universita Degli Studi di Parma (G. Maraini. M.D., G. Alherti, M.D.) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 5.67 PROFESSIONAL: 5.67 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects \x\ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Study of inherited visual diseases provides a means by which both normal and aberrant visual processes might be understood. In addition to directly elucidating the pathophysiology of the inherited disease under study, these studies can provide insights into the structure-function relationships of the molecular components of the visual system and their normal physiology. This laboratory is using a number of approaches to study inherited visual diseases affecting the lens and retina. Lens crystallins make up more than 90 percent of the soluble protein of the lens and are heavily modified in most cataracts. The effects that specific modifications of P- and y-crystallin structure produce on crystaUin functions such as stability and formation of macromolecular aggregates are being expressed using SF9 cells transformed with bacculovirus vector containing coding sequences for normal and modified p A3/A1- and p A2-crystallin genes. Regions of the p-crystallin molecule of special interest include the amino and carboxy terminal arms, the connecting peptide, and the Greek key motifs of the core domains. In addition, the interactions of acidic and basic P-crystallins are being studied. A second approach to understanding inherited visual diseases uses principles of positional cloning to identify genes important in human inherited diseases. Human diseases currently undergoing linkage analysis, gene isolation, or characterization of mutations include Usher syndrome, long QT syndrome, cataracts, and a variety of X-linked syndromes. We are currently collecting families with autosomal recessive retinitis pigmentosa and Bietti syndrome in preparation for study of this important group of diseases. Finally, the effects of specific genetic aUerations, including red pigment gene polymorphisms and glutathione S-transferase Ml deletions on the visual process, are being studied. 190 PHS 6040 (Rev. 5/92) Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The long-range objectives of this project in- clude increasing the understanding of inherit- ed visual diseases, with the eventual aims of increasing the diagnostic ability for these diseases and providing a foundation for developing rational therapies based on a thorough knowledge of their molecular patho- physiology. These long-range objectives will be approached by pursuing the specific aims of identifying genes involved in inherited visual diseases and elucidating the mecha- nisms by which mutations in these genes cause disease. Methods Conventional cloning technology is used in preparing sequences for gene expression studies. These include Ugation with T4 deoxy- ribonucleic acid (DNA) Ugase, screerung by NaOH miniprep methodology, and ^^P-labeled DNA probes as well as aUele-specific oligonu- cleotide hybridization to screen for specific single-base settings. Sequence changes are introduced by site-specific mutagenesis using standard methodology. Gene expression is carried out in insect cells (Sf9) using the baculovirus expression system. Protein ex- pression is monitored by standard two-dimen- sional gel electrophoresis followed by immu- noblotting. Association behavior is assessed by elution volume on sieve FPLC. Crystallin and other complementary DN As and genomic fragments are isolated by library screening with cloned genes or oligonucleo- tides using routine methods. Sequencing is carried out by cycUng or using automated florescent technology. Until recentiy, HrJcage analysis has been carried out by conventional Southern blotting. Cell lines from patients and other family members are immortalized by Epstein Ban- virus transformation. DNA is isolated by standard methodology and digested by restric- tion endonucleases. After agarose gel electro- phoresis. Southern transfer is performed and the resulting blot probed with isolated DNA fragments labeled with [^^P] by oligonucleotide labeling. Recentiy, short tandem repeat (STR, microsateUite) markers have been analyzed by polymerase chain reaction performed in the presence of labeled oligonucleotides and analyzed on sequencing gels. Linkage data are recorded on a computerized spreadsheet and analyzed using both two-point and n^ulti- point analysis with the LINKAGE program package. Major Findings (1) The p-crystaUins, their structure, and the mechanisms by which heterogeneity arises among this family of proteins are being inves- tigated. The pA3-crystaUin is identical to pAl except for an additional 17 amino acid N- terminal extension. The same gene is believed to encode and express both polypeptides. In addition, the PA3/A1 coding sequences were inserted into the Bluebac expression vector (Stratagene) and expressed in SF9 cells. In SF9 ceUs, a protein with the same amino terminal sequence as the pAl -crystallin is produced when the baculovirus-infected cells are grown past their prime. This is temporally correlated with the disappearance of the (3A3-crystaUin band, suggesting that the smaller band is created by processing or by degradation of the larger in this system. In addition, clones for the mouse pA2-, pBl-, pB2-, and pB3-crystal- Uns have been isolated and sequenced in preparation for characterization of their roles in p-crystallin aggregation. (2) An additional crystallin has been constructed in which the amino terminal arm is deleted and replaced by a glycine residue, so that this extension is identical to that found in Y2-crystaUin. This has been expressed in SF9 ceUs (Blubac vector) and has an appropri- ate migration on Laerrdi gels, CD-spectrum, and amino acid sequence. The activity of this P-crystaUin in association into the typical 200- 250 kDa aggregates has been tested using FPLC on superdex 75 and superose columns. The normal pA3 polypeptide readily associ- 191 FY 1994 NEI Annual Report ates into homodimers, but the truncated (3A3 associates minimally if at all. SF9 cells ex- pressing the recombinant crystallins were grown in '^^'S containing medium purified and reassociated with an excess of lens extract containing normal crystallins (unlabeled) using limited urea denaturation followed by dialysis. Association into p-crystallin aggre- gates was assessed by FPLC on sizing col- umns. The recombinant full-length P-crystal- lin peptide associates into both dimers and tetramers, with the dimer peak migrating slightly before the p-Ught peak. The truncated PA3-crystallin, however, migrates slightly behind the p-light peak and does not form obvious tetramers. These data strongly sug- gest that the amino terminal arm of P-crystal- Uns assists in the association of p-crystallins into higher order aggregates. (3) A pA3-crystallin has been constructed in which the entire connecting peptide from the first to the second domain has been re- placed with the corresponding sequence from Y2-crystallin. This tests the hj^othesi: that the connecting peptide, which crystallographic data show is extended in the p-crystallins and curved back on itself in the v-crystallins, is responsible in this fashion for the p-crystal- lins' tendency to dimerize. The p-crystallin with the modified connecting peptide was subjected to the same tests of association as described in number 2 and behaved essential- ly as the normal (unmodified) pA3-crystallin. The secondary structure of the modified P- crystallin is currentiy being confirmed with CD analysis. (4) The mouse pB2-crystallin has been subcloned into a baculovirus vector and ex- pressed in the SF9 cell culture system. The expressed protein is the appropriate molecular mass for an intact pB2-crystallin and forms dimers appropriately. Modifications, includ- ing deletion of the amino and carboxy termi- nal arms, are being engineered into the pB2- crystallin protein; the effects of these modifica- tions on its formation of homodimers and heterodimers with the normal and modified PA3-crystaIlin protein will be examined. (5) Studies of phase transition properties of the y-crystallin gene family have begun in collaboration with Dr. George Benedek, from the Massachusetts Institute of Technology, Boston. The bovine vB-crystallin has been modified at two of the four residues proposed to be critical for phase transition behavior. Phase transition analysis of the expressed, unmodified 72-crystallin has begun in Boston. (6) Ophthalmological diseases have been studied in humans by linkage analysis of restriction fragment length polymorphism markers. Diseases that we have mapped within the past year include Long QT syn- drome, two types of autosomal dominant congenital cataracts, and Usher's syndrome type I. In addition, family collection has begun on Bieti syndrome in anticipation of carrying out linkage analysis. Clinical and genetic heterogeneity of Usher syndrome within the Acadian population has been explored in detail. Genetic analysis confirms the cUnical impression that both type I and U Usher S5mdrome are found in the Acadian population and even within a single extended pedigree. The heterogeneity analysis des- cribed above implies that this must be due to segregation of two different and unlinked genes within this population. Two genes causing Usher sjmdrome type I have been mapped. In the Acadian popula- tion of Louisiana, the genetic locus is on chromosome lip, although in our British families, the gene is on chromosome llq. These findings have been subjected to hetero- geneity analysis using both the HOMOG2 program and the M-test and are sigrvificant at p < .01 under the most stringent analyses. This surprising finding implies that multiple genes can cause the rather specific cUnical findings present in Usher syndrome. The location of the Usher syndrome gene on chromosome lip has been studied in detail using fine linkage mapping and haplotype analysis. The Usher syndrome gene has been localized to a 5 cM interval between the mark- ers D11S861 and D11S899. A YAC contig is being established over this region, and four YAC clones extending from D11S861 to 192 Laboratory of Mechanisms of Ocular Diseases D11S902 form the minimal tiling path over the assembled region, with about 12 additional YAC clones incorporated into this contig. (7) Several large families with autosomal dominant and recessive cataracts have been ascertained and studied clinically and samples collected. Genotyping of microsateUite mark- ers has begun in four of these families with attention initially concentrated in regions around candidate genes. Two families have 5delded significant lod scores, one on chromo- some 2 and one on chromosome 17. (8) The possible association of various genetic mutations and polymorphisms v^th visual function is being carried out. The effect of various polymorphisms in the red pigment genes with visual function in heterozygous females is being studied in collaboration with Dr. Raphael Caruso (Ophthalmic Genetics and Clinical Services Branch), and the effect of deletion of the glutathione S-transferase Ml gene on the frequency of cataracts is being investigated in collaboration vsdth Dr. G. Maraini, from the Universita Degli Studi di Parma. Significance to Biomedical Research and the Program of the Institute Elucidation of the genetic defects causing visual disability will have ttnpUcations far beyond the patient population suffering from the specific syndrome under study. Inherited diseases provide a means by which the molec- ular physiology and pathophysiology of the visual system may be understood, and this knowledge can then be appUed to a broad spectrum of diseases as well as to the normal eye. This rationale also applies to the study of inherited diseases of which visual defects are only a small part. Thus, although our studies of myotonic dystrophy have already resulted in improved diagnostic abUities, the mecha- nism by which cataracts occur in this disease will provide insight into cataractogenesis in other hereditary syndromes as well as age- related and nonspecific cataracts. Proposed Course (1) Studies wiU continue on the structure- function relationships of lens crystallins, concentrating on the effects that modifications of the terminal arms, interconnecting peptide between the two domains, and surface charge and hydrophobic contact sites have on aggre- gation of both acidic and basic p-crystaUins. We wiU also continue to explore the effects that modification of the Greek key motifs have on crystaUin stability and, where applicable, lens transparency. In addition, the effects that modifications of y-crystaUin sequences have on the protein-phase transitions and their rela- tionship to cold cataract will continue to be explored. (2) Sample collection and linkage analysis of a variety of human diseases vdll continue. The main emphasis will be on inherited visual diseases, especially Usher syndrome type 1 and cataracts. Fine mapping and physical mapping of the Usher s3mdrome type IC gene on Chromosome lip is being pursued, and candidate gene analysis will be initiated as soon as it would be meaningful. We are initiating fine mapping and candidate gene analysis of the autosomal dominant cataracts that we have mapped in families ascertained in collaboration with Dr. Muriel Kaiser-Kupfer and Dr. G.N. Rao and linkage analysis of the of autosomal recessive cataracts ascertained in collaboration wdth Dr. J.S. Murty in India. This will be coordinated with a new project categorizing and mapping expressed se- quences of the human lens and the ongoing mechanistic studies on lens crystallins de- scribed above. Together, these projects should provide a coordinated effort to elucidate the mechanisms of cataractogenesis in the human lens. (3) The studies of possible associations be- tween mutations and polymorphisms of genes functioning in the vision process and visual function wiU continue. Sample collections from both Italy and the National Institutes of Health (NIH) wiU continue for the cataract epidemiological study. Patients will be re- 193 FY 1994 NEI Annual Report cruited for these studies through the NIH eye dinic. (4) Families will be studied, and samples wUl be collected for a collaborative study of open angle glaucoma in Barbados. This will be carried out in collaboration with Dr. Christina Leske, from the State Uruversity of New York at Stony Brook, and wiU build on ongoing epidemiological studies. NEI Research Program Lens and Cataract — Molecular Genetics Publications Hejtmandk JF: Neurology of the visual sys- tem, in Conn PM (ed): Neurology. Philadel- phia, J.B. Lippincott Co., in press. Hejtmancik JF, Kaiser-Kupfer MI, Piatigorsky J: Inherited disorders of the eye lens, in Scriver CR, Beaudet AL, Sly WS, Valle D (eds): The Metabolic Basis of Inherited Disease. New York, McGraw HiU, in press. Hejtmancik JF, Ostrer H: DNA diagnosis of endocrinological diseases, in Becker KL (ed): Principles and Practice of Endocrinology and Metabolism. Philadelphia, Lippincott Co., in press. Hejtmancik JF, Piatigorsky J: Molecular and cell biology of the transparent and cataractous eye lens, in Bittar EE (ed): Advances in Molecu- lar and Cell Biology. Greenwich, JAI Press Inc., 1994 Hope JN, Chen H-C, Hejtmancik JF: Aggrega- tion of pA3-crystallin is independent of the specific sequence of the domain connecting peptide. / Biol Chem, in press. Hope JN, Chen H-C, Hejhnancik JF: pA3/Al-crystallin association: Role of the amino terminal arm. Protein Eng 7:445-451, 1994. Nickerson JM, Hejtmancik JF: Molecular biology and genetics of the retina, in Tasman W, Jaeger E (eds): Duane's Foundations of Clinical Ophthalmology. Philadelphia, J.B. Lippincott Co., 1993, pp 1-49. Scott MH, Hejtmancik JF, Wozencraft LA, Reuter LM, Parks MM, Kaiser-Kupfer MI: Autosomal dominant congenital cataract: Interocular phenotypic heterogeneity. Ophthal- mology 101:866-871, 1994. Smith RJ, Berlin CI, Hejtmancik JF, Keats BJ, Kimberling WJ, Lewis RA, MoUer CG, Pelias MZ, Tranebjaerg L: Clinical diagnosis of the Usher syndromes. Usher Syndrome Consor- tium. Am J Med Genet 50:32-38, 1994. Towbin JA, Li H, Taggart RT, Lehmarm MH, Schwartz PJ, Satler CA, Ayyagari R, Robinson JL, Moss A, Hejtmancik JF: Evidence of genet- ic heterogeneity in Romano-Ward long-QT Syndrome (LQTS): Analysis of 23 famihes. Circulation, in press. Hejtmancik JF, Piatigorsky J: Molecular biolo- gy of the eye lens, in Alpert DM, Jakobiec FA, DowUng JE, Ra viola E (eds): Principles and Practice of Ophthalmology: Basic Sciences. Philadelphia, W.B. Saunders Co., 1994, pp 168- 181. 194 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00149-21 LMOD PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Ultrastructure and Func tio n of the Cells and T is sue s of the Eye^ PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: W. Gerald Robison, Jr. Ph.D. Others: Nora Laver M.D. Jorge Jacot Ph.D. Anne Groome B.S. Joe Hackett B.S. Evita Bynum B.S. Joel Glover B.S. Head, Section on Pathophysiology LMOD, NEI Special Volunteer LMOD, NEI IRTA Fellow LMOD, NEI Histology Technician LMOD, NEI Biologist LMOD, NEI Microbiologist LMOD, NEI Biologist LMOD, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Pathophysiology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 6.0 PROFESSIONAL: 2.0 4.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Diabetic retinopathy is the major cause of blindness in adults (20 to 74 years old) in the industrialized countries. Whereas systemic diabetes mellitus results from lowered availabihty and/or cellular recognition of insulin, the complications of diabetes, such as diabetic retinopathy, are caused by the chronic hyperglycemia itself. Exaggerating the hyperglycemic effect by feeding rats a galactose diet, we produced the first rat model for diabetic retinopathy. Intervention studies designed to simulate clinical trials were used to test the possibility of delaying, halting, or reversing retinopathy soon after the earliest capillary lesions could be documented. Weanling male Sprague-Dawley rats were divided into 10 groups, 4 of which received either normal lab chow or a 50 percent galactose diet with or without one of two aldose reductase inhibitors (ARIs: AL-3152 or WAY-121,509), and other groups that received 50 percent galactose for four, six, or eight months and then intervention either by addition of inhibitor or removal of galactose. From rats killed at four, six, eight, 16, 18, and 24 months, one retina was prepared for obtaining electron micrographs of capillary transections; the other was used for whole mounts of isolated retinal vessels. Images of whole and transected capillaries were captured and analyzed using computer hardware and programs specially designed for 1024-x 1024-x 8-bit resolution. Based on several quantitative assessments, including basement membrane thickness, PAS stain intensity, acellularity, dilation, tortuosity, length, and microaneurysms, the retinopathy was graded on a scale of to 10. At 6 months, when intervention was begun, the untreated galactose-fed rats exhibited a 30 percent, statistically significant (p<0.01) increase in capillary basement membrane thickness and grade 1 retinopathy overall. By 18 months, the same group had grade 7 retinopathy, whereas the rats receiving intervention with an ARI-enriched or galactose-free diet exhibited only grade 2 retinopathy, and the rats fed control diet or galactose plus AL-3152 throughout the 18 months showed no retinopathy. At 24 months, the untreated rats exhibited grade 10 retinopathy, and both intervention groups had a grade 8.5 retinopathy. In conclusion, intervention at 6 months delays but does not halt or reverse the progression of galactose-induced retinopathy. We plan to attempt, by dietary manipulation, to produce rat models that develop the diabetic-like retinal angiopathies sooner. Also, using cell and organ culture, we will investigate the possible mechanisms. 195 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The objective of this project is to use special diets in vivo and controlled media in cell cultures of ocular tissues to mimic the diabetic state to determine if diabetic-like tissue chang- es can be prevented by inhibitors of aldose reductase (AR). Methods Weanling male Sprague-Dawley rats were divided into groups, some of which received either normal laboratory chow or a 50 percent galactose diet with or without an AR inhibitor (AL-3152 or WAY-121,509 at 11 mg/kg/day), and other groups that received 50 percent galactose for four, six, or eight months and then intervention either by addition of inhibi- tor or removal of galactose. Rats were killed at four, six, eight, 16, 18, and 24 months. A new enzyme digestion procedure (elastase method), which was developed in this labora- tory, was used on the retina of one of the eyes of each rat to remove aU the retinal tissues except the vessels. This provided a whole mount of the retinal vasculature and thus permitted the recognition of degenerated pericytes ("ghosts") and all the more advanced angiopathies by Ught microscopy. The retina of the other eye was sectioned and examined by electron microscopy. Images of whole and transected capillaries were captured and analyzed using computer hardware and programs specially designed for 1024 x 1024 x 8 bit resolution. Based on several quantitative assessments, including basement membrane thickness, periodic acid Schiff stain intensity, acellularity, dilation, tortuosity, length, and micro- aneurysms, the retinopathy was graded on a scale of one to 10. Tissue cultures of human, bovine, and canine retinal capUlary pericytes and lens epithelial cells were used to investi- gate the mechanism of underlying diabetic angiopathies. Major Findings Vascular whole mounts prepared by our new enzyme digestion procedure exhibited multi- ple retinal angiopathies identical to those typical of human background diabetic retinop- athy in the capillaries of rats fed galactose for 24 months. These did not occur in the retinas of rats fed a galactose diet with an AR inhibi- tor. AR was shown to be present in cultured retinal pericytes: (1) by immunohistochemistry using antibody against human placental AR, (2) by its activity using measurements of xylitol production in cells grown in a medium supplemented with xylose, and (3) by the detection of messenger ribonucleic acid for AR. There was a compromised proliferation rate in pericytes compared with endotheUal cells when incubated in high (30 mM) sugar concentrations, suggesting toxicity of polyol at the cellular level. AR appears to be involved in all the retinal complications of diabetes, from pericyte degeneration to micro- aneurysms. Significance to Biomedical Research and the Program of the Institute Diabetic retinopathy is mainly a disease of retinal capillaries. Recently, potentially benefi- cial treatments and animal models have be- come available. However, demonstration of the earliest vessel lesions has relied on the 30- year-old trypsin digestion method for the isolation of retinal vessels. Until now, basic experimental studies and drug testing on diabetic retinopathy have been limited by the lack of reliable and convenient animal models. Now, besides the alloxan diabetic dog ai\d the galactosemic dog, there is a galactosemic rat model. All this has been possible because AR is involved in diabetic retinopathy. AR, w^hich has been implicated in sugar cataracts, certain corneal healing defects, and peripheral neu- ropathy of diabetic and galactosemic animals, now appears to be involved in all lesions of background diabetic retinopathy. Although 196 Laboratory of Mechanisms of Ocular Diseases the normal physiological role of this enzyme in most tissues remains unknown, under the conditions of high plasma sugar concentra- tions encountered in diabetes and galactose- mia, AR converts these sugars to their respec- tive sugar alcohols (polyols). These polyols are not readily metabolized nor do they pene- trate ceU membranes easily. Thus, once formed at significant rates, they may accumu- late to very high levels in cells, leading to hypertonicity, alteration of ion permeability, and eventual cell death with consequent tissue changes such as cataract formation. Treatment of diabetic or galactosemic rats with potent AR inhibitors such as sorbinil or tolrestat decreases the accumulation of polyols, which in turn appears to prevent the formation of cataracts in lenses, defective healing in scraped corneas, thickening of basement membranes in retinal capUlaries, and de- creased conduction velocity in nerves. We have shown for the first time that the rat can be a good model for human diabetic retinopathy and that demonstration of early lesions can be improved by using a novel vessel preparation method. Pericjiie loss, endothelial cell proliferation, microaneurysms, shunts, occlusions, dilations, and all the other microangiopathies that we found in the galac- tose-fed rat are identical to the histopatholo- gies that characterize human background diabetic retinopathy. Until now, the only other experimental animal model has been the diabetic or galactosemic dog. We have shown for the first time that diabetic-Uke retinopathy in galactosemic rats can be prevented with an AR inhibitor. Proposed Course The following studies are proposed for fiscal year 1995. We will manipulate the rat diets to shorten the time when diabetic-Uke retinal angiopathies appear, thus improving the rat as a model for diabetic retinopathy. As needed, we wdll extend the intervention studies to determine how late one can interrupt the disease process and still obtain beneficial results by treatment wdth various AR inhibi- tors. Also, we wUl examine the early forma- tion of intracellular vacuoles, cell transport systems, the mechanism of basement mem- brane synthesis, and the relations of these changes to AR in isolated retinal cells grown under diabetic conditions. NEI Research Program Retinal Diseases — Diabetic Retinopathy, Sickle CeU Retinopathy, and Other Vascular Abnor- maUties Publications HaUfrisch J, Xue Q, MichaeUs OEIV, Robison WG Jr: Carbohydrate copper interactions in the development of cataracts [abstract]. FASEB meeting, Anaheim, California, April 24-28, 1994, vol. 8, issue 5, p A945. Jacot JL, O'NeUl T, ScandUng DM, West SD, McKenzie JE: Role of nitric oxide in modulat- ing retinal, choroidal, and anterior uveal blood flow in piglets [abstract]. Invest Ophthalmol Vis Sci 35(4):1288, 1994. Laver NM: Diabetic retinopathy: Laboratory investigations. Washington Society of Pathol- ogists Residents' Night. Washington, DC January 20, 1994. Laver NM, Robison WG Jr, Calvin HI, Fu S- CJ: Early epitheUal lesions in cataracts of GSH-depleted mouse pups. Exp Eye Res 57(4):493-498, 1993. Laver NM, Robison WG Jr, Hansen BC: Spontaneously diabetic monkeys as a model for cUabetic retinopathy [abstract]. Invest Ophthalmol Vis Sci 35(4):1733, 1994. Lazarous DF, Scheinowitz M, Shou M, Hodge E, Rajanayagam S, Hunsberger S, Robison WG Jr, Stiber JA, Correa R, Epstein SE, Unger EF: Effects of chronic administration of basic fibroblast growth factor on coUateral develop- ment in the cainine heart. Circulation, in press. Robison WG Jr: AR inhibition and retinopa- thy. Diabetes 43(2):337-338, 1994. 197 FY 1994 NEI Annual Report Robison WG Jr, Jacot JL, Laver NM: Diabetic Robison WG Jr, Laver NM, Lou MF, Kinoshita retinopathy: Pathogenesis and prevention JH: The role of AR in diabetic retinopathy: with various inhibitors of AR. Medico Intera- Prevention and intervention studies, in mericano, in press. Osborne NN, Chader GJ (eds): Progress in Retinal and Eye Research. Oxford, Pergamon, in Robison WG Jr, Laver NM: Sorbinil preven- press, tion of cataracts and retinopathy in the galac- tose-fed rat model of diabetic compUcations Xue Q, Hallfrisch J, Michaelis OE IV, Robison [abstract]. Invest Ophthalmol Vis Sci 35(4):1586, WG Jr: Cataract development and glycation 1994. in rats fed galactose and fructose with margin- al copper [abstract]. FASEB meeting, Ana- heim, California, April 24-28, 1994. FASEB J 8(5):A945, March 18, 1994. 198 Laboratory of Molecular and Developmental Biology Report of the Chief Laboratory of Molecular and Developmental Biology Joram Piatigorsky, Ph.D. In its 13th year, the Laboratory of Mo- lecular and Developmental Biology (LMDB) has continued to explore the molecular basis and consequences of gene expression in the eye and especially the lens. As always, our work strives to understand the eye from a molecular and cellular perspective while using this extraordinary organ as a model for general processes of evolution and development. We have also continued to exploit our findings that the lens crystalUns are expressed outside of the eye, where they perform nonrefractive functions, to link gene expression in the eye with that in other parts of the body and with noneye and systennic diseases. The LMDB now comprises five sections, and their major accompUshments this year are detailed below. In addition to per- forming basic research, which is the primary function of each of the groups, the Transgenic Animal and Genome Manipulation Section, estabUshed last year, also produces transgenic mice as a service for the rest of the National Eye Institute (NEI). The development of this service has been a great success story, as can be gleaned from its report below. Section on Molecuur Genetics This section, headed by Dr. Joram Piatigorsky, has continued to investigate the molecular basis of crystaUin gene expression in the lens and other tissues. An important new develop- ment this year was the discovery that a-crys- tallins can be autophosphyrylated in vitro. This Unks these ubiquitous crystaUins with the enzyme-crystaUins and raises the possibUity that they are involved in signal transduction pathways. Thus, the a-crystallins, which are molecular chaperons, can now be considered in a metaboUc as weU as a structural role. This provides new avenues of approach for investigating the functions of a-crystaUin in the lens as well in nonlens and diseased tissues, for which there is a large and growing literature. A great deal of progress has been made on identifying the cis- and trans-regala- tory controls for crystallin gene expression. Shared and tissue-specific ds-elements use have been identified for numerous crystallin genes. "Regulatory tissues prints," defined as the patterns of czs-elements used for expres- sion of genes in different tissues, have been determined for the mouse aB-crystaUin gene in lens, skeletal muscle, heart, and lung by performing experiments in cultured cells. Apart from its intrinsic interest, this informa- tion may be useful for designing promoters for gene therapy in the future. A particularly exciting development has been the finding that Pax-6, a paired-box homeodomain tran- scription factor, can act as a lens-specific activator for the chicken and mouse aA- and chicken 61-crystallin genes. Because numer- ous eye mutations in species ranging from Drosophila, which have compound eyes, to humans, who have complex eyes, are due to lesions in Pax-6 or its homologue, it seems possible that the diverse crystaUins may be linked by one or more common transcription factors. Pax-6 will clearly be investigated thoroughly next year. Many more findings on the molecular events of crystallin gene regula- tion have been made that wiU also be fol- lowed up, and these can be found in the annual reports form this section. 201 FY 1994 NEl Annual Report Finally, the presence of Dr. Joseph Horwitz, who has been on sabbatical leave from Jules Stein Eye Institute, UCLA School of Medicine, since January 1994, has led to an increased attention to biochemistry. One important development from this has been the finding of pB2-crystaUin in nonlens tissues (retina, testis, brain). This was complemented by the finding that pB2-crystaUin, like a-crys- taUin, can autophosphorylate in vitro. These data suggest strongly that the p-crystalUns, and possibly their Y-crystaUin relatives, have metaboUc nonlens functions in addition to their refractive role in the lens. The second important development in protein biochemis- try has been the discovery that the ratio of the two duck 8 crystaUin/argininosuccinate lyase polypeptides in the tetrameric native protein affects enzyme activity. Kinetic studies indi- cated a cooperative interaction between the 81 and 62 polj^eptides. This provides a regula- tory role for the inactive 81 polypeptide and explains why it continues to be S3mthesized outside of the lens. Transgenic Animal and Genome Manipulation Section This section, headed by Dr. Eric Wawrousek, has continued to provide the NEl investigators with transgenic mice in support of their re- search efforts. This year, 155 new transgenic founder mice were generated from 34 deoxjrri- bonucleic add (DNA) constructs. More than 400 matings of transgenic mice were set up, and more than 4,000 mice were weaned, tagged, tail biopsied, and their DNA isolated and analyzed. This year also marked the successful launching of the group's embryo cryopreservation and banking program, pro- viding the NEl with a reliable mechanism for long-term storage of valuable mouse Unes. So far, 1,326 embryos from five transgenic Unes have been frozen. This section has also been conducting independent research in two areas, generating transgenic mouse models of ocular disease and probing the function of lens crystallins by deleting these proteins in "gene knockout" mice. In the first area, the section has created a transgenic mouse model of progressive ocular inflammation by expressing a modified form of human interleukin-ip in the eye under transcriptional control of the mouse aA-crystaUin promoter. The animals are healthy, reproductively capable, and exhibit a consistent and reproducible pattern of ocular disease. In neonates, the eye is relatively normal, but inflammation with accompanying neovascularization of aU eye tissues becomes evident and progresses to phthisis bulbi in adult animals. This will be a useful model for investigating many of the processes involved in chronic ocular inflammatory disease such as cytokine levels, receptor levels, arachidonic acid metaboUte levels, cellular adhesion mole- cule expression, and systemic effects of the localized inflammation. Sigiuficant progress has been made toward functionally deleting the a-crystaUin gene family. Knockout vectors have been generated for mouse aA- and aB-crystallin and mouse aACRYPB-1, and approximately 20 embryonic stem ceU clones have been isolated in which one allele of the aA-crystaUin gene has been disrupted. Toward generating knockout mice, the group has mastered the skills for inserting ES cells into mouse embryos to generate chimeric mice. Many chimeric mice have been produced from unmodified ES cells, and it is expected that chimeric mice containing the aA-crystaUin knockout ES cells will be pro- duced very soon. The knockout mice will provide a unique resource for stud5mig the function of the a-crystaUins in lens and eye development and shed some light on their function in nonlenticular tissues. Develop- ment of the technology may one day also be a valuable resource for other researchers in the NEL Section on Regulation of Gene Expression This section, headed by Dr. Ana Chepelinsky, has continued to study the expression of genes 202 Laboratoiy of Molecular and Developmental Biology encoding lens fiber membrane channels, which are of utmost importance for maintaining lens transparency. They have mapped several cis regulatory elements in the 5' flanking se- quence of the human major intrinsic protein (MIP) gene. Negative regulatory elements were found within the sequence -2840/ -254, and positive regulatory elements were re- vealed between the sequence -70/ -40 of the MIP gene. A successful collaboration between the LMDB and the Laboratory of Immunology at the NEl allowed the development of trans- genic mice and rats with constitutive expres- sion of interferon gamma (IFN-y) and major histocompatibility complex (MHC) class 11 in their eyes. These animals provide a compre- hensive transgenic model system for elucidat- ing the Unkage between aberrant MHC class II expression and predisposition to autoimmuni- ty, the role of IFN-y in the treatment of in- flammatory eye diseases, and cytokine signal- ing during embryonic eye development. Section on Molecular Structure AND Function This section headed by Dr. Graeme Wistow, has continued to investigate the variety of proteins that can serve as functional crystal- Uns. Work on ^-crystaHin has revealed a natural example of a consensus paired-domain binding site for Pax-6. This site is essential for lens-specific promoter function. Other projects have led to discoveries with significance beyond the lens. /i-CrystaUin, originally discovered in marsupial lens, is expressed abundantiy in retinal photoreceptors of hu- mans and rodents, probably as an enzyme of glutamate or ornithine metabolism. Migration inhibitory factor (MIF), which was isolated as a developmental marker in chick lens, has been shown to have a general role in cell proliferation. It interacts with the retinoblas- toma protein and is essential for progression through the cell cycle. Antisense suppression of MIF halts ceU growth even in transformed mouse cells. Section on Cellular Differentiation This section, headed by Dr. Peggy Zelenka, has made progress in several areas in the past year. Several steps have been taken to follow- up the surprising discovery that postmitotic, differentiating lens fiber ceUs contain cycUn B, an activator of the cycUn-dependent kinase, p24cdc2^ that is normally associated with mito- sis, hnmunocytochemistry has shown that cyclin B is concentrated in the fiber cell nuclei as early as developmental day E6, and bio- chemical analysis indicates that cycHn B and p2^cdc2 complex may play a role in lens fiber cell denucleation, transgenic mice that carry the weel gene under control of the y-crystallin promoter. The product of the weel gene is a kinase that inactivates p34'"''^. Other studies carried out during this year have probed the similarities and differences between lens fiber cell differentiafion and apoptosis. These have shown that cycUn B may be induced during apoptosis in PC12 ceUs as well as during lens fiber cell difierenfiation. Moreover, in apopto- tic cells, cyclin B is complexed with a novel partner that is immunologically related to PCTAIRE-1, a member of the cycUn-dependent kinase family whose function is unknown. Another similarity between lens fiber differentiation and apoptosis that was discov- ered is that the proto-oncogene, c-jun is acti- vated during both processes. However, in differentiating cells, c-jun is downregulated after a few hours, while in apoptotic cells, it remains elevated until the ceUs die. Using a retrovirus vector to introduce c-jun into cul- tured cells, it was shown that elevated levels of c-jun promote cell death in the absence of growth factors. These studies open new opportunities for investigating the relation- ships between cell division and terminal lens fiber ceU differentiation. Z03 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00238-09 LMDB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) P roto-onco g ene Expression During Lens Differentiation and D evelopment PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Peggy S. Zelenka Ph.D. Head, Section on Cellular LMDB, NEI Others: Chun Yun Gao Emmanuel Vacchiano Anuradha Rampalli Jaspreet Arora Vijay Chauthaiwale Graeme Wistow Differentiation M.D., Ph.D. IRTA Fellow Ph.D. IRTA Fellow Ph.D. IRTA Fellow Ph.D. Visiting Fellow Ph.D. Visiting Fellow Ph.D. Head, Section on Molecular Structure and Function LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI COOPERATING UNITS (if any) Department of Surgery and Department of Anatomy and Cell Biology, New Jersey Medical and Dental College (Thomas Lysz, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Cellular Differentiation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 5.6 PROFESSIONAL 5.6 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project investigates the expression of proto-oncogenes and other cell cycle regulatory genes in the embryonic chicken lens to determine their relationship to cell growth, quiescence, and differentiation. The normal developmental profiles of six nuclear proto-oncogene messenger ribonucleic acid (mRNAs) (c-myc, N-myc, c-fos, c-jun, Rb, and p53) and the cell cycle regulatory protein, cyclin B, have been completed. The unexpected finding that cyclin B is present in postmitotic lens fiber cells and that it is complexed with p34"''^^ suggests that lens fiber cell differentiation may involve aberrant progression through the cell cycle. To test this hypothesis, a line of transgenic mice has been produced that overexpresses Weel (a protein kinase that inhibits cyclin B/p34"''^^) under control of the y-crystallin promoter to target expression to postmitotic lens fiber cells. Preliminary analysis of lenses from this transgenic line indicates that lens fiber denucleation may be abnormal. Similarities between apoptosis and lens fiber differentiation prompted us to compare proto- oncogene expression in differentiating and apoptotic cells. The earliest difference we have detected between these two processes is deregulation of c-fos, c-jun, and c-myc expression during apoptosis. We have demonstrated that expression of these proto-oncogenes is strictly regulated by 12(S)HETE synthesis and are exploring the mechanism of this effect through analysis of the c-fos promoter. Candidate genes that may be regulated by these proto-oncogenes are being explored through transfection of cultured cells and deoxyribonucleic acid (DNA) binding studies. We have demonstrated that cyclin B is expressed during apoptosis of postmitotic PC12 cells, where it complexes with p34"'''^ and with a novel, 40K member of the PCTAIRE family. The identity and function of this 40K protein are being investigated. 204 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology Project Description Additional Personnei VuBui Graeme Wistow PhX). IRTA Summer Fellow, LMDB, NEl Head, Section on Molecular Structure LMDB, NEI and Function Objectives This project seeks to determine whether the expression of specific proto-oncogenes is altered during lens cell differentiation, and, if so, to determine the mechanism of gene regu- lation and the function of the corresponding proto-oncogene products in the developing lens. The objective is a greater understanding of the mechanisms underlying lens ceU growth and differentiation. Methods Techniques of molecular biology are used in conjunction v^th traditional techniques of cell biology. Conventional methods are used for analysis of proteins and nucleic acids, includ- ing polyacrylamide gel electrophoresis, ribo- nucleic acid (RNA) and deoxyribonucleic acid (DNA) isolation, polymerase chain reaction (PCR), nucleic acid hybridization, in vitro transaction, in situ hybridization, immunocy- tochemistry, and immunoblotting. DNA /pro- tein interactions are studied using DNase I footprintimg, electiophoretic mobility shift assays, and ultraviolet crossUnking. Studies use lens epithelia and lens fibers of embryonic chickens, explants of embryonic chicken lens epithelia, primary cultures of embryonic chicken lens epithelial cells, and other avian and mammaUan cell Unes. In addition, tiansgenic mice are produced to test the function of proto-oncogenes and cell cycle regulatory proteins in the lens in vivo. Major Findings Terminal differentiation of lens fiber cells is marked by chromatin condensation, abrupt dissolution of the nuclear lamina, vesiculariza- tion of the nuclear membrane, and complete degradation of the nucleus and other organ- elles. Because these events resemble the chromosomal condensation and nuclear enve- lope breakdown associated with mitosis, we have investigated whether a similar biochemi- cal mechanism may be involved by testing for the expression and activity of cycUn B/p34'' ^ complexes in differentiating lens fiber cells. A coupled reverse transcription /PCR (RT/PCR) using RNA from embryonic chicken lens fibers amplified a product of the expected size, which was identified as cycHn B2 by sequenc- ing. In situ hybridization localized cycUn B messenger RNA (mRNA) to the superficial nucleated fiber cells, and immunocytochemis- try with antichicken cycHn B2 antiserum showed positive staining in lens fiber cell nuclei. Immunoblotting of lens fiber proteins purified by affiiuty chromatography on pl3- agarose beads identified both p34'"''^ and cyclin B, indicating that these proteins are present as a complex. Moreover, fiber cell proteins purified on pl3-agarose beads showed histone HI kinase activity, which was enhanced by phosphatase digestion. These results demonstrate that active cyclinB/p34'"''^ complexes are present in differentiating lens fiber cells before denuclea- tion and support the possibility that phos- phorylation of specific nuclear substrates by p34'"^'^ may be responsible for the denucleation of lens fiber cells during terminal differentia- tion. Although denucleation of lens fiber cell differentiation resembles mitosis, other fea- tures such as degradation of DNA to oUgonu- cleosomes resemble apoptosis. This similarity and our discovery of cyclin B in differentiating lens fibers led us to explore the involvement of cyclin B in apoptosis. These studies were performed using neuronaUy differentiated PC12 cells forced to undergo apoptosis by withdrawal of nerve growth factor. Cyclin B 205 FY 1994 NEI Annual Report mRNA increased approximately 10-fold four days after NGF withdrawal, as indicated by competitive RT/PCR. Cyclin B protein also increased during this period, as indicated by immunoblotting. Immunoprecipitation with anticycUn B antibody demonstrated that cyclin B was associated with HIK activity, which reached a maximum five days after NGF withdrawal. When proteins immunoprecipi- tated with anticyclin B antibody were immu- noblotted with anti-PCTAIRE antibody, pro- teins with apparent molecular weights of 34kDa and 40kDa were detected. The 34kDa protein was identified as pSA"'^'^ on the basis of immunoreactivity with antibody against the C-terminal portion of p34^'''=^. The 40kDa protein reacted strongly with an antibody against the C-terminal region of PCTAIRE-1 and failed to bind to pl3-agarose beads, suggesting that it is a member of the PCTAIRE family. Kinase assays of anticyclin B immunoprecipitates from apoptotic cell lysates that were first rmmunodepleted of p34'"^^ indicated that significant HIK activity was associated with the cyclin B/40kDa pro- tein complex. These findings suggest that certain members of the PCTAIRE family may function as cyclin-dependent kinases in apop- totic cells. We are currentiy investigating expression of the 40kDa protein in differentia- ting lens fibers and in apoptotic retinal cells. The relationship between lens differentia- tion and apoptosis has been further probed by Dr. Anu Rampalli, who has compared expres- sion of several proto-oncogenes and cell cycle markers in embryonic chicken lens epithelial explants during differentiation or apoptosis in vitro. Interestingly, proto-oncogenes c-fos, c- jun, c-myc, and -pSi are sequentially upregu- lated during both processes. However, in differentiating cells, these increases in mRNA expression are transient, although in apoptotic cells they are not. Loss of regulation of c-fos and c-jun mRNA expression, which occurs within five hours in culture, is the earliest difference detected between differentiating and apoptotic cells. Moreover, Dr. Emanuel Vacchiano has demonstrated that overexpres- sion of c-jun in chicken lens epithelial cells in the absence of growth factors greatly increases the rate of cell death due to apoptosis. Thus, deregulated expression of c-jun appears to be an early step in the pathway leading to apop- tosis of lens epithelial cells. Two experimental approaches have been used to explore the functional role of proto- oncogenes such as c-fos, c-jun, and c-myc in the lens. Dr. Vijay Chauthaiwale has used a candidate gene approach to determine wheth- er c-myc is involved in regulation of the a- enolase/x-crystaUin gene. The promoter of this gene contains a potential c-myc binding site that is conserved in both duck and hu- man. Dr. Chauthaiwale has demonstrated that c-myc binds to this site by immunoblot- ting with anti-(c-mi/c) antibody following electrophoretic mobility shift assay of lens nuclear proteins bound to an oligonucleotide containing the potential binding site. Immu- noreactive bands were detected by enhanced chemHuminescence. Interestingly, a mutated oligonucleotide containing substitutions at the two central nucleotides of the potential bind- ing site showed unimpaired binding of c-myc but greatiy diminished binding of a faster migrating band. Transfection studies using a a-enolase/x-crystaUin promoter construct bearing this same mutation had previously shown that this mutation increased basal transcription of the promoter and abolished inducibility by c-myc. Together these results suggest that c-myc competes with a negative regulatory factor for occupation of the same site. Dr. Chauthaiwale has now demonstrated that the fast migrating band that is diminished by the mutation comigrates with a band produced by in vitro translated max proteins, suggesting that max /max homodimers may be the negative regulatory agent. This possibility is being explored by means of cotransfection studies with c-myc and c-max plasmids. The other experimental approach used to study the targets of proto-oncogenes in the lens is overexpression of these genes and negative dominant mutations using retroviral vectors. We have used the avian retroviral vector RCAS to overexpress wild-type chicken c-jun, or a deletion mutant of chicken c-jun (junAT) lacking the DNA binding region, to 206 Laboratory of Molecular and Developmental Biology investigate the possible role of c-jun in lens epithelial cell proliferation and differentiation. Both constructs were efficiently expressed in primary cultures of embryonic chicken lens epithelial cells. Overexpression of c-jun in- creased the rate of cell proliferation and great- ly delayed the appearance of "lentoid bodies," structures that contain differentiated cells expressing fiber cell markers. Excess c-jun expression also significantiy decreased the level of PA3/ArcrystaUin mRNA without affect- ing aA-crystallin mRNA. In contrast, the mutated pvotein junA7 had no effect on proUf- eration or differentiation but markedly in- creased the level of aA-crystallin mRNA in proliferating cell cultures. These results sug- gest that c-jun or ;Mn-related proteins may be negative regulators of aA- and pA3/Al- crystallin genes in proUferating lens cells. Studies of the role of 12-lipoxygenase in regulating lens epithelial cell proliferation have made significant advances in the past year. To determirie whether products of this pathway are involved in lens cell proliferation, we measured the effect of 12-Upoxygenase inhibitors on endogenous 12-hydroxyeicosa- tetraenoic acid (HETE) production and epider- mal growth factor (EGF)/insuUn-stimulated DNA synthesis and proto-oncogene expression in cultured neonatal rat lens epithelial cells. Incubation of neonatal rat lenses in EGF plus insulin, which stimulated endogenous 12- HETE production eightfold to 10-fold, also produced a transient induction of c-fos and c- myc mRNAs after two to three hours, followed by a round of DNA synthesis approximately 20 hours later. The lipoxygenase inhibitor cinnamyl 3,4 dihydroxy-a-cyanocinnamate (CDC) strongly inhibited both the endogenous 12-HETE synthesis and growth-factor stimulat- ed DNA sjmthesis with half-maximal inhibi- tion between 10-20 fiM. CDC (10/xM) also inhibited the expression of c-fos and c-myc mRNA and to a lesser extent, c-jun mRNA. The inhibitory effects of CDC on proto-onco- gene expression and DNA synthesis were prevented by 0.3 jiM 12-HETE but not by equivalent concentrations of either 5- or 15- HETE. These findings suggest that endoge- nously synthesized 12-HETE may mediate EGF/insuUn-stimulated DNA synthesis in neonatal rat lens epitheUal cells by regulating proto-oncogene expression. If 12-HETE also regulates human lens epitheUal cell proUferation, inhibition of 12- Upoxygenase activity may provide a means of preventing unwanted proUferation following cataract extraction. We have, therefore, exam- ined expression of this enzyme in human lens epitheUal ceUs. Presence of 12-Upoxygenase mRNA was demonstrated in epitheUal ceUs of adult and neonatal human lenses by RT/PCR and sequencing of the PCR product. The presence of the corresponcUng protein was demonstrated in cultured neonatal human lens epitheUal ceUs by immunoblotting with an antibody raised against human platelet 12- Upoxygenase. A medium derived from hu- man lens epitheUal cell cultures was shov^m to contain 12-HETE by radio immunoassay. Experiments are in progress to test whether inhibition of 12-Upoxygenase blocks DNA synthesis in cultured human lens epitheUal ceUs as in the neonatal rat lens. Dr. Jaspreet Arora has also demonstrated the presence of the 12-Upoxygenase pathway in cultures of rabbit lens epitheUal ceUs (N/N10003 ceUs). This system wUl be used to investigate the mechanism of the 12-HETE efiect on proto- oncogene expression in the lens. Proposed Course The foUovkdng studies are in progress or are proposed for fiscal year 1995: (1) The hypotiiesis that the cycUnB/p34^''^ kinase is responsible for nuclear loss in differ- entiating lens ceUs will be tested by analysis of several Unes of transgenic mice that express the weeV kinase in lens fiber ceUs. This kinase inactivates the cycUnB/p34'"''^ kinase and would be expected to delay or prevent nuclear loss if, indeed, cycUnB/p34'"''^ is required. (2) The finding that cycUn B binds to a novel 40kDa PCTAIRE-Uke protein in apop- totic ceUs wUl be explored further. The identi- ty of the protein will be examined by protein microsequendng, and an attempt will be made 207 FY 1994 NEI Annual Report to clone the corresponding complementary DNA. Expression of this protein will be examined in the lens during differentiation and in a variety of ocular cell types during apoptosis. (3) The effect of c-myc on transcription of the T-crystallin/a-enolase gene wiU be exam- ined further by cotiansfection studies with max and myc plasmids. (4) The collaborative effort with Dr. Thomas Lysz, from the University of Medicine and Dentistry of New Jersey, will be contin- ued to determine whether the 12-lipoxygenase pathway plays a role in regulating DNA synthesis in human lens epithelial cells. (5) The possible role of posttranslational modifications of Rb and p53 proteins in lens cell differentiation will be explored using differential extraction techniques and specific antibodies. The relationship between migra- tion inhibitory factor and modified forms of Rb will be examined in differentiating cells. NEI Research Program Lens and Cataract — Molecular Genetics Publications Dash A, Chung S, and Zelenka PS: Expres- sion of HSP70 mRNA in the embryonic chick- en lens: association with differentiation. Exp Eye Res 58, 381-387, 1994. Gao CY, Bassnett S, Zelenka PS: Cyclin B, p34cdc2^ and Hl-kinase activity in terminally differentiating lens fiber cells. Develop Biol, in press. Lysz TW, Arora JK, Lin C, Zelenka PS: 12(S)- Hydroxyeicosatetraenoic acid regulated DNA synthesis and protooncogene expression induced by epidermal growth factor and instdin in rat lens epithelium. Cell Growth Dijf, in press. 208 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00126-13 LMDB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Crystallin Genes: Structure, Organization , Express ion , and Evolut ion PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: Others: Joram Piatigorsky Sharmilla Basu James B. Brady Sambath Chung Ales Cvekl Melinda K. Duncan Peter Frederikse Rashmi Gopal-Srivastava John I. Haynes, II John G. Ilagan Ph.D. Ph.D. Ph.D. B.A. Ph.D. Ph.D. Ph.D. Ph.D. Ph.D. B.A. Chief IRTA Technician Visiting Fellow IRTA Senior Staff Fellow Staff Fellow IRTA Howard Hughes Medical Institute/ NIH Fellow (Additional peisonnel listed under Program Description.) LMDB, NEI LMDB. NEI LMDB. NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI COOPERATING UNITS (if any) Jules Stein Eye Institute, UCLA School of Medicine (J. Horwitz, Ph.D.); University of Waterloo (Jacob Sivak, Ph.D. and Judith West Mays, Ph.D.); University of Nijmegen (Wilfried de Jong, Ph.D.); National Institute of Diabetes and Digestive and Kidney Diseases, NIH (Emery H. Bresnick, Ph.D.); National Cancer Institute, NIH (John N. Brady, Ph.D. and Fatah Kashanchi, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 15.70 PROFESSIONAL: 13.15 2.55 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The structure, expression, and evolution of the crystallin genes of vertebrates and invertebrates are being studied. The following advances have been made. Pax-6 activates the chicken and mouse aA-crystallin promoter and the chicken 61 -crystallin enhancer. The chicken aA promoter contains a composite element that suppresses activity in fibroblasts by binding USF and AP-1 proteins (JunD and Fra2) and activates promoter activity in lens by binding USF and CREB\CREM proteins. USF also acts as a negative regulator of the chicken aA promoter by binding to another, downstream element. USF also binds to the 6EF1 site of the chicken 61 -crystallin enhancer, where it probably contributes to the activation of this gene. CREB\CREM cooperates with aA-CRYBPl and Pax-6 to activate the mouse aA promoter in lens cells. Binding studies suggest that HSF may also be involved in chicken aA and mouse y¥ promoter activity. Expression of the mouse aB-crystallin gene in different tissues utilizes both shared and tissue-specific control elements, the exact pattern being called its "regulatory tissue print." Elements for lens-specific expression have been narrowed to positions -101/+30 in the chicken PBI gene and -143/+22 in the chicken PBA3/A1 gene in transgenic mouse experiments. aA, aB and PB2 were shown to be able to autophosphorylate, raising the possibility that they are involved in a signal transduction pathway. Close linkage was found for chicken pBl and PA4, and evidence was obtained indicating that mammalian PB2 is expressed in nonlens cells. A CRl element was found in the intergenic spacer of duck 6-crystallin; the duck 61 and 62 polypeptides were shown to interact cooperatively to modulate argininosuccinate lyase activity of the tetramer. The jellyfish J3-crystallin gene has been cloned and shown to contain at least six introns. 209 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Additional Personnel Joseph Horwitz Ph.D. Jules Stein Eye Institute Cynthia J. Ph.D. Staff FeUow, LMDB, Jaworski NEI Marc Kantorow PhD. Staff Fellow, LMDB, NEI Xuan Li Ph.D. Visiting Associate, LMDB, NEI Joan B. PhD. Biologist, LMDB, NEI McDermott Barbara Norman M.S. Chemist, LMDB, NEI Christina M. Sax Ph.D. Senior Staff Fellow, LMDB, NEI Stanislav I. PhD. Visiting Scientist, Tomarev LMDB, NEI Objectives The objective of this project is to understand the structure, organization, expression, and evolution of the gene families encoding the lens crystallins in the animal kingdom. Partic- ular attention is given to the regulation of crystallin gene expression in the developing lens and, in the case of multifunctional crystallins and enzyme-crystaUins, in nonlens tissues. Methods Conventional methods used for analysis of proteins and nucleic acids include polycry- lamide and agarose gel electrophoresis, ribo- nucleic acid (RNA) and deoxyribonucleic acid (DNA) isolation, molecular hybridization (Southern and Northern blots), complementary DNA (cDNA) and gene cloning, DNA se- quencing, recombinant DNA (rDNA) construc- tion and mutagenesis, in situ hybridization, expression of recombinant DNAs in trans- fected cells and transgenic mice, polymerase chain reactions (PCRs), primer extension and SI protection experiments, in vitro and in vivo footprinting, gel mobility shift analysis, chro- matographic purification of proteins, and Western immunoblotting. Major Findings a-Crystallins. -aA-CRYBPl is a zinc-finger transcription factor that binds to the mouse aA-crystallin gene at positions -66/ -57 of its 5' flanking sequence. We have previously cloned a partial cDNA encoding this protein. This year it was shown that the aA-CRYBPl protein exists in different sizes in lens and fibroblasts, suggesting the possibility that posttranscriptional mechanisms regulate its binding or functional activity in a tissue- specific fashion. Moreover, we have estab- lished the full-length sequence for this 9.5 kb messenger RNA (mRNA). The deduced protein contains two sets of consensus C2H2 zinc-fingers (one set at the N-terminal region and one set at the C-terminal region) as well as a nonconsensus zinc-finger motif in the center. The 70 kbp gene has also been cloned and its 10 exon structure established. aA- CRYBPl cDNAs were isolated from adult mouse brain and testis as well as from cell lines derived from mouse lens and muscle. An alternatively spUced aA-CRYBPl cDNA was demonstrated in the muscle cell line, and a unique, smaller cDNA was found in testis. An antisense expression construct derived from an aA-CRYBPl cDNA was introduced into the aTN4-l transformed mouse lens cell line, and this significantiy inhibited expression from a transfected heterologous promoter that used the aA-CRYBPl binding site. This provides the first direct evidence (apart from mutagenesis tests) that aA-CRYBPl is a posi- tive transcription factor contributing to expres- sion of the aA-crystallin gene in the mouse. Transgenic mice experiments have estab- Ushed that the DE-1 (-111\-106) and aA- CRYBPl (-66\-57) regulatory elements of tiie mouse aA-CRYBPl are functionally redun- dant. Elimination of either one alone does not affect the lens-specific activity of the promoter; how^ever, elimination of both simultaneously eliminates promoter activity. Footprinting experiments have shown that these regulatory sites are occupied in lens ceUs that are ex- pressing the aA-crystallin gene and in fibro- blasts that are not expressing the gene. It is not yet known whether the proteins binding 210 Laboratory of Molecular and Developmental Biology to these regions are identical in the two cell types or whether cofactors are involved. Mutagenesis, cotransfections, and immunoshift experiments have provided strong evidence that DE-1 binds the cAMP responsive tran- scription factor CREB/CREM and is thus a functional CRE site. Consequently it has been called DE-1 /CRE. These footprinting experi- ments have also revealed the binding of proteins to two other putative regulatory regions called PEl (involving the TATA box and immediately downstream thereof; -35 /-1 9) and PE2 (+24\+43). Mutagenesis, transfection, and protein-binding experiments have shown that PEl and PE2 are indeed important regula- tory regions for the expression of this gene. A particularly unexpected finding is that site- spedfic elimination of the TATA box of the mouse aA-crystallin promoter fused to the chloramphenicol acetyltransferase (CAT) gene did not prevent this transgene from being expressed specifically in the lens of transgenic mice. Finally, negative-acting and protein- binding elements have been identified within the -1556 /-1 165 sequence of the mouse aA- crystallin 5' flanking sequence. Protein binding, mutagenesis, cotransfec- tion, and immunoshift experiments have indicated that Pax-6, a paired-box/ homeo- domain protein expressed highly in the lens in the early stages of development of the mouse and chicken, is an important transcription factor affecting the lens-specific expression of the mouse and chicken aA-crystaUin gene. Pax-6 also appears to activate the chicken 61- crystallin enhancer. A number of studies have implicated Pax-6 as a universal regulator of eye development in both vertebrates and invertebrates, including both complex and compound eyes. Our studies provide the first evidence for specific target genes activated by Pax-6. The chicken aA-crystallin promoter \ en- hancer (-153 \ +77) has been divided into at least five functional regions (A-E) by protein binding, mutagenesis, immunoshift, and transfection experiments. This region has been shown to be composed of a complex array of positive and negative elements in- volving Pax-6, USE, CREB/CREM and API proteins. Sites A (-148\-139, binds USE) and B (-138\-132, binds CREB\CREM in lens and Fra-2 and \ or JunB in fibroblasts) comprise a composite element that activates transcription in lens and represses transcription in fibro- blasts. Sites C (-128\-101) and E (-56/-41) bind Pax-6 only in lens, which appears to be of central importance for the lens-specific expression of this gene. Site D (-102 \ -93) binds USF and is a negative element. Finally, electrophoretic mobility shift experiments using specific antibodies have provided evidence that heat shock transcrip- tion factors bind to 5' flanking sequences that are involved in the regulated expression of the chicken aA-crystaUin and mouse YF-crystaUin genes in the lens. These regions include positions -157/-136 of the chicken aA gene and positions -53/ -23 of the yF gene. Indirect evidence for the use of heat shock factors for crystallin gene expression has also been ob- tained in stably tiansformed and transfected K562 cells treated with hemin, which is known to induce HSF2, a heat shock transcrip- tion factor known to be expressed in the lens. The aB-crystallin gene contrasts with the aA-crystallin gene in that, although lens- preferred, it is also expressed to a relatively high degree in numerous other tissues and is inducible by heat and other physiological stiesses. In 1991, we identified a strong mus- cle and weak lens enhancer between positions -426 and -257 of the 5' flanking region of the gene. Last year, we identified by site-spedfic mutagenesis experiments four regulatory elements (aBE-1, aBE-2, aBE-3m and muscle regulatory factor [MRF]) v^thin this enhancer. The MRF site is an E-box and was shown by tiansfection and transgenic mouse experiments to be muscle specific and to be activated by MyoD, myogenin, or another member of this family of transcription factors. This year, we showed by DNase I foot- printing experiments that there is another regulatory element (aBE-4) located between aBE-1 and aBE-2 that is used spedfically in the heart. A series of footprinting, mutagene- 211 FY 1994 NEI Annual Report sis, and transfection experiments have resulted in the following conclusions: aBE-1 and aBE- 2 are used for expression in muscle, heart, lung, and lens; aBE-4 is heart-specific and binds a protein identical or related to the serum response factor; aBE-3 is used by muscle, lens, and heart; MRF is used by mus- cle and heart and maybe by lung. Recent evidence suggests that MRF may bind differ- ent proteins in the different tissues. Last year, we reported on another se- quence called LSR (for lens specific region), that is located downstream of the enhancer at position -147\-118 of the mouse aB-crystaUin gene. This element binds nuclear proteins that appear specific to the lens and may account for the high expression of the aB-crystallin gene in the lens. Site-specific mutagenesis and transfection tests have supported the impor- tance of this sequence for lens expression. Experiments with tiansgenic mice have shown that the -164 \ +44 sequence, which contains the LSR, fused to the CAT reporter gene is sufficient to direct lens-specific expression of CAT; by contrast, the -426 \ +44 sequence containing the enhancer directs CAT expres- sion to the lens, skeletal muscle, heart, and, to a much smaller extent, brain and lung. Thus, the differential expression of the aB-crystaUin gene is regulated by a combination of tissue- specific and shared cr's-control elements. Moreover, the shared control elements do not necessarily bind the same nuclear proteins in the different tissues. We have called the combination of regiilatory elenients used for expression of the aB-crystallin gene in any specific tissue its "regulatory tissue print" and, apart from its intrinsic interest, has obvious importance for gene therapy. We have discovered an unexpected auto- phosphorylation ability of the aA- and aB- crystaUin pol5q3eptides. This raises the possi- bility that these crystaUins are involved in a signal transduction pathway that tiltimately covdd affect expression of crystaUin genes in the lens. We have also shown that the deter- gent deoxycholate generates dimers and tetiamers of the aA-crystallin polypeptides and that these small aggregates have approxi- mately 10 times more autophosphorylation abihty than the larger molecular weight aggre- gates present without the detergent. Interest- ingly, the aB-crytaUin polypeptides also form dimers and tetramers in the presence of de- oxycholate, but their autophosphorylation abUity is not increased, suggesting a funda- mental difference between the structure and possibly function of the aA and aB-crystallin polypeptides. Recent collaborative studies with Dr. WUfried de Jong, from the University of Nijmegen, The Netherlands, have shown that the autophosphorylation of aA does not occur on serine 122, the amino acid that is phosphorylated in the cAMP-dependent reaction occurring on this polypeptide. This is consistent with our recent finding that the chicken aA-crystallin polypeptide is also autophosphorylated although it has an alanine substituted for serine at position 122. ^-crystaUins. Our previous fransfection and footprinting experiments have identified numerous confrol elements in the -152 \ +30 sequence of the chicken pBl -crystaUin gene. Transgenic mice have now been produced that have established that the -152 \ +30 sequence is sufficient for lens-specific expression. Further deletions have shown that even the -101 \ +30 sequence is sufficient for lens-specific expres- sion of the fransgene in fransgenic mice. By confrast, the -52\+30 sequence is not capable of expressing the fransgene. Transgenic mice carrying various mutations in the 152 \ +30- CAT transgenes are being produced to identi- fy specific confrol elements. Analysis of the 5' flanking region of the cloned chicken pBl-crystaUin genomic frag- ment revealed, unexpectedly, the presence of the pA4-crystaIlin gene. This gene is arranged head to head with the pBl-crystallin gene, with approximately 2 kbp of spacer DNA separating the two genes. The pA4 gene was sequenced and shown to contain, Hke the pBl gene, six exons, with the first being noncod- ing. A pA4-crystallin cDNA has been isolated from the embryonic chicken lens, indicating that this gene is expressed. The close head-to- head linkage of the pBl and pA4 crystallin genes raises interesting possibilities with 212 Laboratory of Molecular and Developmental Biologi/ respect to the regulatory mechanisms used for their expression. We have shown previously that transcrip- tional activating sequences lie upsteam of the chicken pA3/Al-crystaUin gene between positions -382 and -143, which contains a complex transcriptional enhancer between positions -287 and -254. This year we have performed DNase 1 footprint analysis reveal- ing extensive protein binding in this region. FuU enhancer activity requires sequences upstream and downstream of -270, although sequence -270 \ -254 has some enhancer activity by itself. There is an AP-1 consensus binding site at position -264 \ -258. Multiple proteins in lens nuclear extracts, including members of the AP-1 and ATF\CREB families, bind oligo- nucleotide -271 \ -251 in gel shift assays. Finally, the -143 \ +22 sequence, which lacks the enhancer, fused to the CAT gene in trans- genic mice is sufficient for lens-specitic expres- sion. Thus, the -287/ -254 enhancer is not required to direct expression of the PA3/A1- crystallin gene in the lens. During his sabbatical year in the LMDB, Dr. Joseph Horwitz, from the Jules Stein Eye Institute, UCLA, has been exploring the possi- bility that pB2-crystallin is expressed in non- lens tissues. He had obtained stiong prelimi- nary evidence that this was the case before starting his sabbatical here. This would be of great interest because there is no unequivocal evidence that any of the p/y-crystallins have nonlens functions as do the a-crystallins or the enzjmie-crystallins. At present. Western immunoblotting of retina, testes, and brain from rats support the idea that pB2-crystalltn is present outside of the lens. Amino acid sequencing of the putative pB2 polypeptide from bovine retina provided strong support for the expression of this protein in that tissue. The only caveat that must be kept in mind is the possibility that the retinal pB2-crystallin was derived by contamination from the lens. PCR data support the existence of |3B2 mRNA in testes and brain of the rat, and Northern blots are presentiy being performed. Initial experiments on purified bovine |3B2-crystaUin indicate that this polypeptide is capable of autophosphorylation hke the cx-crystaUin polypeptides; this autophosphorylation reac- tion has not been characterized yet. Auto- phosphorylation ability would open the possi- bility that pB2-crystallin is involved in some type of metaboUc or signal transduction path- way, providing an explanation for its nonlens expression as well as introducing new consid- erations for the role(s) of this polypeptide in the lens. b-aystallin/argininosuccinate lyase (ASL). There are two Unked ASL/6-crystallin genes in the chicken, with the 5' gene being special- ized for expression in the lens. The 5' gene encodes a polypeptide that lacks ASL activity; the 3' gene encodes a polypeptide that has ASL activity. A similar situation exists in the duck, with the exception that the two ASL/ 6-crystaUin genes are equally expressed in the lens in this species. The 5' 61 gene is ex- pressed to a limited extent in nonlens tissues in both species. This year, cloning experi- ments have shown that the 4.6 kbp intergenic spacer of the 6-crystallin genes in the duck is 79 percent identical to the 4 kbp spacer in the chicken, except for the existence of a 615 bp CRl element, highly reiterated in the duck genome. Further sequence analysis revealed that inti-on 3 of the duck ASL/ 62 gene is 80 percent identical to intron 3 of the chicken 61 and ASL/ 62 genes; the identity rises to 93 percent in the region of the chicken 61 en- hancer core located within the third intron. These findings raise the speculation that the CRl repetitive element in the duck intergenic spacer plays a role in the high expression of the ASL/ 62 gene in the lens in this species, perhaps by diminishing the effectiveness of a silencer element that remains active in the chicken. Footprinting and transfection experiments have shown that the chicken 61-crystallin enhancer has at least two functional Pax-6 binding sites. The finding that Pax-6 activates the 81-crystallin enhancer shows for the first time that the a- and 6-crystaUin genes may be expressed by similar mechanisms. This idea is further supported by another recent finding, namely that USF binds to the 6EF1 site in the Z13 FY 1994 NEI Annual Report 81 enhancer. USF participates in the regula- tion of the chicken aA-crystallin gene. The 6EF1 site binds the negative regulator, 6EF1, in nonlens tissues to suppress gene activity. We envision that USF binding to the 8EF1 site activates 81 expression in the lens, and 8EF1 binding to this site represses 81 expression. Native 8-crystallin is a tetrameric protein. In 1979, we showed by SDS-polyacrylamide gel electrophoresis that the five major isoelec- tric forms of duck native 8-crystallin result from differences in the relative amounts of the two major polypeptide bands (about 49kD and 50kD). Current protein sequencing experi- ments have established that the 49kD poly- peptide is encoded by the 61-crystallin gene and the 50kD polypeptide is encoded by the ASL/62-crystallin gene. Moreover, we have demonstrated that the ASL activity of the isolated isoelectric forms of duck 8-crystallin is directiy related to the relative amount of 82 pol3^eptide present in the native tetrameric protein. Our new data indicate that the two 8-crystaIlin polypeptides interact cooperatively to modulate ASL activity. This provides a dominant negative role to the 81-crystaUin pol5rpeptide in the regulation of cellular ASL activity within tissues. Interesting cDNAs. We have cloned a partial cDNA for proxl from an embryonic chicken lens library. This homeodomain protein is knov^m to be expressed at high concentrations in the developing mouse lens; its Drosophila homologue, prospero, is ex- pressed in the lens secreting cone cells of the developing compound eye of this invertebrate. Thus, proxl /prospero is another candidate transcription factor for controlling the high expression of eye genes throughout the animal kingdom, as Pax-6. A genomic clone for proxl has also been cloned from humans that is in the process of being characterized. A zinc-finger cDNA has been cloned from a newborn mouse lens library. The mRNA for this gene is also present in numerous other tissues. It encodes a 555-amino add protein that contains riine C-terminal zinc-fingers and an N-terminal domain found in a subset of C2H2 zinc-fingers known as the Kruppel- associated box (KRAB). The amino acid sequence located between the KRAB domain and the zinc-finger shows an unexpected similarity to human profilagrin, a protein expressed in differentiating epidermal cells. Sequences that hybridized to this cDNA are detectable in 10 other mammalian species. Invertebrate crystallins. We are continuing to explore the structure and expression of the squid S-crystaUin genes, which are descended from glutathione S-transferase. A collabora- tive study with Dr. Richard N. Armstrong, fiom the University of Maryland, has led to the preliminary x-ray structure of squid gluta- thione S-transferase. We have cloned this cDNA from the digestive gland and its respec- tive gene of the squid last year. One of the interesting aspects of the squid glutathione S-transferase is that it is approximately 100 times more active than its mammalian homol- ogues in in vitro experiments. The three- dimensional structure of squid glutathione S-transferase is relevant to lens in view of the family relationship between this metabolic enzyme and the S-crystallins that make up approximately 90 percent of the squid lens protein. A collaborative immunolocaBzation study with Dr. Jacob Sivak, fiom the University of Waterloo, Canada, has demonstrated that during development S-crystalHns accumxilate first in the posterior lens primordium and subsequently in the anterior lens primordium and their respective lentigenic cells and con- necting lentigeruc processes. Examination of adult lens and lentigenic cells of the squid suggested that squid lens crystallins are syn- thesized in the mature squid. Another major interest of this section is the crystallins of the cubomedusan jellyfish. These primitive metazoan organisms have well developed eyes called oceUi, which con- tain cellular lenses. We have previously reported that the cubomedusan lenses contain three crystallins (Jl, J2, and J3) v^th molecular sizes near 35kDa, 20kDa, and 19kDa, respec- tively. Last year, we cloned the cDNAs and 214 Laboratory of Molecular and Developmental Biology genes for Jl-crystallins, which make up a family of three very closely similar polypep- tides, each encoded by a separate intronless gene. This year, we have cloned the cDNA and gene for JS-crystaUin. In contrast to Jl- crystaUin, J3-crystallin appears to contain only a single gene that has at least six introns. Another new and exciting development is the cloning of a genomic fragment, obtained by PCR, for cubomedusan Pax-6. Significance to Biomedical Research and tfie Program of tlie Institute The crystaUins comprise a diverse family of differentially expressed proteins that are re- quired for the optical and functional proper- ties of the transparent lens. Understanding the structure, function, and evolution of these protein families and their genes contributes to our knowledge of embryonic development, eukaryotic gene expression, cell differentiation, molecvdar evolution, cataract, and the visual system. That crystaUins are multifunctional proteins expressed in lens and nonlens tissues adds another dimension of interest and has implications for metaboUsm, ceU biology, and drug and gene therapy. Proposed Course The following studies are proposed for fiscal year 1995. (1) Continue studying czs-acting elements and frans-factors used for expression of crys- taUin genes in the lens and other tissues. (2) Give particular attention to Pax-6 and proxl transcription factors, especially with respect to their possible roles in crystallin gene expression. (3) Continue investigations concerning the possible role(s) of a-crystallins in metabolic reactions such as the regulation of transcrip- tion. (4) Continue the cloning and characteriza- tion of the cubomedusan jellyfish crystallin genes and their putative regulatory sequences. (5) Continue to investigate the structure and possible functions of the cephalopod S- crystaUins and the expression of their genes. NEI Research Program Lens and Cataract — Molecular Genetics Publications Brady JP, Piatigorsky J: A mouse cDNA en- coding a protein with zinc fingers and a KRAB domain shows similarity to human profUaggrin. Gene, in press. Cvekl A, Sax CM, Bresnick EH, Piatigorsky J: Complex array of positive and negative ele- ments regulates the chicken aA-crystaUin gene: involvement of Pax-6,USF, CREB/CREM and AP-1 proteins. Mol Cell Biol 14:7363-7376, 1994. Gopal-Srivastava R, Piatigorsky J: The murine aB-crystaUin/ small heat shock protein en- hancer: identification of aBE-1, aBE-2, aBE-3, and MRP control elements. Mol Cell Biol 13:7144-7152, 1993. Gopal-Srivastava R, Piatigorsky J: Identifica- tion of a lens-specific regulatory region (LSR) of the murine aB-crystaUin gene. Nucleic Acids Res 22:1281-1286, 1994. Hejtmancik JF, Kaiser MI, Piatigorsky J: Mo- lecular biology of inherited disorders of the eye lens, in Scriver CR, Beaudet AL, Sly WS, VaUe D (eds): The Metabolic Basis of Inherited Disease, ed 7. New York, McGraw-Hill Pub- lishing Co, in press. Hejtmancik JF, Piatigorsky J: Molecular and cell biology of the transparent and cataractous eye lens, in Bittar EE (ed): Fundamentals of Medical Cell Biology. Greenwich, JAI Press Inc, in press. Kantorow M, Piatigorsky J: a-CrystaUin/ small heat shock protein has autokinase activity. Proc. Natl Acad Sci USA 91:3112-3116, 1994. 215 FY 1994 NEI Annual Report Piatigorsky J: The twelfth Frederick H. Verhoeff Lecture: gene sharing in the visual system. Trans Am Ophthalmol Soc LXXXI: 283- 298, 1993. Piatigorsky J, Kantorow M, Gopal-Srivastava R, Tomarev SI: Recruitment of enzymes and stress proteins as lens crystallins, in Jansson B, Jornvall H, Ryberg U, Terenius L, VaUe B (eds): Toward a Molecular Basis of Alcohol Use and Abuse. 86th Nobel Symposium. Basel, Birkhauser Verlag, 1994. Sax CM, Cvekl A, Kantorow M, Sommer B, ChepeUnsky AB, Piatigorsky J: Identification of negative-acting and protein-binding ele- ments in the mouse aA-crystaUin -1556/-1165 region. Gene 144:163-169, 1994. Sax CM, Piatigorsky J: Expression of the a- crystallin/ small heat shock protein /molecular chaperone genes in the lens and other tissues, in Meister A (ed): Advances in Enzymology and Related Areas in Molecular Biology. New York, John WUey and Sons Inc, 1994, vol 69, pp 155- 201. Tomarev SI, Zinovieva, Piatigorsky J: Primary stiucture and lens-specific expression of genes for an intermediate filament protein and a P- tubulin in cephalopods. Biochim Biophy Acta 1216:245-254, 1993. Tomarev SI, Duncan MK, Roth JH, Cvekl A, Piatigorsky J: Convergent evolution of crystal- Un gene regulation in squid and chicken: the AP-l/ARE connection. ] Mol Evol 39: 134-143, 1994. West JA, Sivak JG, Pasternak J, Piatigorsky J: Immunolocalization of S-crystallins in the developing squid {Loligo opalescens) lens. Dev Dynamics 199:85-92, 1994. 226 '^ I PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00259-05 LMDB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Molecular Biology of th e Cornea PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Joram Piatigorsky Ph.D. Chief LMDB, NEI Others: W. Todd Kays Ph.D. IRTA LMDB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.05 PROFESSIONAL: 1.05 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects Q (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Aldehyde dehydrogenase class 3 (ALDH3) comprises approximately 40 percent of the cellular protein of the mammalian corneal epithelial cells, an amount reminiscent of an enzyme-crystaUin in the lens. Consequently, we are investigating the molecular basis for the high expression of the ALDH3 gene in the corneal epithelial cells. The results will be compared with those obtained for crystallin genes in the lens and will provide a foundation for eventual gene therapy in the cornea. The complete mouse ALDH3 protein has been deduced from a cloned corneal complementary deoxyribonucleic acid (cDNA). Three 16-18 kbp mouse genomic fragments for ALDH3 have been cloned: one comprises the entire ALDH3 gene, one contains about 13 kbp of 5' flanking sequence, exon 1, intron 1 (3.2 kbp) and part of exon 2, and the third is being analyzed. Northern blots have established that ALDH3 messenger ribonucleic acid (mRNA) is at least 100 times more prevalent in the cornea than in the stomach, bladder, and lung, the only other tissues showing a trace of this gene product. Transfection and transgenic mouse experiments using the chloramphenicol acetyltransferase (CAT) reporter gene have shown that 1050 bp of 5' flanking sequence of the ALDH3 gene gives low-level expression in the liver, but is inactive in all other tissues tested, including the cornea. New promoter/CAT constructs containing intron 1 of the ALDH3 gene have been made and are being tested. The 5' flanking sequence of the ALDH3 gene contains numerous potential control elements, including antioxidant response elements, which will be tested for function. 217 PHS 6040 (Rev. 5/92) FY 2994 NEI Annual Report Project Description Objectives The project objectives are to identify and characterize the genes that are preferentially expressed in the epitheUum and endotheUum of the cornea and to study the molecular basis for their expression in this transparent eye structure. Methods Conventional molecular biology methods of cloning, sequencing, recombinant deoxyribo- nucleic acid (rDNA) construction, transfection, and transgenic mouse production are used. Major Findings Aldehyde dehydrogenase class 3 (ALDH3) comprises approximately 40 percent of the soluble protein of the corneal epithelial cells in the mouse and other mammals, including humans. Such abundance suggests that this metaboUc enzyme may have a refractive function Uke an enzyme-crystaUin in the lens. It is also possible that the high concentration of ALDH3 protects the eye from ultraviolet absorption and /or protects the cornea from oxidative damage. Moreover, the extremely high expression of the ALDH3 gene in the corneal epithelial cells provides a potential source of regulatory sequences for directing gene expression to this tissue, which could be of great value for gene therapy. • Last year, we created transgenic mice carrying a transgene comprising the P-galacto- sidase reporter gene fused to 1.1 kbp of the cloned mouse ALDH3 gene that we thought contained the promoter. However, no expres- sion of the transgene was observed in any of the tissues of the transgenic mice that were examined, including the cornea. We thus performed rapid amplification of complemen- tary DNA (cDNA) ends (RACE) experiments using corneal ribonucleic acid (RNA) and showed the existence of an additional 5' exon, indicating that our construct lacked the cor- neal promoter. This 5' exon does not appear to be present in the human ALDH3 gene on the basis of the published literature. This year, we have characterized a new 16 kbp mouse ALDH3 genomic fragment that was shown to contain approximately 13 kbp of 5' flanking sequence, exon 1 (40 bp), intron 1 (about 3.2 kbp), and part of exon 2 (40 bp). Two additional mouse ALDH3 genomic clones were obtained that were 16 kbp and 18 kbp, respectively. The 18 kbp fragment appears to contain the whole gene on the basis of se- quencing with exon specific primers; the 16 kbp fragment is in the process of being ana- lyzed. The entire mouse ALDH3 sequence was deduced from a fuU-length cDNA that we isolated from a corneal library. The high expression of the ALDH3 gene in the mouse and bovine cornea was corifirmed by Northern blot experiments. A trace of ALDH3 messenger RNA (mRNA) was present in the stomach, bladder, and lung (at least 100 times less than the cornea), and no ALDH3 mRNA was observed in the lens, adrenal gland (except a dubious smear of higher molectilar weight), brain, and Hver. Constructs were made that contain the -1050/ +21 fragment of the ALDH3 gene fused to the chloramphenicol acetyltransferase (CAT) gene in both the forward and backward orien- tation. Transfection tests using corneal SIRC cells and lens N/N1003A cells (both from rabbit) showed that this plasmid has low but detectable promoter activity leading to CAT expression when the putative ALDH3 promot- er is in the correct orientation. Unexpectedly, however, this transgene did not express in transgenic mice, except for limited amounts in the Hver. The cornea was painfully devoid of CAT in the transgenic mice. Consequently, new constructs have been made that contain 1050 bp of 5' flanking sequence, exon 1, the 3.2 kbp intron 1 and the first 5 bp of exon 2 (excluding the ATG translation start codon) of the ALDH3 gene fused to the CAT gene to test the possibility that a corneal enhancer is present in intron 1. At the time of writing, these constructs are being tested for promoter 218 LaboratoTy of Molecular and Developmental Biology activity in transfected cells and will be used to generate transgenic mice. Finally, analysis of the 5' flanking se- quence of the ALDH3 gene has revealed numerous potential regulatory elements, including NF-kB, AP-1, NFI, AP-2, SP-1, GRE, EivF/CREB, ARE, and XRE sites. The pres- ence of ARE and XRE sites supports the idea that the high expression of this gene may be connected to an ultraviolet responsive or antioxidant response. Significance to Biomedical Research and the Program of the Institute The molecular biology of corneal epithelium and endothelium has not advanced to the same extent as that of the collagenous stroma; consequentiy, it should be investigated. The cornea is a tiansparent ectodermally derived tissue like the lens. Thus, comparative studies between it and the lens are of special interest with respect to tiansparency and the regula- tion of gene expression. Moreover, because of our finding several years ago that corneal epithelial cells show taxon-specific gene sharing of metaboUc en- zymes, as does the lens, comparative studies on the cornea and lens are important from developmental and evolutionary perspectives. Finally, the cornea is particularly accessible for gene therapy on account of its exposure to the surface and its association with numerous hereditary diseases. The identification of a functioned promoter with corneal specificity will open the door to gene therapy and genet- ic manipulation in the cornea as isolation of crystallin promoters with lens specificity did in that tissue. Proposed Course The following investigations are planned for fiscal year 1995: (1) Identify the corneal-specific regulatory elements in the ALDH3 gene of the mouse by transfection and transgenic mouse experi- ments. (2) Establish a convenient culture system to analyze the expression of ALDH3 and other corneal genes by transfection. (3) Begin to analyze the hans-factors used for expression of the mouse ALDH3 gene in corneal epithelial and, perhaps, endothelial cells. (4) Obtain a human ALDH3 gene for comparison with the mouse gene and to provide the foundations for gene therapy in human corneas. NEI Research Program Corneal Diseases — Structure and Function, Stroma 219 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00255-06 LMDB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Molecular Biology and Functions of Lens Proteins PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI; Graeme J. Wistow Ph.D. Chief, Section on Molecular LMDB, NEI Structure and Function Others: Vishwas Paralkar Caroline Graham Lorenzo Segovia Jill Richardson Ronit Frilling Cynthia Jaworski Ph.D. B.S. Ph.D. Ph.D. Ph.D. Ph.D. Visiting Associate Biologist Visiting Fellow Visiting Fellow Visiting Fellow Chemist, Section on -Molecula r Gen et i c s — LMDB, NEI LMDB, ^fEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI COOPERATING UNITS (if any) National Institute of Allergy and Infectious Diseases (Christine Kozak, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Structure and Function INSTITUTE AND LOCATION NEI. NIH. Bethesda. MD 20892 TOTAL STAFF YEARS: 6.9 PROFESSIONAL: 5.9 1.0 CHECK APPROPRIATE BOX(ES) r~| (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Crystallins are the major components of the normal eye lens. In a novel evolutionary process crystallins have arisen by the gene recruitment of stress-proteins and enzymes without prior gene duplication. In the lens- specific alternative promoter of guinea pig NADPH:quinone oxidoreductase/^-crystallin we have identified an element that is essential for function. We now find that this element contains a binding site for Pax-6, a "master gene" for eye development. Mutation of the Pax-6 site abolishes promoter function and Pax-6 is required for formation a lens-specific complex. Futhermore, Pax-6 is expressed in the mature lens. Pax-6, thus, has a continuing role in maintenance of lens-specific gene expression and a key role in the gene recruitment of a crystallin. //-crystallin, which was discovered in marsupial lenses, is a novel NADPH-binding protein related to enzymes of ornithine and glutamate metabolism. In humans, in which it has not been recruited as a crystallin, it is expressed most abundantly in photoreceptor outer segments, suggesting an unexpected role in the visual process. Other proteins are essential for normal development in lens. Migration inhibitory factor (MIF), a small protein expressed in differentiating lens cells, is essential for progression through the cycle. Antisense to MIF abolishes proliferation of growth factor stimulated and cancer cells and halts them at the Gl/S boundary. Promoter analysis of the human MIF gene has identified a region required for growth factor response. We have also found that LP2, a lipid/retinoid binding protein we have cloned from bovine lens, is expressed preferentially in differentiated fiber cells. 220 PHS 6040 (Rev. 5/92) LaboratoTy of Molecular and Developmental Biologi/ Project Description Additional Personnel Ph.D. Visiting Fellow, MG, LMDB, NEI Summer Program Ales Cvekl Steven Puopolo Objectives We are investigating the molecular basis of normal lens structure and function. This includes the characterization of crystaUins, their lens and nonlens function, and their mechanisms of expression and recruitment. This has entailed the identification of factors responsible for regulation of lens cell differen- tiation and gene expression. The interplay of such factors is an essential part of normal lens development and function. Methods A wide range of molecular biology techruques are used, including messenger ribonucleic acid (mRNA) analysis, gene and complementary deox5aibonucleic acid (cDNA) cloning and se- quencing, functional gene promoter analysis in cultured cells and in transgenic mice, polymer- ase chain reaction, and antisense technology. We perform some protein analysis and make use of commercial facilities for protein se- quencing. We make extensive use of com- puter-based sequence analysis and molecular modeling. Major Findings (1) Gene Recruitment: Enzyme CrystaUins. Guinea pig ^-crystallin is a taxon-specific crys- tallin, an enz5ane that has achieved high expression in lens through an alternative, lens- specific promoter. This promoter contains an element (ZPE) that binds tissue-specific com- plexes in electrophoretic mobility shift assay. Jill Richardson has shown that both lens- spedfic complex formation and promoter function depend on an intact binding site for Pax-6 in the ZPE. Pax-6 is expressed in em- bryoruc eye and central nervous system and is implicated in the establishment of lens compe- tence. Antisera to Pax-6 specifically abolish lens complex formation by the ZPE. Protein and mRNA for Pax-6 are present in mature mouse lens and in cells that support t,-crystal- lin lens promoter expression but not in other cell types. This suggests that L,-crystallin is a target gene for Pax-6 and supports the idea that Pax-6 is a "master gene" factor for tissue- specific gene expression in the lens. Ronit Frilling has shown that the isolated fragments covering the ZPE region of the t-crystallin promoter can confer lens-preferred expression on a heterologous promoter, the thymidine kinase minimal promoter driving a reporter gene. Experiments in transgenic mice show that sequences upstream of the Pax-6 site, between -498 and -385, are required for sup- pression of promoter function in brain, which is also a site of Pax-6 expression. Although some taxon-specific crystaUins are famihar enz5anes, others were discovered first as crystaUins. ju,-Crystallin is the major component of the eye lens in several Austia- Uan marsupials. In other mammals, including humans, it is expressed at enzymatic levels in lens, but Lorenzo Segovia has shown that it is most strikingly expressed in retinal photore- ceptor ceU outer segments. /x-Crystallin has sequence similarity with bacterial ornithine cyclodeaminases and glutamyl-transfer RNA reductases, suggesting a role in amino-acid metaboUsm that probably involves a gluta- mate-related pathway. This is particularly significant because glutamate is the neuro- transmitter of the photoreceptors. The gene for /i-crystallin has been cloned from both kangaroo, where it has undergone gene re- cruitment, and from human, where it serves only as an enzyme. The human gene for ix- crystallin maps close to a locus for congenital cataract and microphthalmia. /x-Crystallin has also been mapped in the mouse genome. Another major enzyme crystallin (up to 25 percent of total protein) in mammals is i~\- CTystallin found in elephant shrews. Caroline Graham has cloned the complete cDNA se- quence for this protein from two different species. Current results suggest that T|-crystal- 221 FY 1994 NEI Annual Report lin is very closely related to aldehyde dehy- drogenase 1 (ALDHI) and is expressed both in lens and liver. However, a second, closely related ALDHI is also expressed in liver, suggesting that the gene recruitment of Ti-crystallin has led to gene dupUcation and specialization, perhaps as a consequence of adaptive conflict. The elephant shrew genome also contains several processed pseudogenes for Tj-crystallin/ALDHl-like sequences. aB-crystallin is multifunctional, serving as both a major structural protein in the lens and as a small heat shock protein in other tissues in mammals. The gene for aB-crystallin in a bird {Anas platyrhynchos) has been cloned and sequenced. Because only the most important functional features of a gene are conserved among distantly related species, comparison of this sequence with those of mammals can be very informative. Although several function- ally defined elements in mammals are con- served in the duck gene, consensus stress- response elements are not well conserved. Current experiments are aimed at determining whether the bird gene does indeed have the stiess role suggested for mammalian genes. (2) Molecular Markers of Differentiation. Macrophage migration inhibitory factor (MIF) was originally identified as a l5anphokine. However recent work strongly suggests a role for MIF beyond the immune system. We have found that it is expressed specifically in the differentiating cells of the immunologically privileged eye lens and in many other tissues. Now Vishwas Paralkar has shovwi that MIF is an essential part of the mitogenic response to growth factors operating through two differ- ent receptor systems. Both platelet-derived growth factor and transforming growth factor pi stijnulate expression of MIF in NIH 3T3 cells in a delayed early response. When cells treated with either growth factor are grown in the presence of antisense oligo to mouse MIF, MIF protein synthesis and cellular prolifera- tion are abolished. Control oUgos have no effect under identical conditions. When anti- sense oligo is removed the cells regain the ability to proliferate in response to mitogenic signals. Analysis of levels of cycUns E, A, and B suggests that the effect of MIF is localized close to the Gl/S transition in the cell cycle and that MIF is essential for progression into S phase. In promoter analysis of the human MIF gene a region required for induction by growth factors has been localized. This MIF gene region also contains an E-box, a potential myc family-binding site. The gene for MIF has been mapped in human and mouse. LP2 is a member of the lipid /retinoic add binding family of P2 proteins. Cynthia Jaworski has cloned bovine LP2 and has shown that its mRNA is expressed preferen- tially in differentiated fiber cells. Because retinoic acid has now been implicated in fiber cell specific y-crystaUin gene expression, this protein could have a direct role in mediating lens gene expression. Molecular modeling shows that LP2, unlike adipocyte P2, myelin P2 or other relatives, contains two close cyste- ine residues capable of disuilfide cross-Unking. LP2 is thus a potential target for oxidative damage to the lens in aging and cataract. Significance to Biomedical Research and the Program of the Institute We are uncovering fundamental mechanisms in the evolution and differentiation of the lens that may be generalizable to other complex tissues. We have shown how a single gene can have multiple functions and how it can acquire tissue-specific patterns of expression. In examining proteins that have been recruited as crystaUins, we have discovered a novel enzyme that may have an important role in human retinal photoreceptors. Our work in lens has also revealed important markers for cellular differentiation. In particular one of these proteins, MIF, has an essential role in cell proliferation, and antisense treatment to suppress MIF halts proliferation, even of transformed ceUs. Proposed Course (1) To continue to examine the molecular mechanisms for lens preferred expression and gene recruitment of crystaUins. 222 Laboratory of Molecular and Developmental Biology (2) To characterize the function and nonlens role of /x-crystallin, particularly its role in photoreceptor outer segments. (3) To define the role of MIF in the cell cycle and to test the potential for antisense treatment to suppress the growth of trans- formed ceUs. (4) To determine the function of LP2 pro- tein, a marker for differentiation in the lens. NEI Research Program Lens and Cataract — Molecular Genetics Publications Graham C, Wistow G: The predominant cadherin in fetal human lens is identical to N-cadherin and is not a candidate locus for the Marner cataract. Exp Eye Res 59, in press. Lee DC, Gonzalez P, Wistow G: Zeta-crystal- lin: a lens-specific promoter and the gene recruitment of an enzyme as a crystaUin. / Mol Biol 236:669-678, 1994. Paralkar V, Wistow G: Cloning the human gene for macrophage migration inhibitory factor (MIF). Genomics 19:48-51, 1994. Patent G.J. Wistow. US Patent 5,328,990: "Isolation of macrophage migration inhibition factor from ocular lens." 223 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00251-07 LMDB PERIOD COVERED October 1 , 1993 to Septembe r 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Gene tically Engineer ing the Eye with the aA-Cr ystal lin Promoter PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Ana B. Chepelinsky Ph.D. Head, Section on LMDB, NEI Others: Devonne M. Parker Charles Egwuagu Chi-Chao Chan Robert B. Nussenblatt Jorge Sztein B.S. Ph.D. M.D. M.D. D.V.M. Regulation of Gene Expression Biologist Scientist, PHS Head, Section on Immunopathology Clinical Director Visiting Associate LMDB, NEI LLNEI LI, NEI LI, NEI VRRS, NEI COOPERATING UNITS (if any) Department of Cell Biology, Baylor College of Medicine, Howard Hughes Medical Institute (Paul Overbeek, Ph.D.; Michael Robinson, Ph.D.); Imperial Cancer Research Fund, London, England (Clive Dickson, Ph.D.); Gerontological Research Unit, National Institute of Health and Medical Research, Paris, France (Yves Courtois, Ph.D.; Maryvonne Laurent, Ph.D.) LAB/BRANCH Labo r atory of Molecular and Developmental Biology SECTION Section on Regulation of Gene Expression INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.8 PROFESSIONAL: 0.5 0.3 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) We generated transgenic mice and rats expressing interferon (IFN)-y in their lenses to investigate the possible role of this lymphokine in ocular pathogenesis. Embryonic lens and retina differentiation were affected in the ciA-crystallin/IFN-Y transgenic mice resulting in microphthalmia, microphakia, retinal detachment, and persistent hyperplastic primary vitreous in the adult mice. Major histocompatibility complex (MHC) class II messenger ribonucleic acid (mRNA) levels were significantly increased in the transgenic eyes, and MHC class II proteins were expressed in their cornea, iris, ciliary body, choroid, lens, and retinal pigment epithelial (RPE). Constitutive expression of IFN-y and its induction of MHC class II molecules in the eye provide a useful model to study the linkage between aberrant MHC class II expression and predisposition to autoimmunity and the role of IFN-y in the treatment of inflammatory eye diseases and cytokine signaling during embryonic eye development. Fibroblast growth factor (FGF)-3 expression was directed to the eye to investigate how the aberrant expression of this growth factor would affect the developmental program of the eye. The aA-crystallin/FGF-3 transgenic mice presented exophthalmia and aberrant elongation of central lens epithelia at 15 days of embryonic development. The hypertrophic lens mass was extruded through the cornea at 16 days of embryonic development, resulting in cornea perforation. Postnatal microphthalmia was characterized by intraocular hyperplastic glandular structures replacing the normal iris, ciliary body, and lens. 224 PHS 6040 (Rev. 5/92) Lahoraton/ of Molecular and Developmental Biology Project Description Objectives The objective of this project is to understand how aberrant genetic expression of interferon gamma (IFN-y) or fibroblast growth factor-3 (FGF-3) under the control of the aA-crystallin promoter perturbs normal eye development in transgeruc mice. Methods Recombinant deox5Tibonucleic acid (rDNA) techniques used in this study include plasmid construction, oUgonucleotide sequencing. Southern and Northern hybridizations, DNA sequencing, primer extension, polymerase chain reaction (PCR), reversed transcription PCR. Other molecular biology techniques used in the study include immunohistochem- istry, in situ hybridization, and production and analysis of transgenic mice. Major Findings (1) IFN-y. This project is conducted in collab- oration with Drs. Charles Egwxiagu, Jorge Sztein, Chi-Chao Chan, and Robert B. Nussenblatt from the Laboratory of Immunol- ogy (LI) at the NEI. The IFN-y gene is specifi- cally expressed in activated T lymphocytes and natural killer cells. It plays a crucial role in the ontogenesis and phenomenology of the immune response and has potent immunomo- dulatory and antiproliferative effects on tumor cells. The ectopic expression of IFN-y in the lens of transgeruc mice allowed us to study its effect on eye development and its regulation of major histocompatibility complex (MHC) class n gene expression in a nonlymphoid tissue such as the lens. We previously generated FVB/N and BALB/c transgenic mice containing as a transgene the murine aA-crystaUin promoter (-3667+46) fused to the murine IFN-y coding sequence. In both aA-crystaUin/ IFN-y trans- genic mouse Lines, ectopic expression of IFN-y in the lens affected the growth of the whole eye, resulting in microphthalmia and blephar- ophimosis. Lens differentiation was severely affected resulting in microphakia, impairment of lens fiber formation and cataract, thickening of the anterior lens capsule, and rupture of the posterior capsule. Retardation of retinal differentiation into inner and outer neuroblas- tic layers was observed in the transgenic eyes. Serous retinal detachment with presence of macrophages in the subretinal space, persis- tent hyperplastic primary vitreous, corneal vascularization, and absence of a normal anterior chamber were observed in the adult transgenic mice. MHC class 11 messenger ribonucleic acid (mRNA) levels were sigiufi- cantly increased in the eyes of transgenic mice, and MHC class 11 proteins were expressed in the corneas, irises, ciliary bodies, choroids, lenses and retinal pigment epitheUa. Expres- sion of genes coding for IFN-y-inducible transcription factors, interferon-consensus- sequence-binding protein, and interferon response factor 2, absent in the normal eye, were induced in the transgenic eyes. These results indicate that the ectopically expressed transgenic IFN-y is biologically active in vivo. We also derived a transgenic Sprague Dawley rat line using the same murine aA-crystallin /IFN-y construct. The transgenic rat eyes, similar to those from the transgenic mice, present microphthalmia and micropha- kia with cataract formation. However, in contrast to the transgenic mouse, the anterior chamber of the transgenic rat eye is weU formed, and the architecture of the retina is intact with focal retinal serous detachment. The aA-crystaUin/ IFN-y transgenic rat, the first transgenic rat strain generated for vision research, is of particular interest because the rat is a well-characterized species for experi- mental autoimmune uveitis studies. (2) FGF-3. This is a collaborative project with Drs. Paul Overbeek and Michael Robin- son, from Baylor CoUege of Medicine, and Dr. Clive Dickson, from the Imperial Cancer Research Fund. FGF-3 /int-2 is a member of the fibroblast growth factor (FGF) family. To assess whether ectopic expression of FGF-3 would perturb normal lens development, we 225 FY 1994 NEI Annual Report directed its expression to the eyes of trans- genic mice using the murine aA-crystallin promoter. We obtained three lines of trans- genic mice expressing the aA-crystallin /FGF-3 transgene. The anterior lens epithelia of the transgenic mice undergo premature cell elon- gation at day 14 of embryonic development concomitant with major intrinsic protein (MIP) synthesis. The lens mass was extruded through the cornea, resulting in cornea perfo- ration at 16 days of embryonic development. At postnatal day four, intraocular glandular structures that replaced the normal irises, ciliary bodies, and lenses and stained positive- ly for FGF-3 and Muc-1 (a marker for secreto- ry epitheUa) and negatively for connexin 46 and MIP were observed. We observed a marked increase in Muc-1 mRNA levels, while MIP, connexins 46 and 50 mRNA levels were drastically reduced in the adult transgenic mouse eyes. The ectopic expression of the FGF-3 during the embryonic development of the lens induces prematvure differentiation of the central lens epitheUa, expulsion of the lens through the cornea, and postnatal appearance of intraocular secretory glandular epithelia. (3) Acidic FGF. In collaboration with Drs. Overbeek and Robinson and Dr. Yves Courtois, from the Institute for Gerontological Research (INSERM), we obtained three lines of transgenic mice carrying as a transgene the aA-crystaUin promoter fused to the bovine addic FGF complementary DNA. These mice do not present any particvilar phenotype. We are currently studying whether the lens cells derived from these mice are able to give rise to a lens epithelia cell line with differentiated properties. Significance to Biomedical Research and the Program of the institute Constitutive expression of IFN-y, and its induction of MHC class 11 molecules in the eye, provides a useful model to study the linkage between aberrant MHC class n expres- sion and predisposition to autoimmunity and the role of EFN-y in the treatment of inflam- matory eye diseases and cytokine signaling during embryonic eye development. The ectopic expression of FGF-3 will allow us to elucidate the mechanisms underlying eye development. Proposed Course The following studies wiU continue during fiscal year 1995: (1) The effect of IFN-y on the regulation of gene expression in the eyes of the trans- geruc mice will be further characterized. (2) The effect of FGF-3 on gene expression in the eyes of transgenic mice will continue to elucidate the role of FGF-3 in premature lens epithelia differentiation and the appearance of intraocular secretory epithelia. NEi Research Program Lens and Cataract — Molecular Genetics Pubiications ChepeUnsky AB, Robinson M, Overbeek PA, Parker DM, Chan C-C, Jamieson S, Dickson C: FGF-3 ectopic expression induces differentia- tion of central lens epithelia and appearance of secretory epithelia in the eyes of transgenic mice. Invest Ophthal Vis Sci 35(suppl):1988, 1994. Egwagu CF, Sztein J, Chan C-C, Reid W, Mahdi R, Nussenblatt RB, ChepeUnsky AB: Ectopic expression of gamma interferon ex- pression in the eyes of transgenic mice induc- es ocular pathology and MHC class 11 gene expression. Invest Ophthal Vis Sci 35:332-341, 1994. Egwagu CF, Sztein J, Chan C-C, Mahdi R, Nussenblatt RB, ChepeUnsky AB: Gamma interferon expression disrupts lens and retinal differentiation in transgenic mice. Dev Biol, in press. Egwagu CF, Sztein J, Chan C-C, Mahdi R, Nussenblatt RB, ChepeUnsky AB: Transgeruc rat and mouse models for the study of intraoc- 226 Laboratory of Molecular and Developmental Biology ular effects of IFN-y and autoimmunity. Sax CM, Cvekl A, Kantorow M, Sommer B, Invest. Ophthal Vis Sci 35 (suppl):1987, 1994. Chepelinsky AB, Piatigorsky J: Identification of negative-acting and protein-binding ele- Egwagu CF, Sztein J, Chan C-C, Mahdi R, ments in the mouse aA-crystalUn -1556/-1165 Nussenblatt RB, Chepelinsky AB: Transgenic region. Gene 144:163-169, 1994. rat and mouse models for studying the role of gamma interferon and MHC class II in intra- ocular diseases and autoimmunity. Proceed- ings of the 6th International Symposium on the Immunology and Immunopathology of the Eye, in press. PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00253-06 LMDB PERIOD COVERED Octob er 1, 1993 to Septe mber 30, 1994 TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) Regulation o f E x pres sion o f Lens Fiber Membrane Genes PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Ana B. Chepelinsky Ph.D. Head, Section on LMDB, NEI Regulation of Gene Expression Others: Chiaki Ohtaka-Maruyama Ph.D. Visiting Fellow Xiaoyan Wang M.D. IRTA Fellow Devonne M. Parker B.S. Biologist Chris Chon Summer Student LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI COOPERATING UNITS (if any) Harvard University (David Paul, Ph.D.); Columbia University (Jorge Fischbarg, M.D.); Tokio Medical and Dental University, Japan (Sei Sasaki, M.D.) Laboratory of Molecular and Developmental Biology SECTION Section on Regulation of Gene Expression INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.37 PROFESSIONAL: 2.5 0.87 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x| (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project studies the regulation of expression of genes encoding lens fiber membrane channel proteins. We are presently focusing on the regulation of expression of the gene encoding, the major intrinsic protein (MIP) of the lens fiber membrane, that is specifically expressed in the ocular lens fibers and belongs to an ancient superfamily of transmembrane channel proteins. We are studying the cis regulatory elements responsible for the lens specificity and developmental regulation of the MIP gene. We are fusing 5' flanking sequences of the human MIP gene to the reporter chloramphenicol acetyltransferase (CAT) gene and analyzing CAT gene expression in transient assays in primary cultures of lens cells. Deletion analysis of the 5' flanking sequence of the human MIP gene suggests that negative regulatory elements are located within the sequence -2840/-254. The human MIP sequence -70/- 40 appears to contain an important regulatory element for the activity of the MIP promoter. This sequence contains one of the domains that interacts with Spl and AP2 transcription factors by DNase I footprinting analysis; it also forms a complex with chicken lens nuclear extracts that appear as a retarded band in mobility shift assays. Mutations at positions -49/-51 affect binding of this nuclear factor in vitro, and deletion of the sequence -70/-49 abolishes promoter activity in transfected lens cells. These studies will further our understanding of the regulation of the MIP gene expression in the lens. 228 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology Project Description Oblectives The objective of this project is to elucidate the mechanisms involved in the regulation of expression of fiber membrane genes involved in cell-cell communication in the lens. The identification of the cis regulatory elements of these genes and their interaction with trans- acting factors are essential for understanding the regulation of gene expression in the lens. Methods Recombinant deoxyribonucleic acid (rDNA) techniques used in this study include screen- ing genomic libraries; subclonirig; plasmid construction; oligonucleotide synthesis; South- em and Northern hybridizations; DNA se- quencing; primer extension; polymerase chain reaction (PCR); reversed transcription PCR; gel mobUity shift assays; DNA footprinting; meth- ylation interference; preparation of nuclear extracts by subcellular fractionation; in vitro transcription; tissue culture techniques, includ- ing transfection of primary lens explants and cell Unes; analysis of transgenic mice; in situ hybridization. Major Findings (1) Cis Regulatory Elements of the Human Major Intrinsic Protein (MIP) Gene Promoter. We have characterized 2840 bp of 5' flanking sequence of the human MIP gene to identify the cis regulatory elements responsible for the tissue specificity and developmental regulation of the MIP gene. We found that a DNA frag- ment containing the human MIP sequence -253/ +42 fused to the reporter chlorampheni- col acetyltransferase (CAT) gene activates CAT gene expression when transfected into lens cells. However, the -2840 / +42 sequence does not activate CAT gene expression, suggesting that negative regulatory elements are located v^thin the sequence -2840/ -254. Deletion mutants experiments indicated that -70/ +42 sequence contains an active promoter in transfected lens ceUs. However, when se- quences -70/ -49 are deleted, the promoter activity is lost. The human MIP sequence -70/ -40 appears to contain an important regulatory element for the activity of the MIP promoter. An oUgonucleotide corresponding to sequence -67/ -38 forms a complex with chicken lens nuclear extracts that appear as a retarded band in mobility shift assays. Mutations at positions -49/ -51 affect binding of this factor. This sequence corresponds to one of the domains that interact with Spl and AP2 transcription factors by DNase I footprinting analysis. We have generated two lines of transgenic mice containing 2840 bp of the human MIP gene 5' flanking sequence and 42 bp of exon one fused to the p-galactosidase gene as a tiansgene. We are presently analysing p- galactosidase expression in these transgenic mice to determine whether cis regulatory elements responsible for lens-specific expres- sion of the MIP gene are located in that do- main. (1) Cloning of the Mouse MIP Gene. We have isolated one MIP genomic clone from a PI phage mouse genomic library. Subcloning and characterization of this clone will allow us to study the regulation of expression of the murine MIP gene. (3) Cloning of the Connexin 46 Gene. The connexin 46 gene encodes one of the lens fiber gap junction proteins. To be able to study how its expression is regulated in the lens, we are sequencing, in collaboration with Dr. David Paul, the mouse cormexin 46 gene from a clone isolated from a mouse genomic library. (4) Aquaporin Gene Expression in Cornea Endothelial Cells. Although the physiological basis for the maintenance of corneal transpar- ency has been extensively studied, the molecu- lar mechanisms involved in maintaining cornea transparency are poorly understood. Swelling of the corneal stroma is involved in the loss of cornea transparency. In collabora- tion with Dr. Jorge Fischbarg, from Columbia Uruversity, we are characterizing the member of the MIP family responsible for the CHIP28- 229 FY 1994 NEl Annual Report like water channels observed in primary cultures of bovine cornea endothelial cells. Sequencing data indicate that it has a high degree of identity with CHIP28, suggesting that it may be a new member of the MIP family derived from CHIP28 by gene conver- sion. Significance to Biomedical Research and the Program of the Institute Proper lens fiber membrane biosynthesis and physiology are of upmost importance for maintairung lens transparency. Membrane protein synthesis is temporal and spatially regulated during lens development. Lens membranes undergo biochemical and structur- al changes during cataractogenesis and aging. Therefore, studying the regulation of genes encoding lens membrane channels should further our understanding, not only of the mechanisms involved in the regulation of gene expression in the normal lens but also of its disruption during disease. Proposed Course The following studies will continue during fiscal year 1995: (1) Characterization of the cis regulatory elements of the mouse MIP gene promoter and comparison with its human homologue. (2) Interaction of the mouse MIP gene cis regulatory elements with transcription factors. (3) Sequencing of connexin 46 genomic clones to locate the 5' end of this gene. NEl Research Program Lens and Cataract — Molecular Genetics Publications ChepeUnsky AB: The MIP transmembrane channel family, in Peracchia C (ed): Handbook of Membrane Channels. San Diego, Academic Press, 1994, pp 413-432. Chepelinsky AB, Ohtaka-Maruyama C, Wang X: General transcription factors and lens specific expression of the MIP gene. / Cellular Biochem 18(B):388, 1994. Ohtaka-Maruyama C, Wang X, Chepelinsky AB: AP2 transcription factor is involved in the transcription of the lens MIP Gene. / Cellular Biochem 18(C):50, 1994. Saito F, Sasaki S, Chepelinsky AB, Fushimi K, Marumo F, Ikeuchi T: Human AQP-2 and MIP genes, two members of the MIP family, map within chromosome band 12ql3 using two-color FISH. Cytogen Cell Genetics, in press. Wang X, Ohtaka-Maruyama C, Chepelinsky AB: Cis regulatory elements of the human MIP gene promoter. Invest Ophthalmol Vis Sci 35(suppl):1997, 1994. 230 PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00285-02 LMDB PERIOD COVERED October 1, 1993 to September 30, 199 4 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) NEI Central T ransgeni c Animal Pr oduction F acility PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Eric Wawrousek Ph.D. Research Biologist LMDB, NEI Others: Susan DiCamillo B.S. R. Steven Lee B.S. Mariana Gonzalez-Baez Chemist Biologist Biological Science Lab Aide, Stay-in- School Program LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Transgenic Animal and Genome Manipulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: PROFESSIONAL: 0.5 2.5 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The NEI Central Transgenic Animal Production Facility is a research support facility for all NEI intramural researchers requiring the use of transgenic mice in their research programs. We are currently providing transgenic animal support to researchers from four laboratories in the NEI (Laboratory of Immunology, Laboratory of Mechanisms of Ocular Diseases, Laboratory of Molecular and Developmental Biology, and Laboratory of Retinal Cell and Molecular Biology). In our program, there are currently 84 deoxyribonucleic acid (DNA) constructs that are at various stages of completion. NEI researchers using molecular biology techniques to study the eye submit DNA constructs to our section for production of transgenic mice. We create transgenic mice by standard procedures, then biopsy and perform DNA analyses on the mice that are bom from these procedures to identify positive mice. At researchers' request, we mate positive transgenic mice, wean litters, biopsy and analyze DNA from successive generations of transgenic mice, and provide the transgenic animals to researchers for use in their experiments. During the year, we have generated 155 transgenic founder mice from 34 DNA constructs; set up 456 matings of transgenic mice; weaned, tagged, and tail-biopsied 4,527 mice; isolated DNA from 4,708 samples; and performed 4,912 DNA analyses. This year we began an embryo cryopreservation and banking program to provide long-term storage of important transgenic lines without the need to maintain live mice. A total of 1,326 embryos from five transgenic lines have been frozen. In addition to service functions, we also collaborate with NEI researchers on transgenic animal projects. 231 PHS 6040 (Rev. 5/92) FY 1994 NEl Annual Report Project Description Objectives This project is to produce transgenic animals for use in eye research in the NEI, supply ancillary services related to maintenance of transgenic animals, and provide advice and expertise in matters of transgenic animal projects to all NEI intramural researchers using this technology in their research. In addition, we act as a central fadlity for aU transgenic animal work conducted in the NEI intramural research program to coordinate and conserve resources and use severely Umited animal housing space with maximum efficiency. We provide a comprehensive program for short- and long-term storage of transgenic animal lines for both live aivimals and frozen embryos. Methods Standard methods are used for microinjecting deoxyribonucleic add (DNA) into the pronu- cleus of one-celled mouse embryos and surgi- cally reimplanting the injected embryos into foster mothers to enable development. Con- ventional molecular biology techniques are used to isolate and analyze DNA from biopsy samples of transgeruc mice. Data on all trans- genic mice are maintained in a computerized, relational database accessed by programs written within our group. Major Findings Production of New Transgenic Mouse Lines. We have generated 155 new transgenic founder mice from 34 constructs submitted by re- searchers in four NEI intramural laboratories. These constructs are quite diverse in nature and reflect the diversity of research being performed in the NEI. Some of the general categories of constructs are: (1) pro- moter/reporter constructs in which the pro- moter of an eye gene is fused to a reporter gene to assess transcriptional activity in the transgenic mouse, (2) eye-specific or ubiqui- tous promoters driving expression of genes beUeved to be involved in eye pathologies to assess their roles in pathological conditions in a transgenic mouse model, and (3) other constructs for probing normal eye function and pathological conditions in the mouse. Maintenance of Transgenic Mouse Lines. Transgenic mouse Hnes are derived by mating of the original transgenic founder niice and derivation of successive generations of proge- ny, which are then used in biomedical re- search. To generate lines of transgenic mice from our transgenic founder mice, we have set up 456 mouse matings and weaned, tagged, and biopsied 4,527 mice resulting from mat- ings and microinjection procedures. DNA Analyses. Approximately 15 to 30 percent of mice bom from microinjected embryos are transgenic, and approximately 50 percent offspring from a transgenic mating are transgenic sensitive. A rapid, efficient, and reliable method of identifying transgene positive and negative mice is in place in our group. We have processed 4,708 biopsy samples to obtain DNA and performed 4,912 analyses to determine whether the mice were transgene positive. Embryo Cryopreservation and Banking. We have begun freezing mouse embryos for banking of important lines of transgenic mice. Between 200 and 300 embryos must be frozen for each line of mice banked. This year, we have frozen and banked 1,326 embryos from five lines of transgenic mice. Successful recon- stitution of these transgenic lines has been accomplished by thawing a small portion of the banked embryos and transferring them into the oviducts of pseudopregnant foster mothers. Significance to Biomedical Research and the Program of the Institute Transgenic mice are currently the only readily attainable system for studjdng gene expression in the context of an entire, intact animal. Al- though tissue culture can yield a great deal of information in many studies, a true under- standing of how a particular gene affects an 232 Laboratory of Molecular and Developmental Biology organ (such as the eye) or an entire orgarusm can only be obtained by studying that gene in the intact organism. We play a pivotal role in many NEI intramural research projects by providing the technology and expertise to insert into the mouse genes related to normal eye development and pathological conditions of the eye. Proposed Course (1) Continue producing new transgenic mice for NEI researchers as required for their research projects. (2) Continue breeding and maintaining existing transgenic mouse lines needed for ongoing research in the NEI. (3) Continue freezing and banking embry- os from important lines of transgenic mice. This will free some of our limited animal housing space and ensure that important Unes of mice will not be lost due to aging and loss of fertility. NEI Research Program Lens and Cataract — Molecular Genetics Publications As a service organization, we are generally not included as authors on publications result- ing from research performed on the transgenic animals we produce and maintain. 233 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00286-02 LMDB PERIOD COVERED October 1, 1993 t o September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) g-Crystallin Gene Disr u ption in the Mouse PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Eric Wawrousek Ph.D. Research Biologist LMDB, NEI Others: James P. Brady Ph.D. IRTA Fellow LMDB, NEI COOPERATING UNITS (if any) University of Maryland Medical School (Nicholas Ambulos, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Transgenic Animal and Genome Manipulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.8 PROFESSIONAL: 0.8 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neitiier SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The a-crystallins compose a large fraction of the soluble protein in the vertebrate lens, where they are believed to function as structural proteins; are the first crystallins to be expressed in the developing mouse lens; and are a relatively small family of crystallins encoded by only two genes, the aA- and aB-crystallin genes. The a-crystallins exhibit molecular chaperon activity and kinase activity and, at least in the case of aB-crystallin, have been shown to be expressed in a variety of nonlenticular tissues, where their function is unknown. Toward understanding the role of the a-crystallins in lens and nonlens tissues, we are attempting to functionally delete a-crystallins by disrupting their genes in mice. We are using the technique of homologous recombination in pluripotent mouse embryonic stem cells followed by generation of chimeric mice containing the altered stem cells. We have isolated and mapped 15 kb clones containing the aA- and aB-crystallin gene loci from a mouse 129SV library (the same strain as most of the embryonic stem cell lines currently in use). Construction of aA-crystallin, aB-crystallin, and aA-CRYBPl knockout vectors is complete. We are mastering the technologies to (1) effect homologous recombination iri embryonic stem (ES) cells and (2) introduce ES cells into embryos that develop into chimeric mice. In collaboration with Nicholas Ambulos (University of Maryland Medical School), we are sequencing the mouse aA-crystallin gene locus. 234 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology Project Description Additional Personnel Ellen Liberman PhD. Anterior Segment Disease Branch of the Extramural Research Program, NEl Christina Sax Ph.D. Senior Staff Fellow, LMDB, NEl Objectives The objective of this project is to disrupt the a-crystallin genes (aA and aB) in the mouse to study their effect on normal lens and eye development. Disruption of the genes will essentially delete these proteins from the mouse and enable us to analyze the effects these proteins have on expression of other lens proteins, developmental regulation and morphology of the lens and other eye struc- tures, and the role of these proteins in nonlen- ticular tissues. Methods Standard molecular biology techniques were used to clone the a-crystaUin genes from a mouse 129SV genomic library and construct "gene knockout" vectors. Disruption of the genes will be accomplished by the now stan- dard technology of homologous recombination in pluripotent mouse embryonic stem cells, followed by insertion of the genetically altered cells into blastocyst mouse embryos to gener- ate chimeric mice with the gene disruption. Chimeric knockout mice wiU be bred to gener- ate mice with heterozygous and homozygous knockout mutations. Major Findings Gene Knockouts. Construction of aA-crystaUin, aB-crystaUrn, and aA-CRYBPl gene knockout vectors has been completed. These vectors contain large pieces of the respective gene functionally disrupted by insertion of the selectable marker for neomycin resistance and flanked by the negative selectable marker. HSV tk. Use of these positive and negative selectable markers to facilitate selection of appropriately modified cells is currently standard procedure in this field. Following electroporation of the aA-crystaUin knockout vector into Jl mouse embryonic stem cells and selection with G418 and ganciclovir, approxi- mately 200 colonies were picked. PCR and Southern blot analysis of these clones to detect correct homologous recombination is currently under way. PreUminary analysis indicates that 11 of the first 96 clones screened contain the appropriate aA-crystaUin gene knockout. The technology for incorporating the modified cells into intact mice is being mas- tered in our laboratory. We have successfully generated chimeric mice by microinjecting unmodified ES cells into mouse blastocysts and allowing the embryos to develop in pseudopregnant foster mothers. Several chimeras have been bom in which ES ceUs contribute 10 to 90 percent of the cells in the mouse (estimated by coat color of the mice). We are also developing the newer method of creating chimeric mice by simple aggregation of morula stage embryos with clumps of ES cells. Sequence of the Mouse oA-crystallin locus. Approximately 12.7 kb of the 15 kb aA-crys- tallin locus has been sequenced on both strands. Functional Significance of Sequences Flanking the Mouse oA-Crystallin Gene. We are con- structing vectors containing portions of the aA-crystaUin gene locus to search for possible regvdatory elements located far from the promoter region. A basic promoter vector containing the mouse aA-crystallin promoter (-366 to +46) fused to the bacterial chloram- phenical acetyltransferase gene (CAT) reporter gene has been constructed. In transient trans- fection assays with the N/N1003 rabbit lens epitheUal cell hne, this construct eUcits signifi- cantly higher levels of CAT activity than the corresponding promoterless vector. Several large pieces of the aA locus have been inserted into the basic promoter, and 135 FY 1994 NEI Annual Report after completion of several additional con- sti-ucts, all of the constructs will be tested in the transient transfection assay for modulation of promoter activity. Significance to Biomedical Researcii and the Program of tfie Institute Deletion of the a-crystallin proteins, individu- ally or together, will provide a fundamental understanding of how these proteins function during normal lens development and how they may influence the structure and function of the lens and the entire eye. Additionally, it would give us insight into the function of these proteins in nonlenticular tissues that in turn could help us understand some of their more subtie roles in the eye. Proposed Course (1) Continue the gene knockout experiments in embryonic stem ceUs wdth the two addition- al knockout vectors (aB-crystaUin and aA- CRYBP). Produce chimeric mice from appro- priately modified ES cells for each of our three selected genes, and mate these mice to pro- duce lines of knockout mice for investigation. Deletion of a single allele of either aA or aB wdll be useful in assessing gene dosage effect (50 percent reduction of protein), and breeding to homozygosity (deletion of both alleles) will allow us to study the consequences of com- plete absence of the individual protein. It will be extremely interesting to mate eventually aA and aB knockout mice to produce mice which are totally devoid of a-crystallin. (2) Continue collaborative sequencing the aA-crystallin gene locus. Although a consid- erable amount is known about regulation of the mouse aA-crystallin gene, the complete se- quence of the gene has not yet been deter- mined. The complete sequence of the gene and flanking regions will be beneficial for designing and interpreting experiments with this gene. (3) Continue construction of vectors that wiU be used in transient transfection assays to locate regulatory elements spatially distant from the promoter of the aA-crystallin gene. This along with the sequence of the locus will help to identify potential sites influencing levels of gene expression. NEI Research Program Lens and Cataract — Lens Development and Aging 236 PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00291-01 LMDB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Transgenic Animal Models PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Eric Wavi^rousek Ph.D. Research Biologist LMDB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Transgenic Animal and Genome Manipulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.2 PROFESSIONAL: 0.2 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Transgenic mice offer a unique tool for studying normal and pathological states associated with expression, overexpression, or misexpression a particular gene. Aberrant expression of genes believed to be involved in disease can be easily achieved with the well-established transgenic methodologies. The resulting transgenic mice often may be used as models for studying diseases associated with the respective gene. We have developed a line of transgenic mice that expresses a modified form of mature human interleukin (IL)-ip in the ocular lens. The afflicted mice exhibit a progressive inflammation of the eye and neovascularization of many eye tissues resulting in the eventual destruction of the eye. IL-ip messenger ribonucleic acid (mRNA) and protein have been detected at high levels in the eye. There is upregulation of vascular adhesion molecules and significant influx of inflammatory cells, predominantly macrophages. A systemic unresponsiveness to IL- 1-mediated events has been observed in these mice. 237 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Additional Personnel Chi-Chao Chan Igal Gery James Lai MD. Ph.D. Chief, Immunopathology Section, Immunology Section, NEI Chief, Experimental Immunology Section, NEI Howard Hughes Medical Institute Objectives The objective of this project is to create trans- genic mouse models of ocular disease by aber- rantiy expressing proteins believed to be in- volved in maintenance of normal state or initiation and potentiation of a pathological condition. Our first model involves expres- sion of human interleukin (hIL)-l(3 in lens of transgenic mice to generate an abundant supply of identical mice afflicted with an ocular inflammatory disease of defined origin. These mice can be used to study the many parameters associated with progression of the inflammation and test therapeutic regimens for controUing the inflammation and conse- quent ocular damage. Methods Standard methods were used to construct the transgene with the murine aA-crystallin promoter driving expression of a cassette containing the human tissue plasminogen activator secretion signal fused in frame to the coding region of mature human IL-ip. Trans- genic mice were made by microinjecting the transgene construct into FVB/N single cell embryos. Mice used in this study were Fj hybrids of the transgenic FVB/N and DBA/ 2. These mice are heterozygous for a congenital retinal degeneration found in the FVB/N strain and have histologically normal retinas. Major Findings One line of transgenic mice was generated containing the IL-ip construct. This Une con- tains a single complete copy of the transgene. Large amounts of hIL-ip have been detected in the eyes of these mice both at the messen- ger ribonucleic acid (mRNA) and protein levels but has not been detected in other tissues (mRNA by Northern analysis) nor in serum (protein by enzyme-Unked immuno- sorbent assay). The afflicted mice exhibit a progressive ocular inflammation accomparued by neovascularization, resulting in the destruc- tion of the eyes in adult mice. The inflamma- tory infiltrate consists predominanfly of mac- rophages with some polymorphonuclear neutrophils and Ijonphocyte involvement. Increased expression of the vasctilar adhesion molecule intercellular adhesion molecule 1 was evident in eyes of these nuce. Nontrans- genic litter mates exhibit none of these charac- teristics. The transgenic mice are otherwise healthy exhibiting normal reproductive capaci- ty and longevity. A systemic unresponsiveness to IL-1 mediated events was observed in the trans- genic mice. They showed a decreased suscep- tibility to the toxic effects of Upopolysaccha- ride injection. Injection of 40;u-g per gram body weight resulted in lethaUty in only 6.3 percent of transgenic mice compared with 86 percent lethality in nontransgenic mice. Thymocj^es isolated from the transgenic mice were also less responsive to IL-1 in culture than those isolated from normal litter mates. The mechanism by which this systemic effect is induced is currentiy unknown. Significance to Biomedical Research and the Program of the Institute This model is the first instance of successful creation of a transgenic mouse containing the potent cytokine IL-1. This model provides a 238 readily accessible stock of identical mice exhibiting a consistent pattern of ocular in- flammation beginning with nearly normal eyes at four days of age, progressing with age, and resulting in destruction of the eye in adults. The roles of other cytokines, cellular adhesion molecules and arachidonic acid metabolites in progression of the ocular inflammation can be studied as can the estabUshment of the sys- temic unresponsiveness to IL-1. Proposed Course (1) Continue characterization of the IL- ip transgenic line. Quantitate mRNA levels for mouse IL-1, IL-1 receptor and receptor antagonist in the eyes of affected mice. Study alterations in cytokine expression patterns and arachidonic acid metabolites in these mice. Laboraton/ of Molecular and D evelopmental Biology (2) Attempt to ameUorate the inflamma- tion in this model by administration of anti- inflammatory drugs or antibodies to cytokines or cellular adhesion molecules. NEI Research Program Retinal Diseases — Inflammatory Diseases 239 Laboratory of Ocular Therapeutics Report of the Chief Laboratory of Ocular Therapeutics Peter F. Kador, Ph.D. The Laboratory of Ocular Therapeutics (LOT) focuses on the development, evaluation, and mechanism of action of new ophthalmic drugs to treat eye diseases. The LOT research team is examining aldose reductase inhibitors (ARl) and anticataract agents. In pursuing the development of more effective and less toxic ARls, the efforts are progressing the development of an inhibitor urvrelated to previous ARIs. At present, a new inhibitor is being developed using biochemi- cal, pharmacological, and computer molecular design techniques. Studies designed to eluci- date the specific mechanism (s) of how ARIs diabetic compUcations are also being conduct- ed. In studies using galactose-fed dogs, LOT investigators have estabUshed that retinal changes associated with diabetic retinopathy progressed to the proliferative stage and that the dog represents the first animal model to demonstrate clinical and histological changes found in all stages of retinopathy. Studies are now focused on the development of prolifera- tive retinopathy in long-term galactose-fed dogs. Biochemical changes observed in these in vivo studies are being investigated using in vitro tissue culture techniques. Biochemical turnover in these cells is being monitored through nuclear magnetic resonance. Magnet- ic resonance imaging techniques are also being used to measure in vivo ARI efficacy. 243 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00003-21 LOT PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit or} one line between the borders.) Phar macology of Ocular Complications PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Others: Peter F. Kador Ping Ding William Greentree Petra Lachner Yong Lee Martin Lizak Heike Neuenschwander Irina Obrosova Libaniel Rodriguez Katsumi Sugiyama Ph.D. Ph.D. D.V.M. D.V.M. Ph.D. Ph.D. M.D. Ph.D. Ph.D. Ph.D. Chief Visiting Fellow IRTA IRTA Staff Fellow Staff Fellow Special Volunteer Visiting Scientist Staff Fellow Staff Fellow LOT, NEI LOT, NEI LOT, NEI LOT, NEI LOT, NEI LOT, NEI LOT, NEI LOT, NEI LOT, NEI LOT, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Ocular Therapeutics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 7.69 PROFESSIONAL: 7.69 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews Ix| (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Events leading to the onset of various ocular complications are being investigated. Specifically, the role of the enzymes aldose reductase and aldehyde reductase in the onset and progression of retinopathy, cataract, keratopathy, pupil function changes, and iris and ciliary process structure changes associated with diabetes and galactosemia are being studied. In addition, methods for either delaying or preventing the onset and progression of these complications through the pharmacological control of these enzymes are being developed. Events leading to the formation of several types of cataracts are also being investigated, as well as methods for controlling the onset of these cataracts through pharmacological intervention. 244 PHS 6040 (Rev. 5/92) Laboraton/ of Ocular Therapeutics Project Description Additional Personnel Robert Balaban Duane Miller PhD. Ph.D. National Heart, Lung, and Blood Institute University of Tennessee College of Pharmacy, Memphis, Tennessee Objectives To gain insight into the mechanisms by which polyol-induced ocular diabetic complications and cataracts are formed and to develop methods for their regulation. Methods Diabetes can be experimentally induced in animals by injecting streptozotocin. Diabetes- related complications linked to the sorbitol pathway can also be induced in animals such as rats and dogs by feeding them a galactose-enriched diet. Cataract formation and clinical retinal changes in experimental animals can be monitored through fundus photography. Biochemical studies used for the purification of enzymes include column chromatography, polyacrylamide gel electro- phoresis, isoelectric focusing, chromatofocus- ing, and high-pressure Uquid chromatography. Polyol levels were determined by gas-Uquid chromatography. Immunological analyses include the use of enzyme-linked immuno- sorbent assay, radioimmunoassay. Western blots, and immunohistochemical techniques using the coupled antibody DAB-PAP tech- nique. Computational methods for enzyme analysis, inhibitor structure-activity-studies, and pupil-function changes require the use of the National Institiates of Health (NIH) PROPHET computer system and Charm and Quanta Computer Systems from Molecular Design. Major Findings Biochemical Studies. Studies on defining the inhibitor site of aldose reductase and aldehyde reductase and the development of new aldose reductase inhibitors (ARI) are continuing. To facilitate the rational development of more specific and potent ARJs, more specific knowl- edge of the structural and pharmacophoric requirements of the site at which ARIs interact is required. It has recently been reported that cocrystallization of human placental aldose reductase with the inhibitor zopolrestat result- ed in a complex where the inhibitor was almost completely sequestered in the large, deeply eUiptical hydrophobic pocket that forms the substrate site. Zopolrestat's ob- served location, which makes the active site pocket inaccessible to solvent or further pro- ductive binding of substrate, does not support stiucture activity relationships (SAR), observa- tions, and kinetic results, which indicates that ARIs such as zopolrestat are either noncom- petitive or uncompetitive inhibitors. Using an 5-iodoacetamido analog of the alrestatin as an affinity-labeled ARI, we have located an alternative site of interaction on aldose reduc- tase. The affinity-labeled ARI was irreversibly bound to purified rat lens aldose reductase, and the bound residues were identified by mass spectrometry. ModeUng studies of the identified site were subsequentiy conducted on the protein structure of aldose reductase, calculated by Charm force field using the crystal structure coordinates of human placental aldose reduc- tase pubhshed in the Brookhaven Protein Data Bank. Potential interactions of inhibitors at this site were estimated through docking and binding studies using Quanta 3.3. The appar- ent ARI site is composed of a single region independent of the substrate binding site. It contains a number of pharmacophoric ele- ments previously proposed for the inhibitor site. These include the amino acids arginine, serine, and tyrosine; proline; and a lipophilic area containing hydrogen bonding substi- tuents. Multiple three-point attachments that include possible nucleophiUc interactions with either serine or tyrosine are possible. Also present are two carboxylate binding groups that can orient the inhibitor on the hpophUic binding site. This proposed inhibitor site contains a number of pharmacophoric ele- 245 FY 1994 NEI Annual Report ments previously proposed for the inhibitor site, and its location and composition are consistent with reported kinetic data, SAR observations, stereochemical reqmrements, and quantum chemical calculations. Several reports suggest that a metabolite of S-88-0773 (4-[4-N,N,-Dimethylsulfamoyl)- piperazino] -2-methylpyrimidine) increases sugar alcohol levels in normal and diabetic rats and, as a result, speeds up the appearance of the polyol-induced complications. Others report that the compound slows down the appearance of complications by altering redox states of NAD (P) -couples through inhibition of sorbitol dehydrogenase. We have used this compound to investigate its effect on cataract formation. For these studies, young (50g) streptozotocin-induced diabetic rats received normal diet with or without 0.06 percent S-88- 0773 while similar-aged nondiabetic rats received either normal, 20 percent, 30 percent, or 50 percent galactose diets with or without 0.06 percent S-88-0773. Cataract progression was monitored week- ly by portable slit-lamp microscopy. Arumals were periodically killed and polyol levels in the lenses evaluated. Polyol levels were increased in both galactosemic and diabetic rats, and no difference in lens polyol levels between each corresponding group of rats tieated with or without drug was observed in the galactose-fed groups. Increases in tissue polyol levels associated with sorbitol dehydro- genase inhibition were not anticipated in galactose-fed rats because galactitol is not metaboUzed by sorbitol dehydrogenase. Nevertheless, sugar cataract formation was delayed in both diabetic and galactose-fed rats treated with S-88-0773. These results suggest that S-88-0773, or its active metaboUte, delays sugar cataract formation through a biochemi- cally unknown mechanism not related to either sorbitol dehydrogenase or ARI. Magnetization transfer contrast (MTC) enhancement, which generates high-contrast images based on tissue characteristics result- ing from the interaction of water and macro- molecvdes, was applied to document in vivo cataract formation and other structural chang- es associated with diabetic eye compUcations. For these studies, male beagles ranging from six to 24 months of age were fed a diet con- taining 30 percent galactose. All dogs were then placed in a General Electric 2-T Omega magnetic resonance imaging system under anesthesia with muscle relaxant at four-week intervals. M^ and Mq images were acquired using gradient-recalled-echo sequences, with and without the saturation pulses, respective- ly, consisting of rf -irradiation 10 kHz off- resonance from the free-water proton signal. The Tj33, images were calculated from the data using a one-shot Tj-imaging sequence. The acqviired images were compared with photographs obtained with sUt-lamp microsco- py and retioillumination photography. Excel- lent images detailing the fine structures of the anterior segment that includes the lens, cor- nea, iris, dliary body, choroid membrane, and Schlemm's canal were obtained by MTC. In these images the progression of osmotic cata- ract formation in the galactose-fed dogs could be followed from the initial appearance of distinct cortical vacuoles. Distinct fluctuations in lens size and shape were observed as lenses progressed to the more advanced cataractous stages. "F NMR spectroscopy is also being used to measure in vivo aldose reductase activity in the dog lens by measuring the conversion of 3-deoxy-3-fluoroglucose to 3-deoxy-3-fluoro- sorbitol. This work is an extension of the in vivo evaluation of aldose reductase activity in rabbit lenses. Iiutial spatial coordinates for lenses are calctilated from ^H-images deter- mined on a 2.0 Tesla GE Omega-CSI spec- trometer. The SLOOP-technique (Spectral Localization with Optimal Pointspread func- tion) is then used vdth a proton decoupler to measure the accumulation of sorbitol in the rabbit lens. A double spin-echo sequence is used with selective excitation and refocusing pulses and with optimized phase-encoding gradient pulses using one-second repetition times and 25 millisecond echo times. SLOOP experiments indicate that 3-deoxy-3-fluorosor- bitol can be observed in spectra of the anterior 246 hahoratory of Ocular Therapeutics portion of the lens when adequate amounts of 3-deoxy-3-fluoroglucose are administered. Retinal Studies. Vascular changes associ- ated with diabetic retinopathy can be experi- mentally produced in beagles fed a 30 percent galactose diet. In studies designed to clarify the initiating lesions and progression of dia- betic retinopathy, we have documented the progression of retinal lesions from background through the proliferative stage in the dog with ophthalmoscopic, fluorescein angiographic, and histopathologic findings. Initial retinal changes include aldose reductase-Unked formation of pericyte ghosts and subsequent development of aceUular capillaries, micro- aneurysms, and intraretinal hemorrhages. This early retinopathy progresses to include the appearance of occluded vessels, areas of nonperfusion, and intraretinal microvascular abnormalities. Finally, proUferative retinopa- thy develops, including the formation of fibrovascular membranes seen histologically on both the retinal surface and the posterior hyaloid membrane. Diabetes-like microvascular changes in galactosemic beagles have been shown to be arrested in a dose-dependent manner by ARIs. To determine if retinal changes also can be reduced through the marked reduction of galactitol production after early retinal lesions have developed, galactose diet was removed after either the appearance of pericyte ghosts or microaneurysm formation. The subsequent progression of retinal changes over 24 months was then quantitively compared with retinal changes in remaining galactose-fed dogs. For these studies, 10 control dogs were fed a normal diet, while 50 dogs were fed diet containing 30 percent galactose. The galactose diet was removed from 15 dogs after 24 months, at which time pericyte ghosts had developed; another 15 dogs were removed after 31 months when microaneurysms had developed. Eighteen remained on galactose diet. Beginning at 24 months, four to five eyes from each experimental group and two to three eyes from the control group were enu- cleated at six-month intervals. Isolated retinas were quantified as previously described (/ Ocular Pharmacol 9:257, 1993). Significant increases in the endotheUum/ pericyte ratio and decreases in pericyte density were ob- served with the duration of galactose-feeding. Although no reversal of retinal lesions oc- curred, differences in the progression of reti- nal lesions between the galactose-fed and galactose-removed groups became evident after 12 to 24 months. This study suggests that reduction of polyol accumulation at the initial stages of background retinopathy bene- ficially reduces the progression of retinal lesions. Corneal Studies. Specular microscopic studies indicate that the size (polymegathism) and shape (pleomorphism) of the hexagonal corneal endotheUal cells change in diabetics. Similar morphometric changes of the corneal endotheUum have also been experimentally observed in diabetic rats as well as in diabetic and galactose-fed dogs, and concomitant administration of ARIs can reduce these morphological changes. The purpose of this study was to examine whether corneal endo- thelial changes in galactose-fed dogs are reversible by the marked reduction of galac- titol production after stopping prolonged galactose feeding. Ten control dogs were fed a normal diet, while 48 dogs were fed a diet containing 30 percent galactose. The galactose diet was removed from 15 dogs after 24 months, at which time pericyte ghosts in the retina had developed; another 15 dogs were removed after 31 months when retinal micro- aneurysms had developed. Eighteen dogs remained on galactose diet throughout the study. Specular microscopy was conducted on members of all groups after 38 months of study, and the photographs were analyzed in masked fashion on the Bambi image analysis systems. The evaluation of the corneal endo- theUal ceUs revealed significant differences in the ceU size and density among aU galactose- fed dogs and normal, age-matched control dogs. Corneal endotheUal changes were not significantiy reduced in dogs fed galactose for either 24 or 31 months and then receiving normal diet for 14 and seven months, respec- 247 FY 1994 NEI Annual Report lively, indicating that amelioration of endothe- lial cell changes requires therapy before the advent of endothelial morphologic changes. Significance to Biomedical Research and tt)e Program of the Institute Loss of vision from cataract and diabetic reti- nopathy is significant; therefore, methods for the pharmacological control of these ocular compUcations are required. We have devel- oped an animal model that demonstrates advanced retinal vessel changes that are virtually clinically and histologically identical to those observed in advanced diabetic reti- nopathy. Our present studies in dogs demon- strate for the first time that loss of retinal pericytes, associated with aldose reductase, initiates retinal changes associated with both background and advanced diabetic retinopa- thy and that administration of ARIs in preven- tion studies can ameliorate the loss of peri- cytes and subsequent development of micro- aneurysms and retinal hemorrhages in a dose- dependent manner. The successful develop- ment of noninvasive methods for monitoring aldose reductase activity by nuclear magnetic resonance procedures may directiy affect ongoing and plarmed clinical trials where this procedure could serve as a quantitative indica- tor of drug efficacy. Cataract is also one of the major causes oif blindness in the develop- ing world. In addition, loss of vision due to cataract is one of the major health problems of both the diabetic and the aging populations in the United States. Proposed Course These studies will be continued. Discovered ARIs will be pharmacologically evaluated and developed. The inhibitor site will be further probed through the use of affinity labels so that more potent and specific inhibitors may be developed. Studies on the mechanisms through which aldose reductase induces diabetic compUcations in various tissues will be continued. NEI Research Program Retinal Disease — ^Diabetic Retinopathy, Sickle Cell Retinopathy, and Other Vascular Abnor- malities Lens and Cataract — Pathogenesis of Cataract Publications Greentree W, Takahashi Y, Wyman M, Kador PF: Quantitative analysis of retinal vessel changes in galactose-fed dogs: Intervention studies. Invest Ophthalmol Vis Sci 35(4):1589, 1994. Kador PF: Biochemistry of the lens: Interme- diary metabolism and sugar cataract forma- tion, in Viola E, Dowling J (eds). Principals and Practice of Ophthalmology. New York, Basic Sciences, 1994, pp 146-167. Kador PF, Lee YS, Rodriguez L, Carper D, Bartozko-MaHk A, ParmeU L: Characterization of the aldose reductase inhibitor site. Invest Ophthalmol Vis Sci 35(4):2152, 1994. Kador PF, Takahashi Y, Schaffhauser M: Vorbeugung diabetischer Komphkationen im Auge mit Aldosereduktase-Hemmern. Diabe- tes und Stojfwechsel, in press. Kador PF, Takahashi Y, Sato S, Wyman M: Amelioration of diabetes-Uke retinal changes in galactose-fed dogs. Prev Med, in press. Kador PF, Takahashi Y, Wyman M, Ferris F ni: Diabetes-Uke proliferative retinal changes in galactose-fed dogs. Arch Ophthalmol, 110:1295-1302, 1992. Lee YS, Peralstein R, Kador PF: Moleciilar modeling of aldose reductase inhibitors. / Med Chem 8(6):787-792, 1993. Li Q, Lopez JS, Caspi RR, Nussenblatt RB, Kador PF, Chan C-C: Suppression of S-anti- gen induced experimental autoimmune uveo- retinitis in Lewis rats by oral administration with cgs-13080, a thromboxane synthetase inhibitor. Exp Eye Res 57:601-608, 1993. 248 Laboratory of Ocular Therapeutics Lizak MJ, Ceckler TL, Balaban RS, Kador PF: In vivo measurement of magnetization trans- fer in galactosemic dog lens. Proceedings of the Society of Magnetic Resonance 1994 1:205, 1994. Lizak MJ, Mori K, Ceckler TL, Kador PF, Balaban RS: Magnetic resonance imaging of the galactosemic dog eye using magnetization transfer contrast enhancement. Invest Ophthal- mol Vis Sci 35(4):1948, 1994. Mori K, Takahashi Y, Tsuduki S, Kador PF, Akagi Y: Significance of aldose reductase to experimental corneal epitheliopathy. Invest Ophthalmol Vis Sci 35(4):1946, 1994. Neuenschwander H, Julia C, Wyman M, Kador PF: Corneal endothelial changes in galactose-fed dogs. Invest Ophthalmol Vis Sci 35(4):1601, 1994. Obrosova I, Inoue J, Greentree W, Sato S, Rodriguez L, Kador PF: Evaluation of s-88- 0773 on sugar cataract formation. Invest Ophthalmol Vis Sci 35(4):1931, 1994. Ogawa K, Yamawaki I, Matsusita Y, Nomura N, Kador PF, Kinoshita JH: Synthesis of substituted2,4-dioxo-thienopyrimidine-l-acetic adds and their evaluation as ARIs. Eur J Med Chem 28:769-781, 1993. Sato S, Kador PF: Retinal changes in arumal diabetic models. Diabetes Frontiers 5:108-112, 1994. Sato S, Old S, Carper D, Kador PF: Purifica- tion and characterization of recombinant human placental and rat lens aldose reduc- tases expressed in Escherichia coli. Adv Exp Med Biol, in press. Secchi EF, Lizak MJ, Sato S, Kador PF: Pres- ence of polyol pathway in fibroblast. Invest Ophthalmol Vis Set 35(4):1589, 1994. Takahashi Y, Augustin W, Wyman M, Kador PF: Quantitation of retinal vessel changes associated with diabetic retinopathy in galac- tose-fed dogs. Ocular Pharmacology 9:257-269, 1993. Takahashi Y, Wyman M, Kador PF: Retinal vascularization in galactose-fed dogs. Invest Ophthalmol Vis Sci 35(4):1734, 1994. WaldbiUig RJ, Jones BE, Schoen TJ, Heidersbach S, Bitar MS, Van Kuijk FJGM, de Juan E, Kador PF, Chader GJ: Vitreal insulin- like growth factor binding proteins (IGFBPs) are increased in human and animal diabetics: ImpUcations for understanding diabetic reti- nopathy. Current Eye Res 13:539-546, 1994. 249 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00275-03 LOT PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) NADPH Reductases and Polyol Pathway in Ocular Complications PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Sanai Sato M.D., Ph.D. Visiting Scientist LOT, NEI Others: Peter F. Kador E. Filippo Secchi Ph.D. Ph.D. Chief Fogarty Fellow LOT, NEI LOT, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Ocular Therapeutics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 2.05 PROFESSIONAL: 2.05 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) In diabetes the increased flux of glucose into the polyol pathway results in the accumulation of the sugar alcohol sorbitol that is linked to the onset of various diabetic complications, such as cataract formation and retinopathy. The sugar alcohol formation and properties of NADPH-dependent reductases in fibroblasts, the key cells in the formation of fibrous proliferative tissues, has been investigated. 250 PHS 6040 (Rev. 5/92) Laboratory of Ocular Vierapeutics Project Description Objectives Glucose is metabolized into fructose through sugar alcohol sorbitol in sorbitol pathway. In diabetes the increased flux of glucose into the polyol pathway results in the accumulation of sugar alcohol sorbitol. Experimental evidence has demonstrated the link between the accu- mulation of sugar alcohols and the selective loss of pericjiies, the initial lesion of diabetic retinopathy. In the more advanced stages of retinopathy, fibroblasts play a key role in the formation of fibrous proliferative tissues. This project is designed to investigate whether the sugar alcohol accumulation is also linked to the formation of proliferative tissues. Methods Biochemical techniques include affinity chro- matography, chromatofocusing, isoelectric focusing, electrophoresis, immunoblot and gas chromatography for sugar analysis. "F NMR with 3-deoxy-3-fluoro-D-glucose (3FDG) is used to investigate the metabolism of glucose. The National Institutes of Health (NIH) Prophet computer system is used for kinetic analysis and IC50 calculations. Major Findings The crude extract of murine fibroblast cell line L929 cells displayed reductase activity with DL-glyceraldehyde as substrate and dehydro- genase activity with D-sorbitol as substrate, suggesting that these cells contain two en- zymes of the polyol pathway: aldose reduc- tase (and /or aldehyde reductase) and sorbitol dehydrogenase. A nuclear magnetic reso- nance study with 3FDG confirmed the conver- sion of glucose into fructose through sorbitol in mouse fibroblast ceU line L929. Sugar alcohol accumulated in the cells cultured in medium containing 30 mM galactose, and this accumulation was inhibited by aldose reduc- tase inhibitors (ARI). Purification studies demonstiated that the dominant reductase in L929 cells was aldehyde reductase rather than aldose reductase. Two other fibroblast cell Unes estabUshed from dog and human also appeared to possess aldehyde reductase as the dominant reductase in these cells. These results confirm the presence of the polyol pathway in three different fibroblast cell Hnes. However, aldehyde reductase rather than aldose reductase predominates in these cells. Significance to Biomedical Research and the Program of the Institute Diabetic retinopathy is a leading cause of blindness. The development of potent ARIs is of clinical significance in preventing bUndness associated diabetes. Animal studies have demonstiated that ARIs prevent the selective loss of pericytes, the initial pathology of retinopathy. The observation that the polyol pathway is also present in fibroblast suggests that excess amounts of sugar alcohol may also play a role in the formation of proliferative tissues in advanced stages of retinopathy. This evidence provides additional rationales for the use of ARIs in the prevention and intervention of diabetic retinopathy. Proposed Course The evaluation of the polyol pathway v^ be continued with various tissues where diabetic changes occur. Cell culture techniques will be used to investigate retinal pericytes and endothelial cells as key cells in diabetic retinopathy. NEI Research Program Retinal Diseases — ^Diabetic Retinopathy, Sickle CeU Retinopathy and Other Vascular Abnor- malities Publications Obrosova I, Inoue J, Greentree W, Sato S, Rodrigues L, Kador PF: Evaluation of S-88- 0773 on sugar cataract formation. Invest Ophtlmlmol Vis Sci 24(suppl):1931, 1994. 251 FY 1994 NEI Annual Report Sato S, Kador PF: Diabetic retinopathy. I. Retinal changes in animal models. Diabetes Frontier 5:108-112, 1994. Sato S, Old S, Carper D, Kador PF: Purifica- tion and characterization of recombinant placental and rat lens aldose reductases ex- pressed in Escherichia coli. Adv Exp Med Biol, in press. Schaffhauser MA, Sato S, Kador PF: NADPH- dependent reductases in dog thyroid: Compar- ison of a third enzyme "glyceraldehyde reduc- tase" to dog thyroid aldehyde reductase. Adv Exp Med Biol, in press. Secchi EF, Lisak MJ, Sato S, Kador PF: Pres- ence of polyol pathway in fibroblast. Invest Ophthamol Vis Sci 34(4 suppl):1589, 1994. 252 Laboratory of Retinal Cell and Molecular Biology Report of the Chief Laboratory of Retinal Cell and Molecular Biology Gerald J. Chader, Ph.D. Members of the Laboratory of Retinal CeU and Molecular Biology (LRCMB) elucidate new genes and biochemical mechanisms to better understand the underly- ing causes of ocular diseases. With this knowledge, we hope to intervene better in the disease process before substantial damage to vision has been done or to apply rational methods of gene therapy before the terminal stages of the disease have been reached. The approaches taken are molecular biological, molecular genetic, and studies on candidate genes. The following three areas are empha- sized: • Molecular Biology and Molecular Genetics • Gene Therapy of Retinal Diseases • Molecular Immunopathology Following are specific advances within these areas: Molecular Biology and Molecular Genetics (1) Retina-Specific Genes. Several genes that are predominantly or exclusively expressed in ocular tissues have been identified by subtrac- tive cloning. These include an important gene HIOMT that is involved in the maintenance of circadian rhythms within the eye. Retina- specific genes and genes located on the short arm of the X chromosome have been pinpoint- ed. These genes are being chromosomally localized to see if they are linked to eye dis- eases. Genomic cloning and sequencing are being done such that appropriate restriction fragment length polymorphisms and /or microsateUite repeats are generated to allow for disease testing. (2) Retinal pigment epithelium (RPE)- Specific Genes. Cloning of genes unique to RPE and its functioning is of importance because the RPE cell layer is critical for retinal homeostasis. A new 65 kDa protein of poten- tial importance has been isolated from the human RPE. The bovine and mouse genes have been cloned, allowing for study of tissue- specific expression. The gene is highly con- served, and its posttranslational expression is tightly regulated. This gene is the first RPE- specific gene to be reported and characterized. Knowledge of the 5' regulatory sequence should facilitate RPE-specific gene transfer and gene therapy. (3) Interphotoreceptor Retinoid-Binding Protein (IRBP). IRBP is an integral part of the visual cycle. Collaborative studies with Dr. Harris Ripps, from the University of Illinois at Chicago, on a model of experimental retinal detachment have demonstiated that IRBP is intimately involved in rhodopsin regeneration. We also have identified a protein homologue and cloned the homologous gene of human IRBP from the fiuitfly Drosophila melanogaster . The gene maps to an area of the Drosophila genome that is rich in mutants of ocular disease. We hope to pinpoint a specific hu- man population with similar characteristics and examine the gene for defects in specific human families. 255 FY 1994 NEI Annual Report (4) Pigment Epithelium-Derived Factor (PEDF). PEDF is synthesized by human RPE cells and may be import:ant in the develop- ment of retinal photoreceptors. PEDF induces the extension of elaborate neuronal processes from cultured retinoblastoma cells and, as such, is a neurotrophic protein. Because Y-79 cells are thought to be derived from photore- ceptor cone cells, it is hoped that PEDF can be as effective on cone neuron development in vivo. Collaborative studies have also shown PEDF to be a neuron-survival factor. The clinical use of PEDF in retinal transplantation is thus a distinct possibility. The molecular biology of this potentially very important neurotrophic and neuron-survival protein is being studied for appUcation to retinal degen- erations. (2) Gene Therapy. Ribozymes are specifi- cally constructed ribonucleic acid species that can control expression of proteins v^thin cells. By linking these simplified gene forms to appropriate promoters and using a suitable transfer vector, new therapeutic modaUties can be constructed. Gene therapy can then be planned to treat autosomal dominant disor- ders that are unmanageable. Ribozyme con- structs for IRBP have been designed and are being studied in a transfected human retino- blastoma ceU system. Once perfected, ribo- zymes should be useful in conditions such as diabetic retinopathy and retinopathy of pre- maturity, in w^hich the disorders probably involve overexpression of normal proteins such as growth factors. (5) Fatty Add and Tubulin Defects in Retinal Degeneration. In collaborations with Dr. Muriel I. Kaiser, fatty acid uptake and metaboUsm in Bietti's crystaUine retinopathy and a tubulin acetylation defect in a form of atypical retinitis pigmentosa are being investi- gated in hopes of elucidating the specific defects. Sigr\ificant progress has been made in pinpointing the metabolic problems expressed in both these hereditary conditions. Gene Therapy of Retinal Diseases (1) Transgenic Studies. Transgenic studies can help to uncover factors contiolling gene activation in the embryonic period, specifically in retinal photoreceptor cells. Gene analysis systems in transgenic mice and in tiansient transfections in cultured human retinoblasto- ma cells have been established for IRBP. Much of the 5'-flanking region of IRBP has been determined, and enhancer elements necessary for expression are being defined through target mutagenesis studies. Tissue- and stage-specific elements, including TATA and CAAT boxes, are being exactiy defined as to retinal expression. This work is important so that specific molecules can be "gene-target- ed" to the retina with precision. Molecular Immunopathology (1) Immunopathology. Work with Dr. Igal Gery continues to study aspects of the IRBP- induced uveitis seen in models of human uveoretinitis. Studies in the human are also under way, with the final goal of contiolling or preventing at least some forms of uveitis in man. (2) Immunogenetics. With Dr. Rachel Caspi, an IRBP-mouse model for experimental autoimmune uveitis has been established that is very useful for studying the genetics of the disease and its relapsing characteristics. (3) Antigen Presentation. Collaborative work with Drs. Mark de Smet and Robert Nussenblatt has previously demonstrated the presence of a ceU-surface protein of B cells that specifically binds the major immunopath- ological determinant of IRBP. It is thought that this protein may function as a molecular chaperone in antigen presentation. Recentiy, three intracellular proteins fiom human B cells have been uncovered that bind specifically to the immunodominant, uveopathological epi- tope of the IRBP molecule. Two of these proteins are now known to be novel heat shock proteins. Elevation of serum antibody levels to HSP 70 was found to occur during 256 Lahoratou) of Retinal Cell and Molecular Biology ociilar inflammatory episodes in patients with Beghet's disease. These studies could lead to better diagnosis and treatment of forms of ocular uveitis and serve as a focus for gene therapy. 157 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00070-17 LRCMB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit orj one line between the borders.) Vitamin A and Ocular Tissues PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Barbara Wiggert Ph.D. Head, Section on LRCMB, NEI Biochemistry Others: Kalpana Rengarajan R. Krishnan Kutty Todd Duncan Geetha Kutty Ph.D. Ph.D. M.S. M.S. Visiting Fellow Senior Staff Fellow Biologist Visiting Associate LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI COOPERATING UNITS (if any) U. Lund, Sweden (T. van Veen, Ph.D.); U. Illinois Coll. of Med., Chicago (D. Pepperberg, Ph.D., T.-I. Okajima, Ph.D., H. Ripps, Ph.D.); Med. U.S.C. (R. Crouch, Ph.D., S. Hazard, Ph.D.); SLU Inst. F. Kir, Sweden (K. Narfstrom, D.V.M., Ph.D.); U. Hosp., Utrecht, The Netherlands (B. Zonnenberg, M.D., Ph.D.); Medical College of Georgia (S. Smith, Ph.D.); Emory Eye Center (J. Nickerson, Ph.D.) Laboratory of Retinal Cell and Molecular Biology SECTION Section on Biochemistry INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 5.0 PROFESSIONAL: 3.0 2.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) In the toad retinal pigment epithelium (RPE) eyecup it was demonstrated that interphotoreceptor retinoid-binding protein (IRBP) promotes the formation, as well as the release, of ll-cis retinal. In the experimentally detached skate retina system, the introduction of ligand-free IRBP purified from bovine retina to the subretinal space significantly increased the rate of rhodopsin regeneration and more than doubled the amount of rhodopsin reformed in the darkness as compared with controls in which IRBP content of the subretinal space was diluted as a consequence of detachment and no purified IRBP was added back. In the vitiligo mouse model of retinal degeneration, elevated retinoid levels were found in both the RPE and the liver. These results represent the first evidence of biochemical malfunction in this mutant and suggest that in the eye, the RPE is the primary site of the defect that leads to retinal degeneration. A -70 kDa glycoprotein binding both retinoids and fatty acids was purified to homogeneity from Drosophila melanogaster heads. A method was developed, using reverse transcriptase-polymerase chain reaction (RT-PCR), for the quantitation of IRBP message in small amounts of tissue such as a single mouse pineal gland. Three intracellular human B-cell proteins (-40, 72, and 74 kDa) that bind specifically to peptide 1169-1191, an immunodominant, uveitopathogenic determinant of bovine IRBP, were partially characterized by immunoblotting and microsequencing. The 40 kDa protein was identified as actin, and the 72 and 74 kDa proteins were determined to be members of the hsp 70 family. The 72 kDa protein had 40 percent homology with human hsp 72 from lung fibroblasts, and the 74 kDa protein had a high degree of homology with human hsp 78 glucose-regulated protein from liver. Elevation of serum antibody levels to hsp 70 were found to occur during ocular inflammatory episodes in patients with Behcet's disease. 258 PHS 6040 (Rev. 5/92) Laboratory of Retinal Cell and Molecular Biology Project Description Additional Personnel Igal Gery PhI5. Head, Section on Experimental Immunology, LI, NEI Rachel Caspi PhD. Visiting Associate LI, NEI Tatiana Putilina PhD. Visiting Associate LRCMB, NEI Mark de Smet MD. Visiting Scientist LI, NEI Objectives The purpose of this research project is to investigate the role of specific retinoid-binding proteins such as interphotoreceptor retinoid- binding protein (IRBP) in mediating the action of retinoids in both normal and diseased ocular tissues. Methods Affinity chromatography, fluorescence spec- troscopy, high-performance liquid chroma- tography (HPLC), SDS-polyacrylamide gel electrophoresis. Western blotting. Northern blotting, reverse transcriptase-polymerase chain reaction (RT/PCR), slot-blotting, and the enzyme-Unked immunosorbent assay were used to study retinoid-binding proteins. Major Findings The formation of ll-czs retinal in the retinal pigment epitheUum (RPE) and its release to extracellular medium containing IRBP were studied in the RPE-eyecup of the toad. The results not only indicate that IRBP promotes the formation (from a]l-trans precursor) as well as the release of ll-cis retinal but also suggest the preferred use of recentiy incorporated and esteritied aR-trans retinol in the 11-ds retinal synthesis in a "last in /first out" manner. It has been shown that, compared with a normal eyecup preparation, the amount of rhodopsin regenerated and the rate at which it was resynthesized after bleaching were reduced about 50 percent when the skate retina was detached from its RPE and re- placed immediately on the apical surface of the RPE. In current studies, the detachment proce- dure was performed under fluid to dilute the IRBP content of the interphotoreceptor matrix. Fundus reflectrometry showed that allowing fluid to enter the subretinal space exposed by the detachment procedure caused profound deficits in both the rate and amount of rho- dopsin that regenerated after bleaching. The introduction of Ugand-free IRBP purified fiom bovine retina to the subretinal space signifi- cantiy increased the rate of rhodopsin regener- ation and more than doubled the amount of rhodopsin reformed in darkness. It appears that one important consequence of retinal detachment is the dilution of IRBP in the subretinal space, so that its replacement, particularly in cases of extensive rhegmato- genous detachments, may be beneficial for the recovery of retinal function in human patients. The vitiUgo mouse (C57BL/6-mi"7mi"") model of retinal degeneration exhibits a slow- ly progressing loss of photoreceptor cell nu- clei, gradual loss of rhodopsin, and unevenly pigmented RPE. Analyses of retinoid levels and distribution between the neural retina and RPE in these mutant mice showed that retinyl palmitate levels were significantiy higher in the mutant RPE than in control mice. Further- more, ah-trans retinol was elevated approxi- mately fourfold above controls in the RPE of vitUigo mice. To assess possible systemic involvement in vitiligo mice, retinoids were evaluated in liver and plasma. Plasma retinol levels were normal, but mean Hver total vita- min A levels in affected mice were approxi- mately 1.7 times greater than controls. Analy- sis of esterified and unesterified retinoids in Uver showed that retinyl palmitate levels were elevated. These results showing elevated retinoid levels in both the RPE and hver of the vitiligo mouse represent the first evidence of biochemical malfunction in this mutant and suggest that, in the eye, the RPE is the prima- ry site of the defect that leads to photorecep- tor cell degeneration. This study provides the 259 FY 1994 NEI Annual Report first evidence of altered systemic retinoid metabolism in vitiligo mice that is occurring, significantly, under normal dietary conditions. A glycoprotein binding both retinoids and fatty acids has been purified to homogeneity from Drosophila melanogaster heads. This protein, which has an apparent molecular mass of ~70kDa on SDS-PAGE, is similar to the 140 kDa IRBP in that both proteins are glycosylated, both exhibit endogenous cova- lent and noncovalent fatty acid binding, and both exhibit similar binding affinities for 16-[9- anthryloxy] palmitic acid. The Drosophila protein has a higher affinity for retinol than that of IRBP. A method for the radioanalytic estimation of amplification products generated by reverse transcription coupled to the polymerase chain reaction of the IRBP message in mouse retina and pineal using [a-^^P] was developed. This method allows the detection of the IRBP message in small amounts of tissue such as a single mouse pineal gland. Quantitation of the IRBP message using the G3PDH message as an internal standard was demonstrated. Peptide 1169-1191 is an immunodominant, uveitopathogenic determinant of bovine IRBP that causes severe ocular inflammatory disease in the Lewis rat. Three intraceUular-binding proteins, with apparent molecular masses of 72 and 74 kDa, from Epstein-Barr virus-trans- formed human B cells (from both normal subjects and Beh^ets patients) and stimulated with Upopolysaccharide bind specifically to this peptide. These binding proteins could be released fiom the peptide with adenosine triphosphate (ATP), showing that all three contain an ATP-binding site. Partial characterization of these proteins by immunoblotting and microsequencing of peptides obtained by in situ digestion of specific protein bands demonstiated a 40 kDa protein to be actin and the 72 and 74 kDa proteins to be members of the heat shock protein (HSP) 70 family. The 74 kDa protein has a high degree of homology with human HSP 78 glucose-regulated protein from human Uver, whereas the 72 kDa protein has 40 percent homology with a human HSP 72 from human lung fibroblasts and probably repre- sents a new member of the HSP 70 family. It was also shown in a corollary study that elevation of serum antibody levels to HSP 70 occurred during ocular inflammatory episodes in Behgets patients. Large-scale purification of IRBP was continued for studies on the pro- duction of experimental autoimmune uveitis (EAU) in rats and mice and possible modes of suppression of the disease. Significance to Biomedical Researcti and the Program of the Institute Because of its importance in normal photore- ceptor cell physiology, i.e., in facilitating the transport of retinoids during the visual cycle as well as transport of fatty acids that are essential to normal function, abnormalities in IRBP function resulting from changes in concenfration, distribution, or affinity for retinoids or fatty acids could be important either directiy or indirectiy in visual cell pathogenesis. Proposed Course The physiological role of IRBP in the visual cycle, in particular, the mechanism by which it promotes the formation and release of 11 -cfs retinal, will continue to be probed. We will also be seeking to identify the epitopes on the IRBP molecule that bind retinoids and fatty acids as well as those that are required for eliciting the release of 11-ds retinal from the RPE. Continued studies on the vitiligo mouse model of retinal degeneration will assess retinoid turnover in Uver and RPE as well as the consequences of manipulation of dietary vitamin A on levels of IRBP and retinoids and on photoreceptor degeneration in the eye. The Drosophila head retinoid and fatty acid- binding protein will be further characterized by cloning and sequencing and compared with IRBP. The 72 kDa protein, which binds the uvei- togenic peptide 1169-1191 of IRBP and appears to be a new member of the HSP 70 family of 260 Laboraton/ of Retinal Cell and Molecular Biology HSPs will be investigated further by cloning and sequencing. Antibodies to this protein will also be obtained to study its possible role in antigen presentation. We will continue to conduct large-scale purification of IRBP pro- tein for studies of EAU. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Caspi RR, Chan C-C, Grubbs BG, SUver PB, Wiggert B, Parsa CF, Bahmanyar S, BiUiau A, Heremans H: Endogenous systemic interfer- on-gamma has a protective role against ocular autoimmunity in mice. J Immunol 152:890-899, 1994. Duffy M, Sun Y, Wiggert B, Duncan T, Chader GJ, Ripps H: Interphotoreceptor retinoid binding protein (IRBP) enhances rhodopsin regeneration in the experimentally detached retina. Exp Eye Res 57:771-782, 1993. Duncan T, Kutty G, Chader GJ, Wiggert B: A glycoprotein binding retinoids and fatty adds is present in Drosophila. Arch Biochem Biophys 312:158-166, 1994. Kutty RK, Kutty G, Duncan T, Nickerson J, Chader GJ, Wiggert B: Radioanalytic estima- tion of amplification products generated by reverse transcription PCR using [a-^^P] deoxy- ribonucleoside triphosphate. Biotechniques 15:808-812, 1993. Kutty RK, Nagineni CN, Kutty G, Hooks JJ, Chader GJ, Wiggert B: Increased expression of heme oxygenase-1 in human retinal pig- ment epithelial cells by transforming growth factor-p. / Cell Physiol 159:371-378, 1994. Kutty RK, Kutty G, Rodriguez IR, Chader GJ, Wiggert B: Chromosomal localization of the human heme oxygenase genes: Heme oxy- genase-1 (HMOXl) maps to chromosome 22ql2 and heme oxygenase-2 (HM0X2) maps to chromosome 16pl3.3. Genomics 20:513-516, 1994. Kutty G, Duncan T, Nickerson JM, Si JS, van Veen T, Chader GJ, Wiggert B: Light depriva- tion profoundly affects gene expression of interphotoreceptor retinoid-binding protein in the mouse eye. Exp Eye Res 58:65-75, 1994. Kutty RK, Kutty G, Nagineni CN, Hooks JJ, Chader GJ, Wiggert B: RT-PCR assay for heme oxygenase-1 and heme oxygenase-2: A sensitive method to estimate cellular oxidative damage. Ann N Y Acad Sci, in press. Okajima TL, Wiggert B, Chader GJ, Pepperberg DR: Retinoid processing in the retinal pigment epitheUum of the toad (Bufo Marinus). / Biol Chem 269:21983-21989, 1994. Pepperberg DR, Okajima TL, Wiggert B, Ripps H, Crouch RK, Chader GJ: Interphotoreceptor retinoid-binding protein (IRBP). Molecular- biology and physiological role in the visual cycle of rhodopsin. Mol Neurobiol 7:61-85, 1993. Rajagopalan S, Rodrigues MM, Wiggert B, Advani SH, Nair CN, Nickerson JM: Retino- blastoma: Interphotoreceptor retinoid-binding protein mRNA analysis by polymerase chain reaction. Ophthalmic Paediatr Genet 14:117-125, 1993. Rengarajan K, de Smet MD, Chader GJ, Wiggert B: Identification of heat shock pro- teins binding to an immunodominant uveito- pathogenic peptide of IRBP. Curr Eye Res 13:289-296, 1994. Sasamoto Y, Kawano YI, Wiggert B, Chader GJ, Gery I: Induction of unresponsiveness in adult rats by immunodominant and nondomi- nant peptides. Cell Immunol 152:286-292, 1993. Smith MA, Kutty RK, Richey PL, Chader GJ, Wiggert B, Petersen RB, Perry G: Heme oxy- genase-1 is associated with the neurofibrillary pathology of Alzheimer Disease. Am J Pathol 145:42-47, 1994. 161 FY 1994 NEI Annual Report Smith SB, Duncan T, Kutty G, Kutty RK, Wiggert B: Elevation of retinyl palmitate in eyes and livers and of IRBP in eyes of vitiligo mutant mice. Biochem J 300:63-68, 1994. Wiggert B, van Veen T, Kutty G, Lee L, Nickerson J, Si JS, Nilsson EG, Chader GJ, Narf Strom K: An early decrease in interphoto- receptor retinoid-binding protein gene expres- sion in Abyssinian cats homozygous for he- reditary rod-cone degeneration. Cell Tissue Res, in press. 262 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00297-01 LRCMB PERIOD COVERED October 1, 1993 to Sep t ember 30, 1 994 TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) Microtubule Stability as a Factor in Retinal Degenerations PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Susan Gentleman Ph.D. LRCMB, NEI COOPERATING UNITS (if any) Genetics & IVF Institute, Fairfax, VA (R. Sherins, M.D.) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 1.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Instability of the microtubules of the connecting cilia has been suggested as a cause or complicating factor in some cases of retinitis pigmentosa. These microtubules are normally highly acetylated on Lys40 of a tubulin, a posttranslational modification that appears to be restricted to polymerized microtubules; the function of this acetylation is as yet unknown. We have described the case of an infertile male subject with a rod- dominant retinal degeneration whose sperm flagella show severe morphological abnormalities and greatly reduced acetylation. Currently, the project is directed toward characterization of tubulin acetyl transferase (TAT) using bovine retina and brain as the source. TAT has been purified about 1,000-fold and is in a complex of about 300 kDa. SDS-PAGE analysis of the complex shows four major and several minor bands ranging from 30 to 120 kDa. The major bands are N-terminal blocked and will require digestion and purification of peptides before sequencing can be done. Arrestin (S-antigen) copurifies with the TAT complex from retina and is a substrate for the acetyltransferase activity. A 70 kDa band of x-immunoreactive material is also found in the TAT complex from both brain and retina. The partially purified TAT complex from Y-79 retinoblastoma cells is under evaluation for use in future studies of the molecular biology of the TAT complex. 263 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Additional Personnel Muriel I. Kaiser MD. Chief, OGCSB, NEI W. Gerald Ph.D. Chief, Section on Robison Pathophysiology, LMOD, NEI Objectives Characterization of the components of the microtubule-associated acetyl transferase activity will be used to obtain molecular probes for analysis of deoxyribonucleic acid (DNA) from human subjects with retinal degenerations in which microtubule instability is suspected. Methods An assay of acetyl transferase activity in crude tissue fractions has been developed. An acetyl coenzjone A agarose affinity column, with attachment through the phosphate using phosphoramidate chemistry, has been made. Conventional techniques of protein chemistry and purification are used. Major Findings (1) Sperm flagella from an infertile male subject with a rod-dominant retinal degenera- tion were evaluated for alpha tubulin iso- forms. The level of tubulin acetylation was 30 percent if the normal population, although tyrosinated tubulin level was normal. The tubulin acetyl transferase-specific activity of these flagella was also significantly lower than normal. (2) The acetyl transferase activity from bovine brain and retina has been purified about a thousandfold from the subcellular fraction containing cold-stable microtubules (27,000 xg pellet) by high-salt extraction, anion-exchange chromatography, gel filtration by high-performance liquid chromatography, and affinity chromatography on AcCoA-aga- rose. SDS-polyacrylannide gel electrophoresis (PAGE) analysis of the purified fractions shows four major bands and several minor bands from both brain and retina. The major bands from brain have been eluted from gels for N-terminal analysis. These bands appear to be homogeneous but all are N-terminally blocked and cannot be sequenced without further processing. (3) One band in the retinal complex is acetylated in vitro by incubation of the puri- fied complex with AcCoA. This protein has been identified as arrestin (S-antigen) both by N-terminal sequencing and by immuno- blotting. (4) The purified complexes from both brain and retina were analyzed by immuno- blotting with a panel of monoclonal antibodies against a variety of microtubule binding proteins (MAPs). A 70 kDa band of x immu- noreactivity was found in both preparations. Treatment of the complex with alkaline phos- phatase appeared to increase the immunoreac- tivity of the 70 kDa band to the antibody x-l, which specifically recognizes the unphos- phorylated form of x. (5) The acetyl fransferase complex from Y-79 retinoblastoma cells was partially puri- fied and characterized by immunoblottrng. Arrestin immunoreactivity copurified with the complex. The 70 kDa band of x immunoreac- tivity in the complex from these cells also reacted with the MAP2 monoclonal antibody HM-2 and did not show an increase in x-Uke immunoreactivity with dephosphorylation. Significance to Biomedical Research and the Program of the Institute The microtubule acetyl fransferase complex is a mvdtifunctional complex containing the enzyme with apparentiy a broad specificity and microtubule-binding proteins as well as other proteins, some of which are tissue spe- cific. The single band of x immunoreactivity suggests that a particular isoform is used by this complex. Therefore, a genetic defect in one splice site of x might have major effects on the function of the complex. However, the 264 Laboraton/ of Retinal Cell and Molecular Biology data from Y-79 cells indicate that other MAPs may substitute for the x-like form, although possibly not as weU. The association of arres- tin with the complex in retina also suggests that it may play a role in the trafficking of arrestin in photoreceptor ceUs. Therefore, the retina might be particularly sensitive to mal- function of the complex. Proposed Course The current direction of the project is to con- tinue the characterization of the components of the acetyl transferase complex. With this information, we intend to obtain molecular probes suitable for screening human material. (1 ) Protein sequence of components of the complex will be obtained by digestion and purification of peptides from the proteins eluted from SDS-PAGE. The N-terminal blockade in most of the proteins of the com- plex requires that initial protein sequence data be obtained from internal peptides. Sequences will be matched to those in the available protein databases {e.g., Swiss Prot) for identifi- cation. (2) Peptide sequences not matching se- quence in current data banks wiU be used to design degenerate oUgos for polymerase chain reaction probing of complementary cDNA libraries with the objective of obtaining in- ferred protein sequence for the complete proteins. (3) From the sequence information ob- tained, molecular probes will be made to examine human genes. Family pedigrees of type 2 Usher's syndrome are of particular interest. Several reports from various labora- tories have demonstrated axonemal abnormal- ities at a higher than normal frequency in some of these people. NEI Research Program Retinal Diseases — Photoreceptors and Pigment Epithelium Publications Lloyd RA, Gentieman S, Chader GJ: Assay of tubulin acetyl tiansferase activity in subcellu- lar tissue fractions. Anal Biochem 216:42-46, 1994. 265 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00196-11 LRCMB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Molecular Genetics of the Eye and Ocular Diseases PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Diane E. Borst Ph.D. Senior Staff Fellow LRCMB, NfEI Steven Bernstein Ph.D., M.D. Senior Staff Fellow LRCMB, NEI COOPERATING UNITS (if any) University of Texas, Dallas (R. Hammer, Ph.D.); University of Illinois (H. Ripps, Ph.D.) Laboratory of Retinal Cell and Molecular Biology SECTION Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 2.0 PROFESSIONAL: 2.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Antisense refers to a nucleic acid molecule that is complementary to an expressed messenger RNA sequence present in an organism. We are evaluating the use of various forms of antisense molecules, both catalytic (ribozymes) and noncatalytic in nature, to evaluate the function of specific gene expression in the eye. Ribozymes complementary to interphotoreceptor retinoid-binding protein mRNA have been evaluated in vitro and in vivo. Antisense to aldose reductase is currently being evaluated in vivo. 266 PHS 6040 (Rev. 5/92) Lahoratory of Retinal Cell and Molecular Biology Project Description Additional Personnel Eric Wawrousek PhD. Research Biologist, OSD, NEI Susan DiCamillo B.S. Chemist, OSD, NEI Objectives Antisense and catalytic antisense are being used to downregulate specific functions in ocvdar tissues for the purposes of both basic research and clinical application. This research is designed to define the as- acting elements in the mouse interphotorecep- tor retinoid-binding protein (IRBP) promoter that controls the regulation of the IRBP gene expression. Other factors such as deoxyribo- nucleic acid (DNA) methylation may be in- volved in the regulation of IRBP gene expres- sion; another objective is to define the DNA methylation state of the IRBP promoter in different tissues. Methods This study uses in vitro transcription and assay methodology as well as transgenic expression of antisense constructs in mice and rats. Conventional techniques for clorung and analysis of nucleic acids are used. Transgeruc mice are being used to study the cfs-acting elements of the IRBP promoter. The transgene contains varying lengths of the mouse IRBP promoter fused to the reporter gene, chloramphenicol acetyl transferase (CAT). The levels of CAT activity in these mice show which DNA sequences are impor- tant for the tissue and developmentally specif- ic expression of the IRBP gene. DNA was isolated from different tissues, digested with Msp I or Hpa II, Southern blotted, and probed with fragments of the IRBP promoter to study its DNA methylation state. Ma'ior Findings Most attempts at using ribozymes for down- regulation and "gene therapy" in human immunodeficiency virus have used ribozymes of varying complement length; this comple- ment length gives specificity to the target site of the message. The optimal length for this enzyme-target interaction is unknown, and this is partially confirmed by the high failure rate and exceedingly variable activity rates for engineered molecules against various targets. Using i?j vitro partial duplex transcription, cloned ribozyme templates, and substrate fragments, I have studied the effect of site specificity and varying complement length on ribozyme activity in vitro. I have found that ribozyme activity can be "tuned" in vitro by varying complement length and that this tuning is unique and target-site specific. The current computer-based programs for predict- ing site sensitivity are inadequate to this task; it must be performed empirically. This study is completed. With the help of the above technique, I have generated two constructs containing ribozymes targeted against different sites in the messenger ribonucleic add (mRNA) for IRBP, which have the highest demonstrated in vitro activity and have generated transgenic founder lines expressing these ribozymes in ocvdar and other tissues. The data from this study is preliminary, but there are apparently significant differences in the embryonic sur- vival and tissue-specific expression of transgene and IRBP mRNA in the different constructs. We are currently doing ocular histology studies on outbred animals (Fl generation). With the collaboration of Dr. Harris Ripps, from the Uiuversity of Illinois at Chicago, we have performed electrophysiological studies on these mouse lines. There are apparent differences in some of the transgenic lines in electroretinogram responses, and these appar- ently correlate with transgene expression. Animal Unes apparently expressing the high- est levels of the transgene also demonstrate high embryonic fataUty. Thus, IRBP expres- 267 Pi' 1994 NEI Annual Report sion mav be important in fetal development. This work correlates weU with earlier reports of IRBP expression in fetal tissue of both rats and cows. Aldose reductase (AR) is believed to play a kev role in the development of diabetic retinopathy, neuropathy, and nephropathy. Additionally, AR is apparently involved in cataractogenesis in diabetic humans and galactose-fed rats. Research to date has fo- cused on AR inhibitors in animal systems, these inhibitors must be administered chroni- callv and are expensive; in addition, in one of the most \\'idely studied animal systems, the dog, development of retinopathy requires an extended period of time. Studies in mice are hampered bv the fact that they do not develop histologically documented retinopathy or neuropathy, although they apparently exhibit ner\'e conduction velocity changes. Rats also develop diabetic changes and are an alternative, relatively low-cost mammalian system in which to study progression of diabetic patholog^^ Rat transgenic model svstems are no^v available. Because the mRNA sequence of rat AR is kno\\Ti, we have designed gene constructs containing antisense- based sequence to rat AR. Expressed m vivo, AR deficient rats should show delayed galac- tose-induced cataractogenesis and delayed onset of diabetic histopatholog^^ In collabora- tion with Dr. Robert Hammer, from the Howard Hughes Medical Research Institute in Houston, Texas, 1 am generating strains of rats that express antisense for AR in a variet}^ of tissues. This work is in the animal production stage. Tissue culture correlates of retinoblastoma. We have characterized two mouse retinoblas- toma cell lines in terms of photoreceptor- spedfic gene expression. These Hnes may be useful in the future for in vitro transfection studies using antisense technology' without the need for transgenic mice production. This study is completed. Retinopathy of prematurity (ROP) is one of the leading causes of early childhood blind- ness in the United States. Pathology in this disease results from the development of reti- nal neovascularization in premature or low birth weight infants that can then progress, in its most severe form, to invasion of the vitre- ous body, and either during the rapid period of vessel growth or following involution, to retinal detachment. The only effective treat- ment is surgical, with 25 to 35 percent of the most severely affected infants maintaining vision of 5/200 or less. Dr. Lois Smith, from the Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, has developed a reproducible model of ROP in newborn mice. In collaboration with Dr. Smith, 1 have prepared a series of antisense molecules that I am testing for efficacy of specific inhibition of mitosis. These com- pounds will then be used to determine effica- c}^ of inhibition of ROP in vivo. Msp I and Hpa 11 are isoschizomers that are methylation dependent. Hpa n will not digest the recognition sequence if the 3' cytosine is methylated. Msp I sites of the IRBP 5' flanking region were studied: five in the cow and two in the mouse. One of these sites is in the homologous location and is hypomethylated only in the retina. Similar results have been found for the other Msp I sites that have been studied. Perhaps these regions of the IRBP promoter are important in the regulation of IRBP expression either by being a direct protein-binding site or by some- how influencing the secondary structure of this region. Significance to Biomedical Research and the Program of the Institute hi vitro testing of ribozyme activity may enable the selection of unique ribozymes, with the possibihty of enhanced in vivo action, for use in both gene therapy and basic research. The transgenic animal results may teU us much about the role of IRBP in embryonic development as well as the function of the retina during relative IRBP deficiency^ Gener- ation of transgenic rats expressing antisense for AR may be useful in evaluating the direct Laboratory of Retinal Cell and Molecular Biology pathophysiological Unk between AR activity and pathology in diabetes. Evaluation of antisense molecules with antimitotic activity will likely be useful in direct pharmacological treatment of retinopathy of prematurity. IRBP in adults is expressed only in the retina and pineal gland. Understanding the mechanisms and the nucleotide elements that control IRBP expression is fundamental to understanding retinal development and func- tion. This understanding could help us design drugs that would specifically target the retina and perhaps would also elucidate abnormal retinal function. Proposed Course Evaluation of mouse retinoblastoma ceUs: completed. In vitro evaluation of hammerhead ribozyme activity: completed. Transgenic animal studies/expression of ribo- zyme activity. I am breeding transgenic animal lines expressing the active ribozyme constructs to select for those lines with the highest homo- zygous expression of ribozyme activity. I anticipate sending some of these adult animals to Dr. Ripps for further electrophysiological studies. In addition, 1 am attempting to determine the time of onset of lethality of the ribozyme expression in the prenatal animal. AR antisensel transgenic rats. I am awaiting initial confirmation that Dr. Hammer has indeed generated a strain of rats expressing the AR antisense construct that I sent him. When this is completed, I will begin evalua- tion of in vivo expression of the construct. Iiutial feedings with high galactose will be performed to determine the speed of onset of cataractogenesis vis-a-vis control rat strains. Further analysis depends of the initial delivery on these animals. Determination of antineovascular activity of antisense compounds. Irutial studies determin- ing the specific antimitotic activity are under way. The compounds possessing the highest antimitotic activity wiU be evaluated for their pharmacotherapeutic index in vitro. These specific compounds wdll be evaluated further for their relative antineovascular activity in NEI Research Program Retinal Diseases — Photoreceptors and Retinal Pigment Epithelium 269 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00124-14 LRCMB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Molecular Biol og y of the Retina and Pigment Epithelium PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Gerald J. Chader Ph.D. Chief LRCMB, NEI Others: R. Theodore Fletcher Joyce Tombran-Tink S. Patricia Becerra Timothy Schoen M.S. Chemist LRCMB, NEI Ph.D. IRTA Fellow LRCMB, NEI Ph.D. Visiting Scientist LRCMB, NEI M.S. Biologist LRCMB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 4.0 PROFESSIONAL: 2.5 1.5 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x| (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The title of this project has been changed to more accurately reflect the thrust of the science. The retina and pigment epithelium are neuroepithelial tissues that work in close cooperation. Specific growth and differentiating factors are found in the eye that guide development and interactions of individual ocular tissues to form a functional visual system. Studies on this project are focused on an understanding of the molecular biology and molecular genetics of the retina and discovering new genes that are candidates for hereditary retinal degenerations. For example, ocular tissues synthesize a number of growth factors. There now appear to be several systems present that could self-regulate growth and metabolic activity in the retina-pigment epithelium complex and be involved in eye diseases. In this regard, we have cloned and characterized a unique protein secreted from fetal human pigment epithelial cells, called pigment-epithelial derived factor (PEDF), that is "neurotrophic" to cultured human retinoblastoma cells and may affect neural retinal development in vivo. This protein also is a potent "neuronotrophic" agent in that it promotes neuronal cell survival of cultured cerebellar granule cells. Finally, PEDF is "gliastatic" in that it markedly retards glial cell growth. Along with being a candidate gene in retinal degenerations, the uses of PEDF in neuronal transplant in retina and other CNS areas are obvious. 270 PHS 6040 (Rev. 5/92) Laboratory of Retinal Cell and Molecular Biology Project Description Additional Personnel Joan Schwartz PhD. Chief, Molecular Genetics Section, DIR, NINDS Objectives Our objective is to obtain a better understand- ing of the molecular biology and molecular genetics of ocular tissues in health and dis- ease. The study of growth and differentiation factors, be they protein {e.g., pigment epitheli- um-derived factor [PEDF]) or polypeptide {e.g., insulin-like growth factor [IGF-1]) is critical in obtaining a view of the events that control the early development of the eye and also in maintaining normal function in the adult. Methods Molecular biological, genetic, and immunocy- tochemical techniques are used. Tissue cul- ture is performed on cultured cells. In partic- ular, the human retinoblastoma cell line Y-79 is used as a test system for differentiating agents. Major Findings Hereditary diseases are often caused by de- fects in genes that are important in cell divi- sion and differentiation; PEDF seems to be such a gene product. It is secreted from fetal pigment epithelial ceUs and is present in normal adtilt interphotoreceptor matrix. The protein migrates at approximately 50 kDa on sodium dodecyl-sulfate-polyacrylamide gels. Importantly, PEDF causes marked differentia- tion of human Y-79 retinoblastoma ceUs in culture (neurotrophic effect). This is charac- terized by an extensive elongation of neurite- like processes and a gathering of cells into "rosette-Uke" aggregates. Immunocytochemi- caUy, the expression of specific neuronal markers is also enhanced. Thus, PEDF is a unique protein, synthesized and secreted by retinal pigment epitheUal cells, that could direct early development even in early em- bryogenesis. PEDF is also present after the important developmental period and may help to maintain retinal cell viability (neuron survival) in the adult retina. PEDF is also a candidate gene in the retinal dystrophy ob- served in the Royal College of Surgeons rat. After the cloning of the complementary deoxyribonucleic acid for the PEDF gene, we have determined that the protein is a member of the SERPIN (serine protease inhibitor) superfamily of genes. One other member of this family is known to promote neuronal differentiation so it makes it more probable that PEDF has a major, and similar, role in the retina. The recombinant protein (rPEDF) has now been expressed in Escherichia coli cells and has been shown to be an active neuro- trophic agent. The availabihty of relatively large amounts of PEDF should allow for more direct studies on its role(s) in ocular develop- ment and disease. We also find PEDF mes- senger ribonucleic acid in ciliary body and PEDF protein in vitreous humor, indicating that PEDF could be of importance in ocular tissues other than the retina. In work with Dr. Joan Schwartz, National Institute of Neurological Disorders and Stroke (NINDS), we have evidence demonstrating that PEDF is also a potent "neuronotrophic agent," i.e., neuron survival factor in a cul- tured cerebellar granule cell (CGC) system. Very small amounts of rPEDF added to the CGCs are effective in keeping the cells alive for prolonged periods of time. Surprisingly, we have found that PEDF has "gliastatic" properties as well, in that gUal cells in the cerebellar cultures are markedly inhibited. Importantly, this is a long-term effect; a one- time exposure to nanogram quantities of rPEDF inhibits growth of cultured cerebellar gUa for up to 12 weeks. Significance to Biomedical Research and the Program of the Institute Determining the genes that control normal ocular growth, differentiation, and function 171 FY 1994 NEl Annual Report and studying them on a molecular biological and molecular genetics level will aid us in understanding diseases of the eye, especially those of a hereditary, early developmental nature. With such knowledge, rational meth- ods of gene therapy can be appUed to ocular diseases. Because of its potent neurotrophic, neuronotrophic, and gUastatic effects, PEDF seems to be of potential use in retinal and central nervous system transplantations. Proposed Course The molecular biology and molecular genetics of ocular development will be further exam- ined. The factors that affect normal and abnormal growth will be investigated. The fuU PEDF gene will be examined and ana- lyzed to help elucidate its presumptive role(s) in retinal development. The rPEDF protein will be used to elucidate the role of the novel new protein in retinal diseases processes. NEl Research Program Retinal Diseases — Retirutis Pigmentosa and Other Inherited Disorders Publications Arnold DR, Moshayedi P, Schoen TJ, Jones BE, Chader GJ, WaldbiUig RJ: Distribution of IGF- I and -n, IGF binding proteins (IGFBPs) and IGFBP mRNA in ocular fluids and tissues: Potential sites of synthesis of IGFBPs in aque- ous and vitreous. Exp Eye Res 56:555-565, 1993. Becerra SP, Palmer I, Kumar A, Steele F, Shiloach J, Notario V, Chader GJ: Overexpres- sion of fetal human pigment epitheUum-de- lived factor in Escherichia coU: A functionally active neurotrophic factor. / Biol Chem 268:23148-23156, 1993. Gaudet SJ, TsUou E, Chader GJ: Identification and characterization of arylamine N-acetyl- transferase activity from the bovine retinal pigment epithelium. Curr Eye Res 12:271-278, 1993. Li A, Lane WS, Johnson LV, Chader GJ, Tombran-Tink J: Neuron-specific enolase: A neuronal survival factor in the retinal extracel- lular matrix? / Cell Biol, in press. Poggi L, Melchiori A, Pellegrini R, Defilippi P, Noonan D, Campbell MA, Gentieman S, Chader GJ, Albini A: Laminin-induced Y-79 retinoblastoma ceU differentiation occurs in the absence of "classic" laminin adhesion molecules, in Fassina GF, Percaiio M (eds): Cell Adhesion Molecules in Cancer and Differenti- ation. London, Harwood- Academic Publish- ers, in press. Seigel GM, Becerra SP, Chader GJ, Diloreto DA Jr, del Cerro C, Lazar ES, del Cerro M: Differentiation of Y79 retinoblastoma cells with pigment epitheUal-derived factor and interphotoreceptor matrix wash: Effects on tumorigenicity. Growth Factors, in press. Tombran-Tink J, Pawar H, Swaroop A, Rodri- guez I, Chader GJ: Localization of the gene for pigment epitheUum-derived factor to chromosome 17pl3.1 and expression in cul- tured human retinoblastoma. Genomics 19:266- 272, 1994. WaldbiUig RJ, Jones BE, Schoen TJ, Heidersbach S, Bitar MS, van Kujik F, de Juan E, Kador P, Chader GJ: Vitreal insuUn-Uke growth factor binding proteins (IGFBPs) are increased in human and animal diabetics. Curr Eye Res 13:539-546, 1994. del Cerro M, Seigel GM, Lazar E, Grover D, del Cerro C, Brooks DH, DiLoreto D, Chader GJ: Transplantation of Y79 cells into rat eyes: An in vivo model of human retinoblastomas. Invest Ophthalmol Vis Sci 34:3336-3345, 1993. 272 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER 1 ZOl EY 00148-21 LRCMB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Visual Contro l Mechanistns and Hereditary Defeneration PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Gerald J. Chader Ph.D. Chief LRCMB, NEI Others: Paul Wong Tatiana Putilina Ignacio Rodriguez June Lee Timothy Schoen Ph.D. Ph.D. Ph.D. M.D. M.S. Visiting Fellow Visiting Associate Staff Fellow Visiting Associate Biologist LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI COOPERATING UNITS (if any) School of Veterinary Medicine, Cornell University (G. Aguirre, D.V.M., Ph.D.); Department of Zoology, University of Lund, Lund, Sweden (T. van Veen, Ph.D.); Institute Nazionale per la Ricera sul Cancro, Geneva, Italy (A. Albini, Ph.D., D. Noonan, Ph.D.) _^___ LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology ^ SECTION Section on Gene Regulation . INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 5.0 PROFESSIONAL: 4.5 0.5 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The title of this project has been changed to more accurately reflect the thrust of the science. We are studying the expression of specific gene products that could be related to hereditary diseases of the retina. If normal genetic control mechanisms fail, hereditary diseases of the retina such as retinoblastoma or retinitis pigmentosa will resuh. We have now developed new techniques to clone and sequence retina-specific genes at a higher efficiency. We have found several genes that are either expressed exclusively or predominantly in the retina and are using these as candidate genes in specific blinding diseases. Among these are hydroxy-0-methyl transferase, an important gene on the X chromosome involved in circadian control in the retina and a new gene mapping to chromosome Uq close to four important genetic retinal diseases. Similarly, we are investigating the properties of known retina-specific genes such as the interphotoreceptor retinoid-binding protein (IRBP) and their involvement in retinal disease processes. Progress has also been made in identifying apoptosis as a primary and unifying mechanism for cell death in several hereditary retinal degenerations. All these factors and processes could lead to more efficient gene therapy of the diseased neural retina. 273 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Additional Personnel Muriel I. Kaiser M.D. Chief, OGCSB, NEI Objectives Expression of genes in the retinal photorecep- tor neuron in a normal manner is crucial to visual function in the adult. Thus, the factors that code for normal gene control and expres- sion in human retina and in animal models of retinal degeneration are of primary interest. We also have started a major effort to develop new molecular biological techniques such that unique retinal and retinal pigment epithelium (RPE) genes can be identified, cloned, and sequenced to be used ultimately in screening human populations with inherited diseases of the visual system. Methods Standard molecular biological, biochemical, and neurocheirucal techniques are used. Histochemical techniques are used when necessary. Major Findings (1) We have developed new molecular biolog- ical techniques that allow for more efficient identification of highly expressed genes of the retina-PE complex. Each tissue of the body expresses a unique complement of genes that are trai scribed and translated at a high level. In the retina and pigment epithelium, several very specific proteins are highly expressed such that photoreception and the visual pro- cess can take place. In a similar vein, it is often a genetic defect in these tissue-specific genes that results in a hereditary degeneration such as retinitis pigmentosa. We have devel- oped and are using new methods for rapid polymerase chain reaction-based construction of specifically enriched libraries fiom very small retinal samples. This is especially im- portant because tissue samples are limited in studying early development and rare patholo- gy samples. A significant methodological advance we have made involves subtractive cloning on an immobilizing Dynabead base®. In this way, several new genes have been found that are being used as candidate genes for hereditary retinal degenerative diseases. (2) In collaboration with National Eye Institute (NEI) clinicians, we are now applying these techniques to the study of the role of several proteins and genes in retinal disease. Among these are: (a) Fatty acid-binding proteins that may be involved in normal and degenerating retinas such as in Bietti's crystalline dys- trophy. (b) A newly discovered gene on chro- mosome llq that is a prime candidate gene in Bardet-Biedl's syndrome. Best's disease, familial exudative vitreoretino- pathy, and one form of Usher's syndrome. (c) The hydroxjdndole-O-methyl trans- ferase (HIOMT) gene that maps to the short arm of the X-chromosome and is a key enzyme in melatonin production in the retina. (3) Finally, we are beginning to under- stand the mechanism by which photoreceptor ceUs die in a number of hereditary retinal diseases. This process is called "apoptosis" or programmed ceU death. We have found induc- tion of the marker gene TRPM-2 (clusterin) in aU cases of retinal degenerations studied. It thus appears that programmed cell death may be a common mechanism by which many hereditary defects initiate photoreceptor cell death. Significance to Biomedical Research and the Program of the Institute One approach to studying a hereditary disease process in a tissue and its reversal through gene therapy is to first identify the normal complement of unique genes expressed in that tissue. This is also applicable in an early degenerative process, e.g., retinitis pigmentosa. Lnhoratonj of Retinal Cell and Molecular Biology and in other hereditary diseases such as Bietti's crystalline dystrophy or Best's disease in which the disease may be systemic, but the disease primarily affects the retina. In paral- lel, if one knows the common mechanism by which photoreceptor cells actually die in the various retinal degenerations, it may be possi- ble to design strategies by which retinal cells are spared, at least long enough for transplan- tation or gene therapy experiments to be successful. Thus, studying apoptosis and similar processes in the retina wiU lead to better methods for gene therapy in the neural retina. Proposed Course We will continue to study molecular biological and developmental control mechanisms in the retina and pigment epitheUum. In particular, we will investigate gene expression in normal retinas and in retinas affected with specific genetic diseases and focus on subtiactive cloning as a prime method for identif5dng defective or missing genes in retinal diseases. Apoptosis wiU continue to be an important area for study because future gene therapy in retinal degenerations may depend on under- standing how to prevent death of the photore- ceptor neuron. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Diseases Publications Chader GJ: Retinal degenerations of heredi- tary, viral and autoimmune origins: Studies on opsin and IRBP, in Osborne N, Chader GJ (eds): Progress in Retinal and Eye Research. Oxford, Pergamon Press Ltd, 1994, vol 13, pp 65-99. Duffy M, Sun YF, Wiggert B, Duncan T, Chader GJ, Ripps H: Interphotoreceptor retinoid-binding protein (IRBP) enhances rhodopsin regeneration in the experimentally detached retina. Exp Eye Res 57:771-782, 1993. Duncan T, Kutty G, Chader GJ, Wiggert B: A glycoprotein binding retinoids and fatty acids is present in Drosophila. Arch Biochem Biophys 312:158-166, 1994. Fassina G, PagUalunga G, Noonan DM, Chader GJ, Albini A: Modulation of Y-79 retinoblastoma cell differentiation and IRBP expression by dibutyryl cyclic AMP and laminin. M ] Oncol 2:745-751, 1993. Hershfield B, Chader G, Aguirre G: Cloning of a polymorphic canine genetic-marker, which maps to human chromosome-9. Anim Genet 24:293-295, 1993. Kutty G, Duncan T, Nickerson J, Si JS, van Veen T, Chader GJ, Wiggert B: Light depriva- tion profoundly affects gene expression of interphotoreceptor retinoid-binding protein in the mouse eye. Exp Eye Res 58:65-75, 1994. Kutty RK, Kutty G, Duncan T, Nickerson J, Chader GJ, Wiggert B: Radioanalytic estima- tion of amplification products generated by reverse tianscription PCR using [alpha-33P] deoxyribonucleoside triphosphate. Biotech- nicjues 15:808-812, 1993. Kutty RK, Kutty G, Nagineni CN, Hooks JJ, Chader GJ, Wiggert B: RP-PCR assay for heme oxygenase-1 and heme oxygenase-2: A sensitive method to estimate ceUvdar oxidative damage. Ann NY Acad Sci, in press. Kutty RK, Kutty G, Rodriguez IR, Chader GJ, Wiggert B: Chromosomal localization of the human heme oxygenase genes: Heme oxy- genase-1 (HMOXl) maps to chromosome 22ql2 and heme oxygenase-2 (HM0X2) maps to chromosome 16pl3.3. Genomics 20:513-516, 1994. Kutty RK, Nagineni CN, Kutty G, Hooks JJ, Chader GJ, Wiggert B: Increased expression of heme oxygenase-1 in human retinal pig- ment epitheUal cells by transforming growth factor-beta. / Cell Physiol 159:371-378, 1994. Lloyd RA, Gentieman S, Chader GJ: Assay of tubulin acetyl-transferase activity in subcellu- FY 1994 NEI Annual Report lar tissue fractions. 1994. Anal Biochem 216:42-46, Okajima TL, Wiggert B, Chader GJ, Pepperberg DR: Retinoid processing in the retinal pigment epithelium of the toad (Bufo marinus). / Biol Chem 269:21983-21989, 1994. Pineda R, Chang CC, Ni M, Hayden BJ, John- son MA, Nickerson J, Chader GJ: Human retinoblastoma ceUs express beta-crystaUin in vivo and in vitro. Curr Eye Res 12:239-245, 1993. Putilina T, Sittenfeld D, Chader GJ, Wiggert B: Study of a fatty-acid binding-site of interpho- toreceptor retinoid-binding proteins using fluorescent fatty adds. Biochemistry 32:3797- 3803, 1993. Rajagopalan S, Rodrigues M, Polk T, Wilson D, Chader GJ, Hayden BJ: Modulation of retinoblastoma cell characteristics by hexa- methylene bis-acetamide and other differenti- ating agents in culture. / Histochem Cytochem 41:1331-1337, 1993. Sasamoto Y, Kawano YI, Wiggert B, Chader GJ, Gery I: Induction of unresponsiveness in adult rats by immunodominant and nondomi- nant peptides. Cell Immunol 152:286-292, 1993. Wiggert B, van Veen T, Kutty G, Lee L, Nickerson J, Si JS, Nilsson SE, Chader GJ, Narfstrom K: An early decrease in tnterphoto- receptor retinoid-binding protein gene expres- sion in Abyssinian cats homozygous for he- reditary rod-cone degeneration. Cell Tissue Res, tn press. Wong P, Putilina T, Chader GJ, Tenniswood M: The human gene encoding TRPM-2 exists as a single gene locus on the short arm of chromosome 8. Am J Human Genet, in press. Wong P, Taillefer D, Lakins J, Pineault J, Chader G, Tenniswood M: Molecular charac- terization of human TRPM-2 /clusterin, a gene associated with sperm maturation, apoptosis and neurodegeneration. Eur J Biochem 221:917-925, 1994. Rengarajan K, de Smet MD, Chader GJ, Wiggert B: Identification of heat shock pro- teins binding to an immunodominant uveito- pathogenic peptide of IRBP. Curr Eye Res 13:289-296, 1994. 276 PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00260-05 LRCMB PERIOD COVERED October 1, 1993 to S eptember 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Molecular Biology of Out er Retin a-Specific Proteins PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: T. Michael Redmond Ph.D. Research Biologist LRCMB, NEI Others: Suyan Liu M.D., Ph.D. Visiting Fellow LRCMB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology SECTION Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 2.0 PROFESSIONAL: 2.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Retinal pigment epithelium (RPE) cells and photoreceptor cells are functionally and developmentally closely integrated. Derangements of the RPE are involved in certain retinal diseases. However, the RPE is poorly understood at the molecular level. We have cloned and characterized RPE65, a novel developmentally- regulated conserved 65 kDa RPE-specific microsomal membrane-associated protein. The cDNA sequence is being used to overexpress RPE65 protein for functional studies. The potential role of the protein in inducing uveitis will also be studied using recombinant protein. The RPE65 protein is not expressed in cultured RPE even though its mRNA is abundant. In characterizing this example of posttranscriptional regulation, we have identified distinct sequences in the 3' untranslated region of the RPE65 mRNA that control the stability and the efficiency of translation of the RPE65 message. We have cloned and are sequencing a full-length human genomic clone for RPE65. It is at least 40 kilobases in length. Sequence analysis shows that the RPE65 protein is highly conserved between human and cow. We have also cloned the mouse gene. The human gene for RPE65 is localized to chromosome lp31, and the mouse homolog to distal chromosome 3. These do not correspond to any ocular disease gene localized so far. Nonetheless, RPE65 remains a candidate gene for RPE-involved disease. 277 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The retinal pigment epithelium (RPE) and the photoreceptor cell layer of the neural retina form a functionally and developmentaUy interdependent complex. Dysfunction of the RPE, accordingly, is deleterious to the photo- receptors and, hence, to vision itself. In spite of these important considerations, Uttle is known about the RPE at the molecular level. In this laboratory, we are cloning proteins specifically or preferentially expressed in the RPE with a view to understanding mecha- rusms important to the RPE. At present, our major emphasis is on a 65 kDa protein that we have named RPE65. We are also studying other RPE-expressed proteins. Methods Molecular cloning and biochemical and pro- tein chemistry techniques are used in this study. Additionally, we are using automated fluorescent deoxyribonucleic acid (DNA) sequencing and gene mapping techniques. Major Findings (1) RPE65 is a developmentaUy regulated, membrane-associated, nonglycosylated 65 kDa protein restricted to and conserved in verte- brate RPE and is the major protein of the RPE microsomal fraction. The protein displays an affinity for phosphoUpids that is calcium independent. We have cloned a composite 3,115 bp complementary DNA (cDNA) for this protein. (2) We have found that distinct sequences in the 3' untranslated region (UTR) of the RPE65 messenger ribonucleic acid (mRNA) regulate the stability of the RPE65 message and the efficiency of its translation. This is the first example of 3' UTR-mediated regula- tion in an ocular gene. (3) We have isolated a human genomic done for RPE65. It is at least 40 kilobases in length. Much of the gene has been sequenced. Preliminary sequence analysis indicates that the RPE65 protein is highly conserved in evolutionary terms. (4) We have localized the gene for the RPE65 to human chromosome lp31 and to the far distal end of mouse chromosome 3. Nei- ther of these loci matches that of a known ocular disease or phenotjqse. The mouse gene has been cloned to allow us to pursue trans- genic and homologous recombination ap- proaches for studying RPE65 gene regulation and function in the mouse. Significance to Biomedical Researcli and the Program of the Institute The RPE is poorly characterized at the molec- ular level. This is in spite of its pivotal role in the maintenance of photoreceptor function and, hence, of vision itself. We have identi- fied RPE65 as a conserved RPE-spectfic mole- cule that is developmentaUy expressed. cDNA sequencing demonstrates that it is a novel protein. The function of this protein, although not yet clear, may be related to its affinity for phospholipids. Elucidation of the basis for its posttianscriptional regulation in vitro may have significant bearing on the cvdture of RPE ceUs. This is of some clinical significance because RPE ceU transplantation is receiving much attention as a possible mode of inter- vention in treating some retinal diseases. In addition, because of its RPE specificity, the RPE65 gene can be considered a potential candidate gene for retinal disease. At present, however, neither its human nor mouse chromosomal locations match those of any mapped disease loci. This may change as more disease loci are matched. Again, in view of its RPE-specific expression, elucidation of its gene structure may uncover RPE-spedfic regulatory elements. The high degree of conservation in the RPE65 protein suggests an evolutionarily important function for this protein. FinaiUy, in view of the involvement of the RPE in uveitis, it is possible that RPE65 is uveitogenic. Because we have cloned the 278 Laboraton/ of Retinal Cell and Molecular Biologi/ cDNA, it will now be possible to overexpress the protein to test this hypothesis. Proposed Course (1) The translational efficiency regulating sequence in the 3' UTR will be characterized, as will the mRNA instabihty region. This wUl provide insights into translational regulation in the outer retina. (2) Analysis of the structure of the human RPE65 gene will be continued. The remainder of the gene wiU be sequenced. Due to its importance as an RPE-specific gene, regulatory regions as well as the promoter will be identi- fied and analyzed. (3) The mouse RPE65 gene will be com- pared with the human RPE65 gene, especially with regard to the promoter region. Trans- geruc and homologous recombination "knock- out" studies win be pursued to understand RPE65 function and regulation as well as to provide potential new models for human retinal disease. (4) RPE65 will be tested as a possible RPE autoantigen. RPE65 protein will be overex- pressed for this purpose. (5) Elucidation of the structure and func- tion of the RPE65 protein will continue. This will involve use of a variety of approaches. We will pay special attention to its possible role in retinoid and /or hpid trafficking. NEI Research Program Retinal Diseases — Photoreceptors and Pigment Epithelium Publications Hamel CP, Jenkins NA, Gilbert DJ, Copeland NJ, Redmond TM: The gene for the retinal pigment epitheUum-specific protein RPE65 is localized to human lp31 and distal mouse 3. Genomics 20:509-512, 1994. Redmond TM, Jenkins NA, Gilbert DJ, Copeland NJ, Hamel CP: The gene for the retinal pigment epithelium-specific protein RPE65 is localized to human lp31 and distal mouse 3. Invest Ophtlialmol Vis Sci 35(suppl):1312, 1994. 279 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00250-07 LRCMB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Molecular Biology of Experimental Autoimmune Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI Molecular Biology Others: Dhirendra Singh Shirley Yu Ph.D. B.S. Visiting Associate Biologist LRCMB, NEI LRCMB, NEI COOPERATING UNITS (if any) Department of Ophthalmology, Miami University, Miami, PL (D. Hamasaki, Ph.D.); Department of Anatomy, Nagoya University School of Medicine, Tsuramai, Showa-ku, Nagoya, Japan (Jiro Usukura, M.D.) Laboratory of Retinal Cell and Molecular Biology SECTION Section on Molecular Biology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 2.5 PROFESSIONAL 1.5 1.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) We have previously determined amino acid sequences of human, mouse, rat, and bovine retinal S-antigen and rat pineal gland S-antigen. Immunogenic sites and four uveitopathogenic sites of S-antigen were also determined. Two of the immunogenic sequences were highly conserved among these species. Many proteins that have a similar sequence with a uveitopathogenic site are in the National Biomedical Research Foundation database. We chemically synthesized many peptides, and some of them induced experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) in Lewis rats. In addition, native yeast histone H3 was also capable of inducing EAU. To understand the role in autoimmunity of infectious micro-organisms that have cross-reactive antigens, we injected Lewis rats with peptide M together with one of six different killed bacteria, either with or without incomplete Freund's adjuvant (IFA). The rats injected with IFA developed EAU. To assess the impact of infection by live micro-organisms, low doses of live E. coli expressing S-antigen and baker's yeast, with a cross-reactive antigen, were injected several times into the rats. The rats injected with either live E. coli or live yeast developed EAU. We conclude that infection by micro- organisms that have cross-reactive antigens can break immune tolerance to self-antigens and induce inflammatory autoimmune diseases. As an extension of our previous EAU research, we speculated that some types of cataracts may be induced by autoimmune insults. To investigate this issue we carried out similar experiments. Three groups of four rats were injected three times with lens homogenate, p-crystallins or a P-crystallin (P-Al) emulsified with complete Freund's adjuvant (CFA). All the animals developed severe damage in lens epithelial cells after 5 weeks from the date of the first injection. The rats injected with a synthetic peptide derived from Salmonella typhimurium protein that has five amino acid residues identical to rat p-crystallin (P-B2) also induced similar damage. Infection of microbes having homologous antigens to the lens antigens can induce the auto-antibodies at high levels that provoke damage to the lens epithelial cells. Thus, autoimmune insult in the lens epithelial cells may be an etiology of an initial stage of cataractogenesis. Direction of our future research will be to focus more on the autoimmunity in the lens cataractogenesis. 280 PHS 6040 (Rev. 5/92) Lahoratoni of Retinal Cell and Molecular Biology Project Description Objectives The objectives of this project are to understand the basic etiology of autoimmune inflamma- tion, including uveitis, and to find possible treatments for human uveitis. Methods Conventional methods for analysis of proteins and nucleic acids are used. These include protein purification, ribonucleic acid (RNA) and deoxyribonucleic acid isolation, character- ization and sequencing, molecular cloning, screening of clones, in situ hybridization, immunocytochemistry, and chromosome mapping. We also synthesized and used oligopeptides and oligonucleotides. Bovine, murine, primate, and human materials are used. Arumal experiments were carried out with Lewis rats and monkeys. T-ceU response and adaptive transfer were done with lymph node or spleen cells of rat. Major Findings (1) Local sequence homology was found between peptide M and several other foreign proteins, including potato proteinase inhibitor Ha, Escherichia coli hjrpothetical protein. Hepatitis B virus probable DNA polymerase, Moloney murine sarcoma virus gag-polypro- tern, Moloney murine leukemia virus Gag-pol polyprotern. Baboon endogenous virus gag-pol polyprotein, and Baker's yeast histone H3. (2) The synthetic peptides of the above- mentioned proteins induced experimental autoimmune uveitis (EAU) in Lewis rats v^dth similar pathology to EAU induced by peptide M or native S-antigen (S-Ag). (3) For the first time, we proposed and showed the evidence that molecular mimicry plays a role in the process of pathogenesis of EAU and perhaps in autoimmune diseases in general. (4) Oral administiation of histone H3 peptide suppressed EAU in the Lewis rats. (5) The suppression of EAU by histone H3 was also found in the EAU induced by the S-Ag. Thus, the tolerance also crossreacted between the peptide that has molecular mim- icry. (6) The T-lymphocytes obtained from rats immunized with peptide M or yeast histone H3 transferred disease, EAU, in the naive rats (adoptive transfer) when stimulated either with peptide M or histone H3. In addition, oral tolerance was also adoptively transferred from rats fed peptide M or histone H3 to the naive rats. (7) Infection by microorganisms that have crossreactive antigens can break immune tolerance to a self-antigen and induce inflam- matory autoimmune diseases. Significance to Biomedical Research and the Program of the institute Uveitis is a leading cause of visual handicap in the United States and throughout the world. Some types of uveitis were suspected to be induced by bacterial and viral infections by many physicians for many decades. How- ever, there is no clear link between infection and disease. Autoimmune processes are thought to play a significant role in the pathogenesis of disease. Molecular mimicry, a process by which an immune response directed against a nonself protein crossreacts with a normal host protein, may play a role in autoimmunity. Here, we have proposed the idea of molecular mimicry and showed evidence that molecular mimicry may play a role in the pathogenesis of EAU. In addition, we have shown evidence that infection is a possible cause of autoim- mune iiiflammation. These findings provide an important clue for understanding the etiology of autoimmune inflammatory diseases in humans. ZSl FY 1994 NEI Annual Report Proposed Course This project will be terminated. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications Li Q, Abe T, Kikuchi T, Nussenblatt RB, Shinohara T, Chan C-C: Corticosteroids enhance S-antigen expression in non-retinal ocular tissue of rats with experimental autoim- mune uveitis. Exp Mol Pathol 60:27-38, 1994. Singh DP, Kikuchi T, Singh VK, Shinohara T: A single amino acid substitution in core resi- dues of S-antigen peptide confers the capacity to prevent experimental autoimmune uveitis (EAU). 7 Immunol 152:4699-4705, 1994. 282 I PROJECT NUMBER DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE ' NOTICE OF INTRAMURAL RESEARCH PROJECT i ZOl EY 00132-13 LRCMB PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Molecular Biolog y of Phototran sduction PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute afniiatlon) PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI Molecular Biology Others: Takanobu Kikuchi Ph.D. Visiting Associate LRCMB, NEI COOPERATING UNITS (if any) Mount Sinai Hospital, Toronto, Canada (Martin Breitman, Ph.D.); Department of Anatomy, Nagoya University School of Medicine, Tsurumai, Showa-Ku, Nagoya, Japan (J. Usukura, M.D.) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology SECTION Section on Molecular Biology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.5 PROFESSIONAL 1.5 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) We have characterized the S-antigen genes from human and mouse, 33K protein genes from mouse and human. The S-antigen genes were approximately 50 kbp in length, contained 16 exons and 15 introns. and were composed of 97 percent intron and 3 percent exon. The 5'-flanking regions of the genes, approximately 1.5 kbp long, had no known regulatory elements for transcription such as TATA, GC, or CCAAT boxes. Regulatory sequences and nuclear factors governing tissue-restricted expression of the mouse arrestin gene were investigated. The results showed that while proximal promoter sequence -38 to +304 are sufficient to direct low levels of retina-specific gene expression, sequences extending upstream to position -209 support higher levels of expression in the retina, as well as detectable expression in the lens, pineal gland, and brain. Within the interval between positions -205 and -185 is a region that contains two direct repeats of the hexamer, TGACCT. The proximal promoter binds three apparently retina-specific nuclear factors, Bpl, Bp2, and Bp3, through overlapping sequences centered between positions -25 and -15. Bpl and Bp3 also recognize a closely related sequence found in the promoter regions of several other vertebrate photoreceptor-specific genes. Moreover, the consensus binding site for Bpl, designated PCEl, is identical to RCSl, an element known to play a critical role in eliciting photoreceptor-specific gene expression in Drosophila melanogaster . The results suggest that PCEl and RCSl are functionally, as well as structurally, similar and, that despite marked differences in the fly and vertebrate visual system, the transcriptional machiner\' involved in photoreceptor-specific gene expression has been strongly evolutionarily conserved. 283 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives The objective of this project is to understand the basic mechanism of phototransduction in the retina and to understand the structure, function, and evolution of the proteins present in photoreceptor rod cells and pinealocytes. Methods Conventional methods for analysis of proteins and nucleic acids are being used. These include protein purification, ribonucleic acid and deoxyribonucleic acid (DNA) isolation, characterization, and sequence determination. Various recombinant DNA techniques are also being used, including a Baculovirus expres- sion vector system, S5mthesis of point muta- tion clones, characterization of promoters and transgenic animals. We have also sjnithesized and used purified oligopeptides and oligo- nucleotides. Major Findings (1) The gene sequences of S-antigen (S-Ag) from human and mouse were determined. It is 50 Kbp in length and has 15 introns and 16 exons. The smallest exon encodes for three amino acids. (2) The intron-exon map sequence of the mouse S-Ag gene has been well conserved. Approximately 97 percent of the S-Ag gene is intron, and 3 percent is exon. (3) The human and mouse S-Ag comple- mentary DNAs (cDNA) have been subcloned into two expression vectors and have been ex- pressed. The products of S-Ag cDNA were purified by column chromatography and were prepared for crystallization. (4) The 5'-flanking sequence of the human and mouse S-Ag genes were determined and demonstrated promoter activity in the in vivo and in vitro transcriptional assays. (5) Although the S-Ag promoter sequenc- es are highly conserved between human and mouse, promoter activity was found at differ- ent locations of the 5'-flanking region in the human and mouse genes. This result suggests that the promoter activity is highly specific to tissues and species. (6) The mouse S-Ag promoter, 1,300 bp in length, was fused with the chloramphenicol acetyl transferase (CAT) gene, and this gene was introduced into transgenic mice. The transgenic animals expressed CAT activity only in the retina and pineal gland. This result indicates that the promoters have a tissue-spedfic enhancer and promoter activity. (7) The opsin promoter was fused with a diphtheria toxin gene. This fusion gene was introduced into transgenic mice, which subse- quentiy lost only the photoreceptor rod cell layer. (8) Several cDNAs of Shuzin, a retinal photoreceptor protein, were isolated from human and cow retinal cDNA libraries (lambda-gtU). The entire DNA sequences were determined. The deduced protein has sequence similarity with TFIID. Its gene was also isolated from a genomic library, and the DNA sequence was determined. It is com- prised of two introns and three exons. (9) Two genes of 33 kDa ROS-specific proteins have been isolated from the retinal libraries of human and mouse. The entire DNA sequence of these genes has been de- termined, and they have four exons and three introns. (10) The proximal promoter sequence positions -38 to +304 are sufficient to direct low levels of retina-specific gene expression. (11) The proximal promoter binds three retinal-specific nuclear factors, Bpl, Bp2, and Bp3, through overlapping sequences cen- tered between posifions -25 and -15. 284 Lahoraton/ of Retinal Cell and Molecular Biology (12) The distal promoter sequence posi- tions -205 to -185 is a region that contains two direct repeats of the hexamer TGACCT. (13) We found a consensus retinal pho- toreceptor-specific site (PCEl). (14) The transcriptional machinery in- volved in photoreceptor-specific gene ex- pression has been strongly and evolutionar- ily conserved. Significance to Biomedical Research and the Program of the Institute Eyes have remarkable properties in function- ing efficiently over a wide range of illumina- tions. Rod cells having photosensitive rho- dopsin are more sensitive to dim Hght and adapt in the dark to increase their sensitivity. However, rod cells cease their sensitive photo- transduction in bright Ught. Cone ceUs in contrast do not operate in dim Ught but are operative in bright Ught. Rhodopsin, trans- ducin, phosphodiesterase, rhodopsin kinase, and S-Ag have been known to be associated with the phototransduction cascade. Rhodop- sin kinase and S-Ag are considered to be the important proteins for Ught-dependent modu- lation of phototransduction. To understand this Ught-dependent modulatory mechanism in rod outer segments, we have characterized S-Ag, Shuzin, and 33 K protein as well as their genes. Interestingly, other signal transduction systems have cascades similar to that of phototransduction (one of the best character- ized receptor-mediated signal transduction processes). In the phototransduction cascade, the shutoff mechanism appears to be modu- lated by the phosphorylation and dephos- phorylation of rhodopsin. Studying this modulation mechanism is important for un- derstanding phototransduction as weU as for imderstanding signal transduction in general. In addition, we think that the night blindness of vision may in part be associated with Ught adaptation. Proposed Course This project will be terminated. NEI Research Program Retinal Diseases — Photoreceptors and Pigment EpitheUum Publications Abe T, Kikuchi T, Chang T, Shinohara T: The sequence of the mouse phosducin gene and its 5'-flanking region. Gene 133:179-186, 1993. Abe T, Kikuchi T, Shinohara T: The sequence of the human phosducin gene and its 5' flank- ing region. Genomic 19:369-372, 1994. Shinohara T, Kikuchi T, Tsuda M, Yamaki K: A family of retinal S-antigens (arrestins) and their genes: Comparative analyses of human, mouse, rat, bovine, and Drosophila. Comp Biochem Physiol 103:505-509, 1993. Usukura J, Khoo W, Abe T, Shinohara T, Breitman M: Cone ceUs fail to develop nor- maUy in transgenic mice ablated of rod pho- toreceptor ceUs. Tissue Cell 275:79-90, 1994. 1S5 Laboratory of Sensorimotor Research Report of the Chief Laboratory of Sensorimotor Research Robert H. Wurtz, Ph.D. Research in the Laboratory of Sensorimo- tor Research concentrated on the con- trol of movement by the visual system. These projects covered a range of topics ex- tending from visual processing to the control of specific eye movements. My group has studied how visual motion filling the entire visual field (optic flow) might be organized to modify the movement of observers as they move through the visual world. Dr. Lance Optican looked at an earUer step in the visual pathway and investigated how visual informa- tion might be encoded in the patterning of neuronal impulses in visual cortex. Dr. Op- tican also developed a model of the control of rapid or saccadic eye movements based on recent work done in the laboratory on a brain- stem structure, the superior colliculus (SC). Dr. David Lee Robinson investigated how these eye movements could be modified by changing activity in the coUiculus during the generation of the eye movements. Dr. Michael E. Goldberg also studied saccadic eye move- ments but in relation to the question of how our spatial stability is maintained in spite of the eye movements that move our fixation from part of the field to another. Dr. Freder- ick A. Miles concentrated on a very different type of eye movement, the vergence eye movement, that allows us to fixate on objects at varying distances from us. A summary of each of the projects is presented below. Section on Visual Motor Integration WhUe moving through the environment, the visual world streams past observers in a pattern depending on their movement. These optic-flow fields provide visual information that can guide self-motion, stabilize posture, and reveal the structure of the environment. My group has studied neurons in the dorsal region of the medial superior temporal area (MSTd) of monkey extrastriate cortex that previously have been shown to respond to the expanding radial motion that occurs with forward movement of an observer. Different directions of observer motion are accompanied by centers of motion located in different areas of the visual field, and, in our current experi- ments, we tested to see whether MSTd neu- rons responded best when centers of motion were located in different parts of the field. About 90 percent of the neurons studied responded differentiy to at least one stimulus with a shifted center of motion as compared with those positioned in the middle of the visual field. The preferred centers of motion were always limited to one area of the visual field for a given cell, and all parts of the visual field were represented. We found that many cells prefer a center of motion in the middle of the field, and those cells respond to motion over a more limited region of the field. 289 FY 1994 NEI Annual Report Using these experiments as a basis, we suggest that each of the MSTd neurons has a center of motion field with a gradient of preferred centers of motion and that there is an orderly arrangement of these neurons, with each region of the visual field being represent- ed by a set of neurons. The responses of individual neurons would be graded accord- ing to proximity of the center of motion of a stimulus to the preferred center of motion for the neuron, just as other visual cortical neu- rons have gradients for the direction of planar motion or for the location of a spot of light. The role of MSTd neurons in interpreting the optic flow fields would not be one of qualita- tive feature matching but rather one of re- sponding to visual motion according to the degree of match between the visual input and the preferred optic flow field of the neuron. These MSTd neurons could contribute to the determination of heading of observers moving through the environment. The organization of this area might also be relevant to the control of posture because the influence of full-field visual motion on posture is well recognized. Section on Neural Modeling In the past year. Dr. Optican's section has worked on two distinct problems. The first problem was how the brain represented visual information. The second problem was how the SC helped control saccadic eye movements. Neuronal Encoding of Visual Parameters information about form and color is grouped into separate "channels" in cortex but rather suggests that all neurons participate in visual processing irrespective of the type of visual parameter involved. Control of Two-Dimensional Saccadic Eye Movements Recenfly, Drs. Douglas Munoz and Robert H. Wurtz identified three types of neurons (fixa- tion, burst, and buildup) in the SC that were involved in controUing saccadic eye move- ments. During a saccade, activity seems to spread through the buildup neurons. Classic models, which control saccades in one dimen- sion, have a resettable integrator within a local feedback loop. Dr. Optican has proposed a new, two-dimensional model of the saccadic system that places the SC inside the local feedback loop. His principal new hj^othesis is that the spread of activity in the buildup neurons represents the resettable integrator of the local feedback loop. One complication in this model is that the SC is not mapped in cartesian coordinates. Rather, there is a retinotopic map in polar coordinates that is warped logarithmically with eccentricity. Because polar coordinates do not follow the rules of vector addition, it is not simple to figure out how the activity in the buildup neurons should spread during the saccade to represent accurately the resettable integrator. Dr. Optican has shown that a vector field can provide the appropriate motor updates for the buildup neurons. Dr. Optican and his colleagues used a visual discrimination task to study the encoding of color and form information in cortical neu- rons. It has been proposed that color and form information is divided into separate channels {e.g., cytochrome oxidase blob and interblob regions) in the cortex. A cluster method of information calculation was devel- oped that made it possible to show that infor- mation about color and pattern rises over time in all neurons in cortical areas V1-V4. Such a result is not consistent with the idea that Section on Visual Behavior Within the Section on Visual Behavior, Dr. David Lee Robinson and his colleagues have been studjdng the changes within the oculo- motor pathways during the course of saccadic eye movements. Most models of the oculomo- tor system hypothesize that the signal for retinal error undergoes an integration before its effect on the oculomotor neurons. Our recent models have proposed that this integra- 290 Lahoratoiif of Sensorimotor Research tion actually takes place within the SC. By electrically stimulating the colliculus before and during eye movements. Dr. Robinson's group members have provided data that support this idea. They have demonstrated that stimulation at any time during periods of fixation leads to rather stereotyped eye move- ments; these have been termed fixed vector saccades because they are constant in terms of direction and amplitude. In contrast, when the colliculus was stimu- lated during the course of a saccade, the direc- tions of these evoked saccades were systemati- cally shifted. Just as the eye began to move, the movement evoked by electrical stimulation was rotated in a direction opposite to the primary saccade by as much as 80 degrees. Excitation applied at progressively later times led to progressively smaller shifts. These data are of considerable importance in understand- ing the neural contiol of saccadic eye move- ments and specifying the collicular contribu- tions to them. Section on Neuro- Ophthalamologic Mechanisms Dr. Goldberg and his collaborators have studied neurons in the lateral intraparietal that have visual responses. The visual responses are sometimes predictive: they occur before a saccade that will bring the spatial location of a visual target into the receptive field of the neuron. There are two possibilities to explain the phenomenon: In one, there is a general increase in retinal excitability, basically an expansion of the retinal receptive field; in the other, the receptive field moves on the retina, like a spotUght of excitability. To distinguish between these two hypoth- eses, stimuli were flashed in the presaccadic receptive field or the postsaccadic receptive field at various times before and after the saccade. When a monkey is programming a saccade, neuronal excitability at the old recep- tive field decreases. Just before the saccade, many cells are unresponsive to visual stimuU that would be expected to drive them during normal fixation. At the same time, stimuli that would be expected to drive the neuron after the saccade drive the neuron before the saccade. This result suggests that the locus of excitability moves like a spotlight across the retina, so that the spatial location that will excite the cell after the movement excites it before the movement. The disappearance of excitability ftom the retinal location that will excite the neuron after the saccade prevents the cell from providing an inaccurate message after the saccade and enables spatially accu- rate processing across saccades. Monkeys with deep cerebellar nuclear lesions have a battery of deficits in their oculomotor performance. The crude dynamics of their saccadic eye movements are normal; the main sequence of saccade peak velocity versus ampUtude is normal, but the accuracy of visually guided saccades is diminished. Monkeys make larger saccades than normal, and this deficit is a function of initial orbital position. Normal monkeys can adjust the ampUtude of saccades for target motion, but after deep cerebellar lesions monkeys do not compensate for target motion. They therefore relatively overshoot centripetally moving tar- gets and relatively undershoot centrifu gaily moving targets. Normal monkeys can change the amplitude of their saccadic eye move- ments in response to intrasaccadic target steps; postiesion monkeys cannot. Finally, the variability of the amplitude of visually guided eye saccades is increased in the lesioned monkey. These data all suggest that the role of the cerebellum in the generation of saccadic eye movements is to fine-tune the transforma- tion from visual stimulus to movement, a fine-tuning that requires an ongoing monitor- ing of the saccadic motor signal during the saccade. Three patients with the unusual finding of torsional nystagmus during vertical pursuit were examined. Magnetic resonance imaging studies in these patients show small arteriove- nous malformations in the region of the mid- dle cerebellar peduncle. Eye movements recorded in one patient demonstrate torsional 191 FY 1994 NEI Annual Report nystagmus that scales linearly in velocity with increasing vertical pursuit velocity and is not present during saccades, horizontal pursuit, or horizontal vestibulo-ocular reflex cancellation. The torsional velocity is decreased when the patient pursues an afterimage target, a case where there is no retinal sUp signal present. These findings suggest that the problem in these patients is in the visual-to-motor-signal coordinate transformation. The linear relation- ship between torsional and vertical pursuit velocity suggests that these findings arise fi:om an inappropriate cross-coupling between vertical visual (or motor) signals, which are used in vertical pursuit, and the torsional eye movement system. Section on Oculomotor Control Using monkeys. Dr. Miles and his collabora- tors have demonstrated that the vergence eye movements resulting from sudden changes in the binocular disparity of large textured scenes have almost machine-Uke consistency and a latency of 52-53 msec, which is less than one-third of the value commonly cited in the Uterature (which was obtained with small targets). They suggest that these disparity vergence responses are mediated by neurons that have binocular receptive fields that are spatially selective (in terms of size, shape, and orientation) and offer new insights into the properties of such (cortical) neurons. These short-latency-vergence eye movements were sfrongly affected by an antecedent saccadic eye movement, whereby disparity steps ap- pUed in the immediate wake of a saccade were much more effective than identical steps delivered only 200 milliseconds later: fran- sient postsaccadic enhancement. This postsac- cadic enhancement should normally help to speed binocular realignment when gaze is redirected to (large?) objects in different depth planes. 292 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00045-16 LSR PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Visuomotor Properties of Neurons in th e Tha lamus PRINCIPAL INVESTIGATOR (List ottier professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: David Lee Robinson Ph.D. Section Chief LSR, NEI Others: Alexander A. Kustov Ph.D. Visiting Fellow LSR, NEI COOPERATING UNITS (if any) Department of Anatomy, Howard University (Robert J. Cowie, Ph.D.) LAB/BRANCH Laboratory of Sensorimotor Research SECTION Visual Behavior Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.3 PROFESSIONAL: 2.0 1.3 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) With each movement of the eyes, there are changes in the state of the neural processes that generate eye movements and changes in the perceptual frame of reference. We have electrically stimulated the superior coUiculus at various times around visually guided eye movements to understand the collicular contribution to these changes. When the colliculus is stimulated during periods of fixation, the amplitude and direction of the saccadic eye movement is rather constant. When the same stimulation is applied while an eye movement is in progress, there is a systematic shift in the direction of the evoked eye movement. Just as the eye begins to move toward a visual target, the eye movement evoked by stimulation of the coUiculus is rotated in a direction opposite to the primary visual saccade by as much as 80 degrees. Electrical stimulation that is applied at progressively later times during the visually guided eye movement will evoke eye movements with progressively less shift. These data suggest that the colliculus participates in the changes in spatial reference during eye movements or that there is a gradual decay in the integration process that the colliculus provides to the oculomotor system. 293 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives Within the oculomotor pathways, there are changes before, during, and after each move- ment of the eyes. These are related to the underl5dng neural processes that generate the movement, and they are related to the plan- ning that must take place before the next eye movement. It has been known for a number of years that the superior coUiculus (SC) plays an important role in the generation of saccadic eye movements. The present studies were conducted to understand how the colliculus relates to the preparation for saccadic eye movements and the compensation for neural computations after their initiation. Methods To localize specific regions of the brain and reliably test these sites, adult monkeys w^ere trained to enter a primate chair, sit quietly, and fixate on a spot of light. They also learned to make rapid, saccadic eye move- ments when the fixation spot was turned off and another Hght appeared. The timing between these two events was varied to change the temporal properties of the eye movements and also modify the direction of the animal's attention. After the animals were adapted to these situations, they were im- planted during sterile surgery with several devices for recording eye movements, electri- cal activity within the brain, and instruments for head restraint. Once the monkeys had recovered from the surgery, we positioned fine wire electrodes into specific brain sites, recorded the discharge patterns of individual cells, and electrically excited these loci. The timing of the electrical stimulation was varied in relation to the onset of target Ughts as well as the time course of the evoked saccadic eye movements. The evoked eye movements and other behavioral responses were recorded and quantified. Major Findings As the oculomotor system plans and generates each eye movement, there are many changes within its pathways and those systems that begin to calculate the next saccade. We want- ed to learn how the SC interacted with these changing requirements for saccade program- ming. When an electrode enters the superficial layers of the SC, the first neurons encountered are very visually responsive. These neurons have relatively small visual receptive fields that have a fixed position relative to the fovea. The location of the receptive field moves with the direction of the eye. At these dorsal sites within the coUiculus, the threshold for evoking saccadic eye movements by electrical stimula- tion is at its highest (100 /xA). If stimulation of these cells occurs while the monkey fixates a central target, then the evoked eye move- ment has a fixed vector; the eye moves in a set direction with a set amplitude. If the stimulation is appUed while the monkey fixates targets in different locations, then there is a slight effect of the initial eye position on the evoked eye movement. Eye movements that start on the side of the stimulating elec- trode are slightly larger than those that start opposite the stimtilation site. Next, we wanted to learn if the planning or execution of an eye movement would alter the movement evoked by electrical stimula- tion. If the same site were stimulated during fixation, after the disappearance of the fixation point, or shortly after the appearance of the eye movement target, then the same, fixed vector saccade was also elicited. Eye move- ments that were evoked by stimulation just before the visually guided saccade had a sUght shift in their evoked direction. If the electrical stimulation was applied during the course of a visually guided eye movement, then the threshold (120 fiA) and latency were in- creased. The characteristics of these evoked saccades were also modified but wiU be con- sidered in a later section. 294 Laboratory of Sensorimotor Research As we advanced the microelectrode deep- er within the SC, we reached a transition point where the neurons began to respond with a brisk burst of activity that was before and time-locked with the saccadic eye movement. The amount of visual excitability of these neurons was less than for the cells above them. When we electrically stimulated at the sites of these cells, the threshold was much less (20 fiA). In addition, the latency of the evoked eye movements was less; they began at least 15 milliseconds (msec) sooner. Just as for the more dorsal sites, the eye movements evoked during periods of fixation had fixed vectors, and there was the same subtie influ- ence of irutial eye position. Stimulation ap- plied at points long before the visually guided saccade evoked the same reproducible eye movement. Eye movements that were evoked by stimulation just before the visually guided saccade to a contralateral target had a sHght shift in direction. When we stimvilated the coUiculus at the sites of eye movement cells during the course of an eye movement, the threshold did not increase as it had at the more dorsal, visual sites. Also, the latencies of the movements were shorter. The directions of these eye movements shifted in a systematic fashion. At the instant of the start of the eye movement to a visual target, the evoked eye movements had a direction that was shifted from the fixed vector by as much as 90 degrees; the shifted direction was opposite to that of the primary visual saccade. As the time of stimulation occurred later and later during the visually guided saccade, the amount of rotation from the fixed vector systematically diminished. The final position of these electrically evoked saccades was on a line parallel to the plane of the visually guided eye movements. The time period during which the evoked saccades were shifted lasted about 80 msec longer than the duration of the executed visually guided saccade. Thus, the absolute duration of the whole process could last as long as 120 msec. As we advanced the microelectrode still deeper, we encountered neurons that had more prolonged activity between the appear- ance of the saccade target and the saccade to acquire it. Electrical stimulation here evoked fixed vector saccades during periods of fixa- tion, shght influences due to initial position, latencies and thresholds similar to the sites just above, and systematic shifts in the direc- tion of movements when they were evoked during the course of the eye movement. These data show that the eye movements elicited by electiical stimulation are systemati- cally influenced by the preparation and occur- rence of eye movements. There are two possible mechanisms for these effects. The direction of gaze changes with each eye move- ment, and the visual scene is also modified; thus, there is the need to establish a new frame of reference. The systematic shift in evoked eye movements may reflect this slower change in the oculomotor frame of reference. Certain studies of spatial localization during the course of saccadic eye movement provide perceptual data to support this interpretation. Alternatively, the systematic shift may reflect the resetting of the neural integrator. In most models of the oculomotor system, it has been hypothesized that the signal for the retinal error is integrated before its effect on the motoneurons of the eye muscles. After this integration has taken place, the neural ele- ments responsible will take some time to return to their state of rest. Recent data suggest that some neurons within the SC participate in the integration; the systematic shift in electrically evoked saccades may reflect the resetting of the integrative functions of these neurons. Significance to Biomedical Research and the Program of the Institute One of the major goals of system neuroscience is to understand how the brain controls move- ment. A thorough understanding of this function of the brain is necessary for any attempts to rehabilitate patients with cential nervous system lesions. Considerable prog- ress has been made in this area from studies of the control of the oculomotor system. The SC is known to be a major participant in the transformation of visual signals to the oculo- 295 FY 1994 NEI Annual Report motor system. The ideas presented here help to clarify the types of signals that the collicu- lus sends to the oculomotor system and the impact of these signals on the control of saccadic eye movements. Proposed Course The most studied aspect of the SC is the presaccadic discharge of neurons within its intermediate layers. Most modeling of this activity describes descending SC projections to preociilomotor centers. However, previous anatomical data have shown that this region also projects to the thalamus. Preliminary data obtained whUe extending our study of coUicular control of eye movement have demonstrated that the region of the thalamus that receives a projection from the coUicular burst neurons contains cells that discharge before eye movements. Our future studies will involve extensive recording from these neurons, characterizing their discharge pat- terns, testing the effects of microstimulation of these ceUs, and evaluating the effects of local- ized inactivations of this region. NEI Research Program Strabismus, Amblyopia, and Visual Process- ing — Visual Processing and Functional Organi- zation, Structure and Function of Central Visual Pathways Publications Cowie RJ, Robinson DL: Head and eye move- ments eUcited from the superior coUiculus of the macaque. Soc Neurosci Abstr 18:788, 1993. Cowie RJ, Robinson DL: Subcortical contribu- tions to head movements in macaques. I. Contrasting effects of electrical stimulation of a medial pontomeduUary region and the superior coUiculus. / Neurophysiol, in press. Cowie RJ, Smith MK, Robinson DL: Subcorti- cal contributions to head movements in ma- caques, n. Connections of a medial ponto- meduUary head-movement region. / Neuro- physiol, in press. Kustov AA, Robinson DL: Changes in sac- cade trajectory from stimulation of the coUicu- lus of the macaque. Soc Neurosci Abstr 20(1):141. Robinson DL, Bowman EM, Kertzman C: Covert orienting of attention in macaques. E. Contributions of parietal cortex. / Neurophy- siol, in press. Robinson DL, Cowie RJ: Attentional engage- ment and the pulvinar. Behav Brain Sci 16:586-587, 1993. Robinson DL, Cowie RJ: Influences of subcor- tical centers on head movements, in Buttner U, Brandt T, Fuchs A, Zee D (eds): Contempo- rary Ocular Motor and Vestibular Research: A Tribute to David A. Robinson. International Meeting Eibsee, 1993. Stuttgart, New York, Georg Thieme Verlag and New York, Thieme Medical PubUshers. Robinson DL, Cowie, RJ: Visual contributions of the pulvinar, in McCormick DA, Jones EG, Steriade M (eds): Thalamus. Elsevier Science Ltd., in press. Robinson DL, Kertzman C: Covert orienting of attention in macaques. HI. Contributions of the SC. / Neurophysiol, in press. 296 -DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00049-16 LSR PERIOD COVERED October 1, 1993 to September 30, 1994 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Cerebral Cortical Mech anisms foi^e MovemeDts and Visual Attention PRINCIPAL INVESTIGATOR (List other professional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Michael E. Goldberg M.D. Senior Medical Research LSR, NET Officer Others: Edmond J. FitzGibbon M.D. Medical Officer Carol L. Colby Ph.D. Senior Staff Fellow Makoto Kusunoki Ph.D. Visiting Fellow Marc Umeno M.A. pre IRTA LSR, NEI LSR, NEI LSR, NEI LSR, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Sensorimotor Research SECTION Neuro-Ophthalmologic Mechanisms Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 5.2 PROFESSIONAL 4.0 1.2 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Three lines of inquiry were followed to determine how the cerebral cortex and its efferent regions control eye movements and visuospatial attention. In the first single, a neuron recording was used to probe the mechanisms whereby the parietal cortex of the monkey achieves spatial accuracy. It is well known that parietal neurons respond to stimuli in a certain location on the retina, the receptive field. The receptive fields of parietal neurons transiently change before a saccadic eye movement, so that they respond, before the eye movement, to stimuli that will be in their receptive fields after the movement. At the same time, the current retinal location becomes unresponsive. These data establish that there is a specific shift of retinal receptive field, rather than a general change of excitability. In the second, the oculomotor performance of rhesus monkeys was assessed while the monkeys performed an oculomotor task before and after electrolytic lesions of the deep cerebellar nuclei. A number of deficits were found that were consistent with the cerebellum playing a role in adjusting the amplitude of saccadic eye movements but not in initiating them: monkeys had increased variability in the amplitude of their saccadic eye movements, they had tended to overshoot targets and this inaccuracy was dependent upon the initial position of the eye in the orbit; they were unable to adjust the amplitude of their saccadic eye movements for target motion; and they could not adjust the amplitude of their saccadic eye movements in a model of muscle weakness. In the third, the performance of humans with superior cerebellar peduncle lesions was studied in a smooth pursuit task. Cross-coupling between the torsional and vertical pursuit systems was demonstrated. 297 PHS 6040 (Rev. 5/92) FY 1994 NEI Annual Report Project Description Objectives This section has concentrated on three aspects of the physiology and phenomenology of higher visual and oculomotor processing in monkey and human, especially as these func- tions relate to the parietal and frontal regions of the cerebral cortex, their afferent regions, and their efferent targets. Previous work in this section has shown that neurons in the parietal cortex discharge in response to visual stimuU and before saccadic eye movements. The neurons contain a predictive aspect to their visual responses: neurons respond to stimuli whose spatial location wiU be brought into their visual receptive fields by impending saccadic eye movements. Work in the labora- tory for this period has concentrated on un- derstanding the mechanism of this predictive response by studying the time course of changes in retinal excitability before and after saccadic eye movements. Although the cerebellum has been known to participate in the guidance of saccade eye movements, its exact contribution has not been clear. To estabUsh the role of the cere- bellum in the guidance of saccadic eye move- ments, and to chart a path for future research, comprehensive lesions were made in the deep cerebellar nuclei of rhesus monkeys; the per- formance of these monkeys was studied in a number of tasks before and after the lesions. Human disease can often provide insights into normal processes. In the course of evalu- ating oculomotor performance in patients with neurological disease, three patients were discovered with torsional nystagmus on up- ward smooth pursuit. AU three had lesions of the superior cerebellar peduncle. The magnet- ic search coil technique was used to study their eye movements quantitatively. Methods Monkeys were implanted with magnetic search coils for the measurement of eye posi- tion, along with devices for temporary re- straint and electrophysiological recording and stimulation. They were trained to perform a number of visuomotor tasks, including fixa- tion, saccades, and smooth pursuit. Microelec- trodes were placed in the lateral intiaparietal area, and single neurons were studied while the monkey performed various visuomotor tasks. In a second set of experiments, micro- electrodes were placed in the deep cerebellar nuclei of the monkeys, neurons recorded, and saccades evoked by electrical stimulation. Electrolytic lesions were made by passing current through the recording microelectrodes until saccades could no longer be evoked from the area. Oculomotor performance was evalu- ated after the lesion, using the same battery of tasks for which normal data had been estab- lished before the lesion. Magnetic search coil limbic rings were placed on one patient with torsional nystag- mus during vertical smooth purstiit, and his eye movements were measured while he performed pursuit eye movements at several velocities. Major Findings Neurons in the lateral intiaparietal have visual responses. The visual responses are some- times predictive: they occur before a saccade that wiU bring the spatial location of a visual target into the receptive field of the neuron. There are two possibilities to explain the phenomenon: in one, there is a general in- crease in retinal excitability, basically an expansion of the retinal receptive field; in the other, the receptive field moves on the retina, like a spotlight of excitability. To distinguish betw^een these two hypotheses, stimuli were flashed in the presaccadic receptive field or the postsaccadic receptive field at various times before and after the saccade. When a monkey is programming a sac- cade, neuronal excitability at the old receptive field decreases. Just before the saccade, many cells are unresponsive to visual stimuli that would be expected to drive them during normal fixation. At the same time, stimuli 298 Laboratory of Sensorimotor Research that were expected to drive the neuron after the saccade drive the neuron before the sac- cade. This result suggests that the locus of excitabihty moves like a spotUght across the retina, so that the spatial location that will excite the cell after the movement excites it before the movement. The disappearance of excitability from the retinal location that will excite the neuron after the saccade prevents the cell from providing an inaccurate message after the saccade and enables spatially accu- rate processing across saccades. Monkeys with deep cerebellar nuclear lesions have a battery of deficits in their oculomotor performance. The crude dynamics of their saccadic eye movements are normal; the main sequence of saccade peak velocity versus amplitude is normal, but the accuracy of visually guided saccades is diminished: morJ