Brain glucose utilization in mice with a targeted mutation in the thyroid hormone a or 6 receptor gene Yoshiaki Itoh*, Takanori Esaki*, Masahiro Kaneshige', Hideyo Suzuki', Michelle Cook*, Louis Sokoloff**, Sheue-Yann Cheng", and Jacques Nunez* *Laboratory of Cerebral Metabolism, National Institute of Mental Health, and *Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4030 Contributed by Louis Sokoloff, June 22, 2001 Brain glucose utilization is markedly depressed in adult rats made cretinous after birth. To ascertain which subtype of thyroid hor- mone (TH) receptors, TRa1 or TRB, is involved in the regulation of glucose utilization during brain development, we used the 2-[*4C]deoxyglucose method in mice with a mutation in either their TRa or TRB gene. A C insertion produced a frameshift mutation in their carboxyl terminus. These mutants lacked TH binding and transactivation activities and exhibited potent dominant negative activity. Glucose utilization in the homozygous TRBPV mutant mice and their wild-type siblings was almost identical in 19 brain regions, whereas it was markedly reduced in all brain regions of the heterozygous TRe1PV mice. These suggest that the a1 receptor mediates the TH effects in brain. Inasmuch as local cerebral glucose utilization is closely related to local synaptic activity, we also examined which thyroid hormone receptor is involved in the expression of synaptotagmin-related gene 1 (Srg1), a TH-positively regulated gene involved in the formation and function of synapses [Thompson, C. C. (1996) J. Neurosci. 16, 7832-7840]. Northern analysis showed that Srg1 expression was markedly reduced in the cerebellum of TRa?Y/+ mice but not TRBPY/PY mice. These results show that the same receptor, TRa‘1, is involved in the regulation by TH of both glucose utilization and Srg1 expression. cerebral glucose utilization | 2-['4C]deoxyglucose | cretinism | synapse Tes hormone is essential for normal postnatal growth and development of the nervous system (1-6). Neonatal hypo- thyroidism impairs development of virtually all tissues, but the most prominent deficits are observed in brain. Neuronal differ- entiation and axonal and dendritic outgrowth are delayed, and myelination is reduced. These morphological changes are ac- companied by deficits in electrical, behavioral, and cognitive functions. In cretinism, for example, learning and memory are severely impaired. In rats, this array of functional deficits is, at least, partially prevented if thyroid replacement therapy is performed within the first 3-4 weeks of life. Maturation of structure and function in the mammalian brain is normally accompanied by profound increases in local rates of glucose utilization (7, 8), but in rats made cretinous by radio- thyroidectomy within the first 2 days after birth and studied later in adulthood, local cerebral glucose utilization (CMRgic) in all brain regions examined was found to be depressed below values in euthyroid controls by 28-58% (9). The greatest decreases were found in the cerebral cortex and throughout the auditory system. Lesser changes were seen in hypothalamic regions, including those involved in the synthesis of thyrotrophin- releasing hormone (TRH). Numerous applications of the 2-[!4C]deoxyglucose method (10) have demonstrated a close correlation between local neural functional activity and local CMR, (11). The findings in cretinism suggest, therefore, an association between the structural, functional, and biochemical abnormalities and the widespread reductions in energy metab- olism throughout the brain. www.pnas.org/cgi/doi/10.1073/pnas.171319498 The action of the thyroid hormone, L-triiodothyronine (T3), is mediated by thyroid hormone receptors (TRs), which are ligand-dependent transcription factors (12, 13). Three ligand- activated nuclear TR isoforms have been identified, TRel, TRBI1, and TR£2, which are produced by alternative splicing of the primary transcripts of the TRe and TRB genes, respectively. Recently, TR83 and TRAB3 isoforms have been identified but have limited expression in the brain (14). Another spliced product of the TRa gene, TRa2 (15), resembles the viral v-erbA oncogene (16) because both molecules do not bind to thyroid hormone. Each TR form has a unique developmental and tissue-specific expression (17, 18). They bind to specific DNA sequences known as thyroid hormone response elements (TRE) that are located in the promoter regions of the T3 target genes (19). A TRE has also been found in the first intron of the rat RC3/neurogranin gene (20). The TR-T3 complex binds to the TRE together with other coregulatory proteins that function as histone acetyltrans- ferases and serve to remodel the chromatin structure and activate gene transcription (21, 22). In the absence of thyroid hormone, TR binds to TRE together with a complex of inhib- itory coregulatory proteins that promote chromatin deacetyla- tion and inhibition of gene transcription (21, 22). The TR- hormone complex activates most genes, although there are other genes, ¢.g., the thyroid-stimulating hormone (TSH) gene, that are repressed. Thus, the transcriptional activities of TR depend not only on the presence of thyroid hormone but also on the specific TRE involved. TRs bind to TRE as homodimers, TRa/TRB heterodimers, or heterodimers with other members of the receptor superfamily (17). Recently, two types of TR mutated mice, i.e., “knockout” and “knock-in,” have been developed. In the knockout mice, TR genes are inactivated, and no receptor proteins are produced (23). In contrast, in the knock-in TRSPV mutant mice, TRBPV is produced, which is a protein that has totally lost T3-binding and transcriptional activities. TRBPV mutant mice were gener- ated by a targeted mutation in the last 14 amino acids of the TRB gene (24). The TRB knockout mice show increased TSH and thyroid hormone production and impaired hearing (23). The phenotype of the TRBPV knock-in mice is consistent with that seen in patients with thyroid hormone resistance syndrome (24-26); they exhibit severe dysfunction of the pituitary-thyroid axis, impaired hearing, retarded growth, delayed bone matura- tion, and abnormal patterns in the expression of T3-targeted Abbreviations: T3, L-triiodothyronine; TH, thyroid hormone; TR, thyroid hormone receptor; TSH, thyroid-stimulating hormone; TRE, thyroid hormone response element; TRH, thyro- trophin-releasing hormone; Srg1, synaptotagmin-related gene 1; CMRgic, cerebral glucose utilization. +To whom reprint requests should be addressed. E-mail: louis@shiloh.nimh.nih.gov. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. PNAS | August 14,2001 | vol.98 | no.17 | 9913-9918 > iC) i) ml 2 fa i=) [4 —] Ww rad genes (24). More recently, mice with the same PV mutation targeted in the corresponding position of the TRa gene have been generated (TRalPV mice). The phenotype of the het- erozygous TRalPV mice is clearly distinct from that of TRBPV mice and is characterized by dwarfism, increased mortality, and reduced fertility (unpublished data). Inasmuch as structural and functional maturation and energy metabolism have been found to be markedly impaired when TH deficiency is established at early postnatal ages (9), we have examined which TR isotype, TRal or TR8, mediates the de- velopmental effects of TH on brain glucose utilization. Materials and Methods Generation and Characterization of Mutant Mice. The method for generating the TRBPV mutant mice has been described in detail elsewhere (24). The TRB?" gene, which was originally found in a thyroid hormone-resistant patient, has a C insertion at codon 448 in exon 10, resulting in a frameshift of the carboxyl-terminal 14 amino acids of TRB (25). The mutant mice with the TRB?” gene have been found to express TRBPV mRNA in the cere- brum, cerebellum, and pituitary as well as in systemic organs (24). Furthermore, both heterozygotes (TRB?”’*) and homozy- gotes (TRB?PY’"Y) exhibit increased total L-thyroxine and TSH, enlarged thyroid glands, increased TSH-secreting cells in the pituitary, and retarded growth manifested as slow weight gain and delayed bone development. These abnormalities are more prominent in homozygotes. The mouse with a targeted mutation in the TRa gene, which produces non-TH binding TRa1PV protein, was also generated by knock-in the same PV mutation in the corresponding position of the TRa gene (unpublished data). Animal Preparation. All procedures performed on animals were in strict accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the local Animal Care and Use Committee. Local cerebral glucose utilization was determined in 4-week- old wild-type mice (n = 3) and in mice with either the TRa?”’/* (n = 4) or the TRB?’/"Y gene (n = 3), as well as in 10-week-old wild-type mice (n = 4) or mice with the TRB gene (n = 3) to assess the effects of the TRB mutation in adult mice. All of the mice were maintained on a 12-h light/dark cycle, with humidity and temperature controlled at normal levels and allowed food and water ad libitum. On the day of the experiment, the mice were anesthetized with halothane (5% for induction, 1.0-1.5% for maintenance) in 70% N20 /30% Oz, and 13-cm polyethylene catheters (PE10; Clay Adams, Parsippany, NJ) were inserted into the left femoral artery and vein. After the skin incision was sutured, a loose-fitting plaster cast was fitted to the lower torso and pelvis and taped to a plastic box to prevent locomotion. At least three hours were allowed for recovery from the anesthesia and surgery before initiation of the measurement of cerebral glucose utilization. Body temperature was maintained by warm- ing of the environment with a heating lamp. Physiological Variables. Mean arterial blood pressure was mea- sured with a Digi-Med Blood Pressure Analyzer (MicroMed, Louisville, KY). Hematocrit was determined in arterial blood samples centrifuged in a Microfuge B (Beckman Instruments, Fullerton, CA). Arterial plasma glucose content was measured in a Beckman Glucose Analyzer 2 (Beckman Instruments). Determination of Local Cerebral Glucose Utilization. Cerebral glu- cose utilization was determined by the quantitative autoradio- graphic 2-['*C]deoxyglucose method as previously described (10), except for the withdrawal of smaller and less frequent blood samples to avoid excessive blood loss in such small animals. 9914 | www.pnas.org/cgi/doi/10.1073/pnas. 171319498 Northern Blot Analysis. Total RNA (10 jxg) was used for Northern blot analysis. After electrophoresis, RNA was transferred onto membranes (Hybond-N+, Amersham Pharmacia), which were hybridized with appropriate probes. cDNA probes for the human wild-type TRal and rat synaptotagmin-related gene 1 (Srg1) (27) were labeled with [a-**P]dCTP in accordance with a random primer hexamer protocol. For quantification, the inten- sities of the mRNA bands were normalized against the intensities of glyceraldehyde-3-phosphate dehydrogenase mRNA. Thus, the blots were stripped and rehybridized with **P-labeled cDNA for glyceraldehyde-3-phosphate dehydrogenase. Quantification of the bands was done with a Molecular Dynamics Phosphor- Imager. Statistical Analyses. Physiological variables and local CMR, in 19 structures of the brain of three groups of young mice—i.e., TRaf'*, TRBPYVPY, and wild-type mice—were compared by a one-way ANOVA followed by Dunnett’s ¢ test for multiple comparison against a single control group. Physiological vari- ables and local CMRg;, in the adult mice with TRB”Y"’ mutation and in the adult wild-type mice were compared by a non-paired t test. Results Expression of the TRa1PV mRNA in the Cerebellum and Cerebrum of TRaIPV Mice. TRB’ gene expression in the brain of the TRBPV mice has previously been described (24). Confirmation of the expression of the TRa?” gene in the brain of TRa?”* mice was obtained by use of reverse transcriptase-PCR. With a primer pair of SN and 3N (Fig. 14 Top) or 5N and 3PV (Fig. LA Middle), cDNA fragments with sizes of 444 bp or 304 bp, representing the expression of the wild-type and mutant alleles, respectively, were found in the cerebellum and cerebrum (lanes 4 and 5 of Fig. 14 Bottom). Expression of the TRalPV allele was further con- firmed by Northern blot analysis with cDNA encoding human TRal as a probe (Fig. 1B). TRalPV mRNA was expressed with a size of 1.8 kb (instead of 5.0 kb for the wild-type mice) only in the cerebellum and cerebrum of TRa?”’* mice (lanes 3-4 and 2-3, respectively). Physiological Variables. Kaneshige e¢ a/. (unpublished data) noted that TRa?’’* mice are dwarfs, and in the present studies their body weights were less than half that of comparably young wild-type sibling mice (P < 0.01) (Table 1). The young mice with TRBPVPY also exhibited slightly but statistically significant (P < 0.01) lower body weights than young control mice, but this difference is less prominent in adulthood (Table 1). Mean arterial blood pressure was lower in TRa?* and TRB?Y’PY mice than in young control mice (P < 0.01 and P < 0.05, respectively), particularly in the TRa?”’* mice. The hematocrit was also slightly lower in the TRa?’* mice than in the young control mice (P < 0.01) (Table 1). There were no statistically significant differences in the physiological variables between the adult groups (Table 1). Cerebral Glucose Utilization. Local cerebral glucose utilization in all 19 brain structures examined was statistically significantly (P < 0.01) lower in the young mice with TRa?”’* than in the young control animals (Fig. 2). The reductions ranged from 57 to 72%. In contrast, young mice with TRB?’/PY showed no differences in local cerebral glucose utilization from that in young control mice (Fig. 3). There were also no significant differences in local cerebral glucose utilization between the adult TRBPYPY and adult control groups. Expression of Srg1 mRNA in the Cerebellum of TRB?Y+, TRBPY?Y, and TRa?“+ Mice. Because local cerebral glucose utilization is closely related to neuronal functional activity in synaptic Itoh et ai. A. RT/PCR Primer 5N — Primer 3N —_— rs Exon | Polya tall 444 bp a. TRat Primer 5N Primer 3PV —_—_ —— b. TRaAPV a j Pola tall 304 bp c. Expression Cerebellum | Vi | 1234 5 123 4 5 Cerebrum Size t 11 J I it | TRa: +/+ PV/+ +e PVi+ B. Northern Blot Analysis Cerebellum Cerebrum a+ 28S 18S <+TRaiPV (1.8 Kb) l TRa: +/+ PV/+ +i+ = PV/+ Fig. 1. Expression of the TRa1PV mRNA was analyzed by reverse transcrip- tase—PCR (A) and by Northern blot analysis (8). (A) RNA was isolated from the cerebellum and cerebrum. Reverse transcriptase-PCR was carried out with the primer pairs of 5N and 3N as shown (Top) and 5N and 3PV (Middle) for the wild-type TRa1 cDNA and TRa1PV cDNA, respectively. The 444-bp and 304-bp fragments represent the expression of the wild-type and mutant alleles, respectively, as shown (Bottom). The genotypes are marked. (8) RNA was prepared from the cerebellum and cerebrum of the TRa**+ mutant mice and their wild-type TRa*’* siblings. Northern blot analysis was conducted with 32P_\abeled human TRa1 cDNA as a probe. The size of the mRNA is marked. regions (47) and cretinous animals exhibit deficiencies in synaptic development and function, we also examined in the cerebellum of the TRa?”’+ and TRB””’’’ mice the expression of Srgl, a member of a family of proteins involved in the regulation of transmitter release (28). Srg/ is a T3-positively regulated gene and is normally highly expressed in several regions of the rat brain (27, 29). Srgl expression was reduced to 40% in the cerebellum of TRa?”’* mice (Fig. 4A) but not at all in the TRB?’’* and TRB?Y’PY mice as compared with the wild-type siblings (Fig. 4B). These results indicate that Srg1 is repressed selectively in the TRa?”/* mice. Itoh et al. Discussion Local Cerebral Glucose Utilization in TRB?Y”’Y and TRa?’/* Mice. Mutagenesis of the two TR isoforms, TRai and TR, in mice have indicated that some effects of TH in tissues may be mediated exclusively by either one or the other and that some effects may require the participation of both simultaneously (23). For example, TRB mediates both the TH negative feedback of TSH production in the pituitary and the activity of the auditory pathway (23, 30). Basal heart rate appears to be under control of TRal, but TRS mediates a TH-induced increase in rate (31, 32). Hypothyroidism, retarded growth, and postweaning lethal- ity are seen in mice deficiency in both TRal and TRa2 (23, 33). Regulation of myosin isoform expression in mouse skeletal muscle requires the participation of both TRal and TRB (34). In contrast, the two TH receptors mediate opposite effects on estrogen-stimulated sexual behaviors (35). Thus, the presence of each receptor isoform, their concentrations, and the TRal/TR8 ratio are probably critical determinants of T3 action (23, 36). The situation is even more complex in brain. There is a variety of possibilities that depend on which receptor is expressed in which of the many brain structures as well as on the stage of development. Jn situ hybridization and immunohistochemistry have provided information on the distribution of the two recep- tors in adult and developing rat and chicken brain (37-39). In the rat, the TRai receptor is present very early in development and is most abundant during fetal life; in fact, it has been shown to be present in stem cells (40). The TRal isoform is normally detectable in all regions of the hippocampus throughout brain development, whereas the TRB isoform is expressed only in selected regions of this structure. TRB increases with develop- ment in the caudate, nucleus accumbens, CA1 field of the hippocampus, and also in some other regions from birth to adulthood. Thus, the distribution of the TR8 receptor in brain is more restricted, and it is expressed preferentially at later developmental stages compared with the TRal isoform. The present results confirm that TH is involved in the development of those processes in brain that regulate glycol- ysis or require the energy derived from the utilization of glucose. The data support that this involvement is mediated solely by the TRal and not by the TR. No changes in glucose utilization were found in any cerebral structures of the TRBYPY mice including structures of the auditory system, even though it has been reported that TR8 is essential for auditory function in both TRB’? knock-in mice and TRB knockout mice (23, 30, 41). In contrast, rates of glucose utilization were markedly decreased in all regions of the brain examined in the TRa?”’* mice, including sensory and auditory cortex, thalamus, amygdala, hippocampus, and cerebellar cor- tex, regions known to be sensitive to TH deficiency. The reductions ranged from —57 to —72% and are comparable to the —36 to —57% reductions previously found in adult cre- tinous rats radiothyroidectomized just after birth (11). TH deficiency because of neonatal hypothyroidism has been re- ported to have more severe systemic effects than TR deficiency produced by double knockout mutation of the a and B isoforms (23, 42), possibly because of TR-independent actions of TH (43). Isoform-Dependent Action of TR Gene Mutants on Local Cerebral Glucose Utilization. The precise mechanism by which the TRalPV mutant receptor selectively interferes with the functions of wild-type TRs in brain is not clear. The lack of decreases in local cerebral glucose utilization in the TRB”’”’?Y mice may have been because of the overwhelming excess of TRa1 relative to TRB in most neurons at early stages in brain development (37-40). Indeed, with a reporter system, mutant TRBPV and TRa1lPV have been shown to inhibit the transcriptional activity of the PNAS | August 14,2001 | vol.98 | no.17 | 9915 Table 1. Physiological variables before measurement of cerebral glucose utilization Arterial plasma Mean arterial glucose Age, Body weight, blood pressure, Hematocrit, concentration, Animal days g mm Hg % mM Young mice Wild-type mice (9 = 3) 32+1 24+ 0.4 96 +3 46+ 1 12.3 + 0.7 TRaP/+ mice (n = 4) 29+1 9+ 1** 70 + 3** 40 + 0.4** 8.8 + 1.2 TRBPYPY mice (n = 3) 3241 17 + 0.3** 84 + 2* 44+2 9.9+0.9 Adult mice Wild-type mice (n = 4) 76243 26+1 98 +5 44+4 8.7+0.2 TRBPYPY mice (n = 3) 79 +3 26+ 1 90+5 39 +3 8.3 + 0.7 The values are means + SEM obtained in the number of animals indicated in parentheses. *, P < 0.05; **, P< 0.01. wild-type TRs in cultured cells. The extent of the inhibition depended on the ratios of mutant/wild-type receptors (ref. 44 and unpublished data). It is reasonable to assume that the ratios of mutant /wild-type receptor proteins also play a critical role in the phenotypic consequences of mutant receptor genes in vivo. TRal is more abundantly expressed in most regions of the developing brain than TR (40). It has been shown that the level of TRBPV mRNA is similar to that of wild-type TRB mRNA (24). Therefore, it is expected that the TRalPV mutant receptor | Young Wild Mice (n=3) -66% ** -58% ** Local Cerebral Glucose Utilization (umol/g/min) Fig. 2. -61% ** FOES eS would be more abundantly expressed than TR&PV mutant receptor in the brain of mutant mice. Thus, in TRB?”’”” mice, the ratio of TRBPV/TRa1 is not high enough to adequately inter- fere with the functions mediated by TRa1. On the other hand in TRa?Y* mice, the TRalPV/TRa1 ratio is high enough to interfere with the functions of TRal, leading to the reduction of glucose utilization. We cannot, however, exclude other mecha- nisms that might explain the selective actions of the mutant TR isoforms on cerebral glucose utilization. Young TRGPY"Y Mice (n=3) [J Young TRa?”* Mice (n=4) * * oO : PS = % oO i t © se ° lr s PSS oe #4 i 2 © -57% ** -72% ** -62% ** -66% ** es ss MAAR RR CS SS SSS Se DSSS SESS SSS SAO ESSE ES Se SSS TT Local cerebral glucose utilization (mol/g per min) in 19 brain regions of young wild-type mice, young TRB?V"Y mice, and young TRa?/* mice. The percent decrease in glucose utilization measured in the TRa?“+ mice relative to their wild-type siblings is shown for each brain region. 9916 | www.pnas.org/cgi/doi/10.1073/pnas.171319498 Itoh et af. Zettiate Young Wild Mouse Fig. 3: TR a?’ Mouse eee ete ig) ONwWOO NH i iL 0 0 8} ie es 8 No QO. & oO Young TR GPvP’ Mouse Quantitative color-coded autoradiographs of representative brain sections from TRa?”+, TRBPV’PY, and wild-type mice. The local rates of glucose utilization are encoded in the color according to the calibrated color scale on the right side of the figure. Relationship Between Changes in Local Glucose Utilization and Syn- aptic Activity. The major effects of neonatal hypothyroidism in brain are retarded outgrowth of neuronal processes, reduced A. TRaiPV mice a. TRe +i+ PV/+ B. TRBPV mice 1.32) °3 4.5 6.7.8.9 2 Sg— > aa ae a it j l Fold of change (mean): 1 1 1 b. GAPDH —— «= au? aw Ge GP Ge ae aw om | Il II J TRB: +/+ PV/+ PV/PV Fig. 4. Expression of Srg1 in the cerebellum of TRa?Y/* (A) and TRBPY’* and TRB’VPY (B) (n = 3 for each group of 12-week-old male mice). RNA was isolated from the cerebellum. Northern blot analyses were carried out with quantifi- cation by a Molecular Dynamics PhosphorImager. Level of Srg1 expression was standardized by the level of glyceraldehyde-3-phosphate dehydrogenase mRNA obtained from the same sample. Itoh et al. neuropil and synaptic densities, and disturbances in the devel- opment of electrical activities (1, 2). Animals made thyroid hormone-deficient from birth have approximately half the num- ber of synapses as euthyroid animals (45), and the size and packing density of dendritic spines are also reduced throughout the brain (46-48). The membrane-associated synaptic protein Srgl, which is believed to have a role in synaptogenesis and synaptic remodeling, has been reported to be under TH control during brain development (27, 29). Srg1 belongs to the synap- totagmin family of proteins that are involved in fusion of synaptic vesicles with synaptic membrane (28, 49), and triggering of neurotransmitter release at the synapse depends, at least in part, on Ca?* and phospholipid binding to synaptotagmin I (28, 49). It is yet to be established, however, whether Srg1 also binds Ca?* and functions as a calcium-dependent regulator of neurotrans- mitter release. TH also regulates the expression of other genes involved in formation and function of synapses, i.e., Krox-24 (50) and RC3/neurogranin (20), during development. Krox-24 is involved in modulation of neuronal connectivity and plasticity, and RC3/neurogranin, which binds calmodulin and is a sub- strate for protein kinase C, may also play a role in synaptic structure and/or function. Little is known about direct genetic influences of TH via the TRal receptor on enzymes involved in glucose metabolism. Several of the enzymatic activities in the glycolytic and oxidative pathways of glucose exhibit TH-dependent increases during brain development (51), but it is unlikely that all of these changes are mediated by direct TH regulation of their gene expression. The effects of TH on glucose metabolism might be indirect. Previous studies have shown that increases in glucose utilization associated with neuronal functional activation are linearly re- lated to the frequency of action potentials in the afferent inputs, confined to the synapse-rich areas in the neuropil, and depen- PNAS | August 14,2001 | vol.98 | no.17 | 9917 NEUROBIOLOGY dent on Na*,K*-ATPase activity (52). Action potentials result from increased Nat influx and K* efflux in neurons, which then stimulate Na*,K*t-ATPase activity to restore ionic gradients across the membrane to resting levels. The increased ATPase activity in turn stimulates glucose metabolism to maintain the ATP levels. It is noteworthy that brain Na*+,K*t-ATPase expres- sion is regulated by TH at the gene level (53), particularly in developing brain (54). The reductions in glucose utilization throughout the brain found in the TRa?Y/* mice may, therefore, me . Eayrs, J. T. (1966) in The Scientific Basis of Medicine Annual Reviews (Athlone, London), pp. 317-339. . 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