Today,
we're going to review the ongoing
research at the Medical Neurology Branch
of the National Institute of Neurological and
communicative Disorders and stroke into
the viral
metabolic and immunologic
studies on the
ideology and pathogenesis
of amyotrophic gland sclerosis and
associated motor neuron disease.
What I would like to do is review
some of the evidence
suggestive evidence at this
point for a viral ideology to
amyotrophic lateral sclerosis and
outline some of the work which we are doing.
In addition,
we will look at some of the evidence for an immuno pathologic
process involved in amyotrophic lateral
sclerosis.
And again,
what work is being done in this field.
And finally,
we are going to look into the metabolic
studies being performed here at
NIH to see if there are any
unique abnormalities,
regardless of ideology,
which might lead to some way to
counteract the disease process.
There is suggestive evidence for a viral
ideology in amyotrophic lateral sclerosis
for the viral ideology
is the unique observation that there is a late
onset progressive muscular atrophy after
early childhood paralytic poliomyelitis.
This has a usual delayed
onset,
approximately 20 to 40 years following the
childhood disease and
rarely involves cases that
may present with the classic Charcot form of a
myotropic sclerosis.
Other workers have tried to see if there's a
relationship between Anteed and polio virus
infection through looking at serum
antibodies to various polio virus types.
No clear cut relationship has been noted.
However,
if we will remind ourselves of multiple
sclerosis,
we will remember that there is a unique and perhaps
more dramatic rise in the antibody in the cerebral
spinal fluid towards specific viruses in
this disease than in the
serum.
We in conjunction with Dr John sever are
looking at the serum and
cerebral spinal fluid
antibody levels which would neutralize
various types of polio virus antibody in a group of
patients with amyotrophic lateral sclerosis
and comparing this to a group of patients
with other neuromuscular disease such as
peripheral nerve disease and
primary myopathies.
This work is ongoing and I will not report the results
today.
But it is important for us to
conceive of a possibility that an early
infection may lead to late disease.
There is an animal model of marine
progressive muscular atrophy.
After neonatal infection with mouse C type
particles,
this produces an infection in
wild mice and can be reproduced in
the experimental situation leading to
paralysis and pathological changes that
are very much like what is seen in the
human disease.
Amyotrophic sclerosis.
Specifically,
there is a marked absence of
inflammatory response in these animals.
Now,
contrary to what we cannot
find in amyotrophic vito sclerosis
virus particles are seen in
the mice as they develop paralysis and
these are localized in the anterior horn cells.
However,
many anterior horn cells show destruction
without evidence for viral replication.
The exact pathogenesis by which paralysis is
produced is currently under study at the Johns Hopkins
University School of Medicine.
Based on the finding in the animal
model.
Some people including Mike Viola and Saul
Spiegelman at Columbia University College of
physicians and surgeons have looked at the
presence of an RNA instructed
DNA polymerase in the brains of patients with a myotropic
sclerosis.
The mouse C type virus particles and
other C type virus particles are associated with an
enzyme called reverse transcriptase
which will make DNA
from an RN A trans
script.
Looking at extracts of brains from patients
with Guamanian Parkinson's
disease.
He myotropic lateral sclerosis complex.
These workers have been able to find evidence
for a unique RNA
instructed DNA plumes in the brains of
patients with this disease.
However,
patients without the clinical
signs or pathological signs for disease
also have evidence for this enzyme.
More recently,
there have been some reports of virus like particles
occasionally seen in the extra neural tissues
of two cases of acute amyotrophy
out of sclerosis.
The exact role of this
observation in the pathology
of the disease is unclear because
many of these patients had intercurrent infections
and it is possible that these viruses may not
represent direct
infection of the
tissues but may be there as
bystanders.
The exact ideology for possible extra
neural viral infection on central nervous
system degeneration is unclear.
However,
we must keep in mind such syndromes as Ray's
syndrome in which infection involving the
liver leading to liver destruction
can cause a severe neurological deficit.
Now,
there is evidence against the virus ideology
in the classic sense for amyotrophic
sclerosis.
Primarily the
patients with
amyotrophic sclerosis
in Guam and sporadic cases
have not produced disease when
inoculated into cerebrally into experimental
animals in experiments
similar to that which has shown a definite
viral ideology for diseases such as
Kuru and Creutzfeld Jakob
disease in man.
In fact,
the absence of passage for any agent
in amyotrophic sclerosis has served as an
excellent control for the other studies.
Highlighting that
the amyotrophic out sclerosis does not
behave like a simple virus
infection and no clear cut virus is
recoverable in animal inoculation
experiments.
But more importantly,
in contrast to the work being done with the
more sensitive methods of looking at RN A
instructed DNA polymerase.
It is important to note
that the brains
of patients with sporadic cases of
amyotrophic lateral sclerosis show no evidence for
the presence of this enzyme.
Thus,
on the balance,
there is no clear cut evidence
for a viral ideology.
But some animal models suggest that it is
possible for a
progressive muscular atrophy type
syndrome,
similar to amyotrophic sclerosis and
associated motor neuron diseases to occur
following a viral infection early in
childhood.
We are searching for other possible
leads in this regard.
The possibility that there may be an immuno pathologic
process involved in amyotrophic lateral sclerosis
is less clearly established
and evidence sort of lines up on the
side against an immune
process.
For this disease.
Serum cytotoxic
against cultured embryo
anterior horns and culture was thought to
be specific for amyotrophic lao sclerosis serum
in the past few years,
groups at the NIH and elsewhere have shown that
the serum cytotoxic against heterologous
mouse embryo neurons in culture
is non specific and most likely related
to heterologous antibodies
directed toward blood group specific
antigens which cross react in the urine
system at the
present time.
There's no definite histocompatibility
linkage.
Some groups have suggested that there is an increase in
H three and seven in
paralytic poliomyelitis patients and
in patients with amyotrophic lateral sclerosis.
Two other groups including Dr Paul Terrasa,
who is probably the father of the histocompatibility
method have shown that there is no
clear cut statistically significant linkage.
However,
some groups have shown an increase in the L
2-21 haplotype
in patients with severe paralytic
poliomyelitis and
amyotrophic lateral sclerosis.
These findings are suggestive that there
might be a unique susceptibility to some
pathological process that it is linked
to the his compatibility antigens or to the haplotype is
not clear yet,
but perhaps further studies with more specific
delineations of histocompatibility
linkages might give us some idea
as to what is going on at the
present time,
there is no specific lymphocyte response as
measured by macrophage inhibition factor or
lymphocyte transformation to brain
antigens or neuromuscular junction
antigens in the lymphocytes
from patients with amyotrophic lateral sclerosis as
compared to controlled patients.
Some recent evidence has
suggested that there may be a role
for complement in the pathogenesis
of atrophy sclerosis in the medical
neurology branch.
Several groups have demonstrated that there might
be borderline increase in C three levels of
patients with amyotrophic sclerosis compared to
other disease groups.
Recently,
one group has
shown an increased C one Q binding
in patients with Amyotrophy out sclerosis compared to
controls,
it is not clear.
However,
if the controls are adequately age or sex
matched,
but there is some suggestive evidence
that there may be an abnormality and complement
binding in patients with aosis,
whether this is due to intercurrent infection or related
directly to the pathogenesis of the disease is
unclear.
These same people found evidence for some
immune complex deposition in the renal glome
in nine of 18 patients with a
sclerosis suggesting that there might
be some intercurrent infection.
Most recently,
a paper from the same group has shown that
there is no evidence for circulating
immune complexes in these patients.
So exactly why there is increased evidence for
deposition without the presence of circulating
complexes is unclear.
And this issue remains to be decided by
future research,
regardless of whether there is a virus or
immunologic ideology.
For the disease.
There might be some metabolic pathway which is
affected,
which can then be understood and
perhaps changed by drug therapy.
The best example of this would be Parkinson's
disease in man where there is
very good evidence that the post encephalitic
Parkinson's is due to a virus infection.
However,
the exact way that we treat
patients is based on metabolic abnormalities
attended in the dopamine colergic
system,
we are searching for a similar
metabolic abnormality in amyotrophic
sclerosis patients because some
patients with hyper parathyroidism may
present with neurological abnormalities
and occasionally be differentially
diagnosed as having amyotrophic sclerosis.
We have conducted a longitudinal study of
calcium metabolism in patients with amyotrophic lateral
sclerosis,
peripheral neuropathy and primary muscle
disease.
We have looked at the seven
day whole body calcium retention
in patients with als compared to other
controls.
This is a measure of the
absorption of calcium by the body.
Its distribution into the metabolically
active pools of bone and
muscle and its excretion from the
body.
In a fixed length of time.
Normally,
patients will maintain after a
given dose on a given diet
of calcium.
A calcium retention ranging
between 30 to
44 patients with
neuromuscular disease are
statistically below
normal.
But patients with amyotrophic lao sclerosis
are lower yet and lower.
Compared to patients with other neuromuscular
disease.
We have been able to show by measuring the vitamin D level
that is not due to an abnormality in the vitamin D
level.
In the blood as measured by 25 hydroxy vitamin D
and serum calcium is
similar in all three groups.
Although in the low normal range,
interestingly,
when we looked at the parathyroid hormone concentrations,
we found that all three groups
showed evidence that the
parathyroid hormone concentration was
borderline elevated with approximately 40
to 50% of the various groups
being above two standard
deviations from the mean
for normal.
Suggesting that there might be some role of
secondary hyper parathyroidism in these
patients in response to
abnormalities in calcium metabolism
or in the regulators of calcium metabolism.
In order to determine whether there was an abnormality in absorption
early of calcium.
In patients with amyotrophic sclerosis,
we looked at the uptake of radioactive
calcium into the plasma pool in
patients with amyotrophic lateral sclerosis.
Compared to those patients with
nerve and muscle disease.
Patients with neuromuscular
disease,
exclusive of als show
quite normal uptake.
Patients with als show an increased uptake into the
plasma pool and a more heterogeneous
distribution.
Thus,
we were able to show that the decreased
retention is not due to
abnormalities in the absorption acutely
of calcium.
In patients with amyotrophic lateral sclerosis.
The reason for the delayed
decreased retention must have some other
basis.
We know that these,
we know that these patients have
normal vitamin D levels,
but we do not know whether they respond adequately
to this level.
In order to determine this,
we gave patients with als dihydrotachysterol
and a control group with similarly
decreased retentions
and followed these patients before and
after treatment with the seven day whole body
retention,
we were able to show that patients with
amyotrophic lao sclerosis showed an increase in
the whole body retention of calcium
47 at seven days,
similar to the controls and into the
normal range.
This was maintained as long as they were on the
dihydrotachysterol therapy and showed that there was
no vitamin D resistance in these patients.
The exact role that calcium
metabolism plays in amyotrophic sclerosis is
unclear.
Is it an epi phenomena or is it
related to the underlying pathogenesis?
Further studies will have to be done to determine
whether patients with A LS respond
adequately to the normal
stimulation of parathyroid
hormone.
Those studies are currently in progress at the medical
neurology branch
because we were unable to show an
abnormality in calcium
levels in the serum or spinal
fluid between patients with als and other
neurological disease.
We looked at other contaminants of calcium
metabolism in these patients
cyclic nucleotide,
cyclic amp and cyclic GMP are
present in the human body in
large amounts,
but more specifically in the central nervous
system in extraordinarily high
amounts compared to other tissues.
In addition,
the enzymes for their synthesis and degradation are also
present in these tissues.
Cyclic amp and cyclic GMP play an important
role in the metabolic
mediation of certain reactions
such as phosphorylase.
In addition,
there is increasing evidence for a role of cyclic amp and
electrochemical mediation of certain
events in the central nervous system.
In human studies of patients with als and
other diseases,
we use the radio immunoassay to measure cyclic
nucleotides and show here the unique
specificity in distinguishing between the two
nucleotides using our
different rabid antibiotics.
When we looked at cerebral spinal fluid cyclic A MP
levels in the same patient at two different
times under identical pharmacological and
physiological situations,
we showed an excellent correlation with
excellent linear regression
for cyclic amp and cyclic GMP.
The black dots indicate those patients who have not
had proven acid treatment and the
white squares show those patients who have had proven acid
treatment.
There is a clear correlation
regardless of pharmacological
manipulations between the
CSF levels of these cyclic nucleotides in the
same patients on two different
occasions.
And we were able to show that there is no
clear cut difference in the plasma cyclic
amp P level between those patients with motor
neuron disease and those without motor neuron
disease.
However,
there is a significant decrease in the CSF
cyclic amp P compared to these patients.
And there's a more grouping of these
patients compared to a more wide range seen in
other groups.
In order to determine whether the cerebral spinal
fluid cyclic nucleotide level
was completely representative of what was going on in the central
nervous system and did not reflect any changes
in the plasma pool.
We
performed the
following experiment in which we gave Glucagon
infusions and raised the plasma
cyclic A MP level 40 fold
without changing the plasma cyclic GMP level.
There was no significant difference between the
elevations in patients with ALS compared to those
without ALS,
the CSF levels did not change at all during the whole
period of observation
significantly.
However,
the CSF glucose levels rose
significantly in both groups to similar
levels indicating that there was
transfer of glucose into the cerebral spinal
fluid at a time when there was no similar
transfer of cyclic A MP.
Thus,
there is a significant barrier to
cyclic A MP entry into the cerebral spinal
fluid under physiological
conditions such as we employed
here,
the main mode of egress for cyclic A MP from the
plasma pool is to be excreted in the kidney.
Now,
this clearly established that the
level of cyclic nucleotides was
unique in our hands.
And if we look at our formal data
here,
correlating the CSF level of cyclic amp and
cyclic GMP in patients with muscle
disease,
peripheral nerve disease.
To those patients with amyotrophic lateral sclerosis,
we show here a significant decrease in
both cyclic amp and cyclic GMP compared
to patients with these other diseases.
Indeed,
patients with other central nervous system diseases such as
multiple sclerosis,
the level might be slightly increased compared to
normal.
This is important because of our recent studies
involving the Wobbler
mutant,
which is a urine progressive spinal muscular
atrophy,
which is genetically based.
We were able to show in conjunction with Dr David loss of the
laboratory of neuro
neuropathology that there
is an 80% decrease in cyclic
GMP in the spinal cord and brain stem
of these patients of these
mice.
Compared to their literate
controls.
At the same time,
there was no decrease in a neurotransmitter such as
Gaba and high energy phosphate
compounds showed no difference between
control animals and those affected by the
disease.
Thus,
motor neuron disease with anterior
horn loss is associated
uniquely with an abnormality in cyclic
GMP concentration
both in the tissue of animals and in the
cerebral spinal fluid of man.
To highlight this further,
we employed a technique where we gave probenecid
infusions to block the escape of cyclic
nucleotide from the cerebral spinal fluid.
Using an intravenous technique as recently described
by doctors Ebert and Cartel.
We were able to show that there is a
increase in cyclic A MP
at four hours after the infusion,
which is maintained at eight hours after the
infusion.
In comparing patients with als to those
with other neuromuscular disease,
we can show that there is a decrease in the cyclic A
MP turnover
and in the cyclic GMP turnover.
Although this is much more marked in the
case of cyclic GMP turnover.
In order to see whether we could change this in any
manner,
we employed a fossil dia inhibitor
called the lasino,
which does not block the adeno receptor.
The Ayin and papaverine,
which are well known drugs do block the adenosine
receptor and do not allow positive feedback for the increase
in cyclic amp and central nervous system tissues.
By giving thol to patients with als,
we were able to show a dose related
increase in CSF cyclic amp
but no significant change in C
GMP plasma levels show no
significant change.
And we believe that it is because we're able to show that there is a
rapid elimination of plasma cyclic
amp from the
plasma pool.
After this drug is being given
to see if there were any other effect of this
drug on the transport of cyclic
amp.
We did probe infusion studies on patients
before and after therapy.
As we can see here,
there is no significant alteration
from the pre treatment level and those
scraps that I showed previously.
However,
after treatment with thain,
there is a significant increase in the CSF
level of cyclic amp,
cyclic GMP increases slightly but not statistically
at this dose of 40 mg per kilogram.
Thus,
we're able to show a significant effect on cyclic A MP
metabolism but not on cyclic GMP metabolism.
And this possibly relates to the fact that the drug
has a 10 fold greater inhibition of
fossil dira for cyclic amp than GP.
The exact locus for the drug action is
unclear.
Although when we take cerebral spinal fluid from patients with
amy sclerosis and
allow hydrolysis to proceed at room temperature at
four degrees,
it proceeds very rapidly for both cyclic GMP and
cyclic cyclic amp and cyclic GMP.
And the presence of thain is a temperature gradient and
decreased hydrolysis.
For cyclic A MP but not for cyclic
GMP.
In summary,
then we are able to show that
diseases affecting the anterior horn cell are
correlated with decreased cyclic amp and cyclic
GMP in the cerebral spinal fluid diseases
affecting the nerve accent or the muscle
are not similarly correlated with changes in
CSF levels.
And diseases affecting upper motor neurons may
actually enhance or increase the level of the
cyclic nucleotide.
If I may spend a moment on hypothesis
and exact locus of action for some
of these things.
I'd like to relate the observations of doctor
Engle who has shown that a
Neil cycles is
correlated best with the vessels
in the spinal cord and is
much less prominent in the actual neurons.
Most of the previous data showing that there
is increased adniel cyclo
and cyclic nucleotides in gray matter compared to
white matter has been based on dissection studies
and homogenates without clear
cut histo chemical localization.
As you can see here,
the distribution of the anterior spinal artery is
such that the anterior horn and the lateral
column are in the distribution of this
artery.
If there is some way
that disease affecting the vessels
may somehow affect nutrition to the anterior
horns,
we may have some
pathological substrate for the
disease,
amyotrophic lateral sclerosis,
the distribution is the same and
the findings would correlate with our biochemical
findings.
It is of interest that most
recently we have been able to show there is increased
Norine in the spinal fluid of patients with
amyotrophic lao sclerosis.
And how this relates to any possible small vessel
abnormality is unclear.
So these metabolic studies may point the way to some
pathogenesis of the disease.
The exact ideology is unclear,
but we must remember that viruses can attack vessels
and perhaps we can tie everything together in a
similar fashion to Parkinson's disease.
Thank you.