Predicting human dose-response relationships from multiple biological models: Dr. Saul Tzipori
September 28, 2000
USDA Center at Riverside
Riverdale, Maryland
Introduction | Agenda | Speaker list and presentations | Meeting handout PDF
Dr. Saul Tzipori's presentation transcript (slides not included):
Dr. TZIPORI: Our own approach is not so much for
dose-response in relation to risk factors and to see where there is a
correlation between infection in humans or, for that matter, in any other
animal species, and tissue culture. Our approach is to develop animal
models for a variety of different reasons, mostly for drug testing. And
also, over the years we've developed other animal models which were useful
to look at pathophysiological aspects, clinical aspects, house
pathogen interaction, and so on.
It's no coincidence that diseases in relation
to Cryptosporidium first emerged in the mid-70s, that they were
first observed in calves and in humans, which is also an indication where
this particular infection is significant. To this day, cryptosporidiosis
is most significant in calves, perhaps other ruminants, as well, and in
humans. And there are certainly very few species other than humans
and bovine that contract and develop symptoms the way these two species
do.
And so quite clearly, calves became a very useful
tool to look at cryptosporidiosis, and to this day, calves are the most
useful, in fact, the only system that we have in which we could produce
oocysts in sufficient quantities to carry out biological and biomedical
investigation. Just to give you an example, one infected calf can
shed up to 10 to the 10 oocysts in its course of infection, and when
you
think about it, that would--especially some of the ID 50 that were presented
in humans--you could infect the entire U.S. population with a single
calf.
Humans do produce as many oocysts as calves do,
and of course people with AIDS and HIV produce many, many folds more of
infections over many, many months. The models that have been used
over the last several years, in fact, since 1980, are calves and other
ruminants. This was followed by a demonstration of infection in
neonatal rodents. It was possible to infect neonatal mice and neonatal
rats, Guinea pigs and so on.
The next level of development was the immunosuppressed
mouse, which was developed by Mark Heley and others. And that was
used again for testing of drugs and their efficacy in this particular
model. Subsequently, immunodeficient mice were used. The
nude was used. Subsequently, the skid mouse, which can become infected,
and
over the course of several weeks, they begin to shed oocysts in the stools
and that was one of the ways of detecting the infection.
We have taken the skid mouse, which, as I said,
the infection appears after several weeks, and given it antigamma interferon
antibodies and were able to establish an infection with shedding of oocysts
within five to six days, and so that kind of made the model somewhat more
useful and more rapid to test drugs. We were able to use it in two
ways, by using it over the first two weeks. That was kind of equivalent
to the acute phase. But the skid mouse with the antigamma interferon
also gave us the opportunity to look at the chronic infection in which
the hepatobiliary tract is involved, as well.
And that's a very important component in relation
to testing of drugs against people with HIV in whom the infection progresses
to the hepatobiliary tract, which creates different kind of problems.
For instance, you can't really treat such animals or humans with chronic
infections with orally administered drugs, which are confined to the GI
tract. And I think that may explain some of the failures of the
efficacy of some of the drugs that have been tested, because you can
treat
a patient, and because he can't ever clear the infection because it's
always located in the hepatobiliary tract, it really becomes very difficult
to clear such an established infection.
We also had a pig model, which we've used and
utilized and exploited extensively. It's the germ-free piglet free
of microorganisms, as well as Cryptosporidium, because the problem
with using animals other than laboratory animals such as rodents, is
the
fact that they have their own indigenous ability to become infected.
And that's, of course, the limitation of using calves for experimentation. So
we had to resort to using pigs, animals that are derived by cesarean,
maintaining plastic oscillators to test and evaluate drugs.
The advantage of the pig was the fact that they
would develop diarrhea, very significant symptoms, as you know, which
impact the efficacy of drugs, because with diarrhea, you get a sort of
much faster transit through the GI tract and, therefore, reduce markedly
the efficacy of therapy.
The very last model, which will be the subject
of my presentation, is the gamma knockout mouse, which is an extremely
sensitive and useful tool. The nice thing about our approach is
to try to use adult, or at least young adult animals, versus neonates
in some of the earlier models that have been described, because you can
do much more with an adult mouse.
We follow the shedding of oocysts in the stool
three times a week, and then we look for--especially in the gamma knockout
mouse, there is a considerable reduction of body weight as a consequence
of infection, which also--and finally, it sort of helps the cell and
the parasitiferous membrane to form around it.
And if you look here, in fact, the membrane hasn't
quite joined. One tip is here and another tip there. I just
think it's fascinating. So in our search for a way to propagate
type 1, we started to receive, mostly from--and we tried to adopt them
to propagate in the pig. And these are some of the isolates.
Some of them were received from different sources,
and you can see that it gives the HIV status from the patient. That's
from Uganda. Of course we don't know if they came from children.
But this is a number of passages in the pig. You can see we've gone
with this one up to 11 times and it maintains his type 1 status. And
this is our newest, which has been passaged nine times in the pig.
The reason we can only do it in pigs is the fact
that the type 1 very readily and very quickly becomes overwhelmed when
it is contaminated or it is infected with type 2. And this is true
in calves, as well. As soon as there is exposure to type 2, however
small the number of oocysts are, the genotypes here and you can see that
in of the locations from calves.
The second characteristic was--and this is kind
of unfortunate--we find out that our technicians, as soon as they get
exposed to type 1, become infected very quickly and we've been agonizing
again over ways of trying to prevent infection, and I suspect, and hopefully
we will be able to test this soon with Dr. Chappell to see whether really
the infectious dose of type 1 is likely to be much--the ID 50 of type
1 is likely to be much lower. Because we didn't see this phenomena
in the past when we were working strictly with type 2.
The other thing that emerged from some of these
studies that we have done, in which case our technicians became exposed,
that we thought if animals become exposed to a mixture and type 2 predominates
in animals, perhaps when people become infected with a mixture, type 1,
theoretically, should predominate. But we were wrong and that wasn't
the case. Those that became exposed to a mixture, type 2 predominated,
as well. So the question is how does type 1 survive in nature, especially
when 70 percent of people, even in places like Uganda, which is probably
where the level of hygiene is not as highly maintained. And this
is really one of the questions that we are trying to address right now.
There are other characteristic differences.
For instance, the level of homology between the two, at least in one gene
that we have looked at with Dr. Honorine Ward, the level of homology was
only 69. I think it was a GP40. And again, this means this
combined with the fact that the type 1 does not infect rodents, which
indicates that there must be some ligan receptor issues which are going
to be quite distinct from those of type 2. So if that's the case,
we also suspect that there might be some antigenic differences which,
at the moment, we're working on to try to erase some antibodies to see
whether we could have antibodies that would help us distinguish between
type 1 and type 2.
Other sets of experiments that we have conducted
recently are to try to see whether those two types recombine, if there
is a recombinant or they cross over, but we have been unsuccessful in
trying to show that there is any genetic recombination between the two,
which is really, when you think about it, the definition of a species,
because the species, by definition, is supposed to be one which is reproductively
independent, maintaining a reproductively independent cycle.
So are we dealing with two species or two different
variations thereof of C parvum? I think this needs to also be addressed.
And it has all sorts of implications that I'm not going to go into right
now, but whenever we try to mix the two and try to see what really came
up, what the outcome was, we were able to see that there was either one
or the other. Never was there a recombination of the two.
So that was kind of the first part of what I
wanted to mention to you. So there are some differences that we
recognize, and I want to repeat them. There's the fact that there
a receptor ligan difference, which could also indicate that there are
maybe antigenic differences, at least minor. They do not cross.
Genetically, they don't cross. The rate of decay of the oocyst
is much faster, and for some reason, type 2 always seemed to overwhelm
infection
over type 1.
The species of mammals that are infected, that
can be infected with type 1 include the monkeys--we have been able to
isolate type 1 from macaque in one of the primate centers. People,
of course. Pigs and calves. We can infect calves very mildly.
The problem with calves is that as soon as you infect calves with type
1--and for some reason they get exposed to very few oocysts of type 2--then
there is again a loss of type 1. So we have to continue to maintain
type 1 in piglets for the purpose of being able to have access to type
1.
And another point of comparison. We find
that type 1 appears to induce a milder disease than type 2. I'm
not sure that this is the case in humans, because the amount of information
that we have from Uganda, the numbers are not sufficient to really derive
such conclusions, because the studies that we're doing right now, they
are cross-sectional, and you need to be able to follow infected children
for a period of time to decide or to determine whether there are such
differences. But at least in pigs and in calves, definitely the
disease is milder.
I'm going to now cover the area that was really
the purpose of this presentation, which is the dose-response. This is
the IOWA strain, we started in the gamma knockout mouse. This is
the level of shedding in logs, and this is days after challenge.
And you can see that when you do the 1000 oocysts, mice started shedding
within five days. Mice that were infected with 100 oocysts starter
later. Mice that were infected with 10 and five, much later.
And those that were infected with one, again much, much later. And
we always include the GCH-1, which is our own strain with 10 oocysts,
and it's the red one.
So the peaks appear earlier, depending on the
size of the dose. So that the pattern you're going to see with regard
to all the isolates that we've tested, and they've been listed by my predecessors.
We used the IOWA, we used the UCP, which is an isolate but which we get
from Dr. Joe Crab from Imusel. We have also our own GCH-1. We
have the MD, which was described previously, which is a sheep or a deer
isolate from Scotland, and the Texas isolate, which was also described
earlier on.
This is the Texas one again. You can see
that there is a dose-response here, which relates in terms of days after
a challenge and the appearance of--and you can see that sort of somehow
the level of shedding declines to a very low level and some of them, of
course, there a quite a few mortalities there associated with infection
with high doses, but those that receive lower dose continue to go up. And
again we have the same variation and inconsistencies that were described
previously.
What really we are interested in here at this
point is the appearance of the first infection, and the level perhaps
is not as significant the fact that they all--how many of the seven mice
that we infect become infected? And when the first appearance of
oocysts in the stools appear, and the level is sort of almost secondary,
because it's very hard to make it accurate. And as my predecessors
indicated, there are so many different reason why.
Here are body weights, and you can see they are
divided into two groups. The bottom, these here are the higher doses
and these are the lower oocyst doses, including the GCH-1, which is again,
it's interesting, what was said, that isolates that have been propagated
in the laboratory over a long period of time seem to lose something, and
that's certainly the case with our GCH-1. And we're not quite sure
how to address this, because really it's a well-characterized isolate,
we did all our studies with it, but it seemed to have lost some of its
kick.
This is the Moredun. You can see there
is a dose-response, and you can see that those that received a single
oocyst also become infected. There's a single oocyst here and there's
a single oocyst there, and you can see they start shedding, as well. Next
one please.
These are different sections in the GI tract
and you can see all sides of the small intestine become infected, and
that's really what unique about the gamma knockout versus other mice,
in which the infection tends to be in the distal ileum, in the cecum
and the colon, and that's why there isn't really much clinician manifestation,
because the small intestine is spared, and I think that's really what
is significant in terms of the severity of disease or manifestation of
any symptoms.
In the case of the mouse, it's not diarrhea,
but there's a considerable weight loss, as you noticed and, of course,
death. And you can see here in the proximal, the Moredun is much
more severe, that the level of infection in the proximal small intestine
is much more serious than the gamma knockout, when indeed there are major
differences in terms of a loss of body weight and survival between those
two strains. The more proximal the infection is with Cryptosporidium,
as is the case with other infections, the more severe the diarrhea tends
to be.
Here, it shows the mucosal score, which is scored
over the seven or eight sides that were in the previous slide, and here
it shows you the total score. And of course these numbers indicate
the number of surviving mice. The MD, only three out of seven survived
by the end of the experiment, which was 16 days. 10, only three,
and with one, five. So this is really--although the MD isolate
is not particularly--the level of shedding is no different and the appearance
of shedding is no different, but it appears to be more pathogenic to
mice
than isolates.
And you can see here that this is probably statistically
not much different, I think, than the MD. And you can see that UCP
with one oocyst, all seven survived compared with five of MD here, and
of course they all survived on the GCH-1, as I mentioned to you earlier. There
seems to be less pathogenic isolate than the others that we've tested.
This is again the distribution in different sides
of the difficult isolate. Here again, you can see this is with the
Texas strain. It's divided again into two groups. Those that
received high dose tend to lose weight. Don't forget, these are
weened mice. They are four weeks of age, and they're supposed to
continue to grow, and that's why you can see over a period of less than
two weeks, there is a sort continuous sort of body gain compared with
those that are infected with higher doses, which remain stunted.
We've done each one of those studies four times,
and I was going to point out some of the differences between them, but
I'm just going to skip that. This is again showing high dose versus
low dose or versus the GCH-1. You can see there's considerable
and statistically significant difference between mice that receive high
dose
and low dose.
This is where we took the GCH-1 studies from
all the experiments and we pooled them, and you can see this is with 10
oocysts and this is with one oocyst, and you can see that there are some
differences, and again relates to the age of the oocyst. And I think
age doesn't really tell us the whole story. There's a lot of things
about the ability and the infectivity of oocysts which we don't understand.
You take one batch and you use it one day and it's great, and you use
the same batch the next day and it's different, and a week later, it's
different still. But particularly, there is a time factor, but
that's by no means the only factor.
This is really the summary, if you like.
This is--you can see, you can, in fact, most, well, about half the mice,
which is a good indication of ID 50. And the reason we repeated
the GCH-1 twice, it appears twice here because when we first started these
experiments which, before I forget, these studies are being funded by
a grant from the FDA to do this work in parallel with the volunteer studies
that Doctor Chappell and Doctor Keshens are conducting the investigation
in Texas. And hopefully, she may shed some light on some of these
studies. I know that they haven't analyzed the MD isolate yet,
but let me get back to the GCH-1 while we have two.
When we started, we infected these mice with
what we consider to have been a suspension with a single oocyst, and they
all were shedding. So that kind of was surprising. And that's
why we went to point one to see what was going on. And I think
what we discovered subsequently was that mice were infecting one another,
because
we thought if we start the shedding, if we start monitoring the shedding
early enough, we'll be able to pick up those that are infected from the
ones that are uninfected, because it's going to take time for those that
have no oocysts in their inoculum to become infected.
And that really gave us all kinds of ideas of
other possible mechanisms of transmission. So in this case, we
put each mouse in a separate cage, and that's why we were able to get
the
percentage of the --
Here we have one where this just shows a pattern
of shedding over days--GCH-1, IOWA, Moredun and you can see the Moredun
shedding by day nine and then it goes and it continues to go higher.
The Texas was also pretty high. It started, in fact, earlier and
higher, and then it also--but again it subsides down there, and the IOWA
and the GCH-1 are less.
Here is a summary. A single oocyst is inversely
proportional to initial dose.
Dr. COLEMAN: One of the beauties of web technology is that
you'll be able to see his slides on the web when we get it posted. But
do we have a couple questions?
AUDIENCE PARTICIPANT: [Off mic.] I think in terms of
the two different xenopi and occasions on which we will see, essentially,
one xenopi dominating in one segment and later on the other one
dominating--appear that it would be possibly a numbers game by either--or
ability to replicate. But perhaps there may be an active mechanism,
as well, going on, similar to what where one strain--by active production
of certain chemicals and juices. Because that can have some consequences
as to when it may be happening and why you see so low levels of one.
You would expect maybe a near balance? You don't have a balance,
they just go away.
Dr. TZIPORI: Okay. The suggestion here of mechanisms,
how one sort of predominates in one system and one in another. I
really couldn't answer that. It's a puzzle. And there must
be some good reason. There may be different sites in the--there
may be also in terms of timing, maybe one predominates over the initial
period and--the fact remains that clearly these two are very different. And
I kind of--one is tempted to think of them maybe as two separate species.
AUDIENCE PARTICIPANT: You've done some work and did some correlations
about infectivity, what is the relationship between infection in the gut
and what the gut and either onset or total amount of oocysts shedding?
What would be one of the best markers if one were looking? We've
seen in a number of studies that you're looking at 48 hours. What
sort of, what is the validity of those markers in terms of predicting
oocysts shed?
Dr. TZIPORI: You mean in our system?
AUDIENCE PARTICIPANT: Yes.
Dr. TZIPORI: I guess the previous model, which was a neonate.
The marker was to test the--to enumerate the number of oocysts present
in the GI tract after 48 hours. And the question is, essentially,
why are we doing all of this other stuff? What's the point of doing
multiple counts and body weights and mucosal score?
AUDIENCE PARTICIPANT: No. Flip it on its side. What's
the purpose of doing the 48-hour study?
Dr. TZIPORI: Well, I think if you --
AUDIENCE PARTICIPANT: [Off mic.] How do you correlate--what
marker, if any, correlates well with any of these bilateral parameters
that you've measured, which include things like either onset or total
amount of any of those other things.
Dr. TZIPORI: Well, it depends what we're looking for.
For instance, until you actually sacrifice the animals and , what we thought
we may be able to see that different isolates may be colonize a different
section of the GI tract, which may explain why one isolate is more virulent
than the other, as I tried to explain earlier on. So this is something
that we like doing. But that really could wait till the end, and
it doesn't necessarily--you can't use that as a measure of timing or--it's
a measure for that purpose, and in this case, really, we weren't able
to demonstrate a significant difference between the isolates. The
shedding is very important, because it tells you the onset of shedding
in relation to the dose. It tells you that there is a dose-response
relationship in this model, and that the earlier shedding, and that really
needs to be correlated with the studies, because the purpose of this study
is to see how this model correlates to the human situation, and I think
until we put the results side by side and say, okay, the Texas onset was
that many days after challenge versus the mouse. The minimal, the
ID 50 for the Texas was, I think was the lowest, right? It's that
much in the human and that much in the mouse. That's why we do that.
Because ultimately, we will be able to put all this information together
when we have everything, which would include the relationship between
onset of shedding and the type of isolate. I think the body weight
also illustrates yet another parameter that tells you the extent of disease
in the GI tract. Some--for instance, the GCH-1, our own, which appears
to be a milder isolate in terms of virulence. There was no loss
of body weight. And I think that probably will reflect well in
human volunteers, not so much in terms of body weight loss but more in
the area
of the extent of diarrhea.
