So, what I'd like to talk about today is some ongoing
research, a program that I've really enjoyed being a part of
for over 10 years now, looking at risk factors
for childhood asthma. And my disclosure slide.
I'm a consultant for Merck, EraGen Biosciences,
who did some of the work and collaboration to develop
viral diagnostics, and GlaxoSmithKline.
As an overview, I'm going to start off by talking about
some of the recent epidemiology in asthma.
Epidemiology was never my favorite subject in school,
but it's actually provided some key insights into the roll
of the environment in asthma, some of the disparities when you
think about how asthma's sprinkled across the globe, and
even across the United States. Next we'll talk a little bit
about viral infections in infancy.
These have long been regarded as important risk factors
in asthma. And I think with new tools now
we have new insights into the relationship with asthma and
also a host and a viral factors that contribute to the severity
and the long term consequences of these illnesses.
And finally we'll talk a little bit about a newer topic,
how pets and microbial exposure might influence immune
development in early life of the relationship of the
development of allergic diseases and asthma.
And we'll finish there. And along the way, I'd like to
highlight some work that's been done by three medical students
while they were in medical school that have really helped
with these studies. From my perspective as an
investigator it's wonderful to have students in the laboratory,
medical students, for a couple of reasons.
Number one, it's great to see them get the first exposure
to research, in many cases. And secondly, it's always
interesting because you ask questions that at first seem
a little silly, but in fact are quite insightful and
quite important. And I think that this has really
helped my research program to move along.
So first of all, getting at the epidemiology, it's quite clear
that asthma, like a number of other inflammatory diseases,
really is growing in prevalence. And you can see that graph here
from the National Health Interview Survey.
The CDC, who does this survey, actually changed the definition
of how they define asthma. This is done by phone survey.
You can see about a doubling over a 15 year period of time.
This is asking about current asthma, which is very similar
to asthma in the last 12 months. You can see things may have
leveled off, but they've leveled off at a very high rate.
And the question is, why are we seeing more asthma.
And this parallels increases in a number of other allergic
diseases as well. This is a study, very ambitious
study, that was conducted to look at how asthma was
distributed across the world. And to do this, obviously
you're going across many different cultures,
many different languages and there are difficulties
with this. In fact, in some languages
there is no word for wheezing, for example.
But they tried to level the playing field by actually
showing videos when they did these surveys.
And what you see here are a number of symbols.
The stars are places where you see wheezing
in 13 to 14 year-olds, that's greater than 20%
of the population for the past 12 months.
You can see that this is concentrated
in the United States, in the United Kingdom
and in Australia and New Zealand.
With higher rates also in western Europe.
And asthma is really quite low, less than 5% in eastern Europe,
in places, in rural places in India and in China.
And this really provided some very important clues as to the
importance of the environment and local lifestyle factors
in who gets allergic diseases and who does not.
And it led to a hypothesis that I think really changed
the way a lot of people think about preventive advice
in infancy. And this is called the so-called
hygiene hypothesis. And the idea here is that
at birth the immune system is immature.
You can take one of two turns in response to the type
of environmental exposures in early life.
One leads to tolerance and health, and the other leads to
a so-called Th2 bias. So this is an immune system
where the T cell, the memory T cells tend to
overproduce cytokines like an IL-4 and IL-5
that promote IGE synthesis and the eosinofilica inflammation
that's very typical of allergic diseases and asthma.
And the theory is that the type of environmental exposures
early on is what steers you down one of these two pathways.
So if you have exposure to lots of other children in daycare,
or maybe you have a lot of older siblings.
If you're exposed to infections early on, microbes, animals,
think about the third-world environment, this might be
something that's important in immune development and
help to steer you toward health. Conversely, if you have a
situation where you have an only child, maybe no exposure to
infections, a relatively sterile environment, then this immature
immune system doesn't have normal development, and instead
it develops this immune bias and a greater risk of allergies
and asthma. Well, to be able to evaluate
these possibilities, it's very helpful to evaluate
the immune response and early environmental exposures,
even before you start developing signs of allergies and asthma.
And in 1998 and 1999, Rob Lemanske and I
in the Department of Pediatrics with help from Pete Schult
in the Virology Division here in Clinical Virology,
Ron Gangnon of Biostatistics and Kathy Roberg
our lead coordinator, started the COAST study,
or Childhood Origins of Asthma Study.
And the goal here was to have a prospective birth cohort study,
so we'd enroll these families even before birth and
collect information on immune development,
on viral respiratory tract infections and other
environmental and lifestyle factors that might have an
influence on immune development in early life.
Each of these children were at high risks for developing
allergies and asthma, because either the mother or the father
had allergic diseases or asthma. And this shows an illustration
of the first year of the COAST study and some of the procedures
that were done. So, since they were prenatal
enrollment, we were able to collect samples of blood
from cord blood. This would otherwise be
discarded with the placenta. We could take these mononuclear
cells and look at specific immune responses.
And then children generally are seen by a well childcare visits
at two months, four months, six months, nine months
and a year. Each time these children had
one of these visits, one of our coordinators would
go to the doctor's office and collect a sample
of nasal mucus so we could look for respiratory viruses.
In addition, whenever the families noted illnesses at home
they would call our coordinators and we would meet them at home,
at McDonald's, at the car wash, wherever, and get a sample of
nasal mucus and some information about this acute illness.
And then yearly, these children were seen and
provided a small sample of blood so we could continue
the immune studies using the mononuclear cells
and look for evidence of specific viruses.
So for this trial, in which we had I think 1,500
to 2,000 nasal specimens in the first couple of years,
as well as another similar trial in four inner cities, we decided
that we needed to have a new type of viral diagnostic.
The types of virus we were interested in was virtually
every respiratory virus and they're listed here,
over 160 serotypes of viruses at the time, and more have been
discovered since that time. To be able to find these viruses
has always been somewhat of a problem.
PCR based diagnostics are helpful, but we decided to
partner with EraGen Biosciences to develop what's called the
Respiratory MultiCode Assay, which uses multiplex PCR.
And Magnolia Prince actually helped us with the initial
testing of this assay and then the processing of our nasal
samples from the COAST study. So the steps are shown here.
Actually the chemistry is unique in that there are two
nonstandard bases. So there's Iso-C and Iso-G that
don't bond to any other bases, natural bases in the genome,
but bind very well to each other.
Generally, most of these viruses are RNA viruses, so you do
a reverse transcription step and then a PCR-based step here.
These steps incorporate these non-standard bases, which
gives you a little better avidity of binding.
And also some unique tags. So this is a target-specific
extension. The PCR product here
acts as a template. And then you can tag specific
parts of the viral genome from viruses
to bind to colored beads, which then can be read out
on a Luminex machine. So it gives you the capacity
of analyzing up to 80 different PCR reactions.
We used about 20 for this assay. And this is the readout
showing specific detection of respiratory viruses.
For this study, what we did was to take 20 copies of every
CDNA target specific for each virus, and then amplify it
with specific primers. Each one should light up
a particular primer set and bind to a specific colored bead.
And what you can see here is that 20 copies of CDNA
produced a very nice signal-to-noise ratio.
And in each case we had just one viral bead that lit up
with the viral copies of the viral genome.
So a very sensitive, very specific assay,
and really adaptable for a large clinical trial.
And this shows the results of viral diagnostics on the
samples that were obtained during both well child visits
and sick visits of the first year of the COAST study.
So it's grouped. Here on the left, these are
the viruses that were detected in kids with no respiratory
symptoms, then mild symptoms, moderate and severe.
And the first thing you'll notice is that
the isolation rates go up but they don't start with zero.
And actually this is quite typical now of a number
of studies showing up to around a third of healthy infants
have respiratory viruses that you can detect
at any one time. Now some of these kids
are just getting over colds. Their symptoms are gone but
maybe a little bit of the virus lingers on.
A small percentage of these kids will get a cold.
But a lot of these are really asymptomatic infections.
You'll see that in children with more significant illnesses
you see about 90% recovery of viruses in these specimens,
which is very good. And also the type of virus
that you find varies with the severity of illness.
So rhinoviruses in yellow were the most common viruses
overall. Viruses, such as RSV, were
really found predominately in the kids with a greater
degrees of illness. This is also true
for multiple viruses. Some of these children had two,
three and even four viruses at once.
Most of those kids were in daycare.
And the 1998-99 season was actually fairly light
for influenza in the light blue here.
And this can vary dramatically from year for year.
And of course you know every ten years or so,
this is just a huge problem. You can also look at this data
from the perspective of the child.
Those were means according to the type of illness.
In this graph, each of these lines is actually one child.
So this is the birth date. This is calendar year
on the bottom, the birth date, and the one year birthday here.
And each symbol is a viral sampling.
The ones that are colored actually have various viruses
detected. If it's open that means
there's no virus recovered. And you can just see that some
of these kids just had a very difficult first year.
Here's a child here who had five severe illnesses in a row.
Now keep in mind this is taking place at a time when the immune
system is rapidly developing. Obviously, the lungs and
the respiratory tract are rapidly developing.
And you just wonder, what are the long-term
consequences of having these sorts of illness patterns.
That actually the sickest child had 13 moderate to severe
illnesses in the first year. So to address this question,
we actually looked at the history of viral illnesses
in the first three years of life and compared that
to an asthma evaluation in these kids at age six.
These kids are now between eight and nine years of age
at present. The asthma evaluation
took into consideration a number of things:
wheezing in the last 12 months, the type of medications that
infants and children were on, whether they had asthma
controllers or asthma reliever medications.
But it was blinded in terms of the virology
for the first three years. And what we showed with this
analysis is, on this graph here, what I have in the upper graph
is really the life history for the first three years
of children who went on to develop asthma.
And the lower graph is the history of the first three years
of infections related to wheezing
in kids who did not have asthma. And the first difference
I think that you can see is that children with asthma
had a rougher time in their first three years.
Really, virtually every virus has increased compared to
the non-asthma group here. I think the other thing that
stands out is that rhinoviruses cause most of these wheezing
illnesses in the asthma group. And in fact, if you look from
year one, to two, to three, you see an increasing rate of
respiratory wheezing illnesses in the asthmatic group
versus a reduction in wheezing each of those years
in the non-asthma group. So not only a different pattern
of pathogens, but also a different pattern of development
in terms of the first few years. The two viruses that are most
likely to cause these wheezing illnesses early on
are respiratory syncytial virus, which is kind of this prototypic
virus that causes bronchiolitis and rhinovirus.
So what we wanted to do was to look at potential interactions
between these viruses and the rate of asthma
at six years. So on the bottom here are just
representations of kids who wheezed with neither virus
in the first three years. And they had a low rate
of asthma at age six. Kids who wheezed only with RSV,
and it looks like there is somewhat of an increase
in asthma out at age six. And then kids who wheezed
with rhinovirus. And it was these children
who really seemed to have the highest rate of wheezing
at age six. And if you wheezed with both
viruses, it seemed very similar to children who wheezed
with rhinovirus alone. So in summary I think what
these studies are demonstrating is that childhood asthma
is really associated with a different pattern
of respiratory viral illnesses during infancy.
Rhinovirus infections seem to be very indicative
of an increased risk of asthma out at age six.
And the frequency of illnesses actually increases
in the first three years. Now, there are a number
of other studies that show that severe infections with RSV,
those leading to hospitalization or acute care visits,
also indicate risk for asthma.
So one other question when you look at this data base here
is whether there are other factors that modify the severity
of respiratory viral infections in early life.
We'll go back to this graph showing the COAST data.
One thing that you'll note is that rhinovirus infections,
for example, can be found in infants with no illness,
with mild, moderate or severe illnesses.
And the proportion is fairly similar across this range.
We know rhinovirus is a pathogen.
This was shown back in the '50s. You give it to someone
and they get a cold. So it suggests that there
may be either host factors that protect kids, or
don't protect kids from getting more severe illnesses,
and things like interferons, and so forth.
And there may also be viral factors.
For rhinovirus, it stands out among other viruses in that
there are so many different serotypes of rhinoviruses, 101
that are traditionally grouped into the rhinovirus category.
And if the pattern follows, what we see with influenza,
and RSV, and others, it's likely that some of those
serotypes are likely to be more pathogenic,
and others less pathogenic. So that may be also account
for why we see rhinoviruses in both of these
different categories. So we started to look at
these two factors within the COAST population.
One way to look at host immune factors is to
take peripheral blood cells that we get from cord blood
and then at yearly intervals and separate these out
from peripheral blood, incubate them in the laboratory
with various stimuli. So we picked PHA, which is
a very potent T cell stimulant for infant cells,
and also one type of rhinovirus and one type of RSV.
And then, we analyzed the production of a number of
different cytokines, including so-called Th2 cytokines
that have been implicated in allergies and asthma
like IL-5 and IL-13, and then protective cytokines
like interferons, interferon alpha and gamma,
and IL-10, which appears to have anti-inflammatory effects.
So we have found that interferon synthesis from
infant's blood here does have some predictive values.
So what we did here was to take the interferon alpha responses
from cord blood cells and group them in four categories.
These are the children with lowest response.
So these are cortiles, the next highest, next highest
and highest response. And there's some effect
on the proportion of kids who developed moderate to severe
rhinovirus illnesses in the first three years.
But where you really see a protective effect is when
you look at the more severe rhinovirus illnesses, so those
associated with wheezing and lower respiratory symptoms.
And here you see there's about a 50% reduction across these
interferon and alpha categories in those who wheezed
with rhinovirus, going from 39% in the lowest category
to 19% in the highest category. So, clearly, host factors
do make a difference in particular interferons.
This is looking at blood cells. What about airway cells?
This is where the infections really occur.
So we've moved forward to address that question as well.
Do airway immune responses differ in wheezing children?
So for these studies, which were conducted by Brigitte Smith,
we looked at wheezing status at age six and compared this to
the parental report of wheezing in the past 12 months.
We then took the nasal lavage samples that were obtained
during scheduled study visits and also during wheezing
illnesses, and we took the samples that were positive
for rhinoviruses and analyzed these samples
for the content of cytokines. This just shows that process.
Actually, we came up with a technique to sample
nasal mucus in kids. There's a couple of ways
traditionally that this has been done.
One, you can put a swab in the nose, which
doesn't feel very good. Two, you can take some liquid
and squirt it into the nose and take a bulb syringe
and suck it out. And we did that in kids
when they were infants. But when kids started to
be able to blow their noses, we developed this nose blowing
technique, which some of the kids really, really like.
What you do is you actually take a plastic Ziploc bag,
you squirt a little bit of saline in the nose,
just a nasal spray and have them blow their nose
into this plastic bag. And the kids love to see what
comes out, the various colors, and once in a while an object
or two. But you can take this nasal
lavage and analyze this not only for viruses, but also
for cytokines and Brigitte used a so-called Luminex,
kind of a multiplex technology to look at a number of cytokines
that were involved in the acute response to viral infections.
Also, interferons and IL-8, which is a chemo attractant
for neutrophils, which is the most frequent cell
that comes into the airway with these studies.
So, she asked us several questions.
First of all, is there a difference in the types
of cytokines that you find in the nose
during periods of illness versus periods of health.
And you can see that sick kids tended to have more IL-6
in the airway, slightly more IL-10
and also interferon gamma, and a similar trend
with interleukin-8. Secondly, if you evaluated
the wheezing children over a year or so,
these children with a history of wheezing would have illnesses
in which they wheezed, and they would have illnesses
where they didn't wheeze. So she compared these cytokine
levels in wheezing illnesses versus non-wheezing illnesses
to see if this immune response was different in the two groups.
And actually it wasn't, it was quite similar within
these wheezing children whether or not
they had wheezing illnesses. But what did seem like it was
different was if you looked at the wheezing children
versus the non-wheezing children during times of illnesses.
Here you found IL-6 in both groups
and IL-8 in both groups. But what seemed to be
trending different anyway is an increased amount of IL-10
and increased amount of interferon gamma
in children without wheezing. Again suggesting that
interferon synthesis may be important in limiting
the severity of airway disease in these children.
So the other question is whether there are virus-specific
factors that help to determine whether a child becomes very ill
or not so ill. And actually this analysis
is ongoing. I'll show you some early results
that looked very interesting. With rhinoviruses, there are
typically 20 or so rhinoviruses that circulate through
a community in any particular season.
And given the large number of viruses, we anticipate
we're going to have to analyze somewhere between 750
and 1,000 samples before we know which ones are really
the more pathogenic ones. But Wai Ming Lee and our
virology core has started to do this analysis and had
some very unexpected results. What he did was to look at
the genotype in each of these viruses that we're detecting
by PCR. I'll show you how he did that.
He calls this the molecular typing assay.
And of the 102 rhinoviruses that he has analyzed so far,
surprisingly only about half of them corresponded to
known strains of rhinovirus and fully 53, or more than half,
appear to consist of new strains with at least 13 and
up to 33% sequence difference compared to the closest
reference sequence. So this just shows
his molecular typing assay. This is the five prime end
of a rhinovirus. It has about 7,500 bases total,
and there are certain conserved areas that are really
pretty much the same in every rhinovirus.
And these are used for the design of PCR primer
so you can find these viruses. But the interesting thing
is there's a stretch of about 220 bases here
in between these two primers that's actually different
for each strain. So Wai Ming developed a library
of what these sequences were for all 101 known rhinoviruses
and then compared the field isolets to these.
And what you do is when you look at the genetic divergence
among the known strains is you see something like this.
Most strains differ from each other
with at least 10% of the genome. This is, again,
the five prime end that I showed you before.
So anything more than 10% divergence
represents a new strain. And actually rhinoviruses,
when you group them together, there's two groups.
The A group, which is the big group right here.
And the B group. And again, this is just
a genotypic phylogenetic type grouping.
This is a phylogenetic analysis of the new strains
compared to the known strains, previously known strains
of rhinovirus. In the past, again, there were
rhinovirus A strains, in red, and rhinovirus B strains.
The new ones that Wai Ming found are shown here, so it looks like
another branch off of the A tree,
another branch here. But importantly, this new group
of rhinoviruses, which we call rhinovirus C for now,
seem to be as different from A as they are from B.
And these rhinoviruses are in the enterovirus family.
They're also distinct from known enteroviruses.
So he's continuing this work. The implications are
that instead of having 101 cold strains, we think there
may be upwards of 150 to 200. So anyway, let's think again
about the hygiene hypothesis and some of these findings
that I've shown you here with the COAST study.
You know, infections were, at least in the theory, were
supposed to be a good thing. You have infections and it helps
to strengthen your immune system and bring you toward tolerance.
But I don't think we're finding that.
At least not for respiratory infections.
What we're finding, if anything, is that certain
types of respiratory infections, for example, with rhinovirus
and probably RSV, actually put you more at risk
for developing asthma in the long run.
But actually, this hygiene hypothesis has evolved since
it was first proposed in 1989. And now, instead of focusing on
infections, most investigators are looking at
microbial exposures and animal exposures in early life.
And this work has come out of careful study
of western European farm houses. Actually, farming is quite
different in western Europe compared to here, at least
the traditional farming. Here you see a very traditional
house in Austria. So here's the house.
There's a little anteroom right there, and then
it's the barn. In the morning, the farmer
gets up around 4:00, makes himself a cup of coffee,
goes into the barn, does the milking,
goes back in and finishes his breakfast, and then
will go out into the fields. So you can imagine this is
very close contact between the family that lives in the house
and the animals that live in the barn.
And actually study of these homes and comparing them
to urban houses has shown a number of protective factors
associated with the rural lifestyle.
And this includes contact with animals, especially if children
are brought into the stable in the first year or two
of life. And pigs, for some reason, seem
to be particularly beneficial. Actually, there's a study
from Africa that shows if you grow up with a pig in your house
your very unlikely to have allergies or asthma.
So we're currently recommending that to all of our high risk
families. Cats and dogs, also,
in some studies. So how does this happen?
Well, the thought is that these animals bring
into very close contact very high levels of microbes,
such as gram negative bacteria, which have LPS.
It's known that this can activate innate immune responses
and may promote immune development in infancy.
Other factors that are associated with protection
are consumption of fermented beverages.
So, there are certain societies kind of like the Amish
and Mennonites here, that have a very traditional
lifestyle in Europe, fermented mare's milk is one
thing that can be consumed. If you drink it in early life,
you're unlikely to have allergies.
So these are very interesting epidemiologic associations
that the question is, what are the real mechanisms
involved? And although in COAST
there were relatively few kids who really grew up on a farm.
We had eight or nine kids, most kids were really raised
in Madison. We did have lots of families
that owned cats and dogs, so it provided us the opportunity
to look at effects of early pet ownership on allergic
outcomes like atopic dermatitis, wheezing, allergen specific IGE,
and also effects on immune development,
in particular those cytokine responses I showed you earlier.
This work was initiated actually by a medical student
Claudia Reardon, who's now one of our psychiatry residents.
When you look at pet exposure in the home, you think
with allergic families they'd be less likely to have pets.
Well, in Madison, on average 50% of households have pets;
of our allergic families it was 55%.
So they've been ignoring advice from allergists for a long time.
In our cohort, 68 had dogs, 33 had both a cat and a dog,
51 had cats only and about half of the families
had neither a cat nor a dog. So what's the relationship
between pet exposure at birth and subsequent atopic disease?
Again the COAST study allowed us a very nice way to look at this
in a prospective fashion. And in fact, when we looked at
the percentage of kids who have atopic dermatitis
in the first year of life and in the third year of life
we saw a significant reduction in atopic dermatitis
in kids who grew up with a dog in the home.
This was also true for allergen specific IGE,
although, in this case, the effect was really
most noticeable at year one. And by year three,
it was only a trend. We saw the opposite pattern
when we looked at wheezing in kids.
So a non-significant trend toward less wheezing
if you had a dog in the house. But this was a more substantial
effect by year three. So we know that dog exposure
is associated with a reduction in atopic outcomes in the
first three years of life. Is there an immunologic
explanation of this? First, it's important to realize
just how much is going on in the first three years
with immune development. And this is just a sample
of that concept right here. This looks at PHA-induced
cytokine responses. Remember, PHA is a very potent
mitogen that causes T cell cytokine production.
And we looked at this in cord blood year one
and year three. These are all the kids
in the cohort here. And you can see
this is a long scale. Just a marked increase
in IL-5 synthesis in the first three years.
Less pronounced, but still about a one log increase
in IL-10 secretion between years one and three.
This is very characteristic of most of the responses
that we've looked at. So a lot going on,
and the potential that environmental factors are going
to effect this development. So what Claudia did--
This is not Claudia here. But what Claudia did
was to take house dust samples from the bedrooms of a subset
of kids in our study. This was started when
the children were about three years of age.
We had a little over 100 kids who had never moved.
The families had never moved in the first three years.
So what we looked at were cat and dog proteins
This is a major dog allergen and that's a major cat allergen,
just as a quantitative measure of cat and dog exposure.
And then endotoxin, which is a measure of exposure
to gram negative bacteria, a microbial exposure.
What we found were significant relationships between
the amount of dog protein that was in the bedroom dust,
not in the living room dust, and IL-10 development
in these children. So at age one, a positive
relationship, and less marked at age three here.
So it looks like this was kind of a transient effect.
When we looked at IL-5, we saw something similar,
again a fairly reasonable positive relationship between
the amount of dog protein in IL-5 in the first year.
And this seemed to be fading by year three.
Notably, there were no associations between
owning a cat or this cat protein
and either atopic outcomes, the types of allergic diseases
these kids got, or cytokine responses
in the first three years. So we'll go back to our model
with the hygiene hypothesis. Again, it's been suggested
that both microbes and animal exposure were important
in helping to shape that early pattern of immune development
in a healthy way. I think what we have found
with the Madison cohort is that exposure to dogs
actually has an effect on immune development.
And this actually is broader than what I've shown you.
It extends to a number of cytokines including
IL-5, IL-10, IL-13. Probably the interferons.
We're still in the process of looking at this.
And these changes are associated with a tolerance
in these children. So this work is ongoing.
Obviously these kids are still growing.
And we're looking to see how long these effects will last
or whether new effects will surface,
whether continuous contact with the animals are necessary,
and what are the real long-term effects of viruses.
But what we'd really like to know here--
What we'd really like to get is some practical advice.
And people ask me, should I get a dog?
We're thinking about having a baby, should we get a dog?
And I guess that would be one approach.
There's certainly no rationale to get a cat.
( laughter ) But what we'd really
need to know is what are the mechanisms here?
It's not going to be practical to recommend pigs, or even dogs
for every household. But if we knew what it was
with this microbial exposure or maybe it's not
microbial exposure. ( laughter )
There are other things that happen when you have dogs
and cats at home. Maybe it's lifestyle factors.
We don't know. But I think this is
really going to be a goal, to find out how these early
exposures to viral infections, how these early exposures
to microbial products and to animals really effect
immune development. And can we use this to
develop preventive strategies to help families
with allergies and asthma. And with that I'd like to stop
and acknowledge my colleagues especially Rob Lemanske, who's
the head of Pediatric Allergy here at UW, and really
started the COAST study. Wai Ming Lee is the head
of our Virology Lab, has done a great job.
Again, I'd like to highlight contributions from three
medical students who worked hard, made progress,
I think they learned a lot. And they taught us
a few things as well. So Claudia, Magnolia
and Brigitte. Again, Pete Schult
at the Sate Lab of Hygiene, who helped us all along
with the virology and the folks
at EraGen Biosciences. So thank you very much
for your attention. ( applause )