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,
for childhood asthma. And my disclosure slide.
I'm a consultant for Merck, EraGen Biosciences,
who did some of the work
viral diagnostics, and collaboration to develop
As an overview, I'm going to and GlaxoSmithKline.
some of the recent epidemiology start ofin asthma.ng about
Epidemiology was never
but it's actually provided my favorite subject in school,
of the environment in asthma, some of the disparities when you
think about how asthma's
even across the United States. sprinkled across the globe, and
about viral infections Next we'll talk a little bit
These have long been regarded as important risk factors
in asthma.
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
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 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
with these studies. scFrom my perspective as anped
investigator it's wonderful to
medical students, havefor a couple of reasons.ory,
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.
helped my research program And I think that this has really
So first of all, getting at the epidemiology, it's quite clear
that asthma, like a number
really is growing in prevalence. of other inflammatory diseases,
from the National Health And you can see that graph here
The CDC, who does this survey, Interview Survey.
of how they define asthma. actually changed the definition
You can see about a doubling This is done by phone survey.
This is asking about current over a 15 year period of time.
to asthma in the last 12 months. asthma, which is very similar
leveled off, but they've leveled You can see things may have
And the question is, why are off at a very high rate.
And this parallels increases we seeing more asthma.
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
many different languages different cultures,
with this. and there are difficulties
there is no word for wheezing, In fact, in some languages
But they tried to level the playing field by actually
showing videos
And what you see here whare a number of symbols.s.
The stars are places
in 13 to 14 year-olds, where you see wheezing
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
With higher rates also and New Zealand.
And asthma is really quite low, less than 5% in eastern Europe,
in places, in rural places
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
the way a lot of people think thabout preventive adviceed
in infancy. And this is called the so-called
hygiene hypothesis. And the idea here is that
at birth the immune system
You can take one of two turns in response to the type
of environmental exposures
One leads to tolerance and in early life.
a so-called Th2 bias. hSo this is an immune systemto
where the T cell, the memory T cells tend to
overproduce cytokines like an IL-4 and IL-5
that promote IGE synthesis and
that's very typical of the eosinofilica inflammation
And the theory is that the type alof 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
help to steer you toward health. in immune development and
situation where you have an only child, maybe no exposure to
infections, a relatively sterile
immune system doesn't have environment, then this immature
it develops this immune bias normal development, and instead
and asthma. anWell, to be able to evaluates
these possibilities,
the immune response and early environmental exposures,
even before you start developing
And in 1998 and 1999, signs Rob Lemanske and Isthma.
in the Department of Pediatrics with help from Pete Schult
in the Virology Division
Ron Gangnon of Biostatistics here in Clinical Virology,
our lead coordinator, started the COAST study,
or Childhood Origins
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
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.
of the first year of the COAST And this shows an illustration
that were done. stSo, since they were prenatales
enrollment, we were able to collect samples of blood
from cord blood.
discarded with the placenta. This would otherwise be
cells and look at specific We could take these mononuclear
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,
go to the doctor's office one oand collect a sampleould
of nasal mucus so we could look for respiratory viruses.
In addition, whenever the families noted illnesses at home
they would call our coordinators
at McDonald's, at the car wash, awherever, and get a sample of,
nasal mucus and some information
And then yearly, about this acute illness.
provided a small sample of blood these children were seen and
the immune studies using so we could continue
and look for evidence tof specific viruses.
So for this trial, in which we had I think 1,500
to 2,000 nasal specimens
as well as another similar trial in the first couple of years,
that we needed to have in four inner cities, we decided
The types of virus we were a new type of viral diagnostic.
every respiratory virus inand they're listed here,y
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
PCR based diagnostics are helpful, but we decided to
partner with EraGen Biosciences to develop what's called the
Respiratory MultiCode Assay,
And Magnolia Prince actually which uses multiplex PCR.
testing of this assay and then helped us with the initial
samples from the COAST study. So the steps are shown here.
Actually the chemistry is unique
nonstandard bases. in that there are two
don't bond to any other bases, So there's Iso-C and Iso-G that
but bind very well natural bases in the genome,
Generally, most of these viruses to each other.
a reverse transcription step are RNA viruses, so you do
These steps incorporate these andnon-standard bases, whichre.
gives you a little better avidity of binding.
And also some unique tags.
extension. So this is a target-specific
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.
showing specific detection And this is the readout
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.
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,
and really adaptable very specific assay,
And this shows the results for a large clinical trial.
samples that were obtained of viral diagnostics on the
and sick visits of the first during both well child visits
So it's grouped. year of the COAST study.
the viruses that were detected Here on the left, these are
symptoms, then mild symptoms, in kmoderate and severe.ory
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
of studies showing up to around a third of healthy infants
have respiratory viruses
at any one time. that you can detect
are just getting over colds. Now some of these kids
maybe a little bit Their symptoms are gone but
A small percentage of these kids of the virus lingers on.
But a lot of these are really asymptomatic infections.
You'll see that in children with more significant illnesses
you see about 90% recovery
which is very good. ofAnd also the type of viruss,
that you find varies
So rhinoviruses in yellow wwere the most common viruses
overall.
really found predominately Viruses, such as RSV, were
degrees of illness. in tThis is also trueeater
for multiple viruses. Some of these children had two,
three and even four viruses at once.
Most of those kids
And the 1998-99 season were in daycare.
for influenza win the light blue here.t
And this can vary dramatically
And of course you know from year for year.
this is just a huge problem. every ten years or so,
from the perspective You can also look at this data
Those were means according of the child.
In this graph, each of these to the type of illness.
So this is the birth date. lines is actually one child.
on the bottom, the birth date, This is calendar year
And each symbol and this a viral sampling.here.
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,
consequences of having what are the long-term
That actually the sickest child thehad 13 moderate to severerns.
illnesses in the first year.
we actually looked at the history of viral illnesses
in the first three years of life
to an asthma evaluation in these kids at age six.
These kids are now between
at present. eigThe asthma evaluationage
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
But it was blinded medications.
for the first three years. in terms of the virology
analysis is, on this graph here, And what we showed with this
is really the life history what I have in the upper graph
of children who went on for to develop asthma.ars
And the lower graph is the
of infections related history ofto wheezingthree years
in kids who did not have asthma.
I think that you can see is that children with asthma
had a rougher time
Really, virtually every virus ihas increased compared to.
the non-asthma group here.
stands out is that rhinoviruses cause most of these wheezing
illnesses in the asthma group.
year one, to two, to three, you see an increasing rate of
respiratory wheezing illnesses
versus a reduction in wheezing in the asthmatic group
in the non-asthma group. each of those years
of pathogens, but also a So not only a different pattern
in terms of the first few years. different pattern of development
likely to cause these wheezing The two viruses that are most
are respiratory syncytial virus, illnesses early on
virus that causes bronchiolitis which isand rhinovirus.rototypic
So what we wanted to do was to
between these viruses lookand the rate of asthmaions
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
in asthma out at age six. And then kids who wheezed
with rhinovirus.
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.
these studies are demonstrating Sis that childhood asthmat
is really associated with a different pattern
of respiratory viral illnesses
Rhinovirus infections seem to be very indicative
of an increased risk of asthma
And the frequency of illnesses out at age six.
in the first three years. Now, there are a number
of other studies that show
those leading to hospitalization that severe infections with RSV,
also indicate risk or acute care visits,
So one other question when for asthma.
is whether there are other you look at this data base here
of respiratory viral infections factors tin early life. severity
We'll go back to this graph showing the COAST data.
One thing that you'll note
for example, can be found iin infants with no illness,s,
with mild, moderate or severe illnesses.
And the proportion is fairly
We know rhinovirus similar across this range.
This was shown back in the '50s. is a pathogen.
and they get a cold. You give it to someone
may be either host factors So it suggests that there
don't protect kids from getting more severe illnesses,
and things like interferons,
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
And if the pattern follows, iwhat we see with influenza,.
and RSV, and others,
serotypes are likely to be it's likely that some of those
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
from peripheral blood, and separate these out
with various stimuli. incSo we picked PHA, which isry
a very potent T cell stimulant
and also one type of rhinovirus and one type of RSV.
And then, we analyzed the production of a number of
different cytokines, including
that have been implicated in allergies and asthma
like IL-5 and IL-13,
like interferons, ainterferon alpha and gamma,s
and IL-10, which appears to have
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
So these are cortiles, with lowest response.
and highest response. theAnd there's some effecthest
on the proportion of kids who developed moderate to severe
rhinovirus illnesses
But where you really see in the first three years.
you look at the more severe rhinovirus illnesses, so those
associated with wheezing
And here you see there's about and lower respiratory symptoms.
interferon and alpha categories a 50in those who wheezedhese
with rhinovirus, going from 39% in the lowest category
to 19% in the highest category. So, clearly, host factors
do make a difference
This is looking at blood cells. iWhat about airway cells?.
This is where the infections
So we've moved forward to address that question as well.
Do airway immune responses
So for these studies, which were differ in wheezing children?
we looked at wheezing status conducted by Brigitte Smith,
the parental report of wheezing at agin the past 12 months.s to
We then took the nasal lavage samples that were obtained
during scheduled study visits and also during wheezing
illnesses, and we took
for rhinoviruses the samples that were positive
for the content of cytokines. This just shows that process.
Actually, we came up
nasal mucus in kids. with a technique to sample
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.
when they were infants. But when kids started to
be able to blow their noses,
technique, which some of we developed this nose blowing
What you do is you actually thtake a plastic Ziploc bag,.
you squirt a little bit of saline in the nose,
just a nasal spray
into this plastic bag. and have them blow their nose
comes out, the various colors, And the kids love to see what
or two. aBut you can take this nasalt
lavage and analyze this not only for viruses, but also
for cytokines and Brigitte used a so-called Luminex,
kind of a multiplex technology
that were involved in the acute tresponse to viral infections.es
Also, interferons and IL-8, which is a chemo attractant
for neutrophils, which is the most frequent cell
that comes into the airway
So, she asked us with these studies.
First of all, is there several questions.
of cytokines a difference in the types
during periods of illness versus periods of health.
And you can see that sick kids
in the airway, tended to have more IL-6
and also interferon gamma, slightly more IL-10
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,
where they didn't wheeze. So she compared these cytokine
levels in wheezing illnesses
to see if this immune response versus non-wheezing illnesses
And actually it wasn't, wait was quite similar withinps.
these wheezing children whether or not
they had wheezing illnesses.
different was if you looked at But what did seem like it was
versus the non-wheezing children the wheezing children
Here you found IL-6 duringin both groupsesses.
and IL-8 in both groups.
trending different anyway is an increased amount of IL-10
and increased amount
in children without wheezing. Again suggesting that
interferon synthesis
the severity of airway disease may be important in limiting
So the other question is in these children.
factors that help to determine whether there are virus-specific
or not so ill. whether a child becomes very ill
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
we're going to have to analyze of viruses, we anticipate
and 1,000 samples before somewhere between 750
the more pathogenic ones. we But Wai Ming Lee and ourly
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
known strains of rhinovirus and fully 53, or more than half,
appear to consist of new strains
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.
and there are certain conserved areas that are really
pretty much the same
And these are used for in every rhinovirus.
so you can find these viruses. the design of PCR primer
is there's a stretch of Butabout 220 bases hereng
in between these two primers
for each strain. that's actually different
of what these sequences were So Wai Ming developed a library
and then compared fthe field isolets to these.es
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.
the five prime end This is, again,
So anything more than that I10% divergencefore.
represents a new strain.
when you group them together, Andthere's two groups.ses,
The A group, which is the big group right here.
And the B group.
a genotypic phylogenetic And again, this is just
This is a phylogenetic analysis of the new strains
compared to the known strains,
of rhinovirus. previously known strains
rhinovirus A strains, in red, Inand rhinovirus B strains.ere
The new ones that Wai Ming found
another branch off are shown here, so it looks like
another branch here. of the A tree,
of rhinoviruses, which we call But importantly, this new group
seem to be as different from A rhinovirus C for now,
And these rhinoviruses as they are from B.
They're also distinct arfrom known enteroviruses.ly.
So he's continuing this work. The implications are
that instead of having 101
may be upwards of 150 to 200. So anyway, let's think again
about the hygiene hypothesis
that I've shown you here and some of these findings
You know, infections were, with the COAST study.
supposed to be a good thing. at least in the theory, were
to strengthen your immune system You have infections and it helps
But I don't think and brwe're finding that.rance.
At least not
What we're finding, if anything, is that certain
types of respiratory infections,
and probably RSV, actually put you more at risk
for developing asthma in the long run.
But actually, this hygiene
it was first proposed in 1989. hypothesis has evolved since
infections, most investigators And now, instead of focusing on
microbial exposures and animal exposures in early life.
And this work has come
of western European farm houses. out of careful study
different in western Europe compared to here, at least
the traditional farming.
house in Austria. Here ySo here's the house.ional
There's a little anteroom right there, and then
it's the barn.
gets up around 4:00, In the morning, the farmer
goes into the barn, makes does the milking,coffee,
goes back in and finishes his breakfast, and then
will go out into the fields.
very close contact between the So you can imagine this is
and the animals famithat live in the barn.ouse
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
are brought into the stable animin the first year or tworen
of life.
to be particularly beneficial. And pigs, for some reason, seem
from Africa that shows if you grow up with a pig in your house
your very unlikely
So we're currently recommending that to all of our high risk
families. Cats and dogs, also,
in some studies.
Well, the thought is that Sothese animals bringen?
into very close contact very high levels of microbes,
such as gram negative bacteria, which have LPS.
It's known that this can
and may promote aimmune development in infancy.s
Other factors that
are consumption areof fermented beverages.tion
So, there are certain societies kind of like the Amish
and Mennonites here,
lifestyle in Europe, that have a very traditional
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
We had eight or nine kids, who really grew up on a farm.
in Madison. We did have lots of families
that owned cats and dogs, so
to look at effects of early itpet ownership on allergicity
outcomes like atopic dermatitis,
and also effects wheezing, allergen specific IGE,
in particular those cytokine on immune development,
This work was initiated ractually by a medical student.
Claudia Reardon, who's now
When you look at pet exposure one oin the home, you thinkents.
with allergic families they'd be
Well, in Madison, on average less likely to have pets.
of our allergic families 50% of households have pets;
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
had neither a cat nor a dog. and about half of the families
between pet exposure at birth and subsequent atopic disease?
Again the COAST study allowed us
in a prospective fashion. aAnd in fact, when we looked at
the percentage of kids
in the first year of life and in the third year of life
we saw a significant reduction
in kids who grew up with a dog in the home.
This was also true
although, in this case, fothe effect was reallyGE,
most noticeable at year one.
it was only a trend. And by year three,
when we looked at wheezing We saw the opposite pattern
So a non-significant trend in kids.
if you had a dog in the house. toward less wheezing
effect by year three. BuSo we know that dog exposurel
is associated with a reduction
first three years of life. in atopic outcomes in the
explanation of this? Is there an immunologic
just how much is going on Firsin the first three yearslize
with immune development.
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
in IL-10 secretion about a one log increase
This is very characteristic beof most of the responsese.
that we've looked at. So a lot going on,
and the potential that
to effect this development. envirSo what Claudia did--going
This is not Claudia here. But what Claudia did
was to take house dust samples
of kids in our study. fromThis was started whenbset
the children were about three years of age.
We had a little over 100 kids who had never moved.
The families had never moved
So what we looked at in the first three years.
This is a major dog allergen were cat and dog proteins
just as a quantitative measure and that's a major cat allergen,
And then endotoxin, which is a measure of exposure
to gram negative bacteria,
What we found were significant a microbial exposure.
the amount of dog protein that was in the bedroom dust,
not in the living room dust,
in these children. and IL-10 development
relationship, and less marked So aat age three here.ive
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
Notably, there were no associations between
owning a cat
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.
that both microbes and Again, it's been suggested
in helping to shape that early animal exposure were important
in a healthy way. paI think what we have foundt
with the Madison cohort is that exposure to dogs
actually has an effect
And this actually is broader on immune development.
It extends to a number than what I've shown you.
IL-5, IL-10, IL-13. Probably the interferons.
We're still in the process
And these changes are associated of looking at this.
in these children. with a tolerance
Obviously these kids So this work is ongoing.
And we're looking to see are still growing.
or whether new effects how long these effects will last
whether continuous contact will surface,
and what are the real long-term with teffects of viruses.ssary,
But what we'd really like to know here--
What we'd really like to get
And people ask me, is some practical advice.
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 )
need to know is what are the mechanisms here?
It's not going to be practical
for every household. to recommend pigs, or even dogs
with this microbial exposure But if we knew what it was
microbial exposure. ( laughter )'s not
There are other things
and cats at home. Maybe it's lifestyle factors.s
We don't know.
really going to be a goal, But I think this is
exposures to viral infections, to find out how these early
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 acknowledge my colleagues especially Rob Lemanske, who's
the head of Pediatric Allergy here at UW, and really
started the COAST study.
of our Virology Lab, Wai Ming Lee is the head
Again, I'd like to highlight has done a great job.
medical students contributions from three
I think they learned a lot. who worAnd they taught usgress,
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.
for your attention. ( applause ) So thanery much