112 James Gern

>> 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 role 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)