Welcome everyone to Wednesday Nite @ the Lab, I'm Tom Zinnen, I work here at the UW-Madison Biotechnology Center. I also work for UW-Madison's Division of Extension, also known as Cooperative Extension, formerly. And, together with those folks and our other core organizers, Wisconsin Public Television, the Wisconsin Alumni Association, and the UW-Madison Science Alliance. Thanks again for coming to Wednesday Nite @ the Lab. We do this every Wednesday night several times a year. Tonight, it's my pleasure to introduce to you Robert Lemanske. He is a professor in pediatrics here at the UW-Madison School of Medicine and Public Health. He was born in Milwaukee, and went to high school at Nathan Hale High School in West Allis. Then, he came to UW-Madison and studied chemistry as an undergrad. He stayed here and got his MD degree. He did a residency here in pediatrics, and then a fellowship here in allergy and immunology, and then his mentor told him to go away. Was it that nice, or was it a little more gentle?

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[Robert]
 Close.

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[Tom]
 To go away so you can come back, which is really good. So, he went to the National Institutes of Health, specifically, the National Institute for Allergy and Infectious Disease. And then he came back in 1983 to be on the faculty. And now he's a professor here in pediatrics. He's also the Associate Dean for Clinical and Translational Research. And he's going to talk to us tonight about something that's pressing to a lot of us who have children or grandchildren, "When The Sneeze Becomes a Wheeze," discovering the origins of childhood asthma, and how this will lead to a cure. Please, join me in welcoming Professor Lemanske to Wednesday Nite @ the Lab. 
(applauding)

- Thanks, Tom.

Well, it's a real pleasure to be here, and thanks for coming out in the weather, and my previous schedule for coming here was the night of the polar vortex, so I guess in retrospect I'm glad we called that one off, because I'm not sure there'd be anybody in the audience, but, I'm happy you're all here. So, what I'd like to do in the time that I have is to talk to you about a topic that has been my whole life, and that is childhood asthma. Trying to determine the origins, basically how asthma starts in children, and then going on to if we think we know maybe what's happening, in terms of how it gets started, is there anything we could possibly do to prevent it, and actually keep asthma from developing in early life.

So, to get everyone on the same page, what I'd like to do first is to give you some facts, which I think will be very important for you to consider. First, there's close to 27 million persons in the United States that have this condition. There's over 10 million ED visits--

I'm sorry, MD office visits. We have close to 500,000 hospitalizations for asthma, so the morbidity is quite high. And, unfortunately, we also have mortality. We have over 5000 deaths per year in The United States. It disproportionately affects children, and black children and adults. And, importantly, from a pediatrician standpoint, 50% of children with asthma miss one or more days of school per year, which leads to about 14 million days of school missed in The United States, which is a huge problem for us. In fact, it's the most chronic cause of school days missed, or chronic disease in terms of school days missed

that children are afflicted with. And, in terms of the economic burden, the cost of asthma, at least in The United States, is close to $80 billion, and probably more at this point.

Now, there are two additional important points that I'd like to add to emphasize. First, asthma has been increasing in prevalence in the past twenty years. There's a lot of data to suggest that this might be related to the so-called hygiene hypothesis, which really means that we are now living in too clean of an environment, and we're subjecting our children in early life to living in too clean of an environment, as well.

The second part, is asthma usually begins in early life. In fact, when you ask adults, about 50% of adults with asthma will say that they began to wheeze in the first three to four to five years of life. And, that's really what I'd like to focus on during my lecture, and that is basically how asthma gets started in this early time period in life.

So, the title of my talk, "When The Sneeze Becomes a Wheeze," will be important for me to try to make these connections for you. So, we know from epidemiologic data and from a lot of different observations clinically, and also in the laboratory, that usually kids when they're going to go on to develop asthma, begin to sneeze early in life. And, if you think about the causes for sneezing, one potentially could be allergies, and the other could be the common cold, or respiratory tract infections. So, what I'd like to do in the next few minutes is to go over with you some of the interactions that we have discovered between the development of allergic sensitization, or the development of the allergic response, and how this interacts with our host defense against virus infections in the first four to five years of life, and how this sort of gets the asthma phenotype established to develop in the child by the time they reach school age.

So, let me answer this question first, how do allergies develop?

Well, it begins in a person who is atopic, and an atopic person has the genetic predisposition to the formation of increased levels of the allergic antibody, which is called igE. So, an atopic person really has the genes to become allergic, but that doesn't mean they're necessarily going to become allergic. Then, when the individual is born and begins to interact with their environment, in terms of the foods they ingest, the bugs that they're seeing in their environment, exposure to pets, exposure to molds, exposure to house dust mites and plants, such as tree pollen, or ragweed pollen. They can then turn these genes on. And if they turn these genes on, they develop what's called allergic sensitization. Now, we can test for allergic sensitization in the clinic, or in the laboratory. I'm sure many of you may have been evaluated for allergies, and that's why you're here. We can do the skin test, and I think you can see in the bottom left panels somebody who has multiple allergies, and we can also look for this in an in vitro system, where we can take somebody's blood, isolate the serum, and then we can actually measure the igE antibody that could be specific for foods, could be specific for cockroaches, at least in the inner city, this is a major problem. House dust mites in many parts of the world, and pets. And, we have some data from our laboratory to suggest that if you're going to get a pet, you should get it before the child is born, and our data would suggest that dogs are more protective against the development of allergic diseases, actually, protective against the development of certain allergic diseases and asthma,

perhaps, more so than cats. So, when I talk about that particular laboratory result, it makes me really feel good about saying that dogs are indeed man's best friend.

So, we have the genes; we activate the genes. The person becomes sensitized. And we can demonstrate this by the testing I just went over. But, that doesn't mean yet that the individual has allergic disease. The next step, is the targeting of this particular response

to an organ system. Somebody who has hay fever, it'll be targeted to the nose. Someone who has asthma, it may be targeted to the lung. And then, the thing that we least understand, is the same immunologic mechanism can actually kill somebody when they eat a peanut.

So, that's where we're going right now in terms of research, is trying to understand how this system, this allergic sensitization, this igE antibody formation, gets targeted to a given organ system.

So, that's allergies.

Let's talk about viral infections, and I'm sure many of you in the audience know this, but what causes the common cold?

And, we in our group here, have done a lot of work with the human rhinovirus.

Elliot Dick here, the late Elliot Dick on campus, and Palmenberg, her laboratory, Jim Gern's laboratory, one of my colleagues, have been looking at human rhinovirus species for decades. And we now know that there are three different types of rhinovirus, A, B and C. We knew about A and B for quite some time, but C was just discovered within the last ten years. And, the important part about C, is it binds to a different receptor than does A and B. So, if you work for industry, and you want to try to come up with some medication that could potentially prevent the common cold, you would like to be able to know the receptors that these different viruses bind to, to try and prevent the cold from occurring.

Now, we know that at least in childhood, respiratory infections are the most common cause of asthma attacks in children. In fact, up to 80% of asthma attacks or asthma exacerbations, are usually related to a viral respiratory tract infection, and, most mostly, related to the common cold, or human rhinovirus.

So, related to human rhinovirus, what have been some of the important discoveries that have been taking place here at UW? Well, it's hard for me to believe this, because when I was a fellow in training, this particular publication was really a very important discovery. And this was by Dr. Minor and colleagues, working with Dr. Elliot Dick. And, what they did is they looked in the general population here in the Madison area, and they found that the human rhinovirus was involved with asthma attacks in Madison school children. And, this was really the first observation that connected the rhinovirus species, if you will, with attacks of asthma in school children.

Then, Doctor Busse and I, Doctor Busse was my mentor, we began to do experiments in adults. Infecting them experimentally with rhinovirus, and then challenging them with antigen, to see if the infection would alter the response to the allergen challenge. And, what we found is, lo and behold, the infection did indeed alter the response to the allergen exposure, such that the individuals that we challenged developed an immediate bronchospastic response, which occurs in minutes, but when we followed them out 4 to 6 hours later, they had another episode of airflow obstruction, which is called the late phase reaction, which is really related to inflammation, which is a very important characteristic of asthma, airway inflammation. So, this was the first time that we really could say that an infection in some way altered the response to an allergen exposure.

So, this then led us to begin to think about in early life, when and how does asthma begin?

And, from some work that we performed in an animal model, in which we took rats and we infected them with paramyxovirus, which is a rodent pathogen, and we infected them at an early time in life, at about 3 weeks, which is when they're weanlings. And, we had two strains of rats, one that was an allergic strain, if you will, and one that was a non-allergic strain. And, when we infected them both, the allergic strain 8 to 10 weeks later went on to get asthma in a rat. And, we could tell that by how the airways looked under a microscope, and my colleague Ron Sorknis could do airway physiology in small rodents, and we could clearly demonstrate that the animals developed airflow obstruction, and they also developed airway hyperresponsiveness, or twitchy tubes, which is a major characteristic feature of human asthma.

So, we also learned then, with some additional experiments, that we could alter the immune system from a TH1 response, so to speak, to a TH2, and vice versa. And, by doing so, we could take those animals that were allergic, and make them less allergic, and we could actually prevent them from getting asthma. So, this was a huge observation, and it led us to say these results are so compelling, we have to test it out in the human. So, this led us to propose the COAST study, which is an acronym which stands for Childhood Origins of Asthma. And, this is a birth cohort that I put together in 1998, and it's a prospective study and a high-risk cohort, meaning one or both of the parents have either asthma or allergic disease. And, it's designed to evaluate the interactions among age, patterns of immune dysfunction, and virus and bacterial infections with respect to the subsequent development of asthma and allergic disease. Now, this is how COAST was set up. In 1998, our biostatisticians said that in order to answer the question we wanted to answer, to answer the question we wanted to ask, was to look at which kids developed persistent wheezing when they were 3 years of age. And then, in a second grant, when we were lucky enough to be refunded, we looked at them at 6 years of age, because we could diagnose asthma by age 6, but we could only diagnose persistent wheezing at age 3.

So, we had 289 kids that were enrolled. Again, this was high risk, at least one parent with allergies and/or asthma. We had the kids normally come for what's called "well child" visits, and the coordinators met the kids in the clinics when they were seen by their primary care docs, and they obtained a nasal lavage specimen. They actually put saline in their noses, sucked it out, and then we took this to the laboratory. And, the Wisconsin State Laboratory of Hygiene was kind enough to do virology on these specimens, so that we could see the background carriage rate of viruses in the community of these kids in early life. We also did nasal lavage specimens during symptomatic illnesses. And, when I think back about this, it was absolutely remarkable how we conducted this. This was an RO1 grant, and I only had $250,000 a year to be able to conduct this study until I turned it into a program project grant four years later. But, the coordinators were so devoted to this study, they would actually go into the homes, or go into the clinics when the kids were being seen for sick visits, and they would perform a nasal lavage to be able to detect what the pathogen was that was causing the problem.

In doing so, we were able to record the timing, the severity and the etiology of respiratory illnesses throughout childhood, which was an extremely new and important set of observations. We also, through blood testing, as I showed you previously, were able to do a longitudinal evaluation of the development of allergic sensitization. When did the kids become allergic, if they were going to become allergic, and what did they become allergic to? And, this was very nice for the parents, because they were getting all this testing done for free, and we communicated all this information back to their primary care docs, so they were getting all of this information at the same time. Then, we were able, at age 3, at the end of this RO1, at the end of four years, we were able to look at the development of persistent wheezing. We published these results in 2005, and then, happy to say, a 90% retention rate by the time the kids were 6. We were able to determine which of these kids went on to develop asthma by the time they entered school.

Now, what did we learn in terms of what these kids were wheezing with, in terms of the viral pathogens in early life, and those kids who went to get asthma when they were 6, and those kids who did not? What we're graphing here is the mean number of wheezing illnesses per year. In year one, in black, in year two, in gray, and year three in the open bar. And, the various viral pathogens are listed here. No virus, rhinovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, corona virus, metapneumovirus, adenovirus and enteroviruses, that at that point were not classified. Now, there's two important things about this graph. The first, if you look at the overall respiratory pathogen burden, it's much higher in the kids who went on to get asthma versus those that do not. And, if we specifically look at the number of wheezing illnesses related to rhinovirus from year one, to year two, to year three, it's going up each year. In contrast, the exact opposite pattern is being observed in those kids who don't go on to get asthma. Their chances of wheezing with rhinovirus are becoming less and less from year one to year three. And, the difference between these two patterns, is highly statistically significant.

We also looked cross sectionally at those kids who are allergic at age three versus those that were not. And, a RAST test is an abbreviation for a radioallergosorbent test, and we can actually measure antigen-specific antibody, igE in this case, to various respiratory pathogens. So, if a kid was RAST positive, they had allergic sensitization, if they were RAST negative, they did not. We also looked at the interaction between these two events, and the development of asthma at age six years. And what we find here this is the comparator group, those kids who never wheezed with rhinovirus in the first three years, and those kids who were not allergic at age 3. Those kids who were allergic at age 3, but did not wheeze with rhinovirus, you can see here that this particular development, becoming allergic, does increase your risk of going on to getting asthma. If they wheezed with rhinovirus, but they were non-allergic at age 3, their chances, their odds ratio of going on to getting asthma were substantially increased. And if they had the double hit, if you will, they wheezed with rhinovirus, they became allergic by age 3, their chances of going on to getting asthma were huge.

So, from what I've showed you so far, what have these findings in COAST taught us? Well, there seems to be, in terms of asthma, both inception and having asthma attacks, once you get asthma, that contributing factors are the development of allergic sensitization, in terms of antigen-specific igE antibody formation, and the development of virus-induced wheezing illnesses. So, an important piece that was unanswered when we were doing this particular study in those first six years, is which is the horse and which is the cart? Does allergic sensitization precede the development of virus-induced wheezing, or is it the reverse? Does the virus get in the lower airway, strip the airway epithelium, the surface of the airway cells in the lower airway, and in doing so, allow antigen to penetrate through the mucosa, the airway, activating immune response cells and allowing the kid to become allergic? And, what we found in some very cool modeling that our biostatisticians did, is that definitely allergic sensitization precedes the development of virus-induced wheezing illnesses. Now, there's a tremendous interaction between these two, and we've gone on to look at mechanisms in which this occurs. Now, we know when somebody becomes allergic, their host response to virus infection changes. Their ability to produce interferons, which is a major cytokine, which dampens the response to viruses and prevents, or at least attenuates viral replication, is significantly attenuated. And, there are mechanisms for that, and I can draw them for you on the board if you're interested, but this has now been demonstrated not only by our group but by other groups, and so the development of this sensitization process in some way influences the host response to viral illness.

Now, the next question we wanted to answer is can genetic findings be even more informative? We looked at host response, we looked at the environment in terms of allergic sensitization. We now wanted to drill down to look at genetics, and whether or not either an isolated genetic defect or some type of gene by environment interaction might influence some of the results that I've shown you.

So, when we looked at asthma susceptibility genes and their relationship to rhinovirus wheezing illnesses. And for those of you who aren't geneticists, we looked for the genes that code for asthma development in early life, and what we found is they could be linked specifically with RV childhood wheezing illnesses. And, the genetic locus that seems to be the most important, at least so far by GWAS analysis, meaning you'd look at the whole genotype, is a locus at 17q21. So, you're probably asking, well, what does 17q21 mean? Well, if you look at human chromosomes, and this is a human chromosome in green, and then you look at a specific locus on the chromosome, in this case, the important locus for us was the 17q21 locus or region, and then you can break apart this locus into single nucleotide polymorphisms, or SNPs.

And, what we did, and we published in the New England Journal of Medicine in 2013, is we looked at asthma prevalence based on genotype at a certain locus within the 17q21 region. So, these are kids who when they get one component, one allele from the mother, one allele from the dad, these are kids who are homozygous CC, these were kids who were heterozygous CT, and these were kids who were homozygous TT. Now, the first thing I want you to see, is if you look at the blue line, these are kids that did not wheeze with rhinovirus in the first three years of life. So, regardless of what genotype they had at this particular SNP, their chances of going on to get asthma were absolutely no different.

In contrast, if they were homozygous, they had one T, or if they were homozygous TT, their chances of going on to getting asthma were huge. In fact, kids who were homozygous TT, 90% of these kids who wheezed with rhinovirus in the first three years of life went on to get asthma. And, the importance of that is that if we want to design therapeutic studies,

and you're a parent and we're going to do so some things with your child that you may say, "Well, how do you know my kid's going to get asthma?" Well, if we have things like this, that could help us predict risk, it would be very important for that message to relate to families, to allow them to really feel more confident putting their child in these kinds of studies would be potentially very helpful.

So, from what I showed you so far, how can our COAST findings combined with other results inform us? Well, this is a slide that I put together for one of our journals. We know that there are genes that are important. I've only had time to talk to you about the 17q21. But there's another locus called CDHR3, which is actually a gene that codes for the receptor for human rhinovirus C. We're very very intrigued by this, and our laboratories now are in the process of designing experiments to be able to really drill down on this finding. But, there are other locus that have been described that may influence either asthma development or asthma severity or asthma exacerbations.

Now, in terms of the environment, I went over with you viruses, at least the human rhinovirus. We are now doing some work with bacteria, and in many cases, there seems to be, they can act as co-factors to make the child sicker. And this is a topic for a whole, probably 7-hour microbiome, on what influences certain environments. Particularly, living on a farm seems to very protective, if you as the mom are in the farm working with the animals, and the child, you take them to nurse them or put them in the barn where you're doing other tasks, it's extremely influential in actually preventing asthma. In fact, some of our colleagues in Australia where the barns are linked. Not Australia, Germany and Austria, where the barns are physically linked to the houses. The rates of these kids going on to getting asthma is about 0.

Pretty important. Now, they're trying to figure out what's in the microbiome that's good and what's in the microbiome that's bad. And, we're trying to figure that out too in some of the work that we're doing. Now, also, people can develop allergic sensitization. People can wheeze with different viruses, but that doesn't necessarily make them get asthma. So, we also think that there probably are critical developmental windows, either in the development of the lung or the immune system, in which these events have to interact to be able to trigger the development of the asthmatic phenotype.

So, finally, hearing what I just had to say, I'd like to give you an idea of some of the prevention trials that we're currently involved with here at UW.

So, the two trials we have currently in progress, the one is called the PARK trial. This has been funded by the NIH. We are one of ten clinical centers. And what we're doing here is we're taking kids who have a pretty, not definite signal, but kids who are at high risk. They may have eczema. They have a parent who has allergies or asthma. And we're actually treating them with an anti-igE antibody between the ages of 2 and 4. This antibody is commercially available. It's called Omalizumab. It's been out for a long time, a good safety record and has been extremely valuable to us in treating patients with more severe asthma. So, the thought here, is if we can stop the development of the allergic sensitization process, we can actually prevent the response, the reduce response of the host to viral infection, and ultimately prevent the development of asthma.

The second study we're doing-- Well, we're not there yet with viral vaccines, but there are many centers that are currently thinking about this and doing some preliminary work. But we are also involved here at UW with a trial called ORBEX, in which we are feeding children between the ages of 6 and 18 months with a lyophilized extract from bacteria, so these are killed bacteria that we're having the kids ingest to change their microbiome, and there's data to suggest that if you alter the gut microbiome, you can ultimately, also, alter the microbiome of the lung. Now, I'm the lucky guy that gets to come to talk to you on Wednesday night, but this is just a small number of people who have been my colleagues and friends through the years who have helped to contribute to over 85 publications from COAST. We've also presented data from the COAST project in five continents, which we're pretty proud of. I'd also like to thank the parents and the children. The children now are 18 to 19 years of age. I've actually seen kids in the COAST study, and they asked me, when I get married and have babies, can my child be in the COAST study, so they've been taken very good care of by our coordinators. So, finally, if you're interested at all in knowing more about what we do and not only the studies that I just showed you, but we also have ongoing studies in adults. And if you call this number you'll get the Pediatric Asthma and Allergy Research Program. But, if you're an adult and you want to know more about the trials that we're doing in the adult population, these coordinators will clearly get you to the right place. But, if you have kids who you think might qualify, we'd love to hear from you, so that we can hopefully find a cure for childhood asthma. With that, I'm going to stop, and I'd be happy to answer any questions. 
(applauding)

 at the UW-Madison Biotechnology Center.