9UNP0150SD_Brandon Nathan_WordPress

- I can't tell everyone what a pleasure and honor it is to be back at the University of Wisconsin to give Grand Rounds today. I really appreciate the invitation to have an opportunity to talk to you a little bit about the clinical trial work that we're doing at the University of Minnesota, as well as across the country and internationally. And I'm hoping that I'll be able to bring you up to speed on some of the newest things that are happening in that field. I felt like it was important to kind of give you an idea a little bit about what was happening and what I've done since I left Wisconsin, with a little bit of a twist on the Big 10. And as Dave mentioned, I did go to Rainbow Babies, and I had a great fellowship training experience. We really have enjoyed the upper Midwest, and so we stayed in Big 10 country, which is both good and bad. I had a great fellowship training experience. However, I was inundated with this guy in the state of Ohio which was really annoying, after being here in Wisconsin, as my first Big 10 school, and sort of being partial to Bucky. So I'm really lucky and fortunate about the position I've been able to take over at University of Minnesota. 

We're really enjoying our time up there. But of course, you've got to deal with this Goldy the Gopher all the time. That's why it's such a treat to come back, because I really have a place in my heart for Bucky and Wisconsin. And I thought it was important to also let you know about some of the changes in my life personally, and also as an excuse for not showing up for seminars, which I still feel bad about. But I did have a good excuse. This is Allison Nathan. She is now 2-1/2. And this little Colin here, who was born a few days after the start of seminars. But I do again apologize for not being able to come down for seminars. And here's Colin just a few weeks ago. 

So really, our lives have really been enriched by these kiddos. So what I'm hoping to do today, over the next 45 minutes, or so, is to really be able to bring you up to speed, review a little bit about the pathogenesis behind Type 1 diabetes, and talk a little bit more about past history of prevention trials, and what we're currently doing with Type 1 diabetes TrialNet to try to, again, prevent or provide early therapy for patients that have recently been diagnosed. And along the way, these are the objectives that I'm hoping you're able to obtain. The first is to learn the importance of both genetic and environmental factors, and the pathogenesis of Type 1 Diabetes. The second is reviewing the recent trends regarding prevalence and incidents. And we'll talk a little about the trends that are showing in increased incidents of Type 1 Diabetes. Third, is going to be talking about the different stages during the natural history, or what we believe the natural history to be, that provide, really, an opportunity for intervention. And then lastly, just to be able to recall just a few of the studies that we have ongoing for patients of yours, if they're interested in enrolling in some of these studies. So first off, I thought it was important to talk a little bit about where we've come in Type 1 Diabetes. And this is Frederick Banting, who was the primary discoverer of insulin. 

He and his student, Charles Best, in Toronto identified an extract from the pancreas of dogs that eventually led to the discovery of insulin. And that was a really, obviously groundbreaking change for these patients with diabetes. Unfortunately, we didn't have a way to actually provide a lot of insulin for patients of that time. So we moved to actually giving them pig insulin, and insulin from cows, which again, provided more insulin for the patients that needed it. Unfortunately, there was still limited supply. And there were allergic type reactions to that insulin. And eventually, we were able to come up with recombinant DNA technology, which is how all the insulin is made now. And that's really provided insulin to everyone that's needed it. We have much better ways of testing blood sugars now and monitoring disease. Very small amounts of blood are needed to check blood glucoses. 

We no longer have to test urine for glucose, or even taste urine for glucose, which was what they used to do way back when. We've got insulin pen devices. We've got insulin pumps. And we have wireless or tubeless pumps that provide insulin without any attachments. And then finally, most recently, we have continuous glucose monitors that provide real-time evaluations of interstitial glucose levels to help with glycemic control. So we've really come a long way in terms of how we treat Type 1 Diabetes. And this has been important, because we've also learned more about complications of long-term diabetes, and what those mean for people that don't have good control. And you can see in this graph, from the diabetes complications trial, that your risk of all these microvascular complications: retinopathy, neuropathy, and nephropathy, as well, all go up as your hemoglobin A1C goes up over time. So when we're thinking about diabetes, we really want to design strategies to try to keep blood sugars as normal as possible. Keep your hemoglobin A1C as low as possible for these patients. 

Now unfortunately, that does come at the cost of having frequent hypoglycemic events, so as you're hemoglobin A1C levels get down to a more normal range or physiological range, the incidence of hypoglycemia goes up. So what we're really trying to do is balance, trying to keep normal blood sugars, reducing the risks of complications, but also reducing the risks of hypoglycemic events. So with that as a bit of a backdrop, let's just talk very briefly about Type 1 Diabetes, and then we'll talk a little bit more about some of the studies that we're doing. Type 1 Diabetes is an autoimmune mediated destruction of the beta cells within the islets of the pancreas, which results in loss of insulin secretion. Also you know the diagnostic criteria, or clinical symptoms that are associated with Type 1 Diabetes are polyuria, polydypsia, weight loss, and about 50% of children will present in diabetic ketoacidosis. Probably most importantly when we talk about prevention studies is that we can identify markers in the bloodstream that tell us this is an autoimmune process, so somewhere in the neighborhood of 90-95% of new onset patients will have at least one diabetes auto-antibody in their bloodstream. The reality of living with Type 1 Diabetes is that we can care for it actually quite well right now, and people live really relatively normal lives. But it is a change in their life, no doubt about that. It's a life changing event, that most kids and adults are checking their blood sugars somewhere between four to six times per day. There are risks of hypoglycemia and long-term complications, as we mentioned, as well as acute complications. 

And so we really haven't, although would we have come up with great ways to treat the disease, we haven't found the best ways to prevent the disease from occurring. Now what's been interesting and a phenomenon that's been happening probably over the past 30 to 40 years, is there have been signs that the rates of Type 1 diabetes have increased both in the United States and worldwide. We have, as a country, we really haven't been as good as the Europeans in tracking and monitoring Type 1 Diabetes. They have much better designed registry programs. They're doing much better jobs, I think, in tracking the incidence and prevalence data. But we have a new study that's going on right now, which is tracking the information in about five different centers across the country. So this is some recent data that's come out. In 2001, there was a prevalence of about one and a half cases for every thousand youth up to the age of 18, and the incidence for Type 1 is about 20 cases for every 100,000 person-years. Now when you compare that to previous data, that was back from the 1980s, and some small registry programs in different places across the country. There is an increase. 

It's up from somewhere in the neighborhood from 12-15 cases, which was the incidence back in the 1980s. And the fastest growing population are younger children. More recent data from Colorado, also shows this increase of about two to three percent per year. And when you look globally, that's also what you see in most global registries, about a two to three percent increase per year. And this is some data that comes out of Oxford, which I wanted just to point you to this younger aged group here, this 0-4 years, and their incidence, that's been slowly climbing over the past 20 years. So it is becoming even more of a problem. So I'm going to try to put this into a very brief description of why diabetes occurs and why it doesn't occur, although this is really an exploding area and we're still learning a lot more about it. So what we know is that there are certain genetic markers that predispose you to risk. Previously, it was thought that the HLA complex probably gave you most of the risks, and those was really the primary genes that were responsible for inducing genetic risk. Well, we now know that there are probably many more genetic markers and many more genes that are involved in the pathogenesis here, that we don't completely know yet. 

As I mentioned before, this is an autoimmune disease, and there are several different cell types within the immune system that participate in this. So Type 1 was previously thought to be exclusively a T cell mediated disease. We now know that, as I'll show you in a minute, B cells also play a very important role in the pathogenesis. We also think there is a environmental insult that causes the disease to proliferate, and this autoimmune process to sort of spiral until-- or initiate this autoimmune process, which leads to the eventual loss of beta cell function. And lastly, we now know, or we think again, that this is more of a gradual process that probably occurs at least over months, and probably over years in several people. With that knowledge, that really provides us an opportunity to hopefully intervene in some of these high risk patients, or patients that do have evidence for autoimmunity. So again talking about the risks conferred by genetics. About half of the genetic risk can be explained by the HLA complex, specifically the Class 2 sub-region. And the most important alleles there are the DR3 and DR4 markers, so as we go through some of the studies that we're doing, we'll talk about, that's one of the screening tools that we're using for some patients to look for the highest risk population. There are some other alleles that also impose risk. 

And there are also parts of alleles in the HLA complex that actually provide protection against developing Type 1 Diabetes. There are a few other genes, in particular the insulin gene, or actually an upstream promoter on the insulin gene that probably confers about 10% of the risk for Type 1 Diabetes. And this really speaks to the concept of lymphocytes that escape thymic regulations. So people that have smaller tandem repeats in this promoter region on the insulin gene, they actually have a higher incidence of diabetes. So the thought is that they escape normal regulation, and that they're involved in the pathogenesis for Type 1. There are probably several other genes, CTLA4, which we'll talk about at the very end of the talk, as well as several others that have been proposed. And there has been some associations and some studies, and it hasn't been found in others. So we're still trying to learn more about the genetic risks. I mention that allele. So who's at greatest risk? 

This just shows you, like within families, that the risks of diabetes go up if you have a first-degree family member. Important here is that most new onset cases occur in families that don't have any history of Type 1 Diabetes. It's about probably 85-90% of children that develop Type 1 don't have a family history. However, in terms of trying to learn more about the disease and prevention strategies, this is the population that we really want to target, because these are the highest risk patients that we know of. So in the general population, your lifetime risk is somewhere in the neighborhood of 1 in 300 to 1 in 500. And if you're a child that's born to a mother with Type 1 Diabetes, you have a risk of about two to three percent. And if you're father has diabetes, that risk goes up. So there are probably some gender specific genes that are involved there. That risk is closer to six to nine percent. On average, it's about a five percent chance if you have a sibling with Type 1 Diabetes. 

And if you have an identical twin who has Type 1 Diabetes, your risk is 50%. Now I think that's really interesting because that also speaks to the fact that there is some environmental trigger here. Otherwise, we would expect to see that to be 100%. And if you share this high risk allele marker, your risk goes up by five fold compared to the normal risk. So again this high risk marker is present in almost all patients, or at least Caucasian patients with Type 1. Now this is what you typically would see in most cases, if you were able to get a pathology slide of the pancreas. I felt I had to add at least one pathology slide, just building off of problem conference history. So this is what you would typically see. And these slides come from patients that were very ill during the time of diagnosis, from several years ago, who eventually died. And what you see is this lymphocytic infiltration of the pancreas, specifically in the islets. 

These are the beta islets that are stained red here. And these lymphocytes are primarily T cells, but there's also B cells involved here. And what's happening on a cellular level, in a nutshell here, is that you have these presenting cells. So in this example as a dendritic cell, interacting with naive T cells. And this is sort of how the process begins. The important interactions that they make here are with this MHC peptide, which is where the antigen is presented to the T cell. That binds with this T cell receptor, but there are also very important co-stimulatory, secondary co-stimulatory signaling that occurs. And this example between CD40 and CD154. And that co-stimulatory reaction or communication is very, very important. So there are both stimulatory communications, but there are also inhibitory communications, which I'll discuss more towards the end, when we talk about one of our intervention studies. 

As this process occurs, there is release of cytokines, as in this example, which changes this naive T cell that is more of a TH1 phenotype. This TH1 cell then is responsible, or it starts producing more inflammatory cytokines, which bring other cells into the area, other cytotoxic cells into the area, such as macrophages, more cytotoxic T lymphocytes. And this is what brings that autoimmune process and inflammation to the beta cells, which eventually leads to the destruction of those beta cells. So I mention that B cells, we now know are also very important, primarily acting as, although they do produce antibodies, they're also very important as an antigen presenting cell. So this again, interaction between the MHC receptor and the T cell receptor, as well as its co-stimulatory reaction occurs, which leads to the production of inflammatory cytokines and recruitment of other cells into the area. The environmental factors, again, most of these are still not completely understood. We believe that there are environmental triggers, but we don't know exactly what those are. If there are at least one that's identified, there are probably going to be several more. So viruses have been one of the things that have been implicated particularly Cocksackie viruses, but several other viruses, as well. There are nutritionally proposed risk factors. 

In particular, milk, and that's part of the trigger trial in high risk infants, using a predigested formula, or an elemental formula, versus a cow's milk formula to see if you can prevent Type 1 in those children. And that data is still pending. There has been protective effect, or a belief that there is a protective effect in breastfeeding. And that has been shown in a few studies, as well as timing of solid foods that are introduced. That may also be a risk factor in high risk groups. And there are probably other environmental toxins. So the antibody response, and what we've seen and learned a little bit about in high risk individuals. These are first-degree relatives of patients with Type 1 Diabetes. The more numbers of antibodies that you develop over time, the greater your risk is for developing Type 1. So again, these are first-degree relatives. 

And you can see that at about five years, your risk of developing diabetes is about 25%, or so, if you have one antibody. And at five years, if you have two antibodies these are the antibodies, islet cell antigen, as well as antibodies against insulin. Your risk goes up as the more antibodies that you develop. So if you have three antibodies, your risk of developing diabetes is about 75-80% at five years. And again, that's one of the main things that we're going to be using to design prevention strategies. So if you think about what we believe to be the natural course of diabetes or Type 1 Diabetes, and think about this being your full component of beta cells here, if you start off with someone who's genetically at risk, so let's say they're born to a family with another member with Type 1 Diabetes, or have a high risk marker. You start here with a genetically at risk individual, and then there is some environmental trigger that may initiate this process and get this autoimmune process occurring. And as that occurs, you would start seeing evidence for cellular autoimmunity, specifically those T cells that we discussed earlier in the blood stream, which begins this autoimmune process. You may start seeing markers of that, this Humoral response in the diabetes autoantibodies in the bloodstream. And when you get down to about 50% or so of your beta cell mass that's left, you may start seeing some evidence of metabolic decompensation. 

So that would be loss of the first phase insulin response on an intravenous glucose tolerance test. And as things progress and that mass gets a little bit lower, you might be able detect abnormalities on an oral glucose tolerance test, and that they may not have a normal response there. Until eventually, when we think of somewhere in the neighborhood of about 10-15% of your beta cell mass left, is when you present clinically with the normal symptoms that we're all accustomed to. So really, I think each one of these time points represents an opportunity to intervene with a prevention strategy. And that's really been done previously. I was speaking with someone earlier this morning. Most of the studies that have been done have been built off of mouse models, where some of these interventions have been successful in preventing Type 1 Diabetes from occurring in the non-obese diabetic mouse, or the BB rat, which are animals that develop diabetes. And unfortunately, there's been probably close to 200 interventions that have worked in those models. But in humans that hasn't been the case to this point, and we haven't seen that same effect. So all of these have been tried. 

Some of these are prevention studies in high risk individuals, and some of these such as Cyclosporine and Azathioprine have been used in new onset patients. Importantly, these actually have worked, at least on a temporary basis. So the immune effects of Cyclosporine and Azathioprine have worked on a temporary basis. The problem with them is that the effect goes away if you stop them on the medication, or there may be too many toxicities, such as with Cyclosporine, to continue these patients on them. So we haven't quite made the dent we wanted to, but we're getting a little bit closer. This is a some data from Kevin Herold's group at Yale using OKT3, which is Anti-CD3, this is more recent data. This is from their initial study in 2002. And in using this intervention in new onset patients, what they found was that the C peptide levels were preserved in patients that received the Anti-CD3 or OKT3 versus the control group, where their C peptide levels declined over one year. Now unfortunately, those levels look like they start decreasing over 24 years. So even though there was a difference between the control and the intervention group out here at two years, there was evidence that this effect seemed to wean over time. 

So we don't think it is going to be the exact thing that we need, but perhaps maybe there's going to be another intervention at 24 months that may preserve further beta cell function. So that is a backdrop. Let me talk to you a little bit about Type 1 Diabetes TrialNet and some of the work that's going on up at the U of M. So Type 1 Diabetes TrialNet has three main focuses. One is to prevent disease, so treat patients prior to the development of T cell autoimmunity. The secondary component is delaying the onset of disease, so this would be again in individuals that have evidence for autoimmunity, have evidence for antibodies in their bloodstream, and can we delay or prevent the disease from occurring in that population. And thirdly is ameliorating the disease in patients that have developed it. These, again, are patients that have a much smaller, or reduction in their beta cell mass. Can we actually treat these patients to preserve their remaining beta cell population, to prevent long-term complications, and also hypoglycemia. So the first study I'll talk to you about is the natural history study and the Type 1 Diabetes genetics consortium. 

And these studies are really aimed at getting a better idea and understanding of what's happening with the natural history for Type 1, and understanding the importance of genetic contributions. This is very important. Number one, it may help us better define alternative therapeutic strategies. And as an example of this, many of the monogenic forms of so-called MODY disease, we've learned much more about those forms of disease over the past 20 years, specifically the genes that cause them, and those that allowed us to actually intervene with some alternative therapies, that have provided an alternative to them compared to insulin and are very effective. Secondary is to help us better identify high risk individuals. Third would be to help us to better understand the differences between individuals from different ethnic backgrounds. Fourth we know that actually some of the co-morbidities and complications are very different in different individuals, even with similar types of control. So perhaps this would also allow us to better understand the risks of complications long-term. And lastly, to identify gene interactions or gene environment interactions that are important to the pathogenesis. So along these lines, this is the Type 1 Diabetes Genetics Consortium. 

Again, the goal is discovering how these different inherited genes might contribute to the risk of diabetes. And this study is targeting families with at least two siblings with Type 1 Diabetes, or children of specific ethnic backgrounds, specifically African American and Latino backgrounds. It involves a single blood draw and they do all kinds of gene array analysis, and this will be ongoing analyses to look at contributions from other gene types. The natural history study is, again, really helping to better define the natural history of the disease. So the objectives for the natural history are: number one, characterizing the risk for developing Type 1 Diabetes in at-risk population. Number two, is understanding the pathogenetic factors that are involved in the development of Type 1. And third, is to develop clinical trials or be able to offer clinical trials for individuals that have evidence for autoimmunity. So eligible participants for this study are again, looking at first-degree relatives between the ages of 1 and 45. So this would be in your parents or other siblings, or if you have other children and you have Type 1 Diabetes, between the age of 1 and 45, you would also be eligible. And we also enroll second-degree relatives, a little bit younger age there, so even extended family members, where may still be an increased risk. 

The screening for this study really involves a single blood draw, again. And it's really just looking for presence of autoantibodies. If you do have presence for autoantibodies, you would then be eligible for the secondary phase, which is more extensive metabolic testing. If you don't have evidence for antibodies, you can come back and have yearly follow-ups to repeat those antibodies. So if look at the kind of the course of this study, phase one is just the screening for the auto antibodies. If you have evidence, on two occasions, of autoantibodies, or two separate autoantibodies that are present, you move into the phase 2, which is a risk assessment, where you have OGTT performed, a normal glucose tolerance test, a hemoglobin A1C. Some patients will have an intravenous glucose tolerance test and some blood stored for HLA typing, etc. Phase three is for patients that have normal metabolic testing, but want to continue to be followed up very closely. Again, hopefully, we'll identify our patients before they develop DKA and prevent episodes of DKA, so we want to keep a really close eye on them. And most importantly is that offering the opportunities for prevention studies, and which is the next study that I'll talk about. 

So the next study, which is the oral insulin trial, is really looking at trying to prevent diabetes from occurring in high-risk individuals who already have evidence from autoimmunity. So this kind of comes back. This study is really built off of the diabetes prevention trial one, and the DPT1, what it's job was, or what the goal of that trial was, was to enroll first- and second-degree relatives of patients with Type 1. The average age is about 11, so there were a lot of young children in this study, as well as adolescents. And they were all screened for autoantibodies. And individuals who had autoantibodies were eligible, and they went into one of two arms. They either received insulin by subcutaneously daily, as well as intravenous insulin once a year. Or they were in a close observation group, which was the control group. The second arm was an oral insulin or a placebo group. Now the oral insulin does not metabolically active, but the thought was that, again, it may induce a state of immune tolerance and change or modulate the immune system. 

So the hypothesis here is that we might be able to rest the remaining beta cells by giving subcutaneous insulin, or change or modulate the immune system to prevent further destruction of the beta cells in the oral insulin group. And unfortunately, what we learned is that neither arm of this study did anything, actually. So there was no difference over time in either of the patients that received subcutaneous insulin, or insulin about the development of Type 1 Diabetes. However, as often happens in clinical research, it provided a spark for an additional trial. And that really came from this Post Hoc analysis, which is, obviously you don't want to base your findings on a Post Hoc analysis, but it did at least provide a stimulus for thinking about designing a new intervention study. The Post Hoc analysis showed that there did look like there was a benefit in patients that were in the oral insulin trial who had very high titers of autoantibodies against insulin. Now these patients actually had antibody levels that were above 80-90 units per ml. They were initially the group that was targeted for this study, but because they wanted to try to improve enrollment, they allowed some other patients with lower levels of antibodies into the study. But when they separated this group out, what they saw that there was a projected five year delay in patients who had high levels of insulin autoantibodies who received oral insulin compared to the control group. So the oral insulin studies really built off of this Post Hoc analysis in looking at these patients, and asking the question, well what about if we just target this specific group who have high levels of insulin autoantibodies, and give them oral insulin. 

Can we delay the development of diabetes in this group? And the concept behind that, and I see Dr. Lamansky in there, so I have to be really careful about talking about all of this immune stuff. The concept behind this is that this oral antigen or repeated exposure of the oral antigen that is seen by the lymphocytes here, will in fact, induce a state of immune tolerance. So you have these lymphocytes here that see these proteins from the oral insulin that are broken down. They do not recognize this as a foreign protein anymore, and they are sort of reprogrammed to circulate in the system and are programmed to interact with these autoimmune cells, or these cytotoxic cells that are present in the pancreas and the islets, and telling these cells to actually induce a state of tolerance, decreasing the inflammation in the pancreas that's currently going on and inhibiting further beta cell autoimmunity and destruction. So who's eligible for this study? These, again, are high-risk family members between the age of three and 45, if you're a first degree. Or between the ages of 3 and 20 if your a second-degree family member. You have to have elevated insulin autoantibodies with one other autoantibody within the past six months. 

And you can't have evidence for metabolic compromise on that. Okay, so moving on to the next study, which is looking at again, a little different population. And this study, which is called the NIP Study, is looking really at modifying the immune response in children that are at risk, before evidence for autoimmunity occurs. And I was thinking that a good segue into this study was letting you know about my new adventures, as a Minnesotan and ice fishing experiences. And this is a northern that I caught a few weeks ago. It's the first one that I've ever caught. And I was thinking this is a nice segue. Number one, I can show off my fish. And number two, was this northern pike here, although it doesn't have extremely high levels, it does have relatively good levels of DHA, which many fish sources have. And DHA is an intervention study that we're actually looking at for these high risk infants. 

That's what the NIP study is. So the NIP study is basically looking at whether DHA, which is an Omega-3 polyunsaturated fatty acid, whether supplementation with that at high doses will block the initial pro-inflammatory events, and prevent development of autoimmunity in children that are high risk. So these are really targeting infants, before the age of six months. And the background on DHA, as I mentioned, it's a Omega-3 fatty acid. It's rather ubiquitous in the body. It's a very important component of cell membranes. It's found in tissues throughout. We know that it probably plays an important role in neurocognitive development, visual development. It's supplemented in almost all of the standard formulas now, and there are measurable levels in breast milk as well. When you look at what's happened with our diets over the past 200 years, and this is perhaps one of the theories behind maybe why the incidence of Type 1 is starting to increase a little bit. 

If you look at our diets over the past years, and the ratio of Omega-6 fatty acids to Omega-3 fatty acids. Back in the 1800s, it was close to about a 1:1 ratio. And as things have progressed and our food sources have changed, that ratio has changed pretty dramatically now to somewhere in the neighborhood of 20:1 or 30:1 of Omega-6 to Omega-3. And why we think this may be important is that the Omega-3 fatty acids have many beneficial effects. It's actually part of many cardiovascular disease prevention programs, or for patients that have early coronary artery disease, they're on Omega-3 fatty acids, because they have an inflammatory effect, they have beneficial effects on lipid panels. They are antithrombotic, and they have a vasodilatory effect. And Omega-6 fatty acids do the opposite thing. They're all derivatives of arachidonic acid, which lead to a lot of inflammatory mediators. So some of the sources of Omega-3 fatty acids: canola oil, soybean oil, which can be found in mayonnaise, not that, you know, you don't want to take a lot of mayonnaise in. Varying sources of fish, salmon, canned tuna, flax seeds, some leafy green vegetables, kale, and really interestingly which I just found recently, Chia also is an important source Omega-3, so if you really feel like you need some more, you could always just go down to Walgreens and get a Chia Pet and have some Chia that way. 

That was a joke. 
(laughter)
 So what's the evidence that this might actually have a beneficial effect? Well, right now it's been based off of initial studies which have been done up in Norway, where the incidence of Type 1 Diabetes is even greater than what we see here in the United States. And there are a few studies that came out of there over the past 20 years that have at least spurned this concept or this idea. First, was that infants that were born to moms who lived in fishing villages, compared to moms that lived centrally, away from the coast, those infants actually had a lower risk of developing diabetes than the infants that were born more centrally. So the concept was that these people are eating more fish and that may have an effect. So not a lot of data there, but maybe an inkling. And they followed up this study by looking at cod liver oil supplementation, which is what they used for Vitamin D supplements. And so, cod liver oil is a very important source of Vitamin D. 

It also is an important source of Omega-3 fatty acids. They found that pregnant moms who were supplemented in this small county in Norway, that their infants had a decreased risk, about a 63% reduction, although just approaching the conference interval limits there. And then they did a follow-up, study looking more nationally, and although they did find that infants who took cod liver oil during their first year, they also had a reduced risk of Type 1 Diabetes, again. Very borderline on significance, but they also found in the study that there was no effect in the moms when they repeated this study. So there was some evidence there but it wasn't really great evidence. At least, maybe it provided a clue that something was happening there. More recently, and I think which provided more fodder for our study is this DAISY study, which is again looking at high risk children. These are all infants that are born in Denver. This is where the study is. And in this study, they're actually giving the parents food questionnaires that they fill out so they actually know sort of what their intake of different food sources, how much Omega-3 they're taking in. 

They had a separate group, where they're actually monitoring red blood cell membrane DHA content. And they followed these children for about 6.2 years, and then they recently just published their data in JAMA, so it's just a couple of months ago. And what they found was that when you look at risks of developing evidence for autoimmunity for these antibodies, and how that's associated with dietary intake of Omega-3, polyunsaturated fatty acids. That there is a reduction in the risk of autoimmunity with increased intake of Omega-3 fatty acids by about 55%, and that association goes up even greater. It's a stronger association when you look at children that developed more than one autoantibody. That was down to 70% with a stronger P value. Now this was just for the food questionnaire. And when they looked at that cohort that they're following, just for red blood cell membrane content, they found that same trend. So if you look at the DHA content in the red blood cell membranes, you see that same association between higher levels of DHA and a reduction in the evidence of autoimmunity. So this was very exciting. 

And I think provided more steam and more enthusiasm for the NIP study. So this was really a randomized double-blinded placebo controlled trial, looking at DHA versus placebo. There's two entry points. One is for pregnant mothers in their third trimester who are carrying a high risk infant. That means that they themselves have to have Type 1, or the father of the child has to be Type 1, or they have another child with Type 1. They're randomized to 800mg of DHA or a placebo. And once the baby is born, if the baby is eligible, which I'll talk about in a second, the mom, if she's nursing, would continue on her study of capsule. Or if she's not going to nurse, then we actually provide study formula for those infants that either have a standard amount of DHA or a study amount of DHA in it. And the high risk infants, again, to be a high risk infant, you can't have one of the protective alleles that I mentioned before. You have to have either DR3 or DR4 positive. 

Or, if you have another sibling with Type 1 diabetes, you would also be eligible. So those are the two entry points, both moms in the third trimester and infants before the age of six months. The objectives of this study, which is in a pilot phase right now, are to actually demonstrate that we can detect higher levels of DHA in the red blood cell membranes of infants that are on study drug, as well as a reduction of some of the inflammatory mediators that are typically associated with autoimmunity. Eventually, as we launch into a full scale trial, we'll be again, reduction in appearance of autoantibodies. So over the last six or seven minutes here. I'm going to talk about some of the intervention studies that we have for new onset patients, so the goal of these studies is really to try to preserve the remaining beta cell function and the remaining beta cell population in newly diagnosed patients. Not only to do that, but also to induce a state of immune tolerance for those patients, so that there is not ongoing autoimmunity, and ongoing inflammation and ongoing destruction of beta cells. Again, we're really trying to balance off the immune response so in patients that have autoimmune disease, they have a tip toward more of a TH1 response. Some of the these more inflammatory destructive cytokines, like interferon gamma, IL-1, IL-2 beta, and tip that more toward IL-2, IL-3 response, which incurs some more of the protective cytokines. So two studies that we've already finished off, the first one was actually targeting T cell mediated autoimmunity. 

This was using Daclizumab and MMF that target the T cell population, specifically Daclizumab inhibits IL-2 stimulation of the T cell by binding of the alpha receptor of IL-2. And then MMF, which inhibits the salvage pathway of purine synthesis, As you all know, there are many patients in the transplant population on MMF. It's sort of a mild or immunosuppressive that kind of keeps the immune system in check. The second study was actually targeting those B cells, which we now know are important. Using Rituximab or Anti CD20 to deplete the B cell population. So again, here's how Daclizumab works. It binds to this sub-unit of IL-2 receptor on T cells and inhibits their activation, via IL-2. There also is evidence that some of these auto-reactive T cells go through apoptosis without stimulation of IL-2, so we're hoping that go this is going to be an effective intervention strategy. So there are three arms of the MMF, DZB trial. The three arms include active MMF and DZB. 

The second arm is active MMF and placebo DZB. And the third arm is placebo for both of those agents. And again, if effective, we're trying to see if it preserves beta cell function. And we do that using mixed meal tolerance tests and looking at C peptide responses for these patients over time. So we enrolled over 120 patients nationwide. We're hoping that we'll have some two-year follow up data in January of 2009. And I think we'll hear more about the results of that study at that time. Anti CD20. Again, what it does is it binds to CD20 on B cells and mediates depletion of B cells. This is a very effective treatment in Hodgkin's lymphoma. 

We think it may be effective in Type 1 Diabetes, as well, because it has been effective in other T cell mediated autoimmune diseases. And in this study, patients got four IV doses of Rituximab one week apart, or they got placebo. There are some side effects with Rituximab as there are with Daclizumab, specifically transfusion reactions and risks for infection after the infusion, so they're monitored very closely for these possibilities. Again, primary outcome which is all these intervention studies are looking at, are what's the C peptide response after you give them a mixed meal. And there are 68 patients that were enrolled, which was the target number and we'll probably have data, at least one year data on this, later this year. The current studies we have are using another drug called Thymoglobulin, which you may have encountered in your transplant population, or some autoimmune processes like aplastic anemia. And Thymoglobulin is basically purified globulin from rabbits that were injected with human thymocytes. So you can imagine that the serum that you get from the rabbits are full of different antibodies against various markers on T cells. And that's the idea, you get antibodies against several different T cell membranes, CD3, CD4, CD8, as well as HLA heavy chains. When you give this back to patients, you get a really profound reduction in T cell counts, so about 85-90% reduction in T cell counts. 

There probably is also some effect on B cells and how they actually proliferate, so it's a very powerful immunosuppressive agent. Now when the T cells come back, they do come back, and when they come back, what happens is that over these two months that they start repopulating and that the make-up or the population of the cells appears to be different. There's a higher percentage of CD8 positive regulatory cells, which is a positive factor in transplant studies. And we think that there are more regulatory cells that may repopulate the immune system after Thymoglobulin. So not only where we hoping to sort of knock the immune system down a little bit, but repopulate it with cells that may not again participate in that autoimmune reaction. It has been used in several other autoimmune diseases like aplastic anemia in transplant patients. And it has been effective in some mouse models, as well, but as I mentioned before, the mouse model doesn't always translate to the human model. So that's why we're performing this trial. Really, in the Type 1 population, there have been a few case reports, interestingly over 20 years ago, that shows that it may actually be beneficial, but we don't have long-term follow-up there. So for this study, patients come into the DCRC. 

They're hospitalized there for five days. They get four courses of Thymo, a total dose of 6.5mg. Thymoglobulin, for anyone that's actually used it, can be really nasty infusion reactions, so you can have cytokine release syndrome, or you can have later effects from serum sickness. It is a very potent immunosuppressive agent and there are side effects. That's why they're monitored in the hospital while we're giving it. Again we're looking for C peptide levels one and two years out. They only centers that are doing it now is UCSF, which is where Steve Gittleman is, who is the author of this study. They've enrolled two patients, we've enrolled one patient so far. We're really targeting older patients right now. Down to the age of 18. 

They have to have diabetes within 55 days of being enrolled, have one evidence of autoantibody, and have evidence for endogenous production of insulin still. So in that sort of honeymoon phase that when that develops within the first week or two after diagnosis. They have to be EBV Seronegative and their randomized two to one to Thymoglobulin versus placebo. They do get methylpred prior to their infusion or placebo steroids, again, to try to decrease some of the reactions that are going on. And they're followed for 24 months. Then lastly, is an intervention study using CTLA-4. Now CTLA-4 is a really important inhibitory signal that's typically expressed on T cells. So when there is a reaction, when there is inflammation, when there is autoimmunity, CTLA-4 comes out and is expressed probably about 24-48 hours later. We think that in Type 1 patients, there maybe decreased expression of this protein on the T cell. So the concept or goal would be by giving actually CTLA-4 immunoglobulin, we may actually be able to, again, induce a more regulatory state and decrease this autoimmune reaction. 

And how it works is on the B cells, which function as the presenting cell here, this interaction with both CD80 and CD86, they normally interact with CD28, which induces this activation of the T cell to participate in inflammation autoimmune response. CTLA-4 is a negative protein, so by giving CTLA-4, we block this response that occurs, this co-stimulatory signaling that occurs here and it also probably has effects at the T cell receptor and the MHC complex of the B cell. So we think it blocks the stimulatory cascade. It decreases the amount of IL-2 that's then produced. And it probably has effect on self-cycle progression as well. Again, some pretty good evidence in mouse models that this is going to be effective, so if you make knock-out mice that do not have CTLA-4, they get this really profound lymphocyte proliferation, multi-organ destruction, and lymphocytes just kind of go haywire. And if you give these mice CTLA-4 immunoglobulin, you can actually prevent this from occurring. CTLA-4 appears to inhibit release of TH1 cytokines, but it doesn't inhibit TH2 cytokines. And this is in a model of autoimmune encephalomyelitis. It has been shown to be very beneficial in a number of transplant models, as well as in prevention of autoimmune diabetes in the NOD mouse. 

It's trade name is Abatacept, so it's a commonly used agent in children with rheumatoid arthritis, multiple sclerosis, psoriasis, and it's also being used in a number of other trials for autoimmune disease. So this study goes down to younger kids, because it is better tolerated. It doesn't have the same kind of reactions. The rest of the inclusion criteria is very similar. It's, again, two to one intervention, and we're looking at targeting 108 patients. There are some risks for infections, but they're not quite as profound as Thymo. And that's just getting off the ground right now. So in the future, where I think things are going both in TrialNet and in other branches of research and clinical diabetes work. Well, there are probably going to be more immunomodulatory regimens, such as using Anti-CD3, plus the addition of exendatide, which is a peptide 1 antagonist, that preliminary data shows that it may actually help promote proliferation of beta cell repopulation, or increase the number of beta cells. There are vaccinations coming down the pipe using glutamic acid decarboxylase, again, as a way to induce immune tolerance in patients that have evidence for autoimmunity. 

And what we'll probably do here through John Wagner's lab at University of Minnesota is trying to enhance that T cell regulatory population, that you can pick out of the peripheral blood population, enhance that population, give it back to patients. And then for patients with long-standing diabetes, we've got islet transplants, which are getting better, but aren't quite where we want them to be. Xenografts using pig islets, which is coming down the pipe. There's been evidence from Dr. Bernard Herings' group from the University of Minnesota, showing that they can actually do that in primates, and transplant islets from pigs into primates. Stem cell transplants. There's one study that came out in JAMA last year about an initial study of a small group of patients that shows a lot of promise there. And then also just improved insulin delivery systems. So we're looking toward closing that loop of insulin delivery and glucose monitoring. So just to review, the studies that we have out right now, genetically at risk we've got the NIP study we're using Omega-3 fatty acids, Humoral autoimmunity, oral insulin, so people who have evidence of autoimmunity, using oral insulin, that group. 

New onset patients, Thymoglobulin, CTLA-4, which are currently there and then trying to increase our knowledge of what's going on from a natural history standpoint through Type 1 diabetes Genetics Consortium, as well as the natural history study. I think this is Billy Leroy, who is one of the first patients to get insulin. And you can just sort of see how wasted he was here prior to getting any insulin. This was really the only treatment before we had insulin was actually starvation. He gets put on insulin and you can see the really dramatic results, so this is really life changes for patients with Type 1. And I think we're approaching that next phase of really changing the course of diabetes with some of the trials that we're doing, and some other trials that are going on elsewhere. And it's a little cheesy, but I do miss the UW Band. And I really feel like we are kind of marching toward a cure here. So hopefully, we'll see that happen all in our lifetimes. I just wanted to acknowledge everyone from the university, allowing me to skip clinic today and come down to be able to talk to you about some of our studies. 

We have a great group that we work with, and really appreciate your attention and the invitation to come down today. So thank you. 
(applause)