Thanks very much Tom for that introduction and welcome to this session. When Tom first asked me to do this, I kind of assumed it was 6 o'clock in the evening and he said what would I like to talk about and I said 6 o'clock in the morning, probably coffee but anyway what we're going to talk about this morning there is really some issues around airways inflammation some lessons we have learned about whether we should be looking for new targets in molecules for development of new treatments for asthma and also I think a slightly more sophisticated way of looking at the research that's coming out at the moment. I will include a little bit of our work but I'm also going to draw on some recent literature that you may have seen or may not have seen to try and illustrate my points.

Now, about 15 years ago we developed this concept that inflammation was very important in asthma, up until then really the main focus of asthma research was in the physiology of the airways and the way the airways contracted and we had a view of asthma that was focused on the smooth muscle and many of the treatments we are going to be using are focused on the smooth muscle. And there is a sort of essential dogma that has arisen in the last 10 to 15 years has been that what we are seeing in asthma is an inflammatory process in the airways of asthma and that the outside airway, the smooth muscle, and the supporting structures don't really play such a huge part in the disease. So if you have a normal airway and you constrict it with irritant or histamine you have a small amount of narrowing whereas if you have inflammation in your airways or mucous when this narrows, you get a disproportionate affect on the airways lumen and therefore you get airway obstruction. Now there is quite a lot of truth in this, but I'm going to try and show you that in fact it is not quite as simple as that. You'll see during this meeting and other meeting's cartoons along this sort of line, which just show us where a lot of the epibiology has gone. And now instead of just concentrating on the mast cell and the way the antigen triggers the release of inflammatory and the bronchospastic mediators, we now understand a lot about the regulatory framework that this exists in. In particular we know a lot about the T-cell, the way that it regulates the B-cell to produce IgE and the way that it produces other cellular mediators or cytokines, which activate and recruit other inflammatory cells, which are the eosinophils. We are going to also learn about new mediators, particularly leukotrienes and the effect that they have obviously on the inflammatory process in particular the blood vessels as well as their effect on mucous production as well as on smooth muscle. And lately certainly our interests have been in the epithelium, and we're very interested in the way that the epithelium produces a number of cytokines and growth factors, which we think, contribute to the inflammatory process.

Now this has allowed us within the last 10 or 15 years to develop some new agents to that effects these processes in particular leukotriene receptor antagonists, some treatments which are into the T-cell, either cytokines which are pro TH1 cytokines or specific treatments such as immunotherapy which we think will work at this level. There is some anti-cytokines which are involved in targeting the IgE production process, anti-IL4, anti-IL13, anti-IL5 which deals with the eosinophils specifically it actually may have some other effects as well and of course anti-IgE which is now coming into clinical practice. I'd like to emphasize that anti-IgE does not just work on the IgE that's in the serum and gets to the mast cell, it also has direct effects on other cells including T-cells, B-cells and eosinophils. Just one slide on anti-IgE which is a drug that is going to be useful, well as an academic my interest in this is I think it is very useful, that it is going to tell us more about the role of IgE and allergy in the maintenance of asthma than it may necessarily about the right way to treat asthma. It certainly has effect on effector mechanisms; I think it is going to make us think again about the importance of mast cells and basophils in asthma. The other areas that I think that anti-IgE will be useful will be in combination of other therapies, for example giving it in combination with immunotherapy is something which is interesting both in a safety point of view and also for efficacy issues. Of course we know that the clinical trials with the focus of more severe asthma, but I guess most of us would like to see if this treatment works in a broader range of patients, we don't have the data to answer that and we really need that information before we can judge whether this is going to be effective.

Within our own work, we've looked at cytokines in quite some detail, and I'd just like to draw your attention to a couple of things, we tend to talk about asthma as being characterized by the TH2 cytokines in particular IL4, IL5, but in fact if you look in the airways of patients, you look at the messenger in a resting lavage, you see it's quite a different pattern, you see it's quite a lot of IL13, when we first discovered this we didn't know if IL13 was important or not and we certainly didn't follow it up. It is now turning up that this is actually quite an interesting molecule and one we think may be important in particular in the remodeling processes. I'd also draw you're attention to the fact that there is also quite a lot of TNF, TNF alpha present in the airways of patients with asthma and that is something again that we really haven't focused on at the time because we were very fixated on this ideal of the T-cell and the TH2 cytokines. What is true is if you take humans and you allergen challenge their airways and you go back 24 hours later as you can certainly see the IL5 and IL4 particularly if you stimulate the cells, and I think that this model is much more typical of what you are seeing in the animal models where you see the TH2 cytokines, IL4 and IL5 very prominently. So I think that we just have to remember the allergen challenge models in animals may not tell us everything about maintenance, about the maintenance of ongoing asthma. Now a few words on IL4, IL4 is interesting not so much because it helps to make IgE but because it has other actions. It has actions on endothelial cells and it has important action on the production of mucous and I think mucous is a very unexplored area in asthma. Perhaps one of the reasons why it is targeting IL4 may be helpful is it has other effect on the production of mucous as well as on the inflammatory process and on IgE production. Some of you would have seen this study published by Larry Borish and colleagues looking at the effect of the soluble IL4 receptor in adult asthma, it is a good study but it has major problems with them. If you read the paper you will have spotted it, if you take these patients all who have had inhaled steroids but have moderate to moderately severe asthma, they had they're doses stabilized and then the inhaled steroids were withdrawn as soon as they started the treatment. And what happened is that a very high percentage of the patients dropped out of the study, at 28 days into the study, half of the patients basically had fallen out of the study. So when you come to look at the rest of their data, you have to remember that you're only looking at those who tolerated this treatment and the withdraw of steroids. That said, you could see that there is a stabilization of lung function, so the placebo patients, they're lung function gets worse and this is protected by the administration of soluble IL4 receptor and conversely that these patients have much less in the way of symptoms than those who are on placebo but what you'll notice is even with this huge difference say a 7 fold difference almost in the amount of symptoms, this is still not statistically significant and that's because so many patients fell out of the study. IL5 is another interesting molecule and this again is previously demonstrating that if you give IL5 on it's own, you can demonstrate an increase in eosinophils and the sputum and also an increase in bronchial activity, so this one cytokine on it's own is a very narrow target, it can increase the eosinophils and increase bronchial activity. Now this therefore seems a very logical target for anti-asthma therapy, we should perhaps have read the literature more carefully because in the animal studies what we found was that the anti-IL5 bronchial antibodies were very good at reducing eosinophils in this allergen driven model, but what they didn't do, it had no effects on bronchial activity whereas steroids, in this case dexamethasone, also abolished the eosinophils and also reduced the bronchial activity. So we should really have taken away a lesson from this, that IL5 is terribly important in the eosinophils, but that the eosinophils in IL5 may not be that important in bronchial activity. When this study was done, it was a multi-centered study looking at the effects of anti-IL5 on the human late phase response, it was shown very clearly that the actively treated groups had a reduction in sputum and eosinophils down to virtually nothing when they were treated with anti-IL5. When they looked at the allergen challenged responses, you could see here that you have first of all, a baseline value which is the lowest of these curves, but the ones who've been treated with the anti-IL5 there was really no difference in the early response to allergen challenge but a fairly marginal response in the late phase, you could argue that there is a difference in the area under the curve, but it's certainly not very impressive compared to what you would see with an inhaled steroid. And so by targeting down to this very narrow target, the IL5, we've lost the sort of broad effects we had with inhaled steroids. Well several people say, well that's it that's the end of IL5, it's not interesting it's to narrow, it doesn't do enough, it's not interesting. Actually somebody then went on to do another study looking at different effects of IL5 in fibrosis, now the biology of this is just off the wall. We wouldn't have expected IL5 to be relevant to epithelial fibrosis and so I think a lot of people would not have done this experiment and so we are grateful to these investigators for doing something, which is really out in left field. This is a mouse model of sub epithelial fibrosis, which involves the sensitization of ovalbumin, you then wait quite a long time and re-challenge with intra-trachial ovalbumin. Now in this system, what we've got here is we got the control animals, we got the sensitized animals, which produce lots of eosinophils, and if you give the anti-IL5, you loose the eosinophil response. If you use a control montelukast, then there is no effect. So what happens to the fibrosis, well it is very interesting, what happens here is the baseline level of reticulum, this is what happens if you are sensitized and therefore allergen challenged and if you give anti-IL5, you don't get that response. So this is really interesting you didn't know, you didn't appreciate that IL5 or indeed eosinophils had perhaps a great deal to say towards fibrosis, so here we have a tool that we have developed for one reason which may actually prove to be interesting, it is going to be interesting to go back and explore this to see whether this has a benefit to remodeling rather than the allergen challenge model of which it was originally tested in. Just one additional point, this has no effect on the mucous cell hyperplasia, so these animals even though they got a reduction in the eosinophils, and a reduction in fibrosis they still get goblet cell hyperplasia in response to allergen and that's perhaps where the IL4 strategy will come in. Maybe we should be thinking of combining some of these cytokine therapies into a cocktail to try and block the whole asthma process.

Well that's where we dealt with the research being sort of done so far in a number of the clinical trials that cover this, but there are a number of other areas where I think this work is sort of going. The first of these is the interest in one of the ways of blocking this recruitment of cells, what we got to look for here is molecules which are target able which are critical for the recruitment of eosinophils, but do not play a part in the regulation of the immune system. So the difficulty here has really been to separate out the things that are important for cells, cell contact in developing antibody response, which we don't want to influence, but what we do want to get out is the molecules that are involved here that effects granulocyte recruitment. There is interest in targeting some of the mast cell proteases and there is agents being created to target them and there is also the interest in the chemokine area. Chemokines are in biology terms different from the cytokines, they are very promiscuous, they interact with many different cells, and many of them are produced by different cell types and that presents a completely different problem for developing new therapies. I suppose our focus in asthma should be really on chemokines that are produced by the epithelium, that's where a lot of the damage is taken place and these 5 molecules and some of those related to these molecules are the ones of particular interest. You can stain them very easily in the epithelium; this is IL8 in bronchial epithelium demonstrating that it is fair in large quantities and ready to role as soon as there is any insult that hits the epithelium. From my point of view, the whole message is about chemokines in asthma are that these all are very promiscuous, they work across multiple receptors, but there are certain things that look promising, the first of these is that eosinophils responds to several chemokines all of which seem to operate within the CCR3 receptor and so therefore this receptor which is fairly specific is not on a lot of cells, but it receives signals from a lot of these chemokines, this is the way you sort of tackle this situation. So unlike the cytokine issue where you tackle the cytokine directly, for the chemokines you need to think about the bottleneck in the system, which in this case is the receptor off the cell. And there is also some interest in targeting TH2-cells and that what's the difference between TH2-cells and other lymphocytes, well these ones carry CCR3, just like the eosinophils and CCR4 so those two targets look like the main things to go for if you want to take out the TH 2-cells. Now all of this so far, we've been talking about really ways in which the interior of the airway gets distorted and damaged as a result of the inflammatory processes. And I'd like to close my talk in this last series of slides by looking at things which have turned up which are different which tell us that there are really other issues going on, in particular things going on in the smooth muscle, which may explain some of the anomalies that we have picked up over the last few years. Just a brief word about the collagen, the collagen is deposited underneath the bronchial, basement membrane here which has been the focus of interest for us for the past twelve years now, when I would have to say that we have not really got it clear in our minds whether this is important or not. It's certainly there, but it's not clear whether this is driving some of the symptoms that are there in asthma or whether it is just an epiphenomenona. What we can say is that we understand a lot more about the biology of this now and the way that these different inflammatory cells maintain the collagen production. Our view of the eosinophils in the mast cell has been historically really concern with producing mediators that cause smooth muscle contraction and mucous secretion. Jerry Gleich and colleagues demonstrated that the eosinophils had an important role to play in causing damage to the epithelium and really what we've been looking at is the relationship between these cells and the ongoing effect of the inflammatory process and the way that that influences fibroblasts and we can show there is a cross talk between mast cells producing various prostheses and cytokines that stimulate fibroblast growth and that fibroblasts in turn feedback growth factors in this case stem cell factors, but there are others that help the mast cell to grow. Similarly there is a cross talk here between the eosinophils and the fibroblasts and you'll notice here that TNF alpha pops up and TGF Beta, these seem to be the two main products that the eosinophils make that help the fibroblasts produce collagen. And lately by looking at gene expression models, it's been possible to show that IL13 is also interesting in terms of stimulating fibroblasts, which is used to produce collagen. This is really where the old observations that we made maybe 7 years ago about IL13 and TNF alpha start to become more interesting again. If you take bronchial epithelial cells and this is what is going on which Steve Holbrook's group is looking at production of growth factors from epithelial cells, you can show that the cytokines, IL4 and IL13 up regulate the production of TGF Beta directly from the epithelium, so here we have another way in which the epithelium is contributing to the inflammatory process and in this case to the remodeling process in the airway. As far as the smooth muscle is concerned, the published data demonstrating that airway smooth muscle is not normal in asthma. We've been going around saying for 10 years or so that the airway smooth muscle is normal it's just that it's in an inflammatory milliard, but in fact if you look at asthma in asthmatic airways and you look at how much their airway smooth muscle could contract, there is a substantially greater contraction in terms of stress, in terms of maximal shortening that's achieved in asthmatic airways and also that the cells are capable in proliferating in vitro to a much greater extent than from non-asthmatic smooth muscle cells. Well there is now some data that starts to explain why this is happening and it won't come as any surprise to see that it's back to our old friend IL13, it turns out that airway smooth muscle cells have receptors for IL4 and for IL13 and that these are functional, that is that they, that if you put IL4 or IL13 onto the cells, it triggers the transduction mechanisms, you get stat 6 plus forilation and also activation of monokines. This paper went on, instead of just showing a sickening process, they look at what this means in terms of the ability of beta agonists to relax airway smooth muscle cells. And it turns out that if you look here on the left hand graph we got the baseline, which is the stiffness of the airways, and as you put isoproteronol on top of these cells, what happens is the orange curve comes down like this and this is the normal relaxation curve that you see with a beta agonist. If you pre-treat these cells with IL13, what happens is the ability to relax the cells is decreased, so you don't drop as much as you did as you were untreated with IL13. But IL4 doesn't do this, there is no difference for IL4, so this is telling us that here there is something a special role perhaps of IL13 acting directly on airway smooth muscle and making the airway stiff and making it difficult for you to relax it with therapy. So ladies and gentlemen, what have we learned about asthma and inflammation over the last 10 or 15 years? And where are we going at the moment? Well I think the first thing to say is there is more to asthma than just inflammation, it's a complex process, it's clearly a lot of issues that we quite don't understand about triggers for asthma and some of the immunology, there is also a lot of this networking between various cytokines and chemokines, some of which looks like it's just a device for raising money from NIH, but other bits of it actually probably do have some important messages embedded in them. I will suggest to you that there are 2 cell types that we need to concentrate on, that I certainly think are important, the epithelium and the smooth muscle seem to be very active contributors to the inflammation and also to the remodeling process, this is not what we've been telling ourselves for the last ten years and I think that we have to sort of eat some of our words and go back to that and think about how we address this and what role these cells are playing and as a matter of principal when you are looking at molecular targets for the future, what you are interested in really is molecules that have multiple actions, so the pleotropic things the things that work on many different cell types, they are good molecules to target. But if you are thinking about receptors, what you want is some sort of bottleneck receptor, something that has a unique and ideally it is specific to one cell in terms of its function. So, I think this is all philosophically where you should be going with new target developments. I hope that's been of interest to you and thank you very much for your attention.