Dr. Amy Hermon-Taylor: MAP Diagnostics
So, my name is Dr. Amy Hermon-Taylor and I’m a family doctor from London. And I’d like to say, first of all, whoever out there fixed the projector, I love you, because my slides are very interactive and mostly based around pictures. So if I didn’t have my slides, I might just have started crying. So thank you very much.
I’m going to talk today about a new MAP diagnostic test, but first of all I’ll just tell you a little bit about myself, so you’ll know where I’m coming from. So I trained originally at University of Cambridge. I did a degree in natural sciences, majoring in pathology and molecular cell biology. And then I went on to St. George’s in London where I trained as a doctor, and I’ve been a doctor since 2001. I did quite a lot of time at hospital before deciding to become a family doctor, what we call a GP in the U.K.
I’m here today speaking to you because of this man, who’s my father. His name is Professor John Hermon-Taylor, and he’s one of the pioneers in the field of MAP and Crohn’s disease. So he’s worked in this field now for 35 years, initially as Professor of Surgery at St. George’s Hospital where he had a big population of Crohn’s disease patients under his care, and when Professor Rod Chiodini, that Will Chamberlin mentioned, sent him a little culture from a Crohn’s patient, which he grew up and grew MAP after two and a half years, he decided to make that his life’s work. So I’ve grown up with MAP and Crohn’s disease as a topic of dinnertime conversation. And as I myself trained and got to be a doctor myself, I really started to understand the science for myself.
And you might very well ask, what could a family doctor have to say in this niche field which spans gastroenterology, veterinary medicine, epidemiology, immunology, genetics, microbiology. And the answer is something very simple, something very important. It’s something that most of you in this room share with me: and that is a fresh pair of eyes.
So this is my take on Dr. Chamberlin’s message about paradigms. This is Stephen J. Gould. He was a Harvard man. He was a paleontologist and a scientific historian, and he said this on the subject of research progress. “New facts, collected in old ways, under the guidance of old theories rarely lead to any substantial revision of thought. Facts do not speak for themselves. They’re read in the light of theory.” In this field, there’s been a lot of progress, particularly in the last five years. So we need to reassess. We can’t just rely on the conclusions of the past. We need to reevaluate things with a fresh pair of eyes.
So what I’m going to tell you, speak to you about today, is diagnostics. I’ve got my summary up here. First of all, why is MAP testing so important? Secondly, what makes MAP so difficult to study? And then I’m going to go on to talking about our new MAP test. So why is MAP testing so important? And I’m going to start off with a meeting from the American Academy of Microbiology which took place in 2007 and they published their reports in 2008. And this was meeting in Salem, Massachusetts where just about everybody who knows anybody; everybody who’s anybody in this field was present, experts in MAP and Crohn’s disease came from across the world.
And this is what they said on the subject of MAP testing. “There is a conspicuous and fundamental barrier to accomplishing the research on MAP as a causative agent of Crohn’s disease: the lack of a reproducible, sensitive and specific diagnostic for MAP. New tools to identify and isolate MAP are critical to every recommended activity outlined in this document, and without them researchers cannot move forward.” And the reason for that is, it doesn’t matter how many studies you do, your data will be only as good as the diagnostic test that you’re using. So if your test is not sensitive, then you cannot rely on the data that you get.
So these are the tests that we currently have. Looking at mainly two different things here. The specificity of the test, so how accurate is it at detecting the MAP organism. So if your test is not very specific, then you will have a lot of false positive results. But we’re doing pretty well with specificity. We’re at a 99 or 100% rate. Now, let’s look at the sensitivity column. The best figure there is 55%, which means that a lot of the samples that are testing negative, are actually false negatives. So we don’t, in a lot of patients, we don’t actually really know whether they do have MAP or not.
So the next thing that I’m going to talk about is why MAP’s so difficult to study. First of all, it’s very difficult to see it under the ordinary microscope because it doesn’t stain easily in the same way as other bacteria do. Secondly, it’s very difficult to grow it. The first time my father grew it, it took two and a half years. And now that we know it’s little foibles, what it likes, what it doesn’t like, we’ve got that down to about 18 weeks for standard culture methods, and that doesn’t really work for a clinical diagnostic test. And thirdly, it’s a very tough organism. It’s difficult to break it open. In order to use modern molecular testing methods, such as PCR which is a test for the bugs DNA, you have to take the DNA out of the bug first. And it’s very difficult to do that because it’s so tough.
My second message on why is MAP testing so important, is patients want to be tested. Now, I’m passionate about patient and public involvement in research and one of the things I’ve done since I’ve got involved in this is to set up a website, social media pages for this research, and put the science out there for people to see for themselves. Translating it bite size for the ordinary lay-person. We now have about 12,000 followers on our Facebook page and Twitter sites, and the commonest questions I get asked are, “Can I be in the vaccine trial?” and “Can I be tested for MAP?” And people will say to me, I’ll fly to the U.K. if I can be tested for MAP. I’ll pay you to be tested for MAP. They really want to know.
So, the next thing I’m going to talk about is this very important paper. And you’ll see in a minute how this links in with the importance of our new test, which I’m going to tell you about. This is a paper from Nature 2012 and it’s one of the big genetic studies; genome-wide association studies, which looked at 75,000 patients and there were actually 100 authors on this paper. So a big body of specialists looked at the genetics of a large number of people. And they said what are the susceptibility genes associated with Crohn’s, what genes do people with Crohn’s have, that people that don’t have Crohn’s don’t have. And then they looked to see what those genes do. And that helps to give us an idea about causality. And they found that there was a huge overlap between the susceptibility for Crohn’s disease and mycobacterial infection. And we already know from other studies that the three most important Crohn’s genes, NOD2, IRTM and ATG16L1 all are involved in pathways by which the immune system handles intracellular bacteria such as MAP.
Now the reason this study is really important, is because of the two potential causes of Crohn’s that Dr. Chamberlin mentioned, so the dysbiosis theory and the infection immune deficiency hypothesis; this data is pointing us towards the latter and away from the former. And it also tells us that the immune deficiency that you get with Crohn’s disease is primary and not secondary to the disease state. Because nobody is born with Crohn’s, but we are born with our genes that predispose us to mycobacterial infection.
Now MAP isn’t mentioned anywhere in this paper, but every time it says the word mycobacteria, the implication falls on MAP because MAP is the only mycobacteria which time and time again has been shown to be associated with Crohn’s disease.
The other very interesting point about this paper, is it also noted that the genes that give you susceptibility to Crohn’s disease are also found much more commonly in a cohort of what we think of as auto-immune diseases, than you would expect from the general population. Which also leads you to the interesting question of whether mycobacterial infection could be involved in those diseases as well. And the two which were most strongly linked were anklosing spondylitis and psoriasis, both of which occur more frequently in people with Crohn’s disease than they do in the general population.
So, back to why is MAP testing so important. This is the first reasons: the experts say so, the patients say so. The third reason is that it’s just not Crohn’s disease. So the genome wide association studies and also some other studies looking at the presence of MAP in some of these diseases directly show that all these diseases listed up here have been shown in some ways or another to be associated with MAP. And I’d like you all to have a look at those diseases on the list up there and ask yourself, “How many people do you know that have one of those diseases there?” And my point in asking that rhetorical question, is that this is something that’s important to pretty much everyone.
I will say quickly about the path and the mechanisms by which MAP could cause diseases, and is it feasible then that MAP could be involved in these other diseases as well. Well, the first possibility is that active infection causes the disease in a susceptible host, and that’s what we believe happens in Crohn’s disease. But if you look at MAP’s distant cousin, TB, although we think of it as a disease of the lungs, that’s not a single tissue in the body which cannot be affected by TB. So it’s possible that there could be active infection by MAP in other tissue as well and our test will allow us to look at that, because it’s the first test ever which allows us to see MAP infection in Crohn’s disease tissues.
The second way that MAP could cause infection, the second way, sorry, that MAP could cause disease, is that it may act as a trigger, so stimulating the immune system to generate a response against the MAP protein, which looks very like a human protein. We call that a molecular mimicry reaction. So our test is designed to answer these questions I posed here. I’m going to go on to tell you about how is works.
So this is the MAP organism down in the bottom here in pink. And we’re targeting the protein on the surface of the MAP organism, which is found abundantly on the cell surface and it’s entirely specific, unique to MAP. We’ve made a monoclonal antibody against this marker. What we do is we take a slice of tissue frmo a normal paraffin embedded tissue block. We apply the monoclonal antibody, and then we also, then we apply a revealer antibody which has a little fluorescent tag on it. So that when you shine the laser onto the tissue, it lights up green, and that enables us to see it using this instrument, which is called a confocal microscope.
Now, with the greatest regret, I’m not actually able to show you pictures from our lab because of the legal requirement of nondisclosure at this stage of the research. So what I’ve done is to take some pictures showing how this technology has been applied, to duct tissues and looking at other markers, so you can see the potential of the technology and what it can show us.
So this technique is called immunofluorescence microscopy. And this particular group looks at a marker in the mouse jejunum. And you can see now on the left you’ve got the intestine of a mouse for which the marker was knocked out. So that’s a negative slide on the left vs. a positive on the right. And you can see with the negative, you see a ghost outline of the tissue, but on the right you see really bright fluorescence where the marker is showing up positive. And we’ve applied this technology to MAP so we can see it in the tissues and we can see the types of cells it’s infecting, we can see the tissue types that it’s infecting and that has a potential to offer us a lot of information about mechanisms of disease in Crohn’s.
We’ve also, interestingly, looked at this technology in a patient before and after three months of AMAT therapy. And having seen the photos myself, I can tell you that the difference hits you in the face. Where before AMAT therapy you see bright, bright green and after, there’s a noticeable reduction mainly in the brightness of the image. It looks like, after three months, the same number of cells are infected but that MAP per cell is much lower.
Now here we’ve got a different study, this time targeting a marker in the rat ileum. And I picked out this slide because here it looks a little bit more like what we see when we’re looking at MAP. We see specific individual cells rather than here where we’ve targeted a marker that’s on many of the cells. What we’ve done with our test to validate it is to actually cut out individual cells using a special technique called laser capture microdissection. And what that does is a little like what you would imagine on Mission Impossible, where they’re breaking into a jewelers. You get your laser, you say that’s the cell I want, and you go zzzzzz, and cut it out. And you pop it into your pot and you run a PCR on it to find out what has your marker found there. Is it really MAP? And it is MAP.
So we can not only apply this to any tissue in the body, but we can also apply it to blood cells. This group here have taken the neutrophils, which are a type of white cell, from a rat and they’ve stained them up using a marker. And again, they’ve chosen a marker which is on all of the cells. What we see is some cells full of MAP that are positive and also the ghost outlines of cells that don’t contain MAP.
So as well as looking at the cells and seeing where the MAP is, we can also do a test using a machine called a flow cytometer, and that’s what this is. What that is, is a machine really that counts the cells. And it looks at how many cells in the blood stream have particular markers. So we’re using this, obviously, to mark for MAP, and it will tell us how many white cells in the blood stream are infected with MAP as a percentage. And also we can label out the cell subtypes and tell how many neutrophils are infected, how many lymphocytes, how many monocytes. We can also get what’s called a mean fluorescence intensity, so not only how many of the cells, but how bright is the signal from each cell. How much MAP, which will correlate with, how much MAP there is in the cell. And that has the potential to tell us a lot about what’s going on.
Flow cytometry is a machine that most labs will have. It’s very widely used for lots of different types of tests. You only need a few mils of blood. It takes about fifteen minutes to run through the flow cytometer. So it’s going to be a quick test; a cheap test. We think that it will prove to be a very specific and sensitive test as well. And therefore it’s ideally suited to answers a lot of the questions that remain in this field.
So lastly, coming back to why this is all important. In order to solve the case of Crohn’s disease. In order to find out, in order to cure it, we need to find out the cause because that puts us on the path. If you know the cause of a disease, then you have your therapeutic target, and that sets you on the final pathway to cure. So the dominant theory at the moment is that Crohn’s disease is caused by an abhorrent response of the immune system to the microbiome. So here’s your microbiome, and here’s MAP. Now, how will we get, from all of these millions of bacteria, how are we going to find which one is important in Crohn’s disease. Well, research is a bit like being a detective. So, if you’re a detective, and you’re looking for a serial killer, you’re not going to look amongst the hoi polloi. You’re going to go for your chief suspect. And before you go back to the drawing board and start combing the streets, you’re going to gather the data on your chief suspect and rule out everything on him before you look at other things. And that’s what we need to do with Crohn’s disease. So MAP is the chief suspect in the causation of Crohn’s disease from the association studies, from the genetic studies, from the immunology studies and from the epidemiology. And 102 years of hypothesis is enough. We need to convict or acquit.
Dr. William Chamberlin: If I could ask a question, okay? We have been looking for a specific, sensitive diagnostic test for MAP, because without that we were going nowhere. We couldn’t prove anything. And what Professor Hermon Taylor has done, is he has a monoclonal antibody, correct me if I’m wrong.
Dr. Amy Hermon-Taylor: Yes, absolutely.
Dr. William Chamberlin: That’s very specific for MAP. That is specific for several antigens on proteins that are specific for MAP. You are using this monoclonal antibody. You have used this monoclonal antibody to look at a number of Crohn’s tissues. You are seeing MAP in the tissues themselves. You’ve got a few patients that you’ve identified as MAP and you saw a fairly robust infection. You’ve treated it with Anti-MAP treatment and that infection has diminished.
Dr. Amy Hermon Taylor: There’s one patient. And we looked at before and afters of AMAT treatment. Well, we looked at about 70 Crohn’s patients in total and 100% of them were positive for MAP infection. And to the degree of infection, so the number of cells that you see appears to correlate with the technical severity of their disease. So the worse the disease, the more MAP you see in the tissues. Sometimes in remission you’ll see very few symptoms, so you’ll see very little MAP there. But the other important thing to say, which you might be about to ask me but I’ll say it first, is that we do, in normal people you will see some MAP as well. Not everybody, but in a few normal individuals you’ll see the odd cell, here or there, of MAP. Nothing like the propensity that you see in Crohn’s disease.
Dr. William Chamberlin: Now when using your diagnostic test to find causality for MAP in Crohn’s disease, now traditionally we use Koch’s postulate, which essentially said, you had a disease, you cultured a suspicious microbe, you cultured it in pure culture, then you gave it back to the patient. And then you were able to culture that in pure culture again. Obviously, we’re never going to do that with humans for Crohn’s disease, at least we shouldn’t be. But a modified Koch’s postulate is you’ve got the disease, you identify MAP. You’ve got it in pure culture, you’re seeing, again, a quantitative, a qualitative and quantitative MAP infection. You treat that patient and you correlate a bacterial response with a clinical response. And that’s the closest thing to a modified Koch’s postulate that we’ll have. Now I’ll say, for me, that will prove that that’s a causal agent of Crohn’s. But most patients won’t care. You get better with treatment and that’s all you’re really after, and I thank you very much Amy.
Dr. Amy Hermon-Taylor: Thank you Will.
Dr. William Chamberlin: I want to say, your father, I consider myself his disciple. You know, it’s great.