Lipkin presentation, CDC Conference Call 9/10/2013

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CDC Conference Call Sept 10, 2013

Dr. Ian Lipkin


Thank you for the invitation to speak with you and with your colleagues. Before I talk about what we’ve done to date, I just want to acknowledge the people who’ve done this work. I’ll begin with the Chronic Fatigue Initiative.


We began, really, with the XMRV/PMLV study which many of you are familiar with. Our colleagues, Dan Peterson and Jose Montoya, began to meet and we’ve continued to work with them in the context of the Chronic Fatigue Initiative.


The work that we do is not supported by the federal government. The work that I will describe today has been supported by the Chronic Fatigue Initiative, which is the Hutchins Family Foundation, the Evans Foundation and, basically, an anonymous donor who supports Jose Montoya’s work.


I don’t know if Doctors Peterson and Montoya or Bateman, Klimas, Levine, Komaroff and Felsenstein are on the call, but if they are, I thank them again for their participation. And then, on this end, we have many people… but I don’t really want to go into that because I don’t really want to be self serving. But I have here with me coordinators Assari and Andrew, and Mady Hornig and many others whom you know too as well, for the work they’ve done previously.


My own interest in Chronic Fatigue Syndrome dates back to the mid to late 1990’s when I was working with Birgitte Evengård,,and one of the questions that came in earlier today had to do with polyclonal B cell activation. Those of you who may have read that paper that I wrote with Bergitte which had been published in 1999, was one of the first to really draw attention to the fact that people with Chronic Fatigue Syndrome who were diagnosed using the criteria at the time did in fact have a physical condition, but we were unable to find evidence of infection with borna disease virus which was the primary question at that time. We did demonstrate unequivocally that the vast majority of people who met these criteria had polyclonal B cell activation, so they clearly had some form of immune activation. But the question was: Why? And then, three years ago, the Director of NIAID,Dr. Fauci; the Director of NIH, Francis Collins and Harold Varmus from NCI asked me to lead a project where we would examine XMRV and PMLV, which had previously been reported by Judy Mikovits and Harvey Alter and others in association with Chronic Fatigue Syndrome.


There were…as you know, we found no evidence of infection with either PMLV or XMRV in that study using methods which were developed in each of those laboratories. Nevertheless, we were impressed, as we began to work with these samples, that many of these patients had evidence of immune activation, and we have used those samples in some of the work that I will be describing here today.


Now I will tell you from the outset that we have not implicated a single agent in Chronic Fatigue Syndrome, nor have we discovered the cause of Chronic Fatigue Syndrome. I wish I had something different to report to you, but we do have some interesting leads that we think merit follow-up. And I will describe for you the data that we do have with respect to our search for infectious agents, our search for biomarkers, our search for insight into causation and the ways in which we might parse the syndrome and develop new methods of prevention and treatment of the disease.


Let me begin first by talking about the work that we have done with Jose Montoya of Stanford. Now, our role here was to try to look specifically for infectious agents, and as many of you will know, Dr. Montoya is a rigorous clinician. He has been working in this field now for a decade or more, and he sent us a batch of samples. Those we characterized using a method which allows us to detect genetic evidence of infection with a wide variety of bacteria, viruses and parasites. And I will tell you now what the agents are that we can detect using this approach so that when I report to you these findings, you will have a better sense as to their significance.


The agents that we can detect using this approach are:


Human Herpes Virus 6

Cytomegalovirus

Herpes Simplex Virus, Type 1 and Type 2

Epstein Barr Virus

Parvovirus

Borrelia species, those frequently associated with a number of other diseases that we have looked at like Chronic Fatigue Syndrome,

Relapsing Fever

Chlamydia

Enteroviruses

Rhinoviruses

Eastern Equine Encephalitis Virus

Porreco Virus

Polisen Virus

LaCrosse virus

Lymphocytic Meningitis Virus

St. Louis Encephalitis Virus

West Nile Virus

Western Equine Encephalitis Virus

And

Influenza A Virus.


These particular tests are not capable of detecting historical infection. The agent must be present in the plasma or the spinal fluid, in the case where we examine spinal fluid, at the time that we do that assay.


Serology, which is looking for evidence of previous infections, which I think much of what we need to do in the future must focus, is not part of this report that I am making to you today. So, when we looked at the samples that we received… And there we have 285 cases and 200 controls. That is to say, 285 people with Chronic Fatigue, we found HHV6 in 4 cases and in 2 controls; that is, 1.4% of the cases and 1% of the controls We then typed these HHV6 and found that they were 4 from HHV6 Type B and 2 from HHV6 Type A So, HHV6, at least with using the methods we have for direct detection of infectious agents, if it accounts for Chronic Fatigue, it would do so in a very small proportion. I should add that Prof. Montoya is a known expert in Human Herpes Virus 6 and there is a possibility that some people may have been pre-selected in some fashion. It’s a potential confound, but in any event I don’t think it’s likely to account for much of what we see there.


The other samples that were assayed using these methods did not reveal any of the agents to which I have referred.


Now these same samples were studied using a high throughput method, which is a method which was pioneered here, with which we have discovered over 500 viruses, so we feel fairly confident that, to the extent of the technology’s capabilities at present, we would have detected everything that would have been present within these samples.


Now, looking at these sample pools, we found a virus known as anellovirus. These come in three different types, known as Alpha, Beta and Gamma, a torque viruses in three quarters of the samples. The sequence was quite variable. I cannot tell you that this is specific for Chronic Fatigue Syndrome. I really can’t tell you what this means.


We found retroviruses in 85 percent of the sample pools. Again, it is very difficult at this point to know whether or not this is clinically significant or not, and given the previous experience with retroviruses in Chronic Fatigue, I am going to be very clear in telling you, although I am reporting this at present in Prof. Montoya’s samples, neither he nor we have concluded that there is a relationship to disease. I’ll repeat that one more time. We found retroviral sequences, but their relationshipto Chronic Fatigue Syndrome at this point is unclear and, in fact, if I were to place bets and speculate, I would say that they are not going to pan out.


I’m going to move on now to talk about some of the other work that we have done with samples that we have received from Dan Peterson, also a well known clinician. I believe he is the clinician who has had the longest investment in Chronic Fatigue Syndrome, in fact, at this point, dating back in excess of 30 years. And the work that I will be describing, using the samples that came from Dr. Peterson, was supported by funds from the Evans Foundation and the Chronic Fatigue Initiative.


Here we examined 60 cases from Dr. Peterson’s practice, and we obtained samples from his banked spinal fluid repository, and those who exhibited what we described as “classical Chronic Fatigue Syndrome,” met the Fukuda as well as the Canadian Criteria, and we found there were those where there was something different, individuals who may have had Chronic Fatigue according to those criteria that may have developed malignancies—lymphoma typically, some years later.


Here we’re studying cerebrospinal fluid. This is a very difficult sample to obtain, as you know, also, we have invested a great deal of time in rigorously examining these specimens. Using the same approach as I’ve described, the multiplex panels that I’ve already talked about with respect to Montoya’s work, we found no genetic evidence of microbial infection in those spinal fluid samples.


We had one patient who had HHV6-B, but that’s a single patient out of 60, and I don’t consider that a statistically significant finding. Now it is possible that if we think that Chronic Fatigue has many different possible causes, that there may be a small subset of people who do have disease as a result of infection with a single agent, but this is certainly not going to be a consistent theme that is going to be helpful to us in terms of diagnostics at this point. We have not yet done—completed—the high-throughput sequencing, so I can’t tell you the results we will obtain with that work, but I can report to you on some other work that we’ve done both in spinal fluid and plasma. With, again, support from the Chronic Fatigue Initiative and the Evans Foundation.


Let me start first with plasma. So in this instance, what we are doing is using plasma collected by Dan Peterson, Jose Montoya, Cindy Bateman, Nancy Klimas, Sue Levine and Donna Felsenstein. We examined 200 cases, 200 controls using Luminex analysis. And I’m going to give you a summary of our findings to date, and then I will move on at the close to describe the findings with the spinal fluid.


Let me summarize by telling you that our data now suggests that there may be differences, substantial differences in bio-profiles, things that we may find between people who have had the disease for three years or less and people who have had the disease for more than three years. We believe that this is going to be important because it may actually have occasions for therapy as well as diagnosis. So, again, there seems to be a significant difference in profiles obtained from people who have had the disease for less than three years and people who have had the disease for more than three years.


In what I would call the “Early Group,” which is less than three years, there seems to be a number of markers suggestive of some sort of allergy aspect. They have increased numbers, typically, of eiosinophils in blood, and when we examine their markers from areas—cytokines—we find that there are definite differences. I’m not going to talk a great deal about the differences between before three years and after three years, because we are still working on this.


So I will confine my discussion to a number of cytokines and chemokines that are up or down in people who have disease, and again, this is important because I think that we may find that there are drugs which can be used to modulate the levels of cytokines; and while these may not get at the cause of the disease, the primary cause which I still believe is likely to be an infectious agent, it may give us some insight in ways in which we can manage and decrease some of the disabilities associated with Chronic Fatigue Syndrome.


So,overall, in cases we see a decrease in levels of IL-17, IL-2, IL-8, TNF,while we see an upregulation in leptin and serpin. Now there are some other cytokines and chemokines that are less well known and because they are obscure, I’m not going to describe them in any detail except to say that there seem to be significant differences in some of these cytokines and chemokines that are associated with pro-inflammatory responses like Il-17 and IL-2 and IL-8 and I think that this is worth following up in larger numbers so that we can understand what this means.


We also find—I feel comfortable in saying this—in people who have had the disease for less than three years, there is frequently an elevation of the cytokine IL-17. This is important because there are a number of drugs that have been used to modulate the level of IL-17. I would caution people listening in not taking from what I'm saying that it makes sense to go ahead and regulate IL-17 if you’ve had Chronic Fatigue Syndrome for three years or less, nor would I recommend that you go off and ask that IL-17 level be measured, so that you can direct, in caravan fashion. Again, this is a work in progress so that you can understand what it is, in fact, that we are doing and where we think it may take us.


I will now talk about spinal fluid. Again, we’re using the bio-sequence strategy, where we are using the Luminex, the same one we used for plasma, to measure levels of cytokines. Now, the pattern in spinal fluid is very different than it was in plasma. And these patients that we’ve received samples from—donated samples—via Dr. Peterson, do not appear to fit this profile in spinal fluid of very different patterns from less than three years and after three years.


We see these patterns consistently throughout the research. So what we found was that patients with Chronic Fatigue Syndrome, these are classic cases, had elevated levels of a cytokine called IL-10, another called IL-13. These are typically referred to as Th 2-type cytokines. We also have an elevation in levels of what are known as four Th1-type cytokines: IL-1 Beta, IL-5 TNF-Alpha and IL-17.


Now, this is compatible with some sort of an immune profile of a particular response that may provide insights as evidence of immunological dysregulation in Chronic Fatigue Syndrome; but again, I don’t want to recommend that anybody go out and get a spinal tap, because this is not going to, at present, have any profound impact on your care.


Now the only thing that we found that was different in patients that had classic Chronic Fatigue Syndrome versus those who had Chronic Fatigue Syndrome which was associated in addition with inducing something different like a lymphoma at a later date was Fgf Beta in those patients who subsequently developed lymphoma. We don’t yet know what this means. This is something that we still need to pursue.


Now, that is a summary of everything that we have done vis-à-vis looking at plasma and spinal fluid from different cohorts of patients obtained through the initial NIH study, Jose Montoya study, Dan Peterson’s study, and the Chronic Fatigue Initiative, which represents some overlap between the NIH study and the CFI study.


So at this point, I would like to turn my attention to describing what it is we are doing at present to try to further deepen our understanding and get us some answers as to what might be triggering these immunological abnormalities. And again, I want to re-iterate that the sourses of study that we are undertaking were pursued in close collaboration with the clinical group comprising Dan Peterson, Jose Montoya, Nancy Klimas, Cindy Bateman, Sue Levine, Donna Felsenstein and on this end, Mady Hornig, David Hirschberg, Brent Williams and Raphael Tokarz the scientists who are doing the work in the laboratory that I will be reporting to you.


We are using a method for detection of viruses and using an unbiased approach referred to, essentially, as high-throughput sequencing. We take plasma samples, we take spinal fluid samples, we have retroviruses, by treating with specific enzymes to degrade those materials which might otherwise interfere with sensitivity, we then extracted and we sequenced them and we looked for an agent and anything we find suggestive of being a potential candidate, we have developed a PCR assay to look for other subjects for evidence of infection, and we find a larger sample set, which at this point is now in excess of 450 patients. So this is a very large sample set to mine by this approach.


In addition, we specifically looked for bacteria using a method called 16S ribosomal RNA sequencing. This is a method that allows us to amplify by polymerase chain reaction PCR a region that we can then sequence to look for variability in the presence of specific bacteria. We did the same thing by using a method called 18S ribosomal RNA to look at fungi. And I know that many people are interested in that—not only in viruses but also in bacteria and fungi.


Now we have not yet completed the collection of fecal material. I think that this is extremely important because the microbiome in our intestinal tract has the potential to polarize and modulate immune responses. It is imperative, then, that we characterize those. These types of studies are extremely expensive to do. They take a long time too.


So, we are only in the early days of doing that, and I have to be very frank with you and tell you that we don’t have the funding that’s required to do this work as rapidly as we need to do it. So that is always a challenge, and as you know, that’s a big challenge with Chronic Fatigue. And I’m not pointing fingers at anyone in particular, but I think it is very important that everybody realize that with sequestration at present, the budget at the National Institutes of Health is very, very, very much diminished and it is extremely difficult to get funding.


The other thing that we are trying to do is to develop immunological assays that have the capacity to detect previous infections with infectious agents. The method we use goes by the name of LIPS (L-I-P-S; Luciferase Immunoprecipitation Systems). It is a very rapid, very sensitive approach whereby we take the protein associated with the agent we think might be important, we test it in a special assay that allows us to find antibodies, to measure the amount of those antibodies, to determine the specificity of those antibodies, and that then gives us a picture of what an individual might have seen at some point in the past. This method requires that we specify the agents we want to test for, so we also have another system which is unbiased, very similar to sequencing, but this is what we call a “peptide array.” It allows us to survey the potential viruses. We haven’t gotten the bacterial one yet. It surveys the potential viruses that might be associated. So we’re looking for direct and indirect evidence—shadows, if you will—of earlier infections.


Now I think that’s really all I can tell you about the data that we’ve generated, the data that we hope to generate, and the challenges that we face going forward. And at this point I think I will close my presentation, unless there is something else that Dr. Unger would like to bring up.


UNGER:


No, thank you very much. I thought that was just fantastic. And perhaps we could move to the questions that have been submitted for Dr Lipkin.


ANNOUNCER:

Great. Thank you, Dr. Lipkin.

We’ll now move on to the questions which have been submitted for our guest. The first question is:

At the recent Invest in ME Conference, it was quoted by a colleague of yours that they have identified several promising pathogens, including a novel pathogen but that the work was early. Can you update us on the work and let us know when it might be published?


LIPKIN:


Thank you for that question.


As I outlined for you during my talk, we do not yet have a novel pathogen, nor do we have a pathogen candidate in which I would have any confidence. So at this point we have no plans to publish anything in this respect. What we will do, nonetheless, is publish these data very soon on biomarkers and profiles of cytokines and chemokines. There I think we have great confidence in the spinal fluid work that we have done with Dan Peterson, as well as other work that we have done with other colleagues under the auspices of the Chronic Fatigue Initiative.


ANNOUNCER:


Thank you. Second Question:


1.How do ME/CFS patients’ microbiomes compare to controls? And what are the implications of the differences? 2. Can the variations be addressed with medication or other methods? How significant are the symptoms?


LIPKIN:


The question here pertains to the microbiome of patients with Chronic Fatigue versus the microbiome of people who do not have Chronic Fatigue Syndrome?


We have done research on the microbiome in autism and we have found bacteria that are important in causing gastrointestinal problems. We also have some recent work that has to do with the microbiome of patients in colon cancer where we have found that there is specific bacteria associated with the aggressive forms of colon cancer. We have also done work of this sort in childbirth and have found that the microbiome differs in children who are stillborn versus those who are born normally. And in women who are at increased risk for HIV infection, in sub-Saharan Africa. So we have methods that are already in place that could be used to address the microbiome of Chronic Fatigue Syndrome; however, as I said earlier, we are still in the process of trying to collect the stool samples that are needed. The first effort that was made toward collection did not yield what I think was sufficient material for us to begin that portion of the study. It was uncomfortable collecting specimens using swabs, so we have shifted to a method where they directly deposit the fecal material in a special cup which will then sent back to us. As soon as that is completed, we will initiate that work.


We have the funding to do approximately ten percent of what’s needed. So again, I re-emphasize the fact that the budget is really inadequate to do this type of research. It is extremely expensive research. And it’s probably inappropriate for me to be passing the hat, but that is precisely what I’m doing.


ANNOUNCER:

Thank you.

Third Question, Dr. Lipkin.


The use of standard measures is important to be able to compare research results between studies. You always cite the Multi-site NIH sponsored study on XMRV led by you as a good example. Dr Lipkin,if we can help more researchers be aware of this approach would it help results be more comparable, and increase the evidence base of ME/CFS research?


LIPKIN:


Yes, let me take that. That’s one more thing that I want to mention with respect to the microbiome. I think that the microbiome is going to be where the action is. That’s my belief, my speculation, so I am really eager to pursue that work.


Now, with respect to similar and standardized measures, I could not agree more. This is why in fact, when we designed the study on XMRV and PMLV which was continued with CFI, we obtained samples from Jose Montoya, Dan Peterson, Cindy Bateman, Nancy Klimas, Sue Levine, Donna Felsenstein, Tony (I can’t believe I’m blocking Tony’s last name—terrible…)


UNGER:


Dr. Komaroff?


LIPKIN:


Yes, Dr. Komaroff. Thank you very much. That’s the problem. You forget your friends’ names and I guess that’s forgiveable.


Anyway, we consulted with them to set up very clear criteria for how we would define a case and how we would define a control. In addition, we specified certain laboratory tests that we required be done before somebody was included, so we would exclude individuals with a history of depression or thyroid disease or elevated liver function tests which would indicate infection of the liver, these sorts of things. So it is critical that we make sure that when we are doing this sort of work we are always comparing apples and apples, not apples and oranges. Or, as you say, we are not going to be able to do the sorts of metanalyses that are required to understand what’s necessary to address the challenge of CFS.


UNGER:


Could I just comment on one thing. Add to that, I think, just looking, that your observation on the time course of the illness is so important as well. And that's something that has not been taken into account in studies of this illness. And so, the more we know about this illness, the more we know about things that actually influence this illness, and that is why it’s so challenging to come up with some consistent findings across studies.


LIPKIN:


Dr. Unger, to reinforce what you just said, we did not decide, a priori, in advance—we did not decide in advance to look at less than 3 years and to look at more than 3 years. We began to examine our data. This came out of analysis of the data. And we analyzed it and we found that this was a break point, where there were some strong differences between the two groups. At some point, I think that it’s very important, given what we now know, that that be inserted into any analysis, certainly of infectious agents and immunological parameters.


ANNOUNCER:


Thank you.


And, Dr. Lipkin, I have one more two-part question for you.

First, is your laboratory studying the polyclonal B cell reactivity you detected in M.E. patients? And Two, Dr. Anthony Fauci, in his immune regulation lab, has published intramural work on B cell dysfunction and autoimmune diseases. Do you know if they’re looking at ME/CFS?


LIPKIN:


Let me take the second part first. Dr. Fauci, as you’ve said, is a rather extraordinary resource and has loads of expertise, not only in microbiology in general but also in cellular and humoral immunology. I do not know whether or not he or anybody associated with him in private is looking at B cell dysfunction in ME/CFS. That would be a question that you would need to pose to him or to other people who work under him?


Now, with respect to polyclonal B cell reactivity, as I mentioned in the very beginning, in work that we published in the 1990’s that is Birgitte Evengård, and I published together, we found evidence of polyclonal B cell reactivity and at that point, we described in the final paragraph of the paper we wrote, and we describe specifically as to whether borna virus could be implicated in Chronic Fatigue Syndrome, we said, in fact, that we were unable to find any evidence of that, genetic or antibody based; but at that point, as many of you will recall, there was a very strong sentiment in some portions of the scientific and clinical communities, not all of it, but in some portions of the community, that this is a psychological illness. And what I said was that, based on our findings, we had very strong evidence that people with Chronic Fatigue Syndrome were ill. It was a real, physical illness, and they deserved a deep dive to find out why they were ill. This was back… you know, we wrote this paper in 1997, a long time ago, and I am now pleased to see that people are paying much more attention to this disorder and what we can do about it.


That said, our evidence suggests, based on the cytokines that I’ve described to you, that there is, in fact, ongoing stimulus to the immune system which results in activation and may well account for many of the symptoms associated with the disease.


The challenge, as I’ve said, is that we have no resources to go after identifying what it is that is stimulating these responses. So, again, I think it is critical that you apply pressure on your congressmen and your senators to generate more research funding for Chronic Fatigue Syndrome research, and if you have the means yourself to do something about this, that you become invested in it because we are partners in this thing. We cannot do this work without your support. We do not have the resources to do what is required. I remember in the very early days of HIV/AIDS when I was still a trainee at the CSF in fact, it was very early days. And in those days there was very little support for HIV/AIDS. It really took advocacy to force that.


Now you’ve got a number of people who are very receptive to this. Tom Frieden is receptive to it. Tony Fauci is receptive to it. But their hands are tied right now because of sequestration. So it is important to that you do something to propose additional resources be allocated to the National Institutes of Health, the Centers for Disease Control, so that this work can proceed, and so that we can get the answer that we need.

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