The following is a transcript of Dr. Lipkin’s talk at the CDC in September. You can watch the full lecture here:
This is actually how I got into this field, I was invited by Brian Mahy, who many of you will remember, and Jim Dobbins who some of you may remember, because of these reports in Japan that there were individuals who developed ME/CFS as a result. That then led to a large NIH study which found no association and Bill Switzer was a critical partner in this work and Judy Mikovits was a critical partner in this work. But in any event, we found no evidence whatsoever of XMRV being implicated. And this was a very expensive, very difficult study to do but it emphasized the importance of getting this sort of work done in a rational way.
Okay, now, for the meat of the talk, now that I’ve shown you all the tools we can bring to bear on the problem, let’s talk about ME/CFS. Right now it’s clinical diagnosis and it is a diagnosis of exclusion, so we make sure that people don’t have other intercurrent infections with hepatitis C, they don’t have hypothyroidism, they don’t have other sorts of problems. And these are data that came from what’s now known as the National Academy of Medicine. It is not a small problem, it’s something like 2 million people in the U.S. alone, and is very expensive because these people can’t work as well as requiring various types of support. The causes of the disorder are not well understood, many patients report what seems like an infectious prodrome with pharyngitis and swollen lymph nodes and feeling feverish and having night sweats and so forth.
So initially when we became involved in this, we figure this is what we’re going to do, was a standard sort of pathogen search. Now this is from the work that we did with Brian and Jim Dobbins, who are not on this paper because for reasons unclear to me, they weren’t able to do that at that time. But they really supported this work, it wouldn’t have happened otherwise. We didn’t find any evidence for Bornavirus infection, but at that point I did make one observation that I think in retrospect was prescient, and that was that these individuals, as we studied them, had high levels of antibodies to things like beta-galactosidase suggesting that they had polyclonal B cell activation.
This is really some of the very first work suggesting that there are immunological abnormalities in these patients. And this work was really done back in 1995. We now have a center for solutions for ME/CFS that’s funded by the NIH, though not as well as it should be. The clinicians are shown in yellow, Dan Peterson, Sue Levine, Cindy Bateman, and Jose Montoya has been replaced by Moneghetti, and then we have Oliver Fiehn from metabolomics. We have John Greally, who primarily focuses on transcriptomics, Tony Komaroff,who develops applications that we use for tracking patients then an admin and Columbia based core as shown here.
So what I’m going to try to do is to summarize som of the things we’ve already done. We found altered profiles for various cytokines and chemokynes in blood as well as in spinal fluid. We did these multi-center studies and so forth.
This is an example of work that we did looking at cytokine expression over the course of the illness, and what we found was that there were individuals who were caught within 6 months to a year or so of the original diagnosis, they had different cytokine profiles than did those individuals who had disease for more than 3 years, suggesting that there might be burnout, I think was the interpretation that we rendered at the time. We really don’t have an explanation for it as yet. And there were differences in levels of stickiness and chemokynes in spinal fluid and our control group here was not only normal controls but of individuals who had multiple sclerosis and you can see here now that there are various abnormalities which we’ve explained elsewhere
The center has three projects; one is broadly considered the microbiology of ME/CFS, which is essentially characterization of bacteria, viruses and fungi, and everything that we can access; feces, saliva, and blood. And the indirect measure of exposure is going to be increasingly important because we’re not really going to find anything as I’ll show you looking at direct measures based on sequencing. Metabolomic and transcriptomic studies that we’re doing with peripheral blood mononuclear cells, and then some translational aims where we look at the impact of somebody standing erect which is a problem for many of these patients, or doing some sort of an exercise test which gives them stress. One of the key features of ME/CFS is that these individuals don’t recover after exercise. And I’m going to show you some data now which I think will explain how this works.
One of the things that we’re using, which is gointo be extremely powerful, is called TruCulture and we actually began to look at drawing samples across multiple sites, their differences and levels of transcripts and proteins as a function of how things are preserved. A group at Institut Pasteur came up with a solution for this where they pull blood directly out of the patient’s arm, put it into these culture vials which can be supplemented with a variety of stimuli, in some instances they represent bacteria or viruses or fungi, and then at regular intervals, you depress this plunger, collect the supernatant, and then the cells below, you can extract and use for various sorts of things.
So this illustrates the way in which you can reduce the variability so, these are individuals who were exposed, who had cells were exposed to lipopolysaccharide and you can see how much tighter the data are, again, I don’t have a pointer, but if you look at the left there, you can see a lot of spread and as you use this other system it becomes very useful. And when you lose that sort of spread, when you get better resolution, you can find individuals that have specific genotypes and phenotypes. So that’s the approach.
So let’s talk about the fecal microbiome; so we’ve actually now done this twice, we had a pilot study that was funded by the Hutchins Family Foundation, which was 50 cases and 50 controls, and then we had a larger study funded by the NIH, which is roughly twice that. And if we look at the Venn diagram showing what comes up as being common, we find that there are 15 different bacterial types that are highly represented. You can see Ruminococcus, Clostridius species,and why is this important? Well, the Clostridius species are associated with inflammatory bowel disease, [inaudible] and so forth. And then you also have these butyrate producers that are reduced. If you have inflammation of the enteric mucosa, you have the opportunity for trafficking of molecules produced by bacteria into the systemic circulation where they can then have an impact. So that is one thing that we’re now pursuing and we do that by using metabolomics in those individuals who have ME/CFS who have irritable bowel syndrome, versus those who don’t.
Again, you can see the butyrate producers reduced, that appears to be common, but there are other bacteria which appear to be coming up as well.
We’ve looked at the salivary microbiome, there again you can find differences between cases and controls. So I don’t know that these are cause or effect, or simply epiphenomenal, but they’re at least observations and give you data that you can test in other populations. And I was meeting earlier with Beth and her team and I think, given that you have saliva, we can examine these sorts of issues. You can then ask questions about predicted metabolic pathway that might be disturbed as a function of different populations and you can see here now that the pyrimidine deoxyribonucleotides salvage, you know, is differentially infected, impacted unfortunately.
We’ve lost, you know, a lot of the information on the left side of the screen but there are inferences that you can make based upon what’s known.
We recently, and by recently I mean the day before yesterday, completed a proteomics pilot. Very, very small so it’s only 40 subjects and 40 controls, but they’re a well-matched population
And this finding has been very, very cool because what we’ve identified is that there are a number of proteins which appear to be differentially expressed in individuals who have ME/CFS. This is not to say that they all have the same cause, but if you use cancer as a paradigm for understanding this, you have multiple ways in which you can get to cancer; tumor suppressor genes, oncogenes, we think something similar is happening here.
So, I’ll give you an example on the next slide which supports the notion that at some point, there might have been an infection that resulted in an immunological trigger. And these are not bad odd ratios, you know? They’re all in excess of 3. Now what are we actually looking at here? These are proteomic studies that reveal differentially, a differential expression of immunoglobulin kappa and light chains and heavy chains, which are present at increased concentrations in individuals with ME/CFS. And you can use these immunoglobulin repertoires to try to understand.
You can actually use these then to develop biomarker diagnostics which allow you to say this individual is at risk for having ME/CFS. And it’s proving to be extremely powerful, we’ve done similar work with metagenomic analyses of feces and with metabolomics.
Metabolomics work we’ve done in collaboration with Oliver Fiehn and Dinesh Barupal at UC Davis. They again are focused really on rigor and granularity so they separately analyze biogenic amines, high and low density lipids, and primary metabolites.
Again, comparing this small group of 50 patients and 50 controls with the larger group of NIH patients, we can look for similarities and here we’re finding those as well. So there are specific abnormalities that are referent to a mitochondria, to phospholipids that are associated with ME/CFS and breakdown differently when you look at individuals who have abnormalities in the bowel and those who don’t.
So you can see here summarized, the glucosylceramide, carnitine plasmalogens, and so forth.
Now what are plasmalogens? I doubt anybody’s heard of them, but they are decreased in ME/CFS. They’ve been observed to be decreased in a variety of dementias as well and in Parkinson’s disease. So this may be some sort of marker for CNS dysfunction.
The last thing I’m going to describe for you in this respect is the epigenetics pilot that we’ve recently done. So we used a system that with bisulphite treated libraries for capturing 5.5 million methylated residues in the human genome. We restricted ourselves to those where we had good data, meaning 20x and we mapped and annotated their location within a specific genes with respect to promoters, non-coding sequences, introns and so on. And these are the results and this again, just like the proteomics work I was showing you is not yet published.
You can see that we’ve got 724 sites in almost 600 genes that met our criteria for methylation. You can see the distribution in the various regions in relationship to those genomes and this is really very interesting, so ME/CFS is marked there in blue and you can see that when we look at percentage of methylation, as you begin to look beyond [inaudible] 60-65%, you get hypermethylation in the ME/CFS group. And this just shows the data in that way. And it’s really, I think this is really some of the most promising data I’ve seen thus far, that has to do with talking about ways in which genomic modification might occur.
And there are a number of different gene circuits that have been implicated in this kind of work, it becomes, as you can see, extremely complicated. But many of these are associated with oxidative metabolism as well as with stress.
We highlight here, the ones where we see the largest changes and if anybody wants to see these data in more detail, I’ll be happy to show you.
35:54 (For animations, please refer to youtube presentation)
So I’m just going to demonstrate for you the ways in which we’re beginning to tease this apart. When we began looking at the most remarkable findings, at least to our mind, we found that they were related to iron metabolism. Now if you look at the methylation differences, you know, they’re modest, 12%, 11%, I don’t know how important that is. You then, we actually then measured RNA abundance so we created real-time PCRS so that we could actually look at the products of those methylated genes, and although no one gene is affected in a great fashion, if you sum them all, they could have a profound effect. So this is where we slip into fantasy, and I’ll show you what we think is going on.
So and this is moving into the cell, in this case we’re talking about monocytes and macrophages and somatic tissues, rather than red blood cells.
So we have our first lesion here, right? We have a lesion at the level of the clathrin coded pits. We have another lesion as these iron molecules are exiting the endosome–
You can see here now on the mitochondria, there’s an effect as it moves into mitochondria.
So in essence, what happens is you have an impact on multiple levels which should have an impact on your ability to metabolize oxygen and this could result in weakness and fatigue.
And we have to do the same thing with all of these circuits we’ve identified using all of these different methods and as we’re putting this thing together, it begins to look sort of like an anemia of chronic disease, with red blood cell damage and so forth, oxidated, stress. And this is a model that’s still in the process of being worked out.
I mentioned earlier to you the fact that we’re trying to build Serochips; this is our first Serochip which is designed to look at autoimmunity.
But more recently we’ve come up with something which is even more expensive but more exciting I think, and that is the entire human proteome complemented or supplemented by other targets that we think are important. Now in our experience, 2-5% of the patients who come through our research clinics, wind up having some evidence of persistent herpes virus infections. Some clinicals have more than others, Jose Montoya at Stanford had large numbers of those. Peterson has them as well. So, working again with this new company, Nimble Therapeutics, which has replaced NimbleGen, we have these 20 thousand odd human sequences. There are 16 mer peptides with 12 amino acid overlap.That’s not as good as what we’ve done in the past where we have 12-mers with 11 amino acid overlap,but you can see when I tell you these are arrays are $3500 each, you know, we had to make some compromises. We’ve added peptides that cover mitochondrial proteins, we have control scramble peptides which are critical for interpretation of singles, of signal, and then we have the reference proteomes of all known herpes viruses, because we’re concerned that they may be associated with persistent infection and the peptides that cover the proteomes. And then, because there’s frequently overlap with tick-borne diseases, we’ve used the refined epitope map that we’ve previously generated that allows us to do this. So we are embarking on a very expensive study that will allow us to examine, now that we haven’t found anything using direct methods, evidence that at some point these patients were also had
So one of the things that I want to highlight here is that, you know, we had the culture and Gram stain, and we had sequencing, and I think sequencing is passe, so what’s next? Right, is to examine the history of immune response so that we can make association between early life infections and [inaudible] quality and these kinds of chips, which are incredibly expensive, but hopefully it’ll come down in cost, will allow us to do this. So in summary of this part, Beth will agree, this is an important disease, it’s under studied. It’s important economically, it’s also important in terms of clinical medicine, all the things that we’ve done thus far provide biological evidence that this is a bonified illness; with cytokine, metabolomic, proteomic, epigenetic, transcriptomic differences, all consistent with some sort of an abnormal immune response oxidative stress, and a dysregulated microbiome. So we’ve come a long way. The high throughput sequencing, thus far, has not identified any one infectious agent so, although we’d hoped that we would find something, we’ve gone home empty there. But the serology will hopefully change that and the future work is going to have to focus on identifying environmental triggers of pathology in people who are genetically susceptible. And this is going to be very exciting.
I couldn’t leave without showing some movies, I’m not going to show you the movies actually though, but so Contagion, you know, Plague Inc. we helped with the game, I did some work on Bond 25 but I can’t tell you about it. I can’t even tell you if they’re still shaking and not stirring the martinis, but it should be cool. I hope it’s going to be cool anyway. If they selected one of the things I gave them. And then Utopia, which is about a flu pandemic that’s supposed to, you know, wipe out the world except for a select few maybe up here on Clifton Drive. And then Three Months, which is the story about an individual who becomes exposed to HIV and worries for 90 days, I don’t know. But you know, it’s not enough yet to pay the bills but it’s fun to work with these people.
And then the people who really do the work, who I’ve tried to identify here. And I’m happy to take questions, thank you very much.