[Gut Microbiome – starts at 25m 52s in the webinar]
So I'd now like to turn to my last topic and that is the topic that many of you already know about, it's our work in on the gut microbiome in ME/CFS. This was published in June in the journal Microbiome(?). This work was a collaboration between Ruth Ley's lab and in in my department and the two people in her lab that worked with us were Julia Goodrich and Tony Walters, again Susan Levine provided the samples from her patients and some controls and Ludovic Giloteaux was the lead author, he is a postdoctoral associate in in my lab.
This work was funded again by a two-year NIH grant that is now expired, again it was a grant, a so-called R21 grant, that's used to explore new ideas in research relevant to NIH. So our study
population were established patients of Susan Levine, she was able to persuade 39 controls, 30 female 9 male, 38 female patients and 11 male patients to provide us with samples, we received both blood samples and faecal samples from these individuals. Of these patients 25 of them reported a sudden onset of ME/CFS, in age the median age was fairly close between the controls and the patient's although there was quite a variation as far as the age between the two, and the body mass index was quite normal though again there was quite a variation from underweight to overweight. Of the controls, 8 of the 39 controls reported some sort of intestinal discomfort such as constipation or diarrhea or stomach ache occasionally, of the patients however 32 of the 47 actually reported that they had some sort of intestinal discomfort.
So let's review the human microbiota briefly, in and on humans there are as many microbial cells as human cells so it's been, that's sparked, that knowledge sparked the interest in the microbiome that has existed in the last few years, along with some new technology that allows this type of microbial cells really to be analyzed quite thoroughly. The majority of these microbial cells are in the large intestine and it is known that they provide enhanced nutrition and protection against pathogens. So many ME/CFS patients complain of gastrointestinal symptoms, so that leads to the question is abnormal gastrointestinal function causing some inflammation that that could be causing some of the sickness symptoms that any CFS patients feel, also does the bacterial gut microbial of ME/CFS patients differ from that in healthy controls? Those are the questions that we asked.
So to explain what we did I need to point out that some bacteria have lipopolysaccharides, abbreviated as LPS, on their surfaces. On the left you will see a diagram of a bacterium, a so-called gram-negative bacterium, and on its outer surface it has these so-called lipopolysaccharides that extend out and can be examined by various assays. So on the right is a diagram of a dysfunctional gut, so if you have some problems with your gut the cells, for example, might allow passage of bacteria from inside the digestive tract to the outside, and this can get into your bloodstream then and if the bacteria get into your bloodstream then the LPS is in there and you then have LPS in your in your blood which then can set off sort of alarm signals to the body that bacteria have invaded and the body will take measures to deal with that.
So we look to see whether LPS and also LPS related proteins are elevated in ME/CFS cases versus controls, so on the left you see the amount of LPS present in controls vs ME/CFS patients. Each of those dots, on the left a red dot, on the right a blue dot for the ME patients and that's the actual measurement that we got to LPS so that you can see there's quite a range there are some patients who have the same amount of LPS in their system as the controls, but if you look at the median and you'll see the line there shows the median, the median indicates that in general the LPS is higher in ME/CFS patients than in the controls.
We also looked, if you look at the far right graph, we looked at a protein called LBP, for LPS Binding Protein, this is a protein in the blood that binds to LPS and that is also raised in ME/CFS and it's significantly different, there again you can see the median line of the level of LBP in the patients and controls and that in the middle there is a receptor in our bodies that detects this LPS bound to LBP that lets the body know that that there are potentially bacteria around, and again it's increased in ME/CFS versus the controls so that's showing something different between patients and controls.
This implies, this data implies, the fact that there's extra LPS and significantly higher levels of these other two, of these two proteins indicates that there is ongoing damage to the gut and this is likely than causing this increased microbial translocation in ME/CFS patients. This observation has been seen in other diseases, so other diseases also have elevated LPS, there's Fatty Liver Disease, it happens in HIV infection and two types of inflammatory bowel disease, Crohn's Disease and Ulcerative Colitis.
So how did we analyze the gut bacterial microbiomes?
Well the patient's themselves actually collected faecal samples, they put them in a preservative and then shipped the samples to Cornell where they were processed. They were processed for DNA extraction and then some of the DNA was then further processed for sequencing on so-called MiSeq sequencer, an automated sequencer that can give you as many as 140000 sequences per sample, so this sequencing data was obtained and then analyzed. So this DNA sequencing, by looking at the actual sequence of the ribosomal DNA, of a portion of the ribosomal DNA, in the bacteria you can identify the family and sometimes the genus and species of the bacteria that is present.
So this happens to be the Fox family and you know that foxes are animals, they are belong to the class mammal, they are a carnivore, they are a member of the Dog family, and then as far as the genus, as far as the genus goes, here's an example of two different species within the genus of Fox, there's an Australian fox and the British Fox. These two species are different but they're in this genus and then of course each one of these, the British fox for example, is an example of the species.
The reason I'm showing this is to indicate that while we can identify the family and sometimes the genus and sometimes the species, if we've only identified the genus of of bacteria we could have two bacterial types in the gut that are is different from one another as that Australian fox and the British Fox. So it so that we can have very different bacteria even though we think they're, we know that they're in the same genus, they can still be quite different in their function. One might be, for example, in the same genus the bacteria, you can have a pathogenic bacteria and you can have normal healthy type bacteria.
So the other issues I should mention is a different species can't always be identified by sequencing. We have to group, if two bacteria have 97% identical sequences we say that they are the same species but we don't actually know, the same group, we don't actually know that they're, the same species.
So what we found is, as we kept, as you look at more and more sequences per sample, we could find that the ME/CFS patients had fewer species in their gut microbiome than the controls so, as you get out to 30,000 sequences on the right, the controls had more, different types of species and gene genera and family members then the ME/CFS patients. This means we have a loss of species richness in ME/CFS microbiomes.
We also found that 24 families and genera differentially abundant between ME/CFS and healthy individuals and this is statistically significant. So I'm going to put a red triangle here, these are all the species that have a lower amounts in patients than in the controls so this is again an indication of the reduced diversity in the patients versus the controls. Among those species that are reduced we see that the members of the Ruminococcaceae are significantly higher in healthy individuals then in the patients and, the reason this could be important is that this is a beneficial type of bacteria, it produces an anti inflammatory protein and butyrate which is an anti-inflammatory fatty acid so this abnormality is also seen in Crohn's disease and Ulcerative Colitis.
So we attempted to use this information to see whether we could identify and distinguish between patients and controls and we, actually we're able to see that we could just identify patients pretty well if, in this particular cohort 55% of the cohort were patients and we were able to identify 53% of the cohort as patients. So that's actually quite high, it's over 90%, unfortunately however though we also identified a number of normal, 12% of the normal people, where classified as ME/CFS. Nevertheless we were able to identify 30% as being normal so this gives a total value of 83% correct classification by microbiome in combination with the levels of those three molecules in the blood.
So I'd like to mention that there are limitations of bacterial microbiome analysis, we can't really tell whether strain is differentially present, for example you need to use other methods to determine whether you have a bad E.coli or an ordinary E.coli, now these bacterial studies don't reveal what eukaryotic pathogens might be present and also it doesn't indicate whether viruses are present in one and not in the other, other types of analyses are needed for that. So to conclude we have less bacterial diversity, we have association of the abundance of specific groups with patient or healthy status, we can classify 83% of the samples correctly and we see that anti-inflammatory species are reduced in patients.
I now like to briefly describe a case report that was led by Betsy Keller who is a exercise physiologist at Ithica College. Now she had a pair of identical twins come to her, one of them had ME/CFS and one of them did not. These were male twins aged 34 and the ill twin had been sick for four years with ME/CFS. She performed two successive cardiopulmonary exercise tests on these individuals, she found that the ill twin on the first day had a lower maximum vo2 than the well twin, and I'm not going to go into what that means except to say that that does mean that the ill twin is less physically fit as you might expect for someone who's been sick for a few years, but what was more interesting is that on the second exercise the ill twin reached anaerobic, the necessity to use anaerobic, metabolism at 13% lower than he did in the first exercise.
So that abnormality, that that reduction in the anaerobic threshold, indicates exertion intolerance in the ill twin and this is a significant result because ill people can usually repeat these results, for example someone with multiple sclerosis who took these two tests would be highly likely to be able to repeat their results from the first to the second, they would not have had this decrease in anaerobic threshold.
So we decided to look at the gut microbiome in these individuals, and again just like the whole cohort that we looked at the ill twin had reduced gut microbiome diversity and also exhibited changes like the larger study in the relative amounts of different types of bacteria, so each color there shows a particular bacterial family and the ill twin clearly has a different composition than the well twin. So this is interesting because in this case the genetics of the two individuals being tested are identical but yet, because one of them has ME/CFS and the other is well, there are differences seen in the gut microbiome.
So what attempts are being made to alter microbiomes in various diseases? In various diseases people are trying to use diet, pre- and probiotics, and antibiotics, and faecal transplants and a number of people have written to me asking whether one of these interventions might work in ME/CFS and the fact is that I don't know because we need more studies to find out whether these interventions could be useful.
One, I would like to mention however that they're not necessarily completely innocuous to try these interventions and on this slide I'm putting up just one example of a review article which examined the effect on inflammatory bowel disease, in this case Crohn's disease, a table about Crohn's disease which as I mentioned earlier has some similarities in its gut disruption to what is seen ME/CFS. A number of different interventions and number of different pre- or probiotics were used and in some cases these are beneficial but note that in some cases they actually did harm to the patients, so we can't just automatically assume that prebiotics or probiotics are always going to be beneficial, and that's why we definitely need more studies to find out what might be beneficial to alter the gut microbiome in ME/CFS.
We now have abundant evidence for biological disruption in ME/CFS. We have aberrant functioning of white blood cells, abnormal physiological responses to exercise, abnormal gut microbiota, altered metabolism and, something I haven't talked about, that there are anomalous images from brain scans. That is actually some work going on at Cornell Medical School as well as a number of other locations, that reveal abnormalities when you use MRI or patch(?) and other types of scan.
So that's why it's unfortunate that inappropriate images of ME/CFS have given the distorted perception to the public of this disease not being as serious as it is. I'm just showing you six of the images is accompanied the many dozens of articles about our microbiome study across the country and the world. These are actual illustrations that is unfortunately often the type of illustrations often used because of the idea that we have a chronic fatigue rather than an actual disease, and I would like to suggest that it be very important for the patient organizations to be able to provide appropriate images to editors who need illustrations of what Chronic Fatigue Syndrome or Myalgic Encephalomyelitis is in one of their articles.
Finally I'd like to say that you can get additional information about our microbiome study because I gave a longer talk about that at this Invest in ME meeting and there's a DVD that you can order. I also will be doing another webinar next week on September 8 at a virtual conference on microbiology and immunology. This conference is aimed at researchers and MDs, in fact MDs can get CME credit for attending this conference, so it's going to be in much more depth and detailed than what I was able to describe either here or at the Invest in ME meeting.
I'd also like to mention that we will be, people from my lab and myself, will also be talking at the IACFS meeting in Fort Lauderdale. And finally I'd like to announce for the first time that our Vice Provost for Research has authorized the formation of a center for ME/CFS, we're going to call this symptom center, the Center for Enervating Neuro Immune Disease. [Enervating – causing you to feel weak and lacking in energy]
This name was actually suggested by a patient given that it's very confusing, but to refer to use as a name for such a center given we've got ME/CFS and SEID as well as other names that various people have suggested, so we're going to call our center the Center for Enervating Neuro Immune Disease.
This center will include people from both Ithica and Weill Cornell Medical College, and at the moment we have actually three researchers, three laboratories, that at Weill Cornell that are studying ME/CFS, we have eight researchers, eight labs, at Cornell and Ithica who either have or have applied for grants for ME/CFS research, and we have one at Ithica College, and we have five collaborating physicians who are working with these groups so we would like to actually increase the number of people at Cornell who are working on ME/CFS and we think that by having this Center we should be able to attract even more interest and increase the researchers who are working on this very important disease.
OK, so that's my last, that's my last slide.
[End of part 4]
