A Different Look at the Immune System
The ultimate goal of this work is the development of a non-subjective clinical tool for diagnosing patients with ME. Lombardi et. al.
Last month Health Rising looked at a potential ion channel biomarker coming out of the NCNED in Australia. This month brings yet another potential biomarker hailing from the Nevada Center for Biomedical Research (formerly WPI) at the University of Reno. Time will tell if either turns out. In the meantime it’s good to see ME/CFS research centers using innovative techniques to look at this disease in entirely new ways.
Immunosignatures use antibody activity to get an idea of what the immune system is reacting to. The idea is that B-cells are involved in some way in most immune responses. Finding which peptides or antigens the antibodies in a person’s blood bind to could tell us what the immune system is reacting to. If ME/CFS is, at least in part, an immune disease, immunosignature research could tell us much. This approach has been used to provide diagnostic biomarkers for cancer, Valley fever, Alzheimer’s disease and others.
Talk about casting a large net. First antibodies are incubated with or exposed to thousands or in the case of this study over 100,000 randomly generated peptides (short-chains of amino acids). Because so many different peptides are used, the immunosignatures produced by this technique can be highly sensitive (i.e. highly accurate in identifying patients). They’re far more sensitive than say an ELISA blood test that might sum the contributions of several antibodies.
The technology used to produce immunosignatures is also very robust; a single drop of blood blotted on some paper and sent through the mail can suffice. It’s also potentially much more stable than the cytokine studies that have, at times, been so variable in ME/CFS. Because antibodies are much more stable than cytokines (they last longer in the body), they might yield better results.
Mol Neurobiol. 2016 Dec 15. [Epub ahead of print]Humoral Immunity Profiling of Subjects with Myalgic Encephalomyelitis Using a Random Peptide Microarray Differentiates Cases from Controls with High Specificity and Sensitivity. Singh S1, Stafford P2, Schlauch KA3,4, Tillett RR4, Gollery M5, Johnston SA2, Khaiboullina SF1,6, De Meirleir KL1, Rawat S1, Mijatovic T7, Subramanian K1, Palotás A8,9, Lombardi VC10,11.
In this study, Lombardi and the Nevada Center for Biomedical Research (NCBR, formerly the Whittemore Peterson Institute) exposed the antibodies in ME/CFS patients’ and healthy controls’ blood to a very large number of peptides (125,000); far larger, in fact, than some cancer studies (20,000). Forty-one ME/CFS patients and healthy controls participated. An important strength of this study: two sets of patients participated – one from the U.S. and one from Europe.
- Immunosignatures have been used to produce highly accurate diagnostic markers in several diseases
- The NCBR’s results suggested the immune systems of ME/CFS patients were highly different from those healthy controls
- Antibodies found in ME/CFS patients’ serum reacted to two different types of peptides: those associated with humans (suggesting an autoimmune process may be away) and those associated with pathogens
- The antibodies also appeared to be reacting to an amino acid pattern found in many of the peptides highlighted in the ME/CFS patients
- That finding suggested that a core immune process may have been found
- Future work will attempt to validate the finding
- Gut studies are also underway to determine if the microflora problems found in ME/CFS are a result of the illness or a central cause of it.
The first step was to identify the peptides that the antibodies in the chronic fatigue syndrome patients’ blood reacted to. The next step was to determine which human or pathogenic proteins they were likely triggered by.
That involved matching their randomly generated peptides with naturally occurring peptides. Since the randomly generated peptide sequences were so short – far shorter than sequences associated with naturally occurring proteins – the gaps were filled in using a “BLAST” search of a human protein database, and peptides associated with pathogens including bacteria, viruses and the human endogenous retroviruses (HERV) the NCBR has found in ME/CFS before.
The search indicated that antibodies were likely reacting to an array of human proteins (an autoimmune process was underway) and a mishmash of viral and bacterial proteins. Proteins associated with HIV, human herpesvirus-2, canine distemper virus, a rodent paramyxovirus and a porcine rotavirus showed up. Plus, seven of the top sequences were associated with a human endogenous retrovirus protein. A closer look indicated that these seven sequences clustered together in one part of a protein.
That was unusual, but even more unusual was the fact that a similar peptide “motif” also appeared in at least 40 of the top 233 peptides. Finding that this recurring amino acid motif was present in almost 20% of the peptides highlighted in ME/CFS probably made Lombardi almost fall out of his chair. It suggested that some sort of core immune reaction was taking place in ME/CFS.
If I understand them correctly, these motifs are short amino acid chains attached to proteins that regulate their homeostasis; i.e., they regulate protein production, utilization and eventually protein degradation. While proteins themselves tend to have very stable structures, these motifs are more “flexible” and, if I understand them correctly, can change over time – perhaps in response to some event.
These motifs, then are important regulators of proteins – the worker molecules responsible for almost every activity in our bodies. Antibodies attacking these motifs, could, it would seem, have important consequences for many processes.
In an email, Lombardi suggested the antibodies could be reacting to a pathogen, even possibly a common pathogen that ME/CFS patients are reacting unusually to. Or they could be reacting to an auto-antigen – a piece of a human protein – which the body is now attacking. Or the disease process in ME/CFS could be producing a unique protein that the immune system is reacting to.
Lombardi noted that the same approach has identified antibodies to the amyloid beta and tau proteins in Alzheimer’s disease. These proteins are naturally produced; i.e. they’re not “mistakes”, but because they’re not being properly cleared in people with Alzheimer’s disease, the immune system is attacking them. The same process could be happening in ME/CFS. That is, the proteins ME/CFS patients’ immune systems are reacting to could be “normal” but quantities might be too high. (That, of course, brings to mind the mysterious factor in the blood that Ron Davis’ experiments suggest are stomping on the energy of ME/CFS patients’ cells. That factor appears to be a protein of some sort.)
In the paper the Lombardi team suggested that if the recurring motif is validated it could “represent a critical discovery in resolving the pathophysiology of ME”.
The immunosignature approach was clearly cutting-edge stuff. When I asked Lombardi if any other ME/CFS research groups had attempted such a thing, it turned out that the NCBR was not alone; in fact they came within inches of collaborating with a Canadian group doing the same thing. That group under David Patrick presented at the IACFS/ME conference.
Plus, the Center for Innovations in Medicine (CIM) lab they contracted with to run their samples liked the project so much they asked to be partners in it. I don’t know how often researchers want so much to be part of an ME/CFS research project that they’re willing to, if I’m reading this right, forgo their fee, but it’s clearly a good sign.
Lombardi explained how it happened:
A couple of years ago, I contacted Steven Johnston and Phillip Stafford at ASU to run my samples at their core facility on a fee for service basis, but they liked the idea so much that they asked to be partners in our study. Shortly after we generated our first data, a group of Canadians contacted ASU to do a similar project. At first, we all agreed to collaborate, but the Canadians had some issues that delayed their participation so we eventually continued our study without them.
Apparently, they were primarily interested in using the technology to follow treatment with Rituximab. Although they have yet to publish their results, from what I saw from their presentation at the Florida IACFS/ME conference, their results and our results are confirmatory. Vincent Lombardi
That lab, by the way, is something else. The Center’s motto “Innovation requires that we put aside what we think we know and start fresh” could have come straight out of genetic pioneer and now ME/CFS researcher Ron Davis’ mouth. According to their webpage, the Center was the first to implant DNA into mitochondria to create a “gene-gun” able to shoot micro-projectiles into the cells of living animals to find a way to make cells resistant to pathogens, and five or six other technical feats. Getting those guys on board was clearly a major coup for the NCBR.
Bigger studies are clearly needed, but the trend line right now is good. Lombardi mentioned that the Canadian group the NCBR almost collaborated with appears to have produced similar findings. If that finding shows up in the published Canadian study, that would make three groups of widely separated ME/CFS patients (U.S., Canadian, European) all with similar findings – a strong and unusual result for this field.
Particularly interesting is the possibility that this unusual motif may be showing up in three different patient groups – something Lombardi thought was impossible given the supposed heterogeneity in this field. Chronic fatigue syndrome (ME/CFS) might not so heterogeneous after all… From Lombardi:
So the conserved motif is somewhat surprising because we have always looked at ME/CFS as being such a heterogeneous disease, but it might be less heterogeneous then we initially thought, at least from an immune perspective.
This is exciting stuff – still preliminary, of course, but exciting nevertheless. It’s particularly gratifying to see a top lab on board. Lombardi and the team at Arizona State University are writing a grant to do a much more comprehensive study using many more ME/CFS patients and controls from other diseases.
Keen to develop a diagnostic test for ME/CFS, Lombardi stated “Coming up with a really good robust diagnostic for ME would really help the patients and move things forward. I believe this is critical!“
To that end, the grant application will include a proposal to receive blinded samples from an outside group to validate their assay. If that checks out, it sounds like we could be close to a diagnostic assay similar to that which was produced for Alzheimer’s. That would clearly be a game-changer.
HERV and Gut Update
Earlier Health Rising reported on the NCBR’s endogenous retrovirus (HERV) findings in ME/CFS. The NCBR has found HERV’s present in dendritic cells that have infiltrated the gut lining of ME/CFS patients. Despite an extensive search they’ve been unable to find them in non-ME/CFS subjects they’ve been unable them in anyone else – they seem specific to ME/CFS. Then peptides associated with these endogenous retroviruses showed up in the ME/CFS patients in this study
The HERV findings are nothing if not intriguing, but Lombardi is being cautious. Research in other diseases that HERV’s show up in such as multiple sclerosis and cancer haven’t been able to determine if they are a part of the pathology or an artifact.
That research is going to pay off in another way. Those dendritic cells appear to be a marker for gut issues in ME/CFS. Lombardi is going to use the presence of those cells to attempt to determine whether the gut flora problems are the result of ME/CFS or are helping to cause it. Is there something about the disease that is compromising the gut flora and mucosal barriers in the gut or are the gut flora themselves responsible for that.
HIV replication in the small intestine, for instance, causes inflammation and changes in the gut flora, which results in systemic inflammation. Even though the gut flora of HIV/AIDS patients is different, it’s HIV, not the gut flora which is responsible.
Contrast that with Clostridium difficile infections in which bacteria in the gut themselves are causing the mucosal barrier breakdowns and other problems. With HIV an infection outside of the gut is causing the gut issues; in C. difficile infections a part of the gut flora is causing the problems. Lombardi is trying to figure out which is the case in ME/CFS.
The news appears to be good at the Nevada Center for Biomedical Research. The NIH grant they scored reaped dividends and they’re working with a top lab to develop a diagnostic immune signature. Plus, they’re trying to answer fundamental questions about the gut. The NCBR’s XMRV finding didn’t pan out but it did raise the profile of the disease considerably and introduced new researchers to the field. Time will tell how this all turns out but this time they may be onto something more lasting.
HERE IS AN INTERESTING ARTICLE RELATING TO HERVS AND AUTOIMMUNITY ETC
Thanks for this article, Cort!
And thanks for that article, lin. It was great!
Thanks for this most interesting article. Perhaps you might consider doing a survey to see if people struggle with the grey typeface. I sometimes have to copy and paste and change to black in order to read. Just a thought.
Thanks Hanora – we will fix that!
I feel the same way, Hanora. I detest the grey font. Unfortunately, it seems to be the default choice for most web programs. You have to go out of your way to change it to something all of us can easily read!
I am seeing it as black, not grey Hanora and very high contrast (an issue for those of us with light sensitivity. On all desktop computers that browsers can increase text size if you are having issues (many phones too). You can do so through the view menu or using Ctrl+ and/or Ctrl-. I often find the need to do this.
I don’t want to bash any researchers’ work — or Cort’s work in bringing us the news and explaining it as simply as possible. But I would humbly suggest — for patients’ sake — that the site pull back from covering something as news just because a lab says it is. These people above told you there may be some autoimmune reaction to a chain of amino acids. So what? We’ve known for ages that autoimmunity could be at play here and so could gut issues. This study does not advance those ideas. It’s nice that the researchers are examining the ideas. But they have not produced a biomarker — they are simply searching for one like everyone else. That’s not news. News would be that they found one.
And the reason this pisses me off is because I am too damn sick to keep reading about people doing BIG things that are, in reality, the same work we’ve been anticipating for ages without an actionable result.
Hmmm….I do understand the worries (and the many exciting findings that have not panned out) and the need for caution – but I think there is potentially an actionable result here – the peptide signature that showed up in the ME/CFS patients vs the controls. That so far as I understand it is the immune signature. A true biomarker would need to be validated by comparing ME/CFS patients to other diseases. The next grant is designed to test that. I was also encouraged that the next grant proposal will include some sort of blinded test that will help accomplish that. Plus I think it is encouraging that three sets of patients from different locations – perhaps not a huge sample set in itself – have apparently gotten similar results – something we rarely see in the immune system.
We have a couple of things in process that could produce biomarkers: the NCNED ion channel work, the NCBR’s and Arizona State Universities immune signature work and Dr. Naviaux’s metabolomics works. Each are in the preliminary stages but each is moving onto a more comprehensive testing stage – and then we will see! 🙂
So much research coming out… but looking in so many different places. It is getting confusing, but I’ll take the shotgun approach to research than no research at all. Hopefully, all of these studies will converge and make more sense in hindsight. A biomarker find at least seems realistic now as opposed to just a few years ago.
I think we may have more researchers doing more sophisticated approaches and using new methods. The more possibilities that are present the better chances of one or more working out – that’s for sure.
Then don’t read it. Some people are still desperately hopeful and any research anyone does is valuable to us.
I read this quite differently.
Firstly they identified what appears to be a specific and unique peptide chain. That does mean a potential biomarker if the study is confirmed on a larger scale.
Secondly (again if confirmed in other studies) – it will mean a specific targeet for the auto-immunity rather than a loose theory.
Thirdly given that there is a mounting view amoung some that certain autoimmune diseases are causes by peptides shared with both the human host and microbial life, it potenitally also identifies an infecton that might be a trigger.
In response to the other comment, during the ‘discovery phase of a disease’ as Ron Davis pointed out, you must first gather data before being able to produce a hypothesis likely to be confimed in experimentation. This raft of different types of study going on now builds that database on which more focussed hypothesis and research projects rely.
To sort my own health out, I had to form a number of hypotheses as to the cause of my symptoms, investigate the lot and then reform a smaller number of hypotheses from the gathered data.
Cort, it recently occured to me too that failure of dendritic immune cells might well be a factor in our disease. Myhill et al showed mitchonddrial dysfuncton in Neutrophils (another type of immune cell) – since Leukocytes use very little energy at rest, but masses of it when responding to infection – a similar dysfunction woulod well be present in Dendritic cells.
I think the Dendritic cells that are present in the gut in CFS are immature. The mature cells are being redistributed somehow: https://www.ncbi.nlm.nih.gov/pubmed/26441989
Thanks for the info Cort. So many new research projects of recent. I’m encouraged by the efforts of these docs to try to help us. Without them, our talk is just hypothesis. They get to the science of it and prove it out.
Fascinating article, thanks Cort! Wonder how the finding of differences in immune systems in those sick 3 yrs or less vs those 3 yrs + fits in with this? Would that be apples & oranges bc of the difference in what’s being looked at? This sounds promising with the patient groups coming from different regions of the world & yet showing similar findings. I wonder how many long term vs short term patients it included.
Great question Hezza. I have no idea how the cytokine studies you’re referring to would match up this antibody data. The immune system is so complex that I really don’t know…
Cort, where these different group of patiënts all being tested in the same lab again, just like the XMRV saga?
I hope they will not make the same mistake again.
I believe they all were tested in the same lab – that Arizona State University lab. That lab, however, appears to be well regarded – both the Canadians and the NCBR sought them out to do this test, and they appear to have pioneered a number of new techniques.
It’s a good question, though, whether true validation of a biomarker wouldn’t require another research group using a different lab. I don’t know for sure but I imagine that it would…
Cort, once again I am grateful for breaking down the articles into something that is more understandable to me. And what I get out of this is the shot gun approach is indeed a good thing. I recall a number of years ago some of the researchers saying it was important to do big data analysis and let the clusters fall into place. I also find it exciting that this research seems to verify Davis’ findings. So glad you are here. Thanks again.
Hi – very interesting write up, also uplifting to read the positivity .
A very old saying, It’s just a GUT feeling. Always after eating, after a so
called sensible diet to suit this dreadful condition, (Gluten Free – Lactose free (because of reaction). …… Almost immediately Always feel very very ill…….
This is why the Write-up on the Gut, in this case is so very true to life…..
Thank you to all involved, ??
Love the article. Gives me renewed hope that research does continue and even if very slowly, we are moving forward to find a biomarker which hopefully leads to an understanding of the etiology and treatment of ME.
Yes, indeed it’s frustrating waiting and waiting for some hope of change in our lifetime. I’ve gone from healthy at 42 to still sick at 61. That’s a very long time to wish and hope for adequate treatment and heaven forbid a cure to happen before I die-most likely at a far younger age than if I didn’t have ME/FM.
Research goes slow. It’s partly political, partly financial, partly not enough researchers willing to become involved in a disease that nobody really understands and therefore not too many instant rewards or accolades from the rest of the medical community unless one finds the cause! There just isn’t a lot of money for ME research.
Speaking of the GUT…..have any of you found that if you do not eat at all, you feel better? Before becoming disabled 15 years ago from this disease I remember feeling good if I did not eat. I would go a few days feeling great, only to begin eating again and feel lethargic. People would ask if I was anorexic, and I questioned that. But I was non eating on occasion because it made me feel good, well I guess normal then. Since becoming too ill to do anything much more than sleep and lay in bed, intestinal issues became a larger problem. I had zero motility in large intestine and eventually had a total colectomy with ileorectal anastamosis. Still have motility and CNS problems but not as severe. I have now been diagnosed with Crohns disease, esophageal issues, acid reflux and hate to eat because I always feel terrible afterwards. Just curious.
Cort, any idea how this might tie in with the ion channel work in Australia and Dr. Ron Davis’ research? It’s way too complicated for me to even think about!
Gael i am part of professor Pete Smiths ion channel and nk killer cell cytotoxic % tests it was complicated for me at first but i have worked it out my nk killer cell %count is 4.01%the ref range is 13.8-34.8 so severely low an indication of infection.some low readings are hereditary. Dont take to much notice of what Cort says he bends the story to suit who is paying him the most.Prof Smith is the overall consultant for the ncned
Yes I too feel better if I do not eat. Or if I get sick and vomit severely or the other exit I feel normal for a few days then it returns. Feel as though something has been flushed out of the system?
Thanks Anne. Either flushed out or our system is not having to process so many things at the same time? I just dread eating although I of course have to, I just wish it would not make me feel so much worse than I already do. I think I can’t get worse, yet I do. Gentle hugs.
Michelena, yes. I think that CFS is a connective tissue disorder which has been neglected in this aspect of study while infections have been OVERstudied. Connective tissues are about more than skin and tendons, they’re about gut motility, ATP cells, mast cells, inflammation, etc. etc. https://www.youtube.com/watch?v=d8IfclFVEOU
Thank you Ly,
Interesting article. I am familiar in my co-morbid issues with connective tissue problems. Along with the IBD and Crohns I have osteoporosis, osteoarthritis, scoliosis(38 degree S curve), Costochondritis, severe pain in mylein sheath body wide, Lupus, Raynauds, sjogrens, lipomas, esophageal disorder problems swallowing, white matter lesions in pons and midbrain, etc etc I could go on and on. I cannot help but believe all of this must be connected to my ME/CFS. I feel like one big blob of disease!
Hugs (very gentle hugs) back!
it “hurts” me when people are writing that you post everytime something else and break you down. I am a verry severely ill me/cfs patiënt (98% bedridden, strugling to just eat, … Your information gives me hope where I have left nothing else thn that. So I really want to thank you for your posts and I always look forward for one from you and youre information. Who else would informate us on an understandeble level. did you see the research that I copyd and past?
Thanks! No worries 🙂
Konijn, I agree with the posts giving other people hope. I for one cannot get enough testing because MD’s just won’t due it. Any research or reason is valuable to me…especially when it’s broken down to make sense. My memory and being in a brain fog all the time enjoys being able to understand these. The negative comments really bother me.
Interesting post, thanks. I think 20% of patients seems a low, unless it’s just a subgroup. Still it’s pretty interesting that they found indications that something could be going wrong with “housekeeping” in the cells at least for some patients.
I have mixed connective tissue disease and a host of waxing & warning immune disfuncttion for last 10 years. Atypical P-Anca’s 1:625 and it felt like I was dying. A very compassionate (now retired) astute Internist/Pulmonary MD & another excellent Rheumatologist saved my life. When Rheum. added Cellcept 3,5 yrs plun Rituximab IV Q Mo. for a year then Simponi last 2.5 years I finally got my infections under control and a fighting chance. CF/ME runs in my family and I was really healthy until Mono and pneumonia were contracted. No depression just anxious to find a cure-if possible. The years the CF/ME patients suffer and the initial multiple drugs did not help at all. Grateful and hopeful that research is getting closer to the truth & hopefully shorter diagnosis period & appropriate treatment. Happy to volunteer in the larger group studies and help with the research. I’m and R.N. 25 years. Thanks to all who have not given up and keep fighting.
Thank you, Cort, for your thorough analysis of the research. I appreciate you SO much!
the following is taken from the paper:
“When this sequence is considered in isolation, we have observed it within several other proteins, in particular, the bacteria genus Burkholderia and also in the human protein calcium voltage-gated channel protein CACNA2D3 (Table 2). Further studies will be required to identify with greater certainty the native antigen to this conserved motif.”
So possibly the target is a “calcium voltage-gated channel protein”. You refer to the recently published study by researchers in NCNED in Australia; they found low intracellular calcium levels. Also, Fluge and Mella have proposed that some people with ME/CFS have an autoimmune disease since they improve when treated with rituximab. Is ME/CFS an autoimmune disease which results in low intracellular calcium levels?
How much did/does it cost to run this “peptide test”? Are there any examples of it being used currently as a diagnostic test for other diseases?
Hi Cort, heads up that the in-website Print Friendly service is having some issues. At least it looks like Print Friendly is the service you use. When I tried to download a PDF or print this or this post “Major Breakthrough in Chronic Fatigue Syndrome (ME/CFS)? Aussies Believe Biomarker Found” and the images say “full size,” they are missing and just the text that goes w/ the pic is there, but it’s overlapping the article text. When I select “small images,” they show up but overlapping text and the text is compressed vertically of to the right or left. However, when I go to PrintFriendly’s website and plug in the web address for Health Rising’s article, it shows up just perfect and I can print it. But I can’t use the print function on Health Rising b/c of above issues. Maybe there is an update needed for the print plug-in on Health Rising? Formatting issue? Idk, I’m not a web page expert. It may affect many or all other articles/posts, these are just the first 2 I’ve tried printing in a couple months or so. Thanks for all you do to keep us informed!
Thanks for letting me know. I’ll have Stavya check it out
Can you guys explain to me the procedure on how to apply biomarker to check fatigue?