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.

antibodies immunosignature ME/CFS

Immunosignatures have been developed for several diseases. Antibodies are the key

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.

The Study

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 S1Stafford P2Schlauch KA3,4Tillett RR4Gollery M5Johnston SA2Khaiboullina SF1,6De Meirleir KL1Rawat S1Mijatovic T7Subramanian K1Palotás A8,9Lombardi 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.

The Gist

  • 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.
Plus, the open-ended nature of the search is compelling. Because no one has identified the central immune or other pathways at play in ME/CFS, doing a broad-based search as this study did (and Ron Davis is doing) may provide the best chance of success. When in doubt search everything and then begin to winnow down your results. Luckily, we have the tools and statistical techniques that are helping to make a once impossible task look more and more feasible.

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.

A similar amino acid motif showed up in many of the peptides highlighted in ME/CFS patients

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.


A consistent finding from as many as three different groups of ME/CFS patients strengthened the findings

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 NCBR is ploughing new ground in ME/CFS

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.

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