Mark Berry continues his series of articles on the 9th Invest in ME International ME Conference in London, with the emphasis shifting from autoimmunity to pathogens and the gut …
This is the second in a series of articles reporting on the 9th Invest in ME International ME Conference (IIMEC9), held as usual in the Lecture Theatre at 1 Birdcage Walk in Westminster on May 30th, 2014.
You can view the full conference programme (with biographies of the speakers) here, and the Conference Journal is available as a PDF document here. Invest in ME have posted a conference report by Dr. Rosamund Vallings, and Phoenix Rising tweeted the conference live.
In Part 1, Autoimmunity and ME, I summarised the first three presentations of the conference, from Professors Jonathan Edwards, Angela Vincent and Jonas Blomberg, all of which focused on autoimmunity.
The next three presentations, from Professors Mady Hornig, Carmen Scheibenbogen and Simon Carding, all echoed autoimmune themes introduced earlier, but now in the contexts of pathogen-hunting, an investigation of the role of EBV, and the beginnings of an exploration into an exciting new frontier: gut microbiota.
Pathogen Discovery in ME
Professor Mady Hornig – Associate Professor, Center for Infection and Immunity (CII), Columbia University Mailman School of Public Health, New York, USA
Mady Hornig began by reviewing the ‘3 strikes hypothesis’ (Genes, Environment and Timing), with a whistle-stop tour through models of causation of disease. She focused on the ways that microbes – or indeed autoantibodies – can be implicated in causing disease, and briefly reviewed the progression of models of causation. First came Koch’s postulates (1890), then Rivers’ 1937 discovery of the adaptive immune response (and Fredericks and Relman’s identification of molecular markers for it in 1996).
In reviewing Witebsky’s Criteria (1957), Hornig noted that in that era the very concept of what we now know as ‘autoimmune disease’ was considered to be a horrifying and ridiculous concept – the shocking idea of the body attacking itself was then known as ‘horror autotoxicus’. Lastly, Hornig highlighted Rose and Bona’s update to Witebsky in 1993, reflecting the discovery that autoantigen-specific T cells may induce disease independently of autoantibodies.
Crossing the Blood Brain Barrier
Revisiting a topic touched upon earlier in the day by Professor Vincent, Hornig now pondered the important question of how an autoimmune attack on the brain might be feasible. Many of us probably carry antibodies in our blood to brain tissue, she noted, and these antibodies don’t cause disease in us. The blood-brain barrier (BBB) should prevent most molecules from passing from the blood into the brain.
However, it’s known that tight junctions can loosen or disintegrate in response to cytokines, viral proteins, hormonal effects and other causes. Furthermore, the circumventricular organs of the brain (including the hypothalamus and certain brain stem organs) are not protected by the blood/brain barrier. So it is actually possible for parts of the brain to be attacked by autoimmune agents in the blood.
Another question to consider is why some people get sick in response to the same stressors, and others don’t. Epidemiological observations of ME/CFS prompt Hornig to wonder whether something might be able to affect the capacity of antibodies to bind to their target receptors.
One good candidate explanation is that redox conditions, or oxidative stress, may either prevent or exacerbate the binding of autoantibodies to certain proteins. This could be a factor in determining who gets sick. For example, in people whose bodies are under oxidative stress, the autoantibodies may be more likely to bind to receptors in certain organs, thereby causing disease symptoms.
Reasons to suspect autoimmunity in ME/CFS
Next, Hornig reviewed the reasons to suspect autoimmunity as an important process in the causation of ME. There are a variety of observations pointing in this direction, she noted, including abnormalities in cytokine levels; changes in the numbers and functioning of Natural Killer (NK) cells; associations of ME/CFS with viral infections; mitochondrial apoptosis and necrosis pathways; and much more besides – not least, Fluge and Mella’s findings, with a significant proportion of patients apparently making great progress after treatment with a drug (Rituximab) which ‘resets’ the B cells and is therefore used to treat autoimmunity.
PANDAS and HSP70
Hornig explained her group’s interest in PANDAS, a bacterial autoimmune neuropsychiatric disorder in which exposure to streptococcus leads to obsessive-compulsive disorder, anxiety and nervous tics (described by Swedo in 1994). Investigating PANDAS in mice (she showed videos of the ‘back-flipping mice’ described in Simon’s Phoenix Rising article on Hornig’s work) it has been found that the sera of affected mice binds to two proteins in the cerebellum and two in the striatum, with Heat Shock Protein 70 (HSP70) being a particularly important target.
A very quick review of the CII’s “pathogen de-discovery” work noted that the group’s multicentre blinded analysis that finally put an end to the XMRV controversy in ME/CFS was not their only ‘de-discovery’ of suspected pathogens. They had similarly debunked an association between bornavirus and ME/CFS in the 1990s (but did find evidence of polyclonal B-cell reactivity) and also debunked the proposed association between MMR and measles vaccination and autism.
But importantly their work in autism did not stop there, and they did find reduced ileal enzymes and transporter mRNA, as well as altered adherent bacterial community structure, in autistic patients with gastrointestinal disturbance, as compared with controls who have GI disturbances only. As a rough bottom line of these findings, it seems clear to Hornig that a subset of patients on the autistic spectrum have autoantibodies against neural proteins, and these are the ones with the gastrointestinal disturbances.
Ongoing ME/CFS Studies
Professor Hornig now reviewed the group’s ongoing studies into ME/CFS: the 200 patient/200 control study in association with the Chronic Fatigue Initiative; the 150/150 study with the NIH; the 400/400 study with Stanford and Dr Montoya; the 60/60 study of cerebrospinal fluid with Dr Peterson’s patients; and a 10/10 study of some of Dr Peterson’s ‘unusual’ cases.
The group are pursuing a staged strategy, starting with MassTag PCR to search for pathogens in the blood, then studying the microbiome, and RNA sequencing, and they are hoping in the future to do some longitudinal analysis following patients over a longer period of time.
So far – and much to their surprise – they have found very little from their search for HHV and parvovirus in ME/CFS patients’ serum, and very little from the sequencing. In the Stanford/Montoya study, they have found very little in the way of viruses in patients’ serum – a little HHV-6B and some annelloviruses – but they are now looking in the PBMCs and hoping to find more there.
The most interesting and potentially the most important finding of Lipkin’s group is the identification of a distinction between the profiles of patients in the first three years of illness and those who have been ill for longer than this.
In patients with a more recent illness onset, they are seeing increased levels in plasma of a variety of pro-inflammatory cytokines. Many of the cytokines that came up are suggestive of allergic type reactions – increased IL4, IL10, IL13, IL1a, reduced eotaxin – but interestingly they are seeing ‘similar but opposite’ patterns in the study of Dr. Peterson’s ‘complex CFS’ patients.
Summing up their findings to date, Hornig said that in ‘classic’ ME/CFS patients they are finding many increases in cytokine levels, and she stressed that identifying the ‘clinical phenotype’ is critical in ME/CFS research – ME/CFS is very probably a heterogeneous population (a cluster of similar disease states but with important differences) and we will have to unpick that mixture in order to make progress.
The Microbiome and Autoimmunity
As she picked up the pace in the limited time, Hornig’s overview of how gastrointestinal issues and the microbiome may relate to autoimmunity was too rapid for me to take much in the way of meaningful notes, but key points she made included a caution that there are many factors involved here: an emphasis on the fact that some of the possibly relevant bacteria in the gut are essential for brain function (for example, some are required for the production of serotonin), but the right balance of gut bacteria is crucial; and a particular interest in pathways relating to tryptophan, as dysfunction of this pathway would affect working memory.
Finally, Hornig offered an update on the very latest state of play with their ME/CFS research work. They were just about to start a pilot study through the Chronic Fatigue Initiative (so this should be well under way by the time of writing), searching in white blood cells, and ‘hot off the press’ their study of 180 metabolites has found that short duration ME/CFS patients (those in the first few years of illness) are showing significantly reduced levels of ADMA (asymmetric dimethylarginine). This could be quite significant, because this would cause oxidative stress – and as she noted earlier, that could be a key factor in promoting autoimmunity.
Answering questions from the audience, Hornig reiterated a theme of several of the researchers present at the conference, emphasising that she thinks ME/CFS is a highly heterogeneous disorder. She also thinks that screening for certain types of antibodies is the first key step to take towards unpicking the puzzle.
Asked whether she thinks that disturbances in the microbiome (gut) are likely to be the cause, or the consequence, of the disease, Hornig noted the Australian research looking into this question right now, but said there is so much work to be done here that she can’t really say.
As to what she thinks of the option of fecal transplants, she seemed (cautiously) quite positive: beyond the ‘yuck factor’ she was impressed by recent findings in the US, in patients infected with clostridium difficile, in which people whose digestive tracts were ‘just shredded’, and who were immunosuppressed (and so at the highest imaginable risk) were seeing a 90% cure rate – even with a pretty dodgy selection of donor (a local nurse, apparently), so there was probably a far lower risk factor than one might have expected from such treatment.
Dr. Charles Shepherd raised a question which may present something of a challenge for the autoimmune theory of ME/CFS, however, asking the conference audience in general whether anyone had tried plasma exchange. This is the classical ‘first try’ for the treatment of autoimmune conditions, exchanging part of the patient’s plasma with plasma from a healthy subject.
As Dr. Vincent had explained earlier, this should result in noticeable (if not permanent) improvement, due to the dilution of the autoantibody population in the patient’s blood. Shepherd recalled trying it a bit in the 1980s, with no effect, and this was echoed by another physician in the audience who had tried plasma exchange with several of his patients with no effect.
EBV and ME/CFS
Professor Carmen Scheibenbogen – Professor for Immunology and Deputy Chair, Institute of Medical Immunology, Berlin Charite, Germany
Professor Scheibenbogen was next up, returning to the Invest in ME conference following her 2013 presentation on her investigations into immune profiles of ME/CFS patients. This year she focused on her group’s studies of the role of Epstein-Barr Virus (EBV, also known as Human Herpesvirus 4, or HHV4) in ME/CFS.
Their clinic for adults with immune deficiencies had about 300 CFS patients registered with them, and they had decided to focus their study on EBV because a subset of these patients had EBV infection at the onset of their illness.
This was typically an EBV infection later in life, and their illness was characterised by fever and recurrent pharyngitis of cervical lymph nodes – “it feels like the EBV infection is ongoing”. These patients have elevated levels of both IgM and IgG antibodies, EBV is detectable in their blood, and they tend to improve at least somewhat when treated with valacyclovir.
Starting with a review of some potentially relevant factors concerning EBV, Scheibenbogen reminded us that EBV is part of the family of human herpes viruses, and as such is an evolutionarily extremely old and well-adapted virus. It tends to live for a long time in human hosts, mostly as a latent infection, perhaps because it is valuable to the virus not to make us sick. It therefore hides in a non-active form but can be reactivated from time to time.
When EBV does reactivate, patients experience a sore throat but there is nothing visible on the skin. There may be some fatigue, but the absence of obvious signs of infection means that the situation is often misdiagnosed as a common cold. It may be a specific subtype of people who suffer from reactivation of EBV.
Most humans are infected with EBV – latent infection in adults is at about 98% these days – and most infection occurs in early childhood. About 80% of this is through saliva, typically from the child’s parents. This early childhood infection rate used to be even higher, and improved hygiene practices are thought to have reduced the rate of early infection.
When EBV infection occurs during adolescence, it can often be a severe infection – mononucleosis, or glandular fever, can last for months or even years. Late EBV infection has been associated with autoimmune diseases such as multiple sclerosis (MS) and systemic lupus erythematosus (SLE), and it is often reactivated when patients are immunodeficient. Chronic active EBV infection can cause further complications and can even lead to lymphoma.
Active EBV infection is usually diagnosed by detection of associated antibodies: VCA-IgM, VCA-IgG, EA-IgG and EBNA-IgG. What we all have (or nearly all of us) are markers for latent infection, or for early infection and/or reactivation.
Studying EBV-Specific Cell Responses in ME/CFS
Their study aimed to characterize EBV-specific B cell and T cell responses in their ME/CFS patients. The second project that Scheibenbogen presented on concerned an EBV seroarray developed with JPT Peptide Technologies of Berlin, Germany.
They had looked at specific EBV antibodies, and in the ME/CFS patients they studied, they found elevated levels of EBV-IgM (a marker for EBV reactivation), and a notable absence of EBV-EBNA antibodies. Importantly, they found that although HSV and CMV antibodies look normal, EBV-specific memory B cells are diminished in many of their CFS patients, and completely absent in a subset of those patients. Scheibenbogen reminded the audience that B cells are divided into naïve, memory and plasma B cells – and the memory cells are necessary for a healthy immune response.
In considering what might be causing this deficiency of EBV-specific memory B cells, Scheibenbogen first wondered whether this might be a response to the lateness of EBV infection – perhaps the later in life you are infected, the more difficult it is to mount a long-lasting immune response. Frequent EBV reactivation may also be related to this deficiency.
They found elevated levels of EBV in a subset of patients (about 10%), suggesting that these patients suffer from an ongoing EBV infection, but in most patients they don’t find this. They also found some EBV in non-B cells, which is odd and they have no explanation for this. They found no evidence of lytic replication, and thus no evidence of reactivated EBV, in accordance with the findings of other groups.
Findings from the EBV Serum Peptide Array
Scheibenbogen then explained the EBV serum peptide array which they have developed in collaboration with JPT Peptide Technologies of Berlin, Germany. The array measures antibodies against more than 2000 peptides from EBV by incubating serum samples on peptide-coated slides.
They carried out a statistical evaluation of the results from this array in a variety of different cohorts – multiple sclerosis patients, healthy controls, patients with depression, ME/CFS patients from their own clinic and from Fluge and Mella’s cohort in Bergen, Hodgkin’s lymphoma patients with and without fatigue, and lupus patients. They see different response patterns in different cohorts, and in particular an enhanced antibody response against EBV peptides in their CFS patients as compared with healthy controls.
The results of their analysis paint a complex picture though: which peptides activate depends on age and a variety of other factors, and they observed distinct patterns in the different patient cohorts. The German and Norwegian patients produced different patterns, and they also saw a difference between the patterns of those who responded to Rituximab and those who didn’t. They repeated their analysis with a second array and were able to reproduce their first set of findings, and they are hopeful that the work they are doing may eventually provide a basis for diagnostic tests.
Gut Microbiome and ME/CFS
Professor Simon Carding – Professor of Mucosal Immunology, University of East Anglia; Leader, Gut Health and Food Safety Programme, Institute of Food Research, Norwich Research Park, Norwich, UK
The last presentation before lunch came from Professor Simon Carding, an expert in the study of the immune response in the gut who is now bringing that expertise to bear in the context of Invest in ME’s project at the University of East Anglia (UEA) and Institute of Food Research (IFR), exploring the role of ‘leaky gut and the intestinal microbiota in the pathophysiology of ME’.
The conference notes state that the Foundation project examining the gut microbiota in ME patients is now underway, having started in October 2013.
Carding began by introducing Daniel Vipond, the PhD student funded with the help of Invest in ME, saying that his research work is now ‘virtually ready to go’ .
Vipond was present at the conference and available to answer questions at the end of Carding’s presentation, although disappointingly no (public) questions were forthcoming, perhaps due to the audience’s eagerness to take a break from an intense morning session and get started on the customary excellent food!
The forward-looking UEA/IFR project has been in the pipeline for some time, and Carding noted that during the two years since the project was instigated there has been an ‘explosion of interest’ in gut biology, which is surely one of the most fascinating frontiers in medical research. His presentation was primarily a general introduction to the subject area, peppered with some fascinating facts about the extraordinary world of the human gut.
Details about the project(s) investigating the gut in ME patients were thin at best, however, and sadly the one slide summarising that work flashed across the screen too quickly for me to take anything in. I’m hoping to track down some more details about it all in due course.
About the Gastrointestinal Tract
Carding explained that by the ‘gastrointestinal tract’ we mean the small and large bowel (the ‘gut’), and introduced its structure and barrier function. At about 9 metres in length, the GI tract is the ‘largest immune system in the body’. More antibodies are produced there than anywhere else in the body, and it also hosts the body’s second largest collection of neuronal cell bodies, after the brain, prompting the labelling of the gut as ‘your second brain’. The surface area of the gut is huge – about the size of a badminton court – but about as thin as a human hair.
The barrier function of the gut is crucial, with Intestinal Villi playing a crucial role. A variety of layers provide protection: mucus on the surface of the epithelium keeps microbes away from the function; immune cells promote and maintain junctions; and microbes are also part of the barrier, preventing colonisation by pathogens.
Microbiota and the Microbiome
The term ‘microbiota‘ refers to the many and varied organisms that make their home in the gut – bacteria (bacteriodetes and firmicutes), archaea, and eukaryotes (including fungi and protozoa). These microbes are tiny, but their numbers in the human body are enormous, outnumbering human cells by a factor of between 3:1 and 10:1.
We carry 300 non-human genes for every human gene – less than 1% of the DNA we carry in our bodies is human! Whereas the term ‘microbiota’ refers to these microorganisms living in us and on us, the term microbiome refers to their overall ‘genetic landscape’.
Each of us carries around 1 kg of gut bacteria, occupying a volume of about 1.5 pints. We each host between 300 and 1000 different species, and they are very active – they need 50 to 65 grams of fuel every day – and all that activity metabolises an awful lot of gas. Carding raised some chuckles when he noted that all of us produce this gas – between 1 and 4 litres of it per day is a healthy figure!
Investigating the Microbiome
The process of investigating the human microbiome begins with extraction of DNA, followed by analysis of the 165 ribosomal DNA (165 rDNA) by polymerase chain reaction (PCR) and pyrosequencing to determine the order of nucleotides in the DNA. Then bioinformatics tools are deployed, to work on organising and analysing the vast quantity of biological data. The whole process takes several months.
Next comes the metagenomics – the process of translating the sequence data into the functions of the organisms being analysed, through computational analysis of the sequences, investigation of the protein coding regions of the bacterial genomes, and other techniques.
Variability in the Human Microbiota
The human intestinal microbiota is highly variable between individuals – even monozygotic twins have distinct microbiota. Seventy-five species are present in over half of us, and 57 species are common to over 90% of us. Overall, three clusters predominate: bacteriodes, prevotella and ruminococcus, with bacteriodes and firmicutes predominating in most people.
Our microbiotas are shaped by who we are and what we eat. Diet is a major driving force in shaping the human microbiome, and to illustrate this Carding showed a pie chart comparing the microbiomes of people from Burkina Faso (eating a rural diet) and Europeans, demonstrating the radically different proportions of dominant species resulting from the food we eat.
The nature of our microbiota also changes throughout our life, with particularly notable changes at birth, when we transition to solid food, and at old age.
Viruses in the Gut
Our microbiota also contains lots of viruses – most of them living inside bacteria. There are a vast number of them, most of them phages. Temperate phages are stable over time, whereas lytic phages have extremely high mutation rates, changing over 100,000 nucleotides every day. Twins and our mothers share more similarity in their gut viral population than unrelated individuals, but our viral populations (viromes) are unique to the individual.
Our mothers are a major source of these gut viruses. Babies get exposed to them in the reproductive tract. Infant nutrition, hygiene measures, gestational age, antibiotic exposure, and intensive care all have an influence on the origin and development of our microbiota. More interest can be expected, as time goes by, in the exploration of the viruses in the human microbiome.
Microbiota: Good and Bad
Our microbiota are far from being purely parasitic organisms: they have protective functions (including limiting pathogens), structural functions (including fortifying barriers and inducing IgA production), and metabolic functions (including metabolising dietary carcinogens and enabling digestion).
We would be in a real mess without these organisms, as demonstrated by mice raised in germ-free environments. They have defective epithelial barriers, defective lymphoid tissue development, and immature immune systems, with defective production of immunoglobulin (IgA) resulting in increased susceptability to infection.
That’s not to say that the functions of the microbiota are purely positive either, though, as Carding explained in his slide on harmful microbiota – ‘The Enemy Within’. Heliobacter pylori can cause stomach cancer and ulcers; enterococcus and clostridum can also cause disease; and several other links between intestinal microbiota and human disease are also known. In diabetes, rheumatoid arthritis and hypothyroidism, the link is probably not simply to one bug that causes the disease; the problem is more about the population of the gut as a whole.
Microbiota and the Brain
Carding’s next theme was the ‘Microbiota-Gut-Brain Axis’ – the bi-directional communication between the gut and the brain. Biological mediators of stress are known to have an impact on both the composition and function of the microbiota.
Environmental stress too can damage the gut, and since in the healthy gut the microbiota modulates brain function and thus behaviour, a damaged gut is capable of having a profound effect on the brain. Physiological abnormalities in the gut have been associated with neurodevelopmental disorders, and there is increasing evidence of a clear link between what’s in our gut and what goes on in our central nervous system (CNS).
Is This Where the Cause of ME Lies?
Having spent the bulk of his presentation introducing the field of gut biology and explaining some of the important themes that may be significant in ME, Carding had little time left before lunch to go into detail about the Invest in ME-funded project to explore the intestines of ME patients. A couple of slides flashed across the screen, but unfortunately I missed most of this information.
One of these slides presented the key research questions that Carding and his group hope to address, but the only one I caught is: Do alterations in intestinal barrier integrity and microbiota exist in ME/CFS patients? ‘Leaky gut’, he said, can be caused by a wide variety of factors, and it can be detected byconfocal laser endomicroscopy – and the prognosis for patients is good if it is detected. So they will be assessing the permeability of the gut in ME/CFS patients.
There is already evidence to suggest that investigating the gut in ME patients may prove fruitful, and Carding highlighted some previous findings showing that many ME patients have gastrointestinal disturbances.
Dr. Henry Butt at the University Newcastle, Australia, presented the first evidence of altered fecal microbiota in 2001 (Butt HL, Dunstan R, McGregor NR, Roberts Tk: Bacterial colonosis in patients with persistent fatigue. In Proceedings of the AHMF international clinical and scientific conference. Sydney, Australia; 2001), and these findings were recently confirmed by Sheedy et al (Sheedy JR, Wettenhall RE, Scanlon D, Gooley PR, Lewis DP, McGregor N, Stapleton DI, Butt HL, DeMeirleir KL: Increased d-lactic acid intestinal bacteria in patients with chronic fatigue syndrome. In Vivo 2009, 23:621–628). A 2010 review of Gut Inflammation in Chronic Fatigue Syndrome, by Lakhan and Kirchgessner, has further detail about these findings.
Looking ahead to possible therapies that may be helpful to address this gut disturbance, Carding noted that stool transplants are already looking like a safe and effective treatment, and the evidence around probiotic therapy seems to suggest some benefit from that.
Carding finished by listing the other members of the team on this project: Tom Wileman, Bharat Harbham, Eleanor Cottam, Amolak Bansal (who will be selecting the patients for the study from his clinic’s roster of over 1000 patients), and PhD student Daniel Vipond.
It’s early days yet for the study of the gut microbiome in general, let alone for the importance of the gut microbiome in ME/CFS, but this is an exciting field and there is every reason to expect that it will be an increasingly significant one for ME/CFS research. Invest in ME’s strategic work to establish a centre of excellence in this field in East Anglia may well pay dividends in the next few years. This is certainly a project worth watching and we’ll do our best to bring you more information about it in the coming months and years.
Coming Up in Part 3…
If past performance is anything to go by, and with a house move ahead of me, Part 3 of this series can’t be expected until September or October at the earliest! But as and when I complete that article, I’ll be reviewing the first three sessions of the afternoon, from three more big name professors based in three different continents.
Professor Sonya Marshall-Gradisnik from Griffith University (Australia) presented on our current knowledge of immunological biomarkers in ME; Professor James Baraniuk from Georgetown University (US) informed and entertained as always with insights from his brain imagining studies; and Professor Julia Newton from Newcastle University (UK) presented on dysfunction of the autonomic nervous system (ANS) in ME. Three big names in the world of ME/CFS research, so there’s plenty to look forward to in the next article … when it eventually arrives …