Attachment 1. A New Era in ME/CFS Research

Transcription

1 Attachment 1. 5 th Invest in ME International ME/CFS Conference 2010 A New Era in ME/CFS Research On 24 th May 2010 I was privileged to attend the 5 th ME conference in London. The conference was attended by delegates from around the globe and some of the leading ME/CFS international researchers presented their work. The conference was ably chaired by Professor Malcolm Hooper, and after his opening words, the first speaker was: Professor Leonard Jason, DePaul University, Chicago. He provided an excellent overview of the illness, covering epidemiology and diagnosis. He discussed CFS in the context of fatigue in general in relation to physical and psychological causes. He pointed out that case definitions can over- or under-estimate prevalence, and talked of other diagnostic pitfalls. In one study cited, he explained how trainees responded more positively to the term ME than CFS when making a diagnosis. With broadening of the case definition between 1988 and 1994, there was a doubling of the rate of diagnosis. He described the cardinal symptoms as post-exertional malaise, problems with short-term memory and non-refreshing sleep. If none of these symptoms occur, the diagnosis may not be accurate. Depression can have a similar set of symptoms, but several of the ME/CFS symptoms will not be found in depression (e g., lymphadenopathy, sore throat). Onset in primary depression is usually more gradual. One study by Jason using discriminant functional analysis provided 100% accuracy in discriminating between ME/CFS and major depression. It is considered too, very important to get the correct measures to avoid diagnostic confusion. Accuracy is vital epidemiologically. A change in prevalence as reported by the CDC with a large upward trend has been reported over the past 20 years. This is partly due to flawed epidemiology in the 90s, less physicians

2 making a diagnosis, less resources and disagreement among physicians. An earlier random community based study by Jason had found that 90% patients had never been diagnosed. Looking at severity and intensity of symptoms is important. Just fatigue alone does not differentiate between ME/CFS and major depression. When attempting a diagnosis, structured interviews and standardised procedures are required. How a question is asked is important too. The threshold of disability needs to be established. Screening tests need high sensitivity and specificity. If there is a 5% error rate, there will be patients diagnosed as having the illness who do not have it. A study by Reeves, using empirical criteria was cited in which a 62.8% level of a past history of childhood abuse was reported among CFS patients. Jason s group used traditional criteria and found no increased incidence of childhood abuse. The Canadian criteria are more specific. Post-exertional malaise, non-refreshing sleep and neuro-cognitive symptoms must be present. The reason the Canadian definition is not yet being widely used may be due to difficulties in translating from one definition to another. The paediatric case definition was discussed and has 97% accuracy (cf Fukuda definition 76% accuracy). Under-diagnosis is a key concern. In making a diagnosis it is important to measure severity and frequency of symptoms. Assessment of stamina and endurance is critical. The patient may control and limit activity to minimize fatigue. Categories of questions need to be reliable and accurate with scoring rules, in order to make a valid diagnosis. The finding of biological markers will be the key to betterment. Professor Nora Chapman, University of Nebraska, discussed her work looking at persistent enterovirus infections. She said there are over 100 serotypes of enteroviruses. Her talk mainly focused on the coxsackie B group. (CVBs). She described 6 subtypes, which migrate into the cell. Her main studies involve infection in heart muscle. (cardiomyopathy and myocarditis). However a wide variety of tissues can be implicated. The initial illness is often flu-like. Much of her studies reported were on mice, which she describes as having similar disease to humans. There is an acute phase, with the virus clearing in 7-21 days, followed by a continuing chronic phase with accompanying auto-immune responses. (post-viral phase). It is difficult to culture the viruses from heart muscle due to the low level of infection. Persisting infection may well be related to CFS. Coxsackie B virus RNA can be identified, but it is a very

3 common virus, but it can be associated with symptoms. In mice the virus can cause malfunction of the heart muscle leading to cardiomyopathy. This virus is a hit and run virus, and may be killed by virus specific antibodies. The virus can also concentrate in the GI tract with cells breaking down leading to risk of transmission of infection to others. By day 28, the virus can be detected by RT-PCR, but it has stopped killing the cells. It is likely that this is due to a small particle of the genome may be missing (a terminal deletion -TD). There is alteration of the heart cells, leading to heart re-modelling and persistent late stage disease. The cells are not killed. This finding was confirmed in a human post mortem in a patient who had died of severe endocarditis. TD viruses make infectious viral particles, which prolong the infection. CVB3/TD virus has more negative strand RNA than normal for these positive strand RNA viruses. Cell cultures have been obtained from the heart and pancreas. Deletions are anti-genomic. Once deletions are generated, wild-type virus genome will be regenerated. The function of the cell is altered. Quiescent cells provide an environment which leads to the generation of low level persisting enteroviruses. The TDs cause alteration of enterovirus replication leading to persisting infection. Replication leads to alteration of cell function. The immune system does not readily clear the infection because there is too low a level of production of viral DNA. She summarized the talk by covering the possible issues relating to ME/CFS: 1. Patients do often have muscle and/or GI infections 2. Severe acute infections are likely to lead to TD viruses in differentiated tissues, such as muscle. 3. Expression of CVB proteins alter the function of the cells and generate chronic inflammation. 4. Sensitive assays will be required to confirm persistence. Dr John Chia, Torrance, California also discussed the implications of enterovirus infection in ME/CFS. In the diagnostic approach to this illness, many patients are found to have a history of onset following water-borne infection, respiratory illness, appendicitis, vaccination, tick bite etc. Past history often includes allergy or asthma. There should always be a detailed review of medical records, coupled with lab testing in making a diagnosis. Almost any system can be infected with an acute enteroviral infection (muscle, heart, CNS etc) leading to many and varied

4 symptoms. Fever is the predominant initial symptom and many do have GI symptoms. Recurrent shingles is a common accompanying illness. Tentative diagnosis of enteroviral infection is therefore made on suspicion following a flu-like episode. There will be few physical signs. Tests done include: neutralizing antibody for enterovirus (EV), qrt-pcr for EV RNA in whole blood (low sensitivity), immunoperoxidase staining, stomach and other biopsies, such as tongue and colon. Correlation of symptoms with results is necessary. He described findings of a patient who had died in 1994, when EV-RNA was found in muscle, heart and brain tisuues, and an Oxford study when EV RNA was found in serum. In the USA in 2002, 38% EV RNA was found in peripheral blood lymphocytes (PBLs) of ME/CFS patients compared to 4% in controls. Gastric biopsies were studied. Slides were shown of a number of positive biopsies. Patients did not always have GI symptoms, but those with the worst looking gastrum were the more severely ill. There were 82% positive in ME/CFS patients and 20% in controls. There were some positive gastric tissue cultures. There was positive clinicopathological correlation. EV was also found in biopsies from throat, sinuses and fimbria, the latter suffering from pelvic pain. Treatment was then discussed. Chia uses initially a symptom based approach. Specific antivirals were discussed such as pleconaril and more general antivirals such as acyclovir and cidofivir. Immune modulation may be appropriate with ampligen, IV immunoglobulin, interferon and herbal immune boosters. Use of steroids is frequently reported in the history and these should be avoided. A shift in Th2 makes one vulnerable with viral persistence, and this occurs with steroid use, in runners, with vaccination and pregnancy. The viral load may gradually drop with a Th1 shift. The herbal preparation used by Chia is Oxymatrine, initially sourced from China. He has found a 52% positive response with this treatment. Treatment was initially for 3 months, but relapse did occur in 2/3 of patients on stopping treatment. Most then responded positively to further treatment. Patients do not tolerate high doses, so initial dosing needs to be very small, increasing gradually to 3-6 tablets daily. Some patients are intolerant or suffer some side effects initially. There may be an initial worsening of CFS-like symptoms. There has been a shift to Th1 noted in positive responders, but not to non-responders. Equilibrant has been formulated from oxymatrine giving up to 88% improvement and seems better tolerated than the original oxymatrine.

5 Dr. Paul Cheney, Asheville, N Carolina, has seen over 8000 ME/CFS patients. He argued that the illness is linked to oxygen toxicity. He described the most prominent symptom as being postexertional fatigue. 99% patients also have cognitive disturbances and 5% do not suffer pain. 60% have mood disturbances. He described several phases of the illness initially misery (associated with being very ill) and then a phase of dynamic dysfunction (the push/crash) eventual adaptation to the illness occurs. Functional recovery may be associated with successful adaptation. Exercise ergometry shows there is oxygen dysfunction, with a serious flaw in oxygen utilization. MRI of the brain shows an increase in ventricular lactate in 70% patients. Interestingly too there are changes in the fingerprint with crevassing and smoothing out. (Scurvy has similar effects). This may represent oxidative stress. There is also increased RNaseL activity in his ME/CFS patients. He then gave an overview of the studies done looking at the incidence of XMRV. CFS 67% Prostate cancer 18-44% Transplant patients with upper respiratory infection 10% Healthy blood donors 3.8% Sputum 3.2% Benign prostatic tissue 6% At the Cheney clinic He feels there is a strong family correlation in ME/CFS and this may relate to XMRV. There could be early transmission. He then went on to discuss cardiac issues in ME/CFS. When heart failure is looked at generally, 54% patients have only diastolic but not systolic dysfunction. While no specific study has been done, he finds the major cause of death in ME/CFS patients has been heart failure. Diastolic dysfunction causes symptoms of orthostatic intolerance with fatigue and weakness. Peckerman had looked at cardiac output in ME/CFS using various methods, and found lowered stroke volume and output, worsened on standing. A diagnosis of Type 1 Diastolic Dysfunction (DD) made by cardiologists describes what the heart looks like when there is no energy in it! The dysfunction shown in ME/CFS compared to other diseases falls between mild shock and

6 traumatic shock. Stroke volume is often dramatically reduced. Cheney describes the diastolic heart failure in ME/CFS as a failure of sufficient free energy (G). A diagnosis of DD can be made by echo-cardiography using a diastolic inflow Doppler, a diastolic tissue Doppler or a pulmonary vein inflow Doppler. The latter is the most sensitive. DD is seen frequently in CFS, with the atrium collapsing on standing leading to lower pressure. It can be likened to a hole in the heart with evidence of a patent foramen ovale (88% CFS patients). Left ventricular cavitation also occurs. There is left ventricular dysynchronicity, which also has a major effect on stroke volume. The Everest III studies (Comex 97) showed findings identical to CFS, and this was created by the altitude experience. Cheney found that administration of oxygen in CFS causes worsening of symptoms. The IVRT (an energy specific diastolic parameter) is equivalent to oxygen toxicity. He concluded therefore that CFS is an oxygen toxic state. Dr Jonathan Kerr, University of London, spoke on his study of single nucleotide polymorphisms (SNPs) in CFS/ME and subtypes of the illness. He explained that there are over a million SNPs in the human genome, and they can be used to subgroup patients. 8 gene expression subtypes have now been identified based on expression of 88 human genes, some of which have therapeutic potential. It may be possible to develop the use of a predictive subtype test in CFS/ME based on SNPs located within these 88 human genes. 500 SNPs were tested (1-8 SNPs per gene) in 108 ME/CFS patients compared with 17 with depression and 68 healthy blood donors. ME/CFS associated SNPs were analysed between the 3 groups (the CFS patients had been previously subtyped using gene expression). 21 SNPs were significantly associated with ME/CFS when compared with the other groups. 148 SNP alleles had a significant association with one or more ME/CFS subtypes. For each subtype associated SNPs tended to be grouped together within particular genes. He also looked at Ancestry Information Marker (AIM) SNPs which detect ancestry. 4 subjects were of Asian origin while the rest were W European. AIM SNP data also revealed a high degree of overall heterogeneity of subjects. Using 2 consecutive methods to reduce complexity in this multidimensional dataset, 84% correlation between gene expression-based CFS subtypes and SNP-based CFS subtypes was revealed.

7 This study provides evidence that human SNPs located within CFS/ME associated genes are associated with particular gene expression subtypes of CFS/ME. Further work is required to develop this into a clinically useful aid to subtype-specific diagnosis. Dr Nancy Klimas, University of Miami, started her talk by telling us she worked with Gulf War illness as well as ME/CFS, and pointed out that there was great cross-over between these 2 illnesses. She then went on to discuss the importance of biomarkers. Biomarkers need to: - define the group - define the subgroup - define markers of improvement and decline - look at pointing (illness mechanisms) She focused then on mediators as biomarkers. These could be: - Immune - Neuro-endocrine - Autonomic - Mitochondrial function - Infectious agents - New pathways identified through genomic studies When looking at the immune system, one can look at: Immune activation DR CD26 expression Th2 shift Pro-inflammatory cytokines TNF1 Functional defects NK cell dysfunction CD8 abnormality Perforins

8 Grazymes Macrophage abnormality Antibody production Examples show that NK function is worse on bad days. Il-6 is higher in CFS. Il-5, a type 2 cytokine is elevated in CFS. Big studies are needed badly to establish a wide range of findings. Cytokines in Klimas studies do show a consistent picture with decrease in NK cells and decreased CD26 receptors when the cell is activated. Several cytokines, chemokines, other growth factors and physiologically important peptides are activated. Studies with neuropeptide Y (NPY) show great promise. NPY is cleaved by peptidases, and stored in the sympathetic nerve terminals, and releases with catecholamines involved in the immune system, CNS, endocrine system etc. The NK cell count in CFS/ME can be 50% decreased. These are normally 5-15% of the lymphocytes, and usually attack cancer cells. The intracellular perforin is down which affects activity. One study induced a relapse on an exercycle in both CFS/ME and GWI, and then looked at genes and perforin levels. The levels worsened with exercise. This was equivalent to dynamic remodeling. Immune abnormalities will lead to autonomic abnormalities and vice versa. It is necessary to look at which pathways actually go wrong. 8 abnormal pathways are immune related and also relate to the genes that are turned on. In conclusion, Dr Klimas summarised a number of useful putative biomarkers: 1. NK function defines severity, but methodological issues limit use 2. Perforin is reproducible 3. CD26 a potent biomarker 4. NPY bridges autonomic and immune activity 5. Cytokine multiplex methods 6. Th2 cytokines 7. ROC curves point to cytokines as some of the most putative biomarkers 8. Patterns which suggest directed therapies 9. Genomic putative biomarkers explain multi-symptoms and multipathway illness 10. Exercise induction increases utility of methods Biomarkers which are mediators are potential targets for treatment.

9 Professor Brigitte Huber, Tufts University, USA presented her work with retroviruses, particularly focusing on the Human Endogenous Retrovirus (HERV- K18) with which she has worked for 10 years. She described this as a retrovirus which is in our genome, and which is very different from XMRV. HERVs represent footprints of previous exposures, with accumulated mutations in the germ line. They are transmitted vertically through successive generations. Approximately 8% of the human genome is composed of HERVs, and she looks on this as an untouched goldmine. HERV-K is the youngest family of HERVs and is found in primates in Africa. HERV-K18 is localized in CD48. It is found in old world but not new world monkeys. There is very low basal expression. Expression is induced by EBV, HHV6 or by treatment with IFNα. The envelope gene of HERV-K18 encodes a super antigen (SAG). SAGs cause massive T cell activation, and T cells promote cytokines. The hallmarks of SAGs are bacterial or viral microbial products. EBV activates the HERV in the cell forming a SAG, which then has the effect of Th shift and symptoms suggestive of CFS/ME. EBV goes into a reservoir. It is possible to develop EBV lymphoma. HERV=K18 could be a risk factor for CFS/ME. Three alleles have been identified HERV-K18-1,2,and 3. There is differential allele distribution. Abnormalities were seen in a study of 1000 MS patients and similar trends seen in CFS/ME. A study is underway looking at CFS/ME patients compared to healthy controls, and will also compare those who developed CFS/ME following EBV and non-ebv onset. Follow up will be 6 monthly. The study will also look at whether HERV levels correlate with severity. 128 patients have already been screened, and 105 patients will each be assessed by 3 physicians. Baseline medical records will be reviewed. Of these, 45 are non-ebv onset. 49 patients have had a 6 month follow up, and 25 have had 12 month follow up. Looking at HERV expression using Taqman qrt-pcr, there was a slight increase in the non-mono group, but a high increase in the EBV onset patients. Severity of symptoms was associated with HERV expression in the EBV group. Looking at the HERV-K18 in RNA, it was upregulated in the CFS/ME groups compared to controls, particularly in the EBV related group. Levels fluctuate over time, and there may be a correlation with symptoms. HERV-K has the potential to become an important bio marker in CFS/ME. Huber then presented her work looking at prevalence of XMRV. She used the Taqman qpcr for XMRV integrase. There was titration of the genomic DNA from WPI 1282, This is considered a very sensitive assay, which detects a single cell. Then they added 5mcg control (negative) DNA which could then detect 1 cell in 500, CFS/ME patients from 3 cohorts were tested. All samples were negative for XMRV integrase. They then used the same

10 nested PCR as WPI there were still no positives, although there were some similar sequences thought to possibly due to contamination. Dr Judy Mikovits, Whittemore Petersen Institute (WPI), Reno, USA discussed the implications of XMRV research for ME/CFS. The XMRV was reported by Silverman in 2006 in 10% prostatic cancer patients. The cancer was of an aggressive, familial variety. It tended to occur in young men. RNaseL activity was decreased by more than 2/3 in these patients, and this may relate to XMRV. The virus was detected in the stroma and not the tumour cells. 50% of a tumour consists of an inflammatory response. The work continued for 3 years when WPI constructed an infectious clone, and sequenced it and found it to be a novel retrovirus. Every cell has the potential to become infected because of XPR-1, a transporter on the surface. XMRV may be a promoter. WPI has a repository of cells collected over 3 years. RNA, DNA, protein and frozen cells have been collected. Patients for study were selected using the Fukuda and Canadian criteria. Age range was with a mean of 55 years. 67% were female. There were 218 healthy controls from the same geographical regions. The rationale for study included RNaseL dysfunction, low NK cell count, innate immune activation (increased cytokines, activated T cells) and chronic active infections of many pathogens. Could these patients be infected with XMRV? Integrated provirus is quiescent in the cell, but when multiplying it can be detected. Detection methods used: Serology antibodies Western blot of viral proteins (gag, env) Proviral DNA (infected cells) detected by PCR (DNA) and RT-PCR (virion RNA) Isolation of virus in cell culture 101 patients 68% showed positive evidence in DNA by single round and nested GAG. No contamination was seen. The group then used antibodies against SFFV ENV and MLVs. Then aimed to detect XMRV protein expression in activated PBMCs in CFS patients. In normal controls there were no positives, but 19 of 30 patients were positive. Cell free transmission occurred in 10 of 12

11 patients, and none in controls. PCR was abandoned as was not detecting the virus. Using culture, it was possible to detect the virus in the 2 nd round for GAG and ENV gene. If unstimulated, 54% were detected with DNA, stimulated 72% detected with DNA, and nested GAG 89% with DNA. Negative studies suggest that PBMCs are not the main reservoir of XMRV. Activation of PBMCs promotes XMRV spread. Antibodies to XMRV env should be able to be tested in ME/CFS. Looking at prostate cancer again, XMRV is similar to MLV expressed primarily in malignant epithelial cells. There is no evidence for an infectious virus yet published for prostate cancer. The positivity seen may only be one group and may be regional. This may be the case in ME/CFS, and methodological differences, diagnostic differences and regional differences may occur. There is some question as to whether autistic spectrum disoder may be a related condition. Family studies are going on looking at parents who are positive for XMRV. One family also had twins with Niemann Pick C disease, and both parents were XMRV env/antibody positive, but no active virus was detected. Associations with such diseases at present are a huge question mark. In conclusion, Dr Mikovits said that there are XMRV footprints in the human plasma. Single round DNA/PCR on unstimulated patients are not stand alone assays. In the role of pathogenesis, hormones and inflammation can increase the replication of XMRV. Cortisol and androgens can turn on the expression of the virus. It is important not to give these patients steroids. Chronic infection with XMRV may lead to immune deficiency, which in turn leads to an increased viral load and a decrease in XMRV antibodies. The conference finished with opportunity for networking and the overall feeling was extremely positive after a very worthwhile and efficiently organized day. I would like to thank ANZMES and Cathay Pacific for enabling me to attend this inspiring event. Rosamund Vallings, MNZM, MB BS