Research: ME-CFS/FMS/Lyme disease/MPS/etc. (2nd collection)

Discussion in 'Fibromyalgia and ME & Chronic Fatigue Syndrome' started by fight4acure, Dec 22, 2009.

  1. fight4acure

    fight4acure Member

    Posted originally by Lisaloo! Thank you!

    http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008519

    A little hard to completely understand

    Background In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV.

    Methodology Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected.

    Conclusion XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not.

    Introduction Top A recent study by Lombardi et al. [1] describing a gamma-retrovirus infection in 68 of 101 chronic fatigue syndrome (CFS) patients was notable not only for its claim of a new viral aetiology of a hitherto controversial disease, but also for the fact that proviral DNA could be amplified from the peripheral blood mononuclear cells (PBMC) of 3.75% (8/218) of the healthy controls. This follows an earlier claim that 1.7% (5/300) of healthy Japanese blood donors carried antibodies to the same virus [2]. The virus in question is a recently discovered retrovirus, Xenotropic Murine Leukaemia Virus (MLV)-Related Virus (XMRV).

    In the original identification of XMRV in prostate cancer stromal cells, Urisman et al. [3] confirmed by sequence analysis that XMRV is not a laboratory contaminant, as is often the case with claims of new retroviral associations with disease. It shares >90% sequence identity in gag and env (two of the three viral structural genes) with other xenotropic MLVs.

    An association between XMRV and prostate cancer was strengthened with the demonstration of XMRV protein expression in malignant epithelial cells [4]. However, these results have not been duplicated in studies conducted in Europe [5]–[7]. Both prostate cancer and CFS have been linked to an Arg to Gln mutation at codon 462 (R462Q) in the RNaseL gene, an interferon-induced ribonuclease [8]. On activation, RNaseL destroys single stranded cellular and viral RNA, thereby preventing viral replication, blocking protein synthesis, triggering cellular apoptosis and providing an innate anti-viral response. The two US studies are of interest, not only because this would be a further example of a virus association with cancer, but because they represent the first demonstration of a gamma-retrovirus able to infect human cells, over-riding the intrinsic immune mechanisms that were believed to protect humans from MLV infection.

    The XMRV sequences derived from prostate cancer tissue are identical to those from CFS patients, but differ from xenotropic MLV sequences, endorsing a genuine cross-species transmission. However, the claim that XMRV is preferentially found in prostate tumours from patients homozygous for the R462Q variant [3] is not borne out by the second prostate cancer study to find XMRV in patients [4], nor was the genetic variant detected in CFS patients carrying XMRV [5].

    The finding of Lombardi et al. of a 67% XMRV infection rate among CFS patients, if confirmed, would have a serious impact on understanding the pathogenesis of this complex and debilitating disease and its treatment. Therefore, it was important to determine if CFS sufferers in the UK were carriers of XMRV. We have screened DNA extracts from the blood of CFS sufferers by PCRs targeted at an XMRV-specific sequence and at a sequence conserved amongst most murine retroviruses (MRV).

    Methods Top Patients All patients gave written informed consent for the use of their DNA to test aetiological theories of CFS, and the study was approved by the South London and Maudsley NHS Trust Ethics Committee. The study recruited 186 patients (62% female, age range 19–70, mean 39.6±11.3years) from consecutive referrals to the CFS clinic at King's College Hospital, London. All patients had undergone medical screening to exclude detectable organic illness, including a minimum of physical examination, urinalysis, full blood count, urea and electrolytes, thyroid function tests, liver function tests, 9 a.m. cortisol and ESR. Patients were interviewed using a semi-structured interview for CFS [9] to determine whether they met international consensus criteria for CFS. All subjects met the CDC criteria [10]; patients with the Fukuda-specified exclusionary psychiatric disorders, or somatisation disorder (as per DSM-IV), were not included. The patient set studied is a well-characterised and representative sample of CFS patients who have been described previously: all were routine clinic attendees, referred within the UK National Health Service, who had taken part in prior studies of neuroendocrine functioning [11] and/or of cognitive behaviour therapy [12]. As is typical of the patients seen in this tertiary care centre, they were markedly unwell. Few were working, and 19% were members of patient support groups for CFS/ME [12]–[14]. The levels of fatigue in this sample were high (mean Chalder Fatigue Scale, 26.3±5.4) [15], as were levels of disability (mean Work and Social Adjustment Scale, total score 28.2±7.2) [16]. The mean GHQ-12 score [17] was 19.7±8.1. Patients had been unwell for a median of 4.0 y (range 1–28 y). Of note was that 45% said their illness definitely related to a viral illness and 45% said it might relate to a viral illness. Overall, we conclude that this sample is typical of CFS patients seen in specialist clinical services in the UK. We also know from collaborative studies that our patients resemble those seen in other specialist CFS services in the United States and Australia [18].

    PCR detection of XMRV and MLV sequences.DNA was extracted from EDTA whole blood using a standard phenol-based organic deproteinisation procedure [19]. DNA concentrations were determined by absorbance at 260 nm (A260). Each sample was amplified in three nested PCRs using primers targeted to an XMRV-specific sequence, to a sequence conserved amongst most MLV and, as a control for sample addition and PCR-inhibition, to a human beta-globin (hBG) sequence (Table 1). Each first-round reaction was performed in a 25 µl volume containing 0.5 units TaqGold (Applied BioSystems, Warrington, UK), 1 x TaqGold reaction buffer (Applied BioSystems), 1.5 mM Mg2+, 200 mM each dNTP, 2.5 pmol each primer to which 5 µl DNA extract or control was added. Reaction conditions were one cycle of 94°C, 8 minutes, 35 cycles of 94°C 30 seconds, 55°C 30 seconds, 72°C 30 seconds and one cycle 0f 72°C, 7 minutes. Second round reaction mixes were identical to the first round and the sample was a 1 µl transfer from the first round reactions. Second round reaction conditions were as for the first round over 30 cycles. PCR amplicons were visualised on a 1% agarose gel stained with ethidium bromide. Each PCR run consisted of test samples, six negative (water) and two positive controls. The positive control was a dilution of a plasmid with a full-length XMRV (isolate VP62) insert, generously gifted by Dr R. Silverman. To validate the sensitivity of the PCR, an end-point dilution of the plasmid was performed. To determine specificity of the PCR, a sample of human DNA from the LNCaP prostate cancer cell line (American Type Culture Collection, code CRL-1740) was amplified with the XMRV and MLV primer sets. To ensure integrity of the DNA extracts, three randomly selected samples were titrated to end-point using the hBG PCR to determine if the PCR copy number equated with the A260. To determine if the DNA extracts exhibited low level non-specific inhibition of PCR, 10 samples were subjected to 30 cycles of the first round hBG PCR (reaction mix and conditions as above) followed by 40 cycles of a nested real-time SYBR-green PCR using the SYBR-green Fast PCR kit (Roche, Lewes UK) according to the manufacturer's instructions.

    Table 1. Oligonucleotide Primers.

    doi:10.1371/journal.pone.0008519.t001 Results Top Nested PCR Validation Based on A260 of the purified plasmid, both primer sets (XMRV, MLV) were able to amplify a single target copy added to the reaction. Amplification of 600 ng of LNCaP cellular DNA added to XMRV and MLV PCRs yielded no non-specific bands when viewed on an ethidium bromide-stained agarose gel. Quantification of DNA samples from three randomly selected test samples by end-point dilution PCR with the hBG primer set showed concurrence of the PCR-determined copy number with A260, thus indicating integrity of the DNA preparations. Nested real-time amplification of 10 samples showed no evidence of non-specific inhibition as determined by the slope of the amplification curves and the height of the signal plateau.

    PCR Analysis of Test Samples Input DNA ranged from 10 to 600 ng (1.6×103 to 1.1×105 cell equivalents) as determined by A260 of which 149 samples had an input of >100 ng and 106 samples >200 ng. None of the 186 test samples analysed yielded a specific PCR product with either the XMRV or MLV primer sets and no non-specific PCR products were observed. A specific hBG product was amplified from all 186 test samples. The positive control was amplified in each run by the XMRV and MLV primer sets. A stained gel of the XMRV and MLV PCR products is shown in figure 1 and a representative sample of our results with CFS DNA and MLV primers is shown in figure 2.

    Figure 1. PCR products of the XMRV VP62 clone.

    Primers are generic to MLV (lanes 1 and 2) or specific to XMRV (lanes 4 and 5). The sizes of the respective fragments are shown. Lane 3–200 bp molecular size ladder.

    doi:10.1371/journal.pone.0008519.g001Figure 2. Nested PCR from the DNA of 8 CFS patients.

    Products of generic MLV primers (including XMRV) are shown. Lanes 1–8, CFS patient DNA (2nd round); lanes 9 and 10, XMRV 2nd round and 1st round positive controls; lanes 11 and 12, DNA of uninfected cell line LNCaP; lanes 13–18, water controls.

    doi:10.1371/journal.pone.0008519.g002 Discussion Top Unlike the study of Lombardi et al., we have failed to detect XMRV or closely related MRV proviral DNA sequences in any sample from CFS cases. There have been numerous claims for an infective aetiology to CFS over the years, not least because, as in this sample, many patients report that their symptoms were triggered by an infective episode. Prospective epidemiological studies have confirmed that certain infective agents, for example Epstein Barr virus, are unequivocally associated with subsequent CFS [20], even if the mechanisms are unclear and almost certainly multi factorial. Nearly two decades ago, sequences from another retrovirus, the human T-lymphotropic virus type ll, were amplified from the PBMCs of 10/12 (83%) adult and 13/18 paediatric CFS patients, but not from healthy control subjects [21]. However, subsequent studies carried out on small numbers (20–30) of CFS patients, failed to confirm evidence for HTLV (type 1 or 11) [22]–[25] or other retroviruses, including the closely-related simian T lymphotropic virus type l, the prototype foamy virus, simian retrovirus, bovine and feline leukaemia viruses [26] and HIV-1 [23].

    The Lombardi paper is the first to study a significantly larger number of people than that in any previous study and to detect a virus only recently discovered. Our study resembles that of Lombardi et al. in certain respects. Both studies use the widely accepted 1994 clinical case definition of CFS10. Lombardi et al. reported that their cases “presented with severe disability” and we provide quantifiable evidence confirming high levels of disability in our subjects. Our subjects were also typical of those seen in secondary and tertiary care in other centres.

    Our own study also differs from that of Lombardi in other respects. Firstly, the PCR operator was blinded to the provenance of the DNA samples. In fact, with the exception of the PCR controls, all 186 DNA test samples originated from CFS patients. Care was taken to grow the XMRV plasmid in a laboratory in which no MLV had been cultured and no MLV vectors used and the PCR was carried out in a CPA-accredited Molecular Diagnostics Unit which processes only human tissue. Multiple (six) water (negative) controls were included in every run to detect low level contamination and a PCR to amplify a sequence that is conserved in most murine leukaemia viruses was included in order to expose any circulating MLV contamination and to detect any variant of XMRV that might be circulating in the UK CFS population.

    Based on our molecular data, we do not share the conviction that XMRV may be a contributory factor in the pathogenesis of CFS, at least in the U.K.


  2. fight4acure

    fight4acure Member

    (Originally posted by Victoria in the Lyme disease message board)
    (Victoria wrote:
    This is from lyme info at yahoo groups, thought it was interesting:)


    The Allergy Research Group is an allergy oriented, hypoallergenic
    nutritional supplement company founded in 1979 by Stephen Levine, Ph.D.
    Some LymeInfo readers may already be aware of this company.

    They publish a newsletter titled Focus and the March 2009 issue has two
    articles which may be of particular interest.

    One is titled, "Dissolve Biofilms with Fibrinolytic Enzymes: One
    Nutritionist's Novel Approach to Autism Spectrum Disorders".

    Here, a nutritionist believes biofilms play a huge role in Autism,
    Lupus, Lyme Disease, Multiple Sclerosis and any autoimmune-type chronic
    infection. The use of fibrinolytics to help dissolve the fibrin in
    bacterial biofilms is discussed.

    The second is titled, "Two Doctors Report on the use of Lumbrokinase in
    Lyme Disease: This Enzyme Complex Helps Potentiate Antibiotics and
    Antimicrobials"

    Lumbrokinase is a group of six, novel proteolytic enzymes derived from the earthworm Lumbricus rubellas. This article talks about how Lumbrokinase may help break up the biofilms in patients who don't seem to improve on antibiotics or herbal antimicrobials alone.

    You can view both articles from this issue of Focus from the Allergy
    Research Group's website starting here:

    http://www.allergyresearchgroup.com/Focus-Newsletter-sp-11.html

  3. fight4acure

    fight4acure Member

    (Originally posted by Lisaloo)
    UK NHS Choices site
    http://www.nhs.uk/news/2010/01Januar...-in-doubt.aspx

    Virus link to CFS 'in doubt'

    “Serious doubt has been cast on the theory that...chronic fatigue syndrome is caused by a new retrovirus,” The Guardian reported. The newspaper said researchers from London have failed to replicate findings from the US that suggested a possible role for a virus called XMRV in causing CFS, also known as ME (myalgic encephalomyelitis).

    In the new study none of the 186 UK CFS patients tested carried the XMRV virus, in contrast to the US study in 2009, which found that about two-thirds of 101 CFS patients tested had the virus. Why the two studies have different findings is not clear, but the results of the UK study do not support an association between XMRV infection and CFS in UK patients. This highlights the importance of different research groups repeating experiments in different populations.

    CFS is a complex disease, and its causes are not well understood. Although an association with XMRV has not been established, this does not rule out the possibility that viral infection is involved. Much more research will be needed in this area.

    Where did the story come from?

    The research was carried out by Dr Otto Erlwein and colleagues from Imperial College London and King’s College London. The researchers were funded by the South London and Maudsley NHS Foundation Trust, the Institute of Psychiatry and the National Institute for Health Research Biomedical Research Centre. The study was published in the peer-reviewed open access journal PLoS ONE.

    The Guardian, Daily Mail and The Independent reported the story. In general, the coverage is balanced and accurate. The headline of the Daily Mail story that “British experts say ME virus is a myth” might be taken to mean that this research excludes any role for viral infection in CFS/ME, but this research only looked at one virus (XMRV).

    What kind of research was this?

    This cross-sectional study investigated whether people in the UK with chronic fatigue syndrome (CFS) were infected with the xenotropic murine leukaemia virus-related virus (XMRV). In 2009, a case control study from the US found that more people with CFS carried the virus than people without the condition. The researchers in this study wanted to see if XMRV was similarly common in people from the UK with CFS.

    A cross-sectional study design is appropriate for determining how common a particular trait is among a certain group of people. However, neither this study, not the original case-control study could prove whether XMRV potentially caused CFS, as neither would be able to establish whether people with XMRV had been infected before they developed CFS or after. The current study would also not have been able to say whether the XMRV virus was more or less common in people with CFS than in those without it, as it did not include a control group of people without the disease.

    What did the research involve?

    The researchers enrolled 186 people with CFS who were living in the UK. These people had been medically examined and diagnosed with CFS according to standard criteria, and other potential causes of their symptoms had been ruled out. Blood samples were taken and tested for the presence of DNA from XMRV or a related virus called murine leukaemia virus (MLV). A number of control tests were also carried out to show that the DNA in these samples was intact, that any positive findings were not a result of contamination of their experiment and that their test would identify XMRV if it was present. The inclusion of these controls is important for ensuring that the experiments were working well and were reliable. The researcher who carried out the DNA tests did not know which of the samples came from people with CFS.

    The participants, all of whom had been referred to a CFS clinic, were mainly female (62%) with an average age of 39.6 years. They had been unwell for an average (median) of four years (range one to 28 years), and had high levels of fatigue. Few participants were working and about a fifth (19%) belonged to CFS/ME support groups. Just under half of the participants (45%) said that their CFS definitely related to a viral infection and 45% said that it might relate to a viral infection. The researchers suggested that the characteristics of their sample were typical of those seen in CFS patients attending specialist clinical services in the UK.


    What were the basic results?

    The researchers did not identify XMRV or MLV in the blood from any of the 186 CFS patients tested. Their control tests showed that the DNA being tested was intact, that there was no contamination in their experiments and that when XMRV was present (in a positive control sample containing XMRV DNA) their test detected it.


    How did the researchers interpret the results?

    The researchers concluded that they “found no evidence that XMRV is associated with CFS in the UK”. They suggested that the reason for the differences between their findings and those from the US might be due to differences in how common XMRV infection is in the different countries.


    Conclusion

    This study suggests that the XMRV infection is not common in CFS patients in the UK. A previous case-control study from the US found that about two-thirds of the 101 CFS patients tested carried XMRV, compared to about 4% of 218 healthy controls. This led the researchers from the US study to suggest that XMRV might be the cause of CFS in these patients. The reason for the differences between the US and UK studies is not clear, but the authors of the UK study suggest that it could be due to XMRV infection being more common in the US than in Europe.

    The findings of this current study highlight the importance of different research groups repeating experiments in different populations. The study does have some limitations in that it was relatively small and all participants came from one CFS centre in London. Further studies in more participants from different centres in the UK would be useful in determining whether these findings are typical of the UK as a whole.

    Even if this study had found significant levels of XMRV in CFS patients, it would not have been able to prove the virus actually caused the condition. This is because, like the original US case-control study, it could not establish whether people with XMRV had been infected before they developed CFS or after.

    The current study would also not have been able to say whether the XMRV virus was more or less common in people with CFS than those without, as it did not include a control group of people without the disease.

    The results of this UK study do not support an association between the XMRV virus and CFS in UK patients. The researchers do not rule out a role for all viruses in CFS, and say that “prospective epidemiological studies have confirmed that certain infective agents, for example Epstein Barr virus, are unequivocally associated with subsequent CFS, even if the mechanisms are unclear and almost certainly multi factorial”. CFS is a complex disease, and its causes are not well understood. Much more research will be needed in this area.

    Links to the headlines

    Research casts doubt over US chronic fatigue virus claim. The Guardian, January 06 2010

    British experts dash ME breakthrough hopes following American promise of new treatment. Daily Mail, January 06 2010

    Scientists' claim to have found the cause of ME is 'premature'. The Independent, January 06 2010

  4. fight4acure

    fight4acure Member

    (Originally posted by TigerLilea:) Thanks for posting this!!!

    Official Statement from the Whittemore Peterson Institute Regarding UK Study

    The Whittemore Peterson Institute (WPI) has reviewed the paper entitled “Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome.” This study did not duplicate the
    rigorous scientific techniques used by WPI, the National Cancer Institute and the Cleveland
    Clinic, therefore it cannot be considered a replication study nor can the results claim to be
    anything other than a failure not just to detect XMRV, but also a failure to suggest meaningful
    results.

    The scientific methods used by WPI are very exact and require specific techniques to ensure
    accuracy. Differences in techniques employed by Erlwein et al. not only explain their failure to
    replicate the WPI study, but also render the conclusions meaningless. These differences
    include, but are not limited to the following:

    1) blood sample volumes and processing;
    2) patient criteria/population differences;
    3) number and type of tests done to assure accurate results, including white blood cell
    culture;
    4) use of a molecular plasmid control in water versus a positive blood sample; and
    5) different primer sequences and amplification protocol used to find the virus, which
    were not validated by a clinical control.

    To see the complete statement, go to http://www.wpinstitute.org/news/docs/WPI_Erlwein_010610.pdf
  5. fight4acure

    fight4acure Member

  6. fight4acure

    fight4acure Member

    (originally posted by QuayMan)

    Funding Biomedical Research into ME

    Invest in ME

    me solutions

    Two charities are joining forces to fund research into
    ME/CFS.

    ME Solutions and Invest in ME are working together
    to maximise the opportunities to fund research into
    ME/CFS. The research project is:

    The role of XMRV in modulation of
    NK cell cytotoxicity and NK cell gene
    abnormalities in ME/CFS patients and
    normal blood donors

    The project will be carried out by Dr Jonathan Kerr
    and his team from St. George's University, London,
    and Dr Amolak Bansal of the Department of
    Immunology, Epsom & St Helier University Hospitals
    NHS Trust.

    Background to the project

    A newly discovered gamma-retrovirus, Xenotropic Murine
    Leukaemia Virus - like virus (XMRV) has recently
    been found to be present in the blood of 68 of 101
    (67%) ME/CFS patients as compared with 8 of 218
    (3.7%) normal healthy controls (Lombardi et al,
    2009).

    XMRV has been cultured from T, B and NK cells, but
    primarily targets NK cells. NK cell dysfunction has
    previously been found to be abnormal in ME/CFS,
    despite their numbers often being largely unaffected.

    Defects in the innate immune system are thought to
    play a key role in the pathogenesis of ME/CFS and
    these abnormalities may leave individuals
    susceptible to XMRV infection.

    This study will relate the presence of XMRV in NK
    cells with ME/CFS-associated abnormalities
    previously demonstrated in NK cells and
    ME/CFS-associated gene abnormalities.

    Plan of Investigation

    A sample of clinically-diagnosed (according to the
    Fukuda and Canadian criteria) ME/CFS patients and
    age-and-sex matched normal controls will be
    recruited.

    XMRV status will be determined and NK cells
    obtained and tested for ME/CFS-associated gene
    abnormalities in NK cells. XMRV status will be
    related to ME/CFS gene expression changes.

    Donations

    ME Solutions and Invest in ME welcome sponsorship
    and donations for this two year project which we
    hope will begin as soon as possible.

    The links below will allow online donations to be
    made.

    every click:

    https://charities.everyclick.com/purchase?d=D&cn=Invest-in-ME&cid=275467&ctId=1114035&cy=GBP

    or:
    http://bit.ly/6wiQt0

    JustGiving:

    http://www.justgiving.com/mesolutions/donate

    or:
    http://bit.ly/4uMEOk

    For further information about making
    donations to this project please contact:

    ME Solutions:

    info@mesolutions.org.uk?subject=Biomedical%20research%20into%20ME

    or

    Invest in ME

    info@investinme.org?subject=Biomedical%20Research%20into%20ME

    ````````````````````

    Support ME Awareness

    https://www.paypal.com/uk/cgi-bin/webscr?cmd=_flow&SESSION=zyxENvZ-kzUKdmy4OvTR4qvBfwviauuMYUqEbrFCYWtPGfqckwCVQQj8KkC&dispatch=5885d80a13c0db1ffc45dc241d84e953ae3a912d7415d1a97451b677930c8a71

    or:

    http://bit.ly/8zIEUd
  7. fight4acure

    fight4acure Member

  8. fight4acure

    fight4acure Member

  9. fight4acure

    fight4acure Member

    Hi Victoria, how are you doing? Fighty is back! =:)

    Bumpity bump bump bump, trying to keep research updated on these nasty chronic painful illnesses we have.

    :(