1. Patients launch $1.27 million crowdfunding campaign for ME/CFS gut microbiome study.
    Check out the website, Facebook and Twitter. Join in donate and spread the word!
The Chronic Fatigue Initiative and Interview with Mady Hornig
In a follow-up article to the recent IACFS/ME conference presentation in San Francisco, and after speaking at length with Dr. Mady Hornig, 'searcher' delves deeper into the impressive work being completed by the Chronic Fatigue Initiative, and focuses in on those cytokine results ...
Discuss the article on the Forums.

December Blood Prodcuts Advisory Committee Full Transcript

Discussion in 'XMRV Research and Replication Studies' started by Esther12, Jan 11, 2011.

  1. Esther12

    Esther12 Senior Member

    Messages:
    5,156
    Likes:
    5,081
    http://www.fda.gov/AdvisoryCommitte.../BloodProductsAdvisoryCommittee/ucm239304.htm


    Long.

    It's a nice, clearly written piece though (which can be hard with transcripts).

    I doubt there will be anything in there we don't know, and a lot of it is over my head, but I'm enjoying bits (the Coffin/Lo bit sounds like a good, honest discussion between intelligent researchers. Nice to have people like that working on CFS, even if their main interest is the virus).
  2. urbantravels

    urbantravels disjecta membra

    Messages:
    1,333
    Likes:
    506
    Los Angeles, CA
    It's so nice to finally have the exact words. I'm especially appreciative of having Harvey Alter's statements word-for-word at last.
  3. Esther12

    Esther12 Senior Member

    Messages:
    5,156
    Likes:
    5,081
    Re-reading the BWG bits... it sounds dubious to me. False positives, inconsistent detection of the known positives. It could be that they'll be able to improve their testing and then this will all work out, but I'm feeling less hopeful since the BWG news. But, there are quite a few different supporting lines of evidence for XMRV... it would need a collection of confidences to explain them all. What a mess.
  4. Deatheye

    Deatheye Senior Member

    Messages:
    160
    Likes:
    18
    I'm looking at the animal test. Seems quit interesting. No Idea if there is anything new in there but it seems easier then a paper...

    In an understandable language what could this mean for us? Or does this anything say at all in an understandable language? xD

    What is the proper context? Anything in there that tells us that?

    Seems like they are going to check longer infection time / chronic sympthoms in theye apes or what ever they are called I always forgett theyr name. They seem to see ongoing low replication and want to check what that causes later on. Also very interesting that they intend to check how much the virus mutates during replication inside the apes. Which hoepfully gives some answers towards what genetical differences one can suspect to see inside the virus.

    Anything anywhere known about this study?

    How did they define a negativ control? I mean it seems they are still a lot of problems finding this nasty little thing. So how do they now those samples really are negativ?

    First time I see Mikovits comment... But by fare did not read all... going trough it starting with what interests me the most. What does she tell us with that?

    That's from Dr. Coffin and really states a basic principal which in my opinion seems hard for a lot of people. I think something like this should be teached at school / university again... Not just memorizing data... people need to start using theyr brain again...

    Many studies seem to be on hold cause they need a hoigh troughput test.

    This is interesting in case anyone has connections to such a group, maybe he could point them in this direction?

    Sad that, whoever was this, didn't seem to get the chance to talk..
    EDIT: forget that later on the patient, seems a woman is allowed to talk. Anyone knows who she is? But please don't toll if you didn't get the agrrement from the person.

    Overall they learned much about CFS in this conference. Shared Knowledge.
  5. biophile

    biophile Places I'd rather be.

    Messages:
    1,350
    Likes:
    4,116
    So much for the IAP assay, a pseudo gold standard?

    http://www.fda.gov/AdvisoryCommitte.../BloodProductsAdvisoryCommittee/ucm239304.htm

    It's way too much to read, so I did a selective text search for "ALTER" and I noticed this ...

    Then again later on during his now famous [we need to get to the bottom of this serious medical disease regardless whether XMRV is the causative agent or not] speech:

    I guess "IPA" is a typo or synonym. Did any of the "XMRV is contamination" articles in the news express any concern whatsoever over the sensitivity and accuracy of the IAP assay used to detect contamination in the recent Retrovirology papers? Apparently the IAP assay has been a major point in discussion/criticism elsewhere of the proposed Dusty Miller study, with Mikovits & Ruscetti allegedly (in a personal communication posted online) also denouncing its use as well.

    As for Esther12's pessimism, the subject of HGRV in ME/CFS is interesting and perhaps promising but I'm mostly still fence sitting for now as well. I have donated to the WPI on multiple occasions because they deserve a fair shake as a ME/CFS research institute and I want the HGRV issue settled ASAP.

    My current Alfred E Neuman avatar and quote "What ME worry about HGRV?" is to reflect the smiling idiocy of indifference surrounding how ME/CFS is treated despite the prolonged suffering and burden it causes on individual patients and society as a whole, with the possibility that decades of unjustified psychogenic dismissal has allowed a family of retrovirus to propagate while egos and ideologies are clashing.
  6. oceanblue

    oceanblue Senior Member

    Messages:
    1,174
    Likes:
    343
    UK
    I hadn't heard of the now-famous speech, but I like it.
    And the fact that Lo has done the IAP test on his samples does give more weight to the not-[mouse]-contamination-in-this-case argument.
  7. urbantravels

    urbantravels disjecta membra

    Messages:
    1,333
    Likes:
    506
    Los Angeles, CA
    I've gotta figure that some of these folks (Alter, Lo, Ruscetti, Mikovits) are preparing rebuttals on the contamination stuff, to be submitted to Virology. That is just my guess. Anyone know more?
  8. urbantravels

    urbantravels disjecta membra

    Messages:
    1,333
    Likes:
    506
    Los Angeles, CA
    The famous speech became famous, in the only form we had it - which was hastily liveblogged summary notes provided to the community by someone who attended the meeting - because it was HARVEY ALTER, standing up and directly rebutting all the accusations about contamination, and then saying that he's learned a lot about this disease, it looks like a viral disease, and if it isn't XMRV we need to find out what it is.

    So not only was an elder statesman of virology forecefully defending his research, but he was going even further and saying the hunt for the cause of ME/CFS must not stop, even if XMRV doesn't pan out. I think those were very powerful words of hope for our community - that the reality of our disease is being validated, that the validation doesn't have to depend on this one particular theory of causation working out, and that we will NOT BE ABANDONED if XMRV doesn't pan out.

    May it be true.
  9. oceanblue

    oceanblue Senior Member

    Messages:
    1,174
    Likes:
    343
    UK
    Let's hope so. Alter seemed to be saying that if it wasn't XMRV it could be another specific pathogen (because of the viral-like trigger for so many); I'd like to think he'd also still be interested if it turns out there are broader explanations such as flawed immune response to infection, or indeed any other cause. I agree - having researchersof the calibre of Alter onside is a huge boost.
  10. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    I was out of action while the advisory committee was reporting and I only managed to lurk and scan rather than analyse and summarise...and I haven't seen the good 'summary of key news' that we normally produce. But scanning through this transcript makes it look like a good place to start...I don't have much time to spend on highlighting/commenting much myself now (but will make a start and others might follow up...) but my main feeling is that there's a lot of very positive evidence and quotes from a variety of places that could do with bringing together.

    I realise I'm going over old ground but...didn't see that summary and I did miss a lot of the earlier comment....

    Here' the section from Maureen Hanson:

    Agenda Item: U.S. Study

    We will move on to the next U.S. study, Maureen Hanson, from Cornell.
    DR. HANSON: Thank you.
    I am going to be calling this study the 20/20 study. It's a small study, funded by an exploratory grant from the NIH, an R21. This study is entirely unpublished. It's still in progress. We haven't been able to complete all the tests at this point.
    I would like to start with acknowledging my collaborators. In my own lab, I have a postdoc, Li Ling Lee, and a part-time technician on this project. We have been assisted by David Ruppert, a statistician. The clinician who has provided the samples is David Bell. He has been assisted by his son, David Bell.
    Forty subjects were selected by Dr. Bell. Dr. Bell presided over one of the outbreaks of CFS that occurred in the mid-1980s in his area of western New York. We have 10 what we call severe CFS, 10 recovered CFS, and 20 healthy controls.The samples were received at Cornell. We were blinded to the health status of the individuals providing the samples. The Bells administered a number of survey instruments to test the health status of these healthy controls and the severe and recovered CFS. I had to be unblinded in September in order to present -- in August, actually -- to present the talk at the XMRV meeting at NIH. But my lab members are still blinded because the study is not yet finished.
    I would like to describe the very severe CFS. These people are very ill. They are housebound. They are often bedbound. They have less than three hours daily of upright activity. The recovered CFS all met the CDC definition of chronic fatigue syndrome at one time, but they all consider themselves either recovered or nearly recovered. They have many more hours of upright activity, 13.5. Six of these 10 were part of the outbreak that occurred in Lyndonville, New York. Of the healthy controls, all of them live in the same geographic area, western New York. They were screened so that they had never lived with a person with CFS, fibromyalgia, or prostate cancer. However, some of these people are close friends of CFS patients. These people have 15.5 hours of upright activity, and they appear to be completely healthy.
    If we graph the scores on the survey instruments, you can see that the blue here, the severe CFS patients, have significantly different scores than the healthy controls. But what's interesting is that the recovered patients, although they consider themselves recovered and perfectly healthy, actually have some lower scores on many of these instruments, significantly lower on the SF-36 test.
    These people do feel well. They feel well enough to donate blood, and a number of them, in fact, have done so.

    We are still working out our assays for looking for the MLV-like viruses. We have a number of assays that we have tried, and I'm going to mention these. But we're still not sure which are the best ones to use. We have results from all the different assays, but we are still, as I said, in progress.
    We have made PBMCs from blood collected in EDTA and made nucleic acids from these PMBCs that are immediately frozen or cultured for 5 to 10 days. We make RNA with TRIzol and make cDNA. We make DNA with CTAB. Then we do PCR on these samples. We observe DNA bands on the gel. We never just conclude from the size of the band that we have a gag sequence. We always sequence it before we conclude that we have a positive gag sequence.
    We have also recently been doing PCR assays on whole blood. We are using the Qiagen kit -- again, making DNA, doing PCR, observing the bands in the gel, and sequencing.
    We have also been looking for virus in plasma by incubating it with the prostate cancer cell line LNCaP cells. Again, we make RNA, convert it to cDNA, make genomic DNA, and do PCR.

    We do take precautions against contamination. We have been using mouse mitochondrial DNA controls, using cox2 primers. We are actually thinking of switching to the ones that Lo described, because he has validated how sensitive those are. We also do all of our work in labs that have never had any sort of mouse cell line or mouse work done in the lab. In fact, we put our hood into a plant growth room. All of our blood is collected in this former plant growth room. We UV-irradiate all of our tubes before we use them. We carry out our first reaction PCR in one of these hoods that has a UV light in it to clean up any DNA that may be in there. We have a second hood for our second PCR. This is all in a lab that's separate from my lab, where we work with the amplicons.
    This is just an example of some of our results. This is actually genomic DNA from these LNCaP cells, incubated with plasma and then assayed after four transfers. We have not had very good luck in any of our assays with just a single round of PCR. Usually you get a gel like this, which is blank. But in second-round PCR, we have been able to detect gag sequences. This is after six transfers. These are different samples. They show again that we can detect gag when we sequence this. We often, like Dr. Lo described, get nonspecific human DNA bands of different sizes. Whenever we sequence a band that is the wrong size, it turns out to be some accidental amplification of human sequences.
    Our sequences that we have obtained are very much like the ones described by Dr. Lo and the Alter group. We, in fact, have one of their blood donors up here. In comparison to XMRV, our sequences look a lot more like polytropic MLV. One thing in particular is this deletion right here in XMRV in comparison to polytropic viruses. We don't have that deletion, nor does the Lo, Alter paper either. This deletion was first described in the prostate cancer study, present in the glyco-gag region of XMRV.
    This is a summary of our current data. We have 7 out of 10 severe CFS patients, 7 out of 10 recovered CFS patients, and 4 out of 20 healthy controls. These are only the numbers where we have done two different tests and gotten positives. We actually have some additional samples that are positive, but we only did it once, and so I'm not reporting that here at this talk. You notice that we have rather a high healthy control number here. It's my suspicion that this may reflect the fact that the samples were taken from an outbreak area, and although these people weren't living with people with CFS, as I said, many of them were friends and associates of people with CFS.
    Why are there so many reports of failure to detect these viruses? One thing I would like to point out is that some of the negative reports -- the people used primers that span this deletion. A primer that spans this deletion is not going to amplify the virus that either Dr. Lo reported or that we are reporting here. This primer will not work. But it was used in at least two of the negative studies. The other possibility is that all of the labs with negative reports optimized their PCR conditions for detecting VP62 XMRV or XMRV from a culture and they didn't optimize for any MLV-like virus that might be present. In my lab we haven't had any XMRV in the lab. We have just optimized according to what's in the samples. We have tried lots of different conditions to figure out good conditions to use to look for virus in the samples, not using XMRV as our control.

    [This prompts a thought. Hue at al's phylogenetic analysis of the published XMRV strains doesn't apply to these PMLVs, does it? So, as and when Hanson's study is published, it looks clear it will be a confirmation of Lo and Alter's results regarding PMLV sequences, and one to which Hue et al's arguments don't apply. Indeed they show the expected sequence variability within the patients they re-tested, and promise forthcoming publication of a paper regarding that data elsewhere in this transcript.]

    For example, just to illustrate what different results you can get with different conditions, here's a case where we have four samples, and these two samples are negative under these conditions and these two are positive. But using these conditions, we get all four of these samples positive -- these, incidentally, being the conditions that were used in the Lombardi et al. paper. The other thing that we have noticed is that really very small differences in your methods can make a huge difference in the results. We have three different brands of PCR machines in our lab. We have seen differences in which PCR machine is used, in our own lab, as to what results we get as far as detecting things or not detecting things.

    I would like to end by presenting some results that were not -- these data were not obtained in my lab. These were obtained by Rachel Bagni's group at NIH Frederick and at the WPI. Samples were sent from Dr. Bell to these groups to look for antibodies in the sera of these same people. Of the seven severe CFS samples in which we detected virus, at NIH six were detected as having at least one XMRV-reacting antibody. The WPI detected the seventh. Clearly the NIH and the WPI tests are a bit different. You are going to be hearing about what those tests are later on in this session. Of the seven recovered CFS, NIH detected four of them as having an antibody to at least one of these antigens, and WPI detected the other three. Of the four controls -- this was quite reassuring to us -- in which we detected virus, NIH detected three as having one antigen and WPI also detected one of those three.
    I would just like to mention that, of course, antibodies are less specific than PCR, and an antibody that reacts to XMRV should also be likely to be reacting with an MLV-like virus.




    Our conclusions are:

    • We have recovered patients who feel well, but they are actually significantly different from healthy controls.
    • We have gag sequences similar to polytropic MLV.
    • We have been able to infect LNCaP cells with patient plasma and get gag sequences after four or six transfers.
    • Our virus sequences are highly similar to those reported in the PNAS paper by Lo et al.
    Thank you.
    DR. HOLLINGER: Thank, Dr. Hanson.

    [ My Summary: Forthcoming publication: Blinded study of 10 severe CFS, 10 recovered CFS, 20 healthy controls from the same area. Testing for MLVs and XMRV by multiple methods. Early results released so far: Severe and recovered were both +ve 7/10 (70%), Healthy were +ve 4/20 (20%) and each positive was positive on two different tests. Control figure is likely high because controls were drawn from the outbreak area.]

    Questions for Dr. Hanson from the committee? Dr. Coffin?
    DR. COFFIN: You do not have a virus that goes with these sequences at this point. Is that correct?
    DR. HANSON: We only have gag sequences at this point. We don't yet have the virus.

    DR. COFFIN: I will ask another one of the same questions I asked him. The sequences that you report, are those based on bulk sequencing of bands or are they based on cloning or limiting dilution of PCR?
    DR. HANSON: These are based on bulk sequencing. However, we don't merely just take the sequence that the facility gives you. In addition to getting the actual text sequence, we also get the actual traces. We know from our other projects in the lab -- my lab works on RNA editing as well -- that if there is about 15 percent of a different sequence, we can see it just in the bulk sequences. So in some of our bulk sequences, we can see some single-nucleotide polymorphisms. Sometimes we can see a G in the provirus and see a G and an A in the cDNA. We can see that. I think if we had more than 15 percent of a different sequence, we would actually be able to see it in the bulk sequences.
    DR. COFFIN: But you do see some.
    DR. HANSON: We do see some, but single-nucleotide polymorphisms, not --

    We are at the limits of detection. We will have a sample positive one time and then negative another and then positive the third time. The PCR is really tricky. As I said, sometimes when we got inconsistent results, we discovered that one person was using one PCR machine and another person was using the other. So you really are at the limits of detection with the PCR. That's why we want to get at least two positive results before we count a sample as positive.
    DR. HOLLINGER: So basically they were tested multiple times?
    DR. HANSON: Yes.
    DR. HOLLINGER: Dr. Nelson?
    DR. NELSON: I was just thinking of the causative criteria, the Koch hypothesis, et cetera. You had a group who had severe disease and another one who had recovered and then another one that was healthy. I guess this is totally qualitative data that you have. But I would just wonder, is there any way to quantitate whether or not the signal you get is different. Does that correlate with the then, obviously, the other criterion is temporality. In order to establish a cause, the exposure has to come before the outcome. I just wonder if there is any way to get at that, in terms of the age of the patients or when symptoms occurred or the age of the controls versus the cases. Can you see any way to look at these two criteria namely, temporality, which I think is the most important, and a quantitative relationship?
    DR. HANSON: First of all, as far as characterizing the patients, we were quantitative in giving them survey instruments, so we do know their health status. Many of these patients have
    DR. NELSON: But what about the virus?
    DR. HANSON: Many of these patients have been ill for 25 years. Many of even the recovered people got sick in that 1984-to-1987 outbreak and then they recovered, most of them five or six years later. As far as the quantitative PCR assay, we are not there yet. I know there are groups, represented here, trying to develop a quantitative PCR assay. We don't have a quantitative assay for the virus at this point.
    DR. KLIMAS: Congratulations on such a nice study. It's really well done.
    Your question is a very good one. The idea of using this epidemic group and comparing it to a cross-section of everything else that is out there would be very interesting. I think that would be good. I think it's very interesting that these so-called recovered patients are equal to the ill patients in the numbers you are calling positive.
    At the XMRV meeting, I came away thinking that the LNCaP cell line, the prostate cancer cell line, wasn't as good for MLV as it was for XMRV. Did I hear that wrong?
    DR. HANSON: That is the conventional wisdom. We have actually never seen what is called classic XMRV. We have only gotten these polytropic-like sequences from the LNCaP cells. I can't explain that at this point, but, in fact, what we are seeing is polytropic-like virus in the LNCaP cells.

    Let me also say one thing about epidemic versus non-epidemic. Not all of these individuals were people from the outbreak. There were people who you would call sporadic cases that appeared since then. There are some more recent people mixed in with the severe and the recovered. It wasn't just the 1985 outbreak.
    DR. KLIMAS: Did they scatter into the positives?
    DR. HANSON: With the small numbers that we have, I don't think we would have any statistical power to tell you that.
    DR. HOLLINGER: Thank you very much.
  11. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    ...and Dr Mikovits on the UK Study...

    Agenda Item: U.K. study

    We'll move to the third talk in this section. It's a U.K. study. Dr. Mikovits will present this.
    DR. MIKOVITS: Thank you. Today I have been asked to talk about a study we presented at the XMRV workshop for a group of patients in the U.K. Since the first isolation of XMRV from the blood of CFS patients, my collaborators and I at the NCI, SAIC, and Whittemore Peterson Institute have been working to develop more sensitive assays for detection of infectious virus. The rationale for these studies is that in our work over the past year we have developed more sensitive methods for both the biological and molecular amplification of human MLV-related viruses, which in this talk will be called HMRV, in the blood and plasma. We have developed these technologies.

    These methods were used to determine the incidence of HMRVs in a U.K. cohort of ME/CFS. Importantly, this cohort was diagnosed using the more rigorous Canadian Consensus Criteria. Those are the criteria that Tony Komaroff and the study you heard from Dr. Lo, David Bell, as well as Dan Peterson from the original Science paper, used throughout their patient populations. It's important, because of the heterogeneity of the disease that we heard from Dr. Hewlett earlier, that the cases were similar.

    Let's talk a little bit more about the study cohort we used from the London area of the U.K. They had gotten diagnosis of what they call myalgic encephalomyelitis. Or, often, post-viral fatigue is how it's diagnosed in the U.K. primarily. All of these patients do meet the Canadian Consensus Criteria of CFS that are used in those studies that we talked about today and in our original study and throughout the WPI. The disease duration in this patient population was 9 to 26 years, with greater than 50 percent of the patients actually housebound, and many bedbound.

    The onset of disease could often occur in childhood or puberty. We won't go into the possible reasons for that today. In addition to that profound post-malaise/fatigue that is really the sine qua non diagnosis of CFS that meets the Canadian Consensus Criteria, the other symptoms included severe cognitive dysfunction, multi-joint pain, the onsets of new and frequent migraine headaches, vertigo, dizziness, lymphadenopathy, profound mitochondrial dysfunction, which might explain the energy. Many of these patients have GI disturbance and dysbiosis, an inability to absorb nutrients, and medications as well. They have chronic infections. As we have often heard, well, those CFS patients have everything. Yes, they do, and that's the point. A healthy immune system doesn't have chronic EBV or chronic HHV-6. We see shingles in a 30-year-old often in these patient populations.

    Importantly, many report a flu-like onset. They knew the day they got sick, and didn't recover. The current age of the study participants that were used was 19 to 70. Interestingly, in the 50 that were done randomly in this first pilot study, equal numbers were male and female. The study design we used is similar to that which we used in other studies done at the WPI. We have the blood drawn by Phlebotomy Services International, which is an independent certified phlebotomist group that goes around the world. PSI codes and ships those samples. In this case they were shipped to the NCI, where they were processed in a laboratory that had no previous XMRV work nor any previous murine research -- a human lab that had not done XMRV research previously. The plasma and the PBMCs were isolated two days after the blood collection. That was largely a matter of the shipping of the samples from the U.K. All the samples were tested in two independent labs, blinded. We blinded in 50 healthy controls taken from blood donors by our collaborator, Jonathan Kerr in London, in the mid-2000s, 2005 to 2008. We didn't have fresh draws from those blood donors, but these were blinded into the study, as those were the controls that we had available.

    [This distinction between the control group and the patient group negates the study's capability to conclusively rule out contamination - but the above does show that any such contamination would have to be systematic in PSI's blood collection process: that's literally the only link in the chain that could explain these results by contamination. This further evidence confirms plenty of previous evidence (eg. the successful detection of WPI +ves by Blomberg using tests that failed to find XMRV in his own samples) that says that any contamination would have to be taking place during the blood collection or storage process, and the strong results under blinding in this study confirm that it can't be during the subsequent handling or preparation of the samples. How on earth the supposed contamination of the collected blood is supposed to systematically (via PSI's processes in both UK and US) introduce both XMRV virus, and suitable antibodies, I don't know...]

    The samples were tested for the four methods. I'll go through it very carefully:

    • For plasma XMRV RNA.
    • For cell-free transmission from the plasma to the LNCaP cells, which we have heard about earlier. I'll describe that assay in detail.
    • We looked for plasma antibodies to HMRV viral proteins.
    • We Western-confirmed the positive cases from those transmission studies. Finally, we did sequence characterization of the HMRV isolates.
    First, we'll talk about the plasma PCR. We had never before done direct plasma PCR. We had been working with the Blood Working Group and thought that perhaps delayed processing, which had been done in other studies, might increase our ability to see viral RNA which may have been associated with other blood components and actually released into the plasma. When we did this plasma RNA from 140 L of the plasma from 48 percent or 24 percent of the patients, we could see in this top, using the Lombardi nested primers and conditions, a very strong band for the gag. These were all sequence-confirmed to be gag of XMRV, but this amplicon wouldn't distinguish the polytropic sequences. It was very small.

    Secondly, we used the Lo primers, went back to these samples that had not been previously frozen and thawed, because we aliquot them into .5 mL aliquots. You can see that two patients who were negative using our PCR were positive using the PCR protocol of Dr. Lo, suggesting that that also sequenced. But we didn't see it as polytropic. Maybe that's just our phylogenetic analysis. But all were confirmed by sequences and again highlight that subtle differences in PCR protocols can give you really big differences in results, as you would have found far fewer of these patients positive by the Lo protocol. Importantly -- and, unfortunately, it doesn't show well here -- we used 5 femtograms of DNA from the murine cell line that we have in the lab. We could see no mitochondria-specific amplicon, as Dr. Lo described.

    The other thing that we do in all of our studies you see here number 2767 -- those are patients from the original WPI Science study, where we consistently and over time -- over three years' time -- can both detect plasma viremia and isolate from that patient. We carry this sample throughout these studies. We do that with several samples in every study. The control samples, as I mentioned, that we blinded in from Dr. Kerr -- you can see that very few actually had XMRV or HMRV RNA in the plasma. But importantly, two out of 50 that were reportedly from the healthy blood donors -- was 4 percent of the population there in the U.K. I do want to remark that of the two negative studies that had come out at this time from the U.K., they had absolutely zero incidence in controls or patient population of XMRV.

    Since we are not a PCR lab and neither we nor the Science paper nor our work focuses on PCR, we went to doing the culture techniques that you have heard about today. I'll describe them in a bit more detail to show the isolation and characterization -- that these were indeed representing infectious virus. In the assay that we used in the Lombardi study, shown in the top line here, we take plasma or activated -- this is dividing peripheral blood and mononuclear cells -- from the patients, and we co-culture them on the prostate cancer cell line, which was responsive to androgens and inflammatory cytokines. This is important because we know we have characterized the LTR, in Steve Goff's lab and Bob Silverman's lab, and we know that there are hormone-responsive elements there that would be an on switch to make the virus replicate more in the cells that were responsive to androgens.

    We culture these for 21 to 42 days. Dr. Hanson mentioned four passages. This is a lot of cultures. Carefully looking at other negative studies, they might culture them for a week. We follow them, in addition, to PCR by Western blots from antibodies.

    These are monoclonal antibodies which were described in the original Science paper. This rat monoclonal to the envelope of this spleen focus-forming virus, which is a polytropic, xenotropic virus -- importantly, this antibody was characterized by Sandy Ruscetti all the way back in 1982.
    But this surface unit I show you here in the Western for the transmission of three of these U.K. patients -- this antibody recognizes all polytropic, xenotropic, and ecotropic viruses. This antibody -- and that may be why our numbers were so high, because our original paper didn't originally rely on just the PCR, when, in fact, this antibody could detect all of the viruses. I'll refer you to that paper. If you look at Figure 1, you will see PCR-negative patients who clearly we could culture virus in, detect it from the antibody, and sequence whole virus.

    This is the assay, which is quite labor-intensive and cumbersome. You know that it took us quite a while to do these 50-odd samples and 50 controls. We have been developing -- and you heard this from Dr. Le Grice at the last BPAC meeting in July, so I won't go into detail -- an assay in which an MLV vector has an inactivated green fluorescent protein in it. That vector is packaged by either XMRV or any MLV-related virus. You then infect those cells with that virus, if it's in the plasma, and in only 4 to 18 days, you can see green cells, representing infectious virus. In order for this vector to go from inactive to activated, it needs both reverse transcriptase and integrase. So it's important that this assay is an assay for infectious virus.

    We show here that you can also quantitate it by flow cytometry and clearly see and count the green cells. Hopefully, that has been speeding things up a lot. When we use it in the U.K. samples, here is a positive control. Only 11 percent are positive. But that is due to viral interference and other things about this assay. But clearly a negative and clearly a positive. Both of these samples, if you go back and look at that first figure, were plasma PCR-positive, suggesting, but not proving, that it's infectious virus. Now we can see that 78 percent, 39 out of the 50, were positive in this infectious assay. This is just showing you other numbers that were negative and positive in the same assay, as you can see here. When we confirmed all of the samples -- and I show you here only the positive -- we confirmed by Western analysis, using an anti-MVL envelope. This is a xeno, so it is not the monoclonal I showed you, and then a Gag antibody as well. You can see that we can detect both Gag proteins and envelope proteins in these Western analyses, confirming that we had, in fact, transmitted the virus from the plasma of these patients to the LNCaP -- we call these DERSE cells.

    Importantly, I show you that 2767 positive control that we carry throughout these studies. We next amplified a wider range, shown in the box on the top, of the envelope. When you look at small amounts of envelope, maybe due to the diversity that is wider than we originally anticipated -- when we actually did a PCR in the pol-pro region, extending down 600 base pairs of product into envelope, and then we sequenced -- I show you here, representative - three of these U.K. samples, we could see that they were indeed more similar to XMRV than to the polytropic viruses we have been hearing about this morning.

    Interestingly, this patient, U.K. 1023, was negative in all of the other assays, but we could actually, from the LNCaP, which was a DERSE assay -- it was only 2 percent, but we could actually see by Western that there was indeed virus there, and we could clone it out. We are doing full-length sequencing of as many of these viruses as we can from single cells at this time.

    We next talk about the serology assay in these patients. This is the assay that you heard from Maureen Hanson and that was described in detail in the Science paper. We use a cell line that expresses the murine spleen focus-forming virus envelope, that same region that I showed you. The antibody recognized all known polytropic, xenotropic, and ecotropic viruses. We take a plasma from a patient sample and incubate it with the non-expressing cell line. You see nothing in this histogram, which shows increasing fluorescence and density of the binding of a secondary fluorescently labeled IgG. But here, with the patient sample on the envelope-expressing cell line -- clearly suggested that there is antibody to the envelope in the patient's plasma. You can compete that using that monoclonal antibody. If we co-culture the monoclonal antibody with the patient sample, you see that you can compete either at 1-to-10 or 1-to-50, demonstrating the specificity of this antibody and that indeed the patient samples do contain antibodies to spleen focus-forming virus envelope.

    This is how the shift looks. We did this in all of the patient samples and controls. When we compare the detection of antibody reactivity with virus isolation from the plasma, you can see a concordance there in essentially all of them. There were only five samples where we could isolate virus, but could not detect presence of an antibody -- we don't know why that is -- and a few samples where we could detect antibody and not actually isolate virus.

    In summary, then:

    • We could detect gag in the RNA in the plasma in 58 percent of the 50 patients.
    • We could transmit 78 percent of the patient samples to the LNCaP cells.
    • We see antibody reactivity in 68 percent of those 50 patients.
    • We could sequence the envelope products, showing that the predominant HMRV in this U.K. cohort is indeed XMRV.
    We conclude that multiple methods are necessary to detect evidence of XMRV infection. In this case, in a very well-defined cohort, similar to the positive studies, we could detect it in greater than 70 percent.

    With that, I'll thank my collaborators and funding and you for your attention. DR. HOLLINGER: Thank you.
    Questions?
    DR. COFFIN: The virus is growing out in the DERSE cells. How much of that have you sequenced?
    DR. MIKOVITS: We have sequenced large parts of the envelope and the gag. We --
    DR. COFFIN: I'm just curious, because one expectation in those cells is that what might happen -- the virus that grows out may not actually be the virus that you originally started with.
    DR. MIKOVITS: That's why we --
    DR. COFFIN: It could be a recombinant that has picked up useful sequences from the vector, like the LTR. It would be nice to see if that's happening.
    DR. MIKOVITS: Indeed, and that is why we also run those LNCaPs without that vector. We have been sequencing the virus out of the LNCaPs where I showed you the Westerns. We run both assays, because we recognize that that might happen. It would be interesting if it did indeed happen.
    DR. RUSCETTI: Can I follow up on the Coffin question?
    DR. HOLLINGER: Could you give your name?
    DR. RUSCETTI: Ruscetti, NCI.
    We have done LNCaP and the DERSE cell on several isolates and found no difference, at least in the envelope and gag regions.
    DR. COFFIN: I was thinking particularly about the LTR, which might well have been exchanged.
    DR. RUSCETTI: We are just beginning to work to look at the LTR. We have been pushed into it by Jonathan, who asked us at every meeting to look at it. So we are now looking at it. But we don't have any results for it.
    DR. MIKOVITS: And it could well be a key to the reservoir. The LTR is really a key, maybe, why we can't grow this virus in these cells. We are looking at other cell types right now.
    DR. COFFIN: That harks back, in a sense, to the other talks. To my knowledge, nobody has ever grown a virus that has a polytropic or a modified polytropic-type LTR in it, unless somebody in the room has done that recently and I haven't heard about it.
    DR. MIKOVITS: Usually these are seen with xenotropic, actually mobilizing the polytropic. The polytropic indeed becomes the pathogenesis. But they need the xenotropic to be --
    DR. COFFIN: In mice that seems to be the case.


    [An interesting little exchange...xenotropic mobilising the polytopic...well who'd have thunk it?!...wish Dr Coffin hadn't interrupted there...]

    DR. NELSON: These are pretty convincing data, to me, that this infection is real. You showed antibodies. One issue that remains is, is this the cause or is this the result? It's quite possible that they have something else - the immune system crashes or whatever -- and they get infected with whatever is around, a mouse or whatever. Have these patients been studied for other chronic viral infections, HHV-6, HTLV-2, or EBV? Have these patients had an extensive virologic/infectious disease workup?

    DR. MIKOVITS: These patients in the U.K. have not. It is a psychosomatic disease in the U.K., and they can't get those types of medical treatments easily and maintain their benefits. In our study in Science, the answer is yes. These patients have multiple chronic active infections -- EBV, HHV-6, CMV, as I mentioned, shingles. We see everything -- mycoplasma. It looks to us like an AIDS patient, with an obvious hypothesis being that the retrovirus causes the underlying immune deficiency. But it alone can't cause the disease. It needs the co-pathogens. You can have HIV without having AIDS, but you can't AIDS without having HIV and one of 25-odd co-pathogens.

    DR. NELSON: So it looks like the next step is to there are several repositories, and this disease may be frequent enough that one could identify an infection with this agent. You have 4 percent of blood donors or whatever. These people need to be followed. These people need to be followed to answer the question: Does this infection occur before the chronic fatigue syndrome or afterwards? If it occurs afterwards, then it's just a passenger; it isn't the case. I think that's a critical question.

    There are some repositories, the NHANES, Washington County, Maryland, where there are large numbers of samples that are stored and frozen, to go back and look at incident disease after that. It seems like this should be of some priority at this point.
    DR. MIKOVITS: It is a critical question, but we can't do it with the existing assays. Part of the reason we were developing these, hopefully, more high-throughput assays is so that we could do the large-scale epidemiological studies necessary. I agree completely.
    DR. HOLLINGER: Dr. Coffin, one more.
    DR. COFFIN: In that same vein, it's also not out of the question that this virus infects very large numbers of people and remains in some very-difficult-to-detect form, as it does, apparently, in macaques after they are infected, as we'll see, and that there is something about a condition like chronic fatigue [sic] that allows it to appear and replicate in that fraction of people, despite the fact that it's in almost everybody to begin with, or in a very large fraction of people to begin with

    DR. MIKOVITS: If it were, we might find it a little more easily than we do.
    DR. COFFIN: We might, but it's not so easy to find Epstein-Barr virus if you don't have an antibody response either in infected people. It's very, very infrequent in latent infection, but it's there. It might be that there is -- I think it's a remote possibility, but a possibility that in some people there is some hidden reservoir somewhere that only becomes visible due to some condition associated -- immune deficiency or whatever -- associated with chronic fatigue [sic].
    DR. HOLLINGER: Thank you, Dr. Mikovits.
    Now we'll go back to the one we were going to start with. Dr. Jonathan Stoye is going to talk to us about a "Summary of the Current Research on MLV-Related Human Retroviruses and Disease Association."
  12. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    I would do the same for Dr Villinger's macaque study if I had time, there is loads of important stuff in there, but for now this quote:

    Throughout, it's been striking that the observations in the animal models have supported the emergent hypothesis of XMRV's role in CFS aetiology. The observed T cell activation is as expected. Next we would expect that further exposure to further pathogens - before, during or after XMRV exposure - may turn the poor monkeys' relatively benign experiences into a condition where multiple infections or exposures become permanent immune vulnerabilities, as they give XMRV further opportunities to aid in the subversion of further specialised T cells.

    The key unanswered question in this model, I think, is: do these co-infections, or co-vulnerabilities, tend to happen before, during, or after XMRV infection? Could be some or all of those possibilities.

    They may also need some monkeys with the right vulnerabilities in APOBEC3-related genes to see ME/CFS symptoms...but if they do go on to find a way to induce the virus to replicate chronically - and this is a group that seems to get results - then that would be a huge step towards understanding the key mechanisms of XAND.
  13. Riley

    Riley

    Messages:
    48
    Likes:
    0
    Louisiana
    I don't know if this is the right thread for this, but how do the monkey studies affect the contamination arguments? If they have infected monkeys with xmrv doesn't this prove that it is an infectious pathogen and not a contaminant?
  14. Esther12

    Esther12 Senior Member

    Messages:
    5,156
    Likes:
    5,081
    This is something I don't get.

    On one hand there seems to be all manner of work progressing on studying the virus, it's behaviour, animal models...

    on the other hand you've got a lot of good scientists arguing that it doesn't exist, and we're just picking up sequences due to contamination.

    I feel like I'm trying to combine two different realities in my head at the same time when I consider it all. Up until the BWG results I thought it should be easy to settle these basic issues... but it's still ambiguous, and looks like it might stay that was for some time to come.

    @ Mark and others: Thanks for pulling out and highlighting bits. Even of the stuff I've read there are bits I missed the significance of.
  15. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    Yes Riley, and I believe that was already established by the WPI in their original paper, in that by showing it infecting human cell lines and growing/culturing it in those cells, together with the rest of their results, they confirmed it as a human-infectious retrovirus. The monkey studies confirm that it can establish an infection in monkeys, and interestingly show at least 3 patterns of immune response, but with very low levels in the blood, but high levels in tissues like prostate, fairly soon after initial infection. The monkeys immune systems seem to deal with the XMRV, but it establishes a persistent infection in loads of tissues. Notably XMRV infected T4 and T8 cells which are already of interest in CFS as a model of the 'immune abnormalities' many believe lie at the heart of the condition.

    What proves anything is not a contaminant, heck knows! It's not in reasonable doubt that this is an infectious retrovirus, that it can infect human cells, and that it can establish a persistent infection in monkeys. Even if it turned out to be a common lab contaminant, it's a common lab contaminant that keeps showing up more consistently in patients with ME/CFS than in healthy controls - blinded - and a common lab contaminant that happens to be a retrovirus that infects humans...(!)
  16. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    esther: I know what you mean about the two realities: one being a world where increasing levels of exciting detail are fleshed out and the pathogenesis of XMRV is explored - but not published - and another being a world of shock blogs, sceptical journals and Science Media Centres where the general public is successfully distracted with arguments about contamination. There's just so much good work going on, and so much promise, and clearly so much that has been done but not yet published, that you just have to think that this is a case of drawing a line between the scientific investigation, and the public view of it...in the science I have the feeling that XMRV, and the truth of it, is inevitably winning out, but on the media front there's this strange wall of silence and this phony debate which omits all the key facts and all the key players...it sure is surreal...
  17. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    This was encouraging too: early results from Bagni's antibody work as they begin refining and calibrating an antibody test - already they can begin to distinguish WPI XMRV+ samples from controls using their antibody screen...25% vs <2%:

    "If we look at the training set, understanding that this data is taken directly from the training set -- so it's not blinded and we are just classifying how well we did -- for the subjects, of which we had 39, we see that 10 of these actually have reactivity above background for capsid, transmembrane, and surface unit. Then if we look at the donors, there is actually one out of the 77."
  18. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    Finally (think this is the last piece from my trawl through this), I liked this quote from John Coffin - an alternative explanation for the narrow sequence variability found by Hue et al, which seems reasonably consistent with known ME/CFS epidemiology, and a typically fair-minded look at alternative possibilities that might explain a given set of observations...I know he can be annoying lately in his role as a sceptic, but I do generally get the impression he is just trying honestly to cover all the bases, which is of course just what he should do as a good scientist:

  19. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    Dr Hackett of Abbot Laboratories:

  20. Mark

    Mark Acting CEO

    Messages:
    4,504
    Likes:
    1,926
    Sofa, UK
    I suspect Dr Alter's fabulous speech has already been posted in full elsewhere, but it can't hurt to reproduce it again since I've now reached the end of that massive document - what a brilliant statement this is...and very quotable indeed...

    I really am finished with this document now!

See more popular forum discussions.

Share This Page