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Transcription of Judy Mikovits Prohealth Lecture

Sing

Senior Member
Messages
1,782
Location
New England
Thank you from me too, Rachel! Transcription is so important for those of us who missed the presentation, or can't remember or have trouble processing that form of communication, for one reason or another. I am glad we are helping each other maintain A GRIP!

Sing
 
T

thefreeprisoner

Guest
Yeah I almost didn't believe 130wpm myself, but that's what it said on the typing test I took on the internet. (Wish I was up to stenographer speed of 180wpm but hey ho... I am clearly pushing the boundaries already.)

I guess it could have been a bit off-beam. Or maybe in the survey quoted, they measured people's typing speed when they're composing emails rather than typing a set text or something.

Anyway, whatever... that's what 20 years of geekery and smaller-than average hands will do for you.
Please, nobody tell me to get out more ;)

Rachel xx
 
K

_Kim_

Guest
The complete lecture has been posted on ProHealth.

We need some volunteers to transcribe the lecture in 15 minute blocks, picking up where Rachel left off (was that at 27 minutes into it?).

Video 1 (81:50)
Section 1 - complete by Rachel (up to 27 mins....or thereabout)
Section 2 - wherever Rachel left off - up to 40 mins (Kim is transcribing)
Section 3 - 40 mins to 55 mins
Section 4 - 55 mins to 70 mins
Section 5 - 70 mins to the end of video #1

Video #2 (54:21)
Section 6 - start to 15 mins
Section 7 - 15 mins to 30 mins
Section 8 - 30 mins to 45 mins
Section 9 - 45 mins to end of video #2
 
K

_Kim_

Guest
Judy Mikovits
Section 2
(Video #1: 27+ mins to 40:08)

On some of these patients we looked three and four times for the DNA in the unstimulated cells. So this is just that pellet that I made when I sorted all the various samples. I just held one as white cells so that I could make DNA later or RNA later, depending on the technique I wanted to use downstream. So, it’s important that this was in 68 out of 101 samples. It was 68 out of 101 patients and it clearly says that in the paper. So, at any given time, depending on the viral life-cycle, we might not find this virus in the unstimulated group (inaudible). And I give you the example of that is: follow this patient 1118 throughout the talk and you’ll see that this patient, if you only use sequences, would have been called ‘negative’. So, we were concerned because PCR is a technique that is fraught with contamination. If you’re looking for a needle in a haystack, just a few sequences in a million bases, you might make an error in your enzyme and it might put the wrong base in there.

So that…Jaydip Das Gupta in Bob Silverman’s lab, cloned and sequenced three of these patients – and that’s shown here – and what it’s intended to show is: If you compare the isolates that they had from the 3 prostate cancer cases, where they had actually cloned these, you can see, if you compare it to the reference strain, known as VP62, that’s the reference strain of what this virus looks like, the CFS samples here were clearly different, but they were highly similar - 99.7% - there were maybe 8 bases different across the entire 8,000 base pairs. So, this virus isn’t like HIV theoretically. It’s not changing. We don’t find quasi-species in patients when there are lots of different viruses, because HIV mutates so much. Therapeutically, that’s something that we can take advantage of and suggest that it might be easier to develop therapies because the virus is going to be largely the same.

So, Rachel Vagny, my former student at the National Cancer Institute – I asked her if she could construct what is called a phylogenetic tree of this virus so we could understand where it came from (hopefully). And so that’s shown on the next slide. And what a phylogenetic tree is - is you take all of the sequences of all the Murine Leukemia viruses - they’re called Ecotropic viruses – all the families of virus that they’ve ever identified, Mason-Pfizer Monkey virus, all the sequences, and you put them into the computer, and then you put into the computer at the same time the sequences of our 6 isolates – the 3 prostate cancer and the 3 CFS isolates that we had at that time. And you do what’s called ‘blasting’. You ask the computer to find similarities. And when it doesn’t find similarities, you get what’s called a new branch on the tree. So, clearly, these diverge here, and we don’t know when that is in time, but these data suggest that the prostate cancer – that XMRV both in prostate cancer and in CFS – form a new distinct branch. That it’s a new human retrovirus. It doesn’t have any of the sequences of mouse in it. And when we blasted it, also we did the same thing against the human genome - because I told you, we have a lot of endogenous viruses that don’t actually come out of our bodies as infectious particles – we blasted it against the human genome and found that it did not match any sequence in the human genome. So, it’s clearly a foreign, exogenous virus that can now, theoretically, be infectious. And that’s what we’ll show in the next slide.

So, here are our sequences. And you can see, they’re clearly not contaminants. We didn’t have this – we weren’t working with this in the lab, actually, at the time. But we didn’t have this, and maybe spread it through the sample in any way. It was there – clearly different isolates. We now have more than 170 isolates, because we isolate from every single patient in all of our studies. And we’re actively looking for funds and going to sequence those viruses because it might give us clues as to some of the differences in what we see, maybe something, you know the various symptoms, because CFS is quite a heterogeneous disease.

So, at any rate, we next went to – I’ll summarize that – So in summary, what is XMRV then? These data suggest, at this point in time, we have sequences related to XMRV that were not found in any mouse strain. So, it’s a new human retrovirus. The origin of XMRV remains unknown. We don’t know how it got into the human species. We don’t know how long it’s been – 40 years is the guess of John Coffin, who is a mouse retrovirologist working on these families of viruses for more than 40 or so years. And that XMRV is not a mouse virus – clearly from these data. So it’s a new human retrovirus.

So we next asked: Could we find those proteins I mentioned? So we took advantage of.. Sandy Ruscetti, Frank Ruscetti’s wife had been in retrovirology as long as he has, but because they didn’t want to work on the same thing, men usually get the credit for what women do, so Sandy worked on mouse viruses and Frank worked on human viruses and I don’t think they actually ever published together. But we were thinking about it and saying: None of the reagents that were out in the world, so far nobody had found viral proteins from XMRV, even though it had been discovered 2 or so years earlier. In January we started looking. So Sandy had saved a box of antibodies – this is really a tribute to the value of your tax dollars going to basic research – because they created this mouse retrovirology program and put a lot of money into trying to understand – if you can understand how viruses cause cancer in mice, you might understand how it causes cancer in humans. And this was in the late ‘70s and early ‘80s. And somewhere in the early 2000s, they were going to throw out all of these reagents that they developed and Sandy said, “No, I’ll keep them in my freezer.” Frank always says that the reason they’re still married is because Sandy never throws out anything. So, at any rate, she gave us these viruses, I mean these antibodies, and we screened our samples there for protein in our samples. So, we looked at the activated peripheral blood mononuclear cells. And what we do is, we stimulate these to divide, and add T-cell growth factor, or now known as IL2, which was actually the discovery that Frank made that preceded the identification of the first human retroviruses. Retroviruses grow and divide in cells, so you have to divide the cells in order to get the virus to replicate to levels that you can see with the technology of the time. And that’s important in this study too.

So, what we’ve got here is we looked a number of her antibodies – these are all family members of the virus – this particular antibody which you’ll hear a lot about is a spleen focus forming virus. It’s a mouse virus that causes various diseases including a neurological disease and erythroleukemia – red blood cell leukemia. So, its envelope is both a neurotoxin and an oncogene. It causes cancer and causes toxicity. So this virus itself – she had this antibody that was highly specific. It recognizes all known polytropic and xenotropic viruses. We hypothesized that it would recognize this virus and clearly high levels in some patient’s cells, but not in others. Interestingly enough, if you look, and use a panel of antibodies, this is a gag antibody to a gag protein I showed you there that structural gene and this virus, this antibody is a polyphone virus that recognizes the entire MULV. And you can see when you use a panel of antibodies to the viruses, essentially everyone, 68% now of 50 people we tried just one time, you could see their proliferating blood cells. You can see evidence of viral proteins.

So we next asked if we could see this in normal cells, because of course you want to make sure that it’s not in normal people. And you can see clearly here in the 24 normal donors (now up to 60 or 70 that Frank’s done) at the NIH clinical center where they have a good donor program – they’re all negative. So, these proteins, these viral proteins are expressed specifically in the CFS patients and not in normal donors.

So we next asked if we could transmit that. Is there any evidence that it’s an infectious virus? So the first thing we did was we took plasma – so that’s the plasma, the liquid off the white blood cells there – and we took their plasma and [this becomes essentially the key to the whole study] we co-cultured it. We simply put it in a flask with the cells known as LNCAP and that comes from lymph node-cancer-prostate. So this came from a lymph node of a 62 year old man who had metastatic advanced prostate cancer. And these cells grew by themselves in the laboratory so that you could use them as a tool for studying prostate cancer. And, in one of my lives, I developed prostate cancer drugs, because, when my stepfather got ill, I became interested in prostate cancer and had been working on this. So, I knew LNCAP was also deficient in RNase L, and the type one interferon pathway. It had no interferon response. So, we always look for biological multiplication of the virus instead of the multiplication you would use with PCR. So, actually replicate the virus or multiply the virus in cells. You have to find a cell that will grow a lot of virus so that you can study it. So we took that plasma from all of these patients you see high levels – now 84% of the plasmas contain infectious virus that we could not see. I sent all of these plasmas to Bob Silverman and he said, “Sorry Judy, I don’t see the RNA of the virus” there when he looked for the two copies of RNA in the particles which suggested there were very few copies of actual particles of virus in these cells. But again, we could transmit it.

And the next question we asked is: Is this a whole virus? Is this an infectious virus? Kun…Shima, my friend at the NCI who is an expert in Electron Microscopy, did this electron micrograph for me, and what you can see here is the budding of a virus from the cell. It shows you again that it’s not a contamination, it’s actually a transmission, because you’ve got a budding particle. And that particle is called a C-type retrovirus, because in the old days, when we used the word, they called them ‘C’ but they changed the name to gamma, but we’re old-fashioned, so we keep the ‘C’ type. [Ends at 40:08 in video #1]
 
K

_Kim_

Guest
Thanks froufox, I'll add you to the list:

The complete lecture has been posted on ProHealth.

We need some volunteers to transcribe the lecture in 15 minute blocks, picking up where Rachel left off (was that at 27 minutes into it?).

Video 1 (81:50)
Section 1 - completed by Rachel (up to 27 mins....or thereabout)
Section 2 - wherever Rachel left off - up to 40 mins (Kim - completed)
Section 3 - 40 mins to 55 mins (froufox is transcribing)
Section 4 - 55 mins to 70 mins (sproggle is transcribing)
Section 5 - 70 mins to the end of video #1

Video #2 (54:21)
Section 6 - start to 15 mins
Section 7 - 15 mins to 30 mins
Section 8 - 30 mins to 45 mins
Section 9 - 45 mins to end of video #2
 
K

_Kim_

Guest
Garcia beat me to Section 5.

I'll take Section 6.

Okay. This is great!! I'll update the list.

The complete lecture has been posted on ProHealth.

We need some volunteers to transcribe the lecture in 15 minute blocks, picking up where Rachel left off (was that at 27 minutes into it?).

Video 1 (81:50)
Section 1 - completed by Rachel (up to 27 mins....or thereabout)
Section 2 - wherever Rachel left off - up to 40 mins (Kim - completed)
Section 3 - 40 mins to 55 mins (froufox is transcribing)
Section 4 - 55 mins to 70 mins (sproggle is transcribing)
Section 5 - 70 mins to the end of video #1 (garcia is transcribing)

Video #2 (54:21)
Section 6 - start to 15 mins (Advocate is transcribing)
Section 7 - 15 mins to 30 mins
Section 8 - 30 mins to 45 mins
Section 9 - 45 mins to end of video #2
 

froufox

Senior Member
Messages
440
Judy Mikovits talk Section 3 (Video #1: 40.08 to mins to 55.40)

Section 3 (Video #1: 40.08 to mins to 55.40)


And what you can see here, characteristic of a gammaretrovirus, you can see this budding - remember I showed you it takes the cholesterol and buds itself out of the cell to form the outer membrane. And heres that capsid that encloses where the viral RNA is, to protect it. So you can see both immature particles and many mature particles in those LNCaP that have just been exposed to patients' plasma, showing there is infectious virus there. So the next thing...so we were pretty happy with this and we sent it off to Science in early May of last year, and they came back to us and they said, "We're 95% convinced, but show us an immune response...if this really is an infectious, non-self virus, not an endogenous virus, your body will make an immune response."

So again we went to Sandy Ruscetti and um this part was funny too because we were struggling to do this, because you don't want a whole virus infected cell, you need to have just a part of the virus in order to get the noise out of there. And what Sandy had developed when she was studying the spleen focus-forming virus was this antibody again to the envelope protein. And she expressed it on the cell lines - used two cell lines. This is a mouse b-cell line that expresses the erythropoeitin receptor (its just for red blood cells), and when she co-expressed the envelope, you see high levels of the envelope on the surface of these cells. So we took these cells and put them in whats called a flow cytometer where a laser will see the fluorescently tagged antibody on the surface of the cell and count the infected cell as it runs through the instrument, the channel and single cell. So you can see that the cell line went out the envelope protein being expressed, you see the white and the black are superimposed showing that theres nothing reacting specifically with that. If you then take that antibody I showed you, to the envelope, its called 7C10, and expose the cells to it, they all light up, virtually 100% of these cells have the antibodies that are recognising the cells with the envelope protein. If we then take a patient sample and do exactly the same thing, you see there theres an antibody, this is for patient no 1104, thats one of the sequences we have, and there it is, theres the immune response in the plasma showing now we have an infectious virus with particles that can exogenously infect and is non-self.

So, the next step in what happened in the literature is work in prostate cancer again. So this comes from the lab of Ila Singh, whos an MD PhD at Utah, and she was looking at XMRV in malignant prostate cancer tissue in the tumour cells. One of the other reasons why the oncologists in the cancer community weren't excited about Bob's discovery of XMRV sequences was because when they looked at those, they only found them in the infiltrating stromal cells - the microenvironment. But those of us who think a little deeper than most oncologists about cancer, know that 50% of all tumours are actually your immune system, your white blood cells going in to try and clear the cancer because thats their job is to recognise tumour cells. So we werent concerend, we were excited that it was, and it made sense to us that it wasn't the tumour cell itself harbouring the virus, but the immune cells that were inside the tumour.

But Ila showed that XMRV WAS present in the malignant tumour cells and that it was associated with that high grade tumour, that tumour that my stepfather died of, that you get younger and they get really sick really fast. And what was different in the advance in her study is she developed an antibody specifically to XMRV, to the whole virus, another polychromal antibody. And she showed that she could recognise with that antibody, in whats called Immunohistochemistry when you send a biopsy to the lab, they look at it, at a tissue block. So she did that and she showed that 23% of the prostate cancer tissues she looked at had a protein to XMRV, a lot like our study but she saw a lot less DNA sequences than she saw proteins. So this paper came out about a month before our paper but we knew about it from about mid summer when we first met.

So again in her study, the limitation in her study, was that again that there is no evidence of the infectious virus that I just showed you. So we had evidence of infectious virus in CFS...can we see evidence of infectious virus in prostate cancer? So Frank did this, this is again that antibody, looking for the antibody in the patients. And here he used, this is called a prostatic secretion, so they're just looking at the prostatic secretion and when they had a person who had sequences of the virus, positive in the prostatic secretion, you can see there that there are antibodies in that patient, so that patient is infected. In an XMRV PCR negative patient we don't see antibodies, so that person is unlikely to be infected with XMRV. And again in the plasma of this integration here, so that now they have actually found in this patient exactly where the virus integrated into the cell, and that patient has a significant amount of antibody. So in prostate cancer no-one had ever transmitted virus and shown that it was infectious that way. So I show you the exact same study where we took the plasma from the prostatic secretions there and found high levels of the virus when we put it on LNCaP, showing now in both prostate cancer and CFS, XMRV is an infectious virus. And in a significant portion now they are finding in prostate cancer patients.

So why bring that up today, is because if we look and we do a summary table of the technologies that I showed you that we used to find the virus, what you see is that patients here in red are clearly infected when you look at plasma antibody responses, and you look for tramsmissions through infectious particles in the plasma, you can see the red patients both in the prostate cancer and in the WPI patients. These patients were PCR negative, I bring back to you 1118, but we found plasma transmission of that virus that I didnt point out, pardon me when we passed that slide...but ALL of these samples were negative when you did the most sensitive PCR that Bob and everyone developed in unstimulated cells. So those white blood cells, fresh out of the body, not dividing...very low copy numbers of this virus, but clearly these individuals are infected.

So going back to the literature now, two studies have come out since then, and one was in October, right around the time our paper came out. And this was from a German group led by Norbert Bannert and he found a lack of evidence for the virus in over 580 prostate tumour tissues, when he used the sensitive nested GAG PCR techniques that me and Bob and everyone is using right now. And he had developed his own ELISA which is looking for an antibody in the sera - its a similar test to what I showed you for looking for antibodies to that. And he couldn't see any of the evidence of the virus in those sera, and so he concluded, and they concluded that XMRV was not in prostate cancer. And then earlier this year, a similar study came out by a group in England that showed a failure to detect XMRV in CFS. And they looked at 186 DNA samples and they did nested GAG PCR and they found nothing.

So what could be the reasons for the discrepancies in these studies and what we've shown you in the studies of Ila Singh. So first of all, the prevalence of XMRV, thats the distribution around the world, is unknown. The studies that we've shown you today is all we know about XMRV prevalence - that its in the US and in several hundred people including those with both prostate cancer and CFS. But I remind you that retroviruses are not ubiquitous, they're not everywhere. The sensitivity of the assays in these studies were not the same because both of these studies didnt rely on ???, they relied on PCR, they didnt look for infectious virus. Of course the Bannert group didn't know our study because they were under consideration at the same time. And then also that XMRV has an extremely low copy number that I showed you, that even if it is there, you could miss it by these sensitive techniques. And mostly importantly, and something that didn't occur to me until I saw all of this data, was that we don't know anything about the viral reservoir of XMRV. I assumed its lymphocytes because thats what I know about HIV and HTLV1. But what if the plasma virus was coming out of the tissues and then the cells that were actually in the peripheral blood were not the main reservoir of the virus? What if there is another tissue reservoir? We don't know what that is, so these are all possible explanations for why we saw it, and we see a lot of it as you see in the plasma of these people, not a lot by copy number, but certainly there is infectious virus there. So thats what we're thinking.

So if you look at data that suppoorts these arguments, what you will see is the distribution here of HTLV1. Now HTLV1 infected people are 10-20 million across the world, and I bring up this one point that HTLV1 causes a neurological disease known as HTLV1 Associated Myelopathy...they have trouble walking and balance and almost like a paralysis looking disease. And that occurs only in about 20% of the infected individuals. And then of course HTLV1 was shown to be causative, satisfied Koch's postulates as we know them for viruses - for an adult T-cell leukaemia, and this is a very aggressive leukaemia and the mechanisms for how it causes that are still largely unknown. But at any rate 10-20 million people are infected, but you see very few - only sporadic cases occur in the US or Europe and the US incidence is only about 0.2%. They dont even test for it in the blood supply because its just simply not a problem in America, its endemic in the regions that are shown here today.

And the second argument that supports maybe whats different between these studies is the transmission from the actived PBMCs .. so if I take the white blood cells, some of which where I can't see virus and just put them on LNCaP, I can transmit the virus to this indicator cell-line that has shown you because its defective in RNaseL (theoretically because its defective in those, but we learn more about it later), will amplify and replicate high levels of the virus. So there are scientific reasons why there are differences between these studies, but I dont think there is any doubt that XMRV is a new human retrovirus, and since both HIV and HTLV1 are associated with neurological diseases and cancer, and now we have associated them with a neurological disease and cancer, that this a real human pathogen.

So recent publications after those publications (I'm just walking through the literature off the last few years) might give us a clue to the pathogenesis - how XMRV might cause disease. So this paper by Steve Goff's lab shows that XMRV establishes in an efficient infection, and spreading infection, thats enhanced by transcriptional activity in prostate cancer cells. And what that means is, I told you the receptor is on every cell of the body, but clearly every cell doesnt have the machinery necessary to replicate the virus to high levels. In fact we see that the peripheral blood mononuclear cells really don't, and thats why we dont know where the tissue reservoir is. So he simply infected a lot of different cell-lines and he found that the expression was very very low level except in essentially one cell-line and thats LNCaP. So we got very very lucky in that this was the only cell-line I thought about as an indicator cell-line....we could have screened the hundreds of cell lines I know of that we do regularly when we're looking for viruses because if you can't grow it you can't study it.

So LNCaP turned out to be really serendipitous and I think the key technical advance to being able to make that discovery, its just clearly luck. He showed that LNCaP responds to androgens, I told you it lacks interferon and RNA cell anti-viral responses, and I'll show you whats called the promotor, the response elements, that might give us a clue as to the pathogenesis. And then Bob Silverman's lab showed the same thing, he showed that androgens stimulate transcription which is the replication and division of the virus. So here's a clue to the disease, because we know the only two diseases so far that are associated with this retrovirus are prostate cancer, a hormone responsive disease, and CFS, one thats thought to occur primarily in women. Interestingly that I didnt say that I knew is LNCaP is androgen responsive, so you can make it do a lot of good things and thats why we use it in drug development for prostate cancer. (ends at 55.40)
 

sproggle

Jan
Messages
235
Location
Teesside, England UK
Section 4

...the response elements that might give us a clue as to the pathogenesis and then Bob Silvermans lab showed the same thing. He showed that androgens stimulate transcription (the replication and division of the virus). So here's a clue to the disease because we know the only two diseases so far that are associated with this retrovirus are prostate cancer (a hormone responsive disease) and CFS (one that's thought to occur primarily in women).


Interestingly that I didn't say that I knew is LNCaP is androgen responsive. So you can make it do a lot of good things and that's why we use it in drug development for prostate cancer. So lets look at I showed you that organisation of the gag col and envelope of this simple retrovirus. This U3 region is highlighted because this is sort of the on/off switch of the virus. This turns it on to make more of the particle in your genome so this signals your cellular machinery to start making more virus and what Steve Goths lab showed (and he graciously gave me these slides about mid summer) was that there's only three responsive elements that turn on this virus that he can find so far. Two are called glucocorticoid response elements and their shown here. When a protein actually recognises that exact sequence and sits down it tells the virus to turn on replication. And so interestingly enough, what turns on the virus? Hormones. Progesterone, androgen receptor and testosterone and we don't know all the other hormones. There are a lot of oestrogens and oestrogen like compounds even in our environment these days which might tell us maybe there's an oestrogen compound that's not a naturally occurring oestrogens in a plastic in the environment that is actually turning on the virus.
So we don't know all of the things that turn it on at this point. And the other thing that turns it on is cortisol. So what is cortisol? It's the stress hormone and so right there your turning on the replication so it's an on/off switch for the virus with the stress response. When your told that you respond poorly to stress there might be a reason for that if your replicating a retrovirus! (laughs). Sorry I shouldn't laugh.


So then we went back into thinking about this virus, we thought about the clinical research findings that had occurred throughout laboratories around the world throughout the years. What it mentioned in part was we know that CFS is a multi system disorder (and in Spanish I say sequelae) but there's lots of inflammation going on, you have allergies, multiple chemical sensitivities there's a lot of inflammation and increased numbers of activated T cells and the production of these inflammatory molecules I mentioned known as cytokines and kinokines. Also a key dysfunction in the immune system of CFS patients is this low natural killer cell activity and sometimes numbers.
The natural killer cell has two jobs in the body, kill tumour cells and kill virus infected cells. In CFS it's long been recognised (I think first identified by Nancy Klimas and her colleagues more than 20years ago) that natural killer cells in CFS patients don't function normally although the dysfunctions not known, but that again gives us a clue to the pathogenesis. So this suggested to us that this chronic infection with a retrovirus (retroviruses are associated with immune deficiencies) might lead to the creation of actually immune deficiency that has patients succeptible to opportunistic infections and more likely to develop cancer.


So I've schematically drawn our hypothesis on the next slide and I basically just lifted the graph of what happens in HIV and changed it to what we know happens and changed it to all the data that we have so far. In HIV what happens is that there's an early infection, the green line is actually the plasma viral load and it goes up in a spike. This might be a flu like syndrome or it might be nothing at all, you might never know that you were actively infected at this point and get sick. But then you have multiple other infections, stress hormone, advance inflammatory responses that cause these various spikes of the virus throughout a time course which we don't know.


I've heard the incubation period of this virus is 21 days. We don't know anything about the incubation of this virus we've just discovered it! So at any rate, all these events operate to set the viral load higher because every time you divide a cell, that your white blood cells, the cells in your immune system and actually our paper shows its the TB and NK cells are infected. Those cells are getting infected, more and more and more of them and some of them are long live memory cells that you need or they're going to the tissue then and they're infected and they're spreading the virus to other cells and we don't know where that tissue reservoir is and as I said the receptor theoretically is on every cell. Not every cell can replicate the virus but virus can get into every cell. So it's infecting more and more NK cells as the load keeps coming up and at this point something happens to your NK cells, this envelope antigen comes to very high levels like we see in our patients plasma and white blood cells and we know that that in animal models or in animal viruses of this family is actually a noctogene and a neurotoxin. So we hypothesise that the envelope alone is creating some of the neurological sequelae and that they're different from the virus replicate. So it can be sort of the envelopes around a lot more, I showed you the defective particles we less infectious virus and more defective virus but those proteins can affect your body.


So we know your making antibodies but some of the sicker patients don't make antibodies and CFS patients are known to have problems with antibody production for whatever reason, we're not saying that's direct to the virus but you know it's not a great leap of faith because that's what we saw in the early eighties with AIDS patients we had no idea how long those men had the virus.


All of a sudden there were getting Pneumocystis and Kaposi's Sarcoma (a form of cancer that only occurs in older men in Italy) and that's because as you age your immune system loses effectiveness too. So all of a sudden we're seeing a virus that is not endemic in the United States, well actually from these patients they actually indentified HHV8 (Human Herpes Virus 8) which actually is causative for Kaposi's Sarcoma and that virus, I led a drug development program about a decade ago just before I came to California and we were going to make drugs to target Aids associated malignancies and we found as soon as we got the highly active anti-retroviral therapy and got rid of the HIV and silenced that the Kaposi's Sarcoma went away as did the HHV8 so they cut the budget for that drug program and rightfully because there's no need to develop these drugs because they learned that at that point all you have to do is control the retrovirus, get the immune system back to functioning, and also the good news is most of those men their immune systems are functioning well. You can get a lot of them back to at least a level of health even though they have to stay on various drugs the rest of their lives at least they could cure the immune deficiency.


So in summary then of the science part of the talk:


XMRV is the first simple human infectious retrovirus. It's a gamma retrovirus it's not complex so it's the first one known in this family and we know nothing about the pathogenic potential other than the two diseases that we've seen it in. We know that human retroviruses are not ubiquitous I've shown you the distribution can be quite low in various places in the world. We don't know how it spreads across continents.


They're not benign, meaning they cause disease. All three known human retroviruses are associated with the neurological diseases and cancer. And importantly they are not airborne, retroviruses are not contagious you don't get them in the air. We know that for instance with AIDS patients that it's not a problem to kiss AIDS patient and hug AIDS patients and so that knowledge is there for this virus as well. So there's three known now, the complex and now the simple and I've mentioned that a number of times.


Interestingly and something we should think about in light of the replication studies and the other studies as we're going on, I say HIV and HTLV but I've been saying one but there are variants of HIV there's a HIV2 that is less pathogenic, there's a HTLV2 that is less pathogenic in fact hardly pathogenic at all. And these are clearly different and have different pathogenic profiles and just a short extension of that suggests that there could be variants of XMRV there could be subtly different sequences of viruses out there that are associated now with different phenotypes, so the way the disease looks, and different cancers or different neurological diseases. So I know that the scientific community is actively looking for variants so that's another good news about these studies is that there are a lot of exited retro virologists and immunologists who started as soon as these learned this in July to the put the world resources and the best minds on this virus associated with CFS and that's probably the first time that's happened in the world so they're excited about that.




So a lot of the questions that I got, and I wrote this talk around the question that I got, had to do with reasons to be tested. You know we don't have the best diagnostic test yet because we still haven't validated that serology test. That serology test is done in a labratory it's very cumbersome we need to validate it clinically in order to look for antibodies in the population against this virus and that is the number one test when you go look for HGLV. But that said there are opportunities to get tested and you might have your own reasons to get tested. Now generally a physician won't test because there are no treatment options. There are no known anti retrovirals currently that are known to be good for XMRV so why go get a test for it if you can't treat it?


But it can give you additional validation that your illness is an organic illness and that can have a huge psychological boost because you can begin then to think about immune support and things you might do and changes in your life style where you may be able to support your immune system in the meantime while we develop drugs. And importantly you want to protect your personal family and public health, we need to know where this virus is. And it does help, physicians then start to see, physicians like Dr Peterson will know how that might relate to your other infections your other immune issues if you have cytokine profiles some of the tests he does. It might help him or some of the other physicians with your therapy to know that this is a player in the game now.


And again it underscores the more people that are infected, that 3.75% is 10 million Americans, so that I didn't have to say anything the drug companies called me the next day and said Gee we'd like to help! and so we're actively working with them and they are helping because there's another piece of good news which is that there are drugs that were on the shelf that were developed all the way through phase 2 clinical trials so they were shown to be safe in people but they just didn't work as well against HIV as the drugs that were out there so why spend a lot of money developing them? So there are real targets that you can go after that can serve regions between these viruses right now and maybe come up within the next year with a drug and a clinical trial for that drug that would go along way toward treatment.


So right now we recommend to prevent the spread of XMRV, if you have CFS and you wanted to be as prudent even if you didn't get tested say Okay I might be infected. So what would we recommend? The HIV precautions because it's a retrovirus we know it's spread we found it in blood in the body fluid secretion prosthetic secretions so you just want to assume that these precautions that are very stringent, and have prevented the spread of HIV in some countries, that if you don't donate blood or sperm (this virus can infect sperm cells) so if you have CFS or maybe a history of aggressive prostate cancer in your family you might think about not being a blood donor.


Follow the HIV precautions. Don't share toothbrushes because you can have bleeding gums or razors. Use safe sexual techniques and I say here do not breastfeed. It's don't breastfeed after six weeks when the maternal antibodies go away. When they did that in Japan where ATL (that aggressive leukemia) was rising in the late seventies and early eighties, all they did was say Okay no breast feeding! and 40% reduction of ATL rates in Japan. So prevent the spread of this virus and you can reduce the disease and protect your family and your children.


So what are our research priorities?


At the WPI we're actively working with the federal government to develop that next generation of tests we expect than sereological assay ( Rachel will get mad at me) but within a month. She told me yesterday.....
 

Advocate

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Section 6

Judy Mikovits XMRV Q&A
Section 6. Video 2.

Male Questioner: [mostly inaudible] collaboratorsAnd you also brought more interest in the illnessprobably seen

Judy Mikovits: Youre welcome. [applause] Thats interesting, because the reviewers of the paper didnt really know what team they were [CFS is a] poorly understood and complex disease, so they went on to the virology. So they were able to get that reviewed without any kind of bias. And I think that as well. Yes, sir. [pointing]

MQ: What percentage of the population inaudible this virus?

Judy: We found it in 3.75 percent of the U.S. population and it was from across the United States. And in Japan they found it, just screening the blood supply of a couple thousand people, they found it in 1.7 percent. So we dont know the true prevalence.

MQ: So, only in 3.7 percent [inaudible] 96 percent of CFS.

Judy: No, thats the healthy population. Its in 97 (?) percent of the CFS population. I stuck this slide in here and Frank keeps taking it out, but you might have heard in the press after the paper came out that we didnt do all of the tests, all four tests, on all the people prior to the submission in May. We looked for evidence of infection, looked for the virus, to make the point. And it wasnt so much about the CFS.

So what we did after the paper was published, is we went back and we looked with all four assays for evidence of XMRV in those PCR negatives, because now we know that indeed those negative samples may have evidence of infection.

What we found was that 19 of the 33 had antibodies in the plasma. We found transmissible virus in the plasma of 33 of those people, and we looked at that latent virus because the company I used to work for here in Santa Barbara was called Epigenics, and it was developing methylation (?) for epigenetic silencing, and thats what (happens?) to viruses, and so we used Decitibine, which is a (dimethylating?) agent that opens up the genome and turns on the virus, and found that there was latent virus in ten of those people. And when we summed it all up and tabled it out, 99 of the 101 patients in the Science paper had evidence of XMRV infection, so

Another way that HIV/AIDS causality was established was by saying So the statistics of this means theres a 10 to the minus 35 chance that you had CFS in our study without having XMRV.

So Ill go back to the AIDS analogy. You can have HIV We wrote in the paper so its virtually impossible The editor took it out. We wrote, highly significant. The editor took it out. Finally we said, significant. But at any rate, 35 zero (??) would tell me that its virtually impossible to have had CFS without having had XMRV in this study.

So you can be infected with HIV and not have AIDS. We know that. People are being treated [and?] a lot more [???] controllers are coming out. But you cant have AIDS without having HIV. So if we can establish that XMRV is the [?] of events as HIV is to AIDS, which is what were trying to establish through that immune system understanding. So HIV, youll see [??] and it leads to AIDS. XMRV does what to the immune response? To the T, B and NK cells? Which leads to CFS. Because you cant To turn your question around, sir What about, the incidence of CFS in this country is 1 to 2 millionis that more or less what is said? ??? Canadian or Fukuda criteria? So I said 10 million people were infected. Where were the other 8 million? I showed you that only 20 percent [?] infected people were actually sick with one of those diseases.

So you can be infected with retroviruses and be carriers and not be sick.
So thats one reason to be tested so if you If there is a genetic susceptibility, which were looking to, may be a reason known as to why some people get sick and others dont. You certainly want to know if youre a carrier so you can protect your family.

Any other questions?

Female Questioner: (inaudible)

Judy: I dont work with the company. I know that hundreds So they only take samples two days a week because it takes three days to do that, so they dont Theyve done hundreds of samples in the last couple of months, and at least half of them are positive. Or 40 percent. And again, their doctors are looking doctors who are well versed in CFS, so theyre immediately sending off Dr. Cheny, Dr. Klimas, a doctor in Canada, Ellie Stein, maybe Susan Levine in New York. Im not sure because Im not Its illegal for me to know those data because of confidentiality between the patient and the physician. But quite a number and, yes, its there. CFS is a heterogeneous disease. I mean anything based on [??] So everything is not going to be this virus. [??] is a disease, and of course thats what were looking for, but I dont want our thirst for understanding how you can get sick and be sick forever and not have drug targets and not have diagnostics, so certainly there are going to be lots of people who have what MIGHT be called CFS today, and thats why weve also coined the term XAND, for XMRV-Associated Neuroimmune Disease, and that would be because weve seen

I had done a number of studies with family members prior to after the paper came out and prior to now where I just said theres a family member where the children have autism, theres fibromyalgia, theres cancer, and when you look, you find the virus. Weve found the virus in Atypical MS, so its a non-demyelinating MS, it looks like MS, it has some brain lesions like MS on SPECT scans, so Im sorry, Dan, I [? ? ? ], Im not a physician So just looking at families with different types of neuroimmune disease, we started seeing that the virus was there, and so thats why we started thinking it might be involved in a broader spectrum of neuroimmune diseases, with overlapping symptoms In fibromyalgia, pain is the primary symptom, but in a lot of people its body-numbing fatigue, so fatigue goes along with it.

In fact, Cindy Bateman, whos a fibromyalgia expert at the University of Utah She says its CFS with pain, fibromyalgia, and she can distinguish those who get better with fibro with certain therapies and compounds, and it takes the others away who dont respond at all, and puts some in the CFS group. So theres definitely going to be a lot of things where

Female Questioner: Inaudible

Judy: Yes, thats correct. I answered that question based on the samples that came through. XMRV is not going to be everywhere. Its 4%. But the people who are infected are sick.

Q: inaudible So if youre positive youre positive, but if youre negative its not necessarily negative.

Judy: Yes, thats correct. So I answered that question based on the samples that came through there. Everyone who is positive is definitely positive for having the virus. But we dont know what the people are, what the doctors sending in, so the people could not have that disease. So it could be a clearly, distinguishing delineating markerbiomarker-- or diagnostic at that point for various diseases. So a doctor might see a spectrum and say I dont know, Id better check. Because the earlier you catch itJust like cancer. Early detection Make sure the reservoir isMake sure you dont have that virus multiplying, and you can live a normal life. Dont let it get If we keep it down we keep the immune system strong.

Male questioner: So what youre saying is inaudible negative

Judy: Thats correct. If you do it by PCR. If you do it by VIPdx, at least right now, its running along the lines of Weve got the antibody, and weve got three of the four tests. Well license it to anyone. Were a non-profit institute, so... Everyone pays the same royalty, so any diagnostic company could do the gold standard. But right now if you test negative youre not necessarily negative, even at VIPdx. Because we want to go do that serology test. Maybe we cant find it But youve been exposed which would be a good thing because your levels are theoretically low and youve just now made the antibodies and so you can prevent disease, as we did with Magic Johnson. We dont know anything about the immune response. Yes?

Female questioner: inaudiblepregnantbabybreastfeeding

Judy: Well, it is theoretically possible. We dont know. Gamma retroviruses are vertically transmitted, so the egg and the sperm can be infected and you can actually vertically transmit gamma retroviruses. But this is the first Weve only studied it for two months. We dont know. Theoretically its possible. If we make those data it will certainly fall out when we start looking at family studies

The horizontal transmission is the only thing we know about right now. Because of course you have to find somebody who just gets infected in order to understand the disease. And since a diagnosis of CFS is being sick for six months, well, if only, if thats the only thing we can do is to stop that practice, making someone wreck their immune system or be totally sick for six months that would be a great thing with the discovery If thats the only thing that comes of it

Woman questioner: inaudible

Judy: It depends on where it is sent.

Woman questioner: take for example, if VIPdx

Judy: At VIPdx, as soon as we have the serology well go back and do all the negatives. We save them. So well go back and well We do isolate virus from all of them, but it costs a lot, so So if theyre really negative, we wont isolate virus either. Thats correct. At VIPdx were going all the way to virus isolation, because we want to make sure were sure.

Woman: So at a non VIPdx

Judy: Theyre only doing PCR. Thats wrong. [?] 60% probably. Very few people can find them. And participate in our research studies, because as I said, theres no hurry Theres no real reason to pay for a diagnostic test because you can participate in research studies and fund those studies and you get better data. Im not privy to those data. I cant answer the question of where they came from, where they are in the United States Both patients and doctors have to release that information, and thats not the fault of the diagnostic company. So the information is not useable that way. Now were pretty good right now. Weve been as responsive as can be. By the way, if anybody thinks they might have been in our repository If you simply e-mail me, or e-mail me at info@, Im JudyM@WPinstitute.org, we did decode that study over the Christmas holidays, so we can tell you if youre positive or not. We can tell you whether or not you were in the study, because everybody Theres more than 500 samples in the repository. We pulled about a hundred of those for this study so we dont know if I dont remember everybody, but we did decode it so we know who the positives are at this point, and we are sending letters, but you have to ask me first for our insurance protocol, so just ask me and Ill tell you.

Question: inaudible

Judy: It actually uses the cell lipids to make its lipid membrane. Its an envelope virus and it has lipids, so it pulls cholesterol in to make its So it uses all the cell machinery, [it?] codes the enzymes and all the proteins and cells, so the lipid bilayer of the envelope virustheres lipids in it from your cells, and cholesterols a part of that.

Question: Ive met a lot of them inaudible blood problems inaudible

Judy: Yeah, and nobodys ever looked. Its certainly something they could look at and correlate. I cant think of a reason why. You might presume youd have less if youre using it up for another purpose.

Question: inaudible

Judy: We do have a little bit of data on that because we have two children in a study who have a genetic disease of cholesterol. Its called Nieman Picks Disease. Its also known as Childhood Alzheimers. (15:11)
 

garcia

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Section 5

So what are our research priorities?

At the WPI we're actively working with the federal government to develop that next generation of tests. We expect that serological assay (Rachel will get mad at me but) within a month. She told me yesterday that the data were looking really good.

And we want to investigate the prevalence of XMRV. The federal government, the National heart, lung & blood institute actually called as soon as the paper came out and we set up a blood working group to investigate what is the true prevalence. Prevalence means the presence, the distribution, not necessarily the disease, we use incidence with disease, and prevalence of XMRV in the blood supply. Our numbers were small they were only 2 or 3 hundred that’s 4%. And so, but 4% is still 10 million Americans, so you want to look at that, and they actively are. And they’re working on that second generation test as well.

We want to understand those tissue reservoirs and clearly it may not be the PBMC’s. Is it the lymph nodes? Is it bone marrow? It’s possible (I don’t expect it) but it could be the brain. We don’t know at this point.

We are actively working as I said with drug companies to develop anti-retrovirals and immune based therapies.

We want to understand how it’s transmitted. We’ve got a family study going on in the research plan, it’s just getting IRB approval and ready to start so hopefully we can get families who have any number of diseases across the spectrum, fibromyalgia, other neuro-immune diseases, maybe a higher incidence of cancer, but we need healthy people as well, so we’ll take essentially anybody into that protocol. And as I said that protocol will help us investigate the incidence of XMRV in other neuro-immune diseases.

Important questions that the field is working hard to answer and we are as well but we won’t be able to do all this: Is XMRV a causal factor in CFS and possibly some aggressive prostate cancer? And we’ll talk a little bit about how you think about a causal factor. One way to do that is, we have several patients who came to Dr Peterson and they said “I was fine until I got into a car accident. I got a blood transfusion in the hospital and I got CFS” or “I had a surgery and had a blood transfusion.” So if you can identify a blood transfusion exchange of an acute infection that causes the disease and the virus wasn’t there before in the human and it’s there afterwards and it’s in the donor, then of course you’ve got causality and that is one way that causality was shown in HIV as causing AIDS. I think we all know the tennis player Arthur Ashe and that is how he got HIV/AIDS and subsequently died.

So how does XMRV enter the human population? Is it a zoonotic [from animals] transmission? We know its not a mouse, at least not any of the mice we know. It could be a field rodent of some kind, but we’ve never found the virus in another animal. This is the first animal that is the “Xeno” and that is man. So how does it enter the population and when did it enter? What’s the worldwide incidence of XMRV disease that should say or prevalence of XMRV. Where is it? Is it in England, in Europe, at what level? We know it’s 1.7% in Japan because of a study done earlier this summer.

And does it alter the risk of cancer development? Because HIV & HTLV1 both by causing immune deficiencies do.

So a lot of people wanted to know are we working internationally to replicate the studies. Everyone you see on this slide, a lady in Canada, part of the blood group in Canada also had called me since the study came out. We’ve been working with Jonathan Kerr and we have a 5-year RL1 with him, but Ellie Barnes in MRC in Oxford. Norbert Bannert on that German paper, he was working with a advocacy group led by Regina Koch I think and they found a few samples that were maybe positive. So he called me and said “Can we work together and have that antibody?” Again everybody you see on this list, Jonas Blomberg in Sweden. Norway, Germany, the Netherlands, Italy, Spain. We can’t handle the samples we’ve got so far, but we’ll try and we’ll send the reagents out to anybody to replicate the work and find out more about the disease.

We also know of additional incidence studies that we’re not involved with but are occurring at Kiel University again in Germany. And here in the USA, Sam Chow is working and has identified the virus in China and I do know that Richard Huber has had success at finding virus in CFS and other patient groups and of course I mentioned that blood working group that’s working throughout the United States and I didn’t list the number of US collaborators we have. There is a lot interest, a lot of the world’s best labs are working on this and we’re going to get there a lot more quickly than we would because of everything we have learned from HIV.

So what about Diagnostic Tests? I said should your physician or you want to be tested currently (the last time I looked online) there were only 3 companies offering the diagnostic test. Of course the WPI licensed the technology to VIP Dx, (we’re a non-profit institute) who is using our proprietary culture method and PCR in combination along with a Western Confirmation. So we look for both antibody and PCR positivities in the cultured and co-cultured cells. And we use 20mls of blood to do this and make sure of the accuracy of the result and the price is $450. Clongen lab has a real time PCR that is just looking for sequences on 1ml of whole blood and there price is here [$375]. You won’t find this virus in 1ml whole blood by PCR. I think I’ve shown you that with the negative cases in the prostate cancer. And also a company known as Cooperative Diagnostics in South Carolina. We don’t know what their PCR method is but their using a drop of blood on a piece of paper so they tell you if you put a drop of blood on a piece of paper, you don’t need a doctor or anything, just send a cheque and of course they won’t find anything.

Your help is critical really to advance this science. At the WPI we either want your money or your blood. That’s the only two choices! You decide what you’d rather do. But we need you to participate in these research studies and we do have a form online: www.wpinstitute.org and you can email me. We’ve got a form online to register. We’re asking for some clinical characteristics but we’re asking for those more to help us put it in a study. We won’t turn anybody away. We will look for the virus if we can get those studies. We’re waiting for the IRB approval, that’s the human assurance to make sure we’re not hurting you and we’re protecting your privacy. So we expect that this week. Donate funds to the WPI research and clinical programmes that will be established later this year. The clinical programmes will really come of the research and the diagnostics. And then write to your government officials and encourage them to support XMRV research. This is an infectious disease. Why isn’t the national institute of allergy and infectious disease considering this virus? They’ve been pretty quiet haven’t they? We haven’t heard a word from them. So we need our government agencies to look at this virus because it’s an emerging infection as I said of unknown pathogenic potential.

I’d like to thank the people who, we couldn’t have done this study without them. This has been a 3-way collaboration between the National Cancer Institute and its contractor SAIC, Cleveland Clinic and the WPI. As I said earlier when Vinny Lombardi and I together with Max Pfost first saw the few sequences of the virus I called Bob [Sliverman] because obviously we were doing the work with him and then I called Frank [Roscetti] and said you know “I need you” and he said “I won’t go” and I said “I’ll pay your way to San Diego on the beach for a week! Whee!” and he said “Not any more of your schemes Judy! I’m not going to do that again!” So at any rate we met at a restaurant and we showed Bob, and he didn’t know what I was going to tell him. Interesting Frank’s a bit cantankerous because I gave him about a week’s notice because we’d had 3 weeks and I was pretty sure I knew what we had. We had to get a 3-way inter-institutional confidentiality agreement. So Frank called the office in the government and they said: “No we’re not going to do that” and he said “Look we’re talking next Saturday. You can either have a confidentiality agreement or you won’t”, but they got one. So Frank and his lab, Dan Bertolette did everyone of those beautiful Western’s that I showed you, just a magician. Mike Dean & Burt Gold sequenced the entire RNAse-L gene in more than 100 patients. Ying Huang did all of the PCR that we had done totally blinded where samples never came to our labs to show it wasn’t contamination. And of course I’ve mentioned the lab of Sandy Ruscetti, Charlotte Hanson & Jami Troxler who were key in providing all of those reagents without which this study clearly wouldn’t have been done. Cari Petrow-Sadowski developed that immune-response assay in a real hurry this summer and I mentioned Kunio & Rachel who did bioinformatics support and electron micrographs. We couldn’t do it without Dan Peterson’s diagnostic skill. I mean he biased the patients such that we could find the needle in the haystack but these are the patients that come to the institute. They have classic symptoms of CFS. When we have taken patients that have emailed us with exactly those same symptoms, we find the virus every time we look including in Europe, in England, Ireland. We couldn’t do it without the CFS patients and advocates. We do so appreciate the support all along. This was a tremendously difficult year in trying to keep quiet. Knowing what you had and don’t say anything until you are sure you’re sure you’re sure. Every day was just are we sure? And so we were able then just with the small crew you see here and this supportive staff. Vinny, Katy and Max they pretty well have worked 24/7 for at least the last year and we have our lab meetings at the bar. They said they were going to make a drinking game based on my talks, I’m not sure if it’s how many times I said “umm” or whatever! So at any rate, with that I’ll thank you for your attention and take questions.
 
K

_Kim_

Guest
Thanks to Rachel, froufox, sproggle, Advocate, and garcia for getting sections 1-6 transcribed. There are three more sections to go. Any takers?

The complete lecture has been posted on ProHealth.

We need some volunteers to transcribe the lecture in 15 minute blocks, picking up where Rachel left off (was that at 27 minutes into it?).

Video 1 (81:50)
Section 1 - completed by Rachel (up to 27 mins....or thereabout)
Section 2 - wherever Rachel left off - up to 40 mins (Kim - completed)
Section 3 - 40 mins to 55 mins (froufox - completed)
Section 4 - 55 mins to 70 mins (sproggle - completed)
Section 5 - 70 mins to the end of video #1 (garcia - completed)

Video #2 (54:21)
Section 6 - start to 15 mins (Advocate - completed)
Section 7 - 15 mins to 30 mins
Section 8 - 30 mins to 45 mins
Section 9 - 45 mins to end of video #2
 

shannah

Senior Member
Messages
1,429
"Section 5

So what are our research priorities?

At the WPI we're actively working with the federal government to develop that next generation of tests. We expect that serological assay (Rachel will get mad at me but) within a month. She told me yesterday that the data were looking really good."

So what does this mean exactly? Does this mean an FDA approved test?

(I moved this over to the Discussion thread where it probably should be - sorry for any confusion.)