Dr Judy Mikovits On XMRV and CFS from Prohealth; Part I

January 24, 2010

Written by thefreeprisoner

1037-DrJudyMikovits.jpgThanks to ‘thefreeprisoner‘ from the Phoenix Rising Forums for transcribing the first part of Dr. Mikovits talk on Prohealth on Jan 22nd.

Annette Whittemore:

Dr. Mikovits Prohealth/HHV-6 Fdtn XMRV Lecture

…happy to see that the ProHealth organisation was able to get this online so a lot of patients who are too ill to make it are able to follow this online.

First of all I’d like to that Rich Carson, I’d like to think ProHealth for putting this together and the HHV-6 foundation and Kristin Loomis.

It’s a pleasure to be here and to have an opportunity speak to you about the recent discovery of XMRV in Chronic Fatigue Syndrome patients. Thank you for inviting Judy Mikovits today. We’ve made a special effort to learn more about the most exciting news in the world of CFS since the 1980s when major outbreaks of this disease were reported in several locations around the US including three small towns in Nevada.

These reports came on the heels of the discovery of HIV and AIDS. During this time Dr Mikovits was at the NCI working in laboratories that were actively studying this new virus which she began her doctoral program at George Washington University. In fact she wrote and presented her doctoral thesis on HIV latency, presenting it the same day that Magic Johnson announced he was HIV positive. The good news is that he is still healthy after all these years, having had the opportunity perhaps to prevent a high viral load from ever ocurring.

Jumping forward to 1989 brings a critical event to the life of our family. Our daughter became ill and suddenly we found ourselves in a black hole of medicine where noone seemed to agree on anything having to do with a disease that had been dubbed ‘Chronic EBV’. The problem was that she didn’t have EBV or even the antibodies that would have indicated that she’d been exposed to that virus. Like you, we sought answers but instead we found confusing and even nonsensical theories about her illness. And thus began the journey for answers.

All of us have various milestones in that journey. Our first and probably one of our most exciting milestones was meeting Doctor Peterson who at that time was rpomising that he would do all he could to help Andrea and to help this family. Our second milestone was really meeting Dr Mikovits. And that was a wonderful and perhaps prophetic meeting. We were at an international conference and she had come along as a guest of the HHV-6 Foundation which she heard a very important talk about a set of patients who were developing a rare form of cancer. She took a leap of faith with us to develop a medical research institute when we asked her to come to Reno and the rest is history.

Judy brought with her a scientific passion for discovery of truth, and a curiosity, with well-taught skills from the laboratory of Dr Francis Rosetti, the co-discoverer of the first retrovirus HTLV-1. Judy has a fiery temperament and a heart of gold. And when she is not counselling CFS and cancer patients, she teaches students, devises experiments and travels to major conferences and universities to educate others about the intricacies of the scientific methods used to find infectious and replicating XMRV in the blood of CFS patients.

Before I introduce you to Judy, I’d like to ask three things of you.

First, please stay involved and advocate for your rights; medical treatment and adequate funding of research. Your congressmen and senators need to hear from you.

Second, please stay informed and educated. Listen critically to what is said and who is delivering the message. Are they speaking on your behalf?

And third, I want you to know that the WPI is going to continue its promised mission. We are not going to stop until we find the answers, but we can’t do it alone and so we do continue to ask for your support and your help. We appreciate so much all the donations that have come in, all the amazing good wishes that have come our way, and we wanted to thank you today all around the world for letters that have come in to support this effort; that really really helps.

So now it is my good pleasure to introduce you to Dr Judy Mikovits, research director of the WPI.

[applause]

Dr Judy Mikovits:

Well thank you Annette.
I too would like to thank Pro Health and particularly Kristen Loomis and the HHV-6 Foundation for sponsoring this event.

What Annette didn’t say is that it was Kristen Loomis who put us together by asking me to attend that meeting in Barcelona, Spain. I never did get outside to see Barcelona but I saw some amazing scientists and physicians there in the room and in the meetings. So I’m also honoured that you came out on this day and I understand how difficult it is for patients to get here, and I appreciate all the calls and letters we have gotten around the world since the publication of this paper. It’s amazing the response that we’ve gotten and we’re just delighted. We work for you. The institute is a translational research institute. You can tell the architects drew this because they’re making a lot of money. We don’t usually drive Porsche cars [laughter].

But at any rate, this is what building looks like. It is three-quarters built and will be open to serve patients in September this year. So keep your eye out for the opening ceremonies and the ground-breaking there. We’re excited to see patients.

Before we could see patients, Annette knew that she would need to start a research program because there were no bio-markers, diagnostics, treatments or anything. So we started looking with the patients there, with the diagnostic acumen of Dan Peterson. So I came up right after that meeting in Spain in 2006 and spent the summer just meeting the patients.

I’d never heard of the disease before then and it was just eye-opening to me to see how sick these patients really were and to try and understand really what the disease was. So my dear husband when I talked to him about Reno, all he kept saying was ‘Reno? That’s not by the ocean’ [laughter] but I’m a patient advocate as well in Ventura County with a Cancer Support group with Bible Fellowship Christian Cancer Support Group and they were kind enough to let me go to Reno because I said “These people are much sicker than you.” [laughs]

So I’m going to talk to you exclusively about the retrovirus XMRV. All of these slides will be uploaded both to the WPI website and to Pro Health so you can look at them later on.

I understand that I’ve left a lot of the detail of the science because when we see the science you start to understand the implications of this discovery in not only this disease but perhaps a number of old diseases where we might find a new understanding.

So this is the Cleveland Clinic rendition of the retrovirus and of course the electron micrograph that accompanied the publication in Science.

So I’m going to give you a little history lesson. We’re going to walk through the publications.

Interestingly XMRV was identified by Bob Silverman and Joe de Risi in just 2006 so just at the time that we were meeting each other and making these fateful introductions, this virus came out, where Bob Silverman who was an immunologist at the Cleveland Clinic was looking at prostate cancer patients where there was familial prostate cancer. That is, it’s hereditary, but that it’s hereditary in a funny way where maybe brothers-in-law or distant relatives would get it and not direct father-son, things like that. We actually had a case in my own family where my stepfather died very young of prostate cancer and it’s an aggressive cancer, and when you get prostate cancer very young it suggests there’s something else going on environmentally.

So he looked at a single nucleotide change of a varient in an anti-viral gene known as RNase-L. This gene, the protein’s job is just to degrade RNA from viruses and protect you, and turn on the interferon response. But he found a variant in that gene where that single base change that is in about 13% of the population makes this enzyme only about 20% as active, so it dysfunctional because it doesn’t work. So he hypothesised that maybe these men were susceptible to a virus. And he met Joe De Risi at UCSF, who had a technology which is basically a chip. It’s like a chip with a bunch of information on it. The information is just sequences of every known mammalian virus, so 20-30 maybe 70 base pairs of every known virus from [considerable region?].

He simply took the DNA from these men and applied it to the chip, and the red part you see right here shows that it exactly matched [if you want to match in opposite (?)] the sequences from this particular virus. So when they took this out and they sequenced the virus there, they found that there were retroviral sequences in 10% of those tumours, and those with that particular variance, and that those sequences were most closely related to what was xenotropic murine leukemia virus. It’s a gamma retrovirus and we’ll talk more about that later. They just number them alpha, beta, gamma for convenience because the number are known.

Xenotropic means it can no longer infect mice. Xeno means foreign. So what we know from the xeno family of viruses is that they look like murine leukaemia viruses but they lack a receptor and we’ll talk about that later, so that they can’t infect mice. So he named this virus Xenotropic Murine Leukaemia Related Virus because it wasn’t exactly the mouse virus. Clearly it was something different, suggesting that this might be a new virus. So his laboratory did a little more work in the next 2 years. Again we’re just talking about 2007-2008. Usually it takes a year just to get a paper published.

So what he at first identified; they knew that the mouse family of xeno viruses would recognise and bind and actually enter the cell through this receptor. So it sees the receptor; it’s called Xpr-1 and this is a calcium channel type, an ionic receptor. They don’t know the function of it… this is called the G protein it has a particular role in sequencing. We know there’s a loop right here or so, where the mouse virus has two or three amino acid changes and that’s why the virus can no longer infect mice. That’s one of the reasons we know this is not from a mouse. This receptor is on every cell in the body. So it doesn’t tell you a whole lot about the infection or what cells would get infected in the disease.

So the next thing he did was, he molecularly cloned this. He used techniques to write the virus, the entire 8,000 base pairs and put it in a vector which allowed him to multiply it and make it an infectious virus. So he made this infectious virus, he didn’t actually isolate it. Using the infectious clone, he then infected various cells and found that the virus integrated. It inserted itself into DNA preferentially at the start site of genes. And that’s the part of the gene that turns on and off their expressions, so a lot of the differences you see in patients culd be explained by turning on and off the wrong genes when a retrovirus integrates.

So let’s do a little bit of retrovirology 101. This is the genomic structure of a retrovirus. Now, retroviruses have an RNA genome. We have DNA genome. We have nucleic acids; our genetic information is packaged in DNA. This virus has a single stranded RNA genome that’s present in two copies in the virus. So it first has to be reverse transcribed by the enzyme reverse transcriptase. So you have to take the RNA back to DNA and then the integrase gene there, shown here; this is a pall, so all a simple retrovirus will encode is the structural proteins, gag, pall and the envelope, and then the enzymes. They don’t have any extra proteins like HIV or HTLV-1 which are complex retroviruses and they write a bunch of proteins that regulate different parts of your body.

The good thing about this virus is that it’s a simple retrovirus. There’s less that it can do to interact with your cells to have those go wrong. So that’s the first piece of good news. It’s the first ever simple retrovirus known to infect humans. So we can think a lot about that as scientists and how it might cause disease.

So once you make the virus, you go from your genetic information I showed you in the last slide into the envelope protein which has two proteins actually in the surface unit. This is what binds to that receptor, and then the trans-membrane unit that sets itself into the matrix of the capsid. This is the capsid protein there, and that’s known as gag, so you’re gonna see capsid and envelope throughout this talk so you’ll understand that when you have antibodies that develop, these antibodies are recognising these areas of proteins, and this is depicted here as that double stranded RNA nucleus, and the polymerase which simply writes the RNA into DNA and then packages it all up and leaves the cell.

Now let’s talk about how you detect retroviruses, because that’s important in thinking about how we found this virus and how we study it in the laboratory. So the viral life-cycle as I just described; once you have the DNA integrated into the chromosome, once it’s integrated to the reverse transcriptase integrated it into the chromosome. It’s there and it replicates every time your cell divides and your DNA replicates. So if your cell isn’t dividing, theoretically it’s just latent. It’s just there in the DNA and it’s not making more viruses, it’s not making copies of itself. It’s not infecting more cells. This is a good state if you have a retrovirus is to just shut down the transcription.

As Annette said when Magic Johnson was found to sero-convert, they found an antibody in his blood so that’s very shortly after he became infected, so they were able to give him therapeutics to prevent the virus from making many more copies in his body, so theoretically the reason why he never got sick is because he is maintained on those anti-retroviral therapies as well as the immunomodulating therapies. He’s kept that virus down so that he never theoretically will get AIDS. We’ll talk more about that later.

Once the cell starts dividing and you start writing that DNA and transcribing it into all of the proteins we just discussed, the envelope will then package the core of the capsid there, that looks like this, in the RNA genome, that double stranded genome, and it actually uses your cell membrane, cholesterol and lipids to leave the cell then and look like that artist’s rendition of the viral particle. So when you’re looking for retroviruses, and there are only two known, are the HTLV-1 family — there’s a one and a two — and HIV, the human immunodeficiency virus.

As Annette mentioned, Frank Ruscetti discovered this virus and reported it in 1980. At that time there was no PCR so he couldn’t look for an infected cell by a sensitive method so he looked for that enzyme reverse transcriptase because reverse transcriptase is only in retroviruses and not in human cells, so it’s easy to look for the activity of that protein that would then transcribe and make the virus. And sometime maybe if you’ve learned the history; it’s amazing the small small signals they found in the early days to describe the virus.

But then you’re going to do what we call a western blot which is to run out the proteins of a cell on a gel electrophoresis and just blot it and look for antibodies — and we’ll show you those later — for the viral proteins and test for specific antibodies to the envelope and the gag proteins and just look for the presence of virus in infected cells.

The first thing you do clinically is you look for serology — that test that shows you that your system is making an antibody to that virus. That was the test that Magic Johnson got. You have a virus in your body and your immune system’s job, to distinguish self from foreign. So we know this is foreign because you have made an antibody to it and then finally, it’s rarely done clinically, to identify HIV or HTLV-1, is to isolate the virus and actually purify it in cell culture.

So that leads us up to the next paper. So after the first bit of work that Bob did in describing this virus, there wasn’t a lot of excitement about it in the scientific community, because they didn’t know that it was an infectious virus. It was just sequences in prostate tissue tumours, and it wasn’t meaningful to the scientific community because we all have sequences of viruses in our body as we all know, maybe as much as 15% of our genome is made up of viruses that are silenced by our immune system so that they can’t be expressed.

So the work that we did then generated a lot of excitment. What we did was we detected this infectious retrovirus and showed that it was infectious in the blood cells of patients with Chronic Fatigue Syndrome. We’re going to walk through exactly how we did this to show the virus.

At first we did PCR because at the time this paper was done, the only thing that was known was Bob Silverman’s specific PCR technique. So we had not validated or identified any antibodies. It was not known that it was a pall virus, an infectious virus. So that’s what this work was, it was serendipitous really, that we happened to have the patients who had this virus because if we did not have a well-identified cohort of CFS patients and we were just looking at the general population, retroviruses aren’t highly expressed in the general population. HTLV-1 is 0.2% in the US population, and we’ll talk a little bit more about what that means too. Retroviruses are not ubiquitous. It’s not like EBV and CMV where everybody has them.

So we had these well-characterised patients who had been sick for many years. I think it was a large part of why were able to isolate this virus.

We’ll start at the beginning and that’s the cohort, who they are. When it came to the Institute, what we talked about was really important was having a repository of samples from all of the patients so we could look at the RNA for their gene expression, at the DNA for maybe what was different about the genetics of some of you that might make you sick.

Then we look at the plasma for proteins to see if we could identify immune modulators called cytokines that tell your immune system and tell your brain how to function. So we made these samples across RNA, DNA, protein and plasma, and then a culturable cell, so we kept some frozen such that we could grow them up and make more of them any time we wanted of your peripheral blood mononuclear cells; that’s your white blood cells.

So we used patients who came literally from around the world and this was actually not correct in the Science paper because I didn’t know there were international people in the repository at the time. When they come to Incline Village it’s assumed that they are from Nevada, and when we decoded this over the Christmas holidays we found 12 or 15 states, the UK, Ireland, Germany and Australia as well. So we had both international and people literally from all over this country, not necessarily Reno, Nevada, where the associated outbreak that we know occurred there in the early ’80s.

So the inclusion… all you had to do to be a sample in our repository was have a CDC diagnosis of Fukuda criteria or the Canadian definition diagnosis which is more stringent for various immune defects and inflammatory defects. Regardless of severity, the samples in the repository are from people aged 19-75. We don’t have any whole bodies yet of people; though people do offer to donate whole bodies, however I don’t think we need them at this point [laughs].

The study characteristic, like the disease was 67% women, reflecting the gender bias in incidence of CFS. The mean age was 55, but some of these people had been sick since they were children or early 20s or early 30s so they had a long haul with this illness.

The 218 control samples were de-identified samples so we don’t know who these people are. They came from two places; they came from a medical practice in Reno, they came from a doctor identified people as healthy and these were collected from people before I came to the University in 2007, and they were looking at the immune systems of healthy people to identify some of the dunctions so we were able to use those samples under IRB approval. There is also a paternity diagnostic company in Reno where they get samples from all over the world from mum and dad, so we tested from those 100 or so samples too, so we were at least able to zip code match. We have regional areas for the geographic location so that it was matched for location.

This is a PCR gel; I simply run them out for electrophoresis and that gives you a different size so you can look at the exact size of the fragment of DNA that you’re looking at. Again, this was done by Bob Silverman who is our collaborator in the study. Today is actually the first anniversary of January 22nd when I called … we saw some of these data right after the Christmas holidays and we had promised Bob for a long time that we would look at this because RNA cell is a major defect in our patients, whether it’s underactive or overactive, something is wrong with the RNase-L pathway in CFS patients according to decades of research.

We promised Bob that we would simply look, although we had done a micro-ray technology and we had not found the virus there, we had his specific primer pairs so we could go in and look for that gag structural and that envelope gene so that we could see viral sequences in the cells that could make viral proteins theoretically. What we found was that 67% of the patients we looked at, we could find sequences in both the gag and the envelope gene or just the gag depending on the virus life cycle at the time. This was astounding because we only found the sequence in 3-4% of the healthy control population. It’s also interesting; I said 68 out of 101 patients. On some of these patients we looked…

Dr. Mikovits Prohealth/HHV-6 Fdtn XMRV Lecture

[here the video ends]

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