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Lipkin finds biomarkers not bugs

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The CDC PCOCA telephone broadcast on 10 September 2013, featured a lengthy presentation from Dr Ian Lipkin who revealed some stunning initial results from the study that is primarily hunting for pathogens in ME/CFS. Simon McGrath and Russell Fleming (Firestormm) review this exciting and possibly game-changing news...


Dr Ian Lipkin
Lead Researcher
Chronic Fatigue Initiative (CFI)
Pathogen Discovery and Pathogenesis Study

Read the full Lipkin Transcript: Here.

Dr Ian Lipkin has been a human whirlwind in ME/CFS research since he became involved a few years back, and he's just surprised us all by announcing the first results from the world's largest ever biomedical ME/CFS study in a public broadcast!

He started off by lowering expectations: their results thus far do not implicate a single infectious agent and they have not discovered the cause of CFS. But he then wowed the hundreds listening to the conference call by revealing they had found strong evidence for immune overstimulation, both in blood plasma and in cerebro spinal fluid; and that they were now hard at work trying to identify what could be causing these abnormalities.

The big pathogen hunt draws a blank thus far

This study is looking exhaustively for any pathogen: viral, bacterial, fungal or parasitic, to see if a chronic infection could explain ME/CFS. Many of the results are in, but so far there is no clear sign of viruses at least. It is every bit a collaborative effort, with patients provided by long-established physicians including Dr Dan Peterson and Professor Jose Montoya.

The Key Players

Lipkin began by thanking the clinicians who had provided the all-important samples. Effectively it was a roll call of America's top ME/CFS physicians:

Professors Jose Montoya, Anthony Komaroff and Nancy Klimas; and Drs Dan Peterson, Lucinda Bateman, Sue Levine and Donna Felsenstein.

The study itself was run by Dr Mady Hornig (CFI Principal Investigator) at the Centre for Infection and Immunity at Mailman School of Public Health; where Lipkin is also based.

He went on to acknowledged the very necessary support of the Chronic Fatigue Initiative, the Evans Foundation and an anonymous donor supporting Montoya's work.​

First the researchers looked at blood plasma for 285 CFS patients and 201 controls from Jose Montoya at Stanford (these are serious numbers). They searched for genetic evidence of infection using a panel that was able to detect 20 specific bacteria, viruses or parasites; including Epstein-Barr virus, Human Herpes Virus 6, enteroviruses, Influenza A and Borrelia bacteria.

Next they used high throughput sequencing, a method that was pioneered by Lipkin and colleagues that was used to discover over 500 viruses – so they felt fairly confident that if a virus was present, then they would have found it.

But they effectively drew a blank using both methods - only 1.4% of CFS patients were found to have HHV6 infection, but so did 1% of controls.

Other findings...

Lipkin said they also found Anelloviruses in 75% of those studied in plasma samples, but it was not specific for Chronic Fatigue Syndrome (we don't currently have separate figures for patients and controls). Anellovirus is believed to cause widespread chronic infection in the human population, but has not yet been associated to any specific disease. "I really don’t know at this point what this finding means" said Lipkin.

They also found retroviruses in 85% of the sample pools. However, Lipkin expressed caution:

"It is very difficult at this point to know whether or not this is clinically significant. And given the previous experience with retroviruses in Chronic Fatigue, I am going to be very clear in telling you – although I am reporting this at present – in Professor Montoya’s samples neither he nor we have concluded that there is a relationship to disease ...if I were to place bets and speculate, I would say that this is not going to pan out."

Window on the brain: Cerebrospinal fluid samples

Both Lipkin and Hornig were clearly excited to have had access to the 60 cerebrospinal fluid samples from Peterson, and kindly donated by his ME/CFS patients. Cerebrospinal fluid bathes our brain and spinal cord so it gives access to what goes on in the brain, a prime site of interest to researchers, but hard to access; so these spinal tap samples are precious.

However, using the same molecular techniques to track down pathogens as they had applied to Jose Montoya's samples, Lipkin's team again drew a blank.

At the Invest in ME conference in May, Hornig said they had tentative findings that there might just be a novel pathogen or pathogen candidate in these samples - but it was too early to say for sure. In reply to a question yesterday, Lipkin said there is no more to report:

"we do not yet have a novel pathogen nor do we have a pathogen candidate in which I would have any confidence. So at this point we have no plans to publish anything."
Nonetheless, he also said they hadn't finished all the DNA sequencing work on these samples, so this is not yet a dead end.

Immune stimulation and biomarkers

Up to this point we'd heard a lot of very impressive negative findings, based on large, well-defined samples of patients and controls, and using powerful research techniques. But what we most wanted to hear about was positive leads - and Ian Lipkin duly obliged:

blood1.jpg

Plasma is the clear fluid left when blood is separated

They took plasma from the CFI cohort of 200 very well-defined ME/CFS cases and 200 controls collected by clinicians (See 'Key Players' above). Then they searched for cytokines and chemokines, which are regulators and controllers of the immune system.

The big finding was a decrease in levels of Interleukin's IL-17, IL-2, IL-8 and in TNF-alpha - all of which have a role in pro-inflammatory responses. Lipkin believes these significant differences are worth follow-up in larger cohorts to better understand what they mean.

Professor Jonathan Edwards (who is an adviser to the planned UK Rituximab trial) commented on this forum yesterday, that it was a surprise to see TNF-alpha levels decrease, and suggested it could mean that 'inflammation' is too crude a concept for the process.

Lipkin and colleagues also found upregulation in Leptin, a hormone that plays a key role in regulating energy uptake and use, and Serpin, a protein family with multiple roles. More obscure cytokines, and and their cousins chemokines, were also affected but Lipkin didn't give any further detail.

Two new patient types revealed

Ian Lipkin said they found something very surprising in their data: there appear to be substantial differences in profiles between those people who have had the disease for 3 years or less and those who have had the disease for more than 3 years. And that this is important because, "it could have implications for therapy as well as for diagnosis".

In the ‘early group’, who have been ill for less than 3 years, there seem to be a number of markers suggestive of some sort of allergy aspect. For example there are often increased numbers of eosinophils in blood, with more differences in cytokines. But while this is tentative, he was more confident in the finding the IL-17 was elevated in these 'early' patients, compared with reduced levels in patients ill for over three years.

Lipkin said that he thought this 3-year division could be very important and that it hadn't been something they were looking for; but would be looked at in any future work they did.

Cerebrospinal fluid: different differences

Lipkin's team also found differences in cerebrospinal fluid biomarkers between patients and controls. I'm not sure from what he said if there were significant differences between the 'early' group and patients who had been ill for more than three years. However, they did find that patients had elevated levels of the TH-2 type cytokines IL-10 and IL-13 and elevated levels of four TH-1 cytokine: IL-1 beta, TNF-alpha, IL-5, and IL-17. Lipkin said this is compatible with a profile of some particular types of response that may provide insights into immunological dysregulation in Chronic Fatigue Syndrome.

So, overall they found clear differences between patients and controls in both plasma and spinal fluid. Many immune differences have been detected before, but findings are not very consistent and none had used such a large and robustly-defined patient cohort (meeting Fukuda and Canadian criteria), or with such carefully matched controls.

HEALTH WARNING

Dr Ian Lipkin again was very clear that he did NOT recommend anyone act on these provisional findings: he doesn't want anyone to get a spinal tap, or measure the cytokine levels and hope to modulate them with drugs based on this research: it's too early for that.​

The lost years...
Back in 1999 Ian Lipkin published his first paper on CFS, which ruled out a connection between the illness and Borna Disease Virus (one of the many viruses he's discovered). But crucially they found evidence of polyclonal B-cell reactivity. He commented that back then:

"there was a very strong sentiment in some portions of the scientific and clinical communities – not always and not everywhere – but in some portions of the community, that this was a psychological illness. What I said was that based on our findings we had very strong evidence that people with Chronic Fatigue Syndrome were truly ill with a physical illness and they deserved a "Deep Dive" to find out why they were ill."
He noted that was a long time ago and dryly added "I am pleased to see that people are now paying much more attention to this disorder and what we can do about it".
What next?

"I still believe the primary cause is likely to be an infectious agent."
Having found clear signs of cytokine abnormalities, Lipkin wants to find out what is causing these changes; he currently thinks that an ongoing stimulus is causing immune system activation which might be resulting in the symptoms we associate with the disease. And interestingly, he still believes that infection lies at the root of it all - despite not yet finding any infections.

The team - working closely with the clinicians he's already mentioned - plan even more extensive work on a cohort of 200 CFI patients and controls. They will further test for viruses and crucially they will also look for bacteria and fungi by sequencing ribosomal RNA (which should detect unknown bacteria and fungi, as well as known ones).

Previous infection detection

The group has looked for current pathogens in the blood and spinal fluid samples they have - and has so far drawn a blank, though there is more work underway. However, what if the trigger for ME/CFS is an earlier infection that has since been cleared: the 'hit and run' scenario Hornig has described? Lipkin says they are looking for "shadows" of previous infections by searching for antibodies against specific infections, rather than the infections themselves (the body will continue to produce low levels of specific antibodies years after an infection has been cleared (the basis of much immunity)).

They will use a technique called LIPS to look for antibodies against particular known infection. In addition, as in their viral sequencing work, they will use an 'unbiased' system (using peptide arrays) that should detect all potential viruses - or rather antibodies against them. They don't currently have an 'unbiased' system that works on bacteria or fungi.

Fecal future?

What excites Ian Lipkin most is the gut microbiome, the community of micro-organisms that live in our gut - which might seem odd given his focus so far on pathogens and the immune system. However, it turns out that the microbiome can have a profound influence on the immune system and may be important in many diseases. Hornig and Lipkin have already developed techniques to study the microbiome, and have shown it could be a factor in autism and even in the severity of colon cancer.

It turns out the best way to find out what lives in the microbiome is to measure what comes out of the gut: fecal matter, or 'poop' to you and me. Their ME/CFS microbiome project has started, but Lipkin felt there wasn't enough material in the first attempt at gathering samples, so things have been delayed as they switch to a new technique (the 'special' collection cups).

The Big Ask: "we can't do this without you"

Microbiome research is very expensive (analysis, not sample collection), and they currently only have the funds to do 10% of what's needed. In fact, throughout the call, Lipkin was stressing the need for more funds, because good research costs and ME/CFS is woefully underfunded: there just isn't enough money to do the work needed. The current science budget cuts (Sequestration) in the US makes the climate even harder.

"It is probably inappropriate for me to be passing the hat, but that’s precisely what I am doing."

He urged patients to contact their representatives in Congress, demanding more funding for ME/CFS research - pointing out that in the early days of HIV, there was little funding until patients demanded it. He also said that if patients were able to afford it, they should invest in research themselves.

Conclusion

ME/CFS research is coming of age. Ian Lipkin has reported these early - and soon-to-be published results - from a huge study, using 285 of Jose Montoya's patients, the 200 patients from the Chronic Fatigue Initiative, plus the 60 'gold-dust' spinal fluid samples from Dan Peterson's patients. Using state-of-the-art techniques, they have largely ruled out a role for a specific pathogen - though that work is incomplete. And they have strong evidence of ongoing immune stimulation in ME/CFS patients, with abnormalities found in both the blood and spinal fluid. The next stage involves deeper searching for bacteria and fungi, looking for 'shadows' of previous infections and investigating the gut microbiome. These are exciting times!

Thanks to the CDC for organizing the presentation and to Firestormm for providing a transcript of this talk, and above all to Dr Ian Lipkin for taking time out to talk to patients.

If you would like to donate to Ian Lipkin's research, click here. In the comments box, put "for M.E/C.F.S Study" to make sure it goes to the right place.


Simon McGrath tweets on ME/CFS research:



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He only tested plasma for infection didn't he ?, and CSF ? He didn't test white blood cells. And there is activated immune system, both Th1 and Th2. I wouldn't throw out the infection impact just yet. Others are finding infections - this needs to be explained.
He has stated they havent even tested serology and they think they will find some answers there, so infections are still definately in.
 
Many of us do have symptoms of herpesvirus infections -- extreme fatigue, sore throat, swollen glands, and elevated antibody titres.
Those symptoms might not be herpes though, they aren't specific. Of course if you've tested positive then perhaps it is. My point was really not to argue against herpes infection though, it was to point out that secondary infection as a result of a broad immunodeficiency is unlikely, else we'd have a plethora of infections, some with very specific, easily identifiable symptoms.

Not all people with ME/CFS don't get colds. (Daughter and I are both fighting colds now, as a matter of fact) That seems to me to be more of a stage of the illness thing, although I could certainly be wrong. :) In my case, I went through a catching everything phase, an allergic MCS-type phase where I didn't catch common colds, and a more normal but still inclined to catch things phase.

I agree. And there has to be explaination for these immune characteristics from both sets of people. I don't think a broad immunodeficiency at any stage of illness is it though, else we'd be dying more and getting those obvious symptoms from some infections. If we are more prone to certain infections as a result our our immune dysfunction then perhaps infections such as herpes which can embed in our DNA are better placed to take advantage than other infections.

The immune system is far more complicated than up-regulated or down-regulated. It's far from a certainty that if one part fails, another part will compensate, or that if some compensation does occurs, the immune system can maintain that abnormal condition long-term.
Sure, but for the purpose of discussion and given our - well, certainty my - lack of detailed immune system knowledge, it's still useful to talk in such terms I think - particularly when talking in net terms as I was.
 
My point was really not to argue against herpes infection though, it was to point out that secondary infection as a result of a broad immunodeficiency is unlikely, else we'd have a plethora of infections, some with very specific, easily identifiable symptoms.
I don't think it's as simple as that. There are certainly people with severe immune deficiencies who are susceptible to everything -- think the "bubble boy" disorder. But if only part of the immune system is deficient, the person could be susceptible to a limited subset of pathogens, for example intracellular or extracellular pathogens. Also, a limited immune deficiency leaves the victim less able, but not completely unable to fight off infections.

HIV patients get different secondary infections at different stages of the illness because their immune systems can handle some infections early, but as the disease progresses their immune systems become more incapable and they get more and more infections. We could easily be in a analogous position to HIV patients in the earlier stages -- more susceptible to reactivations of herpesviruses, bacterial pneumonia, staph infections, candida, malaria, TB, but not so severely impaired that we suffer from every infection that comes our way.
 
Viruses may well set up the conditions for such feedback loops, such that the loops continue after the viral infection has resolved. But I don't see why the idea of an autoimmune disorder in the absence of a chronic infection is such a controversial issue.


On the one hand it is claimed attack on self for no resean.

On the other hand it was claimed no immune response what so ever to the infection I suspect. That has changed over the years. It is now know, there are immune responses but they do not show in the useal manner.
 
A POSSIBLE BACTERIA OR FUNGI EXPOSURE! LEPTIN HORMONE! IMMUNE INFLAMATION!
Take a look at DR?SHOEMAKER AND SPANOUGLE AND FINDING ON MOLD EXPOSURE! LEPTIN! IMMUNE INFLAMMATION! AND LEAKY GUT. It would make sense mold is an epidemic 25 percent of people have poor detox pathways.

http://www.survivingmold.com/diagnosis/the-biotoxin-pathway
http://sponauglewellness.com/wellness-programs/mold-toxicity/black-mold-used-in-weaponry/

Functional medicine world meets traditional medicine (please work together!)

Hi Dolche,

Have you tried Dr. Shoemaker's protocol? Do you know of anyone getting better on it?
Any ME/CFS patients that found relief with the Shoemaker protocol?
I'm really curious.
 
SOC said:
Not all people with ME/CFS don't get colds. (Daughter and I are both fighting colds now, as a matter of fact) That seems to me to be more of a stage of the illness thing, although I could certainly be wrong. :) In my case, I went through a catching everything phase, an allergic MCS-type phase where I didn't catch common colds, and a more normal but still inclined to catch things phase.​


I wonder if it makes sense to distinguish between “colds” and “flu.” From a symptom standpoint, influenza is more likely to involve the presence of significant fever in adults. Colds are more likely to commence with a sore throat and to involve a runny and/or stuffed up nose. Colds are generally milder than the flu.

All influenza is caused by RNA viruses of the family Orthomyxoviridae, but apparently only 10%-15% of colds are attributed to these viruses. Some 200 other viruses can cause colds.

In other words, if you were somehow very resistant to flu viruses you could still get colds, since 85%-90% of them are not caused by flu viruses.

I don’t know if such selective resistance is possible, but it would be interesting to know if CFS patients who get colds also get the flu with high fever.


[FWIW, my illness apparently commenced with a severe flu, but that was the last time I caught the flu (or a cold) for a decade.]
 
Proper accurate testing as well as well defined cfs study participants could account for there inability to find viruses. Doesn't seem to be a problem for lerner Peterson klimas, as well as being able to treat and improve many of them.

I hate to sound deragotory towards some of the viral testing and I'm seriously calling BS on some of it. I don't think you can test 225 or so people with cfs/me and only come up with 4 cases of EBV and a 2 cases of HHV-6. Whether they were looking a antibody test or viral dna I'm still concerned. It's known that copy numbers for HHV-6 are very low as they just kick the shit out of you when they come rolling through. The study just a few weeks ago showing EBV is now leaving a viral copy within the host cell while it is sends a complete new copy of the virus out to infect a new cell leaving the old alive and well and still infected.

One thing that sticks out at me from the research standpoint are they using this stud to bolster their new arrays that can see 500 different viruses utilizing a high throughput system. The research should find, or at least look at the pathogens and viruses instead from a researcher perspective.

Many of these samples are 4 years old from Dr. Montoya and I imagine Dr. Peterson as well. How many times have they been thawed and re-frozen and thawed again. I'm not doubting the arrays they are using, but is this the place to be using them. As I said before I don't think a 500 virus array in a high throughput system will find HHV-6 on a very consistant basis. I'm sorry, but I find it very, very hard to believe we as patients have such high levels of EBV and HHV6 antibodies checked in a research lab for HHV-6 anyway, but they can only find four cases on their super 500 virus array on a high throughput system.

Are they looking for a cure for cfs/me or are they testing their newly develop arrays? I'm confused to say the least and not trying second guess the researchers intentions, but something don't freaking add up and I would like to know why?

Who was it that recently stated that if you look to quick it is going to get missed. It was a reference on this forum by a doctor. I hope they find something that will slow them do.
 
Those who are complaining about persistant colds and flues since they developed ME/CFS ... are you sure that you have an infection, or are you having a reaction? Are you immune-suppressed or immune-reactive? As a long time sufferer of ME/CFS, I use to think that my symptoms, when I became extremely ill were because I had a virus or an infection (a down-regulated immune system). It was not until I began reading stories at a child daycare (germ factory), a couple of hours a day, that I realized, when I became very ill, it was not an infection or virus causing my symptoms, but that I was having a reaction (caused by an up-regulated immune system). I would become terribly congested; have a low-grade fever; sore throat; cough; suffer from headaches and extreme fatigue. By the end of the “work-week” I would feel so sick, that I use to think I had caught something from the children. The same thing would happen week, after week, but I would miraculously recover over the weekend. Eventually, I had to give up the position. As soon as I did, my problems with what I first thought was a virus or infection disappeared. I still have ME/CFS, but I am now quite aware when my symptoms become worse, I am reacting to something. I do not have a down-regulated immune system, but an up-regulated one. This knowledge has helped me to cope a with my illness.
 
Lipkin looked in plasma and spinal fluid, but the literature that I've just looked at suggests that HHV-6 would be better detected in lymphocytes or brain tissue:
HHV-6 active infections can persist in the brain tissue long after all traces of the virus have disappeared from the blood (Caserta 2004) and can be found in large quantities in the brain tissues with barely a trace in the spinal fluid (Fotheringham 2007).

http://hhv-6foundation.org/what-is-hhv-6/epidemiology-of-hhv-6

This study, that found HHV-6 in 70% of CFS patients, detected it in lymphocytes, not plasma:
http://www.ncbi.nlm.nih.gov/pubmed/1309285

Other research has suggested that HHV-6 is found in large numbers of the general population:
In a US study of lung tissue samples, both variants were found in 67% of transplant patients and 54% of controls (Cone 1996).

http://hhv-6foundation.org/what-is-hhv-6/epidemiology-of-hhv-6
 
Bob just to save me reading those papers - were they active infections that were found? Only that's what Lippers was a seeking in this first instance.

I think so, but it's only one study, and there has been plenty of conflicting HHV-6 research.
I'm not familiar with the wide range of HHV-6 research, as I've never taken a close interest in it.

Here's the results from the abstract, suggesting active infection:
Primary cell culture of lymphocytes showed active replication of HHV-6 in 79 of 113 patients (70%; CI, 61% to 78%) and in 8 of 40 controls (20%; CI, 9% to 36%) (P less than 10(-8], a finding confirmed by assays using monoclonal antibodies specific for HHV-6 proteins and by polymerase chain reaction assays specific for HHV-6 DNA.

http://www.ncbi.nlm.nih.gov/pubmed/1309285
 
I hate to sound deragotory towards some of the viral testing and I'm seriously calling BS on some of it.
Let me clarify a few points:
One thing that sticks out at me from the research standpoint are they using this stud to bolster their new arrays that can see 500 different viruses utilizing a high throughput system. The research should find, or at least look at the pathogens and viruses instead from a researcher perspective.
He says nothing in the transcript about using a 500-virus-detecting array. They screened uisng a panel of 20 viruses, bacteria and parasites. The high throughput approach, as I understand it, is unbiased ie looks for any virus based on the DNA/RNA sequence. However, Ian Lipkin did say his team had discovered over 500 viruses using this method (and you can't discover a new virus using a panel of known ones). The purpose of this stuy was not to test an array.

Many of these samples are 4 years old from Dr. Montoya and I imagine Dr. Peterson as well. How many times have they been thawed and re-frozen and thawed again.
Would be interesting to know if this had happened, or if the researchers had factored this in. I think that DNA viruses like herpes are fairly robust so would survive freezing/thawing cycles - a bigger risk is probably introducing contaminants and finding false positives.

As I said before I don't think a 500 virus array in a high throughput system will find HHV-6 on a very consistant basis. I'm sorry, but I find it very, very hard to believe we as patients have such high levels of EBV and HHV6 antibodies checked in a research lab for HHV-6 anyway, but they can only find four cases on their super 500 virus array on a high throughput system.
So far they have only reported on plasma viral studies, which will only detect viruses that are currently active. If people have dormant HHV6 and EBV (and EBV at least is prevalent but generally dormant), they won't find it in plasma. They have yet to report on the serology studies that should show up antibodies if the patient has a prior (or dormant) infection.

I note Bob study below that found high levels of HHV6 using cultured lymphocytes (rather than plasma) - but it was from 1992 and wonder if there have been a replication since? Still, it was a pretty big study.

It's quite possible that the serology work will find antibodies indicating past/dormant EBV and HHV6 infections. And I agree that it would be odd if they don't find EBV at least.

One final point. The virology work is a collaboration with Jose Montoya, an expert in HHV6 currently running a CFS pilot study treating HHV6. My guess is that Montoya would know how to detect HHV6. But maybe when the paper is published (should be soon) there will be lots of caveats about the limits of the techniques they used.