≡ Menu

Lipkin finds biomarkers not bugs

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…

Lipkin - CFI

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:

plasma and blood

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:

Join our volunteer authors, proof-readers, fundraisers, technicians, or our Board of Directors! Please contact Mark via the forum.​

{ 165 comments… add one }

  • Legendrew September 12, 2013, 12:23 pm

    Great article guys! Exciting to hear about the signs of immune activation and also interesting to me to hear about the increase in Leptin, i have to admit I overlooked it intially but reading the article it stand out to me as a very interesting upregulation to see. It makes a lot of sense to have upregulation of a molecule such as this given the evidence of mitochondrial and ATP production problems!

    I think it's worth mentioning too that these findings given important evidence for both the autoimmune theory that Fluge and Mella are working on and indeed the pathogen theory that Lipkin himself believes. Hopefully some of these abnormalities hold up to further scrutiny!

  • Sasha September 12, 2013, 12:57 pm

    Well done, Simon – great article! And well done, Russ – great transcript!

  • Simon September 12, 2013, 12:59 pm
    Legendrew

    Great article guys!

    …interesting to me to hear about the increase in Leptin, i have to admit I overlooked it intially but reading the article it stand out to me as a very interesting upregulation to see. It makes a lot of sense to have upregulation of a molecule such as this given the evidence of mitochondrial and ATP production problems!

    Thanks!

    Re Leptin:

    the blog

    Lipkin and colleagues also found upregulation in Leptin, a hormone that plays a key role in regulating energy uptake and use

    Yes, I was intrigued by that too, though I that line is pretty-much the extent of my knowledge of Leptin.

    I think it's worth mentioning too that these findings given important evidence for both the autoimmune theory that Fluge and Mella are working on and indeed the pathogen theory that Lipkin himself believes. Hopefully some of these abnormalities hold up to further scrutiny!

    Yes, and I think they key thing is replication, which is very much what Ian Lipkin plans to do. I have to say, these broad findings are probably consistent with many different theories so I will be very interested in what the future work turns out: certainly Ian Lipkin's main focus now seems to be to find what is responsible for these changes.

  • prioris September 12, 2013, 1:38 pm

    Overactive immune system has been known for at least 2 decades.

  • Antares in NYC September 12, 2013, 1:53 pm

    This is remarkable. I think it's interesting that this study makes ME/CFS somewhat similar to MS, particularly in the "hit and run scenario". A virus-like infection comes in shortly, disrupts the immune system, and it's all downhill from there.

    Also, how do we pressure for more research funds? How do we organize and lobby to get these researchers what they need?

  • Legendrew September 12, 2013, 2:04 pm
    prioris

    Overactive immune system has been known for at least 2 decades..

    Indeed it has but we now seem to be getting to the bare bones of the issue with what exactly is wrong with strong evidence to support these initial claims – if nothing else these findings are good clues as of where to look next!

    Sometimes finding nothing at all is the best outcome. In terms of viruses as a central causation, this somewhat conclusively shows little significance in the commonly held perpetrators which means we can move on to newer ideas without the baggage that these old ideas bring with them.

    An analogy I made in a previous thread earlier today seems to also be relevant here I feel with regards to the 'hit and run' idea many people discuss. The reference to autoimmunity is however just a personal opinion I hold and for the purpose of this topic should be interchangeable with immune activation.

    Legendrew

    As an analogy I believe the genetic predisposition is like having petrol liberally doused over an area – it's invisible (but likely a few small hints that go unnoticed) – it then only takes a spark in the form of a viral or bacterial trigger to start the immune activation and through exploitation of the genetic predisposition and suddenly a whole fire breaks out – this is when the symptoms first appear and suddenly it becomes much more difficult to manage. There is little point in identifying the initial virus or bacterial 'spark' as this does nothing to ease the fire. The only option then comes in calming the aberrant immune response and trying to break the autoimmune cycle.

    The interesting part here is the specifics of the immune activation – certainly not all of these will prove to be repeatable but if just a handful do we could be looking at a good trace of why the immune response has turned so aberrant.

  • Nielk September 12, 2013, 2:11 pm

    Thank you Simon and Firestormm for the hard work putting this clear review together and supplying a transcript for us. I had listened in to the meeting but, apparently missed a lot of the details.

    It is clear to me that Lipkin found enough abnormalities/biomarkers for him to be excited to plunge in deeper for further studies. Any study showing differences between patients and controls is promising. It remains to be shown/proven what this will lead to.

    Their finding of the three year marker showing differences in the results is intriguing. I wonder why three? As he stated though, they were not looking for that, it just has seemed to appear when comparing results.

    I wish I could understand in layman's terms what these abnormalities in cytokines mean or what this Leptin issue means.
    I almost wish I had gone through medical school in order to understand and follow these developments.:)

  • Waverunner September 12, 2013, 2:33 pm

    Great news! Thanks for the article. I truly hope that they advance the understanding of the disease mechanisms. It's fascinating that some inflammatory markers were even low. I also hope that microbiome analysis gets a lot cheaper through better implementation of computers and resulting automation.

  • Erik Johnson September 12, 2013, 2:59 pm

    In addition to regulating the hunger-response, Leptin controls the release of toxins from the fatty tissues.

    Lipkins findings of Leptin is consistent with "the elusive biomarker for CFS" that we have known about for about fifteen years now.

  • Legendrew September 12, 2013, 3:03 pm
    Erik Johnson

    In addition to regulating the hunger-response, Leptin controls the release of toxins from the fatty tissues.

    Lipkins findings of Leptin is consistent with "the elusive biomarker for CFS" that we have known about for about fifteen years now.

    With regard to Leptin, I think it is a very interesting find and it's more interesting that it has been found in the past. I'm not sure it will prove to be a causative agent in the disease pathology of ME but certainly it would be interesting to know why it appears to be getting up-regulated – I suspect it could be due to its role in metabolism considering the findings of dysfunctional mitochondria and Lactate build-up in the muscles – presumably through increased anaerobic respiration during trivial exertion.

  • roxie60 September 12, 2013, 3:17 pm

    Help in understanding, so is there an up- regulation in Leptin if you have CFS?

  • roxie60 September 12, 2013, 3:27 pm

    Checked SNPedia and there are Leptin related genes/snps for those who wish to travel down that rabbit hole.

  • Erik Johnson September 12, 2013, 3:27 pm

    As you might expect….It's complicated.
    Leptin stimulates MSH "Melanocyte Stimulating Hormone"
    Which in turn regulates the innate immune system.
    High Leptin and low MSH is seen in illnesses where toxins are stored in the fatty tissues.
    The high Leptin makes weight loss extremely difficult.

    Since CFS researchers have yet to make the connection between this phenomenon and CFS, the connection remains to be explored. But for Lipkin to see it makes it more likely that CFS researchers will take an interest.

  • roxie60 September 12, 2013, 3:41 pm

    I wonder if the genes associated with Leptin will be determined to play any role. For now I checked three of the SNPs associated with Leptin and I have the common variety so they dont explain my expereince. I am one of the 'fighting this battle longer than three years' group. I still suspect when I had mono in 1995 that was the start of it all even though I did not recognize the long periods between crash and recovery as being related. The last 6-7 years I know realize the unmistakable relation. I am just coming out of another crash (6-7 weeks) and there is a difference, more function (although not sure I'll ever get back to 100%), not as sick all the time, symps not as severe. I wish I understood this crash and recovery element to whatever is causing this :confused:. Do others also experience this crash and recovery I am describing? There are some symps I have all the time but when I crash I am more disabled, other symps added and the ones I usually have seem worse then like a cloud lifting I enter a day or week that I realize I am much better. It is so friggen madding the inconsistency. I blame that most for the reason I lost my career, how can I or my employer count on me when I dont know one day to the next if I can function. 😥 Sorry for the derail I just want answers, to heal and get my life back!!!!

  • Erik Johnson September 12, 2013, 3:58 pm

    The variability was in my quote from Dr Ramsay.

    Yes, I know what controls the crash phenomenon, and it is linked to leptin signaling.

  • acer2000 September 12, 2013, 4:45 pm

    It seems that he did find bugs. He reported that he found both anellovirus sequences and also retroviral sequences. He said he didn't know what it meant of if it would lead anywhere. Given the craziness that happened last time a researcher tried to associate a viral cause with CFS I don't blame him for being cautious and wanting more evidence before making any claims. But he did state he felt the cause still seemed like an infection and that all of the findings (bugs or not) should be followed up.

    Also, I have a hard time reconciling the claims about IL-8. Several other studies have shown IL-8 to be significantly elevated in patients with CFS. My own IL-8 is one of the most consistently elevated markers, and I have participated in several of these studies through my CFS doctor (including this one). I'll admit though, I don't know the intricacies of testing for cytokines. Perhaps they vary based on time of day, or processing method or something.

  • Antares in NYC September 12, 2013, 5:23 pm
    acer2000

    It seems that he did find bugs. He reported that he found both anellovirus sequences and also retroviral sequences. He said he didn't know what it meant of if it would lead anywhere. Given the craziness that happened last time a researcher tried to associate a viral cause with CFS I don't blame him for being cautious and wanting more evidence before making any claims. But he did state he felt the cause still seemed like an infection and that all of the findings (bugs or not) should be followed up.

    Also, I have a hard time reconciling the claims about IL-8. Several other studies have shown IL-8 to be significantly elevated in patients with CFS. My own IL-8 is one of the most consistently elevated markers, and I have participated in several of these studies through my CFS doctor (including this one). I'll admit though, I don't know the intricacies of testing for cytokines. Perhaps they vary based on time of day, or processing method or something.

    Acer2000:

    You bring a very good point. I noticed he mentioned the anellovirus and traces of some retrovirus, but qualified it quickly as needeing more research. After the xmrv debacle, you can't blame any researcher for being utterly cautious.

    I think the title is not necessarily misleading, but leaning heavily on Dr Lipkin's cautious approach to the findings.

  • Forbin September 12, 2013, 6:17 pm

    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”.

    Dr. Lipkin is even more emphatic on this earlier in the call…

    "My own interest in Chronic Fatigue Syndrome dates back to the mid-to-late 1990’s when I was working with Brigette Evangard, and one of the questions that came in earlier today had to do with polyclonal B cell activation. Those of you who may have read that paper that I wrote with Brigettea, which was ultimately published in 1999, was one of the first to really draw attention to the fact that people with Chronic Fatigue Syndrome who were diagnosed using the criteria at the time did in fact have a physical condition.

    "Although we were unable to find evidence of infection with borna disease virus, which was the primary question at that point, we did demonstrate unequivocally that the vast majority of people who met these criteria had polyclonal B cell activation, so they clearly had some sort of immune activation. But the question was, why?"

    [my bolding]

  • bel canto September 12, 2013, 7:46 pm

    Montoya's recently released results from a small (10 participants, I believe) study: They obtained blood draws for 25 consecutive days from these patients, who also, I believe, kept journals indicated their fatigue levels. The only finding that correlated directly with fatigue levels in most (not all) of these patients was the hormone leptin. I think that the overall levels were not abnormal, but the changes in leptin tracked the fatigue level changes.
    I imagine the details of this are discussed somewhere in the forum, but I haven't had a chance to track it down yet.

  • Nielk September 12, 2013, 7:59 pm
  • bel canto September 12, 2013, 8:09 pm

    Yes, thanks Neilk!

  • searcher September 12, 2013, 9:25 pm

    As bel canto said, what was interesting about the Montoya et. al study was that leptin wasn't particularly high– instead leptin levels correlated with fatigue in patients but not in controls. Leptin levels themselves do not appear to be a useful biomarker– they will be higher in overweight people even without fatigue.
    From Montoya's paper:
    "The relationship we observed between leptin and fatigue existed even though leptin levels were not abnormally elevated, and there was no statistical difference in leptin values between the CFS and control groups. "
    So, although I think Shoemaker's work is interesting and relevant to CFS patients, I don't think he has made the same conclusion about leptin that Montoya and his team has.

    The Montoya paper on leptin can be found at http://www.translational-medicine.com/content/pdf/1479-5876-11-93.pdf

  • HowToEscape? September 12, 2013, 11:47 pm
    Antares in NYC

    This is remarkable. I think it's interesting that this study makes ME/CFS somewhat similar to MS, particularly in the "hit and run scenario". A virus-like infection comes in shortly, disrupts the immune system, and it's all downhill from there.

    Also, how do we pressure for more research funds? How do we organize and lobby to get these researchers what they need?

    By organizing.

    There is a template for that, the AIDS folks were very well organized and targeted specific actions within a plan. If we just wait for "them" to "do something", we'll wait till we're old and eventually dead.

  • GcMAF Australia September 13, 2013, 12:46 am

    did he look at Lyme??

  • Simon September 13, 2013, 2:43 am
    Antares in NYC

    This is remarkable. I think it's interesting that this study makes ME/CFS somewhat similar to MS, particularly in the "hit and run scenario". A virus-like infection comes in shortly, disrupts the immune system, and it's all downhill from there.

    Also, how do we pressure for more research funds? How do we organize and lobby to get these researchers what they need?

    I think it's worth stressing that the 'hit and run' scenario discussed by Mady Hornig is, at this point, only suggeseted as a possiblity for ME/CFS – and they don't yet have any resultf from their work looking for 'shadows' of previous infections.

    I'm not in the US so don't have any idea of how to press for funds – but it might be worth posting something in the advocacy forum (I couldn't see anything there yet). There is probably discussion of it on the CDC phonecall thread too, but it's rather lengthy.

    Nielk

    Their finding of the three year marker showing differences in the results is intriguing. I wonder why three? As he stated though, they were not looking for that, it just has seemed to appear when comparing results.

    That seems a pretty weird finding to me too: it almost implies some sort of shift in the illness after 3 years, which is hard to fathom. Hopefully things will be clearer when the new paper is published, which Ian Lipkin said would be 'very soon'.

    acer2000

    It seems that he did find bugs. He reported that he found both anellovirus sequences and also retroviral sequences. He said he didn't know what it meant of if it would lead anywhere. Given the craziness that happened last time a researcher tried to associate a viral cause with CFS I don't blame him for being cautious and wanting more evidence before making any claims. But he did state he felt the cause still seemed like an infection and that all of the findings (bugs or not) should be followed up.

    Also, I have a hard time reconciling the claims about IL-8. Several other studies have shown IL-8 to be significantly elevated in patients with CFS. My own IL-8 is one of the most consistently elevated markers, and I have participated in several of these studies through my CFS doctor (including this one). I'll admit though, I don't know the intricacies of testing for cytokines. Perhaps they vary based on time of day, or processing method or something.

    He did find some bugs, but said the Anellovirus was not specific to CFS (ie CFS were not greatly different from controls). It sounds like the retroviral thing was the same in that they found it in 'pooled samples'. Hopefully things will be clearer in the full paper.

    Do you have any refs/links re the elevated IL8 findings elsewhere? Would be good to compare.

    Antares in NYC

    I think the title [Lipkin finds Biomarkers not Bugs is not necessarily misleading, but leaning heavily on Dr Lipkin's cautious approach to the findings.

    I have to admit I took a slight liberty with the title, favouring snappiness over total accuracy – though I think it reflects the gist fo the talk.

  • Simon September 13, 2013, 2:52 am
    Nielk

    I wish I could understand in layman's terms what these abnormalities in cytokines mean or what this Leptin issue means.

    Wish I could too! Broadly, I think they are signs of immune activation. Intriguingly, when it comes to the cytokine findings in spinal fluid

    Ian Lipkin

    this is compatible with a profile of some particular types of response that may provide insights into immunological dysregulation in Chronic Fatigue Syndrome.

    He didn't expand on this and I wish knew more, but clearly Ian Lipkin thinks this could be an important clue.

  • justy September 13, 2013, 2:53 am

    The three year marker difference is fascinating and when it is unraveled may explain why it is often said to those newly ill that the best chances of a full recovery are in the first 2-3 years. It is well known that after this (approximate) time period the illness can become chronic/relapsing/remitting.

    Thanks Simon and Firestormm for the article and the transcript – very much appreciated :thumbsup:

  • Simon September 13, 2013, 3:09 am

    Leptin

    Not surprsingly this has caused a lot of comment

    bel canto

    Montoya's recently released results from a small (10 participants, I believe) study: They obtained blood draws for 25 consecutive days from these patients, who also, I believe, kept journals indicated their fatigue levels. The only finding that correlated directly with fatigue levels in most (not all) of these patients was the hormone leptin. I think that the overall levels were not abnormal, but the changes in leptin tracked the fatigue level changes.
    I imagine the details of this are discussed somewhere in the forum, but I haven't had a chance to track it down yet.

    Leptin appears to have a role in signalling to the brain, is directly affected by the immune system and ties in with the HPA axis too (post by jeffrez). So it ticks all the boxes (but then so do many things).

    The full text of Montoya's new paper (from earlier this year) is here: Daily cytokine fluctuations, driven by leptin, are associated with fatigue severity in chronic fatigue syndrome: evidence of inflammatory pathology

    ABSTRACT

    Background
    …Studies have demonstrated elevated levels of inflammatory factors in patients with CFS, but findings are contradictory across studies and no biomarkers have been consistently supported. Single time-point approaches potentially overlook important features of CFS, such as fluctuations in fatigue severity. We have observed that individuals with CFS demonstrate significant day-to-day variability in their fatigue severity.

    Methods
    …we implemented a novel longitudinal study design to investigate the role of cytokines in CFS pathophysiology. Ten women meeting the Fukuda diagnostic criteria for CFS and ten healthy age- and body mass index (BMI)-matched women underwent 25 consecutive days of blood draws and self-reporting of symptom severity. A 51-plex cytokine panel via Luminex was performed for each of the 500 serum samples collected. Our primary hypothesis was that daily fatigue severity would be significantly correlated with the inflammatory adipokine leptin, in the women with CFS and not in the healthy control women. As a post-hoc analysis, a machine learning algorithm using all 51 cytokines was implemented to determine whether immune factors could distinguish high from low fatigue days.

    Results
    Self-reported fatigue severity was significantly correlated with leptin levels in six of the participants with CFS and one healthy control, supporting our primary hypothesis. The machine learning algorithm distinguished high from low fatigue days in the CFS group with 78.3% accuracy.

    Conclusions
    Our results support the role of cytokines in the pathophysiology of CFS.

    The obvious problem with this is that it's a tiny sample. In any event 78% accuracy in separating high from low fatigue days in CFS isn't too great – and is tweaked for this specific sample so would probably be lower in an independent sample. And 6/10 isn't stunning either (though could be subgroups).

    However, it is a really interesting approach and could be consistent with the Lipkin results. As ever, replication is needed.

    PR thread discussing this paper

  • Firestormm September 13, 2013, 4:38 am

    Replication of Lipkin's preliminary work?

    It is unclear at this point, the extent to which this might (at least in part), represent some means of replication for the Lipkin Study we reported on above; but the NIH grant to the UK Biobank team, led by Luis Nacul, does show some similarity to those areas studied by Lipkin's team:

    It will be a 3 year longitudinal study I believe, ending in 2016, and a significant one at that – though only using plasma and no CSF but the controls will comprise MS patients (as well as healthy) and the ME patient cohort will include those in the severe category:

    DESCRIPTION (provided by applicant):

    A longitudinal study will be conducted of clinical presentation, immune phenotype, gene expression and virus infection among ME/CFS patients and MS and population controls frequency-matched by geographical area of residence, age-group (within 5 years), and sex.

    Clinical samples will be collected for studies of NK cell function virology (herpesvirus infection), and gene expression and for banking as a resource for future ME/CFS research.

    Hypothesis:

    ME/CFS is associated with immune dysfunction, which results from – or predisposes to – herpesvirus infections. Immune dysfunction will present as alterations in NK cell function that may lead to, or result from, alterations in cytokine production and altere expression of diverse immune-associated genes.

    Finally, we predict that the ME/CFS immune phenotype may fluctuate over time and in association with clinical presentation and that the majority of patients clinically characterized as having ME/CFS will show a biosignature distinct from that of controls, and that further alterations will be seen during episodes of clinical exacerbation.

    Activities and objectives:

    i) Collect clinical and other data and venous blood samples at baseline and 6 months or at another time during the 6-month follow-up period when there is a perceived "significant" deterioration in symptoms;

    ii) Analyze blood samples for NK cell phenotype and function;

    iii) Screen samples for evidence of herpesvirus infection and viral load, focusing on Epstein Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus-6 (HHV-6);

    iv) Describe clinical phenotype and fluctuations over time;

    v) Correlate the presence of symptoms and severity with markers of virus activity and immune function;

    vi) Investigate gene expression profiles associated with ME/CFS and how they vary in relation to changes in disease severity, virus activity, NK cell function, and other markers of immune function;

    and

    vii) Securely store blood samples from patients and controls, anonymously linked to clinical and other data, as an open resource for researchers to conduct ethically-approved studies of ME/CFS.

    Recruitment:

    150 ME/CFS cases (50 severe, 100 non-severe), 75 MS controls, and 75 healthy controls will be recruited.

    For immunology and virology, 100 cases, 50 MS controls, and 50 healthy controls will be sampled at 2 time points.

    For gene expression, we will analyze 50 ME/CFS cases, 25 MS and 25 healthy controls, each at recruitment and one follow-up.

    Cases will be selected from UK ME/CFS Disease Register and NHS ME/CFS specialty and primary care services in London and Norfolk, Suffolk, and Great Yarmouth and Waveney, UK; MS controls via the NHS; and healthy controls will be identified by ME/CFS patients (excluding blood relatives) or GPs.

    Outcomes:

    Identification of putative biomarkers for diagnosis, severity, and prognosis of ME/CFS, which can be evaluated in larger (ideally prospective) future studies.

    In the long term, identification of robust biomarkers will allow clinicians to correlate ME/CFS phenotype (including clinical presentation, genetic, immune, and viral markers) with disease severity and prognosis and may reveal new options for interventions research.

  • Simon September 13, 2013, 5:11 am

    Thanks

    Hope they take a good look at Lipkin's work first! He looked for several herpes viruses and found almost nothing. He was looking at plasma not NK cells, but if NK cells were infected I would have thought that some of the herpes virus would have been detectable in plasma. Lipkin also didn't include the severely-affected, but he has looked at 285 samples already with another 200 to go. IF they come back negative too that would surely be a concern for the biobank team?

    But still good to see so many chunky studies appearing, and I'm sure the biobank study will generate lots of valuable data and should confirm (or not) the Lipkin findings, even if they, like he, find no herpes.

    Firestormm

    Replication of Lipkin's preliminary work?

    It is unclear at this point, the extent to which this might (at least in part), represent some means of replication for the Lipkin Study we reported on above; but the NIH grant to the UK Biobank team, led by Luis Nacul, does show some similarity to those areas studied by Lipkin's team:

    It will be a 3 year longitudinal study I believe, ending in 2016, and a significant one at that – though only using plasma and no CSF but the controls will comprise MS patients (as well as healthy) and the ME patient cohort will include those in the severe category:

  • heapsreal September 13, 2013, 5:31 am
    Simon

    Thanks

    Hope they take a good look at Lipkin's work first! He looked for several herpes viruses and found almost nothing. He was looking at plasma not NK cells, but if NK cells were infected I would have thought that some of the herpes virus would have been detectable in plasma. Lipkin also didn't include the severely-affected, but he has looked at 285 samples already with another 200 to go. IF they come back negative too that would surely be a concern for the biobank team?

    But still good to see so many chunky studies appearing, and I'm sure the biobank study will generate lots of valuable data and should confirm (or not) the Lipkin findings, even if they, like he, find no herpes.

    I dont think thats correct in that he found almost none of the herpes viruses. From what i heard of the talk, hhv6 was mentioned but im not sure if he is referring to active infections or not.

    Another thread people have said that its not 'the' cause, which is what he means. Im not even sure if he is referring to a particular test as in spinal fluid? herpes viruses do tend to favour nervous tissue? I think we need some clarrification on this as it does go against what alot of other cfs experts have found and although not everyone has had benefits from treating herpes viruses, it does seem to be a stand out sub set that seems to show some improvement with treatment.

    I would also like to no if they tested for herpes viral particles and or lytic herpes viruses which are mentioned alot by dr lerner?? to me it makes sense that these viruses would be an issue if ones nk function is low, but??

    The whole study hasnt really told us much other then they think theres some infectious cause and there are possible cytokine markers and they need to do alot more work and dont have the money to do it yet.

  • Firestormm September 13, 2013, 5:36 am
    Simon

    Thanks

    Hope they take a good look at Lipkin's work first! He looked for several herpes viruses and found almost nothing. He was looking at plasma not NK cells, but if NK cells were infected I would have thought that some of the herpes virus would have been detectable in plasma. Lipkin also didn't include the severely-affected, but he has looked at 285 samples already with another 200 to go. IF they come back negative too that would surely be a concern for the biobank team?

    But still good to see so many chunky studies appearing, and I'm sure the biobank study will generate lots of valuable data and should confirm (or not) the Lipkin findings, even if they, like he, find no herpes.

    Yeah, but if NK cells are not infected then it will serve to also rule out Herpes virus. So another confirmation of a poor theory – at least in so far as current infections go. Doesn't rule out this 'shadowing' or trigger concept of course.

    And I think it is pretty safe to say that the UK Team are aware of Lipkin's presentation by now :)

    If their study is really only 3 years (I took that figure from the NIH link but it isn't certain and is not mentioned in the actual text), it would be a shame; as they have said they will be looking to:

    v) Correlate the presence of symptoms and severity with markers of virus activity and immune function;

    And that could have resulted in a similar differential to what Lipkin was tentatively suggesting at the 3 year point. That's my presumption of course – because as you know I ain't no scientific nerd :alien: 😀

  • heapsreal September 13, 2013, 5:43 am

    Serology, which is looking for evidence of previous infections, which I think much of what we need to do in the future must focus, is not part of this report that I am making to you today.

    This is in relation to all the viral testing done by lipkin and his crew which might explain the low incidence of viruses??
    Rereading the lipkin report it also says that the agent must be present in the plasma or spinal fluid as well as not being able to detect historical infections(whatever that means).

    http://www.mecfsforums.com/wiki/Lipkin_presentation,_CDC_Conference_Call_9/10/2013

  • Firestormm September 13, 2013, 5:45 am
    heapsreal

    I dont think thats correct in that he found almost none of the herpes viruses. From what i heard of the talk, hhv6 was mentioned but im not sure if he is referring to active infections or not.

    Another thread people have said that its not 'the' cause, which is what he means. Im not even sure if he is referring to a particular test as in spinal fluid? herpes viruses do tend to favour nervous tissue? I think we need some clarrification on this as it does go against what alot of other cfs experts have found and although not everyone has had benefits from treating herpes viruses, it does seem to be a stand out sub set that seems to show some improvement with treatment.

    I would also like to no if they tested for herpes viral particles and or lytic herpes viruses which are mentioned alot by dr lerner?? to me it makes sense that these viruses would be an issue if ones nk function is low, but??

    The whole study hasnt really told us much other then they think theres some infectious cause and there are possible cytokine markers and they need to do alot more work and dont have the money to do it yet.

    The point about HHV-6 was made very clear in both the transcript and Simon's review (see above).

    From the transcript:

    They found in the serum from ME patients and controls forwarded by Montoya:

    So, when we look at the samples that we received and there we have 285 cases and 201 controls, that is to say 285 people with Chronic Fatigue; we found HHV-6 in 4 cases and in 2 controls – that is 1.4% of the cases and 1% of the controls. We then typed these HHV-6 samples and found that they were 4 for HHV-6 Type B, and 2 for HHV-6 Type A.

    So, HHV-6 at least with respect to using the methods we have for direct detection of infectious agents, if it accounts for Chronic Fatigue, it will do so in a very small proportion. I should also add that Professor Montoya is a known expert in Human Herpes Virus 6 and there is the possibility that some people may have been preselected in some fashion. It’s a potential confound but in any evident I don’t think it is likely to account for much of what we see there.

    The other samples that were assayed, using these methods, did not reveal any of the agents to which I have referred.

    These same samples were studied using high throughput sequencing, which is a method that was really pioneered here – with which we have discovered over 500 viruses – so we feel fairly confident that to the extent of the technology’s capabilities at present; we would have detected everything that would have been present within these samples.

    And in the CSF from Peterson:

    Using the same approach as I have described (the multiplex panels I have already talked about with respect to Montoya’s work); we found no genetic evidence of microbial infection in those spinal fluid samples.

    We had 1 patient who had HHV-6 B, but that’s a single patient out of 60 and I don’t consider that a statistically significant finding. Now it is possible that if we think that Chronic Fatigue has many different possible causes, that there may be a small subset of people who do have disease as a result of infection with single agents; but this is certainly not going to be a consistent theme that is going to be helpful to us in terms of diagnostics at this point.
    We have not yet completed the ion tronic sequencing, so I can’t tell you what result we will obtain with that work.

    That's the only references to HHV-6 in the presentation.

  • heapsreal September 13, 2013, 5:56 am

    I understand hhv6 but not much is mentioned of other herpes viruses. As mentioned in my last post it appears that he thinks theres more to learn from serology which they didn't do during these tests. Hhv6 may get a different result if serology testing was used??

  • Simon September 13, 2013, 6:48 am
    heapsreal

    I dont think thats correct in that he found almost none of the herpes viruses. From what i heard of the talk, hhv6 was mentioned but im not sure if he is referring to active infections or not.

    heapsreal

    I understand hhv6 but not much is mentioned of other herpes viruses. As mentioned in my last post it appears that he thinks theres more to learn from serology which they didn't do during these tests. Hhv6 may get a different result if serology testing was used??

    Hi heapsreal

    Serology is testing for antibodies against a virus, 'looking for shadows', as Lipkin put it. Primarliy they are using this technique to find signs of prior infection – however, I think if an active infection is in a particular tissue, but not blood, the anti-virus antibodies (eh anti-HHV6) could still be in the blood. However, I'm not sure if herpes virus are generally not detectable in the blood – I'd just assumed they were.

    Firestormm has already mentioned the low levels of HHV6 found in patients and controls (nb this was a very large and well-characterised cohort, probably much bigger than anything that has gone before). However, they also specifically tested for 4 other human herpes viruses: Cytomegalovirus, Herpes Simplex 1 & 2, and Epstein Barr virus (see transcript). HHV7 and Varicella zoser virus are the only other 2 human herpes viruses they didn't test for specifically, but the hightroughput 'unbiased' testing they did should have detected those if they were there (as I understand it).

    Personally, I think we are learning a lot from this study because it is so large and robust – and there is much more to come, in the serology results and the unbiased search for fungi and bacteria.

  • alex3619 September 13, 2013, 8:37 am
    heapsreal

    I understand hhv6 but not much is mentioned of other herpes viruses. As mentioned in my last post it appears that he thinks theres more to learn from serology which they didn't do during these tests. Hhv6 may get a different result if serology testing was used??

    I think Lipkin looked for active infections for something like five herpes viruses, which are listed in his transcript. The point is he is looking for signs of acute infection. Serological evidence of past infection can be problematic, and typically this means looking for antibodies I think.

    None of these address non-acute infections which are part of the lifecycles of some viruses, including herpes and enteroviral families. These are the tissue infections that keep being mentioned. From my current understanding, nearly all these types of viruses can infect B cells.

  • alex3619 September 13, 2013, 8:48 am

    On Leptin and Serpin I am starting to look into this more closely, though I looked at leptin more than a decade ago. There are many links here with ME pathophysiology. Two that intrigue me at the moment is that leptin can induce hypothalamic inflammation and insulin resistance. If you are obese, have high insulin, glucose or triglycerides; or have disordered stress response or sleep, then leptin could be involved. Its worth some more investigation.

    Serpin is a family I think and not one hormone? It can be involved in regulation of other factors, including energy production. I have barely read anything on this though.

  • Gijs September 13, 2013, 8:54 am

    Business as usual. Nothing has been found. Sound familiar to me. I think the cause of this disease will never been found. It is to complicated. The cytokins Lipkin found are not consistent with earlier findings in other reports.

    For example IL17 are not different by CFS patients compared with controls.

    J Transl Med. (2012) 10(1): 191 Cytokine expression profiles of immune imbalance in post-mononucleosis chronic fatigue
    Broderick G, Katz BZ, Fernandes H, Fletcher MA, Klimas NG, Smith FA, O’Gorman MR, Vernon SD, Taylor R

    There are more reports with different outcomes then Lipkin e.a.

    I do not think Lipkin has found a usefull biomarker or profile.

    Sorry.

  • roxie60 September 13, 2013, 9:12 am
    alex3619

    On Leptin and Serpin I am starting to look into this more closely, though I looked at leptin more than a decade ago. There are many links here with ME pathophysiology. Two that intrigue me at the moment is that leptin can induce hypothalamic inflammation and insulin resistance. If you are obese, have high insulin, glucose or triglycerides; or have disordered stress response or sleep, then leptin could be involved. Its worth some more investigation.

    Serpin is a family I think and not one hormone? It can be involved in regulation of other factors, including energy production. I have barely read anything on this though.

    Do you have links Alex to the Leptin info you reference? TIA

  • Sparrowhawk September 13, 2013, 9:19 am

    I thought Dr. Chia's main point is we're not going to find the viruses unless we're also testing a broad set of tissue samples, kind of to heapsreal's points above about the herpes family of viruses. Yes it's great they are testing cerrebrospinal and plasma, but if there is something we don't catch there, and it's already in all your nerve cells in various tissues, GI tract, etc. their "new happy home" then you may not find the usual viral counts or antibodies because the body can't fight them once they are in the nerve cells?

    I do find the new direction on gut biome encouraging. If we can at least strengthen that area with some of their findings, that should do a lot to give the body a fighting chance to correct any other imbalances on its own.

  • Bob September 13, 2013, 10:58 am
  • Simon September 13, 2013, 11:28 am
    Gijs

    Business as usual. Nothing has been found. Sound familiar to me. I think the cause of this disease will never been found. It is to complicated. The cytokins Lipkin found are not consistent with earlier findings in other reports.

    For example IL17 are not different by CFS patients compared with controls.
    [Cytokine expression profiles of immune imbalance in post-mononucleosis chronic fatigue]
    Broderick G, Katz BZ, Fernandes H, Fletcher MA, Klimas NG, Smith FA, O’Gorman MR, Vernon SD, Taylor R

    There are more reports with different outcomes then Lipkin e.a.

    I do not think Lipkin has found a usefull biomarker or profile. Sorry.

    Thanks. I do get the impression that these new cytokine findings don't quite fit with earlier ones. That said, generally cytokine findings are all over the place in this field.

    Re that Broderick paper: it's very interesting in that it looks at cytokines over time in 300 adolescents who had glandular fever, and compares those that recover with those that get CFS. That's a strong experimental design – except there are not many CFS cases to go at. There were 22 CFS cases at 24 months and just 13 at 12 months – compare that with 285 for the Montoya sample in this study.(Though maybe those figures are not so different from the '3 years and under' subgroup in the Lipkin study?).

    What I would particularly like to see is how consistent findings are between patients eg are patients consistently low for IL2,8 &17 – or are some low for 2, others for 8 etc. And also how big the differences are between patients and controls? Certainly for other studies there has been a high degree of overlap between patients and controls despite group differences. Will be good to see the paper when it's published.

  • Nielk September 13, 2013, 11:31 am

    These abnormalities could just be an outcome from the illness – not necessarily a causative agent.

  • Legendrew September 13, 2013, 11:56 am
    Nielk

    These abnormalities could just be an outcome from the illness – not necessarily a causative agent.

    I think there's little doubt that cytokines are not the causative agent – to me they stand out more as footsteps in the sand which are worth following. Abnormal levels of cytokines mean disruption of normal immune function and possibly abnormal and harmful immune loops. The true question is whether these loops are perpetuated by the continuing presence of a pathogen or are self-perpetuating as is seen in autoimmune activation.

  • voner September 13, 2013, 11:57 am

    Simon, Firestormm….. Thank you for that very well written article And the transcript. you are providing a valuable service to this community.

    I Wonder how the Lipkin study fits in with the autoimmune/rituximab info? I guess we'll see what Dr. Edwards has to say about the transcript information….

    i also wonder if Dr. Chia is ever going to get money to do his tissue sample studies. He is certainly a Lonewolf out there.

  • wastwater September 13, 2013, 12:13 pm

    I wonder if hhv6a aka human b cell lymphotropic virus or African swine fever virus was tested for

  • wastwater September 13, 2013, 12:38 pm

    I followed a link on anellovirus and it came up with torque teno virus,it is a very common virus

  • alex3619 September 13, 2013, 12:47 pm
    roxie60

    Do you have links Alex to the Leptin info you reference? TIA

    Its everywhere. I just gave a summary of a points on a few papers. Run a search and you will be overwhelmed with links. If I find really good review articles I will post those however.

    One key word you might like to know is hyperleptinemia. High leptin has its own name.

    PS Here is a paper that shows in mice some of the impact of hyperleptinemia can be reversed by resveratrol:

    http://edrv.endojournals.org/cgi/content/meeting_abstract/33/03_MeetingAbstracts/SUN-146

    PPS. My initial brief survey found no good reviews of leptin. Instead many papers will discuss it in their opening sections, and some will review a particular aspect of leptin. For example, the following paper discusses in its introduction the impact of leptin on the immune system, but the primary focus is breathing problems:

    http://respiratory-research.com/content/pdf/1465-9921-11-152.pdf

    There are so many links with possible ME issues that a word of caution is advised: these are all possibilities, but they require a lot of research to validate and test them. For example, hypoxia is known to induce increased leptin levels. However leptin also interferes with NO, inducing vasoconstriction. Its all very complicated.

  • wastwater September 13, 2013, 12:59 pm

    Another thing I noticed is Chromosomally integrated HHV-6 about the same as autism rates.