Here is a transcript.

Enjoy.

Men who have excessive faith in their theories or ideas are not only ill prepared for making discoveries; they also make very poor observations. Of necessity, they observe with a preconceived idea, and when they devise an experiment, they can see, in its results, only a confirmation of their theory. In this way they distort observation and often neglect very important facts because they do not further their aim….

Claude Bernard, An Introduction to the Study of Experimental Medicine

This article discusses our experience at the one-day Institute of Medicine workshop on vitamin D and calcium. Both of us had an opportunity to make comments before the committee. Here are Paul’s comments and slides and here are Amy’s comments and slides. Note that our 2009 paper in Autoimmunity Reviews discusses some of the science we allude to in further detail.

On the cab ride to the IOM committee meeting on whether to change the dietary reference intake (DRI) of vitamin D, Amy practiced her speech.

The cabbie had been silent for the whole ride, but broke character by talking to us. “So, let me ask you a question,” he said. “Do you take vitamin D?”

“Actually, no, we don’t,” Amy said. Amy explained briefly how our data suggests that the form derived from supplementation is immunosuppressive, meaning that while it may temporarily improve signs and symptoms of disease, we have found it may do so at the cost of long-term health.

We asked him if he took vitamin D. He said yes and explained that a few years back, he had a partially blocked artery. It scared him, so he searched the internet and found that high doses of vitamin D were being recommended for cardiovascular disease. He wasn’t clear about the evidence, but in his words, “I had to do something.”

Which brings us to this point in time. At least in the United States, rates of chronic disease are rising. One recent study predicted that if current trends continue, all Americans will be obese by 2040. Other studies have shown chronic disease is rising at rates faster than could otherwise be explained by an aging population and/or a general increase in population. One recent estimate says that by 2030, 171 million Americans will have a chronic disease. We have to do something, right?

A committee to evaluate the DRI of vitamin D is convened

The Institute of Medicine (IOM) is a non-profit organization that was first chartered in 1970. In 2008, IOM appointed a committee of experts whose charge is to reevaluate the DRI of calcium and vitamin D in light of recent research. The committee is expected to produce a report including these recommendations scheduled to be publicly released in May 2010.

An IOM committee with the same purpose last met in 1997 and set the current standard of 400 IU of vitamin D per day for adults. But none of the members of the previous committee are on the current committee despite, collectively, hundreds of MEDLINE citations to their names. Perhaps this suggests that the IOM was trying to exclude scientists who most vocally tout vitamin D’s benefits from the committee.

A great deal has happened since 1997. We learned that hormone replacement therapy (HRT) can cause disease (which led to thousands of premature deaths) even while early observational studies seemed to quite erroneously suggest the opposite. Also, evidence-based medicine has come of age.

For those who are not from this planet or from a Western country anyway, it’s hard to really express how enthusiastic the support for vitamin D supplementation is – at least in the popular media. A quick search of Google News for “vitamin D” has led us to conclude that the few articles that allude to vitamin D’s risks are vastly outnumbered by stories repeating the same unchallenged claims about vitamin D’s perceived benefits.

As part of their deliberation process, the IOM committee commissioned a report by the Tufts Evidence-based Practice Center. For this report, the Tufts group used a pre-existing set of criteria to identify only those studies meeting a certain standard of validity. Those studies that made the cut were independently analyzed.

According to the report’s abstract: “The majority of the findings concerning vitamin D, calcium, or a combination of both nutrients on the different health outcomes were inconsistent.” For a variety of diseases, the report repeatedly finds few or no controlled studies showing an association between vitamin D intake and disease.

Interestingly, Dr. Boullion, the sole speaker at the meeting from Europe (Belgium) conceded that he was confident that the European Union would not raise its recommendations regarding vitamin D intake based on vitamin D research to date.

The complete list of presentations including audio and slides is available on the IOM website.

Dr. Barry Kramer sounds an early note of caution

Arguably the most illuminating speech of the day came before lunch. Dr. Barry Kramer, MD, MPH, works in the Office of Disease Prevention, a division of the NIH. His speech was somewhat dryly titled, “Weighing Scientific Evidence” (PDF of slides) but might just as well have been titled, “Hey, wait a second.”

Invoking the work of Leon Gordis, PhD, Dr. Kramer discussed the “Levels of Decision Making,” and how the requisite amount of evidence for a non-conservative (our word) medical decision increases as the number of people it would affect increases. In other words, a person must make decisions for one’s family or even groups of patients with a different standard of evidence than he or she would when making decisions on behalf of the entire nation and possibly the world.

The evidence-based pyramid. Higher levels of the pyramid have higher levels of validity. Note that while Dr. Kramer’s evidence-based pyramid contains a section for ideas and opinions, many evidence-based pyramids make no such provision.

Dr. Kramer argued that some levels of evidence are not sufficient – at least not to make decisions on behalf of millions. The evidence must meet a minimum standard of validity: randomized controlled studies (RCTs), if not double-blind, placebo-controlled RCTs. According to Dr. Kramer, the history of research has shown in the cases of high-dose paclitaxel, encainide/flecainide, torcetrapib, and HRT, of course, that confounding variables have a way of compromising researchers’ most certain conclusions.

A good example of a confounding variable is smoking in alcohol’s relationship with lung cancer. Alcohol consumption is strongly correlated with lung cancer, but only because people who drink are also more likely to smoke. Another commonly cited example: Volvos may be involved in fewer accidents, but that’s probably because people who choose to drive them are generally older and more safety-conscious.

Dr. Kramer said in the case of observational studies with a relative risk of less than two, he could “spit them [confounding variables] out at the rate of one a second.” His slide lists a few obvious confounders for vitamin D studies: health consciousness, health insurance, and access to care.

Dr. Kramer also made what should be an obvious point: surrogate outcomes do not substitute for reductions in mortality or disease. A surrogate outcome is a variable that is a substitute for a “true outcome”, used because it is easier, quicker or cheaper to measure – and the most common one used in vitamin D studies is serum 25-D although bone mineral density, polyps, and PTH levels are also used. But Dr. Kramer said that none of these surrogate outcomes, in his words, “measure up.”

At the end of the speech Dr. Kramer showed the audience a classic Far Side cartoon, explaining, “Especially when you’re dealing with public health issues and millions of people, it pays you not to shoot first, because once you’ve shot, you can’t ask the questions any more, because your credibility is invested in your message. It pays to ask the questions before you shoot.”

We’re not sure if Dr. Barry Kramer heard our five-minute remarks (we never saw him after lunch), but we were, in essence, presenting a set of explanations for how his note of caution could later prove to be well-justified or even prescient.

Researchers affiliated with the Vitamin D Council drive the science on vitamin D

Inarguably the most forceful voices for increasing the DRI of vitamin D come from researchers affiliated with the Vitamin D Council, a California-based organization. At the one-day workshop, a total of seven speakers were affiliated with the Vitamin D Council (only Drs. Hollis and Grant are board members; the remainder are listed as “Vitamin D scientists” on the website), and the balance of other speakers could be fairly characterized as strongly sympathetic to their aims.

Many of the most influential papers on vitamin D are published by this group. We searched the online database, Web of Knowledge, for papers published since 2005 that mention “vitamin D” in the title or abstract, and then we sorted that list by number of times cited. The top four papers on that list are by researchers with the Vitamin D Council – as are a number more in the top twenty.

These researchers have a habit of wholeheartedly agreeing with one another; throughout the day, we would hear at least several times something to the effect, “I agree with my colleague.”

What does a bandwagon look like? If you search for the publications in MEDLINE on vitamin D since 2005 in GoPubMed.com and click on the statistics tab, you see how often Vitamin D Council researchers have co-authored each others’ papers. Below is an annotated screenshot (click for full-size PDF) of the professional collaborations in this relatively close-knit and like-minded group. Researchers affiliated with the Vitamin D Council are in red.

Despite a notable lack of data derived from RCTs, those researchers associated with the Vitamin D Council are pushing the IOM committee to raise the DRI of vitamin D by a huge increase – around 5-6 times the current DRI. To achieve this goal, the Vitamin D Council markets the form of vitamin D derived from food and supplements to the public as a nutrient. What harm can high levels of a nutrient cause, right?

Yet although we’re referring to it as vitamin D in this article so that you know what we are talking about, any molecular biologist would confirm that the two main forms of “vitamin” D are actually powerful secosteroids. The active form of vitamin D, 1,25-D, can also function as a hormone. We suspect that people would be less willing to take extremely large amounts of vitamin D if they were actually told, “We’re giving you high doses of a secosteroid that will adjust your hormonal and immune activity in ways not yet fully understood.”

Yet rather than trying to help the public understand these true properties of “vitamin” D, a number of prominent vitamin D researchers still seem content to refer to it as nothing more than the “sunshine vitamin,” some with impressive consistency.

Did our human ancestors really have extremely high levels of vitamin D?

Late in his talk, Dr. Robert Heaney, a researcher affiliated with the Vitamin D Council, said, “We all agree and it is well-established that humans evolved in equatorial East Africa wearing no clothes.” This assumption is repeatedly invoked to justify supplementation with vitamin D at levels that would leave the average American with a 25-D level similar to that of a present-day farmer who works near the equator.

We’re not sure anyone noticed, but in the next talk, Dr. Michael Holick would undercut this very argument. Dr. Holick said that according to his research, students of African descent need three to five times the exposure to ultraviolet light as Caucasians to “barely raise their blood levels” of 25-D. In short, their skin is “such a good sunscreen.” If ancient man had darkly pigmented skin, (according to a paper by Jablonski et al., man only evolved lighter skin pigment as he left the tropics) then why would he produce the copious levels of vitamin D referenced by Dr. Heaney?

What about climate change? That ancient man evolved in a consistently sunny and hot environment makes no provision for several extended ice ages, which corresponded to key periods in hominid evolution.

What about skin cancer? Say that early man did not hunt and gather at dusk like so many other animals – that early humans did evolve in an unforested environment with no caves, no clothing, and no thick body hair, whiling away his hours sizzling like a big piece of Paleolithic bacon. Why then would just a few burns before the age of 20 dramatically increase the risk of skin cancer? Did humans evolve to get skin cancer?

To clear up the confusion surrounding this issue, we recently contacted Dr. Peter Bogucki, an archaeologist at Princeton University, who is a leading expert on prehistoric man. We asked him to estimate how much sun prehistoric man actually got.

Dr. Bogucki responded, and I trust he won’t mind us quoting him, “You raise a very good question, but I don’t know that there’s a good answer. All we have is skeletal remains. There’s no elemental isotope to track sun exposure.” In the absence of such a marker, our understanding of how much vitamin D early man actually synthesized is complicated by several factors including climate variability, migration, and changes in skin pigment.

Dr. Richard Potts sums up the evidence or lack thereof for inferring how man evolved from specific environmental scenarios:

The study of human evolution has long sought to explain major adaptations and trends that led to the origin of Homo sapiens. Environmental scenarios have played a pivotal role in this endeavor. They represent statements or, more commonly, assumptions concerning the adaptive context in which key hominin traits emerged. In many cases, however, these scenarios are based on very little if any data about the past settings in which early hominins lived.

Dr. Richard Potts, Director of The Human Origins Program, Smithsonian Institution

At this point, it’s probably safe to say that we simply do not know how much sun early man got.

With this in mind, isn’t it a bit less plausible that, when it comes to the ability of the human body to naturally adjust its vitamin D levels for optimal health, current humans are a complete evolutionary bust and must be given truckloads of pills in order to remain healthy?

Dr. Michael Holick speaks on sunscreen and vitamin D

Dr. Michael Holick is a professor at Boston University, a medical doctor, and may be the world’s leading authority on vitamin D. Since 2005, he has authored or co-authored 59 publications appearing in PubMed on vitamin D (26 more than Dr. William Grant, who is second in that category and a frequent co-author) and he has the distinction of being quoted on vitamin D in nearly every magazine, newspaper, television show and website ever. In his 10-minute statement, Dr. Holick was critical of dermatologists, a group which he singled out for advising the public to avoid creating vitamin D by direct sun exposure. As it happens, Dr. Holick receives large amounts of funding from the UV Foundation, which is in turn sponsored by the Indoor Tanning Association.

Entitled The D-Lightful Vitamin for Health, Dr. Holick remarks sprinkled his speech with a number of pop culture references including mentions of Charlie Brown and Don King. And then there was the clip of Darth Vader telling Luke to come to the Dark Side. It has been a while since we have seen the Star Wars trilogy, but we don’t seem to recall Darth Vader’s evil stemming from his unnecessary prudence.

Dr. Holick went on to claim that sunscreen use blocks 99% of vitamin D production in the skin. This claim is a featured part of his argument, because there has to be a reason why what he views as vitamin D deficiency is so widespread. If there’s evidence to back up this statistic, then our search of the literature cannot find it.

What we did find were three small studies, one of which Dr. Holick authored himself.

One of these studies measured the vitamin D3 (a precursor of 25-D) levels of only eight subjects while another performed no intervention but simply measured the 25-D levels of 20 sunscreen users. The third put only 27 subjects into tanning beds rather than into the sun, which could easily introduce bias. All three are by the same lead author, Dr. Lois Y. Matsuoka.

As it happens, several reviews have refuted the idea that real-world use of sunscreen entirely halts cutaneous production of vitamin D. By real world, we mean people putting sunscreen on themselves for extended periods of time while exposed to the actual sun.

Dr. William L. Scarlett writes in his review, “Several large prospective studies have shown that vitamin D deficiency does not result from regular sunscreen use.”

A review by Drs. Wolpowitz and Gilchrest states, “There is no evidence that customary sunscreen use causes vitamin D deficiency or insufficiency in otherwise healthy individuals.”

One research team, studying patients with xeroderma pigmentosum, a genetic disorder in which patients are unable to repair damage caused by ultraviolet light, found that vitamin D levels are maintained even when patients practice at least six years of rigorous photoprotection and not supplementing with vitamin D beyond their normal dietary intake. Most importantly, the researchers also concluded that the clinical manifestations of vitamin D “deficiency” were absent.

In a 2007 review, Dr. Melanie Palm concludes real-world people tend not to consistently or repeatedly apply sunscreen. She writes: “Most people’s real-life experience with sunscreen is that despite its application, they still sunburn or tan after casual sun exposure.” Dr. Palm goes on to explain, “SPF [sun protection factor] is a strictly defined and Food and Drug Administration (FDA)-regulated measurement based on applying 2 mg/cm² of product. Studies have shown that most users apply insufficient amounts of sunscreen to meet this FDA standard, and the true SPF obtained is usually less than 50% of that written on the package.”

Dr. Holick also proudly informed the committee of the manner and amount of his vitamin D intake. If you ask us, this is irrelevant. It’s nice that Dr. Holick believes what he says enough to try it on himself, but this kind of data falls to the very bottom of Dr. Kramer’s evidence-based pyramid – the opinion level that should never be used to guide public health decisions.

In the remainder of his talk, Dr. Holick went on to say that no one living in a latitude north of Atlanta, Georgia can make vitamin D in their skin during the winter months. Based on everything else we have heard, maybe you can understand why we’re a bit dubious of this claim.

It seems that one of the unspoken rules of publishing a study on vitamin D is that you must cite Michael Holick – geez, even we have done it. But in light of the conflicting data related to Dr. Holick’s claims, we have to wonder why the man has been accorded that authority and why more people don’t second-guess some of his more definitive statements.

A concession: vitamin D is not for people with granulomatous disease

From our perspective, one positive statement Dr. Holick made was when he conceded, as actually many of the pro-vitamin D researchers will do, that vitamin D is not for everyone, specifically not for people with granulomatous diseases such as Crohn’s or sarcoidosis.

A granuloma is a ball-like collection of immune cells which forms when the immune system attempts to wall off substances such as bacteria. But it looks like patients with granulomatous diseases are going to have a tough time if Holick and his colleagues succeed in drowning us in vitamin D. Raising the DRI of vitamin D would inevitably mean that vitamin D would be added to another slew of foods.

When Dr. Holick et al. were questioned about the fact that some people have been shown to develop kidney stones after taking extra vitamin D or that people with granulomatous disease could easily ingest excess levels of vitamin D and become significantly more ill, they seemed ambivalent. In their eyes, if a certain number of people are harmed by taking vitamin D, it should not matter, so long as more people benefit. We find this risk-benefit analysis difficult to stomach having seen first-hand the suffering associated with granulomatous diseases.

Dr. Cedric Garland discusses vitamin D and cancer

Another member of the Vitamin D Council, Dr. Cedric Garland, spoke in his remarks about vitamin D and cancer. After his remarks, a committee member, Dr. JoAnn Manson challenged him on his claim that vitamin D is protective against cancer at high levels of intake. She asked him about the Women’s Health Initiative-led randomized controlled study which trended in the opposite direction when it comes to breast cancer among women who start out with high intakes of vitamin D.

Dr. Garland brusquely and repeatedly dismissed the cancer study, saying that the dose of vitamin D administered to subjects, 400 IU – which happens to be the current adult DRI – was “not even a placebo.” In other words he believes that 400 IUs of vitamin D has no biological effect whatsoever. Dr. Manson responded, “I don’t buy it.” Actually, neither do we. To put things in perspective, you’d have to consume 20 eggs or four glasses of vitamin D fortified milk a day in order to get 400 IUs of vitamin D.

Interestingly, when you take a look at the five most frequently cited papers on vitamin D published in the last five years, the first four are authored by researchers affiliated with the Vitamin D Council. But study #5 derives its conclusion based on data collected by the Women’s Health Initiative, the same research group whose data Dr. Garland suggested should have no implication on the IOM Committee’s decision-making. That other vitamin D researchers are more than inclined to analyze data from the Women’s Health Initiative suggests that, although Garland may seem like he is an expert speaking on behalf of the entire vitamin D community, not all vitamin D researchers share his views.

We have taken the liberty of annotating in red several of Dr. Garland’s slides to make points about the presentation of data especially as it pertains to vitamin D.

Below is Dr. Garland’s slide showing a strong and consistent increase in the rate of breast cancer since 1935, which he used as a general indication for why it is important to significantly increase the amount of vitamin D added to the food supply.

However, as you can see below, it is very easy to take that same data and “show” the opposite – that vitamin D consumption has led to a dramatic increase in breast cancer.

Another example: Dr. Garland didn’t mention this publication in his speech, but in a 2008 study, his group found a significant association between “low UVB irradiance and high incidence rates of type 1 childhood diabetes.”

Data derived in this observational manner could just as readily be used to show something else entirely.

As you can see in this graphic above, there is a strong apparent association between states that get more sun and teenage pregnancy. But does sun exposure actually cause teen pregnancy? We certainly hope not!

Obviously, you can try to control for confounding variables, as Dr. Garland did in his ’08 publication, but so too did researchers who repeatedly concluded that hormone replacement therapy was safe. According to Dr. Kramer: “There were literally scores, if not hundreds, of observational studies that showed almost beyond reasonable doubt that hormone replacement therapy would prolong women’s lives, if it were given routinely.”

In the words of Dr. David Ransohoff (who Dr. Kramer quoted in his talk), observational data are “guilty until proven innocent.”

When discussing vitamin D, Dr. Garland put up another thought-provoking chart on the effect of vitamin D and calcium on the development of kidney stones (derived from the Women’s Health Initiative).

Several things about Dr. Garland’s chart are of interest.

• Although the y-axis could easily have gone up to only 10%, it goes all the way up to 55%. This visually minimizes the apparent negative treatment effect of calcium and vitamin D and barely impresses on the viewer that if the trend observed in the study is accurate and significant, approximately 1.2 million Americans will develop kidney stones if they continue taking vitamin D and calcium.
• We probably should not be surprised that on this same slide, Dr. Garland opted to display absolute risk rather than relative risk. Absolute risk is a measure of what portion of a population have a disease in a given time period. Relative risk is that percentage increase divided by the risk in a placebo group, e.g. (2.5%–2.1%)/2.1%. In this case, patients who take calcium and vitamin D have an increased absolute risk of 0.4% of developing kidney stones but a relative risk of 19% of getting kidney stones. So by showing absolute risk, Dr. Garland again downplays the sheer number of people who could be negatively affected by taking extra vitamin D and calcium.

Dr. Reinhold Vieth speaks about safety

In his slot, Dr. Reinhold Vieth was asked to speak on whether there was a safe upper limit/level of vitamin D. As he has stated in at least one paper, his answer was no. In his words, “A prolonged intake of 250 mug (10,000 IU)/d of vitamin D(3) is likely to pose no risk of adverse effects in almost all individuals in the general population.”

Dr. Vieth’s comments echoed those of Dr. Garland, who had earlier concluded, “The benefit/risk ratio for 2,000 IU/day of vitamin D is infinite.”

Obviously, we disagree. We take no comfort in the fact that a person, as demonstrated in case reports, can accidentally take several thousand times the recommended dose of vitamin D and still seem healthy after only several months – which is the only data Dr. Vieth provided. Our attention is directed towards long-term outcomes, time windows which correspond to the slow growth of chronic bacteria and other pathogens that may play a role in causing chronic disease. Also, the full negative effect of immunosuppressants (recall that we have found that 25-D acts as an immunosuppressant) can often only be noted after decades.

25-D vs. 1,25-D and the long elusive search for biological plausibility

Most of the talks had us scratching our heads, trying to figure out why, when 1,25-D is the biologically active form of vitamin D and the sole vitamin D metabolite able to activate the Vitamin D Receptor (VDR), almost every speaker focused on research and recommendations pertaining to 25-D levels. For a brief discussion of the different forms of vitamin D see my (Paul’s) speech.

One of the points both of us tried to make in our own five minute presentations is that the levels of the different forms of vitamin D are jointly regulated by several feedback mechanisms. This means that if one alters the level of one form of vitamin D, levels of the other vitamin D metabolites will almost certainly shift to accommodate the change.

It seems prudent then, that if a study measures 25-D levels, it should measure 1,25-D levels as well. Without the ability to examine the relationship between the two main vitamin D metabolites, how can a researcher fully understand the spectrum of the changes that occur when vitamin D supplementation takes place? Over a decade ago, even the FDA suggested that “1,25-D should be measured in order to support claims of a drug’s osteoporotic activity.” Yet few researchers seem to have heeded this advice. Thus, we would venture to say that studies absent levels of 1,25-D should at least be regarded with less rigor than those studies that test both metabolites.

At some point in a discussion with the Committee, one of the experts mentioned how 1,25-D is difficult to detect. We hope that doesn’t serve as an excuse for not testing 1,25-D. Since most major laboratories – including Quest Diagnostics – can easily perform the test, we would expect any vitamin D researcher would be able to do so as well. The real reason 1,25-D might be “hard” to test is that the 1,25-D test costs more than the 25-D test. But we’re all trying to do the best possible research… right?

The potential significance of 1,25-D is suggested in a forthcoming study published in the Annals of the New York Academy of Sciences. In the study, Dr. Greg Blaney of Vancouver, Canada reported on the 25-D and 1,25-D levels of 100 patients with autoimmune disease.

While many of the subjects had very low levels of 25-D, even more of the subjects (approximately 85%) had levels of 1,25-D elevated above the normal range. Under these circumstances can those subjects with low levels of 25-D but elevated levels of 1,25-D truly be considered vitamin D deficient? They are certainly not deficient in the sole form of vitamin D that actually activates the VDR to transcribe approximately 913 genes, TLR2, and the antimicrobial peptides vital to the innate immune response.

When Dr. Heaney was asked to comment on 25-D’s actions by a member of the committee he admitted that he did not know, biologically speaking, how 25-D exerts any of the myriad beneficial effects that he claimed occur when it is elevated. All he could offer was that he knows that 25-D must be present in patients for them to get better.

Is this what passes for biological plausibility among pro-vitamin D researchers?

Later that afternoon, one committee member asked Dr. Cedric Garland, “Do you have a mechanism to explain the outcomes you’re reporting?”

Dr. Garland proceeded to offer his analysis for how supplemental vitamin D, in his words, “eradicates” cancer. Garland pointed to a stack of his papers and asked that it be passed out. When members of the committee seemed hesitant to do so, he went on to explain the details of his model anyway. Dr. Garland shared that he had developed a novel pathogenesis for cancer in which cancer is caused by gaps between cells, which, in simple terms, he believes form as a body becomes vitamin D deficient. This line of inquiry was clearly only in its infancy and had not yet passed muster with cancer researchers. But even if Garland’s model proves to be valid, one would have hoped he would expose it to great scientific scrutiny before using it as the basis for making unequivocal recommendations regarding vitamin D supplementation.

But as Dr. Garland went on to further describe what he believes are vitamin D’s cancer benefits (he was eventually cut off by a member of the committee), he provided a perfect example of the vitamin D expert that we have trouble following. The reason? He used the broad term “vitamin D” when making claims and by doing so, mixed up research that pertains solely to 25-D or 1,25-D. For example, Garland said that vitamin D is able to “upregulate tumor suppressor genes.” Most audience members probably thought he was referring to 25-D since that was the only vitamin D metabolite he ever mentioned. Yet, only 1,25-D is able to activate the Vitamin D Receptor to express Tumor Metastasis Suppressor 1 and other related genes.

Similarly, another talk that we believe should have discussed 1,25-D levels but did not was Dr. Stephanie Atkinson’s remarks on vitamin D in pregnancy. That is because researchers have realized for some time now that 1,25-D is over-expressed during pregnancy. Placental conversion was demonstrated in vitro in 1979, over-expression of 1,25-D in vivo in 1980, and the dysregulated vitamin D metabolism was described in 1981. If 1,25-D becomes elevated during pregnancy, then isn’t it only prudent that studies on vitamin D and pregnancy should measure it and its relationship to 25-D?

We find the relationship between 25-D and 1,25-D important, because it was by observing relationships between the two metabolites that our group was able to realize that in the majority of cases, when a subject’s 25-D level is low, their 1,25-D levels are actually high (AIDS is an exception because HIV completely co-opts the VDR). And it was these relationships that led to our alternate hypothesis for the low levels of 25-D observed in patients with chronic diseases such as cancer. We have found that when 1,25-D is high, the vitamin D feedback pathways naturally downregulate levels of 25-D. This means that what is now viewed as “deficiency” could simply be a result of the chronic disease process. Under such circumstances, allowing people to create extra 25-D by raising the DRI is not only useless but harmful. We believe that our alternative hypothesis at least deserves consideration by the committee, yet are worried that when they are not presented with data on both 25-D and 1,25-D, they will not be able to recognize the pattern that makes our model plausible.

Vitamin D and the evolving definition of autoimmune/inflammatory disease

We also find it problematic that none of the experts who spoke at the meeting seem to be aware that microbial metabolites have a profound effect on the activity of the Vitamin D Receptor (VDR). The US NIH now estimates that 90% of cells in the human body are bacterial in origin while only a mere 10% of cells in the body are truly human. Thus, many microbiologists now believe that humans are best viewed as superorganisms in which a plethora of bacterial gene products can effect the activity of our own receptors and genetic pathways. Indeed, independent research teams have found that Mycobacterium tuberculosis downregulates VDR activity by approximately 3.3 times. Active Borellia lowers VDR activity by about a factor of 50 and Epstein-Barr Virus by a factor of around 10. HIV completely shuts down VDR activity. It’s quite likely that other pathogens yet to be fully characterized have also evolved ways to decrease VDR activity because by doing so, they slow important components of the innate immune response that might otherwise render them dead. That the experts who spoke before the committee have failed to factor this knowledge into their study designs suggests that they cannot fully account for the actions of the various vitamin D metabolites in an in vivo environment.

Furthermore, no vitamin D researcher, of whom we are aware, makes provision for research which shows that the current view of autoimmune disease – in which the immune system is believed to attack itself – may be running its course. Many microbiologists now believe that at least some, if not all, of the inflammation that drives the autoimmune disease state is caused by the presence of chronic pathogens.

Inflammation is a clear potential link between infectious agents and chronic diseases.

Siobhán M. O’Connor

With this in mind, the claim by many vitamin D researchers that vitamin D can help patients with autoimmune disease by slowing an “over-active” adaptive immune response no longer jives with an emerging view in the microbiology/immunology community – that both the adaptive and innate immune systems should be kept active in autoimmune disease in order to allow the body to best target disease-causing microbes.

The possible presence of pathogens in autoimmune and other inflammatory disease states such as cancer and atherosclerosis makes our group’s findings on vitamin D’s actions more plausible. When the immune system is fighting a microbe, it continually releases inflammatory molecules in an effort to kill the pathogen. If the pathogen dies, endotoxins and cellular debris are generated. This leads to increased symptoms of malaise on the part of a person who harbors such microbes.

It follows that any substance that slows the innate immune response will decrease this battle between man and microbe, causing the patient to feel better. The more the immune response is slowed, the greater the decrease in inflammation and inflammatory markers. But while such measures can make the patient appear as if they are getting better for years, ultimately the bacteria causing their disease are able to spread much more easily and exacerbate the disease state over the long-term.

Our molecular and clinical data shows that 25-D, like the pathogens we describe above, binds the Vitamin D Receptor and slows its activity. Since the VDR largely controls the innate immune response, increasing 25-D levels could easily display the pattern of immunosuppression described above. This begs the question – is 25-D a miracle curative substance or simply an excellent palliative?

If we are correct and 25-D slows VDR activity then we have found that patients who are chronically ill benefit from decreasing their vitamin D intake. This is because their VDR activity already appears compromised by the pathogens they harbor. Yet this should not be interpreted to mean we think healthy people can’t consume vitamin D. However, our data suggest that healthy people can get the vitamin D they need by eating a well-rounded diet that does not include fortified foods and getting sun exposure similar to that of a person taking measures to avoid an increase in skin cancer risk.

Our speeches and the reaction to them

In our speeches, we raised the possibility that low levels of 25-D are caused by the inflammatory disease process and that taking vitamin D suppresses the immune response.

In total, the two of us spoke for 600 seconds, and we’re not sure we convinced anyone of anything. By all indications, a discussion of molecular mechanisms was outside the committee’s comfort zone. Most would probably say that they are uninterested in software emulations of molecular interactions, no matter how provocative or far-reaching the conclusions they imply. If we had to pin the members of the committee down on it, I think they would say that when it comes to our clinical trial, we needed better controlled data such as the kind we intend to generate as a part of our West China Hospital collaboration. For this reason, we opted for a more measured tone.

During Amy’s speech, a Dr. Holick sighting, seated in the front on the left.

During Paul’s speech, there was some tittering in the audience (not the committee). He saw one prominent researcher, who shall remain nameless, chuckling. For a moment, he thought he had spinach in his teeth or was trailing toilet paper from his shoe, and then he realized that, oh yes, he was telling 50 PhDs and MDs that their conclusions have the potential to be very misguided.

After the day’s business concluded, everyone began to file out. One woman though turned to us and said, “What a bunch of rebels!”

Glad we could liven up the workshop for you, ma’am.

Although during our speeches, we asked people to come by and ask us about our work, only Dr. Tony Norman did. He did not seem convinced, but did invite us to submit an abstract for a poster presentation at an upcoming vitamin D conference in Belgium.

Anticipating what is to come

If you ask most Vitamin D Council researchers, they would say that this is the “end game,” and there is already more than enough evidence to raise the level of vitamin D added to the food supply. During the question and answer sessions, some of these scientists such as Dr. Garland were dismissive of evidence to the contrary. It was as if many were saying, “Look – there is no downside here. It is demonstrably impossible that consumption of vitamin D can cause harm. If we don’t have all the requisite evidence, it doesn’t matter. Lives are at stake!” We suspect that even if the committee decides to maintain current vitamin D levels, there are other ways to convince the public to increase vitamin D intake.

But despite the media’s stampede to promote the “sunshine vitamin,” the evidence is ambiguous and the issue of biological plausibility – not knowing how 25-D exerts its claimed benefit – is troubling as well. Dr. Kramer said that the root of science is the art of thinking hard about how you could be wrong. Is this something the vitamin D research community is actively doing? Looking through everything that was presented throughout the day, how many confounding variables might Dr. Kramer have identified? How many surrogate outcomes could he point to?

It is difficult to anticipate exactly what decision the IOM Committee will arrive at. However, from this perspective, it would be hard to see how the group could raise the dietary reference intake in light of such an equivocal set of conclusions in the Tufts report – in spite of considerable pressure to do so.

Will an IOM committee ever emerge from this climate of consensus and consider research that would cause them to lower the DRI of vitamin D?

Here are a few possibilities:

• An evolution in the understanding of disease raises new concerns about the risks of using immunosuppressants. The Human Microbiome Project shows that bacteria are not confined to the surfaces of the body, i.e. skin, mouth, gastrointestinal tract, etc. As chronic disease is increasingly characterized as an infection or at least having infectious components, researchers seriously question if reducing the inflammatory response needed to kill chronic pathogens is in a individual’s long-term interest.
• After continuing to increase vitamin D consumption to historic levels, members of the public and some researchers begin to question the absence of the promised overall drop in rates of disease. In some respects, the decision of the IOM committee is immaterial. All indications are that the vitamin D “experts” are having a great deal of success communicating their message that it’s important to take 4-5 times or more the current DRI of vitamin D. People will take increasing amounts of vitamin D as food manufactures will add increasing amounts to their products. Many of the presenters at the Workshop essentially promised double-digit declines in disease. If this does not materialize, there will be questions. If we are right, this could be the hormone replacement therapy (HRT) saga redux, except with potentially broader ramifications.
• Well-controlled long-term studies show that vitamin D consumption increases incidence and severity of chronic disease. To most people – probably in excess of 95% of people at the workshop – this is not even a possibility, but the history of HRT use proves such unexpected results can emerge, eventually, from well-controlled studies.

Epilogue: The ride home

After the meeting adjourned, we were approached by a nattily attired man in his thirties, originally from Barcelona. He offered us a ride home to New York. His Mercedes SUV looked quite appealing, so we skipped the bus and took him up on his offer.

On the ride home, this fellow – who told us he had a PhD in oncology – told us he agreed with the sentiment of our remarks and expressed disappointment with the lack of rigor of the science presented. The word he used to describe the majority of presentations was “pseudoscience.” He told us that, based on what he saw, vitamin D was harmful and that it was only a matter of time before the hype surrounding vitamin D would fizzle.

Although we felt validated, we wondered why he had attended the conference in the first place. It turns out that he was an entrepreneur, had just bought the patent for a new formulation of calcium, and wanted the discussion at the IOM workshop to help him decide how much vitamin D to add to his product.

He seemed like a honest and honorable guy until, that is, he let us know that despite his negative view of vitamin D, he intended to add high levels of it to his supplement anyway, so long as the medical community and public viewed it as beneficial. Later on, he said, he planned to strategically remove it “just before the vitamin D bubble bursts.”

Well, isn’t that wonderful? Some reassurance about the people behind products aimed at “improving our health.”

In that vein, we couldn’t help remembering the short speeches delivered by members of the Dairy Council as well as a yeast company, whose goal in speaking before the Committee were simply to urge the Committee that, if more vitamin D is added to the food supply, it should be added to the food they market. This would give these interests the ability to claim more health benefits from their food and, of course, make more money.

In sum, our adventure in the nation’s capital left us with a bad taste in our mouths. We’d like to wash it away but we’re worried that by the time we do so, no drink won’t be fortified with vitamin D.

Two weeks ago, I boarded a 747 to travel to Beijing, China in order to speak at the 2009 International Congress of Antibodies. Within minutes of hitting the runway, we were greeted by a crew of officials wearing surgical masks and wielding thermometers. The fever analysis was performed as part of an effort on behalf of the Chinese government to ensure that neither I nor my fellow travelers had the swine flu. Luckily I was cleared and proceeded to my hotel which was also the site of the Conference. Before checking into my room, I had to get my temperature checked yet a second time by the hotel staff. I wasn’t opposed to the routine screenings since, at the time, if anyone on my hotel floor had the swine flu the entire floor would be quarantined.

The door of our hotel was guarded by two cement lions and delicate Chinese flowers in a vase were built into the hotel revolving door. The first night, I was exhausted and fell into a deep sleep due to the jet lag. I woke up to greet my colleague, Dr. Trevor Marshall, whose flight had arrived during the night. He was surprisingly chipper and we set off to explore our surroundings. The hotel was located close to the stadium from the 2008 Summer Olympics, also known as the “Bird’s Nest.” Dr. Marshall was lucky enough to have a view of the stadium from his hotel room window. I had to settle for a view of the boring side of the street. Down the street we discovered a Chinese cafeteria that served the locals and had better food than the hotel, at least in my opinion. It was the first of my many trips there to get a stir-fry or other delicious dishes. We also cheated by discovering a supermarket with American food products that we stocked up on for snacks. Then we went to a fourteen-story building occupying a whole city block. It was full of innumerable electronic devices for sale at low prices. Given his earlier work as a microchip designer, surely this was Dr. Marshall’s mothership.

A Chinese health worker boards the plane to check our temperatures on the way in to Beijing.

The first thing I’ll say about the Conference is that its name – International Congress of Antibodies – is something of a misnomer. The reality is that the Conference was not for antibodies but people.

Conference registration was the next day. Finding the registration took some time. It was in a completely different part of the hotel and most of the hotel staff didn’t speak any English at all. In fact, it was the first time in my life where I truly felt “lost in translation.” Even my elaborate hand gestures often couldn’t help the hotel staff understand what I needed, particularly when it came to setting up my Internet connection, trying to use their business center, or communicating with the housekeeping staff about when I would and would not be in my room.

It quickly became apparent that essentially all guests that stay at the Beijing International Continental Hotel are Chinese and that few tourists stay at the hotel. This meant it was easy to pick out other researchers arriving for the Conference. It was as simple as noting that someone spoke a foreign language, had european or hispanic features, or was gesticulating at the front desk.

The Conference officially began on Thursday morning. The opening talks consisted largely of the Conference sponsors touting their products or companies. Eventually I became even more accustomed to such talks since at least half of the researchers presenting were also associated with biotech companies or other businesses that not only conduct research but also sell tools for antibody detection and creation. Their talks were generally of less interest to me than those that discussed purely scientific work.

The Chinese lion standing guard at the entrance to the hotel

The researchers hailed from around the world, several from Europe and the USA, even some from Latin America. Unfortunately, most of the researchers from Japan were not able to attend the Conference because of an increasing number of swine flu cases in their country that had made the Chinese authorities nervous. Perhaps this worked in my favor. Originally I had been scheduled to speak at a session on Saturday afternoon about antibodies and diagnostic tools. However, there is actually very little about diagnostics in my speech. So I asked one of the Conference organizers if I could switch to speak in a session specifically about autoimmune disease the next morning, which had an empty slot because of a Japanese speaker’s absence. She said yes.

Before Sunday rolled around I perused the halls, introducing myself and getting to know my fellow conference attendees. Most people were very open and eager to talk about their work or China in general. Every lunch and dinner we would all walk in a pack back to the main hotel where a buffet-style meal was served. The food was Chinese and sometimes intriguing – intriguing as in I really didn’t know what I was eating! There was plenty of meat in sauce and green leafy vegetables, and of course, plenty and plenty of rice.

Lunch and dinner served as perfect times to make friends. I met a woman from the UK with a wonderful sense of sarcasm and humor. Two German men who managed biotech companies were also very outgoing and full of interesting stories about Germany and their other travels abroad. I really enjoyed the enthusiasm of a Chinese-born researcher who now works at the University of Toronto. His interest piqued when I told him about the MP. Later, after hearing my talk, he became even more enthusiastic and kindly told me that my speech had exceeded his expectations. In fact, I don’t think I’ve ever been at a Conference where Dr. Marshall’s work was so well-received. Nearly everyone wanted to learn about the Pathogenesis. Many weren’t even familiar with latest estimates released by the Human Microbiome Project which state that at least 90% of cells in the human body are bacterial in origin. Everyone seemed to agree that such a plethora of bacteria could very likely cause a wide variety of autoimmune and inflammatory diagnoses. By Sunday morning I had whetted the scientific appetities of as many people as I could so that they would come listen to my speech.

At the opening dinner of the Congress.

Every session at the Conference was monitored by two chairmen, and in my case, both were very open-minded and intelligent scientists. My speech was the last in the session, but finally I made my way to the podium. As I spoke, I could sense that the colorful backgrounds and strong images on my slides (designed by my colleague Paul Albert) were helping people to capture the important aspects of the speech. Several camera flashes illuminated the room when I put up a slide covered by a prolific number of names of different bacterial species recently determined to persist in saliva.

The main point of my speech was to re-examine the concept of the “autoantibody.” Currently, autoimmune disease is thought to result when the immune system attacks itself. In such a model, antibodies, which are molecules used by the immune system to neutralize foreign objects, are thought to be created in response to our own human DNA.

What I pointed out was that, since the human body is composed of more microbial than human cells, this is not necessarily the case. The antibodies detected in what is now considered to be “autoimmune” disease may actually be created in response to bacteria. Specifically, when the innate immune system is activated by continually trying to kill chronic bacteria it activates the adaptive immune system. The adaptive immune system, which is the branch of the immune system that creates antibodies, then likely proceeds to create such antibodies in response to fragments of bacterial DNA generated by the death of the infected cells.

At the conclusion of the speech, those members of the crowd that hadn’t left in disgust began to lob vegetables towards the stage. Fortunately, some of the produce was fresh, and I had enough for a salad….

Dr. Audrey Tchorbanov and I after I finished my speech

The truth is more boring: the speech seemed to go over quite well. The audience applauded enthusiastically, and a number of hands shot up for questions. So many people had questions that a number of people missed lunch to listen to my follow-up remarks. One of the session’s chairmen, Dr. Audrey Tchorbanov of the Bulgarian Academy of Sciences, seemed impressed with the speech and we posed for photos together. A few other people did the same. A pediatrician from Denmark who had given a very interesting speech himself about sepsis in infants commented numerous times on the groundbreaking nature of Dr. Marshall’s work. I think, as has been the case with every conference I’ve spoken at so far, the audience was pleasantly surprised to hear a speech that discussed how scientific observations and more theoretical work can be applied to clinical care.

To celebrate a successful presentation, Dr. Marshall treated me to a meal that has been a favorite of Chinese emperors since the time of the early Ming dynasty. I forget the Mandarin term for it, but I believe in English it is called grilled cheese sandwich and french fries with ketchup.

Later that day, I returned to a few sessions and then retired to my room to decompress. The wry woman from the UK arranged for a group of us to go out that night in honor of the end of the Conference. There were about eight of us including the biotech guys from Germany and the pediatrician from Denmark. We were also joined by a scientist from Ireland whose thick brogue and lively banter kept us laughing all night. One of the members of our group, a Chinese woman, took us to a lake surrounded by rows of outside bars with comfy couches and chairs. The temperature was perfect – warm and breezy. We celebrated until four or five in the morning when we finally headed back to the hotel. It was encouraging to see that, like me, most of my fellow scientists are able to combine work with play.

If it were even possible to sum up China and her people in a single word, that word would be “ambitious.” You can see ambition in the medal counts from the 2008 Beijing Olympics and in the Beijing skyline, being built ever upwards at a furious pace. But I can also see the urge to succeed in Chinese researchers’ apparent receptiveness to the MP and the science which supports it.

Out with some other scientists and biotech executives

I feel that China may eventually prove to be fertile ground for the MP to flourish. There is no vitamin D added to the food chain. Supplementation with D is not promoted or generally used as a therapy in chronic disease. Because pale white skin is considered beautiful in China, staying out of the sun is common practice for the Chinese. Chinese women in particular walk around with sun umbrellas and wear sunglasses and often long-sleeved shirts. Whereas American magazines advertise tanning salons and creams, Chinese magazines advertise hats and skin-whitening cream.

Perhaps I should not have been surprised then that the Chinese people seemed much healthier than the Americans I’m used to seeing. For one thing, almost no one in China is obese or even fat, and it’s not like these people don’t know how to eat! Also, I was impressed at the lack of acne in the Chinese population. It seemed that every Chinese woman – whether at the airport, the hotel, or at the Conference itself – had perfectly blemish-free skin.

All in all, it was a great trip. I certainly plan to keep in touch with some of the people I met, and I definitely hope to return.

The Congress on Autoimmunity is a biennial event. It features dozens of talks, 1,800 registered delegates, and takes place over the course of five days.

Dr. Yehuda Shoenfeld and I are clearly close buddies.

The meeting has a decidedly international flavor. Participants hail from Germany, Italy, Russia, Italy, South Africa, even Mongolia. For many researchers and scientists in the field of immunology, this is an ideal forum to learn about and discuss advances in their field.

Participants include researchers presenting their work, physicians gaining continuing medical education credits, and vendors hawking seemingly sophisticated technology. Who knows — maybe that five-foot chamber with the three LCD screens and dozens of buttons was no more than a glorified alarm clock. I probably should have gotten a brochure.

As host cities go, you can’t do much better than Portugal’s second largest city, Porto. The weather was cool and, except for the skater punks outside my hotel room, the locals were lacking artifice. (One grizzled fellow told me in broken English that Porto was so named, because once upon a time the city’s fathers said, “Let us call it Porto, because it is on a port.” Now there’s something you can’t get in a guidebook.) And what better place to have a conference on immunology than a city dominated by winding streets and dead ends? More than once, I completed a taxi ride thinking I couldn’t tell if I had been taken to my destination using the world’s most clever series of shortcuts or was simply being ripped off.

Belinda Fenter, me, Meg Mangin, Tom Perez, and Dr. Greg Blaney

The meeting itself was held in a historic waterfront building with high pillars and a floor with curious metal tracks leading between one room and the next. Although as good scientists, we spent a lot of time hypothesizing about how the tracks might have been used in the past, no one we talked to could tell us why they were there.

In his speech during the opening ceremony, Dr. Yehuda Shoenfeld, the President of the Congress, proved himself to be something of a jokester. Shoenfeld said he had a particular fondness for Porto, no small part of it due to the fact that his wife had won a beauty contest held in Porto in 1975. When he wasn’t showing pictures of previous years’ attendees ogling belly dancers or introducing the night’s band – the Anti-Phospholipids – he was telling the audience that the three winners of lifetime service awards each would receive prizes of one million dollars (not true!).

One of the award winners was Dr. Eric Gershwin who, in addition to his studies in the field of autoimmune disease, maintains an informal medical clinic for handicapped animals including tortoises, horses, and skunks among others. Dr. Shoenfeld related that Gershwin is a direct descendant of the famous composer George Gershwin. We learned that Shoenfeld’s son, who incidentally turned out to be the keyboardist for the Anti-Phospholipids, received a gift from Dr. Gershwin: an original copy of “Rhapsody in Blue” signed by the composer himself.

Tom, Meg, and Tom (Meg’s husband) enjoy rabbit and other specialties at a typical Portuguese restaurant.

Shoenfeld confessed it was the first time the son thought his father’s scientific connections were of actual value. Incidentally, the other half of the Anti-Phospholipids was a violinist who presented at the Congress.

On a more serious note, Dr. Shoenfeld also noted how more diseases – including, for example, depression – are now considered by some to be autoimmune in nature. Interestingly, the list of diseases thought to be autoimmune closely parallels those which the Marshall Protocol treats. Several talks even discussed cancer and autoimmune disease. I find it interesting that when it comes to the MP, some people have a hard time accepting the hypothesis that nearly every inflammatory condition can have the same basic pathogenesis. Yet as the “autoimmune community” continues to attribute more and more diseases to their same basic disease model few people raise an eyebrow.

I arrived with a contingent of others who work closely with Autoimmunity Research Foundation (ARF). ARF’s time in the limelight came early in the Conference, on Thursday afternoon. The session on vitamin D was two hours long and was chaired by Dr. Trevor Marshall. In addition to the four of us associated with ARF, we heard from four others including Dr. Howard Amital of Israel and Dr. Maurizio Cutolo of Italy, both prominent researchers in the field of vitamin D. The last speaker slated to speak, a researcher from Iran, actually failed to show, presumably because he didn’t want to share the stage with an Israeli researcher. This is not the first time this has happened, we were told.

As the chair, Dr. Marshall spoke first. Marshall’s talk had a decided focus on bacteria. He began by reminding the audience that bacterial cells outnumber human cells by a factor of 10 to 1. He went on to argue that the microbiota we harbor has evolved to decrease antimicrobial peptide expression by dysregulating the vitamin D receptor.

Liz Marshall, Dr. Joyce Waterhouse, me, and Dr. Trevor Marshall after a trolley ride around Lisbon

Dr. Greg Blaney is an MP physician. Dr. Blaney presented data, serum blood values mostly, from his own sizable cohort of MP patients. Using that data, he articulated a rationale for why 25-D is an inferior marker of inflammatory disease compared to 1,25-D. He showed how, among his cohort, measures of 25-D had a high level of variability and 1,25-D tended to be more consistent with disease status. One of his sickest patients, he reported, had a 1,25-D in excess of 100 pg/ml. Hopefully, his talk will challenge, in at least some small measure, doctors’ and researchers’ practice of testing only 25-D.

Later on, Captain Tom Perez, RPh, MPH spoke. He gave some details of the Marshall Protocol study including statistics on how many patients, by autoimmune diagnosis, experience improvement by time frame.

In my talk, I made a case for why autoimmune diseases such as Hashimoto’s Thyroiditis are much more likely to occur in women than in men, especially during the childbearing years. My contribution had value because I was able to show how one can use the alternate hypothesis for vitamin D to formulate viable hypotheses that logically explain other aspects of autoimmune disease. In the parlance of software engineers, the MP is extensible.

My Citizen Kane moment.

Some people are wary of public speaking. As you can surmise from the above video, I am not such a person. Also, I’ve been longing for the chance to connect with researchers and doctors in a larger forum. In any case, I could have spent much more time talking! I actually was so focused on covering all the main points in my talk that I didn’t even notice the Citizen Kane-like screen behind me.

This Congress marks an important, though certainly not the final, milestone in the increasing acceptance of the Marshall Protocol as a therapy for chronic disease. It was telling that speakers supporting the MP model ranged from a molecular biologist to physician to public health official.

As expected, the other speakers in the vitamin D session offered more traditional perspectives on vitamin D. Prior to my opportunity to speak, Dr. Amital showed data connecting a low level of 25-D to lupus severity. Dr. Cutolo offered a similar conclusion for rheumatoid arthritis.

I take issue with observers who conclude that a low level of 25-D is necessarily a cause of the disease process. They could have just as well identified the measure as a result of the disease state. As epidemiologists repeat ad nauseam, “Correlation does not equal causation.” (I’ve been told that it is a rare epidemiologist indeed who doesn’t have this phrase tattooed somewhere on his or her person.)

Paul and I enjoyed the grilled “steer” at the Conference dinner.

In listening to the speakers in the vitamin D session, I imagine that even the least attentive audience member must have felt a certain amount of dissonance, even whiplash. I was disappointed that the forum didn’t allow for a more spirited dialogue, one we desperately need to have.

Being the penultimate speaker allowed me to at least briefly address the disconnect. I concluded my speech with this impromptu observation: “When it comes to correlating disease incidence with low levels of vitamin D, it’s also incredibly important to consider the alternate hypothesis, which is that the low levels of vitamin D may not be causing the disease but may simply be a result of the disease process.”

After the session concluded, Dr. Cutolo introduced himself and we spoke for about 20 minutes. There’s a lot to like about Dr. Cutolo. He’s a jocular guy, but he’s also a serious scientist and was therefore willing to consider the alternate hypothesis. What seemed to intrigue him most was Dr. Blaney’s point that 1,25-D is a more reliable biomarker of autoimmune disease status than 25-D. Perhaps we can expect future studies of autoimmune disease from Dr. Cutolo to rely on this measure. We also talked about my hypothesis: how a muted immune response during pregnancy could lead women to feel greater well-being.

The Congress concluded with a final group dinner held in a winery overlooking the Douro River. Trevor outed himself as a port aficionado. During dinner, he turned down the waiter’s offers of wine and held up his port glass in order to signal how eager he was to drink the local beverage. Also, we finally got to hear genuine native Portuguese music. The band played a series of “old country” ballads including “Do the Hustle” and “I Will Survive.”

Dr. Blaney shaking his groove thing.

The truth is, I can’t say with certainty what effect our speeches and our side conversations had on our fellow attendees. I had the distinct impression that a substantial minority had come to the Conference to conclude business deals. The sparkling booths were evidence of that. Nevertheless, I think we did succeed in presenting the research and hypotheses that underlie the Marshall Protocol to many of the world’s leading researchers and physicians. Thanks to the Internet, the videos of our speeches have the potential to reach an even greater audience.

Colorful representations of sequenced genomes adorn the walls at JCVI.

You may wonder how a researcher can view and understand a particular bacterial genome. On their own, they cannot. Progress in genetics is a group effort, and requires partnering with one of the handful of heavyweight institutions in the world that have developed resources allowing for genome interpretation. Several such institutions exist in the US. The NIH has bacterial protein sequencing tools at its disposal. The Broad Institute at MIT as well as the Washington University Genome Sequencing Center have also developed tools that allow for genome sequencing.

Many would argue though that the Institution most on the bleeding edge when it comes to genome sequencing technology is the J. Craig Venter Institute, formerly known as TIGR. Headed by transformative iconoclast and entrepreneur J. Craig Venter, the Institute is a non-profit research center that was founded in 2006. It has facilities in Rockville, Maryland and La Jolla, California and employs over 400 people, including Nobel laureate Hamilton Smith.

You can imagine how happy I was to get an email from my former advisor at Georgetown (where I have an undergraduate degree), asking if I wanted to attend a training session in bacterial sequencing technology at the Rockville branch of the J. Craig Venter Institute (JCVI). He was keenly aware of my thirst to gain hands on experience with sequencing technology.

The training was called the “Prokaryotic Annotation and Analysis Workshop.” (As some may know, “prokaryote” is just another name for single-celled bacteria.) This experience marked my first exposure to sequencing technology, and I had little idea what to expect. Would I be able to follow the procedures used to identify protein sequences? Three days isn’t much time, but I was cautiously optimistic.

First Impressions

Last Monday, I boarded a train to Washington DC, took a quick cab over to Georgetown to say hello to some of my old professors, and proceeded to take the Metro up to Rockville. After a solid night’s sleep at the “Sleep Inn,” I took the hotel’s shuttle to the door of the Venter Institute.

The entrance of JCVI has an aura of science and progress. The walls of the lobby and hallways are covered with neatly framed images of sequenced genomes. The individual proteins in such pictures are illuminated in different colors, invoking modern art. The head of educational outreach programs gave us a tour of the grounds, which concluded at the space right in front of Venter’s office (he was traveling at the time and unfortunately not in his office!). Employees of JCVI refer to the space as “the museum” as several objects deeply rooted in scientific history are on display. A glass case on the left side of the room stores letters exchanged between Watson and Crick. A very early model of a sequencing machine used by Rosyln Franklin is on the right. A large statue of a tiger seemingly prowls in the middle of the room – one of several tiger statues that used to be at the building’s entrance when the Institute bore its previous name. If the tiger is the unofficial mascot of JCVI, it’s certainly an appropriate one. This is not the place for the ambivalent.

Do the rules say, “No riding the tiger”? I hope not.

It’s clear that the staff at JCVI take great pride in their accomplishments and with good reason! Copies of Science and other prestigious medical journals containing studies published by JVCI or reports of efforts led by Venter are displayed on tables in several locations. The walls of the hallway leading to Venter’s office are covered with framed newspaper and magazine articles featuring Venter – articles in Wired, People Magazine, and the New York Times. Venter has been named one of the top 100 most influential people in the world by Time Magazine for the last two years.

Before the training began, I had the opportunity to chat with some of the twelve other people in my class. I had already met Dr. Anne Rosenwald, a professor at Georgetown whose research focuses on understanding the genetics of various yeast forms. She also teaches biochemistry. Dr. Rosenwald was attending the session in the hopes of working out a deal with JCVI in which the Center could provide her with genomes that have been analyzed by computers but are still in need of human annotation. Annotation refers to the process of using clues in a DNA sequence in order to name and identify protein coding regions. If such an exchange of information is possible, it would allow her undergraduate students to map a bacterial genome as their thesis project. I hope the partnership works out because I think that while challenging, using JCVI’s annotation technology would provide any undergraduate with excellent preparation for microbiology and molecular biology gradate programs. I certainly wish I could have learned how to sequence a genome as an undergraduate!

Let the learning begin

Two members of our group had travelled to JCVI all the way from South Africa. Researchers at the University of the Free State, they were already using several of JCVI’s programs to sequence and thus better understand the genomes of bacteria isolated from several African caves – bacteria that have never before been classified. I spoke with them about the challenges of mapping completely new genomes. Soon enough, I aspire to study new genomes myself, especially those pertaining to the great mass of unclassified species of bacteria in the human body. I figured their feedback could clue me in to the challenges particular to dealing with unknown organisms.

The South African duo were pleased with what they have been able to learn thus far about their cave-dwelling species. When it comes to JCVI’s sequencing technology, they were old pros and suggested improvements to the software throughout the class. Why had they come to JCVI? I sensed an eagerness on their part to see the hub of progress in person and personally get to know some of the people working with and developing the technology they are using. At the end of the session they kindly invited me to visit South Africa and spend time in their lab. Who knows, I might take them up on the offer at some point as South Africa is one of my top travel destinations.

Site of the learning.

Our classroom provided a comfortable atmosphere in which to learn with shiny new laptops for each of us. Access to the laptop allowed us all to get a chance to navigate our way through a program as the instructor described its features in a lecture. Snacks, coffee, hot chocolate and tea were available at all times and during the class we would break every hour or so to refill our cups and chat.

The first day was spent learning about the process by which JVCI’s technology allows unknown proteins to be named and characterized (annotated). Our teacher, Ramana Madupu, is a full-time employee at JCVI who uses the technology discussed in the lecture in the course of her job.

Let’s discover a new species of bacteria!

Let’s say you are picking your nose. First of all, shame on you. But, let’s say that in spite of your flagrant disregard for common decency, you nobly want to contribute to human progress by determining what kind of bacteria are in your booger. After conducting several basic experiments on the bacterial DNA in your lab, you decide that a bacterial species may be new and unique, so you decide to contact JCVI.

JCVI has you send them a sample of the bacteria in question. A non-profit institution, JCVI will run your genome through its sequencing machine at no charge to you. This service is largely automated and it’s becoming cheaper and cheaper. JCVI’s mandate is to sequence as many genomes as possible and freely share that data with researchers. However, JCVI’s offer to freely sequence and interpret your genome comes with an expectation, namely that upon receiving your results, you will review and manually correct any of the sequence errors. At last check, JCVI’s computer annotation programs claims a 95% accuracy rate.

As we know from high school biology, DNA consists of four nucleotides: adenine, thymine, cytosine, and guanine. A gene is nothing more than a sequence of those As, Ts, Cs, and Gs, one which codes for a particular protein. Genes are the blueprints for making proteins, and in fact a new gene is often referred to, at JCVI at least, as a “putative protein.” (The word putative is used until one has sufficiently conclusive evidence to remove that label.)

Sequencing and beginning to interpret a genome

At the risk of gross oversimplification or misstatement, let me dare to explain the technical process of how a genome is sequenced and interpreted. You start with a genome. The DNA is processed with fluorescent dye. Each base pair (aka a nucleotide) emits a different color. Those colors are read by a machine and interpreted as a sequence of nucleotides. The result is an exceedingly long sequence of As, Ts, Cs, and Gs.

Now the fun really begins. The goal is to take this enormous sequence and begin to determine which base pair sequence codes for which protein and in which biological category. The Prokaryotic Annotation Pipeline aka “the pipeline” to the rescue!

The pipeline is an algorithm-based workflow which automatically predicts to a good, but certainly not perfect, degree of reliability for the name, location, and function of a gene. The pipeline does this by comparing your base pair sequences to sequences of previously identified proteins that exist in a variety of databases.

But how does the pipeline know which segments of your base pair sequence to check against known protein sequences in the hopes of finding a match? There are apparently a lot of fancy statistical algorithms at work here, but one way is to look at the codons, or pieces of genetic code which mark the start and end of a protein sequence. The base pair sequences ATG, GTG, and TTG almost always code for the start of a protein, while the sequences TAA, TAG and TGA almost always indicate the end of a protein. Of course there are always exceptions to the rule, which is why every sequence to come through the pipeline should be checked by a human being. That’s the ideal anyway. By identifying these start and stop codons, the pipeline has a pretty good idea of where one protein coding sequence ends and another begins. At this point, each potential protein coding sequence is referred to as an ORF or “open reading frame.”

The algorithm matching a gene to an existing sequence applies greater weight to matches derived by certain databases. For example, one database frequently used for comparison is Swiss-Prot. Swiss-Prot relies exclusively on manual annotation by humans. At present, humans make fewer annotation errors and are, therefore, more reliable than software. For this reason (and perhaps due to the fact that, at least according to stereotype, the Swiss are highly precise), Swiss-Prot is arguably the gold standard. If a sequence from your bacterial genome matches a Swiss-Prot sequence, the confidence level is high that the match is correct.

During the pipeline comparison process, the software will also run your protein sequences against what are known as “Hidden Markov Models” or HMMs. HMMs are essentially statistical models of the patterns of amino acids in a multiple alignment of proteins which share sequence and functional similarity. Proteins run against HMMs receive a score as to how well they match the model. If the score is high enough you can reasonably expect your protein to have the same function that the HMM represents. For example, if your protein has a high-scoring match to an HMM model for a protein involved in sugar transport, you can be pretty sure that the match protein from your genome has the same role.

After a particular protein sequence is run against HMM models, the software assigns it a putative name and role, based on how much information it believes it has to support such a label. The process of comparing sections of your base pair sequence to as many existing protein databases as possible is also referred to as BLASTING. Depending on the level of evidence at hand, the protein is also given a gene symbol, role information, and sometimes numbers that pertain to its classification.

For example, after one of your proteins is run through the pipeline the pipeline might come up with the following result:

TIGR00433

Name: biotin synthase

Gene symbol: bioB

TIGR role: 77 biotin synthesis

Now, what does this mean? Let’s break this down. The fact that the gene has what is called a TIGERfam ID (TIGR0043) refers to the fact that it had a high scoring match to a protein previously annotated at JCVI. Since JCVI obviously believes their genomes have been well annotated, a TIGERfam match that exceeds the minimum threshold for reliability is generally regarded as a sign that the computer has made the correct match. The name and protein role associated with the highest HMM and other database matches for your protein is also displayed, along with the symbol for the putative gene. In this example, it appears that it is the software’s best guess that your protein is involved in the synthesis of biotin (also known as vitamin B7).

JCVI repeats this process for every Open Reading Frame sequence it detects, and the number of sequences often ranges in the thousands.

How then does one access the proposed annotations generated by the pipeline? After each of your protein sequences has been run through the pipeline, JVCI software condenses them into a digital file that is sent to you. At this point you need to use a web-based program that allows you to manually modify the results. The program is called Manatee, Manual Annotation Tool Etc. Etc. An open source project, it was also created by JCVI software programmers. A bit intimidating for the uninitiated, Manatee is a powerful and exquisite program, which allows a person to assign each putative protein with the correct name and function.

Annotating a DNA sequence

Your goal in using Manatee is to make sure that the protein matches made by the pipeline are grounded by supporting evidence. For example, you can check for “gene model curation” which provides information necessary to ensure that your genes have the correct coordinates and that your set of predicated genes is complete. Other features allow you to look at the raw base pair sequence of your genome in order to identify rare start and stop codons that the computer may have missed, or screenshots that allow you to note if the software accidentally annotated overlapping genes.

The labs at JCVI are first-class.

As the human annotators using Manatee use their good old-fashioned brain power to identify where the JCVI computers may have made mistakes, they alter the names of certain proteins in accordance with such findings. When naming a protein, the goal is always to err on the side of conservatism.

Let’s say, for example, that based on a strong HMM hit, the computer has decided that one of your proteins is a ribose ABC transporter protein (ribose is a sugar). But after further examining the protein using Manatee’s tools you decide that there really isn’t enough evidence to support the conclusion that the sugar transported by your protein is ribose. You then manually change the protein’s name in Manatee so that it is less definitive by calling it only an “sugar ABC transporter”. Then, after using even more of Manatee’s features, you decide that you can’t put together sufficient evidence that the gene in question really transports any form of sugar. Under such circumstances, you make its name even less specific, calling it simply an “ABC transporter.”

As you can see, Manatee is a tool which enables researchers to better make judgments about the role and function of genes by assigning characteristics to those genes. Often enough, evidence to make these determinations is insufficient and attributes are characterized by how reliable the best evidence is. One expands and contracts the attributed qualities as the evidence warrants. When the evidence is equivocal, you say that a protein is “putative.” This apparently is the nature of genetic research, one which requires scientists to pick up on indeterminacy and do their best to fill in the gaps as they go.

Every DNA sequence which emerges from the pipeline is putative. Certain sequences remain so because they fall short of threshold reliability which would allow the software to give it an existing name. Under such circumstances, no name is assigned and no role is attributed. The protein is simply named “hypothetical protein.” If a hypothetical protein from one species matches a hypothetical protein from another, each are given the name “conserved hypothetical protein.” Since the hypothetical protein has been found in two different species, the corresponding sequence clearly exists. But the sequence’s series of base pairs are so different from known sequences that, at this point in time, neither the software nor a human annotator are able to give it a name or role. Ramana (my instructor) commented that as the Human Microbiome Project presses on ahead, she expects to see many more “hypothetical proteins” show up in genomes. In fact, she had just recently finished sequencing about eight Microbiome genomes and was surprised at how few of their DNA sequences matched known proteins. This suggests that the majority of the yet unknown bacteria that inhabit the human body are quite different than those species we have already become familiar with such as E. coli or Tuberculosis.

One might ask, “Isn’t the human annotation process open to error and bias?” The answer is yes. It’s up to the human annotators to decide if they can find enough information to support a software derived match and every human has different tendencies when it comes to such decisions. Annotators like Ramana say that after working with a sufficient number of genomes they usually learn to trust their gut feelings and standardize the process by which they make naming decisions. Even the best human annotators would admit that 100% consistency, from one day to the next, between one annotator and the next, is unattainable.

The Institute had a modern aesthetic.

So what happens when an annotator has finished going over all the proteins in a particular genome? Genomes in which all protein sequences have been given a name and function are considered “closed” and made available through GenBank, ultimately. Any genome with loose ends is considered “open,” with the hope that future researchers will we able to confidently determine what the names and roles of current hypothetical proteins. One way to determine the role of a “hypothetical protein” is to study the protein coding sequence in the laboratory using in vitro techniques such as the creation of gene knockouts. Given this, it should be clear to my readers that sequencing technology does not obviate the need for laboratory research. There remains a lot of work to be done in this field.

About the Comprehensive Microbial Resource (CMR)

JCVI enters as many genomes as possible into a database called the Comprehensive Microbial Resource (CMR). CMR is a free, open-source website that allows access to the sequence and annotation of all completed prokaryotic genomes. CMR is a seemingly invaluable resource. Before genomes are entered into the database they are standardized in a manner that makes them much easier to be compared. Researchers from over 200 sequencing centers currently put sequenced genomes into a database called GenBank. GenBank contains about 600 complete prokaryotic genomes with about 10 new genomes released each month. One of the significant problems with GenBank is that the annotation process at each center that submits genomes to GenBank is done so differently that many of the genomes in GenBank have been named using different conventions. Often, they have also been assigned genes symbols and role names that differ depending on their where they were sequenced.

The goal of the CMR is to take the genomes from GenBank and create common datatypes with the same nomenclature sequence elements annotation methodology. When this has been done individual genomes can be compared much more easily and accurately. There are currently about 400 organisms in CMV but the project’s leaders have ambitiously committed themselves to adding several hundred more genomes to the database in the coming months. One reason that the CMV contains fewer genomes than GenBank is because the project is, thus far, unfunded. Apparently, JCVI has been working on the CMV without grant money for the last two years. The program is so well-designed and useful that it’s hard to believe it could go unfunded. I was told that JCVI has just applied for a new grant that might allow the project to be funded and project leaders should hear back about the decision in a week or two. Fingers crossed!

Tanja Davidson is one the main directors of the CMR, who was our teacher on day three. In fact, our entire third day was spent learning about the CMR, which at first glance, contains a daunting but well-organized number of features. CMR allows the researcher to compare multiple genomes using what are called “cross genome analysis pages.” These tools allow two or more genomes to be compared so that the elements they have in common (or the elements that make them different) can easily be analyzed.

Imagine that doctors report an outbreak of a stomach disease and a bacterial species is isolated from people with the illness. The genome of the disease-causing pathogen is put through Glimmer and the pipeline, annotated by humans, and found to be part of the E. coli family. By using CMR tools, researchers can compare the genome of the new E. coli variant to the genomes of other E. coli species that have not been tied to stomach disease. Most of the genes between the different forms of E. coli should be the same because they are of the same family. But those genes that differ between the recently isolated species and those already in the database can be assumed to be those coding for the proteins that endow the new variant with the ability to cause disease. In this case, the CMV comparison tools greatly narrowed down what would otherwise have been a veritable “needle in a haystack” situation.

Other nice features of the CMV include the ability to access a “Role Category Graph” which displays the different roles of all the proteins in a genome in a colorful pie chart. A tool called “Restriction Digest” allows users to splice genes of interest with various enzymes – a procedure that takes a long time to complete in the lab but only minutes to complete using the CMV. A “Pseudo 2-D Gel” allows users to get an idea of what a genome of interest looks like in another dimension. Each dot of a 2D gel represents a single protein whose location can be compared to others. The comparative tools even allow for the creation of a scatter plot in which two genomes are compared on a two-dimensional plane. MUMmer or (Maximum Unique Match) compares genomes at the nucleotide level, allowing scientists to detect just single nucleotide differences between DNA sequences.

Ready to learn about human annotation!

When it comes to the CMR, Tanja and other JCVI employees welcome feedback from scientists other than those at JCVI. In fact, while we were doing some practice CMV tutorials in class, the pair from South African and Dr. Rosenwald came across a few minor glitches in the system. Tanja was quick to write them down and most of them were already fixed by the time we got back from lunch. Rosenwald and others also offered feedback about new features they might like to see in the CMV and Tanja was again quick to record their suggestion and insights. I could tell she was definitely not just humoring people but actually planning to pass every suggestion by her development team.

The reality is that Manatee and the Annotation Engine project are part of the Institute’s open source initiative, the goal of which is to provide high quality software and services to the genomic community. External involvement and feedback is strongly encouraged because it’s such feedback that drives development and continual improvement of the software. In fact, JCVI doesn’t actually have employees who test their software, so they fully depend on user feedback. Some of us joked that because we were testing the CMV as part of our class exercises we should have been paid to attend the training session rather than vice versa.

Improving the accuracy of the pipeline

Human annotation is a lengthy and laborious process. One of the foremost goals at JCVI is to perfect the pipeline and the computer annotation process such that human annotation is no longer necessary. One Idea currently being tossed around at JCVI when it comes to perfecting the output of the pipeline is a concept referred to as something like “humanitization.” (I’ve searched my notes but can’t find the exact name!) The annotators at JCVI are currently being asked to report exactly how they go about using Manatee in order to annotate a genome. As previously discussed, since there are so many databases to compare and analyze in Manatee, each employee using the program has settled into a pattern of evaluating database information in a certain methodical fashion. The hope is that if some of the best human annotation regimens are recorded and analyzed, they can be translated into logic, which software could duplicate.

If these extra steps do indeed increase the accuracy of the protein matches made by the pipeline, there may no longer be a need for humans to check Manatee’s output. So it’s possible that in the coming years genome sequencing may be a completely automated process. At the current moment, the pipeline’s protein matches are accurate about 95% of the time The stated goal is to get that level of accuracy into the 99-100% percent range. So, as Tanja commented, the human annotators at JCVI who are currently helping programers understand how they navigate Manatee may, by doing so, actually be putting themselves out of a job.

But at least for now, human employees are still an integral part of the annotation system. Four recently hired JCVI employees were attending the teaching session. During a discussion about perfecting the pipeline, our instructor confided that one of our classmates had just been hired with the expectations that he would create the technology to make the pipeline more accurate. What a daunting job! The rest of us regarded him with a certain level of awe over the next two days. Every so often our practice sets would reveal a flaw in pipeline output and the instructor would turn to this particular employee and say something like, “Of course now, you’ll be fixing this problem.” Such comments reflect what seems to be the prevailing attitude at JCVI. Most of their projects are extremely ambitious and half the time I’m not sure if they even know if success is possible when a task is initiated. But the mindset is “No matter how hard this goal seems we will simply have to find a way to get it done!” This type of determined thinking does seem to generate results as there is little doubt that such an attitude was the driving force behind the Institute’s ability to sequence the human genome in record time.

Tanja, our instructor, and Phil, a JCVI employee who was hired to improve the pipeline.

As implied by the above paragraph there are a lot of situations at JCVI that end up pitting humans against computers. As Ramana described, it would be ideal if every genome sent to JCVI could be manually annotated from the onset. At least for now, a well-trained human is able to pick up on subtelties of database comparisons that the computer can miss. But such a scenario, at least over the long term, simply isn’t sustainable. Since genome mapping is growing in popularity over the coming years, humans alone cannot keep up with the number of genomes requiring mapping. Although using computers to annotate genomes slightly compromises accuracy, the technology must be used in order to keep up with demand. Ideally genomes are manually checked with Manatee but there are definitely JCVI/TIGER annotations that are never checked by a human annotator at all.

Sequencing bacteria and the future of medicine

In recent years, mapping genomes has grown in popularity. Scientists working on efforts related to the Human Microbiome Project currently want to map the genomes of every single bacterial species capable of inhabiting the human body, and such pathogens may number in the thousands. But large groups of other scientists are set on better understanding the massive number of bacteria that inhabit our oceans. Since little is known about many regions of the ocean, who knows how many microbes these efforts may turn up? Then, like the two scientists in our group, other research teams seek to map the genome of bacteria that live in obscure land locations such as caves, volcanoes, mines etc. So, the JCVI computers and those at other sequencing centers are relentlessly accumulating DNA data.

Perhaps because they have each personally annotated so many hypothetical proteins about which we currently know nothing, the staff at JCVI are very open to the idea that we are only on the brink, if that, of truly understanding the bacteria capable of making us ill. This correlates with the Marshall Pathogenesis in which essentially all inflammatory diseases are attributed to infection with chronic intraphagocytic metagenomic bacteria that, for the most part, have yet to be clearly named and sequenced. One study I often invoke was conducted by Dempsey and team. This Glasgow-based group found human tissue taken from prosthetic hip joints contained protein sequences corresponding to those of hydrothermal heat vent bacteria. Most of the time when I discuss the study, other scientists are skeptical of the results. The average response is that they would like to see the results repeated or that the sample was contaminated. Ramana had no such reaction. In her opinion, there can definitely be hydrothermal heat bacteria in the human body and she’s confident the sample was not tainted. When we discussed the findings she suggested that the bacteria are probably not killed at high temperatures which, interestingly, was one of Dr. Marshall’s first inferences when analying the data.

A JCVI sequencing machine

The organizers made an admirable effort to serve us savory lunches which we ate in one of JCVIs cafeterias. All our teachers attended lunch and sat among us, meaning that I was able to easily batter them with questions. Alex Richter, one of the program heads, was great about answering my questions in detail. Thanks to his anecdotes, I got a much better impression of what microbiology labs will be doing in the coming years and the tools I will likely need to master as a potential microbiology PhD student. Before attending the training I had wondered if I would be able to understand JCVI’s sequencing technology without a background in computer science. But Richter didn’t seem to think that my lack of computer training is an issue and it’s true that I certainly seemed able to follow the discussions in class. I was encouraged by Richter’s comment that someone good at scientific reasoning (such as, ahem, myself) is also likely to be good at working systematically with computer programs. I’m sure he’s right, but even so I won’t be contributing to the Linux codebase any time soon.

It felt pretty darn good to be in a place where I personally believe that government funding is going towards research that is really going to have an impact on our ability to better understand chronic disease. As the Marshall Pathogenesis continues to spread, it’s clear that bacteria will eventually receive all the scrutiny they are due. At that point, scientists, doctors and patients alike are going to demand a more thorough understanding of bacteria implicated in chronic disease and down to the level of the genome.

It’s great that JCVI is already starting to collect data on never before sequenced bacteria. It’s also good that the Institute is striving to perfect bacterial sequencing technology now, so that by the time the Marshall Pathogenesis gains hold, sequencing results should not only be more accurate but also easier to use. Just as our ability to sequence genes has improved exponentially, so, I believe, will our ability to interpret the data. The tools are just getting better and better. As someone who has the inside scoop about the fact that bacteria are headed for the big time, I feel we’re closer than ever to characterizing the genomes of the pathogens that are capable of making us so ill.

De Grey responded, “Because I am an old man. I am 158.”

Aubrey de Grey

It has been three years since then and at the ripe age of 161 (according to his Wikipedia bio, his birthday is in April), Aubrey de Grey presided over the latest of the Methuselah Foundation’s annual anti-aging symposiums. At the end of June 2008, a group of us with ties to Autoimmunity Research Foundation attended that meeting on the *very* sunny campus of UCLA. Our goal was to get researchers thinking about a bacterial explanation for diseases of the aging, and to get them to begin considering the Marshall Protocol as an anti-aging option.

Aubrey de Grey is always surrounded by people, be they prestigious presenters, researchers, conference organizers, or any of his small army of energized volunteers for which he plays field marshall.

De Grey is an eminently likable guy. Although he talks in an impatient staccato, he seems to be an accomplished listener. You can tell he’s listening because he’s thoughtfully tugging on his beard, as if that were some lever that, when pulled, speeds up his thought process.

It’s hard to read too much into why a 45-year old (you knew he was joking about 158, right?) opts to sport a two-foot beard, but I’m pretty sure it may have something to do with the fact he has fully embraced his– some might say, quixotic– quest.

Those of us associated with the Marshall Protocol know what it’s like to be thought of as quixotic: “I can understand how the Marshall Protocol could treat a single indication or even disease, but dozens of them? There’s no way that could be true….” Yup, we hear that a lot.

De Grey’s engaging personality, enthusiasm, and scientific prowess have made him a singular catalyst for the anti-aging movement. It has also earned the man a sizable following from a group including scientists, doctors, entrepreneurs, drug manufacturers, and young students, all of whom share the belief that the end of aging is at hand and only requires the right mix of funding, technology, and an evolution in social attitudes towards aging. De Grey argues that aging is a disease and a curable one at that.

The first day of the Conference, I attended the press conference with a press pass for Bacteriality. During the sound check, I chatted with a group of graduate students who were filming a movie about aging.  As is my custom, I described Dr. Marshall’s work. When the full implications of the MP became clear to them, the grad students’ expressions peaked, and I arranged to do an interview about the MP for their movie (I have no idea when or where it might come out).

The open-mindedness we saw from the pair of filmmakers was largely consistent with what we saw from others. In my opinion, this perspective is a very sensible one. After all, right now, there are precious few curative therapies for any of the diseases of the aging.

Even at an anti-aging conference, the ARF’s work is unique because, as Dr. Marshall alluded to in his talk, the ARF is studying “living, breathing human subjects” rather than fruit flies, rats or even higher primates.

Lunchtime, which lasted for several hours, provided an excellent opportunity to speak with others present.  Lunch was served buffet style and tables were scattered on one of the campus greens in the open air.  About four tables were in the shade, and naturally those of us with Autoimmunity Research Foundation made an effort to grab the shady seats as quickly as possible.  Since it turned out that most other attendees also sought to avoid the California sun, extra chairs were pulled up to the shady tables and each became a hub of discussion that frequently focused on the Th1 pathogens or the immunosuppressive effects of vitamin D. 

It became abundantly clear that the doctors, scientists, and drug manufacturers present had, until speaking with myself or other members of ARF, simply failed to factor chronic bacteria into the aging process.  The same proved true when it came to the researchers giving official talks (Dr. Marshall aside of course!).  For example, on Saturday morning, Dr. Jerry Shay of University of Texas Southwestern Medical Center gave a talk that discussed DNA repair.  At the end of his presentation, I asked the following question:  “Chronic intracellular bacteria… could they be interfering with the processes of transcription, translation and, most importantly, DNA repair in the nuclei of cells?”  After a brief pause and a quick scratch of his head, he replied that he had simply not considered the possibility. 

Rather, most of the talks fit into the model of aging first invoked by de Grey at the inception of the Conference – a model in which the human body is thought of as a car.  Over the years, cars inevitably accumulate damage and generally end up at the repair shop and finally at the dump.  But, as de Grey made clear, much can be done to keep a vehicle in prime condition.  Periodic oil checks, trips to the mechanic for touch-ups before damage occurs, great care in terms of keeping the car clear of scratches and dents – all this can allow the vehicle to function for a much longer period of time.  In fact, if enough effort is invested into a vehicle, it might be able to run forever. 

The metaphor of the human body as a car suggests that all the “junk” (toxins, debris, waste products, etc.) that causes the tissues to age is created by the body itself.  Yet such a model, in my eyes, fails to take into account the severe symptoms caused by diseases of the aging or the extensiveness of the aging process itself.  Sure, the human body will experience wear and tear over time, but can the purely intrinsic accumulation of “junk” suffice to cause conditions as serious as Alzheimer’s disease or as deadly as cancer and cardiovascular disease? 

Isn’t it more logical that external/environmental factors are also driving these serious states of decline and that, based on the recent findings of biomedical researcher Trevor Marshall, chronic intraphagocytic bacteria are the most obvious culprits contributing to tissue degradation, decay, and biological waste products?

And sure enough, the talks given by other researchers frequently supported the Marshall Pathogenesis, even if the scientists presenting the data have yet to realize why.  The importance of maintaining an active innate immune response in order to defy aging was brought up on several occasions. 

Everyone seemed to agree that the full complement of diseases of the aging were connected to inflammation, a concept invoked numerous times by the Conference’s speakers. It was an exercise in self-restraint not to stand up in my seat and shout to the crowd, “Chronic inflammation is the result of chronic bacterial infection!!”

Chatting with fellow participants in front of my poster.

Take our telomeres – the DNA sequences at the ends of human chromosomes that get shorter with every cell division.  Eventually telomeres shorten to a point where a cell can no longer divide and the body loses its ability to effectively produce new cells.  Several of the speakers correlated numerous chronic inflammatory diseases with the presence of shorter telomeres, including not just cancer and Alzheimer’s but also illnesses like ulcerative colitis and idiopathic pulmonary fibrosis (clearly a Th1 disease!).   Since we know these diseases are caused by pathogens, it’s very likely that the shortened telomeres observed in patients with these conditions stem from the fact that chronic bacteria and viruses can mutate telomeric DNA, causing it to shorten at a faster rate. 

If telomeric DNA is mutated by the Th1 pathogens, then such knowledge can greatly aid scientists who are already working on procedures that would allow for the extension of telomere length (several of whom spoke at the Conference).  For example, in an excellent talk called, “The Struggle to Keep Our Telomeres Long,” Laura Briggs of the Sierra Sciences Research Group discussed a current conundrum faced by telomere researchers. 

Briggs and others have developed techniques that can potentially lengthen our telomeres, but they are faced with the dilemma that extending the lifespan of our healthy cells will also extend the lifespan of our cancer cells (and, I might add, infected cells). As Briggs stated, “This leaves us with the irrefutable conclusion that we will be unable to significantly extend our lifespans without finding a cure for cancer that doesn’t limit our ability to extend the lengths of our telomeres.” 

Well… what if I argued that killing the Th1 pathogens with the Marshall Protocol and maintaining the integrity of the VDR and the innate immune response is very likely the cure for cancer that Briggs seeks? One of Dr. Marshall’s talk’s most provocative points is that in a cohort of over 500 high-risk patients who have been followed for up to five years, there has been no incidence of metastasis. With such knowledge at hand, defying aging by successfully lengthening our telomeres is a much more plausible possibility.

Several talks also focused on what are referred to as “senescent” cells – cells that fail to undergo the usual process of cell death and instead enter a stable and essentially irreversible growth-arrest state.  Senescent cells have been shown to accumulate with age and with age-related diseases, making it clear that the presence of these cells contributes to the aging process and disease in general.   And while the reason behind the formation of senescent cells remains a mystery to the medical community at large, it’s quite probable, at least in my eyes, that senescent cells are simply infected cells.
 
As Judith Campisi of the Lawrence Berkeley National Library and Buck Institute made clear in a speech, cancerous stimuli often foster the development of senescent cells and, as discussed above, the Th1 pathogens are quite prolific during cancer.  But the greatest giveaway that senescent cells are at the mercy of the Th1 pathogens stems from the fact that, as Campisi described, senescent cells are metabolically active and secrete myriad inflammatory cytokines.  In my opinion, nothing screams infection more than the release of inflammatory cytokines, since the inflammatory molecules are released by the immune system in response to infection.  Campisi also described how, although senescent cells are targeted for clearance by the innate immune system, they are able to defy the immune response by secreting high levels of enzymes called matrix metalloproteinases (MMPs).  In my opinion, the creation of these enzymes probably marks yet another way the Th1 pathogens have evolved to alter cellular machinery in order to foster their survival.  

Then there’s stem cells.  If the rest of our cells can be infected by the Th1 pathogens, then why not our stem cells?  Or, can our stem cells be damaged by the cytokines secreted by other infected cells?  Indeed, Amy Wagers of Harvard University presented a talk in which she clarified that much of the aging process results when the stem cells can no longer repair the tissues.  Wagers stated that her work points towards “a discrete set of metabolic regulators and inflammatory cytokines which may alter the signals that stem cells receive from their environment in aged animals.”  Again, the fact that inflammatory cytokines seem to affect stem cell resiliency points to the involvement of chronic infection in stem cell decline. 

It got to the point where some of the talks were frustrating to watch because the data presented was so indicative of infection yet the researchers themselves kept missing the signs.  “They’re so close, but yet so far away,” Dr. Marshall commented at lunch.  I could sense the frustration in his voice.  
That most of the researchers at the Conference stand on the edge of better understanding their results in the context of the Th1 pathogens was best exemplified by a presentation given by Rita Effros of UCLA.  Effros actually presented data showing that chronic viral infection leads to an increase in senescent cells as well as significant decreases in telomere length.  Effros stressed how the constant effort that the body must extend in order to try to keep latent viral infections under control clearly detracts from the energy the body needs in order to effectively manage the waste products that damage the tissues.  Yet Effros is convinced that chronic viral infections cannot be eliminated and that, once present in a host, they cannot be stopped from causing an inflammatory response for the remainder of a patient’s life. 

Of course, those familiar with the Marshall Pathogenesis understand that latent viruses are simply co-infectious agents that are able to take advantage of an immune system greatly weakened by the Th1 pathogens.  So if the Marshall Protocol is used to eliminate the Th1 pathogens and innate immunity is restored, latent viral infections can indeed be quelled.  Nothing proves this reality more clearly than our own patient data (collected from the MP study site) which shows that viral co-infections disappear as patients reach the later stages of the MP.
   
Finally, after a brief coffee break, it was time for Dr. Marshall to speak.  I sat near the front, eagerly awaiting the audience’s reaction.  They seemed interested in all aspects of the talk.  But  when Dr. Marshall put up a slide showing the bacterial species detected from a shot-gun sequencing study on prosthetic hip joints and mentioned that one of the bacteria found was “thermal vent bacteria”, the audience cooed with intrigue. 

The slide showed that bacterial species such as Proteobacter, Methylobacter, and others have, only recently, been detected inside human tissues.  “These are bacteria never previously thought to exist in man,” Marshall stated.  “These are bacteria that nobody is looking for.”  And that’s exactly the point. 
The audience suddenly understood that, not just the aging community, but the medical community at large, is ignoring a vast microbiota of chronic pathogens – the pathogens that patients on the Marshall Protocol kill day-in and day-out, the pathogens that cause the inflammation linked to so many diseases of the aging, and very likely the pathogens that influence telomere length, senescence, stem cell resiliency, and the overall accumulation of the “junk” that contributes to the aging process.

Talking science with Dr. Marshall

Serendipitously, the talk after Dr. Marshall’s focused on centenarians, or people who live to be over 100 years of age and still maintain a relatively decent state of health.  The first slide, presented by Sonya Vasto of the University of Palermo showed a wrinkled, white-haired woman doing the splits.  The image made it quite clear that not everyone succumbs to diseases of the aging at the same rate.   Vasto’s team had spent several years tracking the children of centenarians.  Not surprisingly, the children of centenarians themselves often live to be older than the rest of the population.  I say not surprisingly because it was extremely obvious (at least to me!) that centenarians simply harbor very low levels of the Th1 pathogens.  And since they don’t have high chronic bacterial loads, they pass very few of the disease-causing bacteria to their children who, on the whole, also live longer, healthier lives.  

At least some members of the audience were able to make the connection since when the speech ended, Vasto was asked if, based on Dr. Marshall’s research, centenarians might have evolved stronger immune systems that are better able to combat the Th1 pathogens.  Again, the speaker was essentially forced to concede that she simply had not considered the possibility.

Other talks after Dr. Marshall’s discussed ways in which human beings might be able to better regenerate tissue.  One speaker discussed how researchers are manipulating tissue so that human beings who are victim to heart failure might be able to regenerate a new heart.  Exciting stuff!  But a little voice in my head kept repeating, “What exactly is the point of regenerating an organ if you haven’t corrected the disease process that caused the original organ to fail?”  Of course, I’m not talking about situations where people lose an organ due to acute injury.  I’m referring to a case in which someone loses a heart due to, let’s say, a heart attack. 

Will simply growing a new heart ameliorate the disease?  Absolutely not.  Unless the chronic bacteria that caused the first heart failure are killed, they will inevitably infect the second heart.  Not to mention that the blood vessels of the person with a regenerated heart are still infected with the Th1 pathogens and are still full of the plaque (largely made up of dead bacterial cells) that they generate.  So when one researcher put up a slide stating that cardiac regeneration might  “cure” cardiovascular disease, I silently and strongly took exception. 

I had the same reaction in response to a talk that discussed how the tissue of neonatal animals could be used to help patients with severe gastrointestional disease regenerate new stomach and intestinal tissue.  While I’m thrilled that the ability to regenerate tissue is a viable possibility, I strongly feel that, until regenerative researchers factor the Th1 pathogens into the picture, they will be endlessly perplexed by the fact that regenerated organs might fail much sooner than expected. Like the infected tissues they were grown to replace, they too will become victim to the Th1 pathogens.
 
The poster sessions – where I finally got a chance to present my poster “VDR Receptor Competence Induces Recovery from Diseases of the Aging ” – started at 9:00 pm on both Saturday and Sunday. I lost my voice on Saturday night because so many people wanted an explanation of the figures and molecular models on my poster.  On Sunday night I rested my voice in preparation for a larger crowd at my poster since I figured that Dr. Marshall’s speech had ignited an even greater interest in the MP.  And the crowds came.  I spoke to doctors, researchers and students, all of whom wanted an in-depth explanation of Marshall’s work.   Since returning from the Conference, I’ve already received numerous emails from many of the people at the poster session, asking more profound questions about the MP or simply informing me that they are planning to start the Marshall Protocol themselves.  

When I described the Marshall Protocol as an open-based internet study trial it was refreshing to see most people respond with looks of admiration rather than incredulity.  Whereas most of the researchers I have spoken with at other conferences and events (who were educated before the rise of the internet) are often uncomfortable using the words “medicine” and “internet” in the same sentence, the younger and more technologically savvy crowd at “Understanding Aging” seemed excited by the prospect that we are collecting data in a place where it can be viewed and accessed by millions. 
 
In the same vein, Dr. Marshall’s background in technology was seen as an asset to his work and, for many, seemed to increase the credibility of his research. De Grey also possesses a background in computer technology that again seems to have helped him view aging through a wider lens.   In my opinion, the fact that de Grey and Marshall are both technologically savvy and have both succeeded in running two of the most fast-paced and successful scientific movements is far from a coincidence.

I found the staff and board members of the Methuselah Foundation to be very friendly.  They approached me and made an effort to introduce themselves.  Several told me about the Mprize and wanted to know if Autoimmunity Research Foundation might want to take a stab at winning the competition.  Unfortunately, the Mprize is a contest that intends to reward any research team that can make a mouse live beyond its normal lifespan. 

I explained to several of the Methuselah staff that Autoimmunity Research Foundation will never win the Mprize because the chronic microbiota that cause inflammatory disease in humans are not the same pathogens that infect mice.  Furthermore, as discussed in this article, there are great differences between the Vitamin D Receptor homology of mice and men. 

Whereas in man the Vitamin D Receptor controls expression of the bulk of the body’s antimicrobial peptides that target the Th1 pathogens, the rat Vitamin D Receptor performs no such action.  Based on this knowledge, I hope that the Methuselah Foundation considers the possibility of creating an “Hprize” or a prize given to the research team that first allows a human being to live a longer-than-normal lifespan.  In my opinion, those patients on the Marshall Protocol are already headed to living longer, healthier lives. So if tracked over the next few decades, the entire cohort would win the “Hprize” hands down.

Meanwhile, based on people’s reactions to my poster, I could tell that many were capturing the full scope of Marshall’s research.  Happily, many people expressing the most interest were doctors.  I think I even convinced a neurologist who had previously considered CFS to be a psychosomatic disease of the MP’s validity. 

At the beginning of the Conference I had spoken with a gentleman who had literally pulled a bottle of vitamin D out of his pocket when I had started to discuss the vitamin D Receptor.  He had incorrectly assumed that anyone discussing vitamin D would naturally say something positive about the secosteroid.  But by the end of the Sunday poster session he let me know that he had ditched his vitamin D and was even considering the MP.  

Another doctor was so moved by Dr. Marshall’s speech that, during the dinner before the Sunday poster session, he cornered me in the salad line to let me know how excited he was about the MP’s implications. He said the MP reminded him of a particular Sherlock Holmes short story where Holmes figures out the killer’s identity by looking for a clue in the very place no one had considered. He was referring to an electron microscopy image Dr. Marshall showed during his talk, one of a cell infected by bacteria. The bacteria is inside the human cell– no one thinks to look there! I have seen few people so animated and I was so surprised by his exuberance that I dropped my tray (we were eating in a cafeteria) and my dish shattered on the floor.  Whoops! 

As the night wore on and people continued to swarm around my poster, I realized that although my main aim at the Conference was to spread word about the Marshall Pathogenesis, the energy at the Conference was also having a profound effect on me.  I’m excited that so many intelligent minds are working together to extend the human lifespan and, as an optimist, I think that defying death is within the realm of possibility. 

On the other hand, I think that the Aging Community ought to rethink their approach.  Before trying to lengthen our telomeres, we need to better understand why they become short in the first place.  And before trying to eliminate senescent cells, we need to figure out why they incessantly secrete inflammatory cytokines.  And before trying to regenerate organs, we need a full understanding of why an original organ went bad in the first place. 

In my opinion, all of the above have a single (historically elusive) common denominator: the Th1 pathogens.  And, as Dr. Marshall described in his talk, the tools to eliminate the Th1 pathogens exist here and now.  People can start to eliminate their bacterial loads today.  So as a first step, let’s eradicate the Th1 pathogens from the population.  Then let’s see how our telomeres, tissues, and cell-types respond to massive reduction in inflammation.  At that point, I know that de Grey and every researcher who spoke at the Conference will be able to push the boundaries of aging even further with optimal success.

Where was Dr. de Grey anyway?  I caught a glimpse of him out of the corner of my eye.  Standing near the entrance, wine glass in hand, he was laughing heartily, surrounded, of course, by a group of admirers.  If de Grey continues to push forward aging research as effectively as he did at “Aging 2008″, then he may very well live beyond the normal lifespan.  Yet even with the prospect at hand, he was clearly enjoying the current moment.  After all, that’s the key.  No matter how long we live, enjoying the here and now is what makes life worth living in the first place.  As the Argentine poet Borges once wrote, “Por si no lo saben, de eso esta hecho la vida, solo de momentos….” “In case you don’t know, that’s what life is made up, only moments.”  Except for my applause-worthy dropping of the dinner tray, all the moments I experienced at the Conference were good ones and, whether I live to be 50, 100, 1000, or 1,000,000 years old, I’m thankful for the chance to have shared that with so many new and open-minded people. 

Finally, here’s a short video the ARF put together from the conference. (If you have trouble loading this, try the smaller YouTube version.) Enjoy….

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For those who have access to a high-speed internet connection and fast computer, better version of this video, in High Definition is . Also available: the related abstract and Conference details.

Our group in front of the Karolinska Institute

The Marshall Protocol epitomizes translational medicine, which is why, in my opinion, our poster presentations at the Conference were, for the most part, viewed with great interest and optimism.

The researchers who filled the lecture and poster halls at DMM had travelled to Sweden from the most prestigious universities in the world. It didn’t take long to realize that many of them have spent their entire careers looking for faulty genes that might be able to cause mental illnesses such as autism or obsessive-compulsive disorder.

The problem with such research is that the genetic mutations found in people with mental illness can easily be attributed to the fact that, once inside the body, the Th1 pathogens mutate our DNA. Thus, as a person acquires a significant bacterial load, mutations become more common and can even be passed from generation to generation. This alternate hypothesis for genetic mutations in people with various mental illnesses has been largely ignored by mainstream medicine, a fact confirmed by the talks at DMM. Many researchers spoke of only 5 -10% correlations in the genetic mutations found among patients with a particular mental disease such as autism. As I sat through lectures that discussed such correlations, the reality that only pathogens can cause such a diverse array of mutations became increasingly clear.

One might think that researchers looking for a purely genetic cause of disease would have become discouraged by this point. After all, as one of the researchers who gave a talk admitted, back in 1990 geneticists were sure they would identify direct correlations between specific genes and specific diseases by 2000, allowing them to cure most forms of illness. But the researchers at DMM seemed determined not to see this as a sign of failure. Instead, they put forth a new philosophy – namely the idea that genetic research has failed to generate any successful treatments because it has proved to be much more complex than expected. So, based on this notion of complexity, the geneticists at the Conference have yet again pushed back the date at which they expect to find a way to treat mental illness using gene therapies. Those of us familiar with the MP realize the date will be pushed back endlessly until pathogens are finally recognized as the agents causing the complex mutations observed by so many researchers.

After a talk on the genetics of obsessive-compulsive disorder, I threw my arm up in the air. By that point, I had realized that I would be given one of the few opportunities to ask the researcher a question, only if I did some serious arm flailing. “Have you considered pathogens?” I asked. “Have you considered the fact that perhaps the reason you are finding such random genetic mutations among your study subjects is because the mutations are simply the result of active changes to the DNA created by bacteria?” Dr. Matthew State of Yale University, who had given the talk, was thoughtful in his response. He agreed that certain environmental factors could be contributing to cognitive disease, and that pathogens are among these factors. The audience seemed interested in his response. Perhaps, like me, they were wondering just how complex genetic research will have to get before they are given any concrete explanations from geneticists about how to treat so many severe cognitive diseases.

Much of the research presented at the Conference focused on mice, and how we could poke, prod, and medicate them towards becoming alternately quicker learners and more chipper. And what would a neuroscience conference be without mice running mazes? We were not let down, as many researchers at the Conference had created novel ways to test the cognitive abilities of their furry subjects. In fact, one researcher whose talk hadn’t focused on mice squeezed in some data on rodents at the end of his speech, joking that he wouldn’t be believed unless he verified his data on mice. Again, I found it difficult to see how the implications of such studies on rodents could be applied to human beings, particularly since I’m well aware of the fact that the rodent VDR and immune system is vastly different than that of humans.

All of the speakers gave concise, deliberate presentations, and I have a lot of respect for the sophisticated techniques they have mastered in order to conduct their research. It’s just that the DMM’s aim is to stress translational medicine. At a Conference that is supposed to demonstrate how basic research can drive clinical care, none of the data presented by geneticists can be used to actually help even one person with any type of mental illness.

While the lectures inside the grand auditorium at the Institute focused largely on genetics, three posters lingered in the poster hall that implicated pathogens, rather than genes, as the cause of chronic disease. These posters were, of course, the posters created by Dr. Marshall, Meg Mangin, and myself.

Dr. Marshall lucked out in the sense that his poster slot was #53 – meaning that, because of the way the posters were positioned, his poster was the first one every person saw as they entered the poster hall. Just around the corner from his poster were Meg and her poster. This provided an excellent atmosphere in which people could learn about the MP. First, researchers would wander over to Dr. Marshall’s poster where they would get an in-depth explanation of the molecular science behind the treatment. Then, they would turn the corner and Meg would explain the practical side of the science – the way it has been used to create a medical treatment in which patients are monitored over an open study site.

It didn’t take long for people who spoke with Dr. Marshall and Meg to realize that Autoimmunity Research Foundation was the only group at the conference truly exemplifying translational medicine. For this reason, his work attracted many visitors. It didn’t hurt that Dr. Marshall had two small video players velcroed to his poster that ran videos of L-form bacteria inside the blood of a patient with CFS. The blood, which is absolutely overtaken by long biofilm tubules with bacteria inside, was a rather large wake-up call to those researchers who incorrectly consider the blood to be sterile.

In fact, Dr. Marshall was an endless source of electronic gadgets at the Conference. Out of a meticulously packed suitcase came small video cameras, recording devices, computer cables, and essentially any other device that successfully allowed Paul Albert of Weill Cornell Medical College – who came down to help with the technical aspects of the conference – to videotape large segments of the Conference. If Paul was missing a device, be it a cord or tripod, all he had to do was ask, and Dr. Marshall inevitably had it on him. It’s clear that focusing on molecular biology has not prevented Dr. Marshall from keeping up with the latest electronic trends.

My poster

My poster was on board space #89, which was on the far side of the auditorium. I was glad that my poster was separated from Dr. Marshall’s and Meg’s posters because people who wandered down the left side of the auditorium would hear me speak first and my poster seemed to spark enough interest so that they would continue on down the line to Dr. Marshall’s.

But the hypotheses put forth on my poster interested people in their own right. For starters, I was pleasantly surprised that everyone whom I spoke with seemed to realize that Chronic Fatigue Syndrome is an extremely debilitating disease with a very serious mental component. Nancy Pederson, the associate dean at Karolinska Institute, made a point of coming over to my poster because she is actually studying Chronic Fatigue Syndrome in identical twins. She admitted that her group is not finding any significant genetic trends among their study subjects. So her team is considering pathogens, namely viruses. We proceeded to discuss how the viruses observed in some of her study subjects might just be co-infections, co-infections that are able to take advantage of the immunosuppression generated by bacterial blockage of the VDR. She seemed interested. In her case, and in the case of others, I found people constantly picking up my handouts, my brochures about Autoimmunity Research Foundation, and my business card for Bacteriality.

I discussed several topics that related to my poster, but women in particular seemed extremely interested in understanding why women often feel better during pregnancy, then relapse after giving birth. The reason being that during pregnancy the active vitamin D metabolite 1,25-D rises to extremely high levels, interfering with the activity of the nuclear receptors that control many families of antimicrobial peptides. Because the peptides are produced at a much lower rate during pregnancy, women experience much less bacterial die-off and subsequent relief during that time. However, because women with Th1 disease are immunocompromised during pregnancy, the Th1 pathogens they harbor spread easily, causing them to feel worse after giving birth when 1,25-D drops back into a lower range.

A lot of people admitted that what I was putting forth “just really made so much sense!” After hearing so many complex explanations about the genetics of mental diseases, I guess Dr. Marshall’s hypotheses seemed exceptionally logical. I spent a long time discussing the scientific topics on my poster with Dr. Yujiang Shi from Harvard. After he had absorbed the details of the Marshall pathogenesis, he literally turned to me and stated confidently, “You know, Dr. Marshall is going to win the Nobel Prize.” I said, “Yes, I know!”

The concept of the poster hall is a good one because food is served at the back of the hall, meaning that anyone who wants to eat has to walk by all the posters in the process. Then, they can take their plate of food and wander around looking for posters that catch their eye. I didn’t have time to eat. Before I could even reach for a plate of food, someone had come over to my poster and wanted an explanation. Dr. Karl Deisseroth from Stanford (who later presented an amazing talk about new technology his team has developed to track the action of neurons in the brain) again commented that he was very open to the idea that chronic inflammatory diseases might have a bacterial cause. I spoke to several doctors who seemed eager to try the MP on their patients, particularly doctors who worked with children with behavioral disorders. Most seemed amazed that we were actually putting forth a potential treatment backed by so much scientific data.

Every so often I would glance down the hall and see Dr. Marshall’s yellow shirt and red tie gleaming under his jacket. He seemed to be talking non-stop as well. On the first day, I actually lost my voice for a while because of the fact that I talked so much.

The poster right next to mine that detailed how to create an immunosuppressive drug

Because of its location, my poster was quite close to the poster of the researcher next to me. He was a researcher from Northwestern University who sadly was presenting data that might allow for the development of yet another immunosuppressive drug. I have to say that people walked right by his poster and over to my poster instead. At one point, a good natured older man appeared behind me and tapped my shoulder. He said, “Your poster is getting so much attention, it’s too bad that you’re standing between me and my money.” I realized that he had funded the research for the immunosuppressive drug and that the crowd looking at my poster was largely blocking the poster about the medication. I tried to move over to the side, but at that exact moment one of the deans of the Karolinska Institute barged right in front of the other scientist’s poster to ask about mine. The older gentleman sighed and walked away. Any feelings of sympathy were short-lived as I was happy to see that people were more interested in the MP than immunosuppressive drugs.

A researcher a few posters away had travelled to the conference from the Longevity Institute in Japan. He was working with VDR knockout mice. He was so excited about Dr. Marshall’s work that he spent more time by my poster than his own. At one point he even asked me if I would be willing to put my name on one of his papers. I told him that if I thought the paper was accurate, I would be happy to do so. I’ll see how that works out.

Some of the other talks at the Conference, those that didn’t focus completely on genetics, were actually quite interesting. A speech that focused on estrogen sparked Dr. Marshall’s interest. He was also extremely interested in a talk by Dr. Adriano Aguzzi from the University Hospital of Zurich about prions. Prions are small infectious agents made purely of protein that are believed to propagate by refolding abnormally into altered structures that accumulate in infected tissue, causing cell death and tissue damage. I’ll be writing more about prions in another article.

After the poster sessions and presentation ended on Friday, we had just enough time to return to the hotel, rest for about five minutes, and then get ready for a welcome reception at the Town Hall. The words “Town Hall” don’t spur thoughts of glorious architecture in my mind, but I soon realized that the Stockholm Town Hall is an exception. When we entered the gates, we found ourselves just steps away from the water with slim arches bending gracefully over our heads.

Upon entering the reception hall we were offered glasses of wine. The head of the Karolinska Institute gave an inspiring welcome speech, to which we all responded “Skål!”, which is an all-purpose Swedish toast. At that point I started to realize, “Hey, I’m really here. This is the place where the top minds in medicine get together to advance the understanding of medicine.” A little chill ran down my spine as I gazed out the window at the beautiful buildings across the water that were illuminated in the setting sun.

Next, we made our way along a table replete with a smorgasbord of food which unfortunately consisted of a large variety of delicious looking salmon and other fish. Luckily, there was a large platter of Swedish meatballs that saved me from starvation.

Soon, we were ushered on a tour of the entire Town Hall. Having failed to read anything about the Stockholm Town Hall, I was amazed to walk into a room in which the walls were literally made of gold and a massive image of the maternal figure symbolizing Stockholm gleamed on the opposite wall. After that, we entered the “Blue Hall,” the space in which the Nobel Prize banquet takes place on December 10th every year. We then proceeded to enter an ornate room where the Stockholm city council meets. Finally, we ended up standing at the bottom of a tremendous stone tower that is one of the city’s landmarks.

Dr. Marshall and me at the Andy Warhol exhibit

It was tough to leave and go back to the hotel, but I needed to sleep so that I could present my poster with zeal the following day. After another day of long and successful talks and presenting our posters, we were bused to the Museum of Modern Art. First, we were given a tour of the Museum’s Andy Warhol exhibit, which was very well done and included many multimedia effects in an attempt to create an atmosphere that mimicked Warhol’s lifestyle. Dr. Karl Deisseroth from Stanford alerted me to a video which showed a transvestite slowly eating a banana. We had a good laugh over how eccentric Warhol was. Since Warhol’s art clearly reflects the fact that he suffered from anxiety, depression, and other mental afflictions, I could almost see each scientist there trying to come up with their own explanation for his cognitive issues. My biases are more than a little obvious– I think Warhol was just suffering from a tremendous number of Th1 pathogens.

Dinner was served right on the waterfront with a view of the old Swedish city across the water. Towards the end of the meal, Dr. Kenneth Chen of Massachusetts General Hospital and Harvard Medical School, one of the conference organizers, stood up to give a toast. After his own short speech, he selected other important members of the audience to stand up and give a few words about the Conference. I was half-listening, half-eating my delicious piece of steak, when I heard him ask Dr. Marshall to give a short speech. Dr. Marshall has attended the DMM Conference for the last three years and is thus something of a veteran. Dr. Marshall spoke about the importance of translational medicine, which, of course, is the focus of his work. As his words blended with the clinking of wine glasses and the lights of the beautiful buildings across the river, I felt an immense sense of pride in Dr. Marshall. I admire his relentless drive to push forward his scientific discoveries, and to consistently attend conferences even when some researchers dismiss his work. Now, here he was, being given the same recognition as any other scientist at the event.

Paul and Liz passing through the entrance to the Warhol exhibit

During those rare moments of down time, I chatted with Meg, her husband Tom, and Dr. Marshall’s wife, Liz. I’ve known Meg for three years over the internet so it was great to finally meet her in person. She’s spunky, fun, and full of interesting stories. Her husband Tom took full advantage of being in Sweden. Instead of attending the Conference, he spent most of the time at an Irish pub where he made friends with several Swedish natives, one suspiciously named Jim Beam. In fact, both Meg and Tom love Irish pubs. Meg even has her own special toast, part of which Paul caught on video. We discussed how, to a great extent, there are two different Megs. There is professional Meg who must be very careful about adhering to FDA regulations on the study site, and then there’s fun, laid-back Meg who loves to travel and has eight adorable grandchildren. Tom told me about the fact that Meg answers questions on the MP Board from the moment she wakes up until the moment she goes to sleep. He does all the shopping and cooking. Even when they go out to eat, Meg takes her laptop along so that she can post on the study site during the time when they are waiting for the food to arrive.

The Conference was partially sponsored by Cell Press, a group that runs a number of medical journals. The staff from Cell Press didn’t seem very interested in the MP. For one thing, I think they simply weren’t prepared to hear an explanation for chronic disease that was so different from those explanations offered by the lion’s share of articles their journals feature these days. As Dr. Marshall has commented, the editors of medical journals are petrified of publishing something that may turn out to be wrong, and perhaps the MP seemed almost too good to be true.

Interestingly, the representatives from several drug companies were very interested in my poster. A man from AstraZenica asked me a lot of questions. I told him that if his company wants a heads up, they should start creating drugs that prevent the formation of L-form bacteria. He laughed, but it may not be that long before he realizes that I was completely serious. At the museum dinner, I sat across the table from several drug chemists who told me they were disappointed that none of the speeches had really given them any clues about how to create more effective drugs for mental disease.

Their comments reenforced my belief that, at the moment, the timing is ripe for acceptance of the MP. Drug companies have little to work with in order to create palliative drugs for mental illness or illness in general. Genetic researchers claim they need a few more decades before their work will be able to help patients. Meanwhile, doctors are getting restless. They are tired of telling patients, “I can’t explain your illness and I can’t help you get better.” These doctors are increasingly attracted to the MP, or at least they were at Karolinska. Presenting information about the MP in person made a big difference. It makes me wish that I could sit down with every doctor and give them a 15 minute talk on the MP.

Giving my lecture on the MP to a group of Swedish patients

The day after the Conference should have been a day of rest, but I had made a commitment to give a speech about the MP to a group of Swedish patients and doctors who were interested in the treatment. Aside from having a few difficulties when it came to balancing the microphone and my script in the same hand, it went well. Everyone seemed open-minded. One lady in particular, in the front row, kept nodding her head as I moved from concept to concept, as if all of a sudden her disease was starting to make sense. At the end of the presentation most patients seemed eager to do the MP. However, the Swedish government prevents them from doing any treatment that is not deemed “standard.” My message to the group – go to Norway, a country nearby that allows doctors to do the MP without a problem. I hope they follow my advice despite the obvious hardship such advice entails. I’ll be creating a video version of my speech that should be up on this site soon.

On Sunday, after the conference had finished, all of us took the bright red #47 bus down to the Stockholm Square. By that point, I had realized that everything in Sweden runs smoothly. The buses come exactly on time. There is no traffic, the streets are so clean that sometimes the buildings look as if they are part of a movie set. In Sweden, an elevator is called a “hiss”, a term which makes me smile. It took me a while to learn that in Sweden hiss doors do not open automatically, rather they must be pushed open by the people inside. Particularly during the first days of the trip, I would forget this fact and wait inside the elevator for the door to open, much to the amusement of the Swedish people around me.

The Square was lined with other architectural masterpieces. Again, we could gaze out over the water and watch boats dock into the harbor. I had been looking forward to seeing the Square. After all, it’s the place where almost 20 years ago Dr. Marshall realized that sunlight plays a role in exacerbating chronic disease. He had been told that people in Sweden barely ever get enough vitamin D, but while waiting for a walking tour of the Square, sun streamed down on his face, and he felt physically ill. It was at that point that he started to question whether the current dogma on vitamin D and latitude makes sense.

Paul Albert encouraged Dr. Marshall to re-enact his eureka moment, which he did while standing in nearly the exact same location he had decades before. As I watched him joke around with Liz in an effort to recreate the moment, I was struck by how far he has come in the 20 years since that walking tour. I was struck at how far all of us on the MP have come, in terms of being part of a movement that will change so many paradigms in the medical world. I also marveled about the implications of the Marshall pathogenesis and how the best is yet to come.

Below is a link to a video created by Paul that chronicles some of what I have described above:

Here is a link to a second video clip which contains “outtakes” that did not make it into the main video:

In the following article, I discuss what I will be presenting at Karolinska.

In 2001, Alayne B. spent 8 months staring at a wall. “I went from being a company executive to barely being able to read above a 4th grade level,” states Alayne. “I couldn’t focus/concentrate on anything. My short-term memory turned into about 15 seconds – my nickname was ‘Memento Girl’ after the movie Memento.” The L.A. native would have to write notes immediately after a conversation or she’d forget what had been discussed. She also had to write herself notes in order to remember where she was driving. If she didn’t, she’d forget where she was going and get lost. “Ultimately I had to stop driving because I’d also forget that the notes were on the seat next to me and it’d take me ages to find my way home,” Alayne confesses. “I no longer had any mathematical figuring abilities either.”

Eventually, she started teaching at a private school – a job that was physically demanding, not to mention required increased brain use. “I made it through a few sessions, but would catch pneumonia, etc. and my energy levels were dropping again,” states Alayne. “Plus, I started forgetting words, how to spell, couldn’t understand my own lesson plans, couldn’t figure out the basic math I was also teaching, etc. When students caught a mistake, I’d pretend I’d been trying to catch them up – which they thought was fun. However, that only goes so far. I started teaching exclusively one-on-one in homes, but that also petered out, as I just couldn’t remember what the heck I was doing there.”

In 2005, Alayne was finally properly diagnosed with CFS. Her reading levels fell again to elementary/junior high level, her mathematical abilities to about 0, and her short term memory left her once again. She suffered from high levels of fear and anxiety, her word recall was horrible, and her reasoning abilities were greatly reduced. “I had no choice but to stop teaching even little one-hour tutoring sessions because I was confusing my students AND it would take me 5 days to recover from 3 hours spent away from home (driving, teaching, driving),” states Alayne.

Upon starting the Marshall Protocol in 2005, she was able to grasp enough of the treatment to “see it was the missing puzzle piece.” However, she could not explain it to anyone else, nor remember what she’d read. “I wrote a few notes down and went with it,” she describes. “By that time, I was bed-bound, emaciated, and my boyfriend and I thought I was dying.”

Although the CDC has recently recognized Chronic Fatigue Syndrome (CFS/ME) as a physiological disease, doctors and researchers are only beginning to understand the severe cognitive effects of the illness, such as those described by Alayne.

Alayne

Case histories like Alayne’s are supported by numerous studies that have tested the cognitive abilities of patients with CFS.

Take, for example, a 2007 twin study conducted by researchers at the University of Hawaii. After controlling for genetic and environmental influences, female identical twins, in which one twin met strict criteria for CFS and the co-twin was healthy, underwent a structured psychiatric interview and comprehensive neuropsychological assessment that evaluated 6 cognitive domains. Results indicated that twin groups had similar intellectual and visual memory functioning, but fatigued twins exhibited decreases in motor functions, speed of information processing, verbal memory, and executive functioning.

Similarly, researchers at the Department of Physical Medicine and Rehabilitation, University of Medicine and Dentistry of New Jersey, recently compared the cognitive performance of subjects with chronic fatigue syndrome (11 female subjects, 1 male subject), multiple sclerosis (MS), and healthy controls. After matching subjects for age, education, and verbal intelligence, subjects were given a battery of neuropsychological tests and scores were compared among the groups. Subjects with CFS and MS scored significantly below control subjects on five different tests that examined memory, concentration, word recall, information processing, and other cognitive abilities (CFS subjects scored worse than MS subjects on two of the tests).

“These results indicate that subjects with CFS and subjects with MS show significant impairment on a test of complex concentration when compared with appropriate controls,” state the research team. “The data suggest that subjects with CFS and subjects with MS have difficulty on tasks that require the simultaneous processing of complex cognitive information.”

Interestingly, the vast majority of studies that have tested for various forms of cognitive dysfunction in CFS patients, including those described above, contain only/mostly women as subjects. For starters, more women than men are diagnosed with the disease. In fact, CFS victims are twice as likely to be women.

But while males also suffer from CFS, the cognitive symptoms of women are, for the most part, reported to be more severe then those experienced by their male counterparts. This is not to say that some men with CFS can’t and don’t suffer from severe cognitive dysfunction, only that most of the studies that describe serious cognitive decline in CFS patients focus on women.

While I can’t deny that these studies may suffer from some forms of bias, the notion that women with CFS are more likely to suffer from severe cognitive dysfunction held up when I started research for this paper/presentation. Because I wanted to speak with cognitively impaired CFS patients on a one to one basis, I posted on the Marshall Protocol study site, asking if CFS patients who had suffered or were suffering from cognitive problems could send me an email describing their symptoms. While eight women contacted me, only one male patient volunteered information.

The endometrium

What might explain the hypothesis that women with CFS are more likely to experience serious cognitive dysfunction than their male counterparts? In what ways do men and women differ?

One obvious and rather large difference between men and women is that women have a uterus – an organ that has several internal membranes. One of these layers is the endometrium.

The endometrium is the inner membrane of the mammalian uterus.

The endometrium is the inner membrane of the mammalian uterus. It functions as a lining for the uterus, keeping the walls of the middle layer of the uterine wall separated – thereby maintaining the expanded shape of the uterine cavity.

During the menstrual cycle, the endometrium grows to a thick, blood vessel-rich, tissue layer that is filled with glands. This represents an optimal environment for the implantation of a blastocyst – the mass of cells that eventually forms an embryo if it arrives safely to the uterus.

If no blastocyst is detected, levels of the hormone progesterone (which is secreted in the endometrium) drops, and the endometrial lining is shed during what is known as the menstrual cycle. In the latter case, the process of shedding involves the breaking down of the lining, the tearing of small connective blood vessels, and the loss of the tissue and blood that had constituted it through the vagina. In humans, the cycle of building and shedding the endometrial lining lasts an average of 28 days.

If a blastocyst is able to implant, pregnancy results. In this case, the endometrial lining is neither absorbed nor shed. Instead, it remains as decidua. The decidua becomes part of the placenta; it provides support and protection for the embryo.

The endometrium produces certain hormones which affect other portions of the reproductive system and participate in important feedback pathways. This means that the endometrium is also home to a plethora of receptors whose activity is controlled by these hormones and other molecules.

The vitamin D system in the endometrium

In what is believed to be the first study to investigate the vitamin D system in human cycling endometrium, researchers at the Fondazione Policlinico-Mangiagalli-Regina Elena Hospital at the University of Milano in Italy have recently identified that one of the hormones produced in excess in the endometrium is the active vitamin D metabolite 1,25-D. Not surprisingly then, the research team also confirmed that both cycling and early pregnant endometrial cells express the Vitamin D Receptor (VDR), which is activated by 1,25-D.

One of the body’s most fundamental receptors, the VDR controls the activity of the innate immune system, or the body’s first line of defense against infectious agents. It also transcribes many antimicrobial peptides – peptides that kill bacteria, viruses, and fungi by a variety of mechanisms including disrupting membranes, interfering with metabolism, and targeting components of the machinery inside the cell.

Furthermore, the receptor activates the transcription of hundreds and possibly thousands of genes. Researchers at McGill University have compiled two lists of genes transcribed by the Vitamin D Receptor including 913 genes on the confirmed list and over 20,000 on the putative list. These genes are associated with genes ranging from cancers to multiple sclerosis.

The Italian team was able to gauge the presence and quantity of 1,25-D in the endometrium by using several different molecular methods to track levels of the enzyme 1{alpha}-hydroxylase (1-OHase) – or the enzyme that catalyzes the production of 1,25-D.

The team, under the leadership of P. Vigano, found that the endometrium harbors abundant quantities of 1,25-D, independent of the phase during which it is cycling under normal conditions. They also discovered that the hormone rises to even higher levels during pregnancy when the decidua is formed.

Expression of the Vitamin D Receptor in endometrial stromal cells and early pregnancy decidual cells, as evaluated by the molecular technique, Western blot.

A molecular technique called Western blot analysis showed a 40% increase in the expression of 1-OHase (which correlates with 1,25-D production) in the early pregnant decidua versus the percentage of 1,25-D created in the endometrium under normal conditions that lead to menstruation. The team confirmed these results by using another molecular technique called immunohistochemistry.

In order to confirm that not just 1,25-D, but also the VDR is found in the endometrium, the Italian team used Western blot analysis to look for the presence of nuclear receptor VDR protein. The protein was identified in both proliferate and secretory cells of the endometrium as well as in the decidua. The team confirmed their results by using a rat antibody to the human VDR that, as expected, reacted with both endometrial and decidual samples.

The team also tested whether the endometrium is a site where 25-D – the secosteroid precursor form of vitamin D – can be energetically converted to 1,25-D (the active form of the substance). The team found that the endometrium does indeed “represent a site of local conversion from the precursor to the active form” of vitamin D, and that levels of 1,25-D produced in this manner were similar to those detected in other areas of the body except for the kidneys, where conversion occurs at an even greater rate.

Cellular localization of 1-OHase in the human cycling endometrium and early pregnant decidua as evaluated by immunohistochemistry.

The functional consequences of 1,25-D production and the presence of the VDR in the endometrium were tested by evaluating the expression level of two genes that are transcribed when the VDR is activated – CYP24 and osteopontin. Levels of the genetic material that make up these genes were negligible or very low in unstimulated endometrial or decidual cells (cells that had no 1,25-D). However a “significant and strong increase” was observed in the expression of both genes after 1,25-D was added to the cells. In endometrial cells transcription levels of CYP24 went up to about 2000-fold after about a 1000nM concentration of 1,25 was added; in decidual cells this increase was also very strong. This same addition of 1,25-D stimulated osteopontin expression levels by about 60–70% in both types of cell culture.

The results of the study make one thing clear: because 1,25-D and the VDR are produced at high levels in the endometrium, the substances are over-expressed in women. Why might this be?

Truth be told, nobody knows why the VDR is over-expressed in the endometrium. One possible explanation is that since 1,25-D is the vitamin D metabolite that activates the VDR, it could be that early in human evolution, women acquired the ability to produce more 1,25-D and more VDRs because interactions between the hormone and the receptor would offer them an advantage during pregnancy. Think about it. When the Vitamin D Receptor is activated, the AMPs are produced at a greater rate, causing them to become increasingly active against viral, fungal, and bacterial infection in the fetus.

Furthermore, the innate immune system is strengthened, and hundreds, possibly thousands, of genes are expressed at a prolific rate. This provides an ideal situation for women who are preparing to bear a fetus and eventually conceive a child. Both mother and child should find it easier to fend off new infectious agents encountered during the pregnancy, and genes controlled by the VDR that may affect the success of the pregnancy should work at an optimal level.

Vigano and team seem to agree, stating, “The presence of the enzyme that catalyzes the synthesis of the active form of vitamin D in cycling endometrium and its up-regulation in first trimester decidua, support the possibility that this hormone might be involved in some mechanisms of pregnancy establishment or maintenance.”

Researchers at Children’s Hospital Medical Center in Ohio published a study in Endocrinology confirming that 1,25-D and the VDR are also expressed at high quantities in the placenta. The team found that the extra levels of the hormone and its receptor stimulate the synthesis and release of human placental lactogen – a hormone that modifies the metabolic state of the mother during pregnancy to facilitate the energy supply of the fetus. This supports the hypothesis that elevated 1,25-D and VDR expression are intended to optimize success during pregnancy.

Pathogens hijack the system

But at some point in the history of man, chronic idiopathic pathogens evolved a way to take advantage of the VDR receptor – by creating ligands that block its activity.

Biomedical researcher Trevor Marshall was the first to identify a species of bacteria that creates a ligand capable of binding and blocking the activity of the VDR. After reading a paper that discusses the characteristics of a bacterial species isolated from biofilm deposits on surgically removed human prosthetic hip joints, Marshall noted that the chronic pathogen under study creates a substance – a sulfonolipid called Capnine – that is able to bind the VDR and substantially slow its activity.

A molecular model of capnine blocking the Vitamin D Receptor.

This discovery strongly suggests that other pathogens can create similar substances, each of which can potentially block the VDR and diminish the body’s ability to keep bacteria in check. “There is now proof of concept that bacterial genomes are capable of producing at least one ligand which acts as a strong VDR antagonist,” says Marshall.

One must admit that creating a ligand or other substance capable of blocking the VDR is an exceptionally logical survival mechanism for any form of pathogen. By creating one single substance to block the VDR, they can, in a knockout blow, disable the receptor that would otherwise activate numerous pathways to interfere with their ability to persist in the body – pathways that are activated to an even greater extent by the extra 1,25-D produced in the endometrium, particularly during pregnancy.

“Think about it for a minute,” says Marshall. “If you were a persistent pathogen, wouldn’t it seem a good idea to disable your host’s ability to produce the antimicrobial peptides? And if you discovered that disabling just one receptor, the VDR, would get rid of [many types of AMPs], wouldn’t you try to evolve a mechanism for doing that?”

Because blocking the VDR is such a logical survival mechanism, the idea that most, if not all, of the L-form and biofilm bacteria (collectively called the Th1 pathogens) that cause chronic disease create these substances, is not far-fetched. This suggests that, as a person acquires more and more of these bacteria, their VDRs become increasingly blocked. As the innate immune system slows, it’s much easier for new species of Th1 pathogens, that probably also create VDR blocking substances, to accumulate and enter the body – causing somewhat of a VDR-blocking snowball effect.

Has this bacterial survival mechanism turned what was intended to be a protective situation for a woman (particularly during pregnancy and menstruation) into an environment that now allows pathogens rather than humans to gain the upper hand?

If a woman has acquired a sufficient number of Th1 pathogens, the over-expression of the VDR in the endometrium could suddenly put her at a disadvantage. Since women have more VDRs, they should, in theory, be more impacted by the effects of VDR blockage – a slower innate immune system, fewer AMPs, and reduced gene transcription. “The dysfunction caused by the Th1 pathogens affect women disproportionately,” states Marshall.

Of course, some may argue that the Th1 pathogens cannot cross the placental barrier and enter the endometrium, but evidence is growing that L-form bacteria and other pathogens are able to pass through this protective membrane.

Researchers at Peking University in Beijing recently discovered that the H5N1 bird flu virus can pass through a pregnant woman’s placenta to infect her fetus. Other studies have revealed that other bacterial species such as Borrelia burgdorferi and Mycobacterium tuberculosis are also capable of crossing the placental barrier during pregnancy. If these pathogens can be passed from mother to child during gestation, then why not species of biofilm bacteria and other forms of bacteria that are capable of transforming into the L-form?

Furthermore, if bacteria are unable to infect the endometrium and the placenta, then why do many women who have hysterectomies in order to treat diseases such as cancer or endometriosis find that the procedure affords them symptomatic relief?

For example, the 1994 Maine Women’s Health Study followed approximately 800 women with similar gynecological problems – pelvic pain, urinary incontinence due to uterine prolapse, severe endometriosis, excessive menstrual bleeding, large fibroids, and painful intercourse – for around 12 months. Approximately half of the women had had a hysterectomy while the other half did not. The study found that a substantial number of those who had a hysterectomy had marked improvement in their symptoms following hysterectomy, as well as significant improvement in their overall physical and mental health one year out from their surgery. The study concluded also that, for those who had intractable gynecological problems that had not responded to non-surgical intervention, a hysterectomy was beneficial to their overall health and wellness.

Since the removal of the uterus seems so effective at alleviating Th1 symptoms (an average of 622,000 hysterectomies a year have been performed for the past decade), one can infer that in cases where women feel better after a hysterectomy, the organ was infected – again suggesting that the endometrium is not immune to the effects of L-form and biofilm bacteria.

How might this contribute to the severe cognitive decline seen among women with CFS? It’s possible that women with VDR blockage, who subsequently have a decreased number of AMPs to kill bacteria, and a slowed innate immune system, find that the Th1 pathogens can infect their brains with greater ease. This infectious model seems logical, as nearly all females with CFS report a gradual onset of their mental symptoms – correlating to a gradual increase of bacteria in the brain.

Patients on the Marshall Protocol with diseases ranging from depression to ADD, OCD, anxiety, bipolar disorder, and schizophrenia, are reporting symptomatic improvement after taking bacteriostatic antibiotics aimed at killing the Th1 pathogens. Similarly, almost every patient on the Marshall Protocol is reporting mental as well as physical immunopathology – the name given to the rise in disease symptoms that results from the cytokine and toxin release that occurs when the Th1 pathogens are killed. Symptoms of anxiety, depression, anger, numerous kinds of brain fog, and cognitive deficits are reported along with physical symptoms, confirming that the Th1 pathogens infect the brain as easily as they do the body.

L-form bacteria have also been detected on several occasions in the brains of patients with Alzheimer’s disease. Rolf Zinkernagel at the Institute of Experimental Immunology in Switzerland demonstrated that viruses are able to persist for decades in the brain, so why not other pathogens?

Since, as described by researcher Josep Casadesus in a recent BioEssays paper, every known species of bacteria is probably capable of transforming to the L-form, this means that a plethora of different species of L-form and biofilm bacteria could be causing the neurological symptoms observed in women with CFS, explaining why symptoms are so diverse and differ substantially among individuals.

Of course cognitive decline and “brain fog” are not by any means phenomena unique to CFS. The same type of cognitive deficits observed in patients with CFS are also noted in patients with other Th1 diseases such as sarcoidosis, Lyme disease, and rheumatoid arthritis. Since the basic pathogenesis of these diseases appears to be the same as that of CFS, cognitive decline in these cohorts of patients likely results from the same mechanisms observed in CFS.

PKA

VDR blockage may also directly affect the brain through a pathway that Dr. Marshall will be discussing in his own presentation at the Days of Molecular Modeling conference. This pathway involves the enzyme CYP27B1, which plays a role in the body’s one and only pathway known to produce 1,25-D.

Under conditions where the VDR is activated, this pathway works to carefully limit (through what is known as trans-repression) the amount of transcribed CYP27B1 gene, and in turn the amount of 25-D that is converted into 1,25-D.

Researchers at the University of Tokyo recently published a paper that eloquently describes this feedback pathway in detail, pointing out other molecules involved in the process of limiting the production of CYP27B1. One of these molecules is Camp-dependent protein kinase A (PKA).

Eric Kandel

In his 2000 Nobel Lecture “The Molecular Biology of Memory Storage: A Dialog between Genes and Synapses”, Nobel Prize winner Eric Kandel (who happens to be the person who will be giving the opening talk at the upcoming DMM conference), describes how PKA is involved in broadening action potentials – spikes of energy that allow for communication between neurons in the brain. It also works to strengthen synapses, or specialized junctions through which the cells of the nervous system are able to signal to each other and non-neuronal cells such as those in muscles or glands. These findings provide direct evidence for the role of PKA in the formation of short-term memory.

When one molecule in a pathway is affected, other molecules in the pathway must adjust in order to accommodate new levels of the substance. In cases where high levels of VDR blockage disrupt the trans-repression element that keeps levels of CYP24B1 in a normal range, other molecules in the same pathway, such as PKA are also affected. As levels of PKA are modulated by higher than normal levels of CYP24B1, it is quite possible that PKA can become dysregulated – causing problems in communication between neurons that result in short-term memory loss and other cognitive symptoms.

“We have determined that activation of PKA is regulated by the process of VDR homeostasis in chronic immune disease, pointing towards a putative mechanism whereby immune dysfunction can directly suppress short-term memory,” states Marshall.

However, patients on the Marshall Protocol, who use pulsed, low-dose antibiotics to kill the Th1 pathogens, along with a VDR-agonist, to restore function of the VDR, are reporting not just the return of short-term memory, but also of mid-term and long-term memory. Consequently, it is very important that researchers continue to further investigate the connection between VDR blockage and other forms of cognitive decline besides those associated with short-term memory.

The nuclear receptors

As the Th1 pathogens take hold, the fact that women express more 1,25-D in the endometrium also becomes a problem. As the VDR becomes increasingly blocked, the receptor slows transcription of the gene for CYP24A1- an enzyme that inactivates 1,25-D. “[The production of CYP24A1] is the best documented of the feedback control systems used by the body to limit the concentration of 1,25-D to just that amount needed for proper transcription and activation of the VDR,” states Marshall.

The feedback pathways that control vitamin D metabolism. Graphic by Janet Foutin, taken from a paper by Trevor Marshall

But with a diminishing level of CYP24A1 to keep the body’s level of 1,25-D in check, the level of 1,25-D in patients with VDR blockage starts to rise. Unfortunately, when 1,25-D reaches a certain threshold, it binds many of the body’s other nuclear receptors, displacing the metabolites that are meant to be in the receptors under normal conditions. The receptors affected by 1,25-D include the glucocorticoid receptor, and the alpha and beta thyroid receptors, the adrenal receptors, and the progesterone receptors.

This means that when 1,25-D is high, it competitively displaces cortisol, T3, and other metabolites from their target nuclear receptors. Indeed, in the book Vitamin D: New Research, researcher Joyce Waterhouse PhD and team describe how patients with high levels of 1,25-D do indeed tend to suffer from hormonal abnormalities.

Furthermore, since each of these nuclear receptors transcribes various families of antimicrobial peptides, the displacement of their normal metabolites by 1,25-D causes a decrease in AMP production, generating an immunosuppressive effect (the body’s ability to kill bacteria is slowed without the help of the AMPs).

By “different families of antimicrobial peptides” I mean that there are many different types of AMPs. Although all AMPs are natural antibiotics produced by the body, each family of these peptides works to kill bacteria in different ways, and in different areas of the body.

In a recent article published in BMC Bioinformatics, a team of researchers (who are on the cutting edge of studying the role of the nuclear receptors in the transcription of different families of antimicrobial peptides) found that the glucocorticoid receptor, the androgen receptor, and the Vitamin D Receptor, seem to be in control of 20, 17 and 16 families respectively, out of 22 analyzed.

Excessive levels of 1,25-D affect the progression of chronic disease in both women and men, as 1,25-D is produced in other tissues of the body, including cells of the skin known as keratinocytes, intestinal cells, and in macrophages (macrophage-like differentiated THP-1 cells.) But since the substance is over-expressed in the endometrium, women are again disproportionately susceptible to the immunosuppression that takes place when the hormone displaces ligands from the nuclear receptors, causing a decrease in the production of so many different families of AMPs. Once again, this immunosuppression likely makes it easier for the Th1 pathogens to infect the brain, where they can cause a host of neurological symptoms.

On a similar note, researchers at Tampere University Hospital in Finland have found that several of the nuclear receptors are expressed at different levels throughout the menstrual cycle – for example the androgen receptor, the estrogen receptors, and the progesterone receptor are expressed more abundantly during the proliferative phase of the endometrium than in the secretory phase of the endometrium.

Since the expression of these receptors waxes and wanes during the menstrual cycle, even in healthy women, the activity of the AMPs that they transcribe may fluctuate over the course of a month – allowing for periods when some women may be less able to kill invading pathogens. While men aren’t affected by this surging and waning, it could be part of the reason why women appear to pick up the Th1 pathogens that cause CFS and the neurological symptoms associated with the disease at a greater rate then men.

It could also be hypothesized that women tend to acquire the Th1 pathogens that cause cognitive dysfunction more easily than men because they don’t produce as much testosterone as their male counterparts. Although on average women are more sensitive to testosterone, an adult human male body produces about forty to sixty times more testosterone than an adult female body.

Since males produce more testosterone, the hormone should activate its target nuclear receptor, the androgen receptor, to a greater extent in men than in women. Since the androgen receptor controls 20 families of antimicrobial peptides, this could mean that men produce many of these natural antibiotics at a greater rate than women – giving them an advantage when it comes to fending off infection and targeting the Th1 pathogens.

Then again, the case history of a Marshall Protocol patient suggests that the progesterone receptor may also transcribe antimicrobial peptides – albeit possibly not in the same high quantities as the androgen receptor.

The possibility that the progesterone receptor may also transcribe antimicrobial peptides is supported by the case of Jane T, an MP patient who suffers from a great number of Th1 conditions. One of the symptoms that results from her immunopathology is endometrial bleeding.

A few months ago, Jane’s doctor reported that, after being given a prescription for supplemental synthetic progesterone, her bleeding became stronger and more severe.

Could it be that upon taking the extra progesterone, her progesterone receptors stimulated the production of AMPs? If this were the case, the AMPs might have facilitated her ability to kill more of the bacteria causing the bleeding, leading to a rise in immunopathology in the area. This hypothesis is supported by the fact that her bleeding was reduced when her progesterone dose was cut in half.

Interestingly, the extra synthetic progesterone did not just affect the level of immunopathology in her endometrium. Her symptoms of obsessive-compulsive disorder increased tremendously after her initial dose of high progesterone and also decreased when the progesterone dose was lowered.

This suggests that if AMPs are produced by the progesterone receptor, they are also able to cause an increase in the death of bacteria in the brain. This case history not only sheds light on how intricately different body systems are connected and how easy it is to disrupt the body’s delicate feedback pathways, but also calls for more research into the actions of the nuclear receptors and the AMPs.

Pregnancy

Unfortunately, because of the fact that 1,25-D levels rise to their highest point during pregnancy, the hormone’s ability to cause immunosuppresion by affecting the nuclear receptors is quite prevalent during this time. The subsequent ease at which the Th1 pathogens are able to spread, thanks to the drop in AMP production, may account for part of the reason why many women with CFS often find that occasionally during pregnancy, and almost always after giving birth, they struggle with an increase in cognitive and other symptoms.

According to New York University gynecologist Frederick R. Jelovsek MD, symptoms in CFS and fibromyalgia tend to become worse during the third trimester of pregnancy and during the postpartum period, when women with CFS often report worsening of pain and other symptoms. A 1998 New England Journal of Medicine article also found that in women with multiple sclerosis (a Th1 disease similar to CFS), rates of relapse tend to increase during the first three months postpartum.

However, the majority of women with CFS feel better during pregnancy (at least during the first two trimesters) and report that their disease symptoms seem to improve. Such women are reaping the palliation derived from the decrease in bacterial die-off that occurs when less of the antibmicrobial peptides target the Th1 pathogens. Because less bacteria are killed, immunopathology drops, leading to fewer inflammatory symptoms. However such feelings of “wellness” are short-lived, as after giving birth, such women feel the effects of the new bacteria they have almost surely accumulated during the previous months when AMP production was particularly low.

It doesn’t help that most women are told to take prenatal vitamins that contain vitamin D, which only adds to the excess amount of the substance in the body. “Pregnancy is a time when (these days) the mother is being laced with prenatal vitamins, even though the endometrium is expressing lots of extra 1,25-D during pregnancy itself. So in mothers who start off carrying a sufficient bacterial load, the first pregnancy might well allow proliferation to a point that the subsequent pregnancies could be affected,” states Marshall.

Indeed, a recent study by researchers the University of Hong Kong found that anxiety and depression often increase during pregnancy and are highly prevalent and strongly associated with postpartum depression.

Among a consecutive sample of 357 pregnant women, Lee and colleagues found that more than half (54 percent) had anxiety and more than one third (37 percent) had signs of depression at some point during their pregnancies. Anxiety was more prevalent than depression at all stages of pregnancy.

Between 12 and 17 percent of women in the study were found to have both anxiety and depression at various stages of pregnancy, the researchers report in the medical journal Obstetrics and Gynecology. “Both antenatal anxiety and antenatal depression were found to be more prevalent and severe in the first and third trimesters,” Lee told Reuters Health. Anxiety and depression levels decreased from early to mid-pregnancy, but increased again late in pregnancy.

Since the mechanisms involved in pregnancy are incredibly complex and involve myriad changes – including the production of endorphins, cortisol, and many hormones – the rise in cognitive symptoms in women with CFS cannot be attributed solely to elevated 1,25-D. Yet, it certainly seems to be part of the puzzle.

The cognitive decline seen in women with CFS is serious and must be treated by killing the Th1 pathogens in the brain

While this paper probably raises more questions than it answers, several things are certain. The VDR is at the heart of chronic disease and it is doubtful that the overexpression of the VDR in the endometrium and the tendency of women with Th1 disease to suffer from a greater degree of cognitive dysfunction is simply due to chance.

Furthermore, the cognitive symptoms observed in women with CFS are incredibly debilitating and can only be reversed by a treatment such as the Marshall Protocol that targets the Th1 pathogens in the brain. This means that treatments such as cognitive behavioral therapy, which fail to address the root cause of cognitive symptoms, cannot be used to effectively treat cognitive decline in CFS.

A recent pilot study (Koolhaas, et al., 2008, Netherlands) reported that only 2% of CFS patients are “cured” by cognitive behavioral therapy (CBT), while the greatest share (38%) are actually adversely affected – most reporting substantial deterioration. The study proved to be a stark contrast to claims of psychiatrists and the Dutch Health Council that 70% of CFS patients improve after CBT. Previous studies have also ignored or denied the negative affects of CBT on ME/CFS patients. The pilot study, recently published in the Dutch medical magazine, Medisch Contact, concludes that the previously reported claims of 70% improvement in ME/CFS patients receiving CBT are vastly overstated and misleading.

In contrast, today, after a little over two years on the Marshall Protocol, Alayne feels that her cognitive function is a “gazillion times better.” According to Alayne, her improvements thus far have been absolutely stunning and very obvious.

“On the MP, my brain has been steadily healing over the past 26 months. My parents (academics) see me every 6 months or so and are able to describe the changes for me, which is great,” states Alayne. “I can read books again, although if they’re too intense or convoluted, it takes me time to understand them fully. My reasoning abilities are FAR improved as well, my math skills have returned (although I’m not sure I could teach it now), word recall is FAR better and I find myself at times uttering words I haven’t used for years.”

Alayne finds she can also spell much better again, and despite the occasional dyslexic moment, she no longer has to spell words phonetically.

She’s also able to understand and grasp new concepts far more easily, plus remember them and old ones she never really understood. “My ability to learn new software (that I couldn’t possibly do last summer), has suddenly opened up,” explains Alayne. “I was able to learn three new complicated programs within a few days last fall – ones that had completely stumped me a few months prior.”

Alayne’s level of motivation has increased greatly, up from levels of zero before the MP. “I set myself a schedule or list of ‘things to do’ and actually get them accomplished,” she states. “This is a big change. I still have some difficulties following through with some ‘things’ or projects on a timely basis, but all in all, it’s a lot better.”

In fact, Alayne is now starting a part-time job that will require her to do some freelance work that will stem from her own motivation. She’s also joined another writing group and is taking one of their courses. Since she can now remember numbers and percentages, she’s also able to do small business contracts for other people again.

“Finally, I think I’m more fun to be with,” confesses Alayne. “I’m more social and I’m eager to give feedback during conversations in my classes. I haven’t felt this way for so long.”

“We have seen no sign that the brain doesn’t heal,” says Marshall, who created the Protocol. “The adults recover all their lost faculties as they heal on the MP, and the several children on the MP, who have had a variety of difficulties, also are recovering fully. So our data (at this point) shows that the body heals as bacteria are killed and immune function is restored. All of the body. Including the brain.”

In order to emphasize the severity of cognitive dysfunction experienced by women with CFS, I leave you with the following reports from women who are using the Marshall Protocol to treat their CFS and the cognitive issues that accompany the disease. Happily, these women and others on the Marshall Protocol are experiencing strong immunopathology in the brain and some are at the point where symptoms are beginning to disappear. Complete recovery is expected once all the Th1 pathogens are targeted – a process that takes up to 3-5 years.