Bacteriality

Exploring chronic disease

Category: conferences and trainings

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[1] 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.[2] 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.[3] Also, evidence-based medicine has come of age.[4]

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.,[5] 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.[6]

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[7] 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,[8] 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:[9]

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[10] while another performed no intervention but simply measured the 25-D levels of 20 sunscreen users.[11] The third put only 27 subjects into tanning beds rather than into the sun, which could easily introduce bias.[12] 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 studieshave shown that vitamin D deficiency does not result from regular sunscreen use.”[13]

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.”[14]

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.[15] 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.[16][17]

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[18] 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.”[19]

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.”[20]

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.[21] Placental conversion was demonstrated in vitroin 1979,[22] over-expression of 1,25-D in vivo in 1980,[23] and the dysregulated vitamin D metabolism was described in 1981.[24] 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).[25] 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.[26] 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.[27]Indeed, independent research teams have found that Mycobacterium tuberculosis downregulates VDR activity by approximately 3.3 times.[28] ActiveBorellia lowers VDR activity by about a factor of 50 and Epstein-Barr Virus by a factor of around 10.[29] 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.[30][31][32][33] 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[31]

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.[34] If the pathogen dies, endotoxins[35] 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.[1] 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.

REFERENCES

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  2. Wang, Y. & Beydoun, M.A. The obesity epidemic in the United States–gender, age, socioeconomic, racial/ethnic, and geographic characteristics: a systematic review and meta-regression analysis. Epidemiol Rev 29, 6-28 (2007). []
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  10. Matsuoka, L.Y., Ide, L., Wortsman, J., MacLaughlin, J.A. & Holick, M.F. Sunscreens suppress cutaneous vitamin D3 synthesis. J. Clin. Endocrinol. Metab 64, 1165-1168 (1987).
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  11. Matsuoka, L.Y., Wortsman, J., Hanifan, N. & Holick, M.F. Chronic sunscreen use decreases circulating concentrations of 25-hydroxyvitamin D. A preliminary study. Arch Dermatol 124, 1802-1804 (1988). []
  12. Matsuoka, L.Y., Wortsman, J. & Hollis, B.W. Use of topical sunscreen for the evaluation of regional synthesis of vitamin D3. J. Am. Acad. Dermatol 22, 772-775 (1990). []
  13. Scarlett, W.L. Ultraviolet radiation: sun exposure, tanning beds, and vitamin D levels. What you need to know and how to decrease the risk of skin cancer. J Am Osteopath Assoc 103, 371-375 (2003). []
  14. Wolpowitz, D. & Gilchrest, B.A. The vitamin D questions: how much do you need and how should you get it? J. Am. Acad. Dermatol 54, 301-317 (2006). []
  15. Palm, M.D. & O’Donoghue, M.N. Update on photoprotection. Dermatol Ther 20, 360-376 (2007). []
  16. Chlebowski, R. et al. (2006). The Women’s Health Initiative Randomized Trial of calcium plus vitamin D: effects on breast cancer and arthralgias. Journal of Clinical Oncology, 24. []
  17. This comment was based on Figure 3 of Chlebowski et al on page 1587, strata entitled Baseline total vitamin D (supplements + diet). One can see that those who started out with an intake of 600 IU of vitamin D or higher had a hazard ratio of 1.34 (95% CI, 1.01-1.78), while those who started out low had a risk reduction (the p-value for interaction was significant at 0.003). This is one of the few examples of a randomized trial on vitamin D. []
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  19. Mohr, S.B., Garland, C.F., Gorham, E.D. & Garland, F.C. The association between ultraviolet B irradiance, vitamin D status and incidence rates of type 1 diabetes in 51 regions worldwide.Diabetologia 51, 1391-1398 (2008). []
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  21. Ardawi, M.S., Nasrat, H.A. & BA’Aqueel, H.S. Calcium-regulating hormones and parathyroid hormone-related peptide in normal human pregnancy and postpartum: a longitudinal study.Eur. J. Endocrinol 137, 402-409 (1997). []
  22. Tanaka, Y., Halloran, B., Schnoes, H.K. & DeLuca, H.F. In vitro production of 1,25-dihydroxyvitamin D3 by rat placental tissue. Proc. Natl. Acad. Sci. U.S.A 76, 5033-5035 (1979). []
  23. Steichen, J.J., Tsang, R.C., Gratton, T.L., Hamstra, A. & DeLuca, H.F. Vitamin D homeostasis in the perinatal period: 1,25-dihydroxyvitamin D in maternal, cord, and neonatal blood. N. Engl. J. Med 302, 315-319 (1980). []
  24. Gray, T.K., Lowe, W. & Lester, G.E. Vitamin D and pregnancy: the maternal-fetal metabolism of vitamin D. Endocr. Rev 2, 264-274 (1981). []
  25. Haug, C.J. et al. Severe deficiency of 1,25-dihydroxyvitamin D3 in human immunodeficiency virus infection: association with immunological hyperactivity and only minor changes in calcium homeostasis. J. Clin. Endocrinol. Metab 83, 3832-3838 (1998). []
  26. Turnbaugh, P.J. et al. The human microbiome project. Nature 449, 804-810 (2007). []
  27. Proal, A.D., Albert, P.J. & Marshall, T. Autoimmune disease in the era of the metagenome.Autoimmun Rev 8, 677-681 (2009). []
  28. Xu, Y. et al. Chin. Med. J 116, 1070-1073 (2003). []
  29. Yenamandra, S.P. et al. Expression profile of nuclear receptors upon Epstein — Barr virus induced B cell transformation. Exp. Oncol 31, 92-96 (2009). []
  30. Kivity, S., Agmon-Levin, N., Blank, M. & Shoenfeld, Y. Infections and autoimmunity–friends or foes? Trends Immunol 30, 409-414 (2009). []
  31. O’Connor, S.M., Taylor, C.E. & Hughes, J.M. Emerging infectious determinants of chronic diseases. Emerging Infect. Dis 12, 1051-1057 (2006). [] []
  32. Relman, D.A. Detection and identification of previously unrecognized microbial pathogens.Emerging Infect. Dis 4, 382-389 (1998). []
  33. Rook, G.A. & Stanford, J.L. Slow bacterial infections or autoimmunity? Immunol. Today 13, 160-164 (1992). []
  34. Allie, N. et al. Protective role of membrane tumour necrosis factor in the host’s resistance to mycobacterial infection. Immunology 125, 522-534 (2008). []
  35. Hurley, J.C. Antibiotic-induced release of endotoxin. A therapeutic paradox. Drug Saf 12, 183-195 (1995). []
  • Comments Off on Second-guessing the consensus on vitamin D
  • Filed under: conferences and trainings, featured articles, medical research, vitamin D
  • On Monday, I returned from the 6th International Congress on Autoimmunity held in Porto, Portugal. You can watch my presentation here.

    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.

  • Comments Off on Notes from the 2008 International Congress on Autoimmunity
  • Filed under: conferences and trainings, presentations
  • Though the human genome was fully sequenced in 2001, the most promising work in genomics has just begun and not even in the study of human DNA. Human cells are outnumbered by bacterial cells by a factor of ten to one, and, as the rest of this site alludes to ad nauseam, there is strong reason to believe that bacteria are to blame for many of the chronic diseases from which humans suffer. Genetically speaking, we know relatively little about bacteria that persist in humans. The field is ripe for advances.

    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 theWashington 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 throughGenBank, 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 subtleties 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 analyzing 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.

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  • Dr. Alan Cantwell has investigated the phenomenon of cancer bacteria for over thirty years. A graduate of New York Medical College, Cantwell completed a residency program in dermatology at Long Beach Veteran’s Administration Hospital in Long Beach, CA and then practiced in the dermatology department of Kaiser-Permanente in Hollywood, California, from 1965 until his retirement in 1994. Dr. Cantwell is the author of more than thirty published papers on breast cancer, lymphoma, Kaposi’s sarcoma, Hodgkin’s Disease, lupus, scleroderma, AIDS, and other immunological diseases. These papers have appeared in many peer-reviewed journals, including Growth, International Journal of Dermatology, Journal of Dermatologic Surgery and Oncology, and the Archives of Dermatology. He has also written The Cancer Microbe and Four Women Against Cancer and several books on AIDS.

    1. How did you become interested in looking for bacteria, first in diseases like scleroderma and later in cancer?

    It all started when I was a second year resident in dermatology. I was in the medical library and I came across a paper in the Southern Medical Journal describing a group of people who had been given allergy injections and who subsequently developed deep skin infection with tuberculosis-like germs. It was thought the allergy injection bottles were contaminated with these bacteria.

    At the time, I had a mentally disturbed patient who had been given multiple injections of medications into her buttocks. She later developed deep painful skin nodules in the same areas. No one knew what was causing these nodules that were diagnosed as “panniculitis,” an inflammation of the fat layers of the skin. I thought, “Let’s culture a skin biopsy from one of these deep nodules and see if I can find any TB-like germs.” I was amazed when Eugenia Craggs, the technician at the TB lab, reported that “acid-fast” bacteria were discovered in the skin tissue. I thought “Hey this is just like the article!”

    We also had three other patients with “panniculitis” of the fatty portion of the skin, all of unknown cause. I took biopsy samples and TB-like bacteria were found in all four. These cases were later reported in the Archives of Dermatologyin 1966. At the time my dermatology professor was J. Walter Wilson, who was also a world famous mycologist, an expert in fungal diseases. He was somewhat skeptical about my findings of acid-fast bacteria in all these four patients and he suggested I use a scleroderma patient as a “control.” Scleroderma is a so-called “collagen disease” where the skin becomes hardened. The disease can affect the internal organs and is sometimes fatal. The cause is unknown, and bacteria were never thought to cause this disease. Dr. Wilson said I should check a scleroderma skin biopsy because that would serve as a negative “control” case. I was astonished when Eugenia Craggs called me from the TB lab and told me the skin tissue grindings of the scleroderma sample were positive for acid-fast bacteria, the kind of bacteria found in tuberculosis. She would try and grow the germ in a TB culture. After much searching I was also able to find a few acid-fast rod forms of bacteria in the scleroderma skin biopsy microscopic sections prepared by the pathologist.

    The scleroderma bacterial took a long time to grow and could not be diagnosed as a TB germ or other definite “atypical” mycobacteria. The microbe was highly pleomorphic (various forms). There were round staphylococccal forms, as well as typical acid-fast rod forms. Eventually this isolate became fungal-like and “actinomycete- like.” Despite expert opinion, it was impossible to classify the microbe into a specific species. This case of scleroderma was reported in The Archives of Dermatology in 1966.

    Some time later, Roy Averill, one of the dermatology residents, told me he heard a woman physician being interviewed on a San Diego radio talk show. She was explaining how she found TB-like bacteria in scleroderma in the late 1940s. That woman was Virginia Livingston M.D. She quickly became a dear friend and mentor in my scleroderma research. She told me that scientists at the Pasteur Institute in Belgium also reported finding acid-fast bacteria in scleroderma in 1953, thus confirming her own research.

    I naturally thought all these reports in the medical journals would be recognized by other dermatologists and scientists, and that scleroderma would be recognized as an infectious disease caused by acid-fast bacteria. But after more than a half-century, I’m sad to say that scleroderma is still considered a disease “of unknown etiology” and the bacteria we found are simply ignored. After discovering acid-fast bacteria in scleroderma, Livingston found similar bacteria in cancer. This made her one of the most controversial physicians in America, as detailed in my book, “The Cancer Microbe.”

    2. How did you identify the bacteria in your samples?

    I began my dermatology practice at Kaiser in Hollywood in 1965. Virginia Livingston introduced me to Dan Kelso, a Los Angeles microbiologist who thereafter cultured my skin biopsy samples from scleroderma, and later from lupus erythematosus and a variety of cancers. Depending on the case, sometimes he cultured Staphylococcus epidermidis, or corynebacteria, more rarely streptococci, and pleomorphic bacteria that appeared sporadically as acid-fast bacteria similar to Mycobacterium tuberculosis.

    Naturally I attempted to find acid-fast rod forms in my specially-stained skin biopsy sections, because these forms are the typical forms signifying infection with Mycobacterium tuberculosis or other species of mycobacteria. “Acid-fast” refers to red-stained mycobacteria that can be observed after staining tissue samples with a special procedure and a special dye. At first, I didn’t see the L-form bacteria since they react differently to acid staining. Instead of rod-forms, they appeared as round forms which were only partially acid-fast, staining purple or magenta with the acid-fast stain. It took me many years to finally realize that these partially acid-fast and round forms were bona fide growth forms of mycobacteria. The typical bright red-stained acid-fast rod forms of mycobacteria are unique and easily recognized by pathologists, but unfortunately the non-acid-fast round forms are not recognized and accepted by pathologists. For a long time I passed over these granular and “dusty” tiny forms as meaningless, not realizing that they were, in actuality, what L-forms look like!

    I knew basically nothing about the microscopic appearance of L-form bacteria (also known as cell wall deficient bacteria and “mycoplasma”) until I carefully read the published papers of microbiologist Lida Mattman. Then I realized all the guises that bacteria can undergo, including transformation into “large bodies.” At that point, I went back and looked at my first case of scleroderma and realized that one skin biopsy sample contained large L-form bodies that appeared as yeast and fungal-like forms! These forms, in 1966, were dismissed as “fat degeneration” by one pathologist; and the biologist thought they looked like yeast cells.

    These large L-forms are compatible with what pathologists recognize as Russell Bodies. William Russell (1852-1940) was a well-known Scottish pathologist who first discovered “the parasite of cancer” in 1890. His view of an infectious agent in cancer was dismissed in the early part of the twentieth century. However, I believe Russell bodies are actually large growth forms of cell wall deficient bacteria — and that Russell was indeed recognizing an infectious agent in cancer. More than a half-century later, Lida Mattman was able to transform mycobacteria into “large bodies” by exposing them to antibiotics. For more information on Russell and pictures of Russell bodies, Google my paper “The Russell Body” in the Journal of Independent Medical Research (joimr.org).

    The fact that L-form bacteria have a “life cycle” and can appear in so many different shapes and sizes (pleomorphism) may be why they are so hard to eradicate and why the immune system cannot cope with them. Maybe the large Russell bodies are harder to kill. Or maybe they are easier to kill. I don’t know.

    3. You found bacteria in the tissues of people who died of certain cancers and AIDS and scleroderma at autopsy. What gave you the idea to look for bacteria in autopsies?

    I got that idea from Florence Seibert, a world famous biochemist who developed the tuberculin skin test for tuberculosis, which is still used worldwide. When Seibert heard about the TB-like bacteria discovered in cancer by Virginia Livingston and her colleagues, which included microbiologist Eleanor Alexander-Jackson and cell cytologist Irene Diller, she decided to come out of retirement and help with the women’s cancer research. Seibert advised me to search for bacteria in autopsy specimens and to determine if I could also find them in the internal organs and connective tissue of people who died of scleroderma. She believed this would make my skin research more credible. For the full story of these four remarkable women scientists, read my book Four Women Against Cancer, published in 2005, and available through Internet book sources.

    Alan Cantwell with Eleanor Alexander-Jackson and Irene Corey Diller

    After I decided to look for bacteria in autopsy material, I contacted colleagues in the Pathology department at Kaiser and asked them to provide me with stored tissue autopsy samples, which they did graciously. I was very fortunate to have them assist me in doing this. One of the great things about Kaiser-Permanente is that everything is under one roof. Few private dermatologists would have the easy access to autopsy material that I did at Kaiser.

    4. When did you begin to look for bacteria in people with cancer?

    Never in my wildest dreams did I think I would ever find bacteria in patients with cancer. Before I started my cancer research (which was totally instigated by my friendship with Livingston), it seemed inconceivable that scientists could have failed to recognize a microscopically visible infectious bacterial agent in cancer.

    For a decade I avoided the cancer controversy because I worked for an HMO and I didn’t want to be regarded as a “quack.” Tragically, Virginia Livingston, because of her outspokenness that cancer was caused by bacteria, was widely regarded as a “quack doctor.” However, in the mid-1970s, I found pleomorphic bacteria in patients with sarcoidosis, and also in a patient with lymphoma. I was amazed at how easy it was to detect bacteria in sarcoidosis and lymphoma when the tissue sections were properly stained with an acid-fast staining technique.

    Once I saw for myself that Virginia Livingston was correct about acid-fast bacteria in cancer, I became very enthusiastic about studying bacteria in other forms of cancer, as well as in immune diseases, like lupus. At that point, I finally had enough conviction in my findings, and had the courage to take a stand along with Virginia.

    5. How did you colleagues react to your research?

    Over the years there were very few doctors interested in seeing the bacteria I found in tissue sections. Some would tentatively acknowledge that there were bacteria present. Most were non-committal. With a little arm twisting I convinced several pathologists, who helped supply the autopsy specimens, to put their name on my published papers. But for the most part they didn’t want to get involved. They would say, “Oh Alan, it’s your research…” “Oh Alan, you’ll win the Nobel Prize someday.” Nobody ever wanted to sit down with me and seriously look at the material. I think it’s because finding bacteria in illnesses that are not attributed to infection is highly controversial, and most doctors shy away from controversy. The finding of bacteria in cancer is like opening Pandora’s Box. Once it’s open, a lot of stuff flies out, and pisses off a lot of people. The bacteria aren’t supposed to be there, they are in closet and not supposed to come out.

    Even after I was retired for almost a decade, I never lost interest in trying to uncover bacteria in cancer. In 2003, my partner was diagnosed with prostate cancer. He underwent a prostatectomy, the total removal of the prostate gland. I decided to see if bacteria could be found in his prostate cancer tissue sections after surgery. Prostate cancer is every older man’s worst nightmare, just as breast cancer is every woman’s worst nightmare. I asked the Kaiser pathologist to cut me a section of my partner’s cancerous prostate and to stain it with an acid-fast stain so that I could study it. Sure enough, there were bacteria in the samples. I had a private microscopist photograph the bacteria. One can view the bacteria in prostate cancer I discovered by reading my paper published at the www.joimr.org website.

    6. What’s going on? Why aren’t doctors and researchers taking the idea that bacteria cause cancer seriously?

    As I see it, the identification of simple-to-see cancer microbes would cause havoc in the scientific world and in the cancer treatment industry. It would be the biggest embarrassment to befall modern medicine. Can you imagine the furor resurrecting Russell’s “cancer parasite” — the “parasite” that was thrown out of medical science a century ago?

    It is rare to find a scientist interested in “cancer microbes.” Most physicians are repelled by the idea that bacteria cause cancer. How do you prod scientists to become interested? I’m still not sure.

    A century ago, doctors stopped looking for bacteria in cancer. It’s weird because around that time major diseases like syphilis, tuberculosis, and leprosy were proved to be caused by bacteria. I suppose researchers think, “Well, we looked for bacteria 100 years ago, so there’s no need to look for them now.” But a lot has changed in bacteriology in 100 years. A century ago there was no such thing as an “L-form.” Even now most scientists don’t realize that regular bacteria can change into L-form bacteria, or cell wall bacteria, or mycoplasma, or pleomorphic bacteria, or nanobacteria, or whatever you choose to call these peculiar and little-known growth forms.

    Microbiologists still have a hard time dealing with the fact that bacteria can change so widely in shape and size. How do you get scientists to understand that the tiniest L-forms have the potential to enlarge into a form the size of a red blood cell (or even bigger!). But if you think about it, all human beings were once a microscopic bunch of dividing cells, hardly visible to the naked eye. And we know that these tiny cells can evolve into seven foot tall basketball players. Why then, do we take such a simple view of what bacteria are supposed to do and what they are supposed to look like?

    And the strange part is that using a light microscope you can easily see L-form bacteria. Every scientific paper that I have had published shows pictures of these bacteria. But even when doctors are shown photographs or see these bacteria via a light microscope, they still have a hard time accepting them. It’s bizarre because doctors believe viruses exist, even though most have never seen one. You can’t see viruses. They are too small to be seen with a microscope.

    7. When doctors and researchers claim that there are no bacteria in your samples what explanations do they give?

    When doctors or other researchers try to deny that there are bacteria in scleroderma and cancerous samples their explanations are pretty lame. Maybe something like, “Those aren’t bacteria, those are enlarged red blood cells.” Those “bacteria” are really cell debris, or stain material, or nuclear dust, of mast cell granules, or fat granules— anything but true bacteria. It’s impossible to convince a pathologist, for example, that a “tiny” bacteria can transform into a giant-sized form hundreds of times larger.

    8. Who’s to blame for the fact that bacteria have not been recognized as part of the pathogenesis of cancer?

    Pathologists, dermatologists, infectious disease specialists, oncologists, virologists, microbiologists, and basically all medical scientists who have ignored a century of cancer research pointing to cancer microbes. They have collectively let us down. Unfortunately, pathologists and microbiologists seem to be on two different planets. Pathologists pay little attention to germs in a laboratory, and microbiologists pay little attention to what bacteria do when they infect human tissues that are subsequently examined by pathologists.

    9. What keeps other researchers from finding L-form bacteria in patients with cancer?

    Unfortunately, most microbiologists who have worked with L-form bacteria have not demonstrated how these same forms appear in tissue in human disease when viewed in the light microscope. It’s one thing to describe a microbe in a lab, but what does it look like when it infects the human body? It’s one thing to show these L-forms in pictures taken with an electron microscope that magnifies objects thousands of times. But what do these bacteria look like when view with a “regular” light microscope that magnifies only 1,000 times? As a result, these pleomorphic forms go undetected in diseased tissue. Another reason, of course, is that the pathologist uses a routine stain (the H&E stain) that does not detect these forms. One needs to use an acid-fast stain. This was one of Livingston’s and Eleanor Alexander-Jackson’s most brilliant discovery— the idea that the “cancer microbe” is intermittently “acid-fast” at one or more stages of its growth.

    10. What are some of your concerns about the current medical climate?

    It saddens me greatly that all this great research has been ignored. That is why I wrote The Cancer Microbe (1990), and AIDS: The Mystery and the Solution(1984) and Four Women Against Cancer (2005).

    Every first year med student knows that until you know what’s causing a disease it’s very hard to treat it. In my opinion, hunting for the exact cause of an illness is the most exciting part about being a doctor. The scientists who clued us into the cause of tuberculosis and syphilis, for example, were medical greats because they gave us an idea of what exactly is making the patient ill.

    In my 30 years as a doctor and researcher I’ve never convinced one doctor, not even one, that bacteria cause cancer. My own younger brother is a physician — and I don’t even think he believes me entirely. Two years ago, his daughter-in-law died at age 39 of Hodgkin’s Disease, leaving two small children. I told him, “I wrote about Hodgkin’s Disease!” But he wouldn’t comment. If I can’t convince my own brother — or even interest him in the subject —I feel there is little hope.

    11. What concerns did Kaiser Permanente have about your research?

    A problem with my research was that over a period of years I was finding acid-fast bacteria in patients with a wide array of different illnesses. Some skeptics would say “OK, maybe I can accept that you found bacteria in scleroderma, but come on, in all these diseases?” After several years of productive cancer microbe research, the research committee insisted I be interviewed by a statistician. The committee was concerned because I was discovering bacteria in too many diseases. The statistician insisted that I attempt a statistical study of these bacteria with suitable “controls.” I explained that previous researchers had already determined that all human beings harbor such bacteria, and that these bacteria needed further study as pathogens. It might be impossible to find “negative” controls. At that point I thought, “I’m doomed.” There was no way I could do a statistical analysis of my observations. My research was terminated.

    12. Did anyone try to censor your work?

    In 1984 Virginia Livingston wrote a second book about bacteria in cancer calledThe Conquest of Cancer. She asked me to write a blurb for the back cover of her book. Her publisher took out an ad for her book in the Los Angeles Times Book Review, which included my blurb. Unfortunately, my quote mentioned my association with the Southern California Permanente Medical Group. When the top brass at Kaiser discovered this they were furious. “You can’t do this! You can’t associate our name with a quack like Livingston!”

    At the time I had also discovered that cancer bacteria play a role in the development of Kaposi’s sarcoma, the most common cancer in the newly discovered disease called AIDS. I explained that I had also written a book about AIDS and the bacteria involved in this disease, and that the book was in press and was to be published soon. The Kaiser officials were aghast and told me I was simply not allowed to publish this book. This was at a time shortly before the discovery of HIV and during the period when the precise cause of the immune deficiency was “a mystery.” I had always been well-respected at Kaiser, but I was fearful the Livingston brouhaha and the impending publication of my book might threaten my job.

    Finally my literary lawyer stepped in and worked out a deal with Kaiser whereby I could publish AIDS: The Mystery & The Solution as long as I didn’t mention Kaiser in the book. I had to make sure the printer deleted all references to where I had done my cancer and AIDS research. The thing I had tried to avoid for so long had become a reality: I had inadvertently become a threat to the medical establishment, just like Virginia Livingston.

    13. Tell me about your role model and colleague Virginia Livingston.

    Alan Cantwell with Virginia Livingston

    Virginia was a dear friend whose research formed the foundation of my scleroderma research and subsequent cancer microbe studies. My association with her and Irene Diller and Eleanor Alexander-Jackson and Florence Seibert, changed my life forever. Although she died in 1990 at the age of 84, Virginia still influences me. She is my “scientific soulmate.” These four women are my four greatest heroines in medical science. In Four Women Against Cancer, I describe their amazing cancer research. I knew them all personally, and sadly all of them are now gone.

    14. What do you think about the Marshall Protocol?

    When I heard about the Marshall Protocol I was taken aback. I never thought that a possible cure for chronic disease would happen in my lifetime. I used to tell people that there was no way known to kill L-form bacteria in the body.

    In mid-life Trevor Marshall set out to figure out a good treatment or a cure sarcoidosis because he had the disease himself. That is how — via his own research — that he discovered me and I was made aware of his own admittedly controversial ideas on how chronic diseases might be successfully treated. He certainly, almost single-handedly, revived my scientific career and I am exceedingly grateful to him for his interest and support of the cancer microbe work.

    Having a disease is unfortunate, but it can serve as a great consciousness-raiser. Illness can also bring people together who would have never been brought together otherwise. This interview is a good example of that! From Trevor I am learning about the importance of the “vitamin D receptor” and that Benicar, along with long-term antibiotics can help rev up the immune system and apparently diminish L-form bacteria in patients who are trying his ideas. It’s interesting because Livingston always said that the key to curing chronic disease and cancer is to improve the function of the immune system. In my opinion, the proof is in the pudding. Some people with chronic disease are reporting benefit from the MP.

    Trevor’s not a medical doctor but he obviously is an avid researcher and well-versed and well-trained in biochemistry, pharmacology, molecular biology, subjects that are way beyond my ken. Plus, I went to medical school a half century ago.

    The MP has revealed that the healing process of certain chronic disease needs to go slowly, which in many ways goes against scientific dogma with its “quick cure with a round of antibiotics.” Both Trevor and I believe bacteria are implicated in sarcoid, even though this is still denied by many physicians who consider sarcoid a “disease of unknown etiology” — and all the research pointing to bacteria in sarcoid is ignored. Trevor obviously believes bacterial infection also plays a role in certain other chronic diseases. If you think about it, diseases like tuberculosis, leprosy and cancer all take years to treat. You don’t necessarily expect to get well in one month, one week, or even one year. Similarly, one shouldn’t expect a quick cure in chronic disease, even though bacteria play a big role in these diseases.

    15. What do you feel lies ahead in terms of cancer research?

    I feel that the treatment of cancer will remain dismal until these bacteria are recognized as cancer-causing agents by the scientific and cancer establishments. Only then can better treatment methods be employed that actually are specifically directed against the buildup of these L-forms or are directed towards strengthening the immune system against them, or both.

    IMAGES OF L-FORM BACTERIA BY ALAN CANTWELL

    Click to enlarge and see descriptions.

    Alan Cantwell is a retired dermatologist. He has written two books on the microbiology of cancer, The Cancer Microbe and Four Women Against Cancer: Bacteria, Cancer and the Origin of Life. A number of Dr. Cantwell’s articles, including those which describe the above images in further detail, are published in Journal of Independent Medical Research. He can be contacted via email atalancantwell@sbcglobal.net.

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  • About Amy Proal

    Amy and Zeus

    Amy Proal graduated from Georgetown University in 2005 with a degree in biology. While at Georgetown, she wrote her senior thesis on Chronic Fatigue Syndrome and the Marshall Protocol.