The live presentation was filmed but the room was a bit on the dark side. So, despite a laudable job filming by my colleague, Judy, I decided to put up a slideshow with voiceovers.

We’re not sure if these people are supplementing with vitamin D (there is certainly no vitamin D added to the food chain!) but they’re certainly not getting a lot of sun.

The Vitamin D Council insists that people must expose themselves to sunlight and eat vitamin D-fortified products, yet these people are going about their daily lives without any apparent ill effect.

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

Claude Bernard, An Introduction to the Study of Experimental Medicine

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

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

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

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

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

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

A committee to evaluate the DRI of vitamin D is convened

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

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

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

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

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

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

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

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

Dr. Barry Kramer sounds an early note of caution

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Dr. Michael Holick speaks on sunscreen and vitamin D

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Dr. Cedric Garland discusses vitamin D and cancer

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Dr. Reinhold Vieth speaks about safety

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Vitamin D and the evolving definition of autoimmune/inflammatory disease

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

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

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

Siobhán M. O’Connor

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

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

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

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

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

Our speeches and the reaction to them

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

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

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

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

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

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

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

Anticipating what is to come

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

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

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

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

Here are a few possibilities:

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

Epilogue: The ride home

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

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

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

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

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

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

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

Hello readers!  Suffice it to say I’ve been missing in action for several months.  For much of the time I’ve been traveling.  Some of you may know that I just got back from China where I gave a speech at the International Congress of Antibodies. That will be the subject of my next post when the video of my speech is ready. In the meantime, I finally have time to give you an update of what I was up to before I left for Beijing…just to keep things in chronological order.

Several months ago I travelled to Vancouver Island to stay with Paul’s brother and his wife. It was wonderful to be surrounded by nature again! We took hikes through 200 year old forests and climbed gnarled driftwood on the beach. It was the first time I’ve sat around a bonfire since getting sick. I even got to take a horseback riding lesson and stayed on the horse!

Then, I went to visit my twin sister Sara in Dubrovnik, Croatia where she and her fiancee Tony live.  Not only did I walk the city walls and streets with vigor (old town Dubrovnik is surrounded by an ancient fortress), but I got to see some serious water polo action between my sister’s fiancee’s club team, JUG Dubrovnik, and the top Serbian club team in the world.

Ah… fresh forest air

  The two teams are intense rivals and JUG won their biggest game of the year when I was there.  The whole city went nuts and I got to participate in the celebration… and with the team no less. Sara and I also celebrated our birthday together in Dubrovnik – it was the first time we have been together for our birthday in about 8 years.

After that excitement I returned to New York where I worked on writing two papers for the scientific journal Autoimmunity Reviews.  One of them describes how the interaction between human and bacterial genomes drives the autoimmune disease process: 

Autoimmune Disease in the Era of the Metagenome

For the second paper I worked (with Paul, see below) to explain, as simply as possible, how the Marshall model of vitamin D metabolism contrasts with the current model of “vitamin” D’s properties currently put forth by much of the mainstream medical community.  Among other topics, we point out how the Marshall model is supported by molecular data but certainly makes more sense in the face of research showing that the diseases apparently “helped” by vitamin D supplementation are actually becoming increasingly widespread:

Vitamin D: The Alternative Hypothesis

On my horse before we galloped off into the horizon

Also, I wrote press releases for both articles. These have sparked the interest of at least a few journalists and their publications including Science Daily.

I have also been doing my best to keep up with comments posted on this site. As you may have noticed, my trusted colleague, Paul Albert of Weill Cornell Medical College, has been answering some of the questions himself. I enjoy everyone’s comments but since I’m getting increasingly busy I’m sorry if my turnaround time is a bit slow sometimes. Or if I somehow miss your post or email it’s definitely not intentional! 

Walking with Tony to our apartment on an island near Dubrovnik with Marco Polo’s childhood home in front of us

In the fall, I will start graduate school at University of Illinois at Chicago (UIC) where I’ll be pursuing a degree in microbiology.  The school appeals to me on many levels.  During the interview process the school’s researchers seemed genuinely interested in my work with the MP.  They asked questions, watched my videos, and truly appreciated the personal statement that I had labored over in an attempt to best communicate my passion for exploring the microbial world.  Several researchers are studying biofilm bacteria, which is a topic of great personal interest. 

In particular, one researcher, who graduated from Princeton rather recently, is working on bacterial communication in biofilms.  He’s also very interested in the role of bacteria in chronic disease.  He called me a perfect prospective graduate student, so I hope that I can live up to his impression of me. Obviously grad school will take up much of my time, but I need the expertise that comes with a PhD and I’m excited to better learn how to use some of the latest molecular techniques.  However, I plan to try my hardest to continue to work with ARF on the side.

Happy birthday Sara and Amy!

In less than a week I’ll be heading to Chicago for Sara’s bachelorette party – a three day bacchanalia with her friends from around the world.  Then I’ll take some time to look for an apartment in Chicago and visit my family in the area. As soon as I get back my parents are visiting me in New York.   Another reason I had to take some time off from Bacteriality is because I worked with Paul to design and write the content for Sara and Tony’s wedding website.  

During my free time I’ll be working on another paper for Autoimmunity Reviews – this one will discuss immunopathology. I also plan to help Paul edit what I think is a groundbreaking project on his behalf – the MP Knowledge Base. Paul has been faithfully gathering both old and new content on essentially every topic related to the MP and is organizing it in easy-to-read articles on a very searchable website. His perserverence amazes me. While the site will take a few more months to complete, I’m going to review the major articles and try to ensure the content is in tip-top shape.

Dancing to live Croatian music after JUG’s victory

As I progress towards my grad school days I suspect my posts on this site will become shorter. Perhaps, in many ways, the Knowledge Base will replace the need for Bacteriality. But, if anything, I’ll still use the site to discuss my work at grad school and any conferences or scientific events I attend in the future.

Colorful representations of sequenced genomes adorn the walls at JCVI.

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

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

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

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

First Impressions

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

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

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

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

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

Let the learning begin

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

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

Site of the learning.

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

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

Let’s discover a new species of bacteria!

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

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

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

Sequencing and beginning to interpret a genome

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

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

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

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

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

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

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

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

TIGR00433

Name: biotin synthase

Gene symbol: bioB

TIGR role: 77 biotin synthesis

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

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

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

Annotating a DNA sequence

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

The labs at JCVI are first-class.

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

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

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

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

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

The Institute had a modern aesthetic.

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

About the Comprehensive Microbial Resource (CMR)

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

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

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

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

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

Ready to learn about human annotation!

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

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

Improving the accuracy of the pipeline

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

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

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

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

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

Sequencing bacteria and the future of medicine

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

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

A JCVI sequencing machine

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

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

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

Even those of us who live under rocks have heard of the Human Genome Project, a massive international scientific research project the aim of which was to understand the genetic makeup of the human species. Its primary goal was to determine the sequence of chemical base pairs which make up DNA and to identify the approximately 25,000 genes of the human genome from both a physical and functional standpoint.

The goal of the Human Genome Project was to understand the genetic makeup of the human species.

A working draft of the genome was released in 2000 and a complete one in 2003, with further analyses yet to be completed and published. Meanwhile, a parallel project was conducted by the private company Celera Genomics. Most of the sequencing was performed in universities and research centers from the United States, Canada and Great Britain.

Most researchers would agree that the Human Genome Project was launched in order to answer the long-standing question, “Who am I?” The goal was to identify and sequence every single human gene. By doing so, many researchers were certain they would uncover causes for most of the chronic diseases that plague humankind. At the project’s start, scientists were faced with a multitude of unknown sequences to decipher and understand. Surely such sequences would offer up answers to disease, and specific genes would be found that would correlate with specific illnesses. In a Gattaca-like environment, people would then be informed early in life that they had “the gene” for MS or “the gene” for breast cancer. Scientists would work fervently to identify and change the expression of such disease-causing genes, finally developing enough gene therapies to eradicate human disease. The above scenario has an abiding appeal, largely because the idea that our genes dictate our health is so temptingly simplistic.

Yet, while striving to answer the question- “Who am I?”- those researchers searching for a purely genetic cause for disease have failed to recognize that the question, “Who am I?” can only be answered after the question “Who are we?” has been clarified and understood.

“The question, “Who am I?” can only be answered after the question “Who are we?” has been clarified and understood.”As Asher Mullard of Nature News describes, ‘we’ refers to the wild profusion of bacteria, fungi and viruses that are able to colonize the human body. Such pathogens, and bacteria in particular, number in the trillions. According to one common estimate, the human gut alone contains at least a kilogram of bacteria.

The fact is, at the present moment, human beings serve as communities in which prodigious numbers of bacteria can thrive. Since the average human is currently outnumbered by the pathogens they harbor, the genes produced by these bacteria outnumber human genes as well. According to Mullard, “Between them [the pathogens in our bodies], they harbour millions of genes, compared with the paltry 20,000 estimated in the human genome. To say that you are outnumbered is a massive understatement.”

So by sequencing only human genes, the Human Genome Project has failed to take into account a vast number of bacterial genes that also have the potential to affect the progression of human disease. The fact that many researchers are interpreting genetic data while leaving bacterial genomes and bacteria in general out of the picture is a serious issue.

This is because many of the chronic bacteria we harbor are intraphagocytic – meaning they have developed the ability to live inside the nuclei of our cells. Such pathogens thrive in the cytoplasm, or the liquid surrounding the cellular organelles that allow for DNA replication and repair.

Our DNA sequences are replicated on a regular basis. The process of transcription allows for the synthesis of RNA under the direction of DNA. Since both RNA and DNA use the same “language”, information is simply transcribed, or copied, from one molecule to the other. The result is messenger RNA (mRNA) that carries a genetic message from the DNA to the protein-synthesizing machinery of the cell. In translation, messenger RNA (mRNA) is decoded to produce a specific protein according to the rules specified by the genetic code.

Pathogens in the cytoplasm can likely interfere with the processes of transcription and translation.

Unfortunately, pathogens in the cytoplasm can likely interfere with any number of the many precise steps involved in the transcription and translation processes. Such interference results in genetic mutations, meaning that our DNA is almost certainly altered, over time, by the intracellular pathogens we harbor. The more pathogens a person accumulates, the more his genome is potentially altered.

It is also quite likely that intracellular pathogens disrupt DNA repair mechanisms. Since environmental factors such as UV light result in as many as 1 million individual molecular lesions per cell per day, the potential of intracellular bacteria to interfere with DNA repair mechanisms also greatly interferes with the integrity of the genome and its normal functioning. If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in early senescence, apoptosis or cancer. Problems associated with faulty DNA repair functioning result in premature aging, increased sensitivity to carcinogens, and correspondingly increased cancer risk.

“Lifelong persistent symbiosis between the human genome and the microbiota [the large community of chronic pathogens that inhabit the human body] must necessarily result in modification of individual genomes,” states biomedical researcher Trevor Marshall. It must necessarily result in the accumulation of ‘junk’ in the cytosol, it must necessarily cause interactions between DNA repair and DNA transcription activity”, he continues.

So there is increasing evidence that many of the genetic mutations identified by Human Genome Project researchers are largely induced by bacteria and other pathogens. Rather than serving as markers of particular diseases, such mutations generally mark the presence of those pathogens capable of affecting DNA transcription and translation in the nucleus. This is why, in most cases, the “one gene, one disease” hypothesis has failed to hold water. Instead, geneticists are now stuck examining a perplexing number of different mutations, most of which differ so greatly between individuals that no correlations can be made between their presence and any particular illness. The mutations are nothing but genetic “noise,” induced either by random chance or by the pathogens that such researchers fail to factor into the picture.

As Marshall describes, researchers sequencing human DNA samples often make the assumption that only one genome (the human genome) is present, when in reality, their tests are also likely picking up on the loose bits of multiple genomes (bacterial genomes). So if a person’s genome is sequenced once and then sequenced again, will the same DNA sequences be obtained? Probably not, because each individual sequencing will randomly pull various pathogenic genes into the human mix. Thus, what are currently viewed as “disease-causing” mutations are essentially statistical anomalies that vary depending on when and how a person’s genome is sequenced.

Each individual sequencing of a genome will randomly pull various pathogenic genes into the human mix.

If sequenced genomes are currently just a sum of several random parts, then it’s inevitable that an individual’s genome will change throughout life. Because pathogen-induced mutations, random mutations, and mutations that result from faulty DNA repair accumulate over decades, the genome map of a child will be very different from the genome map produced when the same individual is an adult, with differences increasing as people reach their elderly years. Goodbye the world of Gattaca. Right now, if a child’s genome is sequenced at birth, his or her genetic sequences predict nothing about the common chronic diseases they will encounter, and mutations accumulated later in life largely serve to signal the presence of infection. This means that using people’s genomes to define their identity – as envisioned by futuristic movies in which a person’s genome might serve as their passport and destiny – is not feasible at the moment.

This understanding marks a yet to be fully realized paradigm shift in the way the genome is interpreted. It’s hard not to feel sympathy for those individuals paying hefty sums of money to have their genomes sequenced by certain companies that now offer such a service. Customers are provided with a map of their genome in which the majority of observed mutations serve only to inform them that they harbor numerous intracellular pathogens. As Mullard describes, “Observers have started to question whether the human genome can deliver on its once-hyped promises to tackle disease. To take just one example, anyone so inclined can now pay genetic-testing companies for a preliminary rundown of the genetic variations associated with his or her risk of developing cancer, obesity and other conditions. But the risks identified are often so low or unclear that people are questioning whether the information will actually prompt the changes in health behaviour, such as losing weight, that could make them valuable.”

Finally, bacteria enter the picture – the rise of metagenomics and the Human Microbiome Project

The paradigm shift described above, in which genetic mutations are viewed in a new light, has been largely fueled by a new movement in which scientists are now beginning to use molecular technology to detect and sequence bacteria in lieu of simply trying to grow them in the lab. These tools will allow researchers to bypass the need to culture bacteria, exploring the human microbiome by studying genes en masse, rather than studying the organisms themselves.

Recent studies that have used powerful molecular tools rather than standard cultivation techniques have left scientists slack-jawed at the number of bacterial DNA sequences that correspond to bacteria yet to be named or sequenced. A great number of sequences also correspond to bacteria never thought to have the capability of living on or within the human body. It has recently become all too clear that only a fraction of the bacteria capable of infecting humans grow in the lab, and that we have been oblivious to the presence of the majority of pathogens capable of entering our bodies. This realization that we harbor myriad unnamed and unidentified microbes comes at a time when the Human Genome Project is failing to capitalize on its promise to identify root causes for human disease.

Powerful new molecular tools are revealing the number of bacterial DNA sequences that correspond to bacteria yet to be named or sequenced.

As Mullard admits, “The microbes that swarm in and on the human body have always held a certain fascination for researchers. Since so few of them grow in the lab, it has been difficult to work out exactly who these microbial passengers are and how they interact with one another.” Whereas over the past century, standard laboratory culturing techniques have failed to detect the vast number of pathogens capable of infecting human beings, recent advances in molecular technology that allow for the sequencing of bacterial DNA mean that, at long last, we may be able to successfully identify and sequence the bacteria that cause disease.”

The reality is that the plethora of unknown pathogens that colonize the human body are the previously unrecognized puzzle piece behind chronic inflammatory disease. Enter metagenomics, a relatively new field of research that, thanks to advanced molecular techniques, enables researchers to study organisms not easily cultured in a laboratory as well as organisms in their natural environment.

This year marks the tenth birthday of metagenomics. It was a decade ago that Jo Handelsman and her colleagues at the University of Wisconsin in Madison successfully cloned and determined the functional analysis of the collective genomes of previously unculturable soil microorganisms in an attempt to reconstruct and characterize individual community inhabitants. The team coined the word “metagenomics” to explain their techniques and goals. Since the Handelsmam work, the scope of metagenomics has expanded greatly. Teams of researchers across the world have made efforts to describe the bacteria in environments as diverse as the human gut, the air over New York, the Sargasso Sea and honeybee colonies.

“We can look at the metagenomic analysis so much more deeply, at such a better cost,” says Jane Peterson, associate director of the Division of Extramural Research of the National Human Genome Research Institute in Bethesda, Maryland, which recently launched a five-year initiative to explore the human microbiome.

The five-year initiative is one of several massive projects striving to characterize what is referred to as the human microbiome, the name given to the collection of microorganisms living in and on the human body. The goal of the project is as ambitious as it is exciting – to detect and name every type of bacterial species that is currently capable of inhabiting the human body.

The project is perhaps the best example of a new, and long overdue, shift in thinking among many medical researchers. At long last, microbiome scientists are vastly more interested in studying and identifying the pathogens that inhabit the human body rather than simply examining human genes.

Microbiome scientists are vastly more interested in studying and identifying the pathogens that inhabit the human body rather than simply examining human genes.

Late last year, the US National Institutes of Health (NIH) pledged US$ 115 million to identify and characterize the human microbiome, Also last year, the European Commission and various research institutes committed €20 million (US$31 million) for similar research. Smaller research teams with similar goals are also pledging lesser sums of money towards research that hopes to contribute to a greater understanding of the microbiome. These teams are situated in countries as diverse as China, Canada, Japan, Singapore and Australia.

Since the human microbiome is so diverse, it’s not surprising that an array of different research teams are needed to tackle divergent areas of the project. The NIH’s five-year Human Microbiome Project will spend much of its money identifying where certain bacteria in the body are located. They also plan to compile a reference set of genetic sequences that correspond to each bacterial species.

Although one-quarter of the project’s money has been earmarked to examine the role of the microbiome in health and disease, the Human Microbiome Project will do little to assess the function of microbes during its first year, although it may focus on the topic later. It’s serendipitous that the “health and disease” aspect of the project has been put off, since it’s only a matter of time before the medical community realizes that biomedical researcher Trevor Marshall has already largely elucidated how the intraphagocytic, metagenomic, microbiota of bacteria that cause chronic inflammatory disease are able to survive in the body and evade the immune system. Ideally, the money now dedicated towards examining the role of the human microbiome in disease could be used to pursue research projects related to Marshall’s discoveries.

Since the vast number of bacteria and other pathogens that cause human disease have yet to even be discovered and documented, the primary goal of the Human Microbiome Project is to build up a research community and generate a sequence resource, akin to that developed during the Human Genome Project, so that questions about bacteria and specific disease-causing mechanisms can be tackled at a later date.

“ Under the most ideal of circumstances, the money now dedicated towards examining the role of the human microbiome in disease could be used to pursue research projects related to Marshall’s discoveries.”This year, researchers will collect samples of feces plus swabs from the vagina, mouth, nose and skin of 250 volunteers. 250 people may seem like a small number of subjects for such a massive project, but when one understands that the DNA of every single one of the trillions of pathogens harbored by each subject will be analyzed, it’s easy to see that such an undertaking is actually a monumental task.

How do you effectively study such a vast and unknown community? The ultimate goal is to sequence the complete genomes of hundreds of bacterial species and deposit them in a shared database. Most of the research teams involved in the project will be sequencing short, variable stretches of DNA that code for components of bacterial proteins in order to roughly identify which bacteria are present in each person and how many bacterial species volunteers have in common. Once an estimate of diversity has been attained, the researchers plan to mine deeper by using shotgun sequencing – a molecular technique that will allow them to analyze many short pieces of DNA from all over the microbes’ genomes and reveal which genes are present.

In shotgun sequencing, DNA is broken up randomly into numerous small segments with the goal of creating multiple overlapping sequences.

In shotgun sequencing, DNA is broken up randomly into numerous small segments. The DNA sequence of each fragment is subsequently identified. The process is then repeated in order to create multiple overlapping sequences of DNA. When enough overlapping sequences have been generated a computer program is able to assemble the ends of the overlapping sequences into a contiguous sequence.

Microbiome researchers will initially use shotgun sequencing data from a few bacterial species that can already be grown, and piece together their whole genomes by putting overlapping fragments in order. The Human Microbiome Project plans to provide 600 “reference genomes.” The European project will do another 100, and other sequencing efforts by the NIH and elsewhere will make additional contributions. The hope is that enough research teams are able to set up a broad enough reference database. Then, researchers will be able to predict the genetic capabilities of many currently unculturable species (many of which are in an L-form and/or biofilm-like state) solely on the basis of similarities with known genes.

Creating the database will not be a simple task. According to Peer Bork, a biochemist who heads the European project’s computational center at the European Molecular Biology Laboratory in Heidelberg, Germany, even if many reference sequences are created, fitting together DNA fragments in order to identify unknown species, “is pretty hairy from a computational biology analysis point of view. Even with the immense power of supercomputers to process the sequencing data, it will take some clever analysis to compare the millions of sequence reads that span thousands of species between hundreds of ‘healthy’ and unhealthy people.”

Many different research teams will be working simultaneously in order to build a map of human bacteria.

Yet, the scientists involved in the project appear intent on capitalizing on their promise to sequence the microbiome. Furthermore, each research team will still be allowed to pursue their own pet projects. “Talented people are doing what they think is the most important research to do, rather than being forced to do what somebody else has decided would be the best,” says Ehrlich.

As touched on above, one of the main scientists pushing forward the metagenomics movement is Marshall. Although not directly involved in the Microbiome Project at the moment, decades of in silico and clinical research have allowed the biomedical researcher to create a treatment regimen that effectively kills the intraphagocytic, metagenomic bacteria that microbiome researchers will be identifying in greater detail.

While at first glance it may seem counterintuitive that Marshall’s work has demonstrated how to kill the microbiome before the bacteria that comprise it have even been fully sequenced, one must keep in mind that all bacteria possess certain characteristics. Every bacterial species has a 70S ribosome – a protein region that must be functioning if the pathogen is to survive. Whether or not a species has been named, identified, cultured, or sequenced, if its 70S ribosome is blocked, as it is by the pulsed, low-dose antibiotics championed by Marshall, it will be weakened so greatly that it cannot survive in the presence of an activated immune system.

Every bacterial species has a 70S ribosome – a protein region that must be functioning if the pathogen is to survive.

So Marshall’s treatment protocol – dubbed the Marshall Protocol – already exists, and can kill the pathogens that Microbiome researchers will be identifying. As it perfuses the mainstream, Marshall’s research (when fully appreciated) will represent a quantum leap forward for microbiome researchers. After all, the microbiome community should be quite pleasantly surprised to find out that the disease-causing bacteria they sequence can already be killed.

However, at the moment, patients on the Marshall Protocol have little knowledge of exactly what chronic pathogens they are killing. In a sense, such knowledge isn’t of utmost importance, as specific names are not needed to induce recovery. Yet it would certainly be of great interest for researchers working with various aspects of the Marshall Pathogenesis to possess a greater understanding of the bacterial species any one patient is killing, and what species of bacteria can generally be associated with specific symptoms.

Thus, the database that the Microbiome project intends to provide promises to be uniquely helpful for researchers working on MP-related projects. Such researchers will be able to use the database to get a much clearer idea of exactly which chronic pathogens cause inflammatory disease, the substances created by these pathogens that may lead to receptor blockage, and exactly which bacteria are killed by different antibiotic combinations. As more knowledge is built about the specific pathogens that cause inflammatory disease, other drugs besides the MP antibiotics may be developed that also target them effectively, adding to an already powerful arsenal to render them dead.

Of course, using the Microbiome database to identify the presence and species of bacteria targeted by the Marshall Protocol will require numerous researchers to perform shotgun sequencing of the bacteria in the tissues of patients with many forms of chronic disease. Sequences derived from such patients can be compared with the database in order to match DNA sequences with those of specific bacterial species.

“If periodic shotgun sequencing studies are performed as a patient progresses through the MP, they will undoubtedly reveal that MP patients have high bacterial loads at the onset of treatment and greatly reduced bacterial loads after several years of therapy.”Even better, shotgun sequencing can be used to convince skeptics of the MP’s validity. If periodic shotgun sequencing studies are performed as a patient progresses through the MP, they will undoubtedly reveal that MP patients have high bacterial loads at the onset of treatment and greatly reduced bacterial loads after several years of therapy. Absence of bacteria would, of course, correlate with disease resolution and cure. Such data would provide even the greatest skeptic with the proof necessary to confirm that the MP does indeed reverse inflammatory disease and successfully kill chronic idiopathic bacteria.

The few shotgun sequencing studies performed to date have already helped Marshall flesh out certain aspects of the Marshall Pathogenesis. A recent shotgun sequencing study that detected the species of bacteria present on prosthetic hip joints allowed him to identify (using molecular software) that the chronic pathogen Flavobacter, creates a lipid capable of dysregulating the Vitamin D Receptor (VDR). The discovery finally provided proof of concept for the fact that many of the chronic pathogens we harbor almost certainly increase their survival by creating similar ligands that block the ability of the VDR to activate components of the innate immune response.

The fact that same study also found hydrothermal heat vent bacteria (which clearly cannot be killed by boiling) on the joints reinforces just how much we have yet to discover about the pathogens capable of inhabiting our bodies. Other pathogens detected by the research team include species such as Lysobacter, Methylobacterium, and Eubacteria. “None of these species were previously expected to exist in man” states Marshall. “These are species nobody is looking for, they are not picked up by PCR testing and nobody is culturing them.”

Consider that each species of bacteria detected in the above study has about 1,000 – 4,000 genes. So together they create about 100,000 genes that are active in the body, yet are not even contemplated by the vast majority of mainstream researchers. And those are only the pathogens detected by one molecular analysis.

A continued focus on gut bacteria

As previously discussed, the European Commission has launched a four-year initiative, called Metagenomics of the Human Intestinal Tract (MetaHIT). The project, which contains many different initiatives, overlaps somewhat with the American initiative in the sense that the European team is required to sequence bacterial genomes for a database. American and European results will be put in the same database, one which is freely available for anyone interested in sequencing and identifying bacterial DNA. And who isn’t?

But Tract (Meta HIT) has a different goal than the American Microbiome Project. It will focus on better understanding of the bacteria that inhabit the gut and how they contribute to obesity and inflammatory bowel disease. And, according to Mullard, whereas the Human Microbiome Project is initially comparing people’s microbiota on a species level, MetaHIT aims to find differences in microbial genes and the proteins they express without necessarily worrying about which species they came from.

The bacterium, Entercoccus faecalis, which lives in the human gut, is just one type of microbe that will be studied as part of NIH’s Human Microbiome Project.

“We don’t care if the name of the bacteria is Enterobacter or Salmonella. We want to know if there is an enzyme producing carbohydrates, an enzyme producing gas or an enzyme degrading proteins,” explains Francisco Guarner, a gastroenterologist at Vall D’Hebron University Hospital in Barcelona, Spain. We want to “examine associations between bacterial genes and human phenotypes,” says Dusko Ehrlich, coordinator of MetaHIT and head of microbial genetics at INRA, the French agricultural research agency in Jouy-en-Josas.

This is similar to the approach currently taken by Marshall who is more interested in the observable characteristics of the bacteria, including how they respond to different antibiotics and what substances they produce, than in identifying individual species.

A handful of projects have already tried to characterize the bacteria that cause bowel disease and obesity, including research by Jeff Gordon at Washington University in St. Louis, who found different compositions of bacteria in obese and lean subjects (for details, see my article on obesity). Then, there was the 2006 project by Steven Gill and colleagues at the Institute for Genomic Research in Rockville, Maryland, who threw around some then-hefty numbers when they carried out such a metagenomic analysis of the microbes in two people’s intestines. After 2,062 polymerase chain reactions and 78 million base pairs, the team provided only the briefest of glimpses into the genetic underpinnings of the human gut’s microbes.

According to Mullard, these first surveys involved too few individuals and sampled too few microbes, usually from only the gut or the mouth, to provide an adequate description of the microbiome. But things have changed in the past few years. A few million foreign genes no longer sound so daunting in the face of advanced genetic-sequencing methods that are capable of crunching monumental amounts of numbers. As with the American Microbiome project, thanks to certain cutting-edge technologies, researchers can assess hundreds of millions of base pairs in just a few hours.

A few million foreign genes no longer sound so daunting in the face of advanced genetic-sequencing methods that are capable of crunching monumental amounts of numbers.

An in-depth analysis by Tract (MetaHIT) researchers of exactly what microbes inhabit the gut and what substances they produce will only enhance the knowledge already derived from Marshall’s work, which shows that chronic bacteria in the gut or elsewhere survive largely because they have evolved mechanisms to block the activity of certain receptors that would otherwise activate pathways that would inhibit their survival. A better understanding of what substances gut bacteria create may help Marshall and other scientists identify other ligands that dysregulate the VDR or other receptors.

Combining data derived from Tract (MetaHIT) with that derived from Marshall’s work will also provide an opportunity to better understand exactly what the mass microbes in our gut are up to. Historically, researchers have understood that a great number of bacteria thrive in the gut. However, in the absence of enough data showing how pathogens in the gut survive, or how gut bacteria contribute to disease, they have only been able to guess at the role of the gut microbial community.

Such researchers have proven to be optimists. Over the past decades, the vast majority of them have concluded that, if bacteria are present in large numbers in the gut, they must be doing something helpful. That, or gut bacteria have been assumed to be commensal – helping the human gut in some way and in turn obtaining nutrients from the host. Yet nature shows that commensal relationships are not necessarily the rule. Sure, you’ll find mites on certain birds or orchids growing on trees. But in the majority of cases where two species interact, one usually takes advantage of the other. Furthermore, considering what we already know about bacteria, they are almost always guilty of exploiting the resources of their host. So it may be wishful thinking to assume that the bacteria in our guts are largely friendly helpers.

This isn’t to say that there aren’t species of gut bacteria that can provide a benefit to their host. Yet increasing evidence points to the fact that the vast majority of gut bacteria are actually responsible for causing many bowel diseases previously considered to be of “unknown” cause. When faced with the large number of different inflammatory bowel diseases and the fact that a tremendous number of uncharacterized bacteria inhabit the gut, it’s logical that there’s a connection between the two phenomena. Of course, Marshall’s in silico work, as well as data derived from the MP study site shows that patients who kill large numbers of gut bacteria end up recovering from a number of bowel diseases, providing a good deal of support for the above hypothesis.

This all invokes the rather controversial question, “Do humans really need gut bacteria?” Those patients to spend long periods of time on the MP have killed a great deal of their gut bacteria, yet seem to have GI tracts that function properly. Marshall has conceded that “good” gut bacteria could potentially exist, but as of yet, he has simply seen no evidence of a species that offers humans a benefit.

“Then again, whether or not a certain species of gut bacteria may be considered “helpful” may depend on a person’s set of circumstances.”Then again, as Marshall describes, whether or not a certain species of gut bacteria may be considered “helpful” may depend on a person’s set of circumstances. It’s widely accepted that some gut bacteria help metabolize carbohydrates, causing people to absorb about 15-20% more of the energy from the carbohydrates they ingest. In a country like the United States, where the majority of people are well-fed, or in many cases over-fed, the presence of such bacteria in the gut might provide a distinct disadvantage. People who have access to enough food are usually seeking to lose weight and, in such cases, the presence of bacteria that glean more energy from carbohydrates would contribute to weight gain. The average American would probably be better off without such species in the gut.

But what about people in developing countries who face food shortages and are often limited to eating just the amount of food they need to survive? Under such circumstances, the presence of a bacterial species in the gut that gleans more energy from carbohydrates would be seen as a great advantage, allowing people to acquire more energy from a smaller portion of food. In a world where even the developed world may face food shortages in the future, one can never tell if someday such bacteria would provide a benefit to the entire population.

Scanning electron microscope images of B. thetaiotaomicron, a prominent human gut bacterium, and the intestine.

Yet, possibilities like the one discussed above still don’t answer the questions of whether humans actually need gut bacteria. Bacteria and humans (or our ape-like ancestors) have evolved in tandem for millennia. Are pathogens’ ability to inhabit our bodies an evolutionary adaptation that serves to benefit both humans and bacteria, or is it possible that the ability of microbes to persist in the human body is largely an evolutionary victory for bacteria won at our expense? As more and more diseases are linked to bacteria previously considered innocuous, the latter is becoming an increasingly plausible possibility.

Compare the human body to planet Earth. Creatures including human beings have evolved to live on our planet, yet does the Earth need the presence of such animals to survive? Most people would agree that the Earth would manage just fine without human beings. Although a handful of humans may strive to enhance certain aspects of our natural surroundings, the vast majority of mankind is depleting the Earth’s resources, leading to massive problems such as climate change, pollution, and an accumulation of fake chemicals in our water and food supply. So if we compare the bacteria that inhabit our guts and bodies to the people that inhabit our planet, it’s plausible that both might be better off without alien inhabitants.

Of course, some animals may be seen as beneficial to Earth. The earthworm restores the resiliency of soil, or the honey bee carries pollen from flower to flower. Yet even under these potentially beneficial circumstances, one can still question whether the Earth could maintain a state of homeostasis on it’s own without such help, or quickly evolve different ways to manage without such aid.

Furthermore, there is no question that any bacteria, whether friend or foe, places a burden on the innate immune system. With trillions of bacteria to keep track of, the innate immune system is constantly at work, prioritizing which bacteria to attack and determining where immune system cells should be located. In fact, researchers estimate that 70 percent of the immune system is located in and around the gut. Imagine if gut bacterial load was reduced to the point where much of this burden was lifted? The innate immune system would certainly be able to divert much more strength towards killing pathogens in other tissues as well new pathogens attempting to enter the body. Of course, as of now, such a scenario would only be possible if a person were to remain on low, pulsed antibiotics for a lifetime. Without the help of antibiotics, it seems reasonable to conclude the innate immune system would be over-burdened by the task of keeping the body bacteria-free.

We may be asked to embrace the reality the gut bacteria are not just “friendly” helpers.

Some may argue that probiotics are beneficial bacteria yet, as described in this article. An alternate hypothesis about how they provide palliation must also be factored into the picture.

All this means that Tract (MetaHIT) researchers, MP researchers, and scientists studying gut bacteria in the light of new molecular technology may be facing a paradigm shift in the way gut microbes are perceived. Rather than viewing the majority of them as “friends,” we may unfortunately have to face the fact that many of them are enemies, or at least not necessary for our well-being. It still remains unclear if humans would want to be completely bacteria-free if the option existed, but the possibility that a person would be in better health without bacteria is nevertheless an intriguing possibility. Or perhaps in the future, humans will be able to pick and choose the bacteria that will inhabit their guts, in order to harbor certain species that fit their specific needs.

Competition!

The fact that the Microbiome project and Tract (MetaHIT) plan to generate so much new information on bacteria means that collaboration between the two groups and other smaller groups involved in bacterial sequencing is important. According to Bork, when all the projects are running at speed, reams of data will be generated worldwide. But because different groups are using different techniques to collect samples, extract DNA and annotate data, the data sets could be difficult to compare.

Enter the as-yet-unlaunched International Human Microbiome Consortium. Scientists from several international projects, including the Human Microbiome Project and MetaHIT, have been meeting since late 2005 to figure out how to collaborate on a range of issues such as the compatibility of data and which bacteria to sequence for the reference database. The group is already setting up infrastructure and “beginning to address the tough questions,” says Weinstock. But according to Nathan Blow of Nature News, it is too early to say how well the Consortium will foster collaboration. Its official launch, scheduled for this past April, was postponed for six months to allow the NIH and the European Commission to overcome bureaucratic difficulties.

Another issue being addressed by the Consortium is that of intellectual property. As with other genomic projects, members of the Consortium will be expected to release sequence data into the public domain as soon as they are generated. But according to Blow, this doesn’t necessarily preclude disputes over intellectual property if, for instance, a particular bacterial gene proves to be a useful diagnostic marker for a disease. Another unresolved question is whether a laboratory can have one project that abides by the Consortium’s regulations, and another that doesn’t. “There are grey areas, and I feel that until we have a test case, they will have to be watched very carefully,” says Bhagirath Singh of the Canadian Institute of Health Research, who is helping to develop the Canadian Microbiome Initiative.

“It’s increasingly important for researchers and doctors to start pulling information out of individual laboratories and individual clinic records so that we may compile it.”Intellectual privacy and patent issues aside, optimism for the collaboration still runs high, and having a database of bacterial sequences that is available to other research teams and perhaps even the public would be a great step forward in a medical movement that many believe needs a pick-me-up. It’s increasingly important for researchers and doctors to start pulling information out of individual laboratories and individual clinic records so that we may compile it. Patterns and associations can be detected much more easily when large groups of data are gathered simultaneously and made accessible to as many people as possible. The computer open-source movement, which has spread to many other fields, has seen incredible success in areas where research and data are openly shared. Access to open-source databases will almost certainly augment the pace of major medical discoveries – a pace that, MP aside, can often seem as if it’s at a current standstill.

Participants from microbiome projects around the world have expressed the desire to join and attend the Consortium. Like bulls ready to race down the streets of Pamplona, such research teams will be competing with each other as the search to sequence the microbiome moves forward. After all, each group wants credit for identifying as many new species of bacteria as possible.

“The intention is to work together,” says George Weinstock, a geneticist at Washington University in St Louis, who is helping to organize the Human Microbiome Project, “but for the moment it is more about working in parallel until we can understand how to work together”. Apparently some European researchers feel at a disadvantage because MetaHIT’s operating budget is only a quarter the size of the Human Microbiome Project’s. “This is giving a huge advantage to the Americans,” Guarner says. “They are going to be quicker and they have more equipment.”

Then again, other members of MetaHIT feel that they actually have an edge because money for their project has already been distributed and data collection is under way, whereas the Human Microbiome Project will not announce many of its funding decisions until later this year. “We have an advantage already, we have a show on the road,” says Willem de Vos, a microbiologist at Wageningen University in the Netherlands and a member of MetaHIT.

Color-enhanced scanning electron micrograph showing Salmonella typhimurium (red) invading cultured human cells

For example, in Denmark, a team led by Oluf Pedersen at the Steno Diabetes Centre in Copenhagen is collecting fecal samples from 120 obese volunteers and 60 controls to tease out specific microbial genes that might contribute to obesity. A similar-sized study in Spain, led by Guarner, will compare the microbiotas of patients with inflammatory bowel disease with those of genetically matched controls and examine the effect of drugs.

Others feel that the sharing of data will simply allow the most ingenious teams to get ahead. “If it is an international consortium, it doesn’t matter where the data are generated,” Bork adds. “For example, we can be the pirates here, sitting at the end in Europe, and use American data to make the discoveries.”

As Blow describes, given the number of separate projects, all at such an early stage, it’s almost impossible to make out where the starting line lies or who exactly is edging ahead. But for many of us, the potentially intense competition among microbiome researchers is a welcome change to the increasing number of “consensus conferences” in which researchers with the same opinions fail to consider alternate lines of thinking and generate novel hypotheses. Competition has the potential to speed up output, allowing for a medical community that may stall less and deliver more. Furthermore, when faced with talented competitors, researchers are more likely to consider new hypotheses and break from the norm in order to gain an edge over an opposing team.

“Competition has the potential to speed up output, allowing for a medical community that may stall less and deliver more.”Then again, the fact that trillions of bacterial genomes must be sequenced means that at the current moment there is plenty of work for each research team and multiple ways for every research team to excel. With trillions of microbes to sift through, most researchers feel that there is more than enough of the microbiome to go around. “There’s so much to learn, so much we don’t know and so many adventures,” Gordon says. “There’s enough room for everyone.”

A core microbiome?

How many different bacterial species the microbiome project will uncover remains anyone’s guess. But according to Blow, many of the researchers involved with the project have one impending question.

Is there a core human microbiome?

“One of the things that is obsessing microbiologists is: ‘What is the size of the core microbiome?,’” says Jeremy Nicholson, a biological chemist who studies microbes and metabolism at Imperial College in London.

By core microbiome, researchers like Nicholson are referring to a hypothesized number of bacteria that every person might harbor. For example, if some bacteria are shown to have a beneficial effect on human health, then perhaps everybody needs a certain number of these pathogens to survive. Then again, if all people harbor certain bacterial species, such pathogens may be seen in a different light. If a “core microbiome” is established, then perhaps the bacteria that comprise it contribute a process that happens to every human being. That process is aging.

As Marshall and colleagues discussed at the recent “Understanding Aging: Biomedical and Bioengineering Approaches” conference at UCLA, it’s entirely possible that the bacteria we harbor are able to infect our stem cells – cells found in all adult tissues that act as a repair system for the body by replenishing other more specialized cells. But as people age, stem cells often lose their ability to repair and heal. If bacteria infect the stem cells, it has been hypothesized that they may expedite the rate at which they lose their resiliency, thus accelerating the aging process.

A stem cell derived from the skin.

Remember the above discussion about how certain microbes can allow people to glean 15% more energy from the carbohydrates they consume? While beneficial under some circumstances, Marshall warns that if such a bacterial species can infect nearby stem cells, they will contribute to the aging processes in the gut.

Several studies support the possibility that chronic bacteria can infect the stem cells. A team of German researchers recently showed that patients who had suffered a heart attack (an event most likely caused by chronic bacterial forms in the heart and blood vessels) had stem cells which were only about half as effective at repairing the heart tissue as stem cells transplanted from healthy 20 year-old males. This supports the view that infected stem cells lack many of the healing properties maintained by their healthy counterparts.

Dr Emil Wirosko, one of the foremost experts on L-form bacteria, died before he could publish on the subject. But according to his colleagues, Wirosko believed L-form bacteria are able to infect stem cells.

Then there are telomeres – DNA sequences on the ends of chromosomes that are gradually lost as cells replicate. As they shorten, a cell can no longer divide and becomes inactive or dies – meaning that the length of a person’s telomeres plays a role in how quickly they will age. The fact that people with heart disease, Alzheimer’s, cancer, and other illnesses have been shown to lose telomere sequences at a faster rate than their healthy counterparts suggests that the bacteria involved in causing such diseases may also have an effect on telomere length. As Marshall describes, if pathogens do directly alter our DNA, then the weakened DNA at the ends of telomeres provides some of the easiest genetic material for them to mutate.

The weakened DNA at the ends of telomeres might provide some of the easiest genetic material for bacteria to mutate.

Once again then, the question is posed: What might occur if humans were to become largely bacteria-free? Might they age at a slower rate? The possibility is tantalizing. Data from people on the Marshall Protocol, who are gradually reducing their bacterial loads, will prove to be increasingly insightful in this regard as time wears on.

As previously discussed, the sequencing of the human genome alone does not allow for the Gattaca-like world described earlier in which humans could be identified and catalogued by their unique DNA sequences. Ironically, the human Microbiome Project and Marshall’s work might make that world more of a reality. If it turns out that the bacteria we harbor are a source of disease and a burden on the innate immune system, then the population will seek (like those people on the MP) to eliminate at least the majority of them.

If sequencing procedures then no longer detect bacterial genes along with human genes, it may be possible to sequence a more fully human genome. One must still factor in DNA mutated by other environmental factors or by chance, but nevertheless, we would be closest to actually answering the question “Who am I?”

Will we ever enter a Gattacca-type world?

Perhaps then, after people have eliminated much of their bacterial load, genetic information will prove to be a more valuable human fingerprint, ethical issues aside.

In the meantime, an optimal environment to better the health of humankind will be one in which controversial hypotheses such as that described above are at least put on the table, and new ideas that challenge current paradigms are embraced rather than rejected. Under such conditions scientists can fully live out Mullard’s advice to, “Celebrate their quest to map, catalogue, and understand the human microbiome for the inspiring saga it is.”

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

Aubrey de Grey

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Chatting with fellow participants in front of my poster.

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

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

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

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

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

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

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

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

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

Talking science with Dr. Marshall

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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In general, oxytocin makes people more sociable and less phobic.

Produced naturally in the brain during social interactions, the hormone oxytocin promotes romantic feelings. It’s also the hormone that helps mothers bond with babies and, in general, makes people more sociable and less phobic. Oxytocin is released during orgasm and is also the key birthing hormone that enables the cervix to open and the contractions to work. In situations where labor has to be induced, it is often given to the mother intravenously to kick-start contractions.

Indeed several recent trials have confirmed oxytocin’s “feel good” effects. After testing the hormone on hundreds of patients, Paul Zak of California’s Claremont Graduate University has concluded that its main effect is to curb the instincts of wariness and suspicion that cause anxiety. “It is a hormone that facilitates social contact between people,” argues Zak.

Data collected from the Marshall Protocol study site, as well as information garnered from numerous other clinical studies strongly suggests that people with Th1 disease are often more prone to developing phobias, OCD-like tendencies, and anxiety in general. Of course, there’s a fine line separating such tendencies from the natural feelings that build as a person attempts to deal with the strain of chronic disease. Understandably, chronically ill individuals feel a certain level of depression, anxiety, fear, suspicion, etc. as they try to understand and manage illnesses for which mainstream medicine offers little insight into cause, cure, or means to prevent deterioration.

But as people accumulate an increasing load of the chronic, intraphagocytic metagenomic bacteria that cause inflammatory disease (also called the Th1 pathogens), mental compulsions are directly caused by infection rather than a healthy person’s natural reaction to challenging life events. Many patients on the Marshall Protocol have admitted that they simply didn’t realize the extent of their infection-induced phobias until the symptoms return temporarily as part of the immunopathological response (bacterial die-off reaction). Anxiety, fear, the perception that something is “wrong” when it isn’t, or the feeling that people are conspiring against oneself is often heightened by immunopathology. This suggests that to a large extent, the mental compulsions of people with Th1 disease are not due to circumstance but are instead the result of bacterial infection.

Based on this knowledge, one might hypothesize that levels of oxytocin are often low in patients with chronic disease. And the assumption would be correct. Low levels of oxytocin in the blood have been detected in patients with chronic diseases ranging from sarcoidosis to autism. For example, autistic patients given oxytocin as part of a study in New York found their ability to recognize emotions such as happiness or anger in a person’s tone of voice – reactions that usually elude their mental abilities – improved.

The results of such studies have caused most mainstream researchers to simply write off chronically ill patients as “oxytocin deficient,” with little thought to why the hormone might be low in the first place. But the Marshall Pathogenesis and recent data on the Vitamin D Receptor allow for a reasonable hypothesis that explains why oxytocin is often low in chronic disease.

“The genes that allow for the transcription of the oxytocin receptor are transcribed by the Vitamin D Receptor.”According to researchers at McGill University in Canada, who recently created a database of VDR-target genes, the genes that allow for the transcription of the oxytocin receptor are transcribed by the Vitamin D Receptor. “And very strongly too,” adds biomedical researcher Trevor Marshall.

And there you have it. A small nugget of information allows for a deeper understanding of the pathways that contribute to compulsions and phobias at the molecular level. The Th1 pathogens create substances that dysregulate the Vitamin D Receptor, slowing its activity in the process. The higher a person’s pathogenic load, the more disabled their VDRs become. So as people accumulate the Th1 pathogens, they lose the ability to activate the receptor that would otherwise transcribe the genes necessary for oxytocin production. As transcription of the hormone slows, patients suffer the negative consequences, including an inability to effectively control paranoia, fear, social awkwardness, feelings of exclusion, depression, disconnect with others, and certain manias.

“Another piece of the puzzle falls into place,” Marshall concurs.

In my opinion, such knowledge offers hope when it comes to the future of human relations. Although bacterial load differs greatly from person to person, at the moment, every member of the population harbors at least some of the Th1 pathogens. And in countries where vitamin D supplementation is the norm, nearly everyone has a level of 25-D that is too high. Since elevated 25-D and bacterial ligands block the VDR and subsequently the production of the genes it transcribes, it’s quite possible that nearly everyone in the world currently has lower than normal oxytocin levels. Does that mean that all of us, to different degrees, are a little more anxious, less trusting, a bit more depressed than we would be if we had fully functioning VDRs?

If this is the case, large-scale restoration of VDR homeostasis might allow for a world more willing to compromise and put long held disputes to rest. The possibility is supported by a recent study in which scientists gave doses of oxytocin and a placebo to participants, who were then asked to decide how to split a sum of cash with a stranger. Those given oxytocin offered 80 per cent more money than those given a placebo. And previous research into the hormone by Professor Zak suggests that generous people had higher than average levels of oxytocin in the brain, while “mean-spirited people” have lower than normal levels.

The knowledge that oxytocin levels are regulated by the VDR is an example of knowledge that should allow doctors and researchers to draw a much more accurate line between personality traits and mental tendencies caused by disease. Take extreme shyness. Is extreme shyness simply a result of personality or might it also have an infectious component? In recent trials, researchers at Zurich University in Switzerland managed to ease symptoms of extreme shyness in 120 patients by giving them oxytocin treatment half an hour before they encountered an awkward situation. Such results can only be explained by changes in chemical balance rather than changes in personality, suggesting that, like other chronic mental issues, extreme shyness could result at least partly from infection. It’s of interest that a recent poll found that sixty percent of Britons say they have suffered from shyness, and one in 10 say it impedes their daily life. That’s quite a high prevalence of shyness, suggesting that like almost all other inflammatory diseases, shyness might be on the rise due to vitamin D supplementation, the increased use of immunosuppressant drugs, our sun-loving culture and other circumstances that compromise the integrity of the immune system.

Graeme Obree: deficient in oxytocin?

One could argue that in a world where compulsions can finally be treated, there may be circumstances in which a person would be upset if a mental trait they come to value disappears during recovery. Perhaps a person’s OCD-like tendencies allow them to train beyond the scope of others in order to win an athletic event. If such a person eliminates the pathogenic load in their head, will they still be able to train and compete with such fanaticism? Many would argue that the famous cyclist Graeme Obree, who set the hour record in cycling, was ironically driven to excellence by focusing on memories inspired by his severe depression. There may also be artists whose works are fueled by their phobias. If the MP had existed in the nineteenth century, Van Gogh might very well have become just another artist painting sanguine pictures. Yet when it comes to the majority of people struggling with social anxiety or battling mental compulsions, the ability of the MP to quell a variety of phobias is almost certainly appealing and liberating.

So far I’ve discussed scenarios in which, thanks to VDR activation, oxytocin is naturally able to return to a normal level – a process directed by the body’s own homeostatic mechanisms. Yet, as communicated in a recent article on oxytocin in the Evening Standard‘s website This is London, most researchers seem to have little interest keeping oxytocin levels in the range that would be maintained by a healthy body left to its own ways. Instead, driven by the standard “the more the better” mindset, they seem motivated by the prospects of high-dose oxytocin supplementation. It seems that just like the “experts” who weigh in on vitamin D supplementation, oxytocin proponents are giving little, if any, thought to the possible negative consequences of dousing ourselves with a hormone that under natural circumstances is tightly regulated by the body.

An oxytocin spray has just been successfully trialled at the University of New South Wales and experiments by Dr Eric Hollander at the city’s Mount Sinai School of Medicine found a single intravenous infusion of the chemical triggered improvements that lasted for two weeks. These studies, along with Zak’s work has researchers in the US, Europe and Australia racing to develop commercial forms of the hormone, including a nasal spray. Driven, no doubt in part by the monetary prospects involved in creating oxytocin-containing compounds, such scientists believe that oxytocin can be turned into a “wonder drug” capable of treating a range of personality disorders such as autism, depression and anxiety.

A wonder drug? Wow! I think I just heard the local newscaster regurgitate information about another so-called wonder drug… vitamin D! And whereas vitamin D is fondly coddled as the “sunshine vitamin,” oxytocin will soon be enthusiastically promoted as the “love drug.”

Most scientists seem to have few qualms about high-level oxytocin supplementation.

The comparison would be lovely, if only the view of vitamin D as a helpful “sunshine vitamin” were actually correct. Unfortunately, the majority of vitamin D “experts” have failed to realize that the palliation offered by vitamin D stems only from its ability to slow the innate immune response. So they are content to extol the virtues of the secosteroid’s short-term palliative effects with little regard to the long term immunosuppression – and subsequent rise in bacterial load – it causes when taken in excess.

Zak contends that oxytocin, “is a very safe product that does not have any side effects and is not addictive.” Quite frankly though, how can he pretend to know such information? Clinical studies alone can never reveal the mechanisms by which a compound actually works inside the body and generally do not detect long-term side effects. For example, based on the first generation of clinical and epidemiological studies alone, vitamin D supplementation seemed like an excellent idea. It wasn’t until Marshall used molecular modeling software to mathematically calculate the affinities of the various forms of the secosteroid/hormone for their target receptors that the deleterious effects of 25-D on innate immune function became apparent. So until a researcher succeeds in modelling the effects of the hormone at the molecular level, and proceeds to create a model of the metabolic pathways that regulate its production, it’s madness to think that supplementation with excess oxytocin can simply be assumed to have no negative effects.

Don’t get me wrong, I see possible uses for oxytocin if used in moderation. But the articles I’ve read on the hormone contain quotes from researchers exercising little, if any, caution. According to the Evening Standard‘s article, “The potential uses of oxytocin offer commercial possibilities well beyond individual patients.” Restaurants, for instance, could spray a thin mist over customers to put them at ease. Since researchers at Emory University in Atlanta recently released the results of a study which suggests that oxytocin made rodents more faithful to their partners, some believe that extra levels of oxytocin could be used to prevent extramarital affairs. Others have proposed that oxytocin supplementation could serve as a treatment for alcoholism. Still others argue that oxytocin could be used as a benign form of tear gas, quelling any violent feelings among groups of demonstrators. While it’s plausible that oxytocin spray might succeed in placating a rowdy crowd, does the government really want to disrupt the homeostatic hormonal feedback pathways of large groups of people? And remember the study mentioned above where people given oxytocin gave 80% more money to a stranger? While such generosity may be helpful under some circumstances, it’s possible that if people take too much oxytocin, they may lose their inhibitions to the point where they might give away things they really need. Even some mainstream researchers admit that oxytocin could have potential as a date-rape drug as it is involved in both trust and sexual arousal.

So how about returning to the first scenario discussed in this piece – the scenario in which the Marshall Protocol can be used to restore oxytocin levels to those set by nature. Not that a few extra puffs of oxytocin couldn’t be used under special circumstances, but like vitamin D, it seems to me that hormonal pathways function optimally when left alone.

Yet, I must share this parting thought: could oxytocin spray, if used very carefully, be used to counteract the symptoms of brain immunopathology during difficult times on the MP? While the goal of every MP patient is to allow their hormones to rebalance naturally, those patients who suffer from severe mental infection might be able to use oxytocin in moderation in order to relieve intolerable symptoms. One thing’s clear, when it comes to oxytocin, there remain more questions than answers.

Many people go out of there way to buy probiotics, which can be purchased in myriad forms.

There’s been some discussion on the Marshall Protocol study site about how probiotics, or bacteria that are believed to beneficially improve bacterial composition in the gut, may be palliating symptoms but not improving overall health. This probably seems ludicrous to people who go out of their way to buy yogurt with “friendly” bacteria such as acidophilis, or people who dig into their savings to buy probiotics in numerous forms including little silver pearls.

And yet consider this hypothesis. Whereas it used to be believed that the adaptive immune system dictated the immune response in the gut, recent research has made it clear that the innate immune system – which is active inside the villi of the intestines and stomach – is actually largely responsible for keeping gut bacteria in check.

The innate immune system mounts a response to every pathogen that enters the body, whether “friendly” or harmful.

So when bacteria enter the body – whether described as “friendly” or pathogenic – the innate immune system mounts a response to their presence. That’s its job. It’s as if the innate immune system is a bouncer at a club who must check the ID of each person who wishes to enter.

The response or challenge that the innate immune system mounts towards every bacterium entering the GI track causes the production of inflammatory cytokines and chemokines. According to Marshall, part of this inflammatory response may cause a migration of white blood cells called monocytes from nearby organs of the body, or other parts of the gut, to the area of the GI track where they can face incoming probiotics.

Think about how this response might affect the liver, the pancreas, the kidneys, or even different areas of the gut. Since monocytes engulf bacteria and play an important role in killing the chronic intraphagocytic bacteria that cause inflammatory disease (collectively called the Th1 pathogens), if the white blood cells leave these organs in order to face probiotic bacteria entering the gut, the rate of bacterial die-off in these nearby organs should tone down or subside.

Monocytes may migrate from other organs near the gut in order to face incoming probiotics.

Since we now understand that it is the death of the Th1 pathogens that cause the bulk of a person’s symptoms (inflammatory cytokines and toxins are released when they die), the result of this reduced bacterial die-off should generate a feeling of temporary wellness in these other organs, organs that also affect digestion and detoxfication.

In the same sense, if a patient’s immune system is working to target a pocket of bacteria in one area of the gut, new probiotic bacteria may divert its attention from that area of infection to a different area of the gut, diminishing bacterial die-off in the original area.

Besides slowing the body’s response to bacterial death (known as immunopathology) in organs near the gut or areas of the gut itself, the above scenario unfortunately places an extra load on the innate immune system, which is the same branch of the immune system that works to try to keep the Th1 pathogens under control.

As previously discussed, in cases where patients are infected with Th1 pathogens, the innate immune system is constantly working hard in an attempt to kill them. But because the system must work to mount an inflammatory response each time probiotics enter the body, the ingestion of probiotic bacteria gives the already over-worked system another task, which at the same time diverts its attention away from killing the Th1 pathogens.

If the innate immune system is forced to manage incoming probiotics, then the rate of bacterial die-off in organs near the gut, or other areas of the gut, may slow.

Even in people considered healthy, overloading the innate immune system is still a problem, since if its focus is diverted to dealing with probiotics, it may be less effective at targeting other pathogens that enter the body. Also, since people often begin to harbor the Th1 pathogens well before becoming symptomatic, any feelings of “wellness” they experience after taking probiotics may be due to the same mechanisms described above.

“We now know that the GI tract relies on the innate (Th1) immune system, and the VDR, to deal with intestinal flora,” states Marshall. “A decade ago it was thought that antibodies (the Th2 adaptive immune system) were involved. So it is certain that ingesting probiotics will place a load on the very part of the immune system already weakened by fighting Th1 inflammation in the major organs. Whether this is good or bad is open to interpretation.”

According to chronic disease physician Dr. Greg Blaney, concentrated probiotics, especially if they contain the artificial sugar Fructooligosaccharides (FOS), are most likely to affect the GI tract and surrounding organs in the manner described above. New probiotic blends with extra ingredients added may also be particularly unhelpful.

Indeed, a recent study by researchers at the University of Newcastle in Australia found that treatment with probiotics doubles levels of the inflammatory cytokine Interferon-gamma, confirming that the bacteria do create a Th1 inflammatory reaction upon their entry into the gut.

“ We now know that the GI tract relies on the innate (Th1) immune system, and the VDR, to deal with intestinal flora.”Interferon-gamma also catalyses (by the action of the enzyme CYP27B1) the production of the 1,25-D – the active form of vitamin D that functions as both a hormone and a cytokine. Since the Th1 pathogens create ligands that block the VDR and subsequently dysregulate the pathway that controls CYP27B1, this implies that healthy people might react differently to probiotics than people with Th1 disease, or at least be more negatively impacted by their ability to produce an inflammatory response.

This is because when enzymes such as CYP27B1 are dysregulated by VDR blockage, the body is unable to keep 1,25-D levels in the correct range. As the hormone starts to rise to unnaturally high levels, it binds many of the nuclear receptors – including the glucocorticoid receptor, the alpha/beta thyroid receptors, and the adrenal receptors – displacing the metabolites that are meant to be in the receptors under normal conditions. This upsets the balance of several critical hormonal feedback pathways.

Here at Bacteriality our favorite hormone to scrutinize goes by the name of 1,25-D. So, you can understand our disappointment when we relate that, yet another group of researchers, these from U. of Newcastle, didn’t measure the 1,25-D levels of their subjects. It would have been quite interesting to note if the levels of the hormone/cytokine was higher among those subjects taking probiotics.

The above hypothesis would explain why Dutch researchers recently published a study in the Lancet which found that among patients with predicted acute pancreatitis, more than twice as many patients given probiotic supplements to prevent infection died compared to those who received placebos.

The Lancet study found that among patients with predicted acute pancreatitis, more than twice as many patients given probiotic supplements to prevent infection died compared to those who received placebos.

“The adverse effects of probiotics noted here were unexpected,” Hein Gooszen and colleagues at the University Medical Centre Utrecht in the Netherlands wrote.

In the study of 296 people with similarly acute forms of pancreatitis, one group received a placebo and the other a mixture of probiotic supplements, some commonly available (the probiotics were administered via the mouth by a tube headed directly into the bowels). The number of people who developed infections was similar, but 24 volunteers died in the probiotic group compared to nine in the placebo group. 8 of the subjects in the group given probiotics died from bowel ischaemia, while the others succumbed to pancreatitis.

One must admit that a logical explanation for the deaths described above could be that in the case of the patients given probiotics, macrophages were diverted from areas of the bowel or pancreas where they were striving desperately to fight infection (let’s assume here that bowel ischaemia and pancreatitis are bacterial diseases). Furthermore, when the probiotics reached the GI tract, they may have put such a load on the patients’ innate immune systems that it was otherwise unable to keep their disease states under control. This study at least causes one to raise an eyebrow about the possibility that an alternate hypothesis for probiotics isn’t far fetched.

One must also consider that other explanations for how probiotics improve health remain largely speculative. For the most part, probiotics are assumed to be helpful because they offer some people temporary symptomatic relief – but as high levels of vitamin D or corticosteroids demonstrate, palliation does not always indicate improvement.

Elie Metchnikoff

The first researcher to hypothesize that certain bacteria might play a positive role in the gut was Russian scientist and Nobel laureate Elie Metchnikoff, who, in the beginning of the 20th century, suggested that it might be possible to replace harmful microbes in the gut with useful microbes. He hypothesized that bacteria such as clostridia, which are part of the normal gut flora, produce toxic substances from the digestion of proteins. He believed these compounds were responsible for what he called “intestinal auto-intoxication”, which he linked to physical changes associated with old age.

This led him to propose that milk fermented with lactic-acid bacteria could inhibit the growth of these “toxic” bacteria because of the low pH produced by the fermentation of lactose. Soon, he introduced the idea that a diet high in sour milk fermented with the bacteria he called “Bulgarian Bacillus” could improve digestive health. Friends in Paris soon followed his example and physicians began prescribing the sour milk diet for their patients.

But in 1920, Rettger demonstrated that Metchnikoff’s “Bulgarian Bacillus”, later called Lactobacillus bulgaricus, is actually unable to live in the human intestine. Naturally, the fermented food hypothesis petered out.

After that point, research on probiotics focused on the idea that scientists could isolate bacteria that seemed to be involved in a positive process and add extra amounts to the gut, with the hope of displacing other less desirable pathogens.

For example, Henry Tissier, a researcher at the Pasteur Institute, isolated a bacterium from breast-fed infants and named it Bacillus bifidus communis. He recommended that doctors give bifidus communis to babies suffering from diarrhea in the hopes that it would displace other species that might be causing the problem in the first place. Yet benefit from the treatment remained dubious.

The benefits of giving Bacillus bifidus communis to infants remains dubious.

Later, it was reasoned that “helpful” bacteria should be isolated directly from the gut, and in 1935, certain strains of Lactobacillus acidophilus were found to be very active when implanted in the human digestive tract. Trials were carried out using this organism, and encouraging results were obtained, especially in the relief of chronic constipation. Yet since the liver, kidneys, and parts of the gut not affected by probiotics are involved in the constipation process, one could argue that such benefits are also explained by Marshall’s hypothesis.

In these and other cases, one must question – how can we assume that bacterial species such as those isolated from breast-fed infants or from the digestive tract are necessarily “good”? Can we simply assume that a bacterial species is beneficial because it appears at face value not to be causing any harm?

It’s true that certain bacterial species are competitive, meaning that one species may be able to kill another. Take, for example, Streptococcus, which has been shown to effectively kill Staphylococcus bacteria. In fact, the antibiotic demeclocycline, which was derived from a strain of Streptococcus bacteria, is particularly effective at quelling Staphylococcus infections.

Yet do the bacterial species in common probiotic products possess such competitive properties? To date, there has simply been no evidence or laboratory studies showing that they do. Plus, if probiotic strains are indeed killing other more virulent pathogens, wouldn’t the death of such strains cause a rise in immunopathology rather than a feeling of relief?

“Can we simply assume that a bacterial species is beneficial because it appears at face value not to be causing any harm?”Another reality that probiotic enthusiasists often fail to consider is how horizontal gene transfer affects probiotic bacteria in the gut. Horizontal gene transfer is a process in which organisms swap genetic material by trading plasmids, or circular molecules of DNA that can replicate independently of a pathogen’s other genetic material.

This means that even if a species of bacteria considered to be “helpful” enters the gut, it can easily trade plasmids with other disease causing pathogens – quickly changing it from a potentially harmless organism to yet another pathogen contributing to disease.

This may be especially true for people with high loads of Th1 bacteria in the gut that can all too easily swap their genetic material with probiotic bacteria, rendering them part of the disease process rather than the “cure.”

Horizontal gene transfer allows bacteria to share genetic material

“Increasingly, studies of genes and genomes are indicating that considerable horizontal gene transfer has occurred between bacteria,” states James Lake of the Molecular Biology Institute at the University of California. In fact, due to increasing evidence suggesting the importance of the phenomenon in organisms that cause disease, molecular biologists such as Peter Gogarten at the University of Connecticut have described horizontal gene transfer as “a new paradigm for biology.”

Gorgarten insists that horizontal gene transfer is “more frequent than most biologists could even imagine a decade ago.” In the face of such statements, we may want to reconsider ingesting large loads of extra bacteria that inevitably become part of a pool of pathogens trading genetic material when they actually enter the body.

Indeed, one of the largest meta-analysis studies on probiotics, published in the American Journal of Clinical Nutrition by researchers at the Wageningen Centre for Food Sciences in the Netherlands, reviewed 49 studies on probiotics with lackluster results. 26 of the studies dealt with the prevention or treatment of diarrheal disease, 9 with the prevention of cancer or of the formation of carcinogens, 7 with the lowering of serum cholesterol, and 7 with the stimulation of the immune system. The most widely studied probiotic bacteria were Lactobacillus GG (22 studies), Lactobacillus acidophilus (16 studies), Bifidobacterium bifidum (6 studies), and Enterococcus faecium (7 studies). The team concluded that intake of Lactobacillus GG did shorten the diarrheal phase of rotavirus infection, but that “evidence for the prevention by Lactobacillus GG and other probiotics of diarrhea due to viral or bacterial infections was less strong.” The effects of probiotics on the immune system were “inconclusive” because of the variety of outcome variables reported.

The largest meta-analysis on probiotics reviewed 49 studies on the bacteria with lackluster results.

The team also reported that cholesterol-lowering abilities of probiotics “seem to be transient”, and found that production of mutagens after a meal might be reduced by intake of probiotics, but the relevance of the finding “was unclear”. The study finally concluded that while probiotics may have some effect on rotavirus infection, “other health effects of probiotic bacteria have not been well established.”

A 2005 study by the Food Standards Agency on 11 different types of probiotic bacteria attempted to determine where the pathogens break down as they pass through the digestive system. While the researchers were able to determine that most strains of probiotics survive past entry into the stomach, the data failed to show “if or where probiotics might have an effect,” meaning that mainstream researchers aren’t even sure where probiotics take effect, let alone what they do when they get to their target destination.

Despite doubt cast on the benefits of probiotics, it’s doubtful that Marshall’s hypothesis will gain any credence in the near future. There’s simply too much money at stake, and according to the Associated Press, “the market is ahead of the science. It’s all part of a burgeoning effort to capitalize on an obsession with health foods.” Over 150 food products that have probiotics have been introduced in the market this year – compared to about 100 last year and just 40 the year before that.

In fact, probiotics are a multibillion-dollar global industry. In the United States alone, retail sales of probiotic-containing foods and supplements totaled an estimated $764 million in 2005 and are projected to reach $1 billion in 2010, according to market research firm BCC Research.

Dannon’s Activia yogurt, introduced last year, is among the best known U.S. products. Its first-year U.S. sales totaled more than $100 million. General Mills introduced its competitor, Yo-Plus, under the Yoplait yogurt brand this year.

Other 2007 products include: Kraft Foods Inc.’s LiveActive prebiotic cottage cheese and probiotic cheddar cheese; Nestle’s probiotic Good Start Natural Cultures baby formula; Beech-Nut Nutrition Corp.’s Good Evening prebiotic baby food; and the Swiss firm Barry Callebaut’s probiotic chocolate.

Probiotics have been added to a vast array of products

Probiotic manufacturer Nutraceutics just targeted $100 million in probiotic sales. Meanwhile, New Zealand oral probiotics developer BLIS Technologies is planning to issue new shares to fund an expansion of its business into new international markets in order to boost development efforts that will benefit shareholders if the products take off.

A continent away, dietary supplements with Probi’s healthy bacteria Lp299v will be launched in China in conjunction with the leading domestic health food company Biostime Inc. The list goes on as probiotics continue to be added to a mind-blowing number of new foods and products. As with vitamin D and most other supplements available in dietary form, the mistaken notion that “more is always better” seems to reign supreme.

As stated in a recent article on probiotics in Time Magazine, “Whether or not you’ve ever developed a taste – or even a tolerance – for living things in your lunch, more are on the way. Food companies have been coming to the conclusion that if a few of these superstar bacteria are good for you, then more will be even better.”

Also unnerving is that the FDA hasn’t set any upper limit for probiotic consumption, largely because nobody really knows exactly what they do upon entering the body, so recommending a “desirable” dose is impossible. This means that a person can guzzle tremendous loads of probiotic supplements without ever consulting the advice of a doctor.

When it comes to probiotics, the mistaken notion that “more is always better” seems to reign supreme.

At least, according to Time, the U.S. Food and Drug Administration is “relatively neutral, using the growing popularity of probiotics to caution manufacturers not to pitch the foods as some sort of panacea for any specific disease.” Whether food companies actually follow such advice remains to be seen.

The NIH has declared the study of gastrointestinal bacteria and probiotics a major research initiative. “The fact that there are a number of health implications and a lack of understanding associated with the use of probiotics makes this a very interesting subject to study,” said Crystal McDade-Ngutter, who heads an NIH working group on the topic.

The skeptic in me can’t help thinking that a lot of companies are making a pretty penny off simple palliation. At the very least, the fact that probiotics possess the ability to modulate where and when the innate immune system is activated should give one pause. In the meantime, Marshall prefers not to give advice about probiotics.

“I haven’t stated a position on probiotics,” states Marshall. “There are many on the [Marshall] protocol who are convinced they are helpful, and I would prefer to concentrate on the key issues that folk really need to solve – no vitamin D, plenty of Benicar, and a supportive family/medical environment. Probiotics are a second-order effect, I think (less important).”

“So I tend to leave it to the individual. Most Th1 patients have severe GI tract involvement, and dealing with that takes just about every tool in their arsenal. If probiotics seem to help, then who am I to say no? On the other hand, they do not form part of the base protocol, as any benefit is not obvious to me, whether based on personal experience, or biological knowledge.”

The protein structure of a prion.

Prions have been implicated as the cause of a number of diseases in a variety of mammals, including bovine spongiform encephalopathy (BSE, also known as “mad cow disease”) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. All thus-far hypothesized prion diseases affect the structure of the brain or other neural tissue, and all are considered untreatable or fatal by mainstream medicine.

Although prions have been studied to some extent in the lab for decades, very little research has delved into their actions when inside the human body (in vivo). Thus, many theories put forth about how prions might cause or contribute to neurological disease have been largely speculative.

That is until Aguzzi’s work. At DMM, he presented a series of excellent experiments that studied the actions of prions inside the tissues of animals or human beings. His data confirms that after prions enter the body, they are able to pass through essentially all of the body’s barriers such as the mucosal barrier and the blood brain barrier. They can also bypass both the innate and adaptive immune system. This means that, if a person or animal consumes food containing prions, the small protein molecules can easily pass from the gut all the way up to the brain. As Aguzzi describes, the timing at which prions make their way from the gut to the brain is incredibly precise. For example, when his team exposed a large group of mice to prions in their food, the prions reached the brain in 220 days (plus or minus 3 days) in every single one of the rodents studied.

Years ago, Aguzzi was puzzled by the fact that, if his team infected the lymphatic systems of healthy mice with prions, the prions did not migrate from the lymph system to the brain, meaning that the mice remained healthy. However, when the team wiped out the ability of the mice to secrete immune cells called activated B-lymphocytes, prions in the lymph system were suddenly able to migrate to the brain and cause disease.

Dr. Adriano Aguzzi

This information percolated in Aguzzi’s head, and it was not until decades later that he suddenly realized that, in many infectious states, B-lymphocytes migrate away from the lymph system in order to deal with pathogens in other organs. Under these conditions, so many B-lymphocytes migrate away from the lymph system that the system resembles that of the mice whose B-lymphocytes had been completely knocked out. In somewhat of a “eureka moment,” he realized that such B cell migration is a telltale sign that the host is suffering from a chronic inflammatory disease.

The implications of this connection? It is now accepted that prions are not infectious by themselves, but are only infectious in the presence of chronic inflammation.

Since it is now increasingly understood that chronic inflammatory diseases are the result of infection with an intraphagocytic, metgenomic microbiota (L-form, biofilm, and other persistent bacteria, collectively called the Th1 pathogens), Aguzzi’s work strongly implies that it is only when prions infect a person or animal that harbors the Th1 pathogens that they become effective infectious agents.

Such thinking contradicts previous assumptions about prions, in which the small protein molecules were considered capable of causing infection on their own. Whereas, before Aguzzi’s work, it was simply assumed that prions could fold into tightly packed beta sheets (in which their polymers are connected by hydrogen bonds) on their own, it is now understood that chronic inflammation must be present if such folding is to occur and lead to disease. The altered structure of a folded prion is extremely stable and accumulates in infected tissue, causing cell death and tissue damage. Such stability means that prions are resistant to denaturation caused by chemical and physical agents, making disposal and containment of the particles very difficult.

Indeed, this new view on prions was confirmed by a study in which Aguzzi’s team induced chronic hepatitis in mice. The disease caused the animals’ livers to become inflamed. The mice were subsequently fed prions, and when the rodents’ organs were dissected after death, the team found that the prions had spread directly from the gut to the inflamed tissue in the liver. When the same experiment was performed on a group of healthy mice without hepatitis, no prions were found in the rodent livers after death.

Marshall has several theories about how the Th1 pathogens might interact with prions in order to facilitate their ability to cause disease. Perhaps the Th1 pathogens transcribe enzymes which can actively fold prions into the specific shapes in which they become infectious. Or perhaps proteins, peptides, or lipids from the Th1 pathogens transform human enzymes or proteins into forms which tend to fold the prions and allow them to damage tissues. Nobody knows for sure at this point.

This has led Marshall to believe that prions are just one of the artifacts produced by the [Th1] pathogens. “Something has to make them change shape in the first place, even if they ‘snap shut’ after that. Prions may well propagate the damage being done by the Th1 pathogens more quickly, especially if they are injected, ingested, etc. but, unless the Th1 pathogens are there, the prions are not infectious, and do not spread to the brain,” he states.

A number of other experiments conducted by Aguzzi support the hypothesis that prions become pathogenic only after interaction with the Th1 pathogens. Recently Aguzzi’s team travelled to Slovenia in order to research the effects of prions on sheep, animals that can develop a prion-induced disease called scrapie.

Analysis of frontal cortex samples from the brain of a patient who died of non-cerebral causes (upper row); patient suffering from Creutzfeldt-Jakob Disease, CJD (lower row). Prion protein deposits are visible in the samples of the patient with CJD. Photo from Nature Reviews Neuroscience by Aguzzi.

Aguzzi proceeded to separate sheep into two groups. One group had a chronic viral inflammatory condition called mastitis, while the other group did not. When the milk from both groups of sheep was examined, prions were only secreted in the milk of those sheep who had mastitis. In these cases, macrophages were also secreted in the milk, some or all of which were certainly infected by the Th1 pathogens. This solidified the hypothesis that inflammation of the mammary glands (which occurs in mastitis) is necessary if prions are to infect the mammary glands and end up in an animal’s milk.

A second study by the team examined the milk content of healthy cows that had been infected with the BSE prions that cause mad cow disease. Since the cows were healthy and did not suffer from any inflammatory conditions (they had been kept in what Aguzzi describes as “five star hotels for cows”), the BSE prions were not found in the milk of healthy cows, nor did the cows actually develop mad cow disease.

Results of the above studies were confirmed by yet another experiment that tested the urine content of mice for prions. As Aguzzi describes humorously, several researchers on his team spent two years of their lives collecting rodent urine samples. Some of the rodents were made to suffer from chronic kidney inflammation called nephritis. When prions were introduced into the inflamed kidneys of these mice, they were excreted in the urine. But if prions were introduced into rodents that did not suffer from nephritis, the animals’ urine remained prion-free.

Further research by the team showed that, if inflammation is induced in any excretory organ of the body, prions are excreted in whatever substance the organ excretes.

But perhaps the most exciting aspect of Aguzzi’s research is the fact that his team has developed a florescent stain, called a Luminescent Conjgated Polymers (LCP) stain, that is able to illuminate the polymers created by inflammation. According to Marshall, this stain may be capable of identifying not just prions, but also the protein biofilms (made of protein polymers) that protect the Th1 pathogens in the cytoplasm of infected cells.

Because LCP is made of flexible polymers itself, when it binds bacterial polymers of different shapes, it emits different wavelengths of light depending on the geometry of the polymer under study. Thus, scientists can learn to associate different color wavelengths with bacterial polymers of certain shapes and sizes. For example, during his presentation, Aguzzi shows a slide in which a protein polymer stained with LCP is emitting two different colors. Because of the color difference, he hypothesizes that each end of the protein has a different structure.

The color of the wavelengths emitted by the LCP stain also change in response to the strength between the bonds of certain molecules, or the pH of a a particular environment. The varied spectrums of light emitted by the LCP stain in response to a certain protein or bacterial polymer can also be conveniently compressed into a chart that effectively represents the polymer’s shape and properties.

Indeed, thanks to the stain, Aguzzi presented two slides that Marshall believes show the Th1 pathogens inside various cells. One slide shows the protein polymers indicative of the Th1 pathogens inside the cells of patients with Parkinson’s disease. Another stain reveals amyloid protein in the heart. Amyloid proteins are insoluble fibrous proteins that, according to Marshall, are created by the Th1 pathogens.

If further research proves that Aguzzi’s stain is indeed able to reveal the biofilm surrounding the Th1 pathogens, the stain may allow Autoimmunity Research Foundation to conduct a study that could definitively show the presence of the Th1 pathogens in the blood of people with Th1 disease, as well as the absence of the Th1 pathogens in the blood of patients who complete the Marshall Protocol.

“[Aguzzi's stain] can use spectra to distinguish polymers. This may well be the diagnostic (screening) tool we have been looking for,” states Marshall.

Cellular inclusions in Parkinson’s Disease, which look quite similar to the Th1 pathogens when viewed under phase contrast microscopy.

Essentially, the stain could be applied to a sample of blood. Th1 bacterial proteins would show up as bright spots. By measuring the amount of flourescence given off by the blood, the extent of bacterial load could be estimated. For example, a particular number of photons could be correlated with X number of bacteria. It’s very likely that such staining would reveal that even the blood of people considered to be healthy harbors a certain number of Th1 pathogens, and that nobody is spared from the effects of these persistent bacterial forms.

Clearly, if the flourescent stain used by Aguzzi’s team can identify the Th1 pathogens, the implications of such a discovery are far-reaching. However, the fact that prions cannot cause infection on their own, but only in the presence of inflammation, also offers great hope for the elimination of diseases caused by prions.

If the Marshall Protocol is used to wear away at a host’s Th1 bacterial load, then the person or animal should reach a point at which the Th1 pathogens can no longer facilitate the folding of prions into infectious agents. So by effectively reducing bacteria-induced inflammation, the MP may make render prions harmless.

Does that mean the MP might be able to prevent mad cow disease? Perhaps it’s possible that if the MP were ever adapted to treat animals, it could stop the accumulation of infectious agents that would foster chronic inflammation and the spread of prions.