Presentation - Antibodies and infection in the era of metagenome

Type: Conference presentation
Presenter: Amy Proal
Conference: 1st International Congress of Antibodies
Location: Beijing, China
Date: May 22-24, 2009


So far at this conference we've heard some amazing work which shows how individual pathogens cause human disease. But what I'm going to talk about are pathogens that have gone undetected for a century, bacteria which exist in and on the human body but which evade culture. We're starting to realize that the genomes of some of these bacteria may well have pathogenic activity. A recent initiative, the NIH Human MicrobiomeThe bacterial community in the human body. Many species in the microbiota contribute to the development of chronic disease. Project seeks to characterize all the genomes in the body other than the human genome.

Researchers running the initiative estimate that 90% of cells in the human body are bacterial, fungal or otherwise non-human in origin while a mere 10% of cells in Homo Sapiens are truly human. In fact, the human microbiotaThe bacterial community in the human body. Many species in the microbiota contribute to the development of chronic disease. is so prolific that even as Human Microbiome Researchers and other scientists work as fast as possible to characterize it, to date only a small fraction of the microbiotaThe bacterial community which causes chronic diseases - one which almost certainly includes multiple species and bacterial forms. has been named and characterized.

It follows that humans are best viewed as superorganisms. Due to their small size, hundreds, or even thousands, of bacterial cells can fit inside a human cell. The combined genetic contributions of these microbes inevitably provide myriad gene products not encoded by our own relatively small genomes. In reality, the organism we call Homo sapiens is controlled by a metagenome, a tremendous number of different genomes working in parallel. So the number of genes expressed by our microbial inhabitants numbers in the millions, or possibly billions, while the human genome contains only approximately 30,000 genes. Yet it was only a decade ago that most scientists believed that all bacteria important to man had already been identified. Nowadays, thanks to shotgun and pyrosequencing we're finding DNA from hundreds and even thousands of bacterial species in tissue and blood. Where do they come from and what relevance do they have to disease? This new technology has opened a door to a era of discovery, what some call the era of the metagenome. Whereas scientists previously had to rely on culturing bacteria we can now use software to identify them. They can now be characterized based on their genetic fingerprints. And by studying the genes in their genome, we can figure out how they cause disease processes.

For example, this slide represents the bacteria recently determined to persist in saliva by researchers at the Max Planck Institute. They used 16RNA sequencing to identify the bacterial populations of healthy subjects living in twelve countries scattered over the globe. They identified 101 bacterial genera in the mouth as well as an additional 64 genera previously unknown to science. These results also show a greater diversity in the salivary microbiome within and between individuals then anybody has realized. Just to make things clear, the greater the size of the font the higher the prevalence of the bacteria detected in the study. (Notice Yersinia up there, a pathogen that no healthy person would expect to have in their mouth.) But particularly interesting is that some of these same bacteria can be found elsewhere inside the body. For example, Porphyromonas gingivalis and A. Actinomyces, both of which we know cause tooth decay, have now been identified in artherosclerotic plaque. And that's what I'm going to be talking about. I'm going to be talking about how this microbiota may cause chronic disease and even the antibodies we call autoantibodies.

When it comes to the human microbiota, medicine is now comfortable that bacterial populations exist in the gut and areas of the body in contact with the external environment, such as the mouth, ears, nose and skin. In fact, just a month ago, the onset of type 1 diabetes was tied to the presence of specific bacteria in the murine gut. Yet, now we're finding microbes persist in many other body tissues - joints, blood vessels and in the many tissues that become inflamed in autoimmune disease. Recently, 18 different bacterial taxa were detected in the amniotic fluid, which was previously believed to be completely sterile. And over the past century, chronic bacteria have been repeatedly detected in diseases ranging from sarcoidosis to Alzheimers and yet causality could not be demonstrated. But the number of studies associating bacterial genomes with autoimmuneA condition or disease thought to arise from an overactive immune response of the body against substances and tissues normally present in the body and inflammatory disease states are increasing at a rapid pace.

This chart shows the bacterial species detected when scientists at the University of Glasgow sequenced the bacteria in biofilm A structured community of microorganisms encapsulated within a self-developed protective matrix and living together. removed from prosthetic hip joints during revision arthroplasties - joints that were removed from a body compartment thought to be sterile. But clearly the areas in which these prosthetic hips were located are far from sterile. Note that the prevalence of hydrothermal vent eubacteria - which were previously thought to persist only in the depths of the ocean - is higher than the prevalence of staphylococus aureus, a common biofilm species.

Components of such a metagenomic microbiotaThe community of bacterial pathogens including those in an intracellular and biofilm state which cause chronic disease. can persist in the cytoplasm of the very cells of the immune system that are supposed to kill them, in biofilm communities in which they are protected from the immune response by a self-created polymeric matrix. I'm about to show you a clip of bacteria in the blood of a patient with Chronic Fatigue Syndrome, taken by Dr. Andy Wright of Manchester, UK. It is fresh blood that has been aged for around 6 hours at the time of the video. Notice the heavily infected cytoplasm of this cell, which appears to be exploding from the pressure. Very long biofilm-like strands protrude from infected cells, allowing bacteria to spread and infect surrounding cells. And of course all of these species living in a biofilm-like environment are continually sharing DNA via horizontal gene transferAny process in which a bacterium inserts genetic material into the genomes of other pathogens or into the genome of its host.. So even though wer'e finding hundreds of genera, the actual species are in fact orders of magnitude greater in number again, because of the diversity cause by such gene transfer.

So how does this microbiota persist?

A fully activated immune response should be capable of clearing pathogens from the body. So it's not surprising that many pathogens have evolved mechanisms that allow them to slow the innate immune responseThe body's first line of defense against intracellular and other pathogens. According to the Marshall Pathogenesis the innate immune system becomes disabled as patients develop chronic disease. in order to enhance their survival. One of the mechanisms to achieve this end is subversion of one of the body's most prolific nuclear receptorsIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affect transcription. - the VDRThe Vitamin D Receptor. A nuclear receptor located throughout the body that plays a key role in the innate immune response..

The TACO gene, when expressed, inhibits mycobacterial entry as well as survival. MTB downregulates the VDR and thus expression of the TACO gene in order to survive. Another nasty pathogen, HIV, has been shown to completely take over the VDR in order to transcribe its own genome. It does this by using the VDR to recognize the LTR promoter region of its reverse DNA. So let's take a look at this VDR….

The VDR expresses at least 913 genes, many connected to autoimmune conditions and cancers. The Receptor also regulates expression of several families of key antimicrobial peptidesBody’s naturally produced broad-spectrum antibacterials which target pathogens., including cathelecidin and the beta-Defensins. These play a vital role in allowing the innate immune system to target intracellular pathogens. Furthermore, it transcribes Toll-like-receptor 2, which recognizes gram positive bacteria.

We have shown that at least one bacterial metabolite produced by biofilm bacteria also slows activity of the VDR.

Here is a molecular emulation of the ligand, the sulphonolipid Capnine, sitting in the VDR binding pocket - obstructing other ligands that would otherwise activate the receptor from docking and initiating gene transcription.n You can tell that Capnine is a stable ligand because even as the VDR receptor changes shape it remains steadily in the binding pocket with a high kd - a kd of 6.83.

Slowing the ability of the VDR to produce the AMPs and TLR2A receptor which is expressed on the surface of certain cells and recognizes native or foreign substances and passes on appropriate signals to the cell and/or the nervous system. is such a logical survival mechanism for any form of pathogen that its almost certain that other microbes besides Mtb and HIV have also evolved ways to dyregulate the VDR or the other receptors involved in controlling the innate immune response.

So it appears that the microbiota responsible for autoimmune disease gradually shuts down the innate immune response over a person's lifetime, as it incrementally accumulates bacteria and other pathogens. Eventually, genes from the accumulating microbial metagenome may trigger a clinical disease symptomology such as one of the autoimmune diagnosis, or simply drive the inflammationThe complex biological response of vascular tissues to harmful stimuli such as pathogens or damaged cells. It is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue. associated with the aches and pains of aging. Indeed, the shift towards an increasingly diverse microbiota over a lifetime directly correlates with an increase in diseases and symptoms associated with inflammation.

In essence, wear and tear aside, as the microbiota accumulates over a lifetime, humans go from this…to this. We have been running a large observational trial for patients with various autoimmune diagnoses that uses a VDR agonistA substance such as olmesartan (Benicar) or 1,25-D which activates the Vitamin D Receptor and transcribes the genes necessary for a proper innate immune response. to restart the VDR nuclear receptorIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affects transcription. and very low dose bacteriostatic antibiotics to help eliminate the microbiota. Developed by Professor Trevor Marshall, the treatment is therefore referred to as Marshall's Protocol or the MP for short.

The goal of the treatment is bacterial death. But when bacteria inside the cells are killed some of the cells die too. This reaction called immunopathologyA temporary increase in disease symptoms experienced by Marshall Protocol patients that results from the release of cytokines and endotoxins as disease-causing bacteria are killed.. Some of you might recognize acute phase immunopathology by the name the Jarisch Herxheimer Reaction. When bacteria are killed, the innate immune system produces a cytokineAny of various protein molecules secreted by cells of the immune system that serve to regulate the immune system. and endotoxin release. The subsequent apoptosis leads to the rise in inflammation we call immunopathology. Additional symptoms often arise as the liver and kidneys deal with the endotoxins. Immunopathology has to be controlled so that patients can have a reasonable quality of life. So it often takes seriously ill patients on the MP several years to gradually eliminate their bacterial loads. But as bacterial load drops, patients incrementally report improvement and ultimately objective markers indicating disease resolution.

While not all patients on the MP have been on the treatment long enough to report symptomatic resolution, nearly all the patients who have started the treatment have experienced immunopathology. The ubiquitous nature of this reaction in study subjects, and its absence in their healthy counterparts, serves as proof positive that the VDR agonist allows patients to kill the microbiota. But what causes the antibodies?

Patient A is a 58 year old female who was diagnosed with RA in 1996. She started the MP in August of 2004. Before starting the MP and during the earlier periods of the treatment she presented with 17 positive ANA titers. In March of 2005 her antibody titers jumped from 1:160 to 1:320. But by August of 2005 they were back down at 1:160 until in November of 2006, when her ANA test results came back negative. Since that time, her ANA levels have been tested eight more times and have remained negative.

Also, as reported via an online progress report, patient A noted a steady decrease in her RA symptoms during and after the same period that her ANA test results became negative. Today her symptoms are mild in comparison to those she experienced pre-MP when her ANA titers were high.

Patient A's test results and reaction to the MP again support the hypothesis that RA and other autoimmune disease result from the accumulation of a pathogenic microbiota. It may be that as a patient with autoimmune disease accumulates pathogens unique to their disease state, the innate immune system - to the extent it can - continually attempts to attack the bacteria present. This causes the release of cytokinesAny of various protein molecules secreted by cells of the immune system that serve to regulate the immune system. and chemokines by the innate immune system that in turn send a signal to the adaptive immune system. The adaptive immune system proceeds to generate antibodies from any scraps of bacterial DNA being generated by phagocytosis.

This calls for a re-evaluation of the 'autoantibody'. Just as the antibodies created in response to CMV, Riketsia, Bartonella and other pathogens serve as a way to infer the presence of infection, what we now consider to be autoantibodies in many autoimmune diagnoses may also indicate the presence of pathogens, but pathogens that have yet to be fully characterized and named.

Here's another case history. Patient B, a 49-year-old female, was diagnosed with Hashimotos Thyroiditis in 2004. In September 2004, before starting the MP, her Thyroglobulin Antibodies were at 200 IU/mL After ten months on the MP, during a time when she reported high levels of immnopathology, her antibody results rose to 217 IU/mL. However after 1 year 11 months on the treatment her immunopathology slowed, indicating that her bacterial load had dropped. At the same time, her antibody levels dropped to 51 UI/mL. As with patient A, her symptoms greatly decreased or resolved during the same period that her antibody levels began to drop. Whereas patient B once required supplemental thyroid medication, her thyroid levels have naturally returned to the normal range.

Another 55-year-old female patient was diagnosed with Hashimoto's thyroiditis in Nov 2004. At the time, her Peroxidase Ab count was >1000. Just after starting the MP in Feb 2006, she reported strong immunopathology and her antibody count shot up to 2000. Today, after several years of reducing her bacterial load, her peroxidase antibody count - as of January 2009 - is just 232. As with patient B, her thyroid levels, which were once high, have returned to the normal range.

Note that the antibody titers of patients A, B and C actually went up from their baseline level during early periods of treatment with the MP. These early periods of treatment corresponded to times when each patient reported high levels of immunopathology, because their innate immune systems were dealing with high levels of bacterial debri. This rise in antibody production in response to increased bacterial death further supports our model, a model which explains how antibodies currently perceived as autoantibodies are actually created in response to dying bacteria.

A shift in the way we understand the production of antibodies in autoimmune disease would, of course, change the way such diseases are treated. If we accept that the antibodies observed in diseases like RA, lupus and others are created in response to bacterial death, then treating patients with corticosteroidsA first-line treatment for a number of diseases. Corticosteroids work by slowing the innate immune response. This provides some patients with temporary symptom palliation but exacerbates the disease over the long-term by allowing chronic pathogens to proliferate. to slow the immune response is counterproductive and is likely to exacerbate the disease process by allowing pathogens to spread. For example, Gotlieb et al. found that 74% of sarcoidosis patients who were administered 20 mg of prednisone relapsed after just one year, a significantly higher portion of the time than patients who were not prescribed prednisone. The team concluded that “Corticosteroids contribute to the prolongation of the disease by delaying resolution” and they found no data to support the contention that corticosteroids alter long term disease progression.

Instead, if the antibodies in autoimmune disease are created largely in response to bacteria, patients should be put on therapies that enhance immune function. At last year's International Congress on Autoimmunity in Portugal Captain Tom Perez, who recently retired after 25 years from the FDA, reported partial results of our study showing that among patients who had currently been on the MP for less than 18 months, 49% reported symptom level improvement all of the time, even during peak levels of immunopathology. And 81% of patients reached a point of significant improvement or symptomatic remission after spending between 18-53 months on the treatment. This chart displays the data in more detail and I'd be happy to discuss it later.

Robert KochAuthor of Koch's postulates, a set of rules for establishing a relationship between a causative microbe and a disease. Koch's belief that only one pathogens causes one disease has now been called into question as multiple postulates are increasingly considered out of date. theorized that one pathogen causes one disease. Instead our data indicates that chronic disease result as people gradually accumulate many, many different forms of bacteria and other pathogens. This explains the overlap observed between the symptoms of different inflammatory diagnoses - to the point where sometimes people are diagnosed with different illnesses depending on the diagnosis methodology. The following wheel shows how truly related chronic diseases are. Each “spoke” represents a published study which has demonstrated a significant statistical relationship between patients suffering from one disease and the next.

Our own data confirms this overlap. Each “spoke” on this wheel shows a comorbidity among a random sample of 50 patients in our trial. That's some serious overlap.

This may explain why we also see so much overlap between antibody production in patients with different autoimmune diagnoses and why such antibodies are often polyspecific. Like the bacteria that cause autoimmune disease, antibody production is clearly not governed by a simple “one antibody, one disease” model either.

The key point to take home from this presentation is to understand that the microbiota can persist by suppressing the innate immune system. My colleagues and I have shown that standard bloodwork can be readily assessed for the VDR dyregulation characteristic of this innate immune dysfunction. It is this chronic phase VDR dyregulation that facilitates opportunistic pathogens such as EBV, Borelia, HHV, Clamydia, and Mtb to proliferate. In addition to looking for antibodies to these opportunistic acute infections, it's also very important to look for antibodies indicating underlying chronic pathogens, the antibodies we may now be mistaking for autoantibodies.


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