A letter to the ME/CFS research community (+ doctors, + patients)

October 18th, 2017 by Amy Proal

Dear ME/CFS research community,

My name is Amy Proal. I am a microbiologist who also suffers from ME/CFS. I first became ill with ME/CFS in 2004, while studying medicine at Georgetown University. Almost immediately I began to research the disease from bed and wrote my undergraduate thesis on ME/CFS. Several years later, I obtained a fellowship from Murdoch University (Australia) that allowed me to study the human microbiome. I was awarded a PhD in microbiology in 2011. I’ve published many peer-reviewed papers/book chapters that discuss how microbiome imbalance can drive inflammatory disease processes (commissioned by the J. Craig Venter Institute, the NIH, and the European Autoimmunity Network among other groups).

When I fell ill with ME/CFS in 2004, few, if any, research teams were seriously studying the disease. Now I am thrilled that an increasing number of researchers across the globe are better analyzing the ME/CFS microbiome, metabolome, immune response and more. The results of these analyses have sparked new, exciting dialogue in the the ME/CFS community. By writing this letter I hope to add several of my own hypotheses/observations to the conversation.

Ample evidence suggests ME/CFS is driven by chronic infection

Most studies on ME/CFS, and the general history of the illness, suggest that infectious agent(s)/environmental exposures plays at least some role in driving the disease process. These include (but are not limited to!) early associations with EBV/HHV6, “autoantibodies”/antibodies detected in patients with the disease, and even the nature of early ME/CFS “outbreaks.” In fact, a significant number of ME/CFS patients I know have fallen ill with the disease after travel to a foreign country, or after a severe viral infection (suggesting a lack of immunity against acquired pathogens/toxins?).

The ME/CFS proteome differs from that of patients with Lyme disease and healthy controls

The ME/CFS proteome differs from that of patients with Lyme disease and healthy controls

Most of the latest findings on ME/CFS also make sense when viewed through the lens of chronic infection. Reports of cytokine activation in patients with ME/CFS clarify that the disease is characterized by a sustained inflammatory response. Montoya found this cytokine activation increased with disease severity, suggesting patients may struggle with a growing infectious burden over time. Two research teams have shown that the ME/CFS cerebrospinal proteome differs substantially from that of healthy controls. Since the vast majority of metabolites in the human superorganism are microbial in origin, the findings imply that components of the ME/CFS microbiome may exist in a state of imbalance. Mark Davis and Lab at Stanford recently demonstrated massive clonal T cell expansion in patients with ME/CFS. It’s likely these T cells are activated against an infectious threat. Indeed, patients with Lyme disease (known to be driven by infection) demonstrated a T cell response similar to that of the ME/CFS subjects. Another study analyzing the ME/CFS metabolome demonstrated a sustained hypo-metabolic response in patients with the disease. This  dour-like state is “triggered by exposure to adverse environmental conditions”, as would be expected if the ME/CFS immune system is overwhelmed by a chronic infectious burden.

The recently discovered CNS lymphatic system connects the body to the brain

The recently discovered CNS lymphatic system connects the body to the brain

Other ME/CFS research teams have detected various forms of mitochondrial dysfunction in patients with the disease. While a number of mechanisms could account for these findings, intracellular pathogens are very capable of dysregulating human metabolic pathways. Also, an increasing number of studies have detected infectious agents in the brains of patients with Alzheimers, epilepsy and other conditions characterized by inflammation/immunosuppression. It’s possible that similar pathogens in central nervous system (CNS) tissue could contribute to brain abnormalities reported in patients with ME/CFS. The recent discovery of the human CNS lymphatic system strengthens the likelihood that microbes easily traffic in/out of brain tissue.

The human microbiome persists in tissue and blood

With the above in mind, many research teams have searched for single pathogens in patients with ME/CFS. However, the discovery/characterization of the human microbiome challenges the validity of studying single infectious agents in isolation. Microbes in the human body are now understood to persist in complex communities, where they continually interact with neighboring species. Further, human microbiome ecosystems have now been shown to persist in every human body site/niche (blood, tissue, placenta, amniotic fluid etc). Indeed, just this past month, Stephen Quake and team at Stanford demonstrated the presence of thousands of previously undiscovered bacteria/viruses/fungi in human blood and tissue. These “new” microbes, along with those detected by similar analyses, allow us to study the role of infectious agents in ME/CFS with ample new data.

DNA reads corresponding to known, divergent, and novel microbes detected by Quake and team.

DNA reads corresponding to known, divergent, and novel microbes detected by Quake and team.

In addition, a growing number of inflammatory disease states are now tied to dysbiosis or imbalance of human microbiome populations. This dysbiosis is characterized by massive community-wide shifts in microbial population structure, often resulting in decreased species diversity.

Conditions associated with microbiome dysbiosis include type 1 and 2 diabetes, Crohn’s disease, ulcerative colitis, psoriatic arthritis, among many others. Dramatic, continual alterations in the microbiome were reported during the development of tumors in a murine model of inflammation-driven colon cancer. These changes were directly responsible for tumor development. Urbaniak and team identified different bacterial profiles in breast tissue between healthy women and those with breast cancer. It follows that further studies of the ME/CFS microbiome (particularly in blood and tissue) may identify similar community-wide imbalances in patients with the disease.

We must study microbiome ACTIVITY

While species-based microbiome analyses (like those described above) are extremely informative, they are unlikely to paint a full picture of the ME/CFS disease process. For one thing, the species composition of any microbiome community is complicated by a host of environmental variables; variables that also cause large shifts in the body’s microbial ecosystems. These include geographic location, food consumption, and even time of day (this is particularly true of gut microbiome studies). Many research teams studying inflammatory conditions related to ME/CFS have subsequently been unable to isolate disease-induced microbiome dysbiosis in the face of this “noise.”

To avoid this pitfall, the ME/CFS research community must also study what the microbes in any human ecosystem are doing to drive inflammatory processes. We must examine microbe activity, microbe gene expression, and the myriad ways in which microbes interact with the host immune system, the host genome, and each other.

Microbes persist in complex communities

As mentioned previously, microbes in the human body continually interact, both directly and indirectly (the proteins and metabolites they create are also in constant interplay). Microbial communities exhibit synergistic interactions for enhanced protection from host defenses, nutrient acquisition, and persistence in an inflammatory environment. These include biofilm formation and cooperative signaling via quorum sensing peptides.

Microbes often persist in complex biofilm communitites

Microbes often persist in complex biofilm communitites

Even viruses seldom act as single entities. Virgin and team found that enteric virus activity is regulated by “transkingdom interactions” — processes critical to their infectivity, disease induction, and control. For example, the virus MMTV binds lipopolysaccharide (LPS) on the surface of Gram-negative bacteria. This initiates innate immune responses that culminate in host tolerance, transmission, and viral replication.

Microbes alter their collective gene expression to cause disease

These interacting microbes often subvert the human immune response by collectively altering their gene expression. Analysis of these gene expression patterns (studies of the metatranscriptome) should be a priority for the ME/CFS research community. Time/money can be saved by studying the methods other research communities have developed to analyze these patterns. For example, Yost and team recently performed an excellent gene ontology (GO) enrichment analysis of the oral microbiome during periodontal progression.

Gene expression of stable sites did not change over the two-month study period. In contrast, active sites that progressed to periodontitis were easily characterized by several functional genomic signatures. At the breakdown point these active sites expressed genes associated with ferrous iron transport and response to oxidative stress. At baseline, GO terms associated with potassium ion transport and isoprenoid biosynthesis (among others) were highly enriched.

Ranked species by the number of upregulated putative virulence factors in the periodontitis metatranscriptome (Yost et al)

Ranked species by the number of upregulated putative virulence factors in the periodontitis metatranscriptome (Yost et al)

Progression was also correlated with increased expression of putative virulence factors. In addition, ciliary and flagellar motility, as well as chemotaxis genes that direct bacterial movement, were all active at initial stages of periodontitis disease progression. Viral activity was also detected in all samples, with phage and herpesvirus activity higher in progressing sites as compared to baseline samples.

The team concluded that the entire oral microbial community, and not just a few select pathogens, drives the increase in virulence that leads to periodontitis progression. In effect, under conditions of increasing imbalance and inflammation, the whole community appeared to act together as a pathogen. This was supported by the fact that, in active sites, groups of microbes not usually considered pathogens upregulated a large number of putative virulence factors. S. mitis and S. intermedius, usually associated with dental health, were especially active.

Intracellular “keystone” pathogens may drive microbiome dysbiosis

When it comes to dysbiosis, microbe quantity may be less important than microbe “quality” (what a microbe is capable of DOING). Community-wide shifts in microbiome virulence are often driven by “keystone pathogens.” Keystone pathogens can provoke inflammation even when present as quantitatively minor components of the microbiome. For example, P. gingivalis often comprises just .01% of periodontal biofilms, yet impairs innate immune activity so profoundly that it becomes a central player in biofilm growth and development.

Most characterized keystone pathogens have evolved to persist inside the cells of the immune system. By surviving in this fashion, they can directly interfere with human transcription, translation, and DNA repair processes. Their persistence in the cell cytoplasm further dysregulates the epigenetic environment. If the accumulation of errors resulting from this interference exceeds the capacity of cellular repair mechanisms, serious dysfunction/illness can result.

The millions of proteins and metabolites expressed by intracellular pathogens additionally interact with the host genome, further altering human gene expression in a manner that can promote disease. Even bacterial quorum sensing molecules can dysregulate human pathways. Wynendaele and team found that, in vitro, quorum sensing peptides created by gram-negative bacteria altered human gene expression in a manner that promotes angiogeneisis, tumor growth, and neovascularization in colon cancer. 

The byproducts of human and e.coli metabolism are very similar

The byproducts of human and e.coli metabolism are very similar

The above is complicated by the fact that microbial proteins and metabolites are often identical or similar in structure to those created by their human hosts. For example, the human body and E. coli generate the same intermediate byproducts when metabolizing glucose. The “molecular mimicry” or sequence homology between these molecules makes it increasingly difficult for the host to recognize “foreign” from “self.”

Dozens of recent studies have better characterized mechanisms by which pathogens colonize and survive inside human cells. These include reorganization of the actin cytoskeleton, remodeling of vacuole proteolitic composition, development of “zipper and trigger” mechanisms, among many others.

Different pathogens employ common survival strategies

Identification and characterization of previously undetected keystone pathogens in patients with ME/CFS marks a promising area of research. However, it is likely, and expected, that different keystone pathogens may be detected in different patients with the disease. This is because many keystone pathogens, or intracellular pathogens, employ common survival mechanisms to persist in host cells/tissue/blood. The metabolic dysfunction driven by these different microbes can subsequently result in similar clusters of human inflammatory symptoms.

The ability of different pathogens to dysregulate activity of the Vitamin D Nuclear Receptor (VDR) is an excellent example of how different microbes can drive similar disease processes. The VDR regulates expression of hundreds of genes, many of which regulate inflammatory/malignant processes (eg. metastasis supressor protein 1). The receptor also expresses several families of antimicrobial peptides. Microbes capable of slowing VDR activity subsequently facilitate their survival by slowing the innate immune response.

2014-Proal-NIH2.026Many pathogens frequently linked to inflammatory disease have evolved to survive in this fashion. When lymphoblastoid cell lines are infected with Epstein Barr virus, activity of the VDR is downregulated as much as 15 times. The VDR expresses TACO, a protein critical to intracellular survival of M. tuberculosis; not surprisingly then, M. tuberculosis has also evolved to slow receptor activity. HIVBorrelia burgdorferi, Cytomegalovirus, and Mycobacterium leprae also dysregulate VDR activity to varying degrees. The fungus Aspergillus fumigatus secretes a gliotoxin which significantly downregulates VDR expression. Because disabling the innate immune system via the VDR pathway is such a logical survival mechanism, other uncharacterized microbes may have also evolved to dysregulate receptor activity.

It follows that ME/CFS patients with similar symptoms may not always test positive for the exact same pathogen(s). This trend is likely to continue as an increasing number of analyses examine components of the ME/CFS microbiome. Instead of worrying about these “inconsistencies,” the ME/CFS research community should strive to better characterize even more common mechanisms of pathogen survival.   

Microbes act differently depending on host infectious history and immune status

Pathogens detected in patients with the ME/CFS are also regularly identified in healthy subjects. This is particularly true of studies that have searched for EBV, HHV6, cytomegalovirus and other easily characterized viruses in ME/CFS cohorts. While these “overlapping” results are often viewed as problematic, they make sense in light of research that clarifies how differently microbes can act depending on host immune status, neighboring species, and a wide range of other variables. For example, risk of HIV infection is now understood to vary based on the species composition and activity of other microbes in vaginal/penile microbiome communities.

Many microbes assumed to persist in a “commensal” state are also capable of virulent activity. Like their human counterparts, they evolve in the face of changing environmental conditions. For example, s. aureus can cause a range of illnesses, from skin infections to life-threatening diseases such as pneumonia, meningitis, and endocarditis. However, approximately 30% of the “healthy” human population harbors s. aureus as a member of the normal nasal microbiome. Krismer and team found that s. aureus virulence in these communities was determined by a number of factors, especially the signaling/competitive strategies employed by neighboring microbes.   

The same is true of Escherichia coli (E. coli), which also persists in numerous forms. One study found that “commensal” E. coli could evolve into virulent clones in less than 500 generations. For most microbes, this evolution towards pathogenicity occurs via the acquisition of new genes (a gain of function mechanism), or alteration of the current genome, including gene loss (a change-of-function mechanism). For example, in Pseudomonas aeruginosa the loss of mucA increases its ability to resist pulmonary clearance and evade phagocytosis.

Unique infectious history shapes ME/CFS disease progression

While keystone pathogens may be identified in ME/CFS, composition of the ME/CFS microbiome will likely differ between patients. Even in HIV/AIDS, where an easily detected virus dysregulates immunity, disease symptoms reflect a mix of those driven by HIV, and those driven by “co-infectious” agents able to take advantage of the immunocompromised host. No two patients with HIV/AIDS are expected to harbor the exact same mix of these other “co-infectious” agents.

Influence of chronic CMV on the immune response (Brodin et al)

Influence of chronic CMV on the immune response (Brodin et al)

This same pattern, in which unique infectious history drives symptom presentation may also occur in ME/CFS. A recent seminal study by Brodin and team demonstrates the profound impact infectious history on host immunity. The team performed a systems-level analysis of 210 healthy twins between the ages of 8 and 82. They measured 204 immune parameters, including cell population frequencies, cytokine responses, and serum proteins, and found that 77% of these are dominated, and 58% almost completely determined, by non-heritable environmental influences. Many of these parameters became more variable with age, emphasizing the cumulative influence of environmental exposure.

The team also calculated how acquisition of ONE chronic pathogen — cytomegalovirus (CMV) — conditions the immune response. Identical twins discordant for CMV infection showed greatly reduced correlations for many immune cell frequencies, cell signaling responses, and cytokine concentrations. In general, the influence of CMV was so broad that it affected 119 of all 204 measurements dispersed throughout the immune network. In fact, the lifelong need to control CMV causes approximately 10% of all T cells in CMV+ individuals to be directed against the virus. These and related findings led Brodin and team to conclude that the immune response is “very much shaped by the environment and most likely by the many different microbes an individual encounters in their lifetime.”

Could “successive infection contribute to ME/CFS?”

The above suggests that ME/CFS may be driven by a process I have termed “successive infection.” During the successive infectious process, an “initial event” dysregulates the immune system. This makes it easier for certain microbes to subvert the immune response by acting as polymicrobial entities. Pathogens alter their gene expression in ways that promote community-wide virulence. Infected human cells fail to correctly express human metabolites in the presence of the proteins, enzymes, and metabolites generated by the accumulating pathogenic genomes. Dysfunction due to molecular mimicry accumulates. Intracellular pathogens slow the human immune response, causing the host to more easily acquire other infectious agents. This creates a snowball effect in which the microbiome becomes increasingly dysbiotic as the strength of the immune response decreases.

Successive infection results from a patient's unique infectious history

Successive infection is driven by a patient’s unique infectious history

Eventually, the human host may present with clinically evident symptoms characteristic of ME/CFS or a related inflammatory diagnosis. The unique symptoms any one person develops vary depending on the location, species, and virulence of the pathogens driving dysbiosis, along with the semi-infinite number of ways in which the proteins and metabolites created by these microbes cause dysfunction by interacting with those of the host.

In some cases a specific “trigger” may jump start the successive infection process. For example, between the ages of 3-5 I was repeatedly hospitalized for no less than five severe infectious diseases: viral meningitis, double pneumonia, scarlet fever, measles and german measles (despite receiving the MMR vaccine). My twin sister suffered none of these illnesses and is still healthy today. While I can’t be sure, it’s possible that the pathogens driving these diseases states either persisted in my system, or dysregulated my immune response in ways that made me increasingly susceptible to microbiome dysbiosis over time. For example, the immunosuppressive effects of measles have now been shown to deplete host B and T lymphocytes for up to three years after “recovery.” As the study’s authors state, this profound immunosuppression “resets previously acquired immunity” and  “renders the host more susceptible to other pathogens.”

In other cases, a toxic environmental exposure or the difficulty of enduring a traumatic event may push the immune system to a critical mass such that previously subclinical infections become obvious. Reports of several ME/CFS ‘‘outbreaks’’ over the past decades, in which dozens of people have developed the illness at relatively the same time, may well represent this phenomenon at work. For example, in 2004, many cases of chronic fatigue were reported to occur simultaneously after a water reservoir in Bergen, Norway, was contaminated with Giardia lamblia. Nonetheless, of the approximately 48,000 people who were exposed to the contaminated water, only 5 % of the people went on to develop symptoms characteristic of ME/CFS.

The successive infectious process may even begin in the womb. Infants are seeded in the womb, during birth, and after birth by extensive microbiome populations in the placenta, breast milk, and the vaginal canal, among others. Depending on the health of the parents, these communities may already be dysbiotic. The breast milk microbiome of obese mothers has been shown to harbor a different and less diverse bacterial community than that of healthy subjects (Cabrera-Rubio et al., 2012). The amniotic fluid microbiome can predict perinatal complications prior to infant delivery. 2014-Proal-NIH2.005

Many aspects of “modern” living can additionally drive successive infection. Antibiotic use greatly disrupts the ecology of the human microbiome. For example, C. difficile better exploits other microbes in its community following antibiotic treatment. Antibiotic resistance genes are also regularly transferred from farm animals and produce into the human food supply. The immunosuppressive medications, steroids, and supplements often (paradoxically) prescribed for inflammatory disease further allow pathogens in the microbiome to proliferate. High levels of stress depress the immune response. Electromagnetic radiation from mobile phones and cellphone towers (among other sources) has been shown to lower immunity.

ME/CFS patients should not always be grouped into subgroups

ME/CFS is a spectrum disorder: patients are required to present with four out of eight required symptoms. If successive infection contributes to ME/CFS, this variability in symptom presentation is expected. Furthermore, factoring “unique infectious history” into the disease process helps explain why patients with ME/CFS often suffer from a multitude of symptoms not included in the official diagnostic criteria.

Because patients with CFS/ME suffer from such diverse symptoms, it has been argued that they should be grouped into separately studied ‘’subgroups.’’ In some cases this makes sense. For example, studies that distinguish early-stage/late-stage patients may further elucidate how the ME/CFS immune response changes over time. However, if successive infection contributes to ME/CFS, future research should also focus on better understanding the common pathogenesis shared by all subjects.

“Autoantibodies” in ME/CFS are likely created in response to microbes

A number of autoantibodies have been detected in patients with ME/CFS. These include antiphospholipid antibodies, or antibodies directed against neurotransmitters such as serotonin, adrenals, adrenocorticotropin hormone. This has led some research teams to postulate that ME/CFS may be an “autoimmune” disorder.2013-Proal-Spain.001

However, autoantibodies are notoriously polyspecific. The autoantibodies detected in ME/CFS may actually be created in response to pathogens and possess a high degree of molecular mimicry. In effect, when the immune system generates antibodies in an effort to target pathogens, a proportion that are polyspecific may collaterally target human proteins. For example, Lekakh and team found that autoantibodies with polyspecific activity in the serum of healthy donors were able to cross-react with DNA and lipopolysaccharides (LPS) of widespread species of bacteria including Shigella boydii, E. coli, Salmonella, and Pseudomonas aeruginosa. Another analysis found that B cells infected with Epstein Bar Virus secrete antibodies capable of reacting with dozens of self and non-self antigens including albumin, renin, and thyroglobulin. 

If the above is true, there is no need to “lump” ME/CFS into a category of “autoimmune disorders.” This is especially true in light of the fact that the “theory of autoimmunity” is being called into question by an increasing number of research teams.

What about the human genome?

Mycobacteria alters expression of PTPN22

Mycobacteria alters expression of PTPN22

The discovery of the human microbiome has forced science to redefine the human condition. Our bodies harbor more microbial cells than human cells, and the millions of genes expressed by the microbiome dwarf the approximately 20,500 genes expressed by our human genomes. Humans are subsequently best described as superorganisms, in which the human and microbial genomes continually interact to regulate metabolism. For example, the gene PTPN22 has been connected to rheumatoid arthritis, lupus, and diabetes mellitus. However, PTPN22 expression is also altered by the bacterial metagenome — it is upregulated as part of the innate immune response to mycobacteria.

Under normal conditions, components of the gut microbiome and its corresponding metabolites oscillate in a fashion that exposes them to different gut regions across the course of a day. The host interprets the microbial signals resulting from these interactions and alters its gene expression in a manner that promotes rhythmic homeostasis.

Summary of the dynamic relationship between circadian rhythms, intestinal microbiota, and immune response (Rosellat et al)

Summary of the dynamic relationship between circadian rhythms, intestinal microbiota, and immune response (Rosellat et al)

The above suggests that studies of the human genome in isolation are unlikely to paint a full picture of the ME/CFS disease process.

The ME/CFS metabolome/proteome

Studies of ME/CFS proteome/metabolome may further clarify metabolic dysfunction in ME/CFS. However, data from these analyses must be interpreted to account for the “molecular mimicry” between byproducts of host/microbial metabolism.

As previously mentioned, many human/microbial metabolites share similar structure/functions. Kusalik and team found that 19,605 proteins from the hepatitis C virus (HCV) polyprotein have a high level of similarity to the human proteome. This remarkable similarity persisted even when the team used longer peptide motifs as probes for identity scanning. Another study reported tens of thousands of protein-protein interactions between the genomes of E. coli, Salmonella, Yersinia and the human genome.

This means proteome/metabolome studies must continually ask: “What are we actually measuring?”: aka do samples contain human byproducts, microbial byproducts, or a mix of both?

The same is true of studies that characterize DNA in human tissue and blood. Sample analysis MUST account for microbial DNA and RNA that the human microbiome exudes from infected cells. For example, Stephen Quake and team recently discovered thousands of new microbes in human blood. They derived their results by correctly separating the microbe DNA in their samples from the human DNA in their samples.

ME/CFS research must be supplemented by findings from related research communities

The ME/CFS research community struggles with funding. However, the impact of current grants can be maximized if researchers follow the work of related research communities. Most inflammatory disease states are now connected to microbiome/metabolome dysbiosis. This suggests that common underlying processes may contribute to “separate” disease states.

The high levels of comorbidity and symptom overlap between patients with different inflammatory diagnoses strengthens this assumption. For example, composition of the lung microbiome can predict the onset of rheumatoid arthritis. The figure below demonstrates the profound overlap in disease presentation among patients with a broad range of inflammatory conditions.2014-Proal-NIH2.029

It follows that “big picture” studies of the immune system, nervous system, and microbiome can directly inform ME/CFS research. For example, Davis and team reported massive T cell expansion in patients with ME/CFS. However this same trend was observed in cancer and multiple sclerosis. We can follow how the cancer/MS research communities build on these findings and extrapolate parts of this research towards ME/CFS.

Many research teams are also studying how “acute” pathogens can cause chronic symptoms by persisting in latent forms. These include groups studying Zika, influenza, and other well-characterized viruses. For example, tens of thousands of Ebola survivors have developed chronic symptoms months or years after initial infection, including joint pain, eye problems, extreme fatigue, severe pain, and a host of neurological problems. Ebola virus has even been detected in men’s semen years after “recovery.” A better understanding of these “chronic sequelae” may also benefit the ME/CFS community.

Treatment of infection often temporarily increases disease symptoms

Immunosuppresive therapies represent the standard of care for most inflammatory conditions tied to autoantibody production. Corticosteroids, TNF-alpha antagonists, and rituximab are among the many treatments routinely used to slow immune activity. These treatments often provide short-term symptom palliation but allow pathogens in the microbiome to spread with greater ease.

This pattern is recognized in the context of acute infection. For example, Earn and team recently concluded that using antipyretic medications to suppress fever (and subsequently the immune response) in patients with influenza allowed viral particles to spread more easily between people. Thus, while subjects taking the antipyretic medications felt fewer symptoms, they were actually more contagious.

2014-Proal-NIH2.036In contrast, treatments that SUPPORT or activate the immune system may allow patients to better target pathogens over time. Development of such therapies should be a priority for the ME/CFS research community.

However, most “immunostimulative” treatments are characterized by immunopathology—a cascade of reactions including inflammation, cytokine release, and endotoxin release that occur as part of the immune response to microbial death. The death of intracellular microbes is particularly hard for the host to manage, as the body must deal with debris generated from apoptosis and phagocytosis as well as the remains of the dying microbes that once inhabited the cells. The adaptive immune system may also respond to the presence of this pathogenic and cellular debris, generating antibodies in the process.

Immunopathology resulting from microbicidal treatment has been documented for over a century, with symptom presentation varying depending on the nature of the pathogen targeted. First referred to as the Jarisch–Herxheimer reaction, it was originally observed during therapy of secondary syphilis using mercury. Researchers have subsequently noted this reaction in a broad spectrum of chronic diseases such as relapsing fever, Leptospirosis, Brucellosis, and tuberculosis among others. Short-term immunopathology is also part of common acute infectious illness. When a patient develops the flu, symptoms are generated primarily as the immune system releases a host of cytokines and chemokines in response to the presence of the infectious agent.

More recently, an inflammatory syndrome similar to immunopathology has been documented in HIV/AIDS patients undergoing Immune Reconstitution Inflammatory Syndrome (IRIS) following treatment with Highly Active Antiretroviral Therapy (HAART). This condition occurs as HAART enables the once compromised host to target pathogens acquired during periods of severe immunosuppression. A number of prominent and easy-to-culture pathogens have been linked to IRIS: the herpes viruses, cytomegalovirus, hepatitis B and C, Mycobacterium avium complex, M. tuberculosis, and Cryptococcus neoformans. The presence of IRIS in culture-negative patients is common, suggesting many pathogens that cannot be detected without metagenomic tools might also be involved.

Luckily, immunopathology as a result of HAART or related treatments is temporary in nature. In most cases, immunopathology gradually subsides as an increasing number of infectious agents are eradicated. Eventually, patients often “turn a corner”, where they feel better as the body recovers.

While some ME/CFS physicians may feel uneasy about the temporary suffering induced by immunopathology, other research communities have become accustomed to treatments that cause discomfort. For example, the cancer community has developed a number of treatments that activate patient T cells. The “cytokine storm” resulting from these therapies leads to massive (temporary) symptom increases, and has even resulted in death (are infectious agents being killed!?). However, this risk is considered acceptable, as patients who survive the reaction often enter a state of remission.


PS: Hornig and team have reported distinct alterations in plasma immune signatures (including prominent activation of both pro-and anti-inflammatory cytokines) early in the course of ME/CFS. However these alterations were not observed in subjects with a longer duration of illness..

It’s possible that in early-stage ME/CFS, the immune system actively attempts to “battle” an increasing chronic infectious burden. Over time however, pathogens in the microbiome may disable the immune response to a point where “immune exhaustion” occurs. Immunopathology and cytokine production would subsequently drop. The resulting disease state could be compared to a garden, in which healthy plants become progressively stifled by kudzu vine over time.

This pattern suggests that treatments for ME/CFS should be employed as early as possible. Or, as Hornig writes, “alterations in opportunities for intervention may be transient.” This is because any treatment aimed at targeting infectious agents in ME/CFS will be most successful before the immune system becomes overly compromised.

It often takes patients with ME/CFS years to receive a diagnosis. This delay wastes much of the valuable period during which the ME/CFS immune system may be most responsive to treatment. Physicians must subsequently be educated to better recognize early-stage ME/CFS. Also, the ME/CFS research community should prioritize the development of predictive/preventative treatment options.





Huge discovery: microbes in human blood/tissue vastly more diverse than previously known

September 20th, 2017 by Amy Proal

Last post I described fascinating research on the immune response by the Mark Davis Lab at Stanford. But another Stanford research team, led by Steven Quake, has published the results an equally exciting study. In fact, the team’s discovery marks one of the most important findings in modern science.

Quake and team used new methods to search for the DNA of microbes in human blood and tissue. They found that 99% of microbes identified were previously unknown to science. As this article in Stanford News describes, the discovery clarifies that “the microbes living within us are vastly more diverse than previously known.” 

DNA reads corresponding to known, divergent, and novel microbes detected by Quake and team.

DNA reads corresponding to known, divergent, and novel microbes detected by Quake and team.

To be specific, Quake and team examined microbe DNA fragments in the blood of patients with a range of conditions characterized by immunosuppression (liver transplant recipients, pregnant women etc). They collected over 1,000 blood samples, and found that they contained hundreds of never before discovered bacteria and viruses. In fact, ~3,761 of the organisms detected represent microbes not known to exist before the study was performed. 

These species include thousands of new bacteria, but also new viruses and phages (viruses that infect bacteria). The research team was forced to add new branches to the “tree of life” in order to classify many of these new microbes. Indeed, their findings literally double the total number of anelloviruses found in humans.

Quake and team conclude their paper by stating that these novel microbes “have potential consequences for human health. They may prove to be the cause of acute or chronic diseases that, to date,  have unknown etiology…”



More thoughts: I experienced several “eureka moments” while reading the Quake study.  First, I have long predicted that new microbes would be identified in human tissue and blood.  I began my microbiology career by studying the work of microbiologists/pathologists in the 1960s. The papers/textbooks published by these scientists are seldom discussed in 2017. However, their work repeatedly identified numerous microbes/pathogens in blood/tissue samples taken from human subjects.

Why are these 1960s studies seldom referenced? In the 1970s, the “theory of autoimmunity” gained hold and research on chronic microbes was largely shelved. When the 1960s microbiologists contested this mindset, their work was often dismissed on the basis of contamination (they were told that external microbes in their laboratories had “contaminated” their human samples). I’ve never believed these claims to be accurate, but most of the general scientific community has accepted them.

In fact, the possible contamination of blood/tissue samples by laboratory microbes is an ongoing concern: Quake and team were careful to include a full section in their paper called “Novel Contigs (DNA reads) are Not Artifacts or Contaminants.” There, they discuss the results of several extra experiments aimed at proving exactly that (see the study’s Methods section).

Stanford researcher Dave Relman

Stanford researcher Dave Relman

Furthermore, over the past two decades, several research teams using molecular tools have already identified numerous microbes in the blood. For example, this 2001 study by Stanford researcher Dave Relman found many bacterial species in the blood of healthy subjects. Relman is one of my greatest role models, so I’ve taken his findings and related studies very seriously.

The Quake study also hammers home a point I’ve made in every speech/paper/book chapter since 2005. Imbalances of the gut microbiome, and external body microbiomes (skin, mouth etc) can contribute to chronic disease. However, microbes in the blood reach internal human tissue, and thus may play the greatest role in driving infectious disease processes. This is especially true since many of these blood/tissue microbes persist inside the cells of the immune system. Quake and team echo this sentiment in their paper stating, “Blood circulates throughout the human body and contains molecules drawn from virtually every body tissue.” Quake also told Stanford News the following about his team’s discovery:

“I’d say it’s not that baffling in some respects because the lens that people examined the microbial universe was one that was very biased…For one thing, researchers tend to go deep in the microbiome in only one part of the body, such as the gut or skin, at a time. Blood samples, in contrast, “go deeply everywhere at the same time.”

Last but not least, the Quake team derived their results by correctly separating the microbe DNA in their samples from the human DNA in their samples. This distinction can prove difficult because microbe and human DNA are often very similar in structure. 

Many other research teams studying the microbiome ARE NOT DOING THIS (CORRECTLY SEPARATING MICROBE/HUMAN DNA).  In conjunction with my colleague Trevor Marshall I have warned about this problem in several peer reviewed papers. For example, Marshall and I state the following about correctly identifying microbe DNA in this Current Opinions in Rheumatology paper:

“What are we actually measuring? Genetic science has not yet noticed the elephant in the room – the microbial DNA and RNA that the human microbiome exudes from infected cells. This contaminates the samples of “human DNA” being analyzed.”

Well…Quake  and team noticed the elephant in the room. And doing so made a dramatic difference in the results they obtained! I am extremely excited to see how their new findings impact microbiome research in the years to come.

New Stanford University data clarifies immune dysfunction/infection in cancer, ME/CFS, MS

September 15th, 2017 by Amy Proal

Mark Davis and his lab at Stanford University are on fire! They recently released fascinating data (some unpublished) on patients with cancer, Lyme disease, MS and ME/CFS. Davis discussed this data at a recent Open Medicine Foundation meeting. The talk was recorded and I HIGHLY encourage you to watch it!

New Davis Lab Findings:

Davis starts by confirming that ME/CFS is characterized by high levels of systemic inflammation. In fact, in concert with Dr. Jose Montoya at Stanford, Davis detected elevated cytokines (inflammatory molecules) in the blood of patients with ME/CFS. First, this cytokine activation distinguished the ME/CFS patients from healthy controls: does anyone still want to argue that the ME/CFS is psychosomatic!? (please tell me no). Second, patients with more severe cases of ME/CFS demonstrated greater cytokine activation; indicating that ME/CFS disease progression is characterized by increased immune dysfunction over time. 

In another series of experiments, Davis looked at T cells responses in ME/CFS and related inflammatory conditions. T cells are part of the adaptive immune response: the branch of the immune system that creates antibodies in response to specific microbes or pathogens. Davis used a novel assay developed at Stanford to obtain T cell sequences from the tissues/blood of patients with colon cancer, MS, Lyme disease, and ME/CFS.

In all four diseases, T cells were activated in a manner not observed in healthy control subjects. To be specific, the team observed massive clonal expansion of the T cells – both in tumor tissue and in the blood of patients with MS, ME/CFS, and Lyme disease.

T cell expansion in healthy subjects as compared to patients with Lyme disease, MS, and ME/CFS

T cell expansion in healthy subjects as compared to patients with Lyme disease, ME/CFS and MS. Unpublished data by Mark Davis Lab, Stanford University.

What does this mean? In simple terms, T cell clonal expansion indicates that the T cells became increasingly activated against a “target.” This activation caused the cells to divide and proliferate. As Davis explains, this “target” could be a pathogen or dysregulated human tissue.

Davis leans towards the “target” being a pathogen, stating that antibodies driving T cell proliferation are likely formed “originally against some pathogen peptide.” In some cases, these “pathogen peptides” may cross react with similarly structured human peptides – causing the immune system to accidentally target human tissue. This is exactly in line with the new model of autoimmune/inflammatory disease I’ve described on this site.

Indeed, Davis’ next goal is to further study the activated T cells in his samples. He hopes to correlate the T cell activity with the presence of specific pathogens (and the antibodies created in response to their presence). This could lead to a better understanding of exact microbes involved in driving cancer, MS, ME/CFS etc.

CONSIDERATIONS: Davis’ data strongly suggests that in cancer, MS, Lyme disease, and ME/CFS the immune system is activated against an infectious threat. This threat could be one pathogen, or it could be many pathogens acting together (in a community).

I support the latter possibility: I suspect that T cells are activated in these conditions as part of a generalized response to microbiome dysbiosis or imbalance. However it is very possible that certain microbes in these communities play a larger role than others in driving disease processes (these microbes are often referred to as “keystone” pathogens.”)

Also, the same general pattern of T cell clonal expansion was observed in cancer, autoimmune disease, and infectious disease. This strongly supports what I have long advocated: different inflammatory conditions, commonly studied in isolation, may actually result from the same root causes. This overlap certainly explains the high levels of co-morbidity observed between patients with different diagnoses! If this is true we should be studying these illnesses TOGETHER, with an increased focus on multidisciplinary research.

Finally, the T cell activation Davis observed in patients with ME/CFS could serve as an excellent biomarker for the disease. In my opinion, we do not need to know the exact microbial species involved for the data to be useful. Nor does it matter that other related diseases demonstrate a similar pattern. For the sake of treatment, all we need to know is that patients with ME/CFS show different T cell activity than that of healthy subjects.

T cell expansion in colon carcinomas (tumors)

T cell expansion in colon carcinomas (tumors). Figure: Mark Davis Lab, Stanford University.


My new peer-reviewed paper: Microbes INTERACT to cause chronic inflammatory disease

September 10th, 2017 by Amy Proal

Hello readers!

The image above shows different species of microbes communicating inside communities called biofilms. In many instances this kind of signaling is able to drive inflammatory disease processes. For much more on this topic, please check out my latest peer-reviewed paper published in Discovery Medicine “Microbe-Microbe and Host-Microbe Interactions Drive Microbiome Dysbiosis and Inflammatory Processes.” Then come back here and ask me questions! Or give me feedback/constructive criticism! Thanks.


Abstract: An extensive microbiome comprised of bacteria, viruses, bacteriophages, and fungi is now understood to persist in nearly every human body site, including tissue and blood. The genomes of these microbes continually interact with the human genome in order to regulate host metabolism. Many components of this microbiome are capable of both commensal and pathogenic activity. They are additionally able to persist in both “acute” and chronic forms. Inflammatory conditions historically studied separately (autoimmune, neurological and malignant) are now repeatedly tied to a common trend: imbalance or dysbiosis of these microbial ecosystems. Population-based studies of the microbiome can shed light on this dysbiosis. However, it is the collective activity of the microbiome that drives inflammatory processes via complex microbe-microbe and host-microbe interactions. Many microbes survive as polymicrobial entities in order to evade the immune response. Pathogens in these communities alter their gene expression in ways that promote community-wide virulence. Other microbes persist inside the cells of the immune system, where they directly interfere with host transcription, translation, and DNA repair mechanisms. The numerous proteins and metabolites expressed by these pathogens further dysregulate human gene expression in a manner that promotes imbalance and immunosuppression. Molecular mimicry, or homology between host and microbial proteins, complicates the nature of this interference. When taken together, these microbe-microbe and host-microbe interactions are capable of driving the large-scale failure of human metabolism characteristic of many different inflammatory conditions.

Probably the most important sentence in the paper:

  1. In effect, under conditions of increasing imbalance and inflammation, the whole community appeared to act together as a pathogen.

TOP IMAGE: Property of the Center For Biofilm Engineering. They are an awesome organization, check them out:


The power of patient reported feedback

January 29th, 2016 by Amy Proal

Last year I was invited to give a speech at a scientific conference that examined the role of the microbiome in autoimmune disease – concepts I describe in this Current Opinion in Rheumatology journal article. Our research team had also developed an immunostimulatory treatment for autoimmune disease based off concepts in the paper. Doctors in at least a dozen countries were using the treatment with their patients, often with success.

I didn’t discuss this treatment in my speech, but made the following statement during the last twenty seconds of the talk: “We have developed an immunostimulatory treatment that patients are using in conjunction with their doctors. If you’re interested in any of our case histories find me later.” Continue reading

Of mice and not men: can complex human inflammatory disease be studied in mice?

January 13th, 2016 by Amy Proal

Much of my junior year at Georgetown University was spent in an animal research facility. Along with my undergraduate thesis mentor and several fellow students, I studied the impact of a high-fat (ketogenic) diet in Sprague-Dawley rats. We had read reports in which human children with epilepsy who were fed this ketogenic diet experienced fewer seizures. Now we were attempting to ascertain whether rats eating a ketogenic chow would experience seizures at a different rate than those eating a chow rich in carbohydrates.

I graduated before the research project was complete, but later learned that some differences in seizure incidence between the two groups of rats were identified. Yet the team was never able to figure out the root cause underlying these differences.

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Mothers and microbes, Part 2: The placental, breast milk, and breast tissue microbiomes

December 27th, 2015 by Amy Proal

While the vaginal microbiome has received a great deal of attention from the research community, recent research also indicates that microbes persist in the womb, where they come in contact with a fetus before it is born. Studies demonstrating the presence of microbes in the amniotic fluid have now been bolstered by the discovery of a placental microbiome. Dysregulation of this placental microbiome by pathogens has also been associated with preterm birth and low infant birth weight.

Consistent with the presence of a placental microbiome, naturally-born infants often harbor microbes not commonly found in the vagina. For example, while vaginal communities are often composed of up to 80 percent Lactobacillus, the microbiomes of newborn infants contain high levels of other taxa, such as Actinobacteria, Proteobacteria, and Bacteroides. Infants appear to have acquired these microbes in the womb, and not during the birthing process. Continue reading

Mothers and microbes, Part 1: The vaginal microbiome in health and disease

December 20th, 2015 by Amy Proal

“Like mother, like daughter.” The phrase is often invoked to describe how children resemble their parents. While we know that human genes are passed from generation to generation, an expanding body of research now shows that many microbiome populations are also inherited. The microbes a child inherits are acquired from both parents and even siblings. However, microbial populations inherited from the mother have a particularly strong impact on a child’s development and health.

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Stanford researchers: The immune response is shaped by microbes rather than human genes

December 11th, 2015 by Amy Proal

Two different people are riding the subway. A third person coughs on these individuals over the course of their trip. One person gets the flu, but the other doesn’t. Somewhere nearby, two more people accidentally eat a piece of meat that wasn’t correctly refrigerated. One develops food poisoning, but the other remains healthy. What factors contribute to these different outcomes?

The key factor is the immune response. Immune cells such as macrophages and granulocytes kill invading microbes. Other immune proteins called cytokines and chemokines aid cellular communication and stimulate the movement of cells towards sites of inflammation. Immune growth factors also form part of the immune response by stimulating the proliferation of specific tissues. If profiles of these immune parameters differ between individuals, then their ability to respond to pathogens will also vary. Continue reading

Industry ties deeply influence guidelines for calcium/vitamin D intake

December 3rd, 2015 by Amy Proal

Are you taking vitamin D and calcium for bone health? If so, a new analysis makes it clear that the supplement guidelines you follow are often shaped by money rather than science. In July, Andrew Grey and Mark Bolland (University of Auckland, New Zealand) published an article in the British Medical Journal. Their article, “Web of industry-advocacy, and academia in the management of osteoporosis,” powerfully illustrates how industry ties and financial gain have tarnished the legitimacy of worldwide vitamin D and calcium supplementation guidelines.

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