My favorite 2018 papers that impact the study of chronic inflammatory disease

December 30th, 2018 by Amy Proal

2018 was a great year for science. The following article summarizes findings from my favorite studies published this past year. Many of the studies shed light on how persistent infection can contribute to chronic inflammatory disease processes. They also clarify how activity of the human microbiome + virome + mycobiome (fungi) influence health and disease processes. Topics discussed include the maternal microbiome, the gut-brain axis, brain infection, bacteriophage activity, the circulatory microbiome, novel human structures/pathways, pathogen survival mechanisms…and more. There is also a section on potential novel treatments and treatment-based paradigm shifts (especially relevant to neurological disease).

I broke the article into “areas of study” so that it’s easier to read. Please note that despite the many studies listed below, I could still have added more findings to the list! After reading the article, please respond with other important 2018 studies that you think I might have missed.

1. Studies on chronic inflammatory disease and the human microbiome move WAY beyond just species composition:

The human microbiome (Image:

Several times this past year I’ve been told that studies connecting microbiome dysbiosis (imbalance) to chronic inflammatory disease “haven’t moved beyond association.” I disagree. In fact, for years now, teams have been using computer modeling, metabolomics and a range of other methods to dissociate “cause from effect” in microbiome-based analyses. These three 2018 studies are excellent examples of the trend (while they involve the gut microbiome, similar studies have been also been conducted on microbiome ecosystems in other body sites):

Gut microbiota composition and functional changes in inflammatory bowel disease and irritable bowel syndrome

Lead author: Rinse K. Weersma, University of Groningen and University Medical Center Groningen

The team characterized gut microbiome dysbiosis in patients with IBD/IBS using a computer-based tool called shotgun sequencing. BUT, they also analyzed bacterial taxonomy, metabolic functions, antimicrobial resistance genes, virulence factors, and growth rates. Thanks to this extra information, they were able to identify key bacterial species that may be involved in both disorders.

Pathogenic Functions of Host Microbiota

Lead author: Marius Vital, Helmholtz Centre for Infection Research, Germany

Co-occurrence analysis of individual pathofunctions (Vital et al)

The team studied the gut microbiome, and found that different groups of bacteria often exhibit redundant pathogenic functions (they called them “pathofunctions”). These pathofunctions include the production of common metabolites (like trimethylamine, secondary bile acids, and hydrogen sulfide). This functional redundancy has implications for the study of chronic inflammatory disease tied to microbiome dysbiosis: it suggests that metabolic dysfunction driven by different groups of organisms can result in similar clusters of inflammatory symptoms. 

Distinct Microbes, Metabolites, and Ecologies Define the Microbiome in Deficient and Proficient Mismatch Repair Colorectal Cancers

Lead author: Nicholas Chia, The Ohio State University College of Veterinary Medicine

The team combined tumor biology, metagenomics, metabolomics and modeling approaches to study the impact of the gut microbiome on colon cancer. They demonstrated distinct roles for microbes and their metabolites in colon cancer mismatch repair status. For example, highly influential microbes included many butyrate producers.

2. The immune response is shaped by the microbiome + virome:

One of my favorite papers of all time is Mark Davis’ (Stanford) 2015 paper: “Variation in the human immune system is largely driven by non-heritable influences.” Davis and team found that the human immune response is “very much shaped by the environment and most likely by the many different microbes an individual encounters in their lifetime.” In 2018 many studies added to this topic, including these two:

Microglial control of astrocytes in response to microbial metabolites

Lead author: Francisco J. Quintana, Harvard Medical School

The team found that, in a mouse model of multiple sclerosis, tryptophan created by the gut microbiome interacted with the AHR receptor on microglia/astrocytes. Subsequent changes in gene expression regulated communication between the cell types. The study is a great example of a growing trend: microbial metabolites can control immune signaling.

Interactions between Bacteriophage, Bacteria, and the Mammalian Immune System.

Lead author: Paul Bollyky, Stanford University School of Medicine

Interaction of bacteriophages with mammalian immune cells (Bollyky et al)

The paper clarifies that bacteriophages (phages) directly interact with human cells +  impact/modulate the human immune response. It provides examples of how phages can modulate innate immunity via phagocytosis and cytokine responses. Phages can also impact adaptive immunity via effects on antibody production. The team also presents a computational model for predicting these complex and dynamic interactions. These models predict that phages may play important roles in shaping mammalian-bacterial interactions.

3. Re-evaluating “autoimmunity” to account for persistent infection: 

I’ve published several papers on persistent infection + microbiome dysbiosis as drivers of “autoimmune” disease. So I was glad that many 2018 papers added to a growing body of research documenting “autoantibody” production in response to a range of bacterial, viral and fungal pathogens (as opposed to “self”). These pathogens are not short-term “triggers” but persist as members of complex microbiome communities. These are two of my favorite 2018 studies on the topic:

Translocation of a gut pathobiont drives autoimmunity in mice and humans

Lead author: Martin Kriegel, Yale School of Medicine

The team detected Enterococcus galinarum (a common human gut bacteria) in the mesenteric veins, lymph nodes, spleens and livers of mice made genetically prone to autoimmunity. In these mice, the bacterium initiated the production of “autoantibodies,” inflammation and activated T cells. However, this “autoantibody” production stopped when E. gallinarum’s growth was suppressed with the antibiotic vancomycin or with an intramuscular vaccine. In addition, E. gallinarum–specific DNA was recovered from liver biopsies of human autoimmune patients, and co-cultures with human liver cells replicated the mouse findings.

GDP-l-fucose synthase is a CD4+ T cell–specific autoantigen in DRB3*02:02 patients with multiple sclerosis

Lead author: Manuel Rodriguez, Mayo Clinic and Foundation

The team studied CD4+ T cells from the cerebrospinal fluid of patients with multiple sclerosis. They found that these T cells (which are capable of inducing an inflammatory response associated w/ demyelination) could be activated by an enzyme created by bacteria frequently found in the gut microbiome of MS patients.

Paper highlight: “These tantalizing results [in multiple sclerosis] identify a new autoantigen and suggest that one possible trigger of disease could be cross-reactivity to microbiota-derived peptides”

4. Microbial signaling + electrical activity:

Some bacteria generate electricity (Photo: Amy Can UC Berkeley)

Last year, Gurol Suel and team at UCSD discovered that even bacteria in distant biofilms can communicate via electron channel mediated electrical signaling. You can listen to Suel discuss the findings in our recent interview. In 2018, this study added to Suel’s research:

A flavin-based extracellular electron transfer mechanism in diverse Gram-positive bacteria

Lead author: Daniel Portnoy, University of California, Berkeley

The team found that bacteria in the human gut can generate electricity. The genes that code for this electrical transfer were identified in hundreds of bacterial species, including human pathogens like Listeria, Clostridium perfringens and Enterococcus faecalis. Could interrupting this electrical transfer impact the survival of pathogens connected to chronic inflammatory disease?

5. More evidence for infection + microbiome dysbiosis as a driver of cancer:

Evidence for infection + microbiome dysbiosis as a driver of cancer continues to grow…and grow. An increasing number of tumor microbiomes have been identified, with tumor gene expression changes often tied to pathogen activity. These findings clarify cancer “root cause” mechanisms, and also have major implications for the success + management of cancer immunotherapy. For example, I suspect that part of the “cytokine storm syndrome” associated with immunotherapy results from the death of tumor-associated pathogens (a herxheimer-type reaction). Here are two of my favorite 2018 studies on infection and cancer:

Mycoplasma promotes malignant transformation in vivo, and its DnaK, a bacterial chaperon protein, has broad oncogenic properties

Lead author: Robert Gallo, University of Maryland

Robert Gallo (who helped discover the HIV virus) is now working on infection in cancer. In this study, he identified a strain of mycoplasma that creates a protein called DnaK. DnaK promotes cancer by interfering w/ host cell DNA-repair + cell death signaling.

Paper highlight: “Our work provides an explanation for how a bacterial infection can trigger a series of events that lead to cancer…the study also provides a mechanisms for how some bacterial infections can interfere with specific cancer drugs” 

The Pancreatic Cancer Microbiome Promotes Oncogenesis by Induction of Innate and Adaptive Immune Suppression.

Lead author: George Miller, New York University School of Medicine

The team identified a distinct and abundant pancreatic microbiome associated with progressive pancreatic cancer. A series of experiments in mice showed this dysbiotic microbiome drove oncogenesis (cancer) by suppressing macrophage differentiation and T cell activity. In addition, targeting this cancer-promoting microbiome with antibiotics protected against oncogenesis, reversed intratumoral immune tolerance, and enabled efficacy for checkpoint-based immunotherapy.

Paper highlight: “These data suggest that endogenous microbiota promote the crippling immune-suppression characteristic of [pancreatic ductal adenocarcinoma (PDA)] and that the microbiome has potential as a therapeutic target in the modulation of disease progression.”

6. The human virome as a driver of chronic inflammatory disease: 

Research on the human virome (the extensive communities of viruses that persist in human tissue and blood) is exploding, thanks in large part to projects like the Joint Genome Institutes’s “Uncovering the Earth’s Virome” initiative. A large part of the human virome is comprised of bacteriophages, or viruses that infect bacteria (one study estimates that ~31 billion bacteriophages traffic from the human gut into the body on a daily basis). Even then, the vast majority of the human virome has yet to be identified and characterized. For more background on the human virome, watch this video I recorded on the topic. 

A bacteriophage (Medical News Today)

In 2018 more research teams began studying how the human virome impacts health and disease processes. Because phages modulate activity of the bacterial microbiome this is major consideration. These are several of my favorite 2018 papers on bacteriophages as drivers of disease:

Diagnostic Potential and Interactive Dynamics of the Colorectal Cancer Virome

Lead author: Pat Schloss, University of Michigan

The team used a range of molecular tools to evaluate differences in human colorectal cancer virus and bacterial community composition (they analyzed stool samples). When the bacterial and viral community signatures were combined, both bacterial + viral organisms were found to drive the community association with the cancer.

Paper highlight: “Overall, our data support a model in which the bacteriophage community modulates the bacterial community and, through those interactions, indirectly influences the  bacteria driving colorectal cancer progression”

Murine colitis reveals a disease-associated bacteriophage community

Lead author: Lora Hooper, University of Texas Southwestern Medical Center

The team characterized the intestinal virome in a model of T-cell-mediated mouse colitis. The intestinal phage population changed in the mice with colitis, and transitioned from an ordered state to a stochastic (disordered) dysbiosis. In addition, phage populations that expanded during colitis were frequently connected to bacterial hosts that benefit from or are linked to intestinal inflammation.

Smoking is associated with quantifiable differences in the human lung DNA virome and metabolome

Lead author: Brian Keller, The Ohio State University

The team found that commensal viruses are present in the lower respiratory tract and differ between smokers and nonsmokers. The associations between viral populations and local immune and metabolic tone suggest a significant role for virome-host interaction in smoking related lung disease.

Bacteriophages as New Human Viral Pathogens 

Lead author: George Tetz, Human Microbiology Institute, New York

The team presents evidence that phages can interact w/ eukaryotic (human) cells and proteins to drive inflammatory disease processes. They also point to mechanisms by which certain phages disrupt intestinal permeability and provoke chronic inflammation. For more on the paper and Tetz’s work in general, listen to my recent interview with George. 

7. Microbes/viral interactions impact human health and disease: 

The organisms in any microbiome community continually interact. In fact, I recently wrote a paper that describes how microbe-microbe interactions can contribute to chronic inflammatory disease processes. But 2018 saw a major increase in research that characterized how viruses/phages interact with other organisms in the human body (in both health and disease). Here are several of my favorite studies on the topic:

Methicillin-resistant Staphylococcus aureus alters cell wall glycosylation to evade immunity

Lead author: Andreas Peschel, University of Tübingen, Germany

The team studied phages that can infect strains of the antibiotic-resistant bacterial species S. aureus (MRSA). They found that certain phages encode an enzyme (TarP) that helps S. aureus better evade detection by the host immune system.

Paper highlight: “These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences”

Viruses cooperate to defeat bacteria

Lead author: Rotem Sorek, Weizmann Institute of Science, Israel.

A viral mechanism to thwart bacterial defences (Sorek et al)

The team found that phages (which infect bacteria) can suppress the bacterial immune system (the CRISPR/cas system) during an initial wave of unsuccessful infection. However, although this first phage may fail to replicate, the immunocompromised bacterium often succumbs to subsequent successful infections by other phages.

Microbiome interactions shape host fitness

Lead author: William Ludington, University of California Berkely

The team developed a mathematical approach to study the fruit fly bacterial gut microbiome. They found that microbiome interactions are as important as individual species in shaping these fundamental aspects of fly physiology (development + lifespan).

The Emerging Role of Microbial Biofilm in Lyme Neuroborreliosis

Fabrizio Ensoli, San Gallicano Dermatological Institute IRCCS, Italy

The paper describes how in Lyme Neuroborreliosis, different strains of Borrelia often persist in biofilms. The ability of Borrelia to form into biofilm communities may “explain the low rate of Borrelia detection in the blood of infected patients as well as the ability of the spirochetes to evade the host immune system and resist antibiotic therapy.”

8. Microbes + pathogens alter human gene expression:

I’ve said it a million times and I’ll say it again. The ability of microbes/viruses/fungi and their associated metabolites + proteins to alter human gene expression is at the root of human inflammatory disease. This is especially true of intracellular pathogens, which have direct access to human transcription, translation and DNA repair processes. Here are two of my favorite 2018 studies related to the topic:

Coordinated host-pathogen transcriptional dynamics revealed using sorted subpopulations and single, Candida albicans infected macrophages

Lead author: Christina Cuomo, Broad Institut

The team found that the fungus Candida albicans‘s transition from extracellular to intracellular pathogen was accompanied by a coordinated, time-dependent shift in gene expression for both the host and the fungus. These gene expression changes led to a gradual decline in pro-inflammatory cytokine activation by the host immune system. The findings are an excellent example of how the human immune response changes over time in response to different pathogen survival states.

Variation in gut microbiota composition impacts host gene expression

Lead author: Francesca Luca, Wayne State University

The study found that gut microbes exposed to epithelial cells from the human colon modified the expression of over 5000 human genes. Interestingly the microbial taxa with the strongest influence on gene expression altered the response of genes associated with specific complex traits.

9. Microbes/viruses + their metabolites/proteins can dysregulate host metabolism:

Tying the microbiome + virome to metabolic diseases like diabetes and obesity comes down to mechanism. The genes of our microbial inhabitants greatly outnumber the ~20,500 in the human genome. It follows that the majority of protein/metabolites in the human body are produced or modified by the microbiome. A growing body of research now demonstrates how many of these foreign proteins/metabolites can dysregulate human metabolic signaling pathways. Here are several of my favorite 2018 studies on the topic:

The extracellular domain of Staphylococcus aureus LtaS binds insulin and induces insulin resistance during infection

Lead author: Guang Yang, Beijing Institute of Basic Medical Sciences

Here, eLtaS, a protein created by S. aureus, prevented insulin from correctly binding its target receptor. This inhibited signaling and led mice to develop impaired glucose tolerance. The team then developed a human monoclonal antibody against eLtaS that blocked the interaction between eLtaS and insulin. This restored glucose tolerance in certain strains of S.aureus-challenged mice.

Viral insulin-like peptides activate human insulin and IGF-1 receptor signaling: A paradigm shift for host-microbe interactions.

Lead author: Ronald Kahn, Harvard Medical School

The team found that viruses carry sequences with significant homology to human insulin-like growth factors (the sequences are called viral insulin-like peptides or VILPs). In the lab, these VILPs were shown to bind human and murine insulin receptors, resulting in autophosphorylation and downstream signaling.

Paper highlight: “Furthermore, since only 2% of viruses have been sequenced, this study raises the potential for discovery of other viral hormones which, along with known virally encoded growth factors, may modify human health and disease.”

PS: This study found that a microbial metabolite can also dysregulate insulin pathway signaling.

A selective gut bacterial bile salt hydrolase alters host metabolism.

Lead author: A. Sloan Devlin, Harvard Medical School

The study found that deletion of a single Bacteriodes bacteria gene—and the bile salt hydrolase it expresses—altered (mouse) host metabolism in a manner that impacted weight gain, respiratory exchange ratios, and transcriptional changes in metabolic, circadian rhythm, and immune pathways in the gut and liver.

Study highlight: “Our results demonstrate that metabolites generated by a single microbial gene and enzymatic activity can profoundly alter host metabolism and gene expression at local and organism-level scales.”

10. An improved understanding of pathogen persistence:

Most of the pathogens that drive chronic inflammatory disease persist as members of our microbiome communities. The survival mechanisms employed by these pathogens (to remain alive in the face of the host immune response) have been studied for decades. But in 2018, research teams characterized even more novel mechanisms of pathogen persistence (several tied to bacteriophage activity):

The Pseudomonas aeruginosa Wsp pathway undergoes positive evolutionary selection during chronic infection

Lead author: Daniel Wozniak, The Ohio State University

The team used a burn wound model of chronic infection to study how mixed strains of P. aeruginosa adapt and evolve. They found that certain strains of chronic P. aeruginosa acquired genetic elements that altered their CRISPER-Cas (immune) systems. These mutations allowed these strains to better resist attack from specific bacteriophages. Certain P. auruginosa strains were also capable of surviving as “rugose small-colony variants” (RSCVs): small colonies with an elevated capacity to form biofilms.

Persistence of Systemic Murine Norovirus Is Maintained by Inflammatory Recruitment of Susceptible Myeloid Cells

Lead author: Timothy Nice, Oregon Health and Science University

Graphical abstract of Nice et al paper

The team studied a mouse norovirus. They found that the norovirus capsid (the protein shell of the virus) helped regulate cell lysis and inflammatory cytokine release. The capsid also-triggered inflammation that recruited immune cells like monocytes and neutrophils to sites of replication. This promoted the viruses’ chronic persistence.

Paper highlight: “Infection of continuously recruited inflammatory cells may be a mechanism of persistence broadly utilized by lytic viruses incapable of establishing latency”

Tunneling Nanotubes as a Novel Route of Cell-to-Cell Spread of Herpesviruses

Lead author: Krystyna Bieńkowska-Szewczyk, University of Gdańsk, Poland

Tunneling nanotubes containing herpesviruses (Bieńkowska-Szewczyk et al)

The team found that certain herpesviruses can spread between infected human cells via tunneling nanotubes (TNTs): cytoplasmic extensions of human cells that represent a new form of intracellular transfer for viruses like HIV, mRNAs, and even prions. This spread may allow herpesvirus transmission despite the presence of host immune responses. Also, pathogens travelling via TNTs do not enter the blood, making them hard to detect by standard testing methods.

11. Pathobiont behavior + microbiome-associated pathogens:

Potential pathogens persist in every single microbiome community. These pathobionts can act as commensals OR they can change their gene expression to act as pathogens under conditions of imbalance and immunosuppression. This pathobiont behavior helps explain why many microbes  + viruses tied to chronic inflammatory disease are also regularly identified in healthy subjects. Here are two excellent 2018 studies on the topic:

Streptococcus pneumoniae: transmission, colonization and invasion

Lead author: James Paton, University of Adelaide, Australia

The team found that, in the human body, the bacteria S. pneumoniae can persist as a “highly adapted commensal” or a serious pathogen. This difference hinges on its ability to “evade or take advantage of the host inflammatory and immune responses.” 

Precision identification of diverse bloodstream pathogens in the gut microbiome

Lead author: Ami Bhatt, Stanford University

The team used a new bioinformatics software pipeline to identify the source of bloodstream infections in a group of hospitalized + immunocompromised patients. They found that, in many cases, the same strain of a particular bloodstream pathogen was also identified in a patients’ gut microbiome. This suggests that many hospital-acquired infections are not derived from the external environment, but instead represent a change in host pathobiont activity + location of infection + host immunity.

Future research direction: “The results presented are suggestive of a gut microbiota source for both enteric and nonenteric organisms. However, given that the present study sampled only stool microbiota, we cannot exclude the possibility of the same pathogenic strain colonizing multiple body sites from which the infection may have originated instead.”

12. Antimicrobial activity of “useless” or “toxic” molecules: 

The past year saw expansion of a very important paradigm shift: a growing number of human compounds previously regarded as “useless” or “toxic” are actually potent antimicrobial peptides (peptides that form part of the innate immune response TOWARDS microbes in tissue + blood). Compounds shown to possess this antimicrobial activity include amlyoid beta (Alzheimer’s), alpha-synuclein (Parkinson’s) and even prions. Here are two important 2018 studies on the topic:

Alzheimer’s Disease-Associated β-Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection.

Lead author: Robert Moir, MassGeneral Institute for Neurodegenerative Disease

In 2016 Moir and team showed that amyloid beta, a protein that accumulates in the Alzheimer’s brain, has potent antimicrobial activity against fungal and bacterial pathogens. In this 2018 study, the team found that amyloid beta also protects against herpesviruses commonly found in the brain. In fact, the team showed that amyloid beta can bind to proteins on herpesvirus membranes and clump into fibrils that entrap the virus and prevent it from entering human cells.

Antimicrobial and Antibiofilm Activity of Human Milk Oligosaccharides against Streptococcus agalactiae, Staphylococcus aureus, and Acinetobacter baumannii

Lead author: Steven Townsend, Vanderbilt University

The study adds to previous work showing that breast milk proteins called Human Milk Oligosaccharides (HMOs) have potent antimicrobial activity. This particular analysis found that HMOs possessed both antimicrobial and antibiofilm activity against strains of methicillin-resistant S. aureus and A. baumannii. The absence of these HMOs (and other antimicrobial molecules) in infant formula may help explain why formula-feeding is associated with a number of negative health outcomes.

Infants Are Exposed to Human Milk Oligosaccharides Already in utero

Lead author: Lars Bode University of California, San Diego

The team detected HMOs in samples of amniotic fluid removed from women during and after pregnancy. The findings suggest that HMOs may additionally help control pathogens + organisms capable of persisting in the womb.

Prion-like Domains in Eukaryotic Viruses

Lead author: Victor Tetz, Human Microbiology Institute, New York

PrD enrichment in the proteomes of different viral orders (Tetz et al)

The team used a computational algorithm to show that viral prion-like proteins (PrDs) can be found in a range of human viral pathogens. They also revealed probable functional associations between PrDs and different steps of viral replication + interaction with host cells. Since prions have been shown to have antimicrobial activity, I wonder if some PrDs could play a role in viral defense? It’s also worth noting bacteria have been shown to produce amyloid (a similar pattern).

Paper highlight: The predictive approach employed in this study revealed for the first time a large set of putative PrDs in numerous proteins of the emerging human viral pathogens, including those associated with persistent viral infections, oncogenic processes, hemorrhagic fevers, and others.”

13. Persistent infection as a driver of neurodegenerative disease: 

This year I learned that Harvard’s Rudy Tanzi and Robert Moir started the Brain Microbiome Project. I consider this Project to be the single most important initiative in science at the moment. In fact, several months ago I travelled to Harvard, where Rudy and Rob personally showed me some of their data. While their results have not yet been published, the team has identified a brain microbiome that changes with age and in Alzheimer’s disease. However they are currently doing additional work to rule out any possible sources of contamination. The following 2018 studies support research on the brain microbiome + brain infection in general:

Multiscale Analysis of Independent Alzheimer’s Cohorts Finds Disruption of Molecular, Genetic, and Clinical Networks by Human Herpesvirus

Lead author: Joel Dudley, Icahn School of Medicine at Mount Sinai New York

The team detected a range of persistent viruses in the Alzheimer’s brain. These included herpesviruses, torque teno viruses, adenoviruses, and coronaviruses. They also performed a range of experiments which showed that HHV-6A in brain tissue is capable of regulating host molecular, clinical, and neuropathological networks in a manner that can contribute to inflammation and neuronal loss.

Paper highlight: “This study elucidates networks linking molecular, clinical, and neuropathological features with viral activity and is consistent with viral activity constituting a general feature of Alzheimer’s disease.”

Infection of Fungi and Bacteria in Brain Tissue From Elderly Persons and Patients With Alzheimer’s Disease

Lead author: Luis Carrasco, Universidad Autónoma de Madrid, Madrid, Spain.

The team used next generation sequencing + PCR + immunohistochemistry + antibody testing to search for fungi/bacteria in Alzheimer’s brains obtained from elderly control subjects. They identified range of fungi and bacteria in all brains. However, fungi from frontal cortex samples of the Alzheimer’s brains clustered together and differed from those of equivalent control subjects. The findings suggest that polymicrobial infection (pathogens acting together) may contribute to brain inflammation associated with neurological disorders.

Virus-like particles and enterovirus antigen found in the brainstem neurons of Parkinson’s disease

Lead author: Matthew Hannah, National Infection Service, Public Health England

Parkinson’s disease (PD) case: TEM image. There are intranuclear Virus Like Particles lining the internal face of the nuclear membrane of the nucleus (Hannah et al)

The team used both transmission electron microscopy and immunohistochemistry to study autopsied brain samples obtained from patients with late-stage Parkinson’s disease. They identified virus-like particles + enterovirus antigen in Parkinson’s brainstorm neurons.

Evidence of Human Parvovirus B19 Infection in the Post-Mortem Brain Tissue of the Elderly

Lead author: Modra Murovska, Rīga Stradiņš University, Latvia. 

The team studied brain autopsies of elderly subjects w/ molecular tools, immunohistochemistry + microscopy. They detected Parovirus B19 in brain tissue from encephalopathy + control groups “suggesting virus persistence within the CNS throughout the host’s lifetime.”

Brain glial activation in fibromyalgia – A multi-site positron emission tomography investigation 

Lead author: Marco Loggia, Massachusetts General Hospital, Harvard Medical School

The team used Positron Emission Tomography (brain scans) to show that brain glial cells were more active in patients with fibromyalgia as compared to heathy controls. Similar studies are underway for patients with the related inflammatory condition ME/CFS. The findings support a role for neuroinflammation in both disorders. And, as I state in my recent paper on ME/CFS, “these findings must be interpreted in light of novel infection-based paradigms and in concert with emerging data on the brain microbiome.”

14. Gut-brain axis signaling is a critical modulator of both health and disease processes: 

Microbes and their metabolites control bidirectional signaling between the gut and the brain via pathways collectively known as the gut-brain axis. The gut-brain axis involves various pathways including the vagus nerve, with signaling impacting neural, endocrine, and immune processes. A growing number of chronic inflammatory conditions are tied to disruption of gut-brain axis signaling. These are my two favorite 2018 paper on the topic:

Microbiome–microglia connections via the gut–brain axis

Lead author: Sarkis Mazmanian, California Institute of Technology

The paper discusses how the gut microbiome plays a pivotal role in regulating microglial maturation and function. It also reviews a range of studies which demonstrate that bidirectional crosstalk between the gut and the brain may influence the pathogenesis of conditions ranging from autism, to Schizophrenia, to Parkinson’s.

Paper highlight: Signals originating from the gut microbiota and transmitted to the brain have the potential to alleviate or exacerbate disease pathogenesis, changes that may operate through gut-mediated changes in microglial behavior. Thus, continued exploration of the intersection of microbiology, immunology, and neurobiology holds immense therapeutic promise.”

Lead author: Diego Bohorquez, Duke University

The team discovered a neural connection capable of rapidly transducing sensory signals from the gut to the brain. More specifically, they identified a type of enteroendocrine cell in the gut layer called the “neuropod cell.” These cells can communicate with sensory nerve fibers through direct cell-nerve contact. Neuropod cells can also secrete neuropeptides, and may subsequently convey information about nutrients in the gut to the brain by releasing quick-acting neurotransmitters.

15. Newly identified human structures + organ functions:

In 2018, several research teams discovered “new” human structures or pathways, several of which appear to allow immune cells (and the microbes that infect them) to bypass the classical “blood brain barrier.” The findings also suggest that the human body is much less compartmentalized than previously believed. Here are some of the top 2018 studies on the topic:

Functional aspects of meningeal lymphatics in ageing and Alzheimer’s disease

Lead author: Jonathan Kipnis, University of Virginia

In 2015, Jonathan Kipness and team became one of two research groups to demonstrate the existence of previously undiscovered meningeal lymphatic vessels. These fluid pathways connect the cerebrospinal fluid and cervical lymph nodes directly to the brain. In this follow-up study, the team found that dysfunction of meningeal vessel drainage increased Alzheimer’s pathology in mice.

Paper highlight: “…augmentation of meningeal lymphatic function might be a promising therapeutic target for preventing or delaying age-associated neurological diseases.”

Structure and Distribution of an Unrecognized Interstitium in Human Tissues

Lead author: Neil Theise, Mount Sinai Beth Israel Medical Center

The interstitium is shown above as the light pink layer at the bottom of this image.
Credit: Eric V. Grave/Getty

This study documents a previously uncharacterized fluid-filled lattice of collagen bundles that connects all human tissues. This “human interstitium” may be even be classified as a new organ. And because the interstitium drains directly into the lymph nodes, it may also connect immune cells + microbes to the central nervous system.

Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration

Lead author: Matthias Nahrendorf, Massachusetts General Hospital and Harvard Medical School

The team identified microscopic channels that connect skull bone marrow to the lining of the brain. Under conditions of inflammation these channels transport neutrophils (and possibly associated pathogens) directly from the marrow into the brain.

The vermiform appendix impacts the risk of developing Parkinson’s disease

Lead author: Viviane Labrie, Van Andel Research Institute, Michigan

The team found that, instead of being a “useless organ,” the appendix appears to be an immune tissue responsible for the sampling and monitoring of pathogens. They also found that appendix surgical samples removed from patients with Parkinson’s disease contained alpha-synuclein, suggesting that the Parkinson’s disease process might involve the appendix. Since alpha-synuclein appears to have potent antimicrobial activity, the findings further support a role for chronic infection in Parkinson’s.

16. Molecular mimicry contributes to disease processes:

In both acute and chronic disease, pathogen-associated proteins and metabolites are often identical or similar in structure to those created by their human hosts. This “molecular mimicry” or sequence homology between these proteins and metabolites can make it increasingly difficult for the human host to recognize “foreign” from “self.’ This can lead to dysfunction + signaling interference in patients with chronic disease.

Viruses.STRING: A Virus-Host Protein-Protein Interaction Database.

Lead author: Lars Juhl Jensen, University of Copenhagen, Denmark

The database documents known interactions b/t host and viral proteins. So far it’s documented 177,425 interactions b/t 239 viruses and 319 hosts. Now, consider that most human viruses have yet to be identified/characterized (the extent of these interactions are tremendous!)

Paper highlight: “Viruses act as metabolic engineers of the cells they infect as they commandeer the cell’s protein synthesis mechanisms to replicate…thus it is important to study how their disruption of the host protein–protein interaction networks causes disease”

Extracellular vesicles and viruses: Are they close relatives?

Lead author: Leonid Margolis, National Institutes of Health

The team found that viral vesicles and human extracellular vesicles (EVs) share considerable structural and functional similarity (molecular mimicry). These similarities are so extensive that it is difficult to distinguish EVs from (noninfectious) viruses. Since we now know the human virome is vast, it’s not surprising that components of our basic biology are connected to, or derived from, those of our viral inhabitants.

17. The human blood/circulatory microbiome:

Transcriptome analysis in whole blood reveals increased microbial diversity in schizophrenia

Lead author: Roel Ophoff, University California Los Angeles

Relative abundances of microbial taxa at the phylum level in ALS, Schizophrenia, Bipolar Disorder and healthy controls (Ophoff et al)

The team used high-throughput RNA sequencing from whole blood to analyze the blood microbiome. They identified a range of bacterial phyla in blood obtained from patients w/ schizophrenia, ALS, bipolar and HEALTHY CONTROLS…while making a serious effort to account for contamination. Can we please perform this exact same analysis on blood samples from patients with many more chronic inflammatory conditions!? 

Iron Dysregulation and Dormant Microbes as Causative Agents for Impaired Blood Rheology and Pathological Clotting in Alzheimer’s Type Dementia

Lead author: Etheresia Pretorius, Stellenbosch University, South Africa.

The paper describes mechanisms by which inflammation + oxidative stress (driven by re-activated microbes in blood) can lead to red blood cell deformability and dysregulated nitric oxide (NO) synthesis in Alzheimer’s patients. These processes may impair delivery of oxygen to the brain. The study features actual images of deformed red blood cells in Alzheimer’s (as seen under a microscope).

Identification and removal of contaminating microbial DNA from PCR reagents: impact on low‐biomass microbiome analyses

Lead author: Matt Payne, The University of Western Australia

Reagent-derived contamination (with microbes) can compromise the integrity of microbiome data, particularly in low microbial biomass samples (samples often obtained from the brain, blood, and womb). An analysis by the team found that the majority of contaminating DNA was derived from the PCR master mix. Importantly, this contamination was almost completely eliminated using a simple dsDNase treatment, which resulted in a 99% reduction in contaminating bacterial reads.

18. Novel Treatments + treatment paradigm shifts for chronic disease:

The standard of care for most chronic inflammatory disorders are immunosuppressive drugs or palliative therapies. However, a growing number of chronic inflammatory conditions are now tied to microbiome dysbiosis + persistent infection. Under such conditions, treatments that support or activate the human immune system could improve microbiome health by allowing patients to better target persistent pathogens. Treatments that target pathogens directly are also in development. This paradigm shift in treatment, that I have called for in several papers + articles, is already underway. Here are several 2018 papers related to the topic:

Targeting neuro-immune communication in neurodegeneration: Challenges and opportunities

Lead author: Michal Schwartz, Weizmann Institute of Science, Israel

Brain-immune communication points during aging and neurodegenerative disease (Schwartz et al)

This is definitely one of the most important papers of 2018. It describes how supporting the immune system in patients with neurodegenrative disease could form the basis of novel therapies. The team also contends that, when it comes to these neurodegenerative conditions, “autoimmunity” and immunosuppression are failing paradigms.

Paper highlight: “For decades, it was accepted that the CNS is an “immune-privileged site”…This view ascribed the inflammation in chronic neurodegenerative disease to autoimmunity. As a consequence, attempts were made to treat such conditions with immune-suppressive drugs, all of which failed”


“In conclusion, the development of a therapy that boosts the immune system in a well-controlled way, and thereby restores and/or activates brain–immune communication, is an outcome of a general shift toward the perception of the CNS as a tissue that engages in a constant dialog with peripheral immunity. Such an approach is expected to provide novel treatment modalities in order to harness common immune repair mechanisms to combat Alzheimer’s disease and perhaps other neurodegenerative diseases.”

Epstein-Barr virus–specific T cell therapy for progressive multiple sclerosis

Lead author: Rajiv Khanna, University of Queensland, Australia

This open-label, dose escalation trial evaluated the safety and efficacy of adoptively transferred in vitro-expanded EBV-specific T cells for patients with progressive Multiple Sclerosis. Clinical improvement was seen 7 of the 10 patients, with the greatest benefit for patients that received T cells with strong EBV reactivity.

Anti-herpetic Medications and Reduced Risk of Dementia in Patients with Herpes Simplex Virus Infections-a Nationwide, Population-Based Cohort Study in Taiwan

Lead author: WC Chein, Tri-Service General Hospital, National Defense Medical Center, Taiwan

This retrospective cohort study found that use of anti-herpetic medications in the treatment of Herpes Simplex Virus (HSV) infections was associated with a decreased risk of dementia. The findings could be a signal to clinicians caring for patients with HSV infections, especially since the herpesviruses are implicated in a growing number of neurological conditions.

Consumption of Mediterranean versus Western Diet Leads to Distinct Mammary Gland Microbiome Populations

Lead author: Katherine L. Cook, Wake Forest School of Medicine

Graphical abstract of Cook et al study

The team found that, in primates, eating a Mediterranean diet altered composition of the breast tissue microbiome + associated microbial metabolites in the region. The findings suggest that dietary changes may influence microbiome ecosystems outside the human gut.

Metabolomics activity screening for identifying metabolites that modulate phenotype

Lead author: Gary Siuzdak, The Scripps Research Institute

The paper is an excellent introduction to the field of functional metabolomics: the use of carefully identified metabolites to modulate diverse processes like stem cell differentiation, oligodendrocyte maturation, insulin signaling, T-cell survival and macrophage immune responses. Because metabolites used in this fashion directly modulate the biology of the host, they can be potentially be used to create new treatments for a range of conditions.

Paper highlight: “Applications of Metabolomics activity screening (MAS) could be expanded to disease modulation, biofilm initiation or suppression, drug–exposome interactions, plant biology and immunotherapy. Perhaps what is most intriguing is that rather than identifying metabolites to understand pathways, we can apply metabolites to modulate physiology, thereby turning the tables on conventional thinking.”

Defective respiration and one-carbon metabolism contribute to impaired naïve T cell activation in aged mice

Lead author: Marcia C. Haigis, Harvard Medical School

A perfect example of functional metabolomics at work. The  team found that mitochondrial proteome one-carbon metabolism is particularly blunted in aged T cells. But providing metabolites in one-carbon metabolism to the aged T cells resulted in improved activation and survival. This could could represent a new strategy to develop immunotherapies for a range of chronic conditions.

19. Vitamin D is not a “miracle” and recent RCTs do not support supplementation:

For a decade now, my team has published papers and book chapters that call for an end to high-dose vitamin D supplementation. Our research indicates that the low levels of vitamin D often identified in patients with chronic inflammatory disease may be a result, rather than a cause of the disease process (eg, the concept of “deficiency” is incorrect). These two large 2018 studies support an end to vitamin D supplementation:

Effects of vitamin D supplementation on musculoskeletal health: a systematic review, meta-analysis, and trial sequential analysis

Lead author: Alison Avenues, University of Aberdeen

This large meta-analysis published in the Lancet found vitamin D supplementation does not prevent fractures/falls, or have clinically meaningful effects on bone mineral density.

Paper highlight: “There is little justification to use vitamin D supplements to maintain or improve musculoskeletal health. This conclusion should be reflected in clinical guidelines”

Vitamin D Supplements and Prevention of Cancer and Cardiovascular Disease

First author: JoAnn Manson, Harvard University

This nationwide, randomized, placebo-controlled trial found that supplementation with vitamin D did not result in a lower incidence of invasive cancer or cardiovascular events than placebo.

20. No part of the human body appears to be is sterile: 

Microbial metagenome of urinary tract infection

Lead author: Amalio Telenti, J. Craig Venter Institute, California

Normalized abundance of bacterial genera across the clinical groups using 16S rDNA (Talenti et al)

The team used metagenomic tools to identify hundreds of pathogens + pathobionts capable of persisting in the urine of patients with a urinary tract infection (UTI). On average, samples taken from patients with UTIs contained ~41 bacteria genera, ~ 2 fungal species, and ~ 3 viruses. These and related findings on the bladder microbiome mark quite a paradigm shift from the bladder being “sterile.” I discuss the topic in more detail in this video.

Paper highlight: “Genomic analyses suggested cases of infection with potential pathogens that are often missed during routine urine culture due to species specific growth requirements.”

Implants induce a new niche for microbiomes

Lead author: Thomas Bjarnsholt, Costerton Biofilm Center, University of Copenhagen

The team found that previously sterile implants removed from joints, bones, pacemakers, and skulls of symptom-free patients were colonized by a range of bacterial and fungal organisms. The most prevalent microbes present on the implants were not organisms commonly associated with implant infections, suggesting the implants were colonized by microbes present in a range of human tissues. NOTE: A total of 39 negative control implants were included in the study to assess the false positive rate.

22. Inherited microbes and the maternal microbiome:

The maternal microbiome (the microbiome + virome communities passed from mother to child)…is extremely important. Organisms capable of persisting in the placenta and/or amniotic fluid may “seed” an infant over the course of nine months. After birth, infants acquire their mother’s microbes via the vaginal canal and the breast milk microbiome (among other sources). If these maternal microbiome communities are in a state of balance, infant health may benefit. However, if they are dysbiotic or contain certain pathogens, an infant may be more likely to develop a chronic inflammatory condition.

A Critical Review of the Bacterial Baptism Hypothesis and the Impact of Cesarean Delivery on the Infant Microbiome

Lead author: Jeffrey Keelan, The University of Western Australia

This important paper contends that lack of exposure to vaginal microbes is not a driving factor behind Cesarean delivery changes to the infant microbiome. Instead, it points to evidence showing that “indication for Cesarean delivery, intrapartum antibiotic administration, absence of labor, differences in breastfeeding behaviors, maternal obesity, and gestational age are major drivers of the Cesarean delivery microbial phenotype.”

Paper highlight: “Given the likely importance of amniotic fluid in pre-natal microbiome seeding, this finding is not surprising. Unfortunately, the authors did not investigate placental or amniotic fluid microbiomes from these mothers, as these may have been the true source of the neonatal microbiome.” 

The effect of breastmilk and saliva combinations on the in vitro growth of oral pathogenic and commensal microorganisms

Lead author: Christine Knox, Queensland University of Technology, Australia

The team found that neonatal saliva and breast milk interact to release antibacterial compounds (like hydrogen peroxide). This regulated oral bacteria growth for up to 24 hours, although certain species/pathogens continued to grow.

Maternal gut and breast milk microbiota affect infant gut antibiotic resistome and mobile genetic elements

Lead author: Marko Virta, University of Helsinki, Finland

The team found that antibiotic resistance genes, and related mobile gene elements, can be passed from mother to infant via breast milk.

Paper highlight: “…it seems that infants inherit the legacy of past antibiotic use, as they carry high loads of antibiotic resistant bacteria acquired from their mothers and the environment even prior to being exposed to antibiotics themselves.”

Microorganisms in the human placenta are associated with altered CpG methylation of immune and inflammation-related genes

Lead author: Rebecca Fry, University of North Carolina, Chapel Hill

The team, and found that 1000+ human genes were differentially methylated in relation to bacteria in the placenta. Impacted genes controlled growth factors, immune response proteins + inflammation (NF-kb pathway). These changes may impact pregnancy outcomes and fetal development.

23. Environment modifies human genome risk + the exposome:

How do a range of environmental variables + environmental exposures contribute to chronic disease processes? The following 2018 studies delved into the topic:

Convergence of placenta biology and genetic risk for schizophrenia

Lead author: Daniel Weinberger, Johns Hopkins University School of Medicine

The team found that schizophrenia gene risk loci that interact with early-life complications are highly expressed in the placenta. However, these loci were differentially expressed in placentas from women who suffered complications during pregnancy. They were also differentially upregulated in placentae from male compared with female offspring.

Transcription factors operate across disease loci, with EBNA2 implicated in autoimmunity.

Lead author: Matthew Weirauch, University of Cincinnati College of Medicine

The study found that in Epstein Barr Virus infected cells, EBNA2 (an antigen created by the virus) and its transcription factors modulated the activity of human genes associated with risk for multiple sclerosis, rheumatoid arthritis, type 1 diabetes and other conditions. In fact, nearly half of systemic lupus erythematosus risk loci were occupied by EBNA2 and co-clustering human transcription factors.

The exposome: molecules to populations

Lead author: Gary Miller, Emory University/Columbia University Medical Center

Environmental factors that contribute to the exposure (Miller et al)

The paper is an excellent introduction to the exposome: complex environmental factors that exert pressure on our health (chemicals, pesticides, radiation etc). Technologies that better characterize the exposome are improving, meaning we can better study how a range of environmental exposures impact the human the human immune response and the development of chronic inflammatory disease.

24. Don’t forget about archaea!:

Measuring the archaeome: detection and quantification of archaea signatures in the human body

Lead author: Christine Moissl-Eichinger, Medical University Graz

Archea matter too (darn it!). Archea are single-celled organisms somewhat similar to bacteria. They are increasingly being detected in most human microbiome communities. This team found an almost 1:1 ratio of archaeal to bacterial 16S rRNA genes in human appendix and nose samples. Identification of this archaea abundance and diversity required use of a very specific archaea-targeting methods + tools.

Interview with George Tetz: a look at how bacteriophages can act as novel mammalian pathogens

November 28th, 2018 by Amy Proal

George Tetz, MD, PhD is CEO of the Human Microbiology Institute in New York City. His research focuses on the study of bacteriophages: viruses that infect bacteria. Tetz studies how bacteriophages can contribute to microbiome dysbiosis in a range of human chronic inflammatory conditions. He also examines how bacteriophages can act as novel mammalian pathogens by interacting directly with human cells and the host immune response. His recent findings show an early role for bacteriophage activity in conditions such as Parkinson’s disease, Type 1 Diabetes and “leaky-gut” syndrome. 

Please watch this video of our interview/conversation:

Here is a written transcript of the interview. I have edited the content for purposes of accuracy and clarity:

Amy: George hi. You’re the head of the Human Microbiology Institute in New York City. Tell me about the Organization’s primary goals.

George: Sure. We’ve been working in the microbiome area for over the past 15 years. We set up the Human Microbiology Institute several years ago to combine efforts from our scientific team. Currently we’re pioneers in Phagbiome Research: for instance, we’ve shown that bacterial viruses (named bacteriophages) are actually previously overlooked human pathogens. It’s something people should be concerned about and is definitely an area of study that needs a lot more scientific research.

Amy: Yes. I was very excited when I read your paper “Bacteriophages as potential new mammalian pathogens.” Can you tell me more? And to give people context: We’ve been studying the human microbiome but have mostly been looking at just bacteria. How do we add bacteriophages into the picture?

George: Indeed, the predominant part of microbiome research right now is dedicated solely to bacteria. That’s in part due to a lack of effective methods for studying the whole microbial population. Because genome sequencing using 16S RNA – which right now is the most cheap and broadly spread method used by scientific groups – allows for only identification of bacterial species. 16S RNA sequencing does not allow for the study of many other details associated with other components of the human microbiome. So [several years ago] when I analyzed a variety of data showing that the human microbiome is implicated in the development different pathologies, I noticed that all the analyses lacked one very important component: they lacked bacteriophages.

Right now there are a lot of debates. For example the role and modulation of antibiotic treatment on the early childhood microbiota and the development of different diseases. And that’s definitely an important topic. However the most influential, and most important regulators of microbiota stability are not antibiotics. They are bacterial viruses (bacterophages). And all these studies were lacking a deeper analysis of what was happening with the bacteriophage communities.

Just a little background. Bacterial viruses (bacteriophages) are regular viruses but their hosts are not human cells or eukaryotic cells…they are bacterial cells. That’s actually one of the reasons why they were previously overlooked. No one paid attention to them because they seemed to infect bacteria and “that’s it.” However, as I mentioned, once the concept of microbiome alterations in disease appeared, then bacteriophages as regulators of the microbiota became something the scientific community must care about. It is very complicated because there’s a lot of dark matter (things we have yet to understand) when it comes to bacteriophages. First of all they outnumber the total number of bacterial cells in the human body by 10-fold. There are also a lot of previously unknown or not-yet known bacteriophages at the moment. However if we’re talking about the bacteriophages that are currently well-known and can be studied, we have tried to evaluate their role and implications in different diseases. That is what we currently do here at the Human Microbiology Institute.

Amy: Yes. Bacteriophages obviously play a major role in regulating activity of the bacterial microbiome. Can you explain more specifically how bacteriophages modulate bacterial behavior?

George: Sure. Let me go even broader and talk about how bacteriophages can impact human health. Because their interplay with bacteria is just one way they can affect humans. Our research team has separated how bacteriophages can impact human health into two main pathways. One is direct interplay with the host. The other is indirect: bacteriophages impact the host by modulating the bacterial microbiota.

When it comes to the first mechanism (direct interaction) we see two main components. First, bacteriophages can interplay with human cells. Even in 2017 this interaction remained unclear. Then, our colleagues showed that bacteriophages can directly interplay with human cells and penetrate the human body. And of course, most of the ways bacteriophages do this are still unknown. But there is a lot of current research from Polish Institutes showing how bacteriophages can directly interact and interplay with leucocytes…leading to alterations in cytokine production, and modulation of Toll-like receptors and the human immune response.

Our recent work has also identified a number of of prion-like domains on the surface of bacteriophages. These mis-folded prion proteins lead to the consequent appearance of other mis-folded proteins. And whether it’s amyloid beta in Alzheimer’s or alpha-synuclein in Parkinson’s, this leads to the deposition of highly neurotoxic composites in the human brain…which then leads to the development of neurodegenerative diseases that are unfortunately killing many people.

So the prion-like proteins we’ve identified on the surface of bacteriophages: they’re important because they can act as a “seeding component” or initial trigger for the prion mis-folding. We have some very interesting data on this that should be ready to share in 2019.

With respect to the indirect pathways by which bacteriophages impact human health…first of all, they can drive a decrease or increase in the number of certain bacterial populations in the human gut. For example, in a study we published on Parkinson’s disease, we identified that Lactococus bacteriophages led to a decrease in Lactococcus bacteria…which in turn led to the disappearance of these populations prior to the onset of first symptoms in the Parkinson’s patients. 

To go into more detail, the same concept has been noticed by other scientists in Crohn’s disease and in obesity – where the microbiota is already well-known to be associated with the triggering of those diseases. So we are expanding this research to other neurodegenerative diseases and certain “autoimmune” pathologies, including our latest research on Type 1 Diabetes. 

And a final indirect pathway is that, once bacteriophages kill bacteria, or lead to the disruption of microbial biofilms…that can lead to the release of pathogen-associated molecular patterns such as LPS or bacterial cell-free DNA…which in turn are pretty well-known triggers of cascades of immunological reactions that can affect (and be suggested as triggering factors) in different muti-faceted human diseases.   

Amy: Got it. Wow you’re looking at a lot of relevant topics. In the case of the Lactococcus phages, you found that they modulated the activity of bacteria that produce dopamine, correct?

George: Yes in the Parkinson’s study we compared two patient populations. One with very early-stage Parkinson’s (they were even treatment naive). The other was an age-matched control group. We identified that the Parkinson’s patients had a decreased number of Lactococcus bacteria, and this decrease was due to the highly lytic infection of these microorganisms with Lactococcus bacteriophages. And Lactococcus bacteria play a very particular role in the human gut. First, they are important regulators of intestinal permeability. And increased intestinal permeability is an important mechanism implicated in Parkinson’s that leads to the chronic inflammation. Also, Lactococcus bacteria are important producers of different neurochemicals, which are important components of the enteric nervous system. In particular, Lactococcus bacteria produce intestinal dopamine. And it’s fairly well-known that an initial step of Parkinson’s disease starts not in the brain, but in the enteric nervous system. And then, via the vagus nerve goes up to the brain, leading to alterations that are pretty well-known as Parkinson’s disease. 

So to highlight. In these patients we identified that bacteriophages killed microorganisms that are important regulators of the enteric nervous system…microorganisms that are known to be associated with maintenance and balance of intestinal dopamine. 

Amy: That’s what interested me most about the findings. It’s such a clear mechanism. There’s an actual connection between the dysbosis and neurotransmitter production.

George: Yes. We’re very happy because here at HMI we have not only bioinformatics staff, but also MDs who understand how certain microorganisms are involved in human biology (and what happens when something goes wrong with that).

Amy: That is great. Because you can get the bioinformatics data, but you have the biological knowledge with which to best interpret the data. So in the Type 1 Diabetes study what did you find with bacteriophages there?

George: We have not published yet. But we did a longitudinal microbiome study of children from birth to three years old. All children had certain HLA mutations that made them highly susceptible to Type 1 Diabetes. However, at three years of age, only a number of these subjects converted to islet autoimmunity and Type 1 Diabetes. Other subjects, despite having the altered genes, never developed signs of autoimmunity. What we identified is that the children that sera-converted to diabetes had, initially, very high levels of E. coli in their guts. But when we followed these E. coli populations over time, we found that the diabetic children, BEFORE the appearance of autoantibodies, had a complete disappearance of E. coli in their gut. This was due to the fact that those subjects had an active prophage infection, meaning that bacteriophages were responsible for the elimination of the gut E. coli populations. E. coli are members of the Enterobacter family. And it’s pretty well-known that one of the pathogenic-associated molecular patterns (PAMPs) that can be released from their biofilms is highly immunogenic. That means they can trigger other pathologies and diseases including, for example Systemic Lupus Erythematosus. So to summarize, we found that bacteriophages – through this indirect mechanism – led to the appearance of highly immunogenic proteins that in turn could trigger the “autoimmunity”, and in turn, Type 1 Diabetes.

Amy: Very interesting. So it doesn’t seem that what’s really going on in Type 1 diabetes is “autoimmunity” in the classical sense (a reaction to “self”). It seems that instead we need to better study how the immune system reacts to persistent pathogens… 

George: Yes a lot of research is dedicated to that topic. The most important questions is: “Even in people with altered genetics – why do some people develop these diseases and some not?” What happens in the patients who do develop them? And sometimes a simple microbiome analysis cannot complete that process, so it’s necessary to go into more detail in both humans and animals (research we are doing).

Amy: Right. Because now we treat “autoimmune” disease patients with strong doses of immunosuppressive drugs. How do you think those drugs impacts the survival of bacteriophages that may be driving a large part of the dysbiosis?

George: It’s dark matter (a basically unexplored topic). Because there’s not much data on how current therapies impact bacteriophages. Not in chemotherapy or antibiotics. Meaning that certain alterations that can follow from such therapies, and their impact on bacteriophages, is completely unknown.

Amy: Yes, there are very few studies that even look at the effects of immunosuppressive drugs on the bacterial microbiome. I find that frustrating. Because it’s concerning that our “standard of care” treatments focus solely on shutting down the immune response in “autoimmune” patients…when the microbiome and virome seem to be playing a large role in driving the conditions.

George: On the other hand, patients right now don’t have many other options.

Amy: Oh I know. I’m talking about future treatment development. 

George: One of the most important things in future treatment development is what we do here at HMI. If you can figure out the real cause of the disease, it is possible to develop programs for primary disease prevention. To completely eradicate the condition. But to do that you need to know what is the true, causal mechanism – what actually leads to the development of these pathologies.

Amy: Yes. So if we can better understand “root cause” mechanisms by studying bacteriophages and the entire microbiome…we can probably develop some better “root cause” therapies for “autoimmune” disease. That’s the goal right?

George: Absolutely. I agree with you.

Amy: Ok. I’ve read studies on bacteriophages that stem back over the past century. Why did bacteriophages almost “fall out of favor” in the mainstream research community until recently?

George: Yes. Historically there have been (and there continues to be) phage research centers in the former USSR (in Georgia). There are also a lot of USA and European companies that employ bacteriophages for the treatment of multi-resistant bacteria. There is a lot of sense for doing that. Because bacteriophages are highly selective, meaning certain phages kill only their specific bacterial hosts. Which is why people are trying to use bacteriophages as therapy to overcome antibiotic resistance. Some of these efforts have been very successful. For example, there are a number of bacteria like MRSA or multi-resistant Pseudomonas aeruginosa that infect children with cystic fibrosis. These strains are completely insensitive to the total panel of antibiotics, so the children have no other treatment options. The same is true of diabetic ulcers that are also caused by polymicrobial infections. There are attempts to use bacteriophages topically to eradicate such infections, which is great. 

So to be clear, what we are discovering here at HMI doesn’t go against such uses for bacteriophages (phage therapy). We’re talking about a different question: can bacteriophages (not those used for Pseudomonas, MRSA etc) be overlooked human pathogens?

Amy: Yes. In my opinion the situation is similar to how we use some bacterial species as probiotics. In those cases, we know certain bacterial strains have that capability. But we don’t use known pathogens as probiotics. The same would go for phages. If we better work to understand specific phage targets, we can better know which phages to use for effective phage therapy.

George: Yes extra knowledge of how bacteriophages contribute to human health can add an extra level of safety to the selection of phages for phage therapy.

Amy: Yes. Can you explain your research on bacteriophages and “leaky gut?”

George: Sure. The topic was our first publication in the area, with one paper done in collaboration with New York University. We identified that bacteriophages, acting as regulators of microbiota stability, can increase intestinal permeability. This leads to chronic inflammation. This chronic inflammation can, in turn, increase “leaky gut.” “Leaky gut” is associated with a variety of human diseases including neurogenerative diseases and autoimmune pathologies. 

We took rats and gave them a cocktail of different bacteriophages commonly found in waste water plants. So phages against Pseudomonas, Staph aureus, E.coli…and not (I want to highlight) against Lactococcus species or Fusobacterium species. The results were interesting. After the animals were given the strong bacteriophage cocktail with water (the phages were swallowed, not injected) the phages caused significant alterations in the rat intestinal microbiota (as revealed by whole genome sequencing). Moreover, we measured signs of increased intestinal permeability in the same animals. We found that all rats exposed to the bacteriophages had an increase in intestinal permeability and a decrease of Lactococcus bacteria (bacteria known to be important regulators of gut permeability). And again, we did not give the rats phages that would directly eliminate Lactococcus. It follows that bacteriophages can lead to a cascade of microbiota alterations, finally leading to a decrease in certain bacterial species. 

Amy: Yes and chronic inflammatory conditions are closely tied to the environment. So what you also showed is that phages in our environment – let’s say in contaminated water – could be a contributing factor in chronic inflammatory disease.

George: I don’t want to scare anyone, but waste water contains 10^8 bacteriophages on average. 

Amy: I don’t think that should scare anyone. We must face the reality of that knowledge to move forward.

George: Yes a lot of research must be done in this area.

Amy: Yes because we can easily pick up phages from our environment right? In fact, isn’t there a study which shows that ~30 billion bacteriophages traffic from the gut throughout the body every day?

George: Yes. It was published in 2017 in MBio. And the article also highlighted that bacteriophages can interact with human cells. 

Amy: Yes. So there are billions of bacteriophages in the human body. And they are found outside the gut frequently correct? In the cerebrospinal fluid, the placenta, the brain right?

George: Yes. 

Amy: So bacteriophages persist in most human tissue and blood.

George: Yes. And they can persist in two ways. One state is as free-presenting elements like lytic phages. Or bacteriophages can integrate into bacterial genomes (prophages). In those cases they can regulate how they exit the bacterial cell and lead to productive infection. So there can be bacteria in the cerebrospinal fluid (for example) that have phages within their genomes.

Amy: You are originally from the USSR? Is that how you got into bacteriophage research? 

George:  Yes. I’m originally from St. Petersburg, Russia and my sisters are from Estonia. I started doing microbiome research and then noticed the discrepancy in studies that did not account for bacteriophages. 

Amy: And bacteria can also modulate phage activity right?

George: Of course. Particularly when the phage is incorporated into the bacterial genome. There are a number of ways in which bacteria then interplay with the phages. For example, there are different ways bacteria manage the initial steps of phage infection (in an effort to prevent the phages from penetrating). 

Amy: What about inherited phages? Do you think inherited phages influence how diseases are passed in families?

George: We are working on that. Particularly, taking into consideration the fact that a very important part of the microbiome is translated vertically from the mother. That means a number of bacteriophages, both lytic and prophage, are inherited from our parents as well.           

Another important consideration is how bacteriophages influence the long-term safety of fecal microbiome transplantation (FMT). Particularly because a very big number of prophages (both lytic and lysogenic) enter the gut of FMT patients and colonize them. So in terms of the long-term safety of that…I have no answer yet. More research must be done to find algorithms that should be targeted to the individual patient.

Amy: What about passing phages from person to person. That happens right?

George: Yes just like viruses, bacteriophages are transmitted from person to person. The main difference is that bacteriophages can be passed as lytic phages or together with their bacterial hosts. 

Amy: Yes. Because there have been clusters or outbreaks of certain chronic conditions like sarcoidosis and ME/CFS. I’ve always wondered if there could be a sharing of organisms in such cases. 

George: Yes that poses another question – an important, although speculative question. If there are so-called bacteriophage diseases, can they be transmitted? And can they be contagious or not? From my perspective it can happen. But it may be that you need the “right” recipient for disease development…the person might not just have a certain genetic susceptibility…but also a certain microbiome. That adds an extra level of complexity that is hard to compute and analyze with current bioinformatics methods. But it’s something we work on and keep an eye on.

Amy: OK what’s next for you? 

George: It will be a surprise to everyone:) But something related to neurodegenerative diseases and autoimmune pathologies.

Amy: Great we need more research on that! Thanks for speaking with me today.


Interview with David Paez-Espino (JGI): Identifying novel viruses on Earth and in the human body

June 12th, 2018 by Amy Proal

Hey readers!

Viruses are the most abundant life form on the planet

This blog and my peer-reviewed papers explore how the human microbiome can impact chronic inflammatory disease processes. It is well understood that an extensive microbiome persists in the gut (hundreds of trillions of microbial cells in total!) In addition, extensive microbiome communities have been identified in every other human body site – from the brain, to the liver, to the lungs, to the bladder, to the placenta and beyond. We now realize that no part of the human body is sterile. This includes human blood, which is now understood to contain a microbiome (even in healthy individuals).

However it’s very important to understand that even when all this new human microbiome knowledge is taken together, the global research community has still not yet identified, characterized or studied many of the microbes and viruses in the human body. In fact, almost every new human microbiome study turns up new species of bacteria, viruses and fungi that were previously unknown to science. For example, just a few months ago, Stephen Quake identified thousands of species of bacteria, viruses and fungi in human blood that were simply not known to exist before the study was performed.

Viruses are the most abundant life forms on the planet, but we know particularly little about human viruses. In other words, there are an incredible number of viruses in the human body that have not yet been identified. This is especially true of bacteriophages (viruses that infect bacteria). A recent study estimated that 31 billion bacteriophages traffic the body on a daily basis. The human body also harbors double stranded DNA viruses and RNA viruses. Most of us are familiar with double-stranded DNA viruses like the herpes viruses. We are also familiar with RNA viruses like polio, measles and influenza.

But these well-known RNA/DNA viruses comprise just a tiny fraction of all the viruses capable of persisting in the human body. Many other related viruses are still in the process of being discovered!

It follows that we cannot study human chronic inflammatory disease without understanding that viruses we have not yet identified may play a role in many human disease processes. To do so would be like going to the rainforest, studying only 2% of the animals, and coming to conclusions about how the entire rainforest functions off that information alone.

Despite this fact many doctors have been taught to test for only 10-20 well-known viruses in their human patients. If these viruses are not identified in the patient, it is assumed that a virus (or group of viruses) cannot be driving or contributing to the patient’s disease. We must work hard to change this assumption, because it greatly prevents the medical/research communities from looking at a much broader picture of what might be going on.

David Paez-Espino

In order to best make this point, I interviewed David Paez-Espino. David works at the Joint Genome Institute in Walnut Creek California, in the Department of Microbial Genomics and Metagenomics. He and his colleagues have created new, complex computational technologies that help identify both novel and known viruses. In fact, they have started a project called “Uncovering the Earth’s Virome” (viral communities). This project searches for novel viruses not just in the human body, but in ecosystems across the planet – including the ocean, the soil, the air, and in other animals. These viral sequences are stored in the world’s largest public viral database: IMG/VR.

Over just the past few years, Paez-Espino and team have identified and sequenced many, many new viruses. In a 2016 Nature paper, they unveiled more than 125,000 partial and complete viral genomes, which boosted the number of known viral genes in the world by 16-fold. By the time they published a follow-up paper in 2017, the contents of the IMG/VR database had doubled from the figure referenced in 2016. By January 2018 the database had tripled in size. At that point Paez-Espino stated the following:

“Among the million viral sequences predicted, we have now identified over 34,000 of them targeting several microbial taxa for the very first time, we’ve associated new viruses to known microbial genomes (culturable and unculturable), and the vast majority of the gene content (over 21 million genes in total) remains hypothetical or unknown, meaning that there is tremendous potential for new discoveries out of that gene pool.”


In this interview, David and I talk more about these findings. We talk about how his team has identified all kinds of viral entities and how they focus on the identification of less-studied viral groups, e.g. giant viruses, and virophages (viruses that co-infect eukaryotic cells along with giant viruses). These giant viruses were not previously known to persist in the human body. We also talk about how the study of novel viruses holds incredible potential to improve our understanding of chronic and rare inflammatory conditions.

Here is a short video clip of part of our conversation. The full interview is below and has been edited for accuracy purposes:

Amy: Tell me about your job at JGI and what projects you’re working on now.

David: Over the past four years here at JGI I’ve started working on a project called “Uncovering the Earth’s Virome” – a very ambitious name, I know. We are trying to get a sense of what viruses are out there. To do that we are generating a comprehensive list or viral catalog of viruses from samples everywhere on Earth. I work in the Microbiome Data Science group (lead by Dr. Nikos Kyrpides), where we look for novel or known viruses in environmental samples…all kinds of samples: we have metagenomes, we have metatransciptomes. We also have some isolates. And we don’t just look for viruses – we also try to link the viruses to their specific habitats and to their specific hosts. Everything is computational. But then we try to validate our findings by working with other groups here at Berkeley or beyond.

Amy: Where do get your initial viral data from (that you then use to identify novel viruses)?

David: The JGI sequences many of these samples through collaboration projects (CSPs and FICUS programs). But we also have part of our public repository database (called IMG/M) represented by other groups’ data. We developed a virus prediction pipeline to identify viral sequences directly out of those samples, and have created IMG/VR (the largest virus repository integrated by isolates and microbiome-derived viral sequences). And everything is public. We are mining millions and millions and millions of nucleotides. So we have terabytes of sequences. And this collection of viral data in IMG/VR is very diverse – it includes marine/aquatic samples, terrestrial samples (like soil), human microbiome samples, and even microbiomes from mice, ruminants, air, arthropods etc.

Amy: So let’s say I sent you a blood sample. When it comes to identifying new viruses in this sample: What is your technology able to identify versus what a technique like PCR could identify?

David: If you’re using PCR to identify viruses you’re using an approach where you already know what you’re looking for (you are biased towards viruses that are fully characterized and have been entered in known databases). So PCR is useful if you have a well-known virus already understood to be a human health threat. For example, we could identify Zika virus that way.  

But in our case, we have different approaches. The first is a general global viral discovery pipeline. It does not only rely on genes from known viruses. Instead, we use the genes from known viruses to identify novel viruses from environmental samples: think of these novel viruses as, let’s say, the cousins of viruses already in a known database (their genomes are similar to known viruses but with a certain degree of difference). It’s a homology-based pipeline, but one that’s more sophisticated than the usual BLAST approach. 

A T4 bacteriophage

Our second approach is to target very specific groups of viruses that are unique. These include specific families from single stranded DNA viruses, double stranded DNA viruses, single stranded RNA viruses etc. In fact, we have developed specific tools that can identify hallmark genes associated with many types of viruses. These even include giant viruses. Giant viruses are eukaryotic viruses that have long genomes (sometimes 1 megabase, 2 megabases, even almost 3 megabases in size!). They also have very unique capsid proteins that can’t be found elsewhere. So we can develop and use these unique viral characteristics to create specific models for identifying similar viruses. This same process of identification goes for virophages.

Amy: What exactly is a virophage?

David: A virophages is a virus that co-infects a eukaryotic cell along with a giant virus. In the majority of cases the virophage is a parasite of a giant virus. But in some cases it seems that virophages only use part of the giant virus machinery to replicate within the final host (a eukaryotic cell that is often an algae or protozoa -as currently known). The infectious process is kind of like a Russian doll. There are many different levels of infection.

Amy: Where are you finding these giant viruses and virophages?

David: They seem to be everywhere. We are submitting a paper in which we found virophages in all kinds of environments including the human gut. Finding them in the gut was something people had speculated about but we finally proved it is true. Because most people don’t have the right tools to mine for virophages, or they don’t have the right tools to mine the database, or they don’t have samples from the right habitats or styles of life in the database. So a luxury we have here at JGI is that we can apply novel tools to a very wide variety of different samples – and we can pretty much find everything everywhere:)

Amy: Yes. It seems very important to stay up to date on the latest technologies for viral identification. Because if you don’t know HOW to look for a new virus you probably won’t find it.

David: Yes, if you only study already known viruses you may be missing part of a larger unexplored picture.

Amy: I’m interested identifying novel viruses that might play a role in the disease ME/CFS. Let’s say I had blood samples from patients with ME/CFS. Can I send them to you so that you can use your technology to search for new viruses in the samples? How would that kind of collaboration work?

David: We have two ways to collaborate. First we have official calls for research. About 40-60% of those projects get accepted. But those projects are more along the lines of the DOE (energy, carbon cycling, and environmental viruses rather than human viruses). 

But for identification of novel human viruses: We are developing new tools for the discovery of RNA and DNA viruses. And we need validation of these tools. So we want to apply them to all kinds of samples as opposed to just those from the environment. Also because everything is connected we sometimes find environmental viruses in humans and vice versa. So if you’re interested in human viral discovery we don’t have official calls for that research. But you can talk to me and say, “Hey I have these samples and I’d like to know if your RNA viral pipeline could work for my samples.” Then I can talk to my boss, and if we all have this common interest or a common outcome (a paper, a patent) we can hopefully find a way to collaborate.

Amy: What are some of the most interesting viruses you’ve identified in the human body? Do you mostly have gut microbiome samples?

David: So as I mentioned before, all the data we get is deposited in the interactive IMG/VR database. On the database website you can access the habitat of the samples to see where our human viral samples come from. At the moment IMG/VR includes viruses found in five different human body subtypes: skin, urogenital, salivary, gut, and the respiratory tract. 

One interesting thing we’ve found is that viruses are very specific to their habitats. That means if you find a virus in a marine environment it’s very unusual to find it somewhere else. You can find it across the globe in different oceans but, if it’s a marine virus, it’s a marine virus. The same is true of human viruses. If you have a human virus it’s probably going to be only found in the human body. And if it’s a human gut virus, only in the human gut. So, we saw very few connections between the different body subtypes. The findings are similar to those reported in a separate recent paper: The healthy human phageome. In that study, healthy humans were found to have a certain pattern of viruses always present in the gut. We also found that to be true – there seem to be marker viruses that could indicate the health status of a person. Probably because such viruses are maintaining a balance with the bad bacteria.

Amy: And in disease there might be shifts away from that core viral state?

David: Yes, definitely. For example, we have a very recent paper (in editorial status) where we compare the virome of IBS mice to that of healthy mice. And we found a certain core pattern in the gut virome of the healthy mice and a very stochastic pattern in the gut virome of the IBS mice. Basically, totally different virome patterns identified between the two groups. And those patterns may give us some leads as to what happens in human IBS. Hopefully the paper will be out soon.

Amy: Wow, you have some great papers coming out.

David: Yes. We are also doing work related to phage therapy. We are trying to predict certain phages and then predict their bacterial hosts. And of course, many of these bacterial hosts are harmful to humans. That gives us the opportunity to engineer the phages in ways that might allow them to best kill the bad bacteria.  

So, we predict phages in the human body and then say “This viral (phage) sequence is probably infecting this particular bacterial host.” But we are collaborating with other groups here at Berkeley on the project because, if we do identify a bacterial host, we need to know if we can culture it in the lab. Culturing the bacterium allows us to test it. We need to do that because our computer-based predictions are pretty robust – but we must be 100% sure that what we predict computationally holds up in the lab. Also, we need to confirm that the phage sequences we predict are perfect before attempting to use them as therapeutics.

Amy: That’s great. That’s what the phage therapy community needs right now. Much more information on verified phage/bacteria relationships (that can be easily accessed in a database). It’s also great for our general understanding of the microbiome. Because if we fail to account for the phages in any microbiome ecosystem, we’re unlikely to really understand the community dynamics. 

David: Yes. For example, if you study cell biology there’s another layer beneath that knowledge. You also need to understand molecular biology, because you need to understand molecules in order to understand cells. Similarly, if you want to understand bacteria in a microbiome community, it’s important to understand that the way the bacteria behave is based on their relationship to the phages. For example, there is a theory called “kill the winner.” Sometimes a bacterium grows exponentially in an environment. If its levels come back to equilibrium it’s usually because there is a phage that’s killing it. This equilibrium is built into the foundation of the ecosystem. And without understanding viruses you can’t fully understand that equilibrium. So yes, it makes sense to think that way.   

Amy: Yes that’s even true of an intervention like a probiotic. It would be helpful to understand how the bacteria in a probiotic might be modulated by neighboring phages.

So give me some numbers. I know it’s a ridiculous request. But about how many viruses are you finding in just the human samples you have so far?

David: So, starting with only DNA viruses…we have around a million sequences but some are redundant. So, if you only take the unique viral entities, we have about 400,000. Of those 400,000 about 30,000 or so are unique viral sequences coming from the human body.  

When it comes to RNA viruses, that’s a new ongoing project that we haven’t published on yet. But so far, we have around another 100,000 novel predicted RNA viral genomes. And about 20% of them are coming from human samples.

Amy: Do you see an end in sight?

David: There must be an end in sight, right? But the problem with viruses is that they also recombine. It’s complicated because it’s very hard to define a viral species. For example, there are cases where two closely related viruses are trying to avoid predation by bacteria, and to do that they recombine and create a third virus. Then…that third virus can recombine with another virus. Another example is that two viruses infecting the same host, at the same time, can also recombine. So, I think that the possibilities are countless and it’s going to be very complicated. We are definitely not even close to scratching the surface at all.

Growth rate of virus identification and microbial host prediction for the new release of IMG/VR database. Growth over time in the total and unique number of viral sequences in IMG/VR. (Image composite by David Paez-Espino).

Amy: It’s daunting but also exciting. Because we understand very little about the human microbiome and most ecosystems. So it’s great to realize there’s so much more we can learn about what might be going on.  

David: Right. We have a collection of ~21 million different genes from the viruses we’ve collected so far in IMG/VR. And 80-90% of these viral genes are hypothetical or have unknown functions. 

This same pattern is even true with E. coli. People have been studying E. coli for decades but we still don’t know the function of ~30% of E. coli’s genes. So, imagine how little we know about these novel viruses that are predicted from fragments here or there. It’s very complicated to make any kind of estimation.

Amy: It’s mind-blowing. To clarify: E. coli is one of the most well-studied bacterial species and we still don’t know a great deal about it.

David: Yes. E. coli is so well-studied that you’d think all the metabolic pathways are perfectly covered. But if you actually look at the databases, people still have no idea about what 30% of the E. coli genome is doing. So, these are exciting times, yes.

Amy: It’s clear that no matter what you’re studying related to the human body, you need to account for these viruses and other microbes. Whether you’re an immunologist, a neurologist etc. We need to account for their DNA/RNA and metabolites.

David: We have advanced a lot in science. Especially thanks to early studies of the microbiome that created reference genomes. But, for example, I recently went to a conference that discussed the drinking water microbiome. And they were not even testing for viruses (phages) in the drinking water. They are looking at 16S RNA sequences from about 20 known pathogens. And we’re are able to drink apparently safe water with just that knowledge. The basic necessities are covered. But – if we could understand even more about what microbes/viruses are in that water we could develop even better solutions.

This is especially true when it comes to human rare diseases. The majority of them could be caused by something we are not controlling. Medicine is doing a great job, but still – new knowledge about viruses can add to the picture. For example, now we understand that even some forms of cancer are caused by human microbiome dysbiosis accompanied by proliferation of a certain virus. So many of these novel or rare diseases could be better explained if we understand more about viruses and other microbes. 

This is also true for therapeutics (treatment). Now we can think about phages killing bacteria in a more precise way than antibiotics. Or we can use viruses that can be delivered to specific human tissue along with something that can access your immune system. You can design the virus to deliver something to the immune system “a la carte.” This precise medicine needs to evolve along with the new technologies.

My ideal job is applications. But I am also very pro basic science. For example – the CRISPR/Cas system. We are now engineering human cells from the use of bacterial genes that are designed to be a defense system against phage. So, the tremendous repertoire we have of novel genes can give us many new tools and technologies that we can manipulate at our will in the near future. We have been doing that for years, but the pace of discovery is now way, way faster. It’s a catalytic process: we are speeding up the different steps. 

Amy: Yes. The pace of discovery is incredible. I recently submitted a paper to a journal. They wrote back and asked me to give specific numbers for how many bacterial and viral species persist in the human body. I had to explain this wasn’t possible, and even if I made an estimate the number would change by the next month…or even the next day. 

David: Yes, for example, we have the largest viral database in this field. But even then, we’re only operating with assembled sequences larger than 5 KB. That represents less than 2% of all the sequences we have. Which is pretty much nothing. So, it’s not just the discovery pipeline that’s important. We can also go back and analyze metagenomes from 15 years ago. Because now we have different sequencing technology, we have different assembly technology, we have new binning algorithms, we have more robust models for gene annotation and functional predictions. And everything is still getting improved and improved and improved. So yes, as you said, there are no magic numbers. 

Amy: Definitely no magic numbers:) David thanks for speaking with me. I’m fascinated by your research and very excited to see where your findings lead. 














Re-thinking the theory of autoimmunity in the era of the microbiome

April 16th, 2018 by Amy Proal

Antibodies are Y-shaped molecules that target pathogen antigens

Hey there! Last podcast I mentioned the “theory of autoimmunity” and the range of immunosuppressive treatments that have stemmed from its promotion. I stated that the theory needs to be re-evaluated to account for the discovery of the human microbiome. Today I will go into even more detail on why that’s the case.

First, let’s talk about how the theory of autoimmunity gained popularity and widespread acceptance in the first place. Around the turn of the century, scientists began detecting Y-shaped molecules called antibodies in patients with a range of “acute” infectious diseases: diphtheria, tuberculosis, polio and pneumonia among others. Researchers (like the famous immunologist Paul Ehrlich) soon realized that these antibodies play a key role in helping the human immune system correctly target the bacterial and viral pathogens driving these and related diseases. They came up with a model of antibody activity that still largely holds up today. It contends that:

  1. Antibodies are released by B cells and T cells of the human immune system in response to an infectious threat: most commonly a bacterial or viral pathogen
  2. Antibodies target/neutralize these pathogens by recognizing the shape/size of unique protein molecules on the pathogen’s surface. These pathogen proteins are called “antigens.”
  3. When a human antibody correctly recognizes a pathogen antigen, the two molecules bind together. This “tags” the antigen and associated pathogen for further attack by other cells of the immune system (like white blood cells).
  4. The immune system begins to rapidly produce or “clone” more versions of the antibody that correctly “tagged” the pathogen. As more and more copies of this antibody pour into human blood/tissue, the immune system increasingly recognizes and kills the pathogen.

Today, almost every infectious disease is tied to antibody production. For example, when you get the flu, your immune system releases a range of antibodies in response to the flu virus. If all goes well, the antibody that best “tags” the virus’ specific antigens is “cloned.” More copies of this targeted antibody enter your bloodstream and the recovery process begins.

These same antibodies form the basis of the flu vaccine and related vaccines. In simple terms, a vaccine is made by taking an antibody already known to target a specific pathogen antigen – and injecting a small amount of this antibody into a patient. This “prepares” the patient’s immune system for the pathogen. Then, if the patient becomes infected at a later date, the immune system already knows exactly what antibody it should “clone” and rapidly release. The pathogen can then be neutralized and killed before it has a chance to spread.

OK. But what does this have to do with the theory of autoimmunity? In the 1930s-40s, researchers/doctors began to detect antibodies in patients with a range of chronic inflammatory conditions like lupus, rheumatoid arthritis, and multiple sclerosis. The natural next step would have been to search for chronic human pathogens whose presence could be tied to these antibodies. However, at the time (as described in my previous podcast), the human body was incorrectly believed to be largely sterile.

This confused many research teams working on the topic. How could they explain the presence of these antibodies when there were apparently no persistent microbes in the human body? They were forced to come up with their best guess as to what could be going on. They settled on a new concept that forms of the backbone of the theory of autoimmunity: the “autoantibody.” AKA, they postulated that in patients with these chronic inflammatory conditions, the immune system had somehow “broken down” or “gone crazy.” This confused immune system could then generate antibodies against antigens associated with the body’s own tissues. They named these hypothetical “self-targeting” antibodies “autoantibodies.”

Paul Ehrlich at his desk

At first the theory of autoimmunity and the idea that “autoantibodies” could exist met with resistance. Paul Ehrlich, the famous immunologist who had helped characterize antibodies in the first place, was particularly vocal. He wrote papers condemning the theory and coined the term “horror autotoxicus” to best counter it. Horror autotoxicus contends that the immune system has innate protection mechanisms that simply do not allow it to turn “against itself.”

But the scientific community wanted a consensus on the topic and needed simple guidelines on chronic inflammatory disease that could be given to doctors. Large conferences began to promote the theory of autoimmunity and detractors were increasingly pushed to the fringe. By the 1950s the theory started to be included in medical textbooks. Soon a growing number of “autoantibodies” were tied to different chronic inflammatory conditions. For example, patients with lupus test positive for an “autoantibody” that was named ANA. Rheumatoid arthritis was officially diagnosed if the “autoantibody” rheumatoid factor (RF) showed up on blood tests.

But a major question remained: if the theory of autoimmunity is correct, then what causes the immune system to fail so dramatically that it turns against the body’s own tissues? A number of theories attempting to explain this dilemma have been proposed over the years, none of which has been proven. One involves infection and is still frequently referenced today: the “pathogen/trigger” theory. It contends that certain well-characterized pathogens may infect a patient. Something about this temporary infection goes wrong and “triggers” the immune system to misfire and induce “autoimmunity.” The pathogen itself is somehow killed, but the immune system never recovers and the patient develops a full-fledged “autoimmune” disease.

The “pathogen/trigger” theory might have held more weight if the human microbiome had not been discovered around 2005. As described here, the discovery occurred when research teams started using new molecular tools to search for human microbes. The results of these new analyses were astounding: entire ecosystems of microbes were identified in the human body that had been missed by previous laboratory testing methods. Today we understand that trillions of microbes live in and on us – from the moment we are conceived in the womb until the day we die. These vast microbiome communities persist in every human body site – from the brain, to the liver, to the lungs and beyond.

This means that there are actually thousands, if not more, microbes/pathogens in the human body that could be tied to “autoantibody” release in patients with chronic disease. In other words, “autoantibodies” may just be regular antibodies created in response to pathogens that our testing methods were previously unable to detect. So, for example, when the “autoantibody” ANA is identified in patients with lupus, it could simply indicate that an unidentified pathogen plays a role in driving the lupus disease process. Also, it’s more likely that “autoantibodies” are created in response to chronic, persistent human pathogens than a temporary pathogen that the immune system somehow kills (as postulated by the “pathogen/trigger” theory.)

The possibility that “autoantibodies” are created in response to microbiome pathogens rather than “self” is strongly supported by the fact that “new” bacteria, viruses and other microbes continue to be identified in the human body. For example, Stanford researcher Stephen Quake recently detected thousands of never-before identified bacteria and viruses in human tissue/blood. In fact, 99% of the microbes he identified were previously unknown to science.

Also, for decades, “autoantibodies” have been regularly detected in patients with no signs of autoimmune disease that are instead suffering from an infection. For example, high levels of “autoantibodies” like rheumatoid factor, ANA (and others like ASCA, annexin-V and Anti-PL) have been identified in patients with bacterial, viral, and parasitic infections ranging from hepatitis A/B to Q fever to Rickettsia.

E. gallinarum under a microscope

A more recent study by researchers at Yale came to a similar conclusion. The team studied E. gallinarum, a bacteria they identified in the human gut, liver, spleen and lymph nodes. In models of genetically susceptible mice, the researchers found that E. gallinarum initiated the production of “autoantibodies,” activated T cells, and inflammation. Moreover, this “autoantibody” production stopped when they suppressed E. gallinarum’s growth with the antibiotic vancomycin and/or with a vaccine against the microbe. The team also identified E. gallanarum in the livers of patients with “autoimmune disease”, but not in healthy controls. One media headline on the study read: “The enemy within: Gut bacteria drive autoimmune disease.”

Research by Stanford’s Mark Davis supports the findings. Davis and team used a new testing method to obtain T cell sequences from the tissues/blood of patients with colon cancer, MS, Lyme disease, and ME/CFS. In all four diseases, they found that the T cells were activated and “cloned’ in a manner not observed in healthy subjects. According to the theory of autoimmunity, these cloned T cells would indicate “autoantibody” production. But Davis suggested that associated antibodies are likely formed “originally against some pathogen peptide.” This definitely makes sense since since, especially in Lyme disease, we know pathogens drive the disease process.

Interesting right!? But at this point I should bring up an important concern. Some researchers are still convinced that “autoantibodies” can target human tissue. If this is the case, the situation can easily be explained by a concept called “molecular mimicry.” Molecular mimicry refers to the fact that pathogen proteins and human proteins are often very similar in size and shape. This means that an antibody created in response to a pathogen protein/antigen might accidentally target a similarly structured human protein/antigen. This “collateral damage” could result in an inflammatory response towards that human tissue.

Think of molecular mimicry this way: Let’s say you’re a solider in an army. You and your fellow soldiers all have red uniforms, blonde hair, and stand about 6 feet tall. You confront the enemy on a battlefield only to realize that their soldiers also have red uniforms, blonde hair and are about 6 feet tall! Even though their red uniforms are shaped somewhat differently than your own, at a certain distance the shape becomes blurred. This means that when the battle begins, you have trouble telling who’s on your side and who’s the enemy. Occasionally you end up accidentally shooting a member of your own army. These two armies can be compared to similarly shaped pathogen antigens and human antigens that might generate “collateral damage” in “autoimmune disease.”

Ample research supports this “molecular mimicry” model. For example, one research team found that B cells infected with Epstein Barr Virus secrete antibodies that also react with human antigens like albumin, renin, and thyroglobulin. Another Canadian research team found that proteins created by the hepatitis C virus had a high level of structural similarity with ~20,000 human proteins. Researchers in India identified tens of thousands of possible interactions between proteins created by Salmonella, E.coli, Yersinia, and similarly shaped human proteins.

All this research strongly suggests that in 2018 we no longer need the theory of autoimmunity. It seems Paul Ehrlich was right a century ago when he argued for “horror autotaxicus.” In patients with a range of chronic conditions, activated B cells, T cells and the antibodies they produce are likely targeting newly discovered human pathogens rather than “self.” This means the concept of the “autoantibody” is incorrect. In lieu of “autoimmunity,” we should focus on better characterizing pathogens in the microbiome and better studying their activity and survival mechanisms. For example, Mark Davis is planning 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). Excellent, excellent idea!

Most of the top-selling drugs in the world are immunosuppressive

At this point you might say: “Aw, well it looks like theory of autoimmunity will die over time, no huge rush.” But unfortunately the situation is very urgent. That’s because a large chunk of the pharmaceutical industry is focused on creating drugs based on the theory of autoimmunity. These drugs shut down extremely important parts of the human immune system in an effort to stop “autoantibody” production and related inflammation. In fact, these immunosuppressive drugs are the top-selling medicines in the world, generating billions of dollars of revenue each year.

If “autoantibodies” are created in response to pathogens rather than “self”, these drugs are actually hurting the long-term health and microbiome balance/health of patients with “autoimmune disease.” This is almost certainly why patients taking immunosuppressive drugs tend to get sicker over time, to the point where they often fall ill with a second or third inflammatory condition while taking the medications. In fact, rampant use of immunosuppressant drugs is likely a primary factor driving the current epidemic of chronic disease (the incidence of nearly every “autoimmune condition” is on the rise).

That means we stand at a crossroads. This paper published just last year describes an entire new generation of immunosuppressive drugs currently being developed by pharmaceutical companies. Some of these new drugs appear to shut down the human immune system even more profoundly than the ones prescribed today.

OR, we can move in a new direction of drug/treatment discovery. We can ditch the theory of autoimmunity and instead develop treatments that SUPPORT the human immune system. And/or treatments that better target key pathogens and, in turn, promote balance, health and diversity of the human microbiome. Then, maybe…just maybe we can create a future where new treatments target the ROOT CAUSE of human inflammatory disease instead of just palliating symptoms. And then maybe…just maybe a growing number of patients with chronic inflammatory disease could reach a state of actual recovery and remission.



Antibiotics, immunosuppressive drugs and the downfall of the human immune system

March 31st, 2018 by Amy Proal

Hey! Today I’ll discuss two more factors that can debilitate the human immune system (allowing the microbiome to better cause disease). They are antibiotics (when used too frequently) and immunosuppressive drugs: two of the most common medical interventions of our time. Most of us are familiar with antibiotics. By immunosuppressive drugs I mean medications like Humira, Prednisone, Ritauximab, and Enbrel. These and similar drugs block or disable parts of the human immune response in an effort to temporarily palliate inflammatory symptoms.

It’s important to note that most antibiotics and immunosuppressive drugs were developed before mainstream science “discovered” the human microbiome around the year 2005. In other words, they were created/formulated during a period when the human body was assumed to be largely sterile. The antibiotic Penicillin was discovered by Scottish scientist Alexander Fleming in 1928. By the mid 1940s it was regarded as a wonder drug, especially during World War II when it saved countless soldiers from dying of infected wounds.

After World War II, antibiotics continued to be perceived as miracles, and it’s easy to understand why: diseases like pneumonia, scarlet fever, bacterial meningitis, gonorrhea and a range of ear, skin, urinary and throat infections could finally be treated! Life expectancy increased as antibiotics allowed patients to survive an ever-growing range of these specific bacterial infections. And since the human body was considered sterile, it was assumed that antibiotics only killed the single bacterial pathogen they were prescribed to treat. They were subsequently prescribed with increasing frequency, and for minor infections like colds or sore throats (especially in children).

Alexander Fleming discovered Penicillin in 1928

Then, in the early 2000s, new molecular tools were developed that identify microbes by their DNA/RNA rather than their ability to grow in a laboratory Petri dish. When researchers turned these new tools on the human body, they discovered thousands of microbes in human tissue/blood that had been missed by previous techniques. With each new molecular analysis, mainstream science realized that the human body is the opposite of sterile! Instead, humans harbor vast ecosystems of bacteria, viruses, bacteriophages and other microorganisms. The human gut alone harbors trillions of microbes. These complex microbial communities were named the human microbiome – the “discovery” of which marks one of the most important turning points in the history of science.

Almost every facet of human health and biology must be re-evaluated to account for the human microbiome. When it comes to antibiotics we face a stark reality: Current antibiotics like penicillin have such a broad range of activity that they kill most bacteria in the human microbiome in addition to any single pathogen they are prescribed to treat. The situation could be compared to dropping an atomic bomb on an entire continent in an effort to target a group of rebels in one small city.

What are the implications of this? Research on the topic is still in early stages. But we know two things:

  1. Health, diversity and balance of the human microbiome are key to prevention of human inflammatory disease
  2. Microbiome populations decimated by antibiotics can recover, but sometimes not to their full, previous capacity

For example, in this Ted talk, researcher Rob Knight charts how an antibiotic prescribed to treat an infant’s ear infection impacted several of the child’s early microbiome communities. The antibiotic caused “a huge change in community structure” that “set back many months of (microbiome) development.” Interestingly, the infant’s microbiome eventually recovered to a place similar to that of untreated subjects. But as Knight suggests, it’s hard to imagine that there are no longer-term implications associated with the event (especially if the infant is administered even more antibiotics over time). And, as a society, are we taking more antibiotics? We sure are! From 2000-2015 approximately 65% more antibiotics have been sold globally (accounting for 34.8 billion doses in 2015). In the USA, 47 million of these antibiotics are prescribed annually for viral infections that don’t even respond to antibiotics.

With those numbers in mind, it’s no surprise that a second major antibiotic issue has also surfaced. Antibiotics have been prescribed with such frequency that many major pathogens have developed resistance against them. As discussed in previous podcasts, pathogens can easily change their gene expression to survive under difficult circumstances. Many human pathogens have done exactly that – they have acquired genes or mutated in ways that allow them to remain alive in the face of our most commonly used antibiotics. This has created a range of antibiotic-resistant pathogens often referred to as “superbugs.” Many of these “superbugs” are deadly (Acinetobacter baumannii, Pseudomonas aeruginosa, and MRSA for example). In fact, the CDC estimates that these and related “superbugs” kill at least 23,000 Americans a year (for comparison, about 38,000 Americans die every year in car crashes).

“Superbugs” have developed resistance to most common antibiotics

The situation is regarded as a global catastrophe. According to the CDC, the rate at which new strains of antibiotic-resistant bacteria have emerged in recent years “terrifies public health experts.” Many consider new “superbug” strains to be just as dangerous as emerging viruses like Zika or Ebola. Britain’s chief medical officer, Sally C. Davies, has described drug-resistant pathogens as a national security threat equivalent to terrorism. The problem is compounded by the fact that a large number of bacteria in the human body have yet to even be characterized and named. Who knows what our overly zealous use of antibiotics is doing to these understudied bacteria! It’s very possible that heavy antibiotic use may be pushing MOST bacteria in the human body into a more virulent, resistant state.

Add to this a third major antibiotic problem. Over the past decades, farmers started feeding antibiotics to many of the animals we consume as food (cows, chickens, pigs etc). Why? Antibiotics cause the animals to gain weight, increasing the amount of meat that can be sold to consumers. Again, few people paused to consider the long-term or chronic consequences of saturating our food supply with antibiotics. Instead, approximately 80% of antibiotics sold in the United States are used in meat and poultry production. This has created an amazing atmosphere for the development of virulent pathogens that may better resist antibiotics. In addition, genes conferring antibiotic resistance are regularly transferred from farm animals into the human food supply. If ingested, these antibiotic resistance genes can theoretically be transferred and “taken up” by almost any bacterial species in the human body.

To make matters worse, antibiotics were added to the food supply during the first time in history where the average 1st world inhabitant started eating meat imported from around the world. For example, the USA imported 3.01 billion pounds of beef in 2016, from 22 countries across the globe (from Europe, to South America, to China and Thailand). Consumers are subsequently exposed to meat-associated pathogens from around the world – basically a potential cocktail of global pathogens. For example, a study by researchers in Germany found high levels of S.aureus and E.coli in pork/poultry imported into the EU, with other pathogens like Salmonella and Enterobacteriaceae present in lower amounts.

The above questions the interpretation of studies that tie meat consumption to chronic inflammatory conditions like heart disease. If a negative association between the two is identified, is the meat itself to blame? Or are pathogens in the meat to blame? (or a combination of both and/or other variables). The relatively unexplored possibility that pathogens in meat may promote chronic disease is supported by the fact that heart disease is increasingly tied to infection and microbiome dysbiosis. A recent study even found that lipids or fat molecules in arterial plaque can be created by persistent bacteria. Also, to be fair, the possible impact of food-borne pathogens on chronic disease applies to most foods – for example, what do the range of pesticides and chemicals sprayed on plants across the globe do to the microbes that live in/on the produce we consume?

Now I’ll take the topic one step further, by posing a rarely asked but extremely important question: “Where has the human immune system been during the modern era of antibiotics (1940-today)? Because, as I’ve argued in previous blogs/papers, the healthy, activated human immune system has incredible defense strategies for keeping antibiotic resistant bacteria or “superbugs” in check.

After the turn of the century, a range of chronic conditions were linked to persistent inflammation. This chronic inflammation generally results when the immune system attempts to target pathogens. However at that time (as mentioned) the human body was incorrectly assumed to be sterile. Researchers subsequently developed the “theory of autoimmunity” – the belief that in diseases like Crohn’s, Multiple Sclerosis and arthritis, the immune system “loses tolerance” and attacks its own tissues.

Most of the top-selling drugs in the world are immunosuppressive

By the 1960s-80s the theory of autoimmunity had gained incredible traction. Over 80 different conditions were deemed “autoimmune” in origin. The result? Pharmaceutical companies created drugs specifically designed to shut down key parts of the human immune system. These drugs, which temporarily palliate symptoms, have been prescribed so aggressively that today they are the top-selling medications in the world. For example, the potent immunosuppressive drug Humira is literally the top-selling drug in the world: generating more than 18.4 billion dollars in revenue in 2017.

This means we have been using antibiotics at the same time that we have deliberately compromised the immune systems of a growing chunk of the human population. In fact, it’s no coincidence that most “superbugs”  form or are identified in elderly, sick and/or hospitalized patients; patients much more likely to be on cocktails of immunosuppressive drugs/steroids. This trend escalates when severely immunocompromised patients are gathered in one place: a recent analysis found that pipes under NIH’s ICU rooms tested positive for bacterial plasmids that confer resistance to carbapenem antibiotics. The pipes were described as a “seething hookup zone for antibiotic-resistant bacteria.” Another report found that many veterans of the Iraq War suffered from antibiotic-resistant A. Baumannii infections. But the “superbug” was almost always identified in wounded veterans with complex injuries. Indeed, the report clarifies that,  “A. Baumannii  is not particularly virulent. If you’re in good health and (it) were to get into a wound it might not be a problem. It’s a problem for people who are the weakest of the weak.”

The most common treatments for “non-autoimmune” conditions are also immunosuppressive to various degrees. Opioids, antidepressants and antipsychotics dampen the immune response (especially when administered over long periods of time). Certain forms of birth control have been shown to lower immunity. Cancer chemotherapies substantially weaken the immune system. Indeed, childhood cancer survivors are prone to a wide range of health problems later in life including organ damage, cognitive deficits and high blood pressure. These long-term health issues are similar to those observed in patients administered “classical” immunosuppressive drugs. Even in animals given antibiotics for weight gain – do the overcrowded/unsanitary conditions at many farms favor “superbugs” by lowering animal immunity?

All in all, we are living in an era of mass immunosuppression. This has created a field day for antibiotic resistant and virulent pathogens! Such pathogens can easily thwart the human immune system because our drugs are “knocking it down” for them. We are greatly assisting the enemy.

Most concerning is that the “theory of autoimmunity” no longer holds up in the era of the microbiome. A growing body of research suggests that “autoantibodies” are created in response to microbiome pathogens rather than “self.” In addition, “autoimmune” conditions are increasingly linked to microbiome dysbiosis and not a defective immune response. So we are using immunosuppressive drugs to treat patients based on a failing and outdated paradigm. For more on this topic please read this paper.

To combat antibiotic resistance we must subsequently re-evaluate the “theory of autoimmunity” and the immunosuppressive drugs that have stemmed from its promotion. And we must do so NOW. We cannot afford to wait the decades it usually takes for an outdated theory to be replaced by a new, improved paradigm (Thomas-Kuhn style). Too many lives and the health of the entire public are at stake.

How else can we fix the situation? At least in the short-term, antibiotics and immunosuppressive drugs should be considered last resort treatment options. Patients should exhaust all other treatment options BEFORE being prescribed these medications. We must prioritize diet/lifestyle changes that the average doctor does not yet include in treatment plans. Although this may sound simple, it’s easier said than done when drug companies spend millions of dollars a year marketing immunosuppressive medications directly to physicians.

In the longer-term we should replace immunosuppressive therapies with new treatments that support the immune system and encourage balance + diversity of the human microbiome. This would require another major paradigm shift that I describe more here.

This last paradigm shift is essential because we NEED some antibiotics. When administered in a careful, responsible fashion antibiotics still save countless lives. Antibiotics are also a valuable tool for treating chronic inflammatory conditions that are increasingly tied to infection. For example, this article describes how a young boy with crippling OCD used antibiotics to treat a chronic strep infection. His OCD symptoms diminished to the point where he is able to function in society again.

I have several suggestions for how to best move forward:

  1. Antibiotics should be re-branded as complex, immunomodulatory drugs: Few people realize that antibiotics have profound effects on the immune system in addition to their antimicrobial properties. For example, many commonly used antibiotics (rifampin, cephradine, tetracycline, clindamycin, and penicillin among others) can bind directly into the human PXR nuclear receptor: a receptor that controls how the body detoxifies foreign substances. The PXR in turn modulates CYP3A4 – an enzyme key to controlling the human immune response. These and related interactions may explain why antibiotics like minocycline are regularly used as “anti-inflammatory” or “immunomodulatory” drugs in patients with arthritis and other rheumatic conditions. 

So imagine if every time an antibiotic was prescribed doctors were required to say: “I’m giving you a powerful medication that will impact your entire microbiome and immune system in profound ways still not fully understood by science.” This would give people pause before prescribing and/or popping an antibiotic for the common cold.
  2. We should keep this complexity in mind when developing new antibiotics: The superbug crisis has set off a wave of research aimed at creating new antibiotics. Many research teams working in this vein are trying to develop antibiotics that would target specific bacterial pathogens in lieu of the entire bacterial microbiome. This makes sense, but part of the trend involves taking parts of the human immune system and trying to make them into new antimicrobial drugs.

For example, researchers at Vanderbilt University recently made a fascinating discovery: human milk oligosaccharides (HMOs) in breast milk possess strong antimicrobial and antibiofilm activity against several superbugs (Eg: A. baumannii and MRSA). Several research teams responded to the finding by suggesting that these HMOs should be made into new antibiotics. While this makes sense in theory it also runs a large risk – any pathogen that develops resistance against these HMOs will have developed resistance against a key component of the human immune system itself.

The pathogen A. baumannii

What makes the above scenario likely to occur is that the human body constantly adapts its antimicrobial defenses to keep up with the changing nature (evolution) of virulent pathogens. For example, in this interview, Harvard researcher Robert Moir describes how the human immune system regularly changes the structure of the human antimicrobial peptide cathelicidin in response to different pathogen threats (each different form of cathelicidin is called an oligomer). The flexible nature of these oligomers means that when a pathogen mutates to survive in the face of the peptide, the peptide can change its own structure/function to “fight back.”

Human drugs based on only a single form of a human antimicrobial peptide/molecule would lack this flexibility, making resistance against them much more likely.

Ok that’s it for today. I’ll leave with the following message. Let’s not get so worried about current “superbugs” that we rush into making new antibiotics without fully considering long-term consequences. We’ve already done that once right? As the saying goes, “Fool me once, shame on you; fool me twice, shame on me!” The same goes for phage therapy – another proposed alternative to antibiotics. In this interview, Texas A&M researcher Ry Young does a great job explaining how we can move forward with phage therapy in the most proactive and responsible fashion.

Also, not everyone is surprised by the recent surge in antibiotic resistance and superbugs. Certain communities of researchers have been warning about the possibility since the 1960s. I will also do another podcast that talks about these researchers and several of their key findings – findings that should still be taken into consideration today.

The power of intracellular pathogens: how they “take down” the immune system and dysregulate human pathways

March 12th, 2018 by Amy Proal

A human white blood cell

Hey there! Last podcast I talked about how many “harmless” microbes in the human body are able to evolve to a state where they cause inflammation and illness. I explained that this “shifting” towards virulence (or disease) is much more likely to occur in people whose immune systems are compromised or debilitated. There are many factors that can negatively impact the health of the human immune system. I plan to discuss many of them in greater depth. But today I will focus on one major factor – the ability of key pathogens to slow and dysregulate the human immune system as part of their most basic survival strategies.

The most fundamental way that pathogens “take down” the human immune system is by infecting and living inside human cells – especially human white blood cells. Under conditions of health, these white blood cells traffic the body, where they engulf or digest pathogens that originate from both the internal and external environment. BUT, many key pathogens have evolved to thwart this process. They have evolved to remain alive inside these white blood cells; inside the very cells that are supposed to kill them! They can survive this way for long periods of time in a chronic, persistent state. Doing so clearly gives them a HUGE advantage or “edge” over their human host. If the situation was compared to a human war, it would be as if a soldier were able to inhabit the body of its opponent, and then make the opponent act in its own interest.

There are two main implications of this “intracellular persistence”:

1. The infected cell is unable to perform its basic immune system duties.
2. Any pathogen inside the infected cell can interfere with the way the cell “encodes” its human DNA.

Let me explain this second point in more depth. The center of every human cell has a copy of a person’s DNA. This DNA “codes” for the ~ 21,000 human genes that determine who we are and how we deal with our environment. These genes are eventually “translated” into proteins – proteins that direct our human pathways to correctly regulate the immune system, the nervous system, human metabolism and other critical aspects of human function. It follows that any pathogen able to persist in the center of a human cell can directly “mess with” the cell’s DNA and the vital human processes it regulates. The situation could be compared to a computer hacker. Imagine a hacker that not only breaks into your computer, but gains access to the programming code that directs your operating system and websites. By either directly altering this code, or changing the way the code is translated into programs, the hacker can cause your computer to malfunction in an almost infinite number of ways.

Here’s an example of how a pathogen can dysregulate a human cell in the above fashion. A research team in Louisiana studied what happens when Cytomegalovirus (CMV) infects a human white blood cell. They found that CMV significantly changed how the cell expressed its human genes. In fact, the virus “turned up” the expression of 583 human genes, and “turned down” the expression of 621 human genes. These changes were so profound that the infected white blood cells actually changed into an entirely different type of cell – an “inflammatory” cell that promoted CMV’s ability to infect neighboring cells. No wonder then that, as the paper points out, chronic CMV infection is associated with retinitis, gastrointestinal disease, hepatitis and pneumonia – but also with chronic inflammatory conditions like multiple sclerosis and atherosclerosis (heart disease).

Cytomegalovirus (CMV)

A recent study by researchers at Stanford confirms how broadly CMV can “re-program” the human immune response. The team studied identical twins and isolated cases in which one twin was infected with chronic CMV and the other was not. They tested the activity and function of 204 different parts of the human immune system in each twin. These 204 immune system “parameters” included the amount/frequency of certain immune cells, the ability of such cells to signal, and the concentrations of immune system proteins called cytokines. They found that, in general, 119 of these immune system “parameters” were altered in the twins with CMV. In fact they calculated that, in people infected with CMV, “the lifelong need” to control the virus causes approximately 10% of all T cells in the body to become directed against it. This led the 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.”

OK – but how exactly does this major pathogen interference happen? All human pathways that control the immune response, metabolism, cognition etc are regulated by receptors – protein molecules that control pathway signaling. In very simple terms, receptors function in a “lock and key” fashion. Imagine that you want to get into a very secure house. You might have to first open locks on an outside door. Then you might open or adjust a series of other locks on inner doors and/or other checkpoints before finally entering. Now, picture these locks as receptors associated with a human pathway. Under conditions of health, very specific human proteins bind into each receptor. These proteins are analogous to the keys in my house example. If the correct human protein or “key” fits into the correct human receptor or “lock,” the receptor/protein “complex” changes shape in a way that allows that part of the pathway to correctly move forward. This process happens over and over at each receptor “checkpoint” until the pathway fulfills its final, correct role in the body.

Now consider this: many pathogens have evolved to create proteins that are very similar in size/shape to human proteins (this redundancy is sometimes called molecular mimicry). If a pathogen with this ability survives inside a human cell, its OWN proteins may begin to “bind” into human receptors. Gradually, the human proteins meant to control a particular receptor may be increasingly replaced by these similar “enemy” or pathogen proteins. What happens? The slightly different shape of the pathogen proteins cause the receptor to act/signal differently than it would under conditions of health.

An excellent example of the above (that I’ve discussed in several published papers) is the ability of pathogens to dysregulate activity of the human Vitamin D Receptor (VDR). The VDR is an extremely important receptor located at the heart of every human cell. It controls the expression of almost 1000 human genes. It also controls the activity of very important parts of the human immune response. These include TLR2 (a protein the body uses to identify and target pathogens), and several kinds of antimicrobial peptides (natural antibiotic molecules the body uses against infectious threats).

It comes as little surprise then that the fungus Aspergillus fumigates has evolved to create a protein or gliotoxin that can bind into the VDR and directly slow its activity. This results in tremendous survival gains for Aspergillus – the altered VDR can no longer correctly express many of the human genes it regulates. The expression of TLR2 and the antimicrobial peptides under VDR control also diminishes. Now, Aspergillus can flourish in the face of the host’s weakened immune defenses.

In fact slowing VDR activity (and subsequently parts of the human immune system) is such a logical survival mechanism that many other key pathogens have also evolved ways to dysregulate the receptor. When Epstein Barr virus infects human B cells it dramatically lowers VDR activity. Mycobacterium tuberculosis slows VDR activity by a factor of ~3.3. Borrelia burgdorferi, Cytomegalovirus, and Mycobacterium leprae also slow VDR activity to varying degrees. HIV almost completely “shuts down” the VDR. I should be clear that some of these pathogens don’t create proteins that directly bind into the VDR. They interfere with the VDR receptor in ways a little too hard to explain here:) But you get the overall trend right!?

Now factor this in: the human body’s pathways are tightly interconnected. So any change in human signaling has “flow-on” effects that impact yet other pathways and systems. In the case of the VDR, the receptor’s activity is tied to that of related receptors that control hormone signaling (especially the estrogen-beta receptor). It follows that the “original” intracellular infection may also prevent these related receptors/pathways from functioning correctly.

But let’s go back to the immune system, and continue to use the VDR pathway as an example. Let’s say CMV infects a person’s B cells and lowers VDR activity. The person’s immune response suffers as a result. Now, it becomes easier for that person to pick up OTHER pathogens from the external environment. Or, pathogens already in that person’s microbiome find it easier to act in a more virulent fashion. These “new” pathogens may also infect the person’s cells, where the interference they cause further slows the immune response. A “snowball effect” may ensue, where each “new” pathogen begins to facilitate the survival of the next.

A successive infection “snowball” could drive many forms of disease

In many of my papers I’ve referred to this snowball effect as “successive infection.” When I think of successive infection I literally picture a snowball rolling down a hill. The snowball begins with one intracellular infection, and then grows, as more and more pathogens take advantage of the dysregulated atmosphere in the body. As more pathogens “join” the snowball, the disease state that the snowball represents pushes the human body farther and farther away from a state of health.

It’s important to realize that once the “successive infection” process has started, environmental factors besides infection can “add” to the snowball. These include chemical exposures, stress, immunosuppressant drugs, over-used antibiotics and any other factors that cause either the immune response or microbiome balance to suffer. In fact, any of these other factors can “start” the snowball too.

I realize this is a major claim, but in my mind, a successive infection snowball could drive almost any form of inflammatory disease. That’s because such wide-range of different pathogens and negative environmental exposures can contribute to the process. Pathogens are also able to infect so many parts of the human body (nerves, brain tissue, lung tissue). If the snowball represents disease, the specific illness any one person develops would stem from their UNIQUE infectious history, their unique medication history, their unique chemical exposure history, and the many, many different ways these and other variables can interact with/feed into each other.

If you think that’s a stretch, consider this: almost every analysis of the human microbiome identifies new microbes never before detected by previous studies. For example, just a few months ago, Steven Quake at Stanford identified thousands of “new” microbes in human tissue and blood. Many of them are potential intracellular pathogens we didn’t even realize could survive in humans. This led Quake and team to state that these novel microbes “may prove to be the cause of acute or chronic diseases that, to date, have unknown etiology…”

Also, research teams continue to discover new examples of how pathogens might interfere with human pathways. For example, a team in Indiana just identified viruses that create proteins similar in size/shape to the human protein insulin. They found these viral proteins could bind into human insulin receptors that determine how the body correctly regulates blood sugar levels. The viruses that create these insulin-like proteins are regularly detected in human blood and fecal samples. The team concluded that “since only 2% of (human) viruses have been sequenced, this study raises the potential for discovery of other viral hormones which, along with known virally encoded growth factors, may modify human health and disease.”

So I’ll end on the following note: never underestimate the “power” of intracellular infection. Human microbiome studies that fail to account for possible intracellular pathogens may miss part of a larger, more important picture of disease. For example, the gut microbiome dysbiosis now tied to many inflammatory conditions may be a downstream result of intracellular infection in the blood or other tissues. AKA: the widespread drop in immune system activity driven by intracellular pathogens may “set the stage” for an atmosphere that favors gut microbiome imbalance. This is certainly the case in HIV/AIDS, for example (for more context read this blog post).

Ok phew! That’s it for now. Next podcast I’ll go more into immunosuppressive drugs, antibiotics, and other factors that can also compromise the human immune system and/or the human microbiome. Talk then!

Towards a more nuanced view of the human microbiome: pathogens and evolution

March 1st, 2018 by Amy Proal

Vast microbiome communities persist in human tissue and blood (Image:

Podcast transcript:

People often ask me for a quick description of the human microbiome. I appreciate their interest, although it’s actually a difficult request: the microbiome is extensive and capable of vast range of behavior. Nevertheless I’ll usually say something like this:

“When I refer to human microbiome I’m talking about the trillions of bacteria, viruses, fungi, bacteriophages and other microrganisms now understood to persist in complex communities in all human tissue and blood.”

But beyond that: how do I best summarize microbiome ACTIVITY? Aka: What are these microbes in and on us are doing?? I’ve learned that most friends expect a response along these lines:

“The microbes in and on us are our friends. They work night and day to defend us from disease, improve our metabolism, break down our food, control our digestion, educate our immune systems and beyond.” In such a description, all the microbes we harbor are portrayed as allies or even heroes: organisms that continually devise ways to benefit or support our health and well-being.

This kind of description is often accompanied by a specific mindset: the belief that microbiome pathogens (those that cause disease) are of secondary importance to those that “help” us. And that to strongly focus on the presence of these myriad pathogens is to give microbes in general an overly “bad reputation.”

In my opinion, this portrayal the microbiome is too one-sided: At the very least it should be accompanied by an asterisk stating something like: “this is an attempt capture microbiome behavior under conditions of extreme health.” And it’s true, of course, that people with extensive microbiomes (take Olympians!) can function with incredible vigor and ability.

But the vast majority of us sustain a range of minor to major inflammatory health issues. Some people deal with acne, others have trouble tolerating or digesting certain foods. Insomnia, anxiety, metabolic problems…are all extremely common in today’s human population. Beyond that, we are living in the midst of an epidemic of chronic disease, in which the incidence of nearly every well-characterized inflammatory condition is on the rise (diabetes, obesity, heart disease, the “autoimmune diseases” to name just a few). In fact, an estimated 60% of Americans suffer from at least one chronic condition (and that number only includes people who have been officially diagnosed).

A growing number of these inflammatory conditions/symptoms are tied to microbiome dysbiosis: cases in which a microbiome community shifts towards a state of imbalance. This dysbiosis can be severe: pathogens tend to outcompete their more symbiotic neighbors, leading to drops in community diversity that promote an inflammatory environment.

This means that in the vast majority of people, the microbiome is acting in a nuanced fashion. Some of the microbes we harbor may perform health-related functions, BUT a significant number of others may be acting as pathogens. In fact, certain pathogens are so effective at promoting chronic symptoms that they can “push” the microbes around them towards a dysfunctional state. These pathogens are sometimes called “keystone pathogens.” A good way to remember that is that the activity and gene expression of keystone pathogens are KEY to promoting disease.

In my opinion then, it is vital that we continually recognize and encourage the general public to understand that persistent, chronic, pathogens can also play a central role in any microbiome community. To do so is not giving microbes a “bad reputation.” It is simply accurate. In fact, the current, alarming epidemic of chronic disease begs that we study these persistent pathogens with more urgency than ever before.

It also helps to interpret microbiome activity via the lens of evolutionary biology. When microbes are described as “friendly” people often assume that there is actual intent on their part to help us. In reality, any symbiotic or “commensal” microbe/human relationship is still driven by the most basic tenants of evolution: “survival of the fittest” and “reproductive fitness.” The goal of any microbe in/on our bodies is to stay alive and pass its genes (encoding its particular traits and characteristics) to the next generation. So if a microbe is, for example, breaking down our food, it’s because doing so also benefits its own ability to survive and reproduce. The fact that such behavior also helps its human host is simply an evolutionary coincidence.

In most ecosystems on earth (like the rainforest), commensal relationships are actually rare. This is partly because one species in a commensal relationship has only to slightly change its gene expression and/or metabolism for the relationship to shift, and begin to favor one species over the other. This “shifting” happens very easily with microbes because they can quickly adjust their gene expression patterns: they turn certain genes “on” or “off” in order to functional optimally under different environmental conditions. Microbes can also – through a process known as “gene transfer” – acquire completely new genes: genes that allow them to function in totally new ways. And, as with most organisms, a chance mutation in a microbe’s genetic code (their DNA or RNA) can endow it with yet other novel traits and abilities.

Take E.coli. Strains of E.coli are detected in almost every analysis of the human microbiome, even in studies conducted on healthy people. This makes sense, since people digest seemingly innocuous E.coli strains on a regular basis. But most of us are also familiar with E.coli’s ability to cause severe food poisoning and other forms of illness. This range of E.coli behavior is due to the fact that it can evolve to persist in many, many different states – some WAY more pathogenic (or able to cause disease) than others. Take a study done by researchers in Portugal. The team found that when “commensal” E.coli were exposed to human white blood cells, they rapidly mutated and changed their gene expression: the commensal E. coli evolved, in less than 500 generations, into virulent, or disease-causing, clones. When these virulent clones were injected into mice, the mice suffered an increase in inflammatory symptoms.

This “virulence”, or ability of a microbe to cause disease, is dictated in large part by the activity of genes that express “virulence factors”: molecules that help bacteria, viruses and other microbes better persist inside the cells of the immune system, better penetrate host tissue, better obtain key nutrients from the host, better produce toxins…and better engage in many other behaviors that cause human health to suffer.

E.coli as seen under a microscope.

Every microbiome community (even in healthy individuals) harbors microbes capable of expressing these virulence factors. These include potential pathogens present in the earliest microbiome communities that an infant inherits from its mother (microbes generally acquired via the placenta, vaginal canal, and/or breast milk.) In other words, every human is “seeded’ with potential pathogens from the beginning of life.

It follows that pathogens capable of expressing virulence factors can evolve and “emerge” from microbes already present in a “healthy” microbiome community. Let me give you an example. Researchers in England recently published a paper called “Severe infections emerge from commensal bacteria by adaptive evolution.” The team studied the bacterial species S.aureus. Like E.coli, S.aureus is frequently detected in the nasal (nose) microbiome of healthy individuals. However, S.aureus can mutate and change its gene expression so profoundly that it can also cause life-threatening infections.The English research team found that these life-threatening strains of S.aureus could easily descend from commensal S.aureus bacteria already colonizing a person’s nose. In other words, depending on conditions in the body, S.aureus is able to mutate from a harmless microbe to a major pathogen within the same person. This progression towards virulence involved an increase in the activity of genes that promote toxin production, abscess formation, immune evasion and many other processes that harm the human host.

Also key to the study’s findings was that, in the majority of cases, this within-person evolution towards S.aureus virulence occurred in subjects that had “risk factors for infection”: these included a weakened immune response, co-morbid conditions (the subject was sick with one or more inflammatory diseases), and/or medical interventions (subjects had undergone surgery or a procedure).

That brings me to my main point. To best understand microbe behavior we must not ignore the elephant in the room: the human immune system. The human immune system, and its extensive army of immune cells and antimicrobial molecules, plays the greatest role in determining how the microbes in any human ecosystem act, survive, and behave. Lately I’ve been describing this relationship with the following analogy:

Imagine a school. The children in the classroom are different microbes in a human microbiome community. The teacher and her support staff (like the principal) are the human immune system. Some of the children in the classroom are trying hard to get work done. Others are not so studious and hope to cause trouble. But these potential “bad students” don’t act out when the teacher is in the room and they can be disciplined by the principal and other staff. HOWEVER – if the teacher leaves the room, and her support system dwindles, these “bad influence” students can easily change their behavior to cause trouble. Without supervision they stop doing work. Further, they begin to encourage the better students around them to join them in promoting disorder. Little by little, even the best students find it hard to behave in the unsupervised classroom environment.”

For example, take the bacterial species Lactobacillus (which is added to many common probiotics). It is often regarded as a purely “good” microbe. However, as this article on the vaginal microbiome points out, some strains of Lactobacillus help maintain a healthy, acidic vaginal environment. BUT other strains can put women at a higher risk of acquiring a sexually transmitted infection. Indeed, Willa Huston, a professor at the University of Technology Sydney states the following about Lactobacillus and vaginal health: “It depends on the type of Lactobacillus, it might depend on the function of that Lactobacillus…and perhaps how it’s interplaying with the genome type of the woman.”

If you’re not convinced, let me give you a great example of how a “good” microbiome community can go bad (evolve in a direction of virulence). We’re all familiar with the fact that our teeth can develop cavities. The process that drives this cavity formation is called periodontitis. Periodontitis is now understood to be an infectious condition in which different oral microbes work together to damage tooth tissue and bone.

But aside from teeth brushing, what factors determine if a tooth develops a cavity or remains healthy? A team in Boston addressed this question by studying how the entire oral microbiome changes its gene expression during cavity progression. They found that community-wide gene expression did not change in teeth that remained cavity-free. But, the oral community dramatically altered its gene expression in sites that progressed to periodontitis. These changes involved genes that control oxidative stress, iron transport, sulfur metabolism, intracellular survival, and bacterial movement among many others. Cavity formation was also correlated with increased expression of virulence factors and higher levels of viral activity.

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. In effect, under conditions of increasing immune suppression, imbalance and inflammation, the whole oral microbiome community appeared to act together as a pathogen. This was supported by the fact that, in cavity sites, groups of microbes not usually considered to be pathogens expressed a large number of the disease-promoting virulence factors. Two of these microbes were S.mitis and S.intermedius –  bacterial species usually associated with dental health that are commonly described as “good.”

Single microbes can form into communities called biofilms.

Many cavity forming microbes also team up to persist in communities called biofilms. These biofilm communities are surrounded by a matrix that protects the different microbes inside from the human immune system. Participating microbes use electrical and chemical signals to communicate. This allows them to split up jobs important for their own survival – for example, virulent bacteria may protect the biofilm from outside intrusion, while other bacteria on the inside focus on obtaining nutrients for the community.

Few people would argue that microbes in a biofilm are “helping” their human host. They have instead joined forces in a manner that promotes their own survival over ours. It’s not surprising then that biofilms are connected to an increasing number of inflammatory conditions. In fact, some researchers estimate that the majority of bacteria in the body persist in a biofilm state.

So the way I see it, the human immune system and the human microbiome are actually in a continual state of competition and evolution. In healthy people the immune system has gained “an edge” over the microbiome. It is successfully able to keep microbiome pathogens in check. In inflammatory disease however, the opposite happens. The microbiome – or pathogens (or groups of pathogens) – begin to gain “an edge” over the human immune system…and its various defense strategies. This last situation is especially true in our modern world where an increasing number of environmental exposures and drugs are wearing down the human immune system.

And that’s what I’ll be talking about in one of my next podcasts: What factors most knock down or debilitate the human immune system in our modern society? And how can we help bring our immune systems back to a state of better activity and health? – a state that will help keep our microbiomes in a state of balance.

I will also do another podcast that explains more about how pathogens, and the metabolites they produce, are able to interfere with the human immune system and human pathways that control metabolism.

Interview with Ry Young: Phages and Phage Therapy – Pros/Cons and History

February 17th, 2018 by Amy Proal

Ry Young is a Professor in the Department of Biochemistry and Biophysics at Texas A&M University. He is also director of the Center for Phage Technology. Since the early 1970s Ry has studied bacteriophages: viruses that infect bacteria. He is now one of the world’s leading bacteriophage experts. His lab investigates bacteriophages as models for a new class of antibacterials. He has also participated in several “phage therapy” cases – in which bacteriophages are used as therapeutics to treat pathogenic bacterial infections.

Background information:

Tom Patterson phage therapy case: A recent medical case in which phage therapy was successfully used to treat an antibiotic resistant infection. Please watch this TED Talk for more context.

The interview:

Amy: Hi Ry. Thanks for taking the time to speak with me. You’ve researched phages for decades. How did you get interested in the topic? 

Ry: That’s a tough question. I don’t remember a time when I wasn’t interested in phages. I was a graduate student at MIT in 1968. At the time, one of the founders of the Phage Church was named Salvador Luria. He won the Nobel Prize in 1969, along with Max Delbrück and Alfred Hershey, for work on phage genetics. Luria was my microbiology instructor at MIT and turned me on to phages. So I learned about phages from what they call the horse’s mouth – Sal was a very charismatic person.

Phages attach to the outer membrane of bacteria with a thin “tail,” and inject their DNA into the bacterial cell.

Soon after however, I got my Vietnam War draft notice. I left graduate school to spend three years in the Navy. When I got out I went back to graduate school and started working on molecular biology. But as a postdoc, I got an NIH fellowship and chose to work in the lab of a brand new Harvard faculty member, Mike Syvanen, who was a phage guy. In 1978 I started my own phage lab here at Texas A & M. What I like about phage biology is the rigor of the field. Phages are easy to work with and have powerful genetics. You can posit hypotheses, do experiments, and get quick results.

Amy: So when you started researching phages I assume there wasn’t much of a concept of the human microbiome? 

Ry: No no. We had no concept of that. In fact, when I started working here in 1978, I had little idea that I would be one of the last people to start a phage lab for about 15 years. The entire phage movement, or Phage Church, was disassembling itself during the late 1970s and 1980s. This was based on an attitude in which phages were valued strictly as model systems for genetic experiments; as organisms that could help us work out the fundamental rules of molecular biology. But even then, many phage researchers decided it was time to move away from phage genetics to study other model systems. So the people who went on to found important fields of genetics research – like C. elegans for neurobiology and zebrafish for developmental biology – were mostly all phage people who moved away from the field.

This shift was largely started by a researcher named Max Delbruck, who founded modern phage biology. He was a physicist and reductionist: and if you’re a physicist you study the hydrogen atom and then, when you’ve got a predictive understanding of that…you move up to helium and so on. In fact Delbruck himself moved away from phages in the 1950s. So when I started my lab, I was one of a new wave of researchers who were interested in phages per se, rather than using them as a tool to study something else.

Max Delbruck in the early 1940s.

Amy: I see. I didn’t know that.

Ry: If you’re gonna keep up with phage you should get a copy of a textbook called “The Bacteriophages.” It’s edited by Richard Calendar (he’s retired now in San Francisco). I wrote the book’s one-page foreword in 2006. As part of writing it, I did an analysis of  NIH grants for phage research over the past ~ 30 years. I compared 1972, the height of the phage era, with 2002 (30 years later). I discovered that the number of NIH grants on phages had dropped down enormously. The number of RO1s for real phage research had gone from over 200 in 1972 to less than 10 in 2002. Basically the field had died.

Amy: Yes. But now it seems that the field has recently been reborn. 

Ry: Yes. Which is a real problem because suddenly there’s all these people who want to research phages, but few labs set up for phage research.

Amy: Interesting. Does this situation have anything to do with phage therapy?

Ry: No. The first stirrings of renewed interest in phages (and phage therapy) occurred in the early 2000s. That’s because the terrible antibiotic resistance problem we face was becoming more well-known. Even then, we still didn’t know anything about the human microbiome. So there are two separate current justifications for the renewed interest in phage therapy – One: we’re not getting any good new antibiotics. Two: even if we did we shouldn’t be using them. It’s kind of a double whammy in that regard.

Also, since the time when phage biology mostly died out in the 1980s, all sorts of new technologies have emerged – like next generation sequencing, laser technologies and high resolution fluorescence microscopy. The development of cryo-electron microscopy was especially important, because resolution power became so great that we could finally see the details of the phages. Suddenly, phage-related questions that had previously been beyond anybody’s ability to think about could be studied. Not only that, there’s a huge tide of new genomic information that’s bringing home to everybody what an important role phages play in the biosphere.

Because, back in the 1930s, phage biology was classically so powerful that the phage research community would get ahead of itself. That’s what happened with the original phage therapy. In the 1930s phage therapy was pioneered by a guy named Felix d’Herelle. He was a force of nature and went all over the world promoting phage therapy. So thanks to him, phages became the first widely used biological therapeutic.

But in the 1930s science in general was premature –  we didn’t even know about DNA. And because there were no standards, and no FDA or anything like it, most of these early attempts at phage therapy were kind of hopeless. We’d have been a lot better off if d’Herelle had not discovered phages until the 1940s or 1950s, when the scientific community knew more about molecular biology.

But there are other arguments about why this early phage therapy eventually fell out of favor. Part of what caused the decline was personality driven. And a lot was economics driven, because cheap antibiotics like penicillin were also discovered in the 1930s. No biological treatment like phage therapy is cheap. You can’t mass produce phages like you can penicillin. Also, to treat a patient you need thousands of phages versus one penicillin.

Amy: But in parts of Europe and Russia phage therapy did continue, right?

Ry: The very best person in terms of the history of phage biology and phage therapy is Bill Summers at Yale. He’s a great guy to talk to. He’s gives the world’s greatest seminars. They’re funny but illuminating. He was a phage and virus person way back when he was a junior faculty member, but now he’s become a very prominent scientific historian. He’s written any number of articles and books about phages. He’s the only person who knows the whole story. And unlike some people who mercilessly promote phage biology, he can tell you the underside too. And there are some undersides.

Amy: I want to go into those undersides. But before that let me ask you: Now that the microbiome has been better characterized, how would you describe the role that phages play in the human body?

Ry: Oh I don’t think anybody knows right now. Most of the information we have about phages in the actual body is derived from huge metagenomic experiments. For example, they might sequence your gut microbiome and discover there’s an awful lot of phage sequences in there. But by far the most common phages found by such studies are lysogenic phages – the ones present as prophages inside bacterial DNA. That’s a little misleading because all you can tell from that data is that those prophage sequences are being carried by bacteria. You don’t necessarily know if  the phage in question is even functional, or if it’s actually floating around doing anything.

Bacteriophages seen under an electron microscope.

But I think it’s clear that any microbiome community can’t be understood without understanding the phages too. We know phages are going to play a prominent role in the microbiome – there’s no way it can be avoided. Phages clearly interact, both positively and negatively, with the other species. We also know that the human microbiome changes as people develop. But nobody knows how many of these changes are mediated by phages. One of the things about phages in the gut is that there’s a lot of bacteria in the gut microbiome. This crowded environment allows phages to have a massive and rapid effect. Because phages have to absorb bacteria to kill them, and this rate of absorption is proportional. It’s a two body collision. The more phages and the more bacteria you have, the faster those collisions happen. For example, 200 phages can get made out of one bacterial cell.

So it’s a population density issue: when you have lots of any particular bacteria they become suddenly vulnerable to a single phage particle in the population. Whereas if the bacteria are very dilute, you could have 10^5 bacteria X and 10^5 phage Y (that would be able to grow on X) in the same microbiome community, but they might never encounter each other.

Amy: Right. But phages have been detected outside the gut right?

Ry: Oh yeah. Anywhere where you find bacteria in the human body you’re going to find phages. And for every type of bacteria there’s a different phage profile. Phage biology was really started with the study of laboratory strains of E.coli and closely related bacteria like Salmonella. Probably 99% of phage biology research was done on those microbes in the pre-therapy days (including my own research). And with E. coli, we found that you can go into any sewer or pond and find all kinds of different E.coli phages, in their every possible lifestyle. You’ll find lysogenic phages, and then all kinds of what people call lytic phages. Still, much of the variety of phages for E. coli has still not been tapped – we keep finding new ones.

On the other hand take Staph aureus – a high profile bacterial target (MRSA is surging in hospitals etc). There’s actually very little phage biology for Staph aureus. There’s basically two lytic Staph aureus phages. One is called Phage K (a big myophage). The other is a little podophage (a phage with a short stumpy tail). That’s all there is. No matter where you go in the world, if you look for lytic phages for Staph aureus, you’ll only find Phage K, its cousins, or the little podos.

That makes one ask: Why does Staph have such little phage variety? It’s seems like there was a bottleneck way, way back in evolution and those two phages won. It’s unfortunate in some ways, because we would like to have a lot more different kinds of Staph phages. On the other hand, phage K may have “won” because it evolved in a unique fashion. For a phage to affect a bacterial cell, the bacterial cell wall has to have a receptor that the phage can bind to. The bacterial cell wall receptor phage K and its friends use is a molecule called teichoic acid. And this teichoic acid receptor is essential for Staph: it can’t survive without it. So Staph cannot mutate to lose the receptor.  That’s probably why phage K “won”: it learned to recognize that essential receptor and then all the other phages were out of luck.

Amy: So Phage K outcompeted all the other phages? 

Ry: Yes and because the little podo phage has a very narrow host range, phage K is the only phage that’s useful. So there are a bunch of companies that have made phage cocktails against Staph, and they all turn out to be Phage K (plus friends and family). If fact, phage K has probably been patented 30 times.

Ry:  Do you know about the Bayne-Jones-Eaton report of 1934 that supposedly was the death knell of phage therapy?

Amy: No I don’t.

Ry: In 1934, the American Medical Association commissioned two scientists (Stanhope Bayne-Jones and Monroe Eaton) to survey phage therapy, to see if there was any anything “real” to it. They report they produced was very long – it was published in three separate issues of JAMA. They did a thorough study of all the different reported uses of phage therapy. Their general conclusion was that there was no demonstrated therapeutic benefit. And that’s not saying that phage therapy doesn’t work, just that the science hadn’t been done to show it could. However, the report did have an asterisk which stated something like “maybe we’re being too harsh, it does look like phage therapy works for Staph aureus, but nothing else.”

They came to that conclusion because they didn’t know about molecular biology. And if you don’t know what you’re doing, phage K will still work, because no matter how badly you use it it’ll still work. So there’s plenty of good evidence from thousands of cases showing that phage therapy works for Staph infections – evidence that goes way back to the 1910s – 1920s. But the Bayne-Jones-Eaton report still states that phage therapy has no demonstrated benefit (especially if you just read the top line). Also, that was right about the time when antibiotics started becoming available. So that was it: game over for phage therapy.

Félix d’Hérelle

I recently read the Bayne-Jones-Eaton report in depth while preparing for a two-day FDA workshop on phage therapy in July. I did a historical overview to see what the regulatory landscape was back when it was originally written. My interpretation is that “the fix was in for phage therapy,” because Bayne-Jones had a conflict of interest.

In the report, Bayne-Jones actually refers to the “failure” of phage therapy as the “d’Herelle Phenomenon”. And when you start calling something by somebody’s name, you instantly recognize that Bayne-Jones had something in mind about d’Herelle. At the time of the report, d’Herelle had been in the United States as a professor at Yale. There, he did what he did everywhere: frustrate many of his colleagues – he had a real talent for making enemies. He was self-educated with no college degree, much less a Ph.D. I think he was always very defensive about that. So eventually d’Herelle left Yale and Bayne-Jones took over his position. In modern times that would have instantly disqualified Bayne-Jones as an author of the 1934 report: You take somebody’s job and then write a report on the previous guy’s work!? I don’t think that’s a recipe for fairness. Although the report mattered less because antibiotics were going to wipe out phage therapy anyway.

One consequence of this was that d’Herelle had very sad life. He got nominated for the Nobel Prize about 28 times, but always had enemies high up in the scientific hierarchy that would blackball him. Because by any standard he should have gotten the Nobel Prize. He completely invented the whole area of phage therapy.

Amy: It would be interesting if d’Herelle could see new molecular data on phages.

Ry: Oh gosh yeah. He did a lot of fundamental research with no molecular tools, the lessons of which have turned out to be true a hundred years later. But because he was on the outs with the hierarchy, he ended up gravitating to to the left. The book he wrote about phages was dedicated to Joseph Stalin! Not exactly the way to endure yourself to the academic community. He had a real talent for painting himself into a corner.

That’s how he ended up starting the Phage Institute in the Soviet Union. As a result of that he ended up under house arrest all during World War II, because the fascists took over France. It was almost like a bad airplane novel. So now, in 2018, there’s a new phage therapy era. But to me this era is a rebirth of d’Herelle’s original idea.

Amy: I see. So this current rebirth of phage therapy: What’s the climate here in the U.S.? I know the FDA doesn’t allow it. What are the reasons for that?

Ry: Actually the FDA has been nothing but positive about phage therapy. It’s just that no phage therapy cocktail has been put through FDA tests yet. But the FDA has been very proactive: they’re trying to figure out what to do next. In fact, starting with the Tom Patterson case, they’ve set up a system where if a physician reports an appropriate phage therapy case, the physician can get “expanded access” (what we used to called compassionate use) to use phage therapy. This expanded access can be authorized within a day.

So while the FDA is not going to open the floodgates and say, “OK you can start injecting phage into people, don’t even bother calling us,” they’ve agreed to this expanded access (especially in life and death situations). Most of the time expanded access is used for cancer patients – at the end of life, one might as well give experimental treatments a try. So many potential cancer treatments that aren’t ready yet for clinical trials are also used in the same way.

Amy: But in the USA do you have to exhaust all antibiotic options before being allowed to use phage therapy? 

Ry: I think in general that’s correct. Right now if you want to use something that is not approved by the FDA you have to at least say there’s no other treatment option. That’s not a phage-specific guideline but true for all experimental drugs.

Amy: Might that change in the future because of the current antibiotic resistance problem?

Ry: Yeah I don’t know. I doubt that phage therapy will take the place of antibiotics. In the foreseeable future phages are going to be last resort items, mainly because of FDA approval issues. But there are several other problems with phage therapy. One is that we’re not going to find a broad-acting phage like penicillin. And since you know about the microbiota you could say, “we shouldn’t try to find a phage like penicillin!”

Amy: Yes, that’s a benefit in my mind. Because penicillin acts like an “atomic bomb” to the microbiome. It depletes entire microbiome communities in an effort to target one bacterial species. 

Ry: Right exactly. It would be better if we had antibiotics that targeted the disease bug and not the rest of your microbiota. But one reason we don’t have any small molecule-specific antibiotics is that there was no demand for them in the past. Up until recently Medicine wanted broad spectrum antibiotics.

But now there’s going to be a market for specific antibiotic molecules. I’m confident that pharmaceutical science and the industry are going to try very hard to find mass producible molecules that can target Staph, Psuedomonas etc. Those antibiotic molecules would be “round pegs” that fit fairly well into our established FDA drug testing system. For example, if you can find molecule X that kills Staph aureus, and doesn’t kill your microbiome, that molecule will be of great value. Then, somebody will be able to afford to pay the $100 million to $300 million necessary to put that molecule through the FDA drug approval process. I also think that the lysin technology that’s derivative of phage biology will become relevant, because it’s also a cheaper treatment option than phage therapy itself.

Amy: What is lysin technology?

Ry: Lysin technology is part of the phage story. For 40 years the NIH has been funding me to work on lysis (although I have no stock or conflicts of interest). The last step of the phage infection cycle is called lysis. That’s when the bacterial cell blows up and progeny virons are dispersed. One of the proteins involved is an enzyme called endolysin, or lysin, which means “the enzyme from inside that causes the cell to lyse.” Bacteria are protected by cell walls. But if you degrade the cell wall, which is what the phage does, then the cell blows up and the virus particles come out. So these lysin enzymes have now been developed to work from the outside to target gram positive bacteria like Staph. You take the phage lysin, make a lot of it, and inject it.

Crystal structure of a bacteriophage endolysin.

Lysins are not small molecules, but they’re a lot smaller than a phage. So you can mass produce and patent lysins – two things you can’t do with phages. But I think lysin technology will only be useful for Gram positive bacteria. Gram negative bacteria and mycobacteria have tough outer surfaces that endolysin can’t get at. The mycobacteria have a wax-like coating outside the cell wall that’s very difficult to break open.

Amy: Interesting. Back to phage therapy. Imagine there was a perfect world where we had studied phages in much greater depth. There’s a person with a bacterial infection and you’re trying to find a phage to treat them. You’re allowed to proceed however you choose – how would that work? 

Ry: You have to use multiple phages, otherwise you will probably get resistance very quickly. The word resistance means one thing to you, and a different thing to phage biologists. When phage researchers say “resistance” we are referencing a very specific situation: a phage cannot attach to a bacterium because the receptor it targets on the bacterial cell wall is damaged or missing. To clarify, every phage has a bacterial receptor it targets, and almost all the time those receptors are non-essential for the infected bacterium. So the bacterium can simply delete the receptor by mutation, allowing it to become completely resistant to the phage.

Given that notion, if you want a Pseudomonas phage cocktail to work, you need to have at least three phages that would target every possible Pseudomonas strain they encounter. Each one of those phages would target a different bacterial host receptor. The typical frequency of a knockout mutation for bacteria is one in a million: not a very big number if you’re being treated with one phage (because there are millions of bacteria and phages in the system already, some of which are already resistant). So as soon as you kill off all the bacteria that aren’t, you’re right back where you started within a few hours. But, if you have two different phages attaching to two different receptors, then instead of 10 ^- 6 frequency of a bacterial knockout mutation, it’s 10^-12. If you have three, that’s 10^-18. And nobody has 10^18 bacteria in them, at least no one who’s alive:)

So I’m using your perfect world scenario. In my perfect world there’s a Pseudomonas cocktail that’s ready for strain X of Pseudomonas, and we know its receptor targets. We also know that the bacterial strain we’re trying to target has those three receptors on its surface. Now, we can use that phage cocktail with confidence: we’re probably not going to generate resistant bacteria. That’s my hardliner perspective.

So the use of single phages could create a problem similar to what’s happening with conventional antibiotics? (where treatment can select for mutant bacterial strains) 

Ry: Right. Except resistance to phage happens a lot more frequently and quickly than with conventional antibiotics. If a bacterial cell runs into penicillin, it’s going to die unless it evolves to mutate residues of a particular enzyme in the bacterial cell wall (very difficult). So the mutations that create resistance to antibiotics are actually very rare. The problem is that because we’ve overused antibiotics, and put so much selective pressure in humans, many bacterial disease strains have sucked up genes that were already in the dirt. These are genes that had already evolved to degrade antibiotics.

But bacterial resistance to phages is a major concern. In fact, when I was a graduate student working in the heyday of phage biology, the classical attitude was that the resistance issue rendered phage therapy a joke. My mentor (and other members of the Phage Church) called phage therapy “bizarre” because resistance can occur so quickly. Of course, at the time we were completely unaware of how many more phages existed that we could use for cocktails. This was way before the development of next-generation sequencing.

Amy: To summarize. An upside of phage therapy is the specificity of the bacterial target. But that’s also a downside. And this issue could be addressed by using phage cocktails in lieu of single phages?

Ry: As director of the Center for Phage Technology I get to have official positions which absolutely no one has to listen to. But I always dreamed of being able to make pronouncements:) It’s our considered opinion that no phage should be used in a human unless we know what its receptor is. Period. Full stop. There are plenty of people who disagree with me because they don’t want to go through the trouble of figuring out what the target receptor is. But in the long run I can’t imagine the FDA is going to countenance the use of phages without clear receptor targets. And of course the original history of phage therapy has a bad reputation. There were negative outcomes and bad publicity. So now, when we restart phage therapy 2.0, we need to do it right.

Here’s another problem: right now phage therapy is always used via “expanded access.” When the phone rings in my office it’s always a crisis in which somebody is dying. And I’m not going to tell a dying patient and their family requesting a phage that we need six months to figure out what phage to use. But if we keep operating under these rushed emergency conditions, we’re going to start using phages that all use the same receptor. Then we’re going to get resistance and somebody’s going to die. That could lead to serious legal issues. If someone dies after being given an experimental phage, lawyers could later say, “Doctor yes or no…did you send viruses to this sick man? And were these viruses approved for treatment!?”

Amy: But to use an expanded access treatment the patient has to sign a medical release form right?

Ry: Yeah but that doesn’t help a bit because a patient’s heir’s lawyers are not bound by the patient’s request. So lawyers could still come after you. They wouldn’t come after me because I haven’t got a dime. But they might come after my University. Also, a flipside of the specificity issue is that in order to have any expectation of phage therapy success, we need to know whether the bacteria in a given patient are actually sensitive to the phages we have. That means we need at least one day to screen.

Recently there was a phage therapy case involving a young woman. She had gotten a lung transplant and appeared to be cured from a severe Burkholderia infection. But then the infection came back. So along with 3-4 other labs we tested Burkholderia phages. We tested over 400 phages, but only found three, and all of them were lysogenic. It took us 3 days working 24/7 to do that. Unfortunately she passed away a few days later (for reasons not related to phage therapy, but still).

But if we had already had a refrigerator full of phages, and we had known the receptor for those phages, we could have done some very simple, quick experiments. Or you could use a robot to get the bacterial strain from the patient, and know within 24 hours which of your phages might work against it. And if those phages are already pre-purified they could quickly be in the mail. But right now, no such well-characterized stock of phages for any bacterial pathogen is available. Again, the main reason for that is there’s no market for it. Because as far as I know, with “expanded access” you can’t sell the experimental drug (phage): you have to give it away.

Amy: So again, in a perfect world, it seems like the phage therapy community could use some time to calmly prepare for emergency phage therapy scenarios?

Ry: Yes. We could take highly prioritized pathogens like Baumannii and Pseudomonas and create a well-characterized collection of phages for which we knew the receptor. It wouldn’t even be a multimillion dollar project – more like hundreds of thousands of dollars. Which sounds like a lot of money, but I wonder what Tom Patterson’s bill for eight months in the UCSD ICU was? Maybe something like $10,000 a day? So the money needed to generate phage libraries wouldn’t be that large compared to many related costs. But nevertheless, who’s going to pay for it? And is every reference hospital going to have a walk-in cooler with thousands of phage lysates or purified phages? The other possibility, and the dream of the phage therapy world, is to have phage that works against all targets. For Staph aureus that already mostly exists.

Amy: A superphage?

Electron micrograph images of phage K (American Society for Microbiology)

Ry: Phage K is a “superphage.” You can get a phage K that will eliminate 85% of all the Staph on its own. It would target all Staph, except for the fact that bacteria find ways to fight back against phages that aren’t related to “resistance.” Bacteria also have restriction enzymes and their CRISP-R-associated systems, for example. But I think the dream of a super phage is very close to reality with phage K and its relatives. For other bacteria though, we haven’t found many examples of bacteria with essential cell wall components that phages can be targeted to.

Amy: But we’re discovering new phages every day, right?

Ry: Yes. We’re early in the game. And what’s astounding is that all the technology we’re using to characterize phages (with the exception of molecular sequencing) is really the same that d’Herelle used back in the 1930s. Again, that’s because there’s no current market for phage therapy. But, if people think there could be a market down the road, a lot of progress on how to purify phages could occur. The process could become cheaper than it is now. But currently we make phages for therapy in the same way we do for laboratory experiments. And that’s not something you can do on a large scale.

Amy: It seems to me the FDA will have to “paradigm shift” the way they’re going to evaluate phages and other new biological drugs.

Ry: Right, because it’s not just phages. There’s a lot of other biologicals that are like square pegs in the round holes of a regulatory process geared towards developing organic chemicals. I’m hoping the FDA’s “expanded access” phage decision will increase the climate for public and physician awareness of phage therapy. Also, there’s been some progress.

Amy:  What about pressure from Biotech companies? There is a focus in the Biotech community towards the development of personalized treatment approaches.

Ry: Yes. And phages are the ultimate personalized medicine.

Amy: That makes me think the FDA’s current drug approval process is going to become a problem for a lot researchers and drug companies.

Europe is ahead of us in addressing this issue. Partly because the countries involved are much smaller scale countries. If you’re living next door to the Minister of Health, it’s a lot easier for you to get your ideas across. So Belgium, for example, is pretty far ahead of us in terms of making “expanded access” more regularized. They’re going make it easier for physicians to say “I’m going to use phages.” And there will hopefully be suppliers of good phage. Then, the physician will be able to buy/use phages and have protection against being sued.

I don’t think phage therapy will replace antibiotics though. I want to make sure you understand that. I think at least for the foreseeable future phage therapy is going to be a last resort thing. I would like to see a lot more basic phage biology funded. I think in order for phage therapy to best move forward, we also need a rebirth of basic phage biology research. Because almost everything we currently know about phage biology is based on just phages of E.coli (which is of course what I worked on myself.)

Amy: Yes. A recent study estimated that 30 billion phages may traffic the human body on a daily basis. It does seem like we need more research on all these “new” phages!

Ry: Right. DNA sequencing is nice to have, but it doesn’t substitute for doing experiments that help better understand phage activity and function.

Amy: Did you read the recent Georgia Institute of Technology study which showed that the host innate immune response seems to play a role in helping phage therapy work? Because if that’s the case,  then our widespread use of immunosuppressive drugs and steroids seems like a problem. Especially because such drugs are administered frequently in hospital emergency settings.

Phages interact with the human immune system (Roach et al, Cell Press)

Ry: So I suspect that phage therapy will be mostly useful for reducing initial loads of bacteria, under conditions where a patient harbors enormous amounts of a bacterial pathogen. Because once the bacteria get down to a certain level they don’t collide with the phage anymore. Phages are not small molecules, so they can’t penetrate every body niche to find a bacterial target. So I think that phages will be most useful for somebody who’s got a raging infection. You give them a first round of phages and a knock the bacteria down by two or three logs. Now you’ve got a chance for either the body’s own immune system or antibiotics to take over.

Amy: At least in that scenario antibiotics are being used in a careful situation. As opposed to many other ways in which they are over-prescribed today.

Ry: Yes. There’s a lot of speculation out there that many of our current autoimmune diseases, like Crohn’s, may be due to the fact that our generation (my generation) was the first to be massively dosed with antibiotics all throughout childhood. My generation was convinced that if you didn’t get an antibiotic prescription you hadn’t been treated right. It was the golden age of antibiotics.

Amy: Right. When antibiotics were discovered they were perceived as miracles, which they kind of were, especially during World War II. But that has created a situation where the average person has not been well-educated about how antibiotics work. For example, about the fact that antibiotics can’t kill viruses. 

I think that the American Medical Association is at fault for not educating the public better about antibiotics. I mean they really are. For example, you see all these TV commercials for erectile dysfunction or cancer drugs. There ought to be a way that some of that airtime could be used to educate the public about medical topics like antibiotics. Especially because most drugs advertised in these commercials were developed with NIH funding. So I think big drug companies owe at least that to the public. Also, physicians need to have the courage to say “You have a viral infection. I’m sorry but it can’t be treated with antibiotics.”

Amy: Yes. I know you’re considered a “hardliner” when it comes to the future of phage therapy, but I think the caution you advise moving forward in spot on. We don’t want to create a future situation with phage therapy that could repeat problems we’ve encountered with antibiotics. Understanding and correctly managing phage resistance will be critical.

Thanks so much for taking the time to speak with me. I learned so much from this discussion.   

20 Questions for Gurol Suel: biofilm electrical communication

January 29th, 2018 by Amy Proal

Gürol Süel is a Professor of Molecular Biology and the Associate Director of the San Diego Center for Systems Biology. His research team integrates quantitative biology approaches with mathematical modeling to identify principles of bacterial organization and coordination. Most recently, the team discovered a new form of bacterial communication that arises in biofilms: Ion channel mediated electrical signaling. For more context on this discovery, please read this article in The Atlantic Magazine.

Amy: Thanks so much for speaking with me. Your last few papers on electrical communication in bacterial biofilms blew me away. I think many other people feel similarly! How did you get interested in the topic? 

Gurol: We were very interested in trying to understand higher-order organization and behavior in bacterial communities. That’s because even though bacteria are unicellular organisms they’re not solitary. They actually live in dense communities (biofilms) that have certain features: for example they’re very resilient to stress and antibiotics.

We became interested in biofilm electrical signaling partially because we saw that in biofilms there was some kind of long-range metabolic coordination. We were having a hard time understanding how the bacterial cells could communicate so effectively over such long distances. It seemed that a simple diffusion-based mechanism (like quorum sensing) might not be sufficient to support this communication. My Ph.D. is in molecular biophysics. I also have a background in electrophysiology. For example, I used to do electrophysiology in fruit flies with photoreceptors. So drawing from that experience, I started wondering if there could be a different type of signaling process involved.

We started looking at amino acids critical for slow-range biofilm communication. The most important amino acid appears to be glutamate. Glutamate is very interesting because it’s also an excitatory amino acid in the human brain. In fact, about half of all brain activity is regulated by glutamate. Glutamate is also one of few amino acids that’s charged. So it can’t just get in and out of cells on its own because that’s energetically unfavorable. So you’ve got to have some kind of a proton motor force to actually take up the charged amino acids. And if you’re dealing with a proton motor force then the cell membrane potential is important.

Little by little we pursued this line of thought and realized that we were looking at some kind of ion channel purpose or function in this signaling process. And when you think about ion channels and membrane potentials you think about sodium and potassium – those are the most commonly utilized ions in biological systems. So we looked at both and saw that sodium wasn’t really doing anything. That wasn’t surprising because Bacillus subtilis, the microorganism that we work with, is not known to have any identified sodium channels.

Bacteria form communities called biofilms (Image: The Laboratory of Gurol Suel)

But we saw a lot of beautiful signaling and dynamics going on when we looked at potassium. Then, we took the suggested potassium ion channel and started making mutants. One thing led to another and we realized that these bacteria were communicating using potassium ion channels, in a way reminiscent of what neurons do in the brain with action potentials. Once we uncovered that, we realized that if you have these long-range electrochemical waves propagating through the biofilm, then the waves wouldn’t just stop at the edge but might propagate a distance.

That led us to the Cell paper where we said: What if there’s some kind of motile cells nearby? Would they be sensing or responding? Would they be affected by these types of signals being emitted by the biofilm? Pursuing those questions turned into a fun and intriguing study. Then we said OK – if that’s possible, what if there’s another biofilm further away: could the two biofilms sense each other and interact? That led us to the Science paper about time-sharing of limited nutrients.

Amy: Right. I enjoyed reading those papers because your experiments are so well organized. I’m interested in the redundancy between the way neurons signal and the way you found that bacteria in these biofilms are signaling. Do you think it’s possible that there are any interactions between human nerve signaling and these microbes? That they could tap into each other’s signaling?

Gurol: I mean, we don’t know. That’s the short answer. Because no one’s ever looked at that. Would it be possible? I guess it would be possible. I do know that extracellular potassium signals (the potassium ion channel mediated signals) are not specific to just neurons. We showed it now even in bacteria. But it’s also very important in plants. It’s very important in all kinds of other living organisms – because these ions are very generic, meaning they can affect extracellular charge in any membrane.

So it’s not inconceivable to think that if a bacteria releases some charged particles and there happens to be an excitable cell nearby – maybe a mammalian cell – that the mammalian cell might actually respond to that. I don’t think that’s super crazy. Right now it’s unproven but it’s something we think about.

Amy: Very interesting. What about external electromagnetic fields – do you think that our human electronic devices or any radio frequency networks we’ve set up as humans could be impacting biofilm communities?

Gurol: That’s a very good question. Again I don’t know for sure. But it’s well known that lots of organisms have electro-reception, meaning they can follow electromagnetic fields on our planet. Animals with electro-reception range from whales to birds. So if organisms can sense these fields, what you’re suggesting is probably not super crazy.

There are actually bacteria known to have small particles inside that are sensitive to magnetic fields. We also know there are documented cells that will respond to an electrical field, including bacterial cells. There’s also good work out there clearly showing that just like temperature, pressure and so forth, biological systems are sensitive to magnetic forces and electrical forces. That’s more or less well-established. What we don’t know is whether or not your cell phone is going to have anything to do with your gut microbiome or things like that. Those are the things that, to my knowledge, have not been clearly studied.

Amy: Not really. But interesting future research?

Gurol: Yeah. And as I said, there’s enough strong evidence out there showing that living organisms are sensitive to these physical external properties: pressure, temperature, pH, electrical fields, magnetic fields. That’s not a new concept. So we’ll just have to see. And I don’t know, do birds sit on electric wires because it tingles? Or is it just a good sitting place?

Amy: Haha. There was a study which showed that Interference from human electronics and AM radio signals disrupted bird migration…

Gurol: Yes I wouldn’t be surprised. I think there is enough information out there to at least create a good incentive to look at the topic in greater depth.

Amy: Yes. So I research the human microbiome. If someone asked you to describe the human microbiome knowing what you do now, how would you explain it?

Gurol: Maybe I would say that the human body is an ecological network. We’re existing together with our environment, and our environment is not just about whether it’s cold or rainy outside. Within our bodies we have countless organisms (not countless but lots of organisms) and bacteria are just one of them, in fact we probably have even more viruses than bacteria – nobody’s been able to track down everything. So we harbor a lot of organisms, especially in our gut. And I think it’s a complex interaction that we don’t fully understand. There’s always, of course, a little bit of hype – when we discover something new everyone gets excited, maybe even a little over-excited. But even if 10% of all the stuff out there on the microbiome turns out to be relevant that’s already a lot.

So I would say the microbiome is a little universe of an ecological network within our body. There’s things going on between all the bacteria. But then there’s also things going on between the bacteria and our cells, the human cells. And we have yet to fully appreciate how many and which parts of these processes are super-critical and which are processes that are interesting but maybe not as critical.

The human microbiome is an ecological network

For example, there’s always interesting studies about species composition of the microbiome – studies stating that composition is critical to this process or that process. But we have to remind ourselves that the composition of the microbiome is not a static thing. When I wake up and go to bed my microbiome is probably totally different depending on where I went to dinner and other variables.

So to some degree we can’t be super sensitive to species-specific microbiome changes, because then we would have traumatic, physiological problems throughout the day. That model just wouldn’t work. We also know that the microbiome can recover: you can take antibiotics and really flush out a lot of stuff but then you can very quickly recover your microbiome. So it’s going to be important to discern which processes have an effect but are not supercritical and which processes are actually very important. That’s the kind of stuff that makes it interesting for the future – to see where it leads us.

Amy: Many people research microbes in isolation and outside their natural environment. I know great insight can be gained from that work. But do you think there needs to be a larger push to research how microbes really act in vivo (in their natural environment)?

Gurol: Yeah. So “in vivo” is an interesting term. It’s very important when you’re dealing with something like the study of cell lines in the laboratory. Problems have appeared when people have studied these cell lines and then tried to claim things about cancer: because the cell line was taken out of its natural context. But when you’re dealing with bacteria to say “what is in vivo?” is not even very clear to me. Take the organism we work with – Bacillus subtilis. You have it right now on your eyebrows and on your skin. It’s also in your gut. It’s in the soil. It’s on your tablecloths. If you have fruits and vegetables in your fridge it’s in there. So which of those locations is “in vivo”…right? They are all very different conditions, but are all places where bacteria naturally occur.

So the question of “in vivo” is a blurred, fuzzy, and potentially misleading question: in the sense that we don’t actually know what to call “in vivo.” If you isolate bacteria in the lab it’s more clear what you’re doing. But to say “this is the proper in vivo context” – I’m putting out a little word of caution: these bacteria are capable of surviving under so many different conditions that we have to be careful.

But overall any good scientist would know that if you’re studying a cell you have to understand the context of the cell, meaning the environment of the cell. And if the environment of the cell includes other cells, and those may be the same species, or different species, then obviously that is something that needs to be taken into consideration. But one can still execute very important studies without being shackled to this vague notion of “what is in vivo?”

It’s especially important to remind people of this now, because as a scientific community are very concerned in trying to make sure that our data is reproducible, and that we have reliable results: that if I do an experiment somebody else can reproduce it. But the moment we get into these types of in vivo conditions the problem is very poorly defined. Even the eyebrow skin environment differs between two people based on fat content, pH and other variables. So we’re dealing with a lot of unknowns, and although we can pat ourselves on the back and say “hey we’re studying something in a more natural context,” we have to realize that it comes at a cost – the cost of having less defined conditions and a lack of understanding of certain things. So it’s a balance – there’s no right or wrong – but we have to understand the pros and cons of each of these approaches very carefully.

Amy: OK I see. Still, in any environment, the microbiome community should focus on studying microbe activity in addition to just species, right?

Gurol: Sure. Sure.

Amy: Ok, what about other microbes like viruses, phages, fungi. Do you think that they’re involved in this electrical of signaling as well?

Gurol: We already showed in at least one paper that two very evolutionary distant species of bacteria can communicate electrically – Pseudomonas and Bacillus. One’s gram positive and one’s gram negative, so they’re very different types of bacteria. That is an indication that this signaling is not just limited to a single species of bacteria. Although that would have been a great result too. Because then you would have to ask “how can it be that only one species evolved this thing!?” That would be kind of  interesting on it’s own also. But maybe not surprisingly that does not appear to be the case.

Bacteria use action potentials to generate electrical signals

So we are still at the ground level if you will. These discoveries are still quite recent in terms of scientific years – they’re the opposite of dog years that go extra slow. So time will tell how many other species signal electrically – which ones do, which ones don’t and what that all means. But again it goes back to the earlier question that you asked: how widespread could this be? It has something to do with the fact that the membrane potential is a very fundamental property of any cell. Any living organism that’s a cell has a potential across its membrane. It’s a very ancient set of physiological processes, so it wouldn’t be shocking in hindsight if we found that different species, and even organisms across many different kingdoms, can engage in some form of this communication.

Amy: Agreed. Can you tell me a little more about how you integrate mathematical models along with the study of actual biofilms to get your results?

Gurol: Sure, the mathematical models are quite important. We use them for two main purposes. One is to come up with very specific hypotheses. The nice thing about mathematical modeling is that you can make predictions that are hypothesis driven…more than hypothesis driven: that are mathematically supported. That is very important because not only does it mean that you can define the question you’re asking really well, but once you have experimental results you can also interpret them using the framework of a mathematical model in a way that is much more powerful.

The other reason we’re doing it is that mathematical models allow us to identify concepts, and concepts are very important because a lot of people are interested in “what is this gene?, or “what is this protein?” Those are tangible objects, and so people really like working on those things. Concepts are by definition intellectual things. That means they are not tangible, in the sense that you can’t touch it, you can’t say “here’s the protein.” But concepts are very important because they allow us to bridge across very different types of systems. So if we can use the same mathematical description to describe processes happening in very different organisms that is meaningful. You can say, “There seems to be something that is shared between two systems, and then you can say “Are they trying to solve a similar problem?” Maybe there’s some kind of evolutionary information there.

Hodgkin and Huxley

So identifying concepts is very relevant in finding connections between things that appear on the surface to be different. Like bacteria and neurons: if I you told ten years ago that they might have similarities, then people would chase you out of the room and say you’re crazy. But now we can use the mathematical formulas that were originally developed in the 1950s by the brilliant Hodgkin-Huxley team. They got a Nobel Prize for the work. They identified a mathematical framework and figured out that basic action potentials in neurons are driven by potassium and sodium flux across the membrane. We showed that you can use that same mathematical framework to explain and make predictions regarding electrical signaling in bacterial communities.

Amy: Yes absolutely. How have your results been received? It seems like pretty enthusiastically, but have you had trouble communicating this to people who have only studied microbes in isolation?

Gurol: No, I mean communicating work is a very important part of being a scientist, because knowledge not shared is like knowledge that almost doesn’t exist. If only one person knows about it, that doesn’t help anybody. So communication is critical. And when you’re communicating you cannot blame the audience for not getting something – you have to figure out how to communicate your results so that anybody can appreciate them. So we take great pride in trying to write papers that are accessible, even though obviously we have to deal with the fact that we’re communicating technical scientific work. I think this pays off in that if you’re able to explain WHY something is important, people can make their own judgement on whether it’s critical or not. The dangerous thing is if people don’t understand it and they’re just confused. Confusion leads to frustration. At that point it’s very difficult to decide if something is noteworthy or not.

Amy: I think you did a great job with communication. I like how you put the more complex math towards the back of the paper.

Gurol: We try to make sure that you can read the paper, and then if you are interested there’s more meat you can sink your teeth into. I’m sure there’s still much room for improvement but we try.

Amy: No, you did a really good job. Can you tell me little more about ion potentials and about how biofilms are able to coordinate behavior to time-share nutrients?

Gurol: Sure. So you mentioned our three latest papers that you liked. The first paper describes how potassium ion channels are utilized by bacteria in biofilm communities to send long-range electrical signals. These signals allow them to coordinate their metabolism. That was an exciting finding that set many other things into motion. In the next paper we described how these electrical signals could extend beyond a single biofilm community. If there’s planktonic motile cells swimming around (because biofilms exist in aqueous environments) it’s not crazy to think that they could be recruited into the biofilm community (even different species). We knew that bacterial motility is driven by proton motor force – the cells utilize an electrochemical gradient across the membrane to generate the energy required to churn flagella: swirly appendages that allow bacteria to swim. We said, “Well if you have electrical signals maybe they would have an effect on this process of motility.” And indeed, we found that that electrical signals can direct and attract motile cells from a distance to the community. That’s interesting because now you can see how electrical signaling can turn a single species community into one that recruits other species too.

We followed that finding up by saying, “OK so if there’s two biofilm communities – two physically separated communities at some distance – not super far away but, with respect to the size of a bacteria some significant distance – what would happen?”

We saw that if these communities share the same environment they become coupled – they can sense each other so to speak. This happens in two main ways. One is this electrical signaling which syncs communities under metabolic stress. But there’s also competition between two communities, where resources are limited and there’s only enough to sustain one community. We found that under such conditions, communities started modulating the coupling between them and transitioned to different types of dynamics: they began time-sharing the limited resources. Time-sharing is a well-known concept. Take vacation homes or cloud computing (where it’s called resource sharing). Another example would be if you have one toy and two kids that want to play with it. You have the kids take turns playing with the toy. It turns out this same strategy is being employed by these bacteria. It’s an exciting discovery because time-sharing is a very dynamic process. With vacation homes or cloud computing time-sharing is not trivial – somebody has to set up a calendar saying, “You take August I’ll take June.” So it requires some kind of higher organization or somebody who coordinates things.

Bacteria use flagella to “swim”

But we found that bacteria naturally time-share through their dynamic system behavior. They can actually transition from being in phase where they’re competing a lot (and that’s OK if there’s enough nutrients) to then switching to time-sharing behavior when nutrients are low. In that case, there’s nobody from the outside telling which colony to do what. The process naturally emerges through the interaction between the communities. It’s very interesting then that bacteria are capable of executing what appear to be nontrivial strategies for coping with limited resources.

Amy: It’s extremely interesting. Do you think we’ve underestimated bacteria? With each new study it seems that bacteria are able to do much more than we previously believed.

Gurol: I’m always amazed by any living organism. If you realize how long these guys have been around (especially bacteria) they’ve survived mass extinction, one after the other. Not just the extinction that killed dinosaurs, but also the earlier extinction where even more species disappeared.

So they’re extremely resilient. They’ve been able to colonize from the Arctic to the depths of the ocean. It’s fascinating. You know, sometimes I go to the east coast and I have trouble adjusting to the new environment! So I’m impressed by these bacterias capabilities. Also, I’m sure there’s still a lot we don’t know. It’ll be a fun future to figure out what else they can do.

Amy: I agree. I know this is jumping ahead but do you see any possible therapeutic interventions based on these findings? Maybe you could interrupt an electrical signal in a biofilm-driven illness?

Gurol: Sure. We really like doing basic research and then letting the findings lead us in those directions. But we are certainly not blind to the possibility that our discoveries may also have biomedical implications. We’re trying to be careful – we don’t want to put blinders on and just focus on one thing. But we do obviously realize that biofilms are a major public health threat. Biofilms are responsible for the majority of infections in clinics. There are people dying and billions of dollars at stake. So yes. We’re aware of these problems and are slowly trying to explore those avenues as well.

Amy: What are you going to study next?

Gurol: We’re still trying to understand some of the basics. We’re still very intrigued by this new form of communication between bacteria – what else can it do? At the other side, at the smaller scale, we’re also trying to understand if in addition to ion channels, there are other genes and proteins playing regulatory roles. So we’re diving both in the direction of more detailed depth and more rigorous research. But also going in the other direction to see “How widespread is this and what other interesting behaviors can come out of this?” I suspect this research will lead to more unexpected findings.

Amy: Is there a growing community of researchers who are getting interested in this? Or are you guys working mostly alone?

Gurol: No, there’s a growing community. In fact we’re going to have a first session on biofilm electrophysiology at the upcoming American Biophysical Society Meeting this February. There will be speakers from all kinds of different institutions and from around the world. So there’s definitely a community that’s building and I think this field has a lot of potential. It’s obviously a very small field only in its infancy, but we’re trying to draw in more people and create a connected community.

Amy: Yes. Some of papers you cite are from the 1970s. How did that earlier research play into your findings?

Gurol: As they say, we’re standing on the shoulders of giants. Smart people existed before we came along. There’s been beautiful studies done – going all the way back to Hodgkin and Huxley. In fact, I would go back as far as Luigi Galvani in the 18th century who did this beautiful experiment where he cut a frog in half and used electrodes to make the legs twitch. It really freaked people out and led to books like Frankenstein where the monster is brought to life with electrical currents. So this field goes back hundreds of years. Obviously a lot more people work on electrophysiology in the human brain because that’s an important organ and it’s easy to see the relevance. But people have looked from plants to fungi to bacteria because the ability of cells to use electrochemical potential across the membrane is so ancient.

Amy: Yes. So let’s say you were talking to a student who wanted to study biofilms or microbiology. What kind of skillset would you recommend this student get to best succeed in the field? 

Computers play a central role in the study of microbes

Gurol: First you have to learn how to think like a scientist, because once you understand how that process works you can work on anything. In terms of a more concrete skillset, the more diverse the better. If you have a unique background and have done things that others have not done then, in the future, maybe you can see things from a different perspective. You might discover something new. I’m sure my background in electrophysiology allowed me to think about biofilm signaling a little bit differently.

I’ll also say this, the future is as clear: we’re heading in a direction where being able to communicate in terms of a mathematical language is key. Also understanding large data sets and analysis – that’s not going away. So having no fear of computers and software and mathematics and physics: that will be important, because again these boundaries are blurring. I think being interdisciplinary is very important.

Amy. Yes. I wish I had a physics background. I’m jealous. Before we go would you like to leave people with any other message?

Gurol: We covered a lot of stuff. Hopefully we’ll have more stuff to talk about in the future. But I’m happy that people like you and others are taking note and finding our work interesting. In this day and age it’s very important for people in science and people in science communication to make work accessible and reach out to the public. Because the more we educate the general public and try to help them understand what science is and how it works, the better it is for our society.

Amy: I do. Thank you again for this fascinating conversation.

Immunostimulation: embracing a new treatment paradigm for chronic disease

January 12th, 2018 by Amy Proal

Think back to the last time you got the flu (virus). The fever, the runny nose, the aches, the sore throat – what causes these and related symptoms? Most flu symptoms are not driven by the virus alone. Instead, they result from a “battle” between the virus and the human immune system. Symptoms begin when the immune system recognizes the flu virus and creates inflammatory proteins called cytokines in an effort to target infected cells. If infected cells are successfully killed, more inflammation is generated as toxins and cellular debris enter the bloodstream. In addition, antibodies may be created in response to these cell and viral byproducts, again leading to a rise in inflammation.

Most cold medicines lower symptoms by suppressing parts of the human immune system

We don’t have antivirals capable of killing the flu virus. So we “treat” the illness by letting this immune system “battle” run its course. In most cases, the human immune system “wins” over time, and the inflammation caused by cytokines, toxins and antibodies drops. We begin to feel better and life goes on.

In some cases, patients manage the flu with over-the-counter medicines. These include Motrin, NyQuil, and antihistamines. In other cases a doctor may prescribe steroids or immunosuppressive medicines. These medicines lower symptoms but do nothing to target the virus driving the illness. In fact, these medicines “work” by shutting down various parts of the human immune response towards the virus. They “tone down” the battle between the immune system and the virus so that less inflammation is generated.

The above medications make patients FEEL better. But they may actually impede recovery from the flu by allowing the virus to survive with greater ease. For example, Canadian researchers found that anti-fever medications suppressed fever in patients with the flu, but also allowed flu viral particles to spread more easily from person to person. Indeed, the team estimates that the use of anti-fever medicines by flu patients contributes to a 5% increase in general flu cases and deaths.

Despite these negative outcomes, our entire medical system centers on immunosuppressive treatments that “knock down” parts of the immune system to suppress symptoms. Patients with autoimmune disease are regularly prescribed immunosuppressive medicines like prednisone, rituximab or TNF-alpha inhibitors. Humira – a TNF-alpha inhibitor used to treat arthritis and other “autoimmune” conditions – is the “best selling prescription drug in the world,” with a $38,000/year price tag per patient.

Why this focus on immunosuppressive therapies? Most immunosuppressive treatments were developed before ~2004, during a time when the human body was believed to be largely sterile. Under these conditions the “theory of autoimmunity” gained hold. If inflammation was detected in patients with a range of conditions it was assumed to be a result of the immune system “going crazy” and attacking human tissue.

The discovery of the human microbiome greatly challenges this “autoimmune” model of disease. We now understand that vast microbiome populations persist in every human body site – from the gut, to the brain, to the placenta, to the liver and beyond. An increasing number of “autoimmune”/inflammatory conditions are now tied to dysbiosis or imbalance of these microbiome communities. This means that in “autoimmune disease,” the human immune system may be attempting to target pathogens in the microbiome in lieu of attacking human tissue. Indeed, an increasing number of studies demonstrate that the “autoantibodies” used to diagnose autoimmune disease are often created in response to a range of bacterial, viral and parasitic infections.

This growing association between infection, “autoimmunity”, and inflammation helps explain the poor long-term outcomes associated with immunosuppressive therapies. Patients administered prednisone or TNF-alpha inhibitors tend to feel better in the short-term, but relapse is common, and often expected. Each relapse can require higher doses of immunosuppressive medication to get symptoms “under control.” Meanwhile, patients are at greater risk for developing a second or third inflammatory disease, and are more likely to suffer from acute infectious conditions like tuberculosis. Long-term health outcomes associated with prednisone are so poor that the slogan “pred ’til dead” is commonly invoked (patients who start prednisone often require higher and higher doses of the medicine until they die from the underlying disease).

This begs the question: what if we treated autoimmune disease in the exact opposite fashion? If microbiome dysbiosis contributes to autoimmune disease then treatments that SUPPORT the immune system could target pathogens driving inflammation. By addressing this infectious root cause of inflammatory symptoms, such treatments might induce actual improvement or even recovery.

How might patients with “autoimmune disease” or related inflammatory conditions respond to “immunostimulative” or immune-supporting treatments? Case histories from the turn of the century offer clues. In the early 1900s, mercury was used to treat syphillis: a sexually transmitted infection caused by the bacteria Treponema pallium. Mercury “deliberately stimulated the immune response” in patients with the disease. This resulted in a phenomenon known as the Jarisch-Herxheimer reaction (named after the researchers who characterized it). As the activated immune system targeted Tremponema pallium a “battle” not that different from that associated with targeting the flu virus ensued. Patients suffered a temporary increase in symptoms including fever, chills, myalgia, and headache as cytokines were released and debris from dying bacterial cells entered the bloodstream. However, if patients endured these symptoms they generally “turned a corner,” where symptoms subsided as Tremponema pallium was gradually eradicated.

In the 100 years since the Jarisch-Herxheimer reaction was described in syphilis, it has been further documented in patients administered immunostimulative therapies for a broad range of infectious conditions. These include Lyme disease, leptospirosis, brucellosis and tuberculosis. More recently, the term “immunopathology” has been used in place of Jarisch-Herxhimer to refer to “a systemic inflammatory response consistent with elevated immune activation.”

Immune activation and immunopathology in the treatment of HIV/AIDS

Over the past decade, immunostimulative treatments have been developed to treat HIV/AIDS and cancer. These therapies are also characterized by temporary symptom increases as the activated immune system attempts to target root causes of inflammation. Current HIV/AIDS treatment centers on highly active antiretroviral therapy (HAART). Patients administered HAART receive a cocktail of anti-retroviral drugs, each of which impedes the ability of the HIV virus to replicate and spread. Prior to HAART, the HIV virus survives by dramatically slowing key parts of the human immune response, including the CD4 cells that normally target infectious agents. This means that when the virus is “contained” by HAART, the immune system “wakes up” and identifies pathogens acquired during previous periods of immunosuppression.

IRIS leads to increased symptoms as the immune system “re-activates” (Source: Khan Academy)

What happens next is a form of immunopathology. The activated immune system starts to target pathogens it could not recognize before HAART was initiated. The patient begins to experience temporary increases in symptoms ranging from fever, to malaise, to neurological dysfunction. Symptoms wax and wane with time, but generally decrease as the immune system better targets a range of previously unrecognized pathogens. The HIV/AIDS community has named this process “Immune Reconstitution Inflammatory Syndrome”, or IRIS.

A number of well-known pathogens are linked to IRIS symptoms: the herpes viruses, cytomegalovirus, hepatitis B and C, M. tuberculosis and Mycobacterium avium among others. Often however, symptoms increase despite the fact that no pathogen can be identified on routine blood tests. This suggests that newly identified microbes, like many of the thousands recently detected in tissue/blood by Stanford researcher Stephen Quake, are also being targeted by the activated immune system.

Several key patterns have been observed in patients experiencing IRIS. One is the “unmaking” of infections that can range back to childhood. Let’s say a patient suffered from bacterial meningitis at age eight. The meningitis microbe may re-appear on IRIS-related blood tests thanks to the renewed immune “attack” against its presence. This supports the fact that pathogens acquired throughout life can persist in our microbiome communities, where they may contribute to chronic symptoms.

Second, patients experiencing IRIS often ‘develop’ autoimmune conditions as the immune system reactivates. These include sarcoidosis, diabetes mellitus, rheumatoid arthritis, lupus and Graves disease. This strongly suggests that pathogens targeted by the IRIS immune response also drive symptoms associated with these related inflammatory disease states.

Cancer therapies target tumors by activating the immune system

The latest cancer therapies also seek to activate the immune system. According to the American Cancer Society, these novel immunotherapies “stimulate your own immune system to work harder or smarter to attack cancer cells.” Cancer immunotherapy treatments include CAR-T therapies: treatments that remove disease-fighting T cells from a patient, genetically modify them to better recognize and attack tumor cells, and then add the activated cells back into a patient’s blood.

Cancer immunotherapy activates human T cells

Response to CAR-T immunotherapy results in serious immunopathology. Nearly all patients administered CAR-T therapy experience a rise in symptoms due to what has been named Cytokine Storm Syndrome or CSS. As implied by the name, CSS results when an immune system “battle” between activated T cells and cancer cells causes massive amounts of inflammatory cytokines to be released into the bloodstream. Resulting symptoms are characterized by fever and in more severe cases, renal insufficiency, pulmonary insufficiency and altered mental status. Sometimes CSS is so strong that patients die from the reaction. Again however, if patients endure/survive the treatment they often enter a state of remission or recovery.

Factors driving CSS are debated by the cancer community. Researchers more familiar with the concept of immunopathology regard CSS as an “on-target” effect of CAR T-cell therapy—that is, its presence demonstrates that active T cells are at work in the body.” In other cases however, CSS is described as a poorly understood “side effect” of immunotherapy. For example, the Washington Post recently published an article on CSS titled: “New cancer therapies have perplexing side effects.”

This “side effect” viewpoint fails to consider a growing body of research linking cancer to infection. For example, “dramatic, continual alterations in the microbiome” were directly responsible for tumor development in a model of colon cancer. Another study found significantly altered microbiome populations in human breast tumor tissue. This imbalance was correlated with decreased expression of key antibacterial response genes. Even signaling peptides created by bacteria have been shown to directly induce tumor formation.

It follows that CSS may result, at least in part, from an immune response towards infected tumor cells. Immunotherapy may also target pathogens that control tumor development by altering the activity of human metabolic pathways. If this is the case, CSS may be the cancer equivalent of IRIS in HIV/AIDS. This is supported by case histories showing that some cancer patients undergoing immunotherapy also develop “new” autoimmune/inflammatory conditions.

Immunotherapy results in Cytokine Release Syndrome (Breslin, 2007)

For example, the Washington post describes a patient named Diane Legg’s response to cancer immunotherapy, stating: ”Her therapy knocked back her cancer, and she’s glad she got it. But the drug also gave her “almost every ‘itis’ you can get: arthritis-like joint pain, lung inflammation called pneumonitis and liver inflammation that bordered on hepatitis, in addition to the uveitis.”

Did immunotherapy really “cause” Legg to develop these new illnesses? Or, as with IRIS, did the conditions arise due to the “unmasking” of pathogens acquired during earlier periods of illness? It’s also worth noting that patients undergoing immunotherapy often present with “new” bacterial, fungal and viral infections. One study identified 43 infections in 30 immunotherapy patients in the first month of treatment, with infections causing the deaths of two patients.

More research is needed to clarify how these infections correlate with CSS. To move forward, researchers developing immunotherapy treatments must be trained to understand the complexity and extent of the human microbiome capable of driving inflammation. This poses a challenge, since at the moment the immunotherapy and microbiome research communities are not well connected.

Managing CSS in cancer and IRIS in HIV/AIDS is also a great challenge for doctors administering the immunostimulative therapies. Most physicians are not trained to consider the microbiome beyond the gut. They are also not taught to understand the general concept of immunopathology. Indeed, palliative medicine has gained such traction that early trials of cancer immunotherapy did not even anticipate a CSS response. This New York Times article titled “When drug trials go horribly wrong” describes testing of an early immunotherapy treatment (TGN1412). After infusion of TGN1412, all six human trial volunteers faced CSS leading to “life-threatening conditions involving multi-organ failure.” According to the Times, “the outpouring of toxic molecules when T-cells are activated…could not have been predicted from prior animal studies using the drug.”

Physicians also suffer from a lack of solid immunopathology treatment guidelines. According to the Washington Post “Many doctors are not up to speed on how to spot and handle an immune system revved up by immunotherapy.” In severe cases of both CSS and IRIS, physicians are forced to prescribe antibiotics and antivirals to manage symptoms associated with infection. However these drugs kill only a fraction of bacteria and viruses capable of driving symptoms. This forces many physicians to “dampen down” symptoms with corticosteroids or other immunosuppressants, the use of which counters the point of treatment in the first place.

Immunostimulation can target root causes of inflammatory disease (Source: Ty Bollinger)

For immunostimulative therapies to truly succeed then, medicine would need to embrace an entirely new paradigm. Drug companies would turn their energy towards developing new antivirals, new antibiotics or antibiotic alternatives. They would attempt development of palliative medicines that help symptoms without destroying the immune response. Tests that better detect and characterize pathogens in the microbiome would be prioritized. Physicians and drug developers would incorporate knowledge of the microbiome into all treatment practices.

The success of immunostimulative therapies also hinges on the willingness of institutional review boards (IRBs) to accept immunopathology. IRBs decide whether patients are allowed to enroll in a particular drug trial. At the moment, many IRBs are unwilling to allow patients with “autoimmune disease” or non-fatal inflammatory conditions to test immunostimulative therapies. This is based on a “do no harm” mentality that does not support increasing symptoms in an effort to improve long-term health.

We must prioritize human microbiome health (Image: BioCote)

What these IRB boards may not realize is that most patients with “autoimmune disease” or non-fatal inflammatory conditions are more than willing to feel temporarily worse (even for years) if offered hope of actual long-term improvement. Serious “autoimmune/inflammatory disease” can feel like a living death. For example, a patient named Anne Ortegren recently committed suicide after decades of suffering from the neuroimmune disease ME/CFS. In a letter written before her death she stated, “A very important factor [in choosing to die] is the lack of realistic hope for relief in the future. It is possible for a person to bear a lot of suffering, as long as it’s time-limited. But the combination of massive suffering and a lack of rational hope for remission or recovery is devastating.”

This leads to a final consideration: the greatest hope for immunostimulation in ANY disease hinges on the ability of treatment to be initiated early, or in a preventative fashion. Medicine must learn to support the immune system BEFORE pathogens push the microbiome far out of balance. “Bringing back” the immune system after years of neglect inevitably leads to severe symptoms and complications. For example, the severity of CSS in cancer is directly related to the tumor burden of the patients (patients with fewer, smaller tumors experience less CSS). In contrast, immunopathology in early-stage disease can be easy to treat and tolerate.

That is why I have a vision: In this vision, medicine respects and supports the immune system from the earliest days of life (even in the womb). Microbiome health forms the cornerstone of new therapies, with treatment administered at the first sign of symptoms. New drugs that target pathogenic bacteria, viruses and fungi are central to therapy. The need for immunosuppressive medicines in chronic inflammatory disease drops…to the point where maybe, one day, they are largely regarded as a failed relic of Medicine’s past.