Robert P. Friedland, M.D.

Mason C. and Mary D. Rudd Endowed Chair In Neurology

Professor, Dept. of Neurology

401 E. Chestnut Street, Suite 510, Louisville, KY 40292 • Clinical: 502-589-0802 • Academic: 502-852-7981

On Thu, Mar 15, 2018 at 10:14 PM, <pointreyes@verizon.net> wrote:

eg the lancet p 106 jan 14 1978?

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Sent: Thu, Mar 15, 2018 08:41 PM
Subject: Gut PD AL ALS

We Need to Stay on the Gut Microbiome’s Good Side or Risk Microbiota Dysbiosis

Kate Marusina, Ph.D.

Researchers at the University of Louisville suggest that there are several neural routes by which microbial amyloid may influence the central nervous system. [This figure, which originally appeared in PLoS Pathogens (2017 Dec 21; 13(12): e1006654. DOI: 10.1371/journal.ppat.1006654), is reproduced here with the permission of paper’s authors, Robert P. Friedland and Matthew R. Chapman.]

More than a billion years of mammalian–microbial coevolution has led to interdependency. The effect of microbial activity exerted on the host is emerging as a critical factor in disease pathogenesis.

Gut microbiota dysbiosis—imbalances in the composition and function of the microbes in the stomach and intestines—is found to be associated with diseases ranging from neurologic to respiratory, metabolic, hepatic, and cardiovascular.

Human-associated microbes are no longer viewed as a collection of independent species. Advances in whole-microbiome technologies have shed light on the enormous biological and functional diversity of microbes that exist in the body. Now, intense research in microbiota-mediated disease mechanisms fosters hope of novel therapeutic and preventive strategies for a range of chronic diseases.

Inflammation Caused by Gut Microbes Spreads to the Brain

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Researchers at the University of Louisville have identified potential areas of interaction between amyloid-producing bacteria and the gut. [This figure, which originally appeared in PLoS Pathogens (2017 Dec 21; 13(12): e1006654. DOI: 10.1371/journal.ppat.1006654), is reproduced here with the permission of paper’s authors, Robert P. Friedland and Matthew R. Chapman.]

“Parkinson’s disease (PD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS) are neurodegenerative conditions with remarkably similar qualities,” explains Robert Friedland, M.D., who is a professor and the Mason C. and Mary D. Rudd Endowed Chair in Neurology at the University of Louisville. “These diseases usually occur sporadically, with 1–10% of cases having a clear genetic origin. While amyloid deposits are a characteristic feature, they may not be causative of the disease.”

Dr. Friedland drew an analogy with ailments such as kuru and Creutzfeldt-Jakob disease, which are transmitted by prions—infectious proteins with amyloid features. He pointed out that the primary route of prion infection in kuru and bovine spongiform encephalopathy (BSE, also known as mad cow disease) is through the digestive system. Delivered via the intestines to lymph nodes and to other parts of the body, proteins in a prion conformation stimulate other proteins to adopt the same conformation.

Similarly, amyloid proteins in PD and AD are also self-propagating, and transmittable from animal to animal and within the body. “We now believe that the gut, including the mouth, nose, and intestines, is a site of origin of these neurodegenerative conditions, and microbial factors are responsible for triggering disease,” adds Dr. Friedland.

Dr. Friedland’s collaborator, Matthew Chapman, Ph.D., an associate professor from the University of Michigan, has discovered that curli—extracellular fibers produced by various enteric bacteria—share biophysical properties with amyloid. These naturally occurring amyloid polymers help bacterial cells to bind to one another, forming biofilms.

Subsequent research demonstrated cross-seeding of amyloid proteins between species. The research team believes that activation of the innate immune system by microbial amyloids is a key contributing factor that primes the immune system and affects inflammation in the brain. “Activated T cells may travel in the bloodstream from the gut to brain, causing neuroinflammation,” adds Dr. Friedland. He adds that this mechanism suggests a way of reversing neurodegeneration: altering the composition and activity of gut microbiota.

The collaborators propose a new term, mapranosis, to describe the process of microbiota-associated proteopathy and neuroinflammation, where proteopathy refers to disease processes involving altered protein structures. Given the increasing evidence of relationships between microbiota and amyloid pathology, wider multidisciplinary metaproteomic studies of endogenous microbial amyloids could provide actionable information for future interventional strategies.

Metabolomic Signatures Predict Host Response

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Scientists at Metabolon are studying the mammalian-microbiota relationship and how it may be influenced by metabolites that play regulatory roles. These metabolites can arise from host or microbial biosynthesis or small molecules such as drugs or xenobiotics. All have been reported to affect either the physiology of the host or the collective physiology of the “organ” of the microbiome—which in turn can affect the host.

“Our goal is to develop and sustain enabling technologies and processes for comprehensive and reproducible mapping of metabolic changes,” says Luke Miller, Ph.D., vice president of operations at Metabolon. “If the metabolic change is induced by perturbations in gut microbiota, we should be able to detect it with sufficient precision to produce actionable information for each individual.”

Metabolon is one of the few metabolic profiling companies capable of “Tier1” validation, defined by the Metabolomic Society as the highest level of rigor and confidence in compound identification. According to the company, Metabolon is capable of analyzing nearly 350 types of samples via the standardized process. Data acquisition is performed in parallel on four combinations of ultra-high-performance liquid chromatography and mass spectometry.

Combined with comprehensive bioinformatics tools, the analysis pathway routinely yields 1,000–2,000 validated metabolites. “Multiple biomarkers pinpoint the affected pathway with greater precision,” explains Dr. Miller. Metabolon technologies are well positioned to analyze spontaneous changes in gut microbiome.

Of approximately 250 microbe-specific metabolites, 50–80 routinely appear in plasma. “A collaborative project with Weizmann Institute of Science in Rehovot, Israel, tackled an intractable problem of post-dieting weight gain,” continues Dr. Miller. Many dieting individuals fail to maintain a long-term weight loss, and instead undergo repeated weight regain/loss cycles, known as the “yo-yo effect.” The study identified a metabolomics signature attributed to the intestinal microbiome that persisted after dieting and contributed to faster weight regain in laboratory mice. Specifically, flavonoids remained persistently suppressed after weight loss.

“Metabolomics is a pragmatic tool for understanding and affecting microbiome–host interplay,” says Dr. Miller. “Our analysis suggested that flavonoid supplementation could aid in breaking the yo-yo cycle and in resisting secondary weight gain.”

Metabolon actively fosters academic and commercial collaborations in nutritional supplementation, contributing identification of dysregulated metabolic signatures and following their “normalization” with a supplement intake. A recently published collaboration with the team at Colorado State University discovered an endogenous metabolic signature in an animal model of malnutrition. Rice bran supplementation stimulated depressed microflora and supported the scientific evidence of rice bran’s ability to fight off multiple gut pathogens.

Asthma-Promoting Microbes Are Activated in Infancy

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Siolta Therapeutics has developed a platform that implements a three-pronged approach to disease prevention. 1) Microbial sequencing of infant stool is used to identify children at risk for developing asthma in the first days/months of life. 2) Validation is completed using a novel and noninvasive immune assay, which acts as an early diagnostic. 3) A targeted microbial consortium is rationally designed to encode the maximum functional capacity associated with healthy infants using the minimal number of species for each infant deemed to be at high risk.

“Asthma is a chronic inflammatory disease, affecting over 300 million people worldwide,” says Nikole Kimes, Ph.D., cofounder and executive vice president, Siolta Therapeutics. “We know that the nascent gut microbiota of infants is essential in establishing proper immune function and that disruptions to this community result in early immunological dysfunction and subsequent asthma development.”

Siolta is founded on the groundbreaking research out of Dr. Susan Lynch’s laboratory at the University of California, San Francisco, that found strong correlation between childhood asthma and certain dysregulation of neonatal gut microbiota (NGM). While direct causality is yet to be established, the particular composition of the microbiota in neonates, who went on to develop atopy and asthma at a later age, is difficult to ignore.

“Differences in the gastrointestinal microbial composition of these children were associated with significant metabolic alterations,” explains Dr. Kimes. “The resulting lipid metabolites are known to impact immune response by modulating regulatory T cells.” The asthma-promoting microbiome shows diminished relative abundance of key bacterial species, suggesting that oral supplementation to re-establish “healthy” bacterial composition may prevent or even reverse asthmatic inflammatory cascade.

Siolta is developing live biotherapeutic products based on a precision-medicine approach. First, newborns’ microbial signatures are identified through next-generation sequencing. Next, at-risk status is validated by ex vivo immunostimulatory assays using the fecal metabolic milieu. To replace the depleted bacterial species, targeted microbial consortia are manufactured for oral supplementation.

Dr. Kimes points out that until recently, our view of human microbiology was dominated by studies of single species. “We now understand that gut microbes exist in highly complex and interactive communities.” Siolta’s ecosystem approach utilizes key founder species in synergistic consortia to provide important functional features of a protective gut microbiome. The overall approach is designed to generate novel biotherapeutics for a variety of inflammatory disorders driven by microbial imbalances.

“By restructuring the microbiome, we reengineer metabolism and promote immune tolerance,” declares Dr. Kimes. “The potential for disease prevention is promising and very exciting.”

Carbon Buckyballs Identify Live Bacteria in Biofilms

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At the University of Nevada, scientists have shown that a C60-detector-reporter complex can be assembled in a two-step process. First, the rRNA detector complementary to the 16S rRNA signature sequence of a specific bacterium is conjugated with the C60 buckyball. Second, a DNA reporter is hybridized with the detector. Once this complex is formed, the fluorophore is quenched. When the detector binds to a specific 16S rRNA region, the reporter is released and starts to fluoresce.

Profiling the microbiome using advanced molecular biology techniques has become routine, in part due to PCR-based analysis of the bacterial 16S ribosomal RNA. At the same time, approaches for spatial characterization of the microbiome are limited to fixed assays and spectral imaging.

Live assays could provide significant insights in the spatiotemporal organization of the microbial consortium and lead to rapid identification in field conditions. “A major obstacle for live assays is the cargo delivery,” say Bahram Parvin Ph.D., and Qingsu Cheng, Ph.D., both of whom are professors at the University of Nevada.

“We demonstrated that carbon buckyballs are able to permeate through lipid membranes and bacterial cell walls. Our proof-of-concept studies support the conclusion that buckyballs conjugated with carefully designed RNA capture sequences and fluorescent labels are able to differentiate bacterial species,” say Drs. Parvin and Cheng.

Buckyballs are nanoparticles composed of 60 covalently linked carbon atoms (C60). They seem to enter bacteria by simple diffusion, and are non-sticky to the substrate. In the team’s procedure, C60 buckyballs are functionalized with RNA-detector oligos complementary to 16S RNA of three bacterial species. Next, a DNA sequence with an attached fluorescent reporter is hybridized with the RNA-detector in a way that quenches the fluorescent signal. When the RNA-detector sequence binds to its target, the fluorescent probe is released. It can be easily detected by wide-field fluorescent microscopy.

“Our first applications will be to identify foodborne bacteria,” continues Dr. Parvin. “However, periodontal microbiome and associated diseases is an underserved space. Our technology is capable of detecting bacteria from mouth rinse in about 15 minutes, without any sample preparation.”

The buckyball tagging demonstrates 92% probe efficiency and excellent differentiation between closely related species of bacteria that cannot be distinguished by Gram staining. This technology is especially practical when a microorganism could not be cultured. And, Dr. Parvin’s team is developing a microfluidic system for point-of-care applications. The prototype system included sorting of the bacterial mixture using chemoattractants. The future iterations will include multiple sink wells containing buckyballs functionalized for specific organisms for highly multiplexed assays.

Don’t Let the Resistome Strike Back

Antimicrobial resistance (AMR) has become a global public health threat that causes 700,000 deaths annually, according to a study (“Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations”) released by the Wellcome Trust and the British Government. The study notes that AMR is largely due to the widespread use of antibiotics in agriculture and healthcare. Resistant pathogen strains, the study points out, are being created at an unprecedented pace.

Rethinking Anti-AMR Strategy

The rise of resistant pathogens signals an urgent need for new antibiotics, yet fewer antibiotics are being introduced. The development of antibiotics, which has been in steep decline since 2000, is characterized by lengthening timelines and steep capital commitments. Accordingly, efforts to counter the rise of resistant pathogens have shifted to preserving the efficacy of marketed antibiotics.

A viable strategy in the war against AMR is to reduce antibiotic exposure without compromising infection control efficacy. A complementary strategy is to protect the gut microbiome, a virtual primordial soup of resistance, from antibiotic exposure.

Guarding the Microbiome

Many antibiotics accumulate in the gastrointestinal tract at levels sufficient to disrupt the gut microbiome, providing selective pressure for evolution, transfer, and propagation of AMR, and priming the microbiome for opportunistic infections, such as Clostridium difficile infection (CDI).

Researchers at Synthetic Biologics report that they are developing SYN-004 (ribaxamase), a novel “guardian of the gut” approach to protect the gut microbiome from antibiotics. Ribaxamase is an oral β-lactamase enzyme designed to degrade certain β-lactam antibiotics in the gastrointestinal tract before they can reach and harm the colonic microbiota.

Studies evaluating ribaxamase verified that elimination of antibiotics in the upper gastrointestinal tract protected the gut microbiome from antibiotic collateral damage without affecting antibiotic systemic levels, significantly reduced CDI in at risk patients, and diminished AMR, according to Synthetic Biologics’ officials. Antibiotic inactivation appears to represent a potentially promising treatment paradigm to mitigate the emergence and spread of AMR and extend the duration of efficacy of current and future antibiotics.

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