Microbiota composition modulates inflammation and neointimal hyperplasia after arterial angioplasty.

BACKGROUND: Neointimal hyperplasia is a major contributor to restenosis after arterial interventions, but the genetic and environmental mechanisms underlying the variable propensity for neointimal hyperplasia between individuals, including the role of commensal microbiota, are not well understood. We sought to characterize how shifting the microbiome using cage sharing and bedding mixing between rats with differing restenosis phenotypes after carotid artery balloon angioplasty could alter arterial remodeling. METHODS: We co-housed and mixed bedding between genetically distinct rats (Lewis [LE] and Sprague-Dawley [SD]) that harbor different commensal microbes and that are known to have different neointimal hyperplasia responses to carotid artery balloon angioplasty. Sequencing of the 16S ribosomal RNA gene was used to monitor changes in the gut microbiome. RESULTS: There were significant differences in neointimal hyperplasia between non-co-housed LE and SD rats 14 days after carotid artery angioplasty (mean intima + media [I + M] area, 0.117 ± 0.014 mm2 LE vs 0.275 ± 0.021 mm2 SD; P < .001) that were diminished by co-housing. Co-housing also altered local adventitial Ki67 immunoreactivity, local accumulation of leukocytes and macrophages (total and M2), and interleukin 17A concentration 3 days after surgery in each strain. Non-co-housed SD and LE rats had microbiomes distinguished by both weighted (P = .012) and unweighted (P < .001) UniFrac beta diversity distances, although without significant differences in alpha diversity. The difference in unweighted beta diversity between the fecal microbiota of SD and LE rats was significantly reduced by co-housing. Operational taxonomic units that significantly correlated with average I + M area include Parabacteroides distasonis, Desulfovibrio, Methanosphaera, Peptococcus, and Prevotella. Finally, serum concentrations of microbe-derived metabolites hydroxyanthranilic acid and kynurenine/tryptophan ratio were significantly associated with I + M area in both rat strains independent of co-housing. CONCLUSIONS: We describe a novel mechanism for how microbiome manipulations affect arterial remodeling and the inflammatory response after arterial injury. A greater understanding of the host inflammatory-microbe axis could uncover novel therapeutic targets for the prevention and treatment of restenosis.

The intestinal microbiota in the pathogenesis of inflammatory bowel diseases: new insights into complex disease.

Inflammatory bowel diseases (IBD) are a group of chronic diseases of increasing worldwide prevalence characterized by gastrointestinal (GI) inflammation leading to debilitating symptoms and complications. The contribution of the intestinal microbiota to the pathogenesis and etiology of these diseases is an area of active research interest. Here, we discuss key mechanisms underlying the chronic inflammation seen in IBD as well as evidence implicating the intestinal microbiota in the development and potentiation of that inflammation. We also discuss recently published work in areas of interest within the field of microbial involvement in IBD pathogenesis - the importance of proper microecology within the GI tract, the evidence that the intestinal microbiota transduces environmental and genetic risk factors for IBD, and the mechanisms by which microbial products contribute to communication between microbe and host. There is an extensive body of published research on the evidence for microbial involvement in IBD; the goal of this review is to highlight the growing edges of the field where exciting and innovative research is pushing the boundaries of the conceptual framework of the role of the intestinal microbiota in IBD pathogenesis.

A framework for assessing outcomes from newborn screening: on the road to measuring its promise.

UNLABELLED: Newborn screening (NBS) is intended to identify congenital conditions prior to the onset of symptoms in order to provide early intervention that leads to improved outcomes. NBS is a public health success, providing reduction in mortality and improved developmental outcomes for screened conditions. However, it is less clear to what extent newborn screening achieves the long-term goals relating to improved health, growth, development and function. We propose a framework for assessing outcomes for the health and well-being of children identified through NBS programs. The framework proposed here, and this manuscript, were approved for publication by the Secretary of Health and Human Services' Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC). This framework can be applied to each screened condition within the Recommended Uniform Screening Panel (RUSP), recognizing that the data elements and measures will vary by condition. As an example, we applied the framework to sickle cell disease and phenylketonuria (PKU), two diverse conditions with different outcome measures and potential sources of data. Widespread and consistent application of this framework across state NBS and child health systems is envisioned as useful to standardize approaches to assessment of outcomes and for continuous improvement of the NBS and child health systems. SIGNIFICANCE: Successful interventions for newborn screening conditions have been a driving force for public health newborn screening for over fifty years. Organizing interventions and outcome measures into a standard framework to systematically assess outcomes has not yet come into practice. This paper presents a customizable outcomes framework for organizing measures for newborn screening condition-specific health outcomes, and an approach to identifying sources and challenges to populating those measures.

Personalizing health care: feasibility and future implications.

Considerable variety in how patients respond to treatments, driven by differences in their geno- and/ or phenotypes, calls for a more tailored approach. This is already happening, and will accelerate with developments in personalized medicine. However, its promise has not always translated into improvements in patient care due to the complexities involved. There are also concerns that advice for tests has been reversed, current tests can be costly, there is fragmentation of funding of care, and companies may seek high prices for new targeted drugs. There is a need to integrate current knowledge from a payer's perspective to provide future guidance. Multiple findings including general considerations; influence of pharmacogenomics on response and toxicity of drug therapies; value of biomarker tests; limitations and costs of tests; and potentially high acquisition costs of new targeted therapies help to give guidance on potential ways forward for all stakeholder groups. Overall, personalized medicine has the potential to revolutionize care. However, current challenges and concerns need to be addressed to enhance its uptake and funding to benefit patients.

Sickle cell disease incidence among newborns in New York State by maternal race/ethnicity and nativity.

PURPOSE: Sickle cell disease is estimated to occur in 1:300-400 African-American births, with higher rates among immigrants from Africa and the Caribbean, and is less common among Hispanic births. This study determined sickle cell disease incidence among New York State newborns stratified by maternal race/ethnicity and nativity. METHODS: Newborns with confirmed sickle cell disease born to New York State residents were identified by the New York State newborn screening program for the years 2000-2008 and matched to birth records to obtain birth and maternal information. Annual incidence rates were computed and bivariate analyses were conducted to examine associations with maternal race/ethnicity and nativity. RESULTS: From 2000 to 2008, 1,911 New York State newborns were diagnosed with sickle cell disease and matched to the birth certificate files. One in every 1,146 live births was diagnosed with sickle cell disease. Newborns of non-Hispanic black mothers accounted for 86% of sickle cell disease cases whereas newborns of Hispanic mothers accounted for 12% of cases. The estimated incidence was 1:230 live births for non-Hispanic black mothers, 1:2,320 births for Hispanic mothers, and 1:41,647 births for non-Hispanic white mothers. Newborns of foreign-born non-Hispanic black mothers had a twofold higher incidence of sickle cell disease than those born to US-born non-Hispanic black mothers (P < 0.001). CONCLUSION: This study provides the first US estimates of sickle cell disease incidence by maternal nativity. Women born outside the United States account for the majority of children with sickle cell disease born in New York State. Such findings identify at-risk populations and inform outreach activities that promote ongoing, high-quality medical management to affected children.

Insurance coverage of medical foods for treatment of inherited metabolic disorders.

PURPOSE: Treatment of inherited metabolic disorders is accomplished by use of specialized diets employing medical foods and medically necessary supplements. Families seeking insurance coverage for these products express concern that coverage is often limited; the extent of this challenge is not well defined. METHODS: To learn about limitations in insurance coverage, parents of 305 children with inherited metabolic disorders completed a paper survey providing information about their use of medical foods, modified low-protein foods, prescribed dietary supplements, and medical feeding equipment and supplies for treatment of their child's disorder as well as details about payment sources for these products. RESULTS: Although nearly all children with inherited metabolic disorders had medical coverage of some type, families paid "out of pocket" for all types of products. Uncovered spending was reported for 11% of families purchasing medical foods, 26% purchasing supplements, 33% of those needing medical feeding supplies, and 59% of families requiring modified low-protein foods. Forty-two percent of families using modified low-protein foods and 21% of families using medical foods reported additional treatment-related expenses of $100 or more per month for these products. CONCLUSION: Costs of medical foods used to treat inherited metabolic disorders are not completely covered by insurance or other resources.

Enter at your own risk: how enteroviruses navigate the dangerous world of pattern recognition receptor signaling.

Enteroviruses are the most common human viral pathogens worldwide. This genus of small, non-enveloped, single stranded RNA viruses includes coxsackievirus, rhinovirus, echovirus, and poliovirus species. Infection with these viruses can induce mild symptoms that resemble the common cold, but can also be associated with more severe syndromes such as poliomyelitis, neurological diseases including aseptic meningitis and encephalitis, myocarditis, and the onset of type I diabetes. In humans, polarized epithelial cells lining the respiratory and/or digestive tracts represent the initial sites of infection by enteroviruses. Control of infection in the host is initiated through the engagement of a variety of pattern recognition receptors (PRRs). PRRs act as the sentinels of the innate immune system and serve to alert the host to the presence of a viral invader. This review assembles the available data annotating the role of PRRs in the response to enteroviral infection as well as the myriad ways by which enteroviruses both interrupt and manipulate PRR signaling to enhance their own replication, thereby inducing human disease.

Exploring the Impact of Adult-use Cannabis Legalization on Legal System Referrals to Treatment for Cannabis Use: Do Age and Race Have a Moderating Effect?

PURPOSE: Despite the progression of recreational cannabis legalization, the legal system remains the largest source of referral to treatment for cannabis use. The legal system's continued practice of requiring participation in cannabis treatment programs raises questions regarding the extent to which individuals who interact with the legal system are monitored for cannabis use post-legalization. This article presents trends in justice-system referrals to treatment for cannabis use in legal and nonlegal states for 2007-2019. The relationship between legalization and justice system treatment referrals for black, Hispanic/Latino, and white adults and juveniles was explored. Given that minority and youth populations are subject to disproportionate levels of cannabis enforcement, legalization is expected to have a weaker relationship with justice-system referral rates in white juveniles and black and Hispanic/Latino adults and juveniles compared to white adults. METHODS: Using 2007-2019 data from the Treatment Episode Data Set-Admissions (TEDS-A), variables were created for state-level rates of legal system-referred treatment admissions for cannabis use in black, Hispanic/Latino, and white adults and juveniles. Rate trends were compared across populations and staggered difference-in-difference and event analyses were conducted to determine whether legalization is associated with a decline in justice-system referrals to treatment for cannabis use . FINDINGS: For the study period, the mean rate of legal system-referred admissions in the total population was 2.75 per 10,000 residents. Black juveniles had the highest mean rate (20.16), followed by Hispanic/Latino juveniles (12.35), black adults (9.18), white juveniles (7.58), Hispanic/Latino adults (3.42), and white adults (1.66). Legalization did not have a significant impact on treatment-referral rates in any population of study. Events analyses indicated significant rate increases in black juveniles in legalized states compared to controls at 2 and 6 years after policy change, and in black and Hispanic/Latino adults at 6 years after policy change (all, P < 0.05). While racial/ethnic disparities in referral rates declined in absolute terms, the relative size of these disparities increased in legalized states. IMPLICATIONS: TEDS-A captures only publicly funded treatment admissions and relies on the quality of individual-state reporting. Individual-level factors that may impact decisions regarding treatment referrals for cannabis use could not be controlled for. Despite limitations, the present findings suggest that for individuals who interact with the criminal legal system, cannabis use may still result in legal monitoring after reform. The upward trend in legal system referrals for black (but not white) adults and juveniles several years after states legalize cannabis warrants further examination and may reflect continued disparate treatment of these populations at multiple points along the legal-system continuum.

Peptide YY: A Paneth cell antimicrobial peptide that maintains Candida gut commensalism.

The mammalian gut secretes a family of multifunctional peptides that affect appetite, intestinal secretions, and motility whereas others regulate the microbiota. We have found that peptide YY (PYY1-36), but not endocrine PYY3-36, acts as an antimicrobial peptide (AMP) expressed by gut epithelial paneth cells (PC). PC-PYY is packaged into secretory granules and is secreted into and retained by surface mucus, which optimizes PC-PYY activity. Although PC-PYY shows some antibacterial activity, it displays selective antifungal activity against virulent Candida albicans hyphae-but not the yeast form. PC-PYY is a cationic molecule that interacts with the anionic surfaces of fungal hyphae to cause membrane disruption and transcriptional reprogramming that selects for the yeast phenotype. Hence, PC-PYY is an antifungal AMP that contributes to the maintenance of gut fungal commensalism.

Plasma Gut Microbe-Derived Metabolites Associated with Peripheral Artery Disease and Major Adverse Cardiac Events.

Cardiovascular diseases are associated with gut dysbiosis, but the role of microbe-derived metabolites as biomarkers or modulators of cardiovascular disease are not well understood. This is a targeted metabolomics study to investigate the association of nine microbe-derived metabolites with lower extremity peripheral artery disease (PAD), a form of atherosclerosis, and major adverse cardiac events (MACE). The study cohort consists of individuals with intermittent claudication and ankle-brachial index (ABI) < 0.9 (N = 119) and controls without clinically-apparent atherosclerosis (N = 37). The primary endpoint was MACE, a composite endpoint of myocardial infarction, coronary revascularization, stroke, transient ischemic attack, or cardiac-related death. Plasma metabolite concentrations differed significantly between the PAD and control groups. After adjustment for traditional atherosclerosis risk factors, kynurenine, hippuric acid, indole-3-propionic acid (IPA), and indole-3-aldehyde (I3A) concentrations were negatively associated with PAD, whereas indoxyl sulfate and 3-hydroxyanthranilic acid were positively associated. Hippuric acid, IPA, and I3A correlated with ABI, a surrogate for atherosclerotic disease burden. Those in the highest I3A concentration quartile had significantly improved freedom from MACE during follow-up compared to those in the lowest quartile. This study identifies specific indole- and phenyl-derived species impacted by gut microbial metabolic pathways that could represent novel microbiome-related biomarkers of PAD.

Diagnostic guidelines for newborns who screen positive in newborn screening.

BACKGROUND: Recent expansion of the newborn screening panels has presented an interesting challenge to specialty care centers, especially the clinical genetics community. Some of the conditions in the core and secondary newborn screening panels have extremely variable clinical presentations; others are so rare that only a handful of newborns have been diagnosed with them to date (Region 4 Collaborative MS/MS project-http://region4genetics.org/msms_data_project/data_project_home.aspx). Definition of some disorders is problematic-does continued abnormality of the screening analyte constitute diagnosis or is further testing necessary? METHODS: A work group of the New York Mid-Atlantic Consortium for Genetic and Newborn Screening Services (region 2), one of seven regional collaboratives funded by the Federal Health Resources and Services Administration and administered by the Maternal and Child Health Bureau (U22MC03956), has developed guidelines for the confirmation of diagnosis of the conditions in the newborn screening panels for use by the specialty care centers. DISCUSSION: The diagnostic guidelines are a work in progress and are being reviewed and revised regularly as our understanding of the newborn screened disorders improves. The aim is to make it a relevant guide for specialty care physicians and other healthcare professionals in the diagnostic workup of these patients.

Intratumoral accumulation of gut microbiota facilitates CD47-based immunotherapy via STING signaling.

Most studies focus on how intestinal microbiota influence cancer immunotherapy through activating gut immunity. However, immunotherapies related to innate responses such as CD47 blockade rely on the rapid immune responses within the tumor microenvironment. Using one defined anaerobic gut microbiota to track whether microbiota interact with host immunity, we observed that Bifidobacterium facilitates local anti-CD47 immunotherapy on tumor tissues through the capacity to accumulate within the tumor microenvironment. Systemic administration of Bifidobacterium leads to its accumulation within the tumor and converts the nonresponder mice into responders to anti-CD47 immunotherapy in a stimulator of interferon genes (STING)- and interferon-dependent fashion. Local delivery of Bifidobacterium potently stimulates STING signaling and increases cross-priming of dendritic cells after anti-CD47 treatment. Our study identifies the mechanism by which gut microbiota preferentially colonize in tumor sites and facilitate immunotherapy via STING signaling.

Microbial Colonization of Germ-Free Mice Restores Neointimal Hyperplasia Development After Arterial Injury.

Background The potential role of the gut microbiome in cardiovascular diseases is increasingly evident. Arterial restenosis attributable to neointimal hyperplasia after cardiovascular procedures such as balloon angioplasty, stenting, and bypass surgery is a common cause of treatment failure, yet whether gut microbiota participate in the development of neointimal hyperplasia remains largely unknown. Methods and Results We performed fecal microbial transplantation from conventionally raised male C57BL/6 mice to age-, sex-, and strain-matched germ-free mice. Five weeks after inoculation, all mice underwent unilateral carotid ligation. Neointimal hyperplasia development was quantified after 4 weeks. Conventionally raised and germ-free cohorts served as comparison groups. Conclusions Germ-free mice have significantly attenuated neointimal hyperplasia development compared with conventionally raised mice. The arterial remodeling response is restored by fecal transplantation. Our results describe a causative role of gut microbiota in contributing to the pathogenesis of neointimal hyperplasia.

Fecal microbiota transplant rescues mice from human pathogen mediated sepsis by restoring systemic immunity.

Death due to sepsis remains a persistent threat to critically ill patients confined to the intensive care unit and is characterized by colonization with multi-drug-resistant healthcare-associated pathogens. Here we report that sepsis in mice caused by a defined four-member pathogen community isolated from a patient with lethal sepsis is associated with the systemic suppression of key elements of the host transcriptome required for pathogen clearance and decreased butyrate expression. More specifically, these pathogens directly suppress interferon regulatory factor 3. Fecal microbiota transplant (FMT) reverses the course of otherwise lethal sepsis by enhancing pathogen clearance via the restoration of host immunity in an interferon regulatory factor 3-dependent manner. This protective effect is linked to the expansion of butyrate-producing Bacteroidetes. Taken together these results suggest that fecal microbiota transplantation may be a treatment option in sepsis associated with immunosuppression.

Microbiota control acute arterial inflammation and neointimal hyperplasia development after arterial injury.

BACKGROUND: The microbiome has a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia development has been linked to inflammation, a direct role of the microbiota in neointimal hyperplasia has not yet been established. Germ-free (GF) mice are an invaluable model for studying causative links between commensal organisms and the host. We hypothesized that GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally raised (CONV-R) mice. METHODS: Twenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers at five days. Systemic inflammation was assessed by multiplex immunoassays of serum. CONV-R mice treated in the same manner served as the control cohort. GF and CONV-R mice were compared using standard statistical methods. RESULTS: All GF mice remained sterile during the entire study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia development compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media) area. The collagen content of the neointimal lesions appeared qualitatively similar on Masson's trichrome staining. There was significantly reduced Ki67 immunoreactivity in the media and adventitia of GF carotid arteries 5 days after ligation. GF mice also had increased arterial infiltration of anti-inflammatory M2 macrophages compared to CONV-R mouse arteries and a reduced proportion of mature neutrophils. GF mice had significantly reduced serum IFN-γ-inducible protein (IP)-10 and MIP-2 5 days after carotid ligation, suggesting a reduced systemic inflammatory response. CONCLUSIONS: GF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which is likely related to altered kinetics of wound healing and acute inflammation. Recognizing the role of commensals in the regulation of arterial remodeling will provide a deeper understanding of the pathophysiology of restenosis and support strategies to treat or reduce restenosis risk by manipulating microbiota.

Unc93b Induces Apoptotic Cell Death and Is Cleaved by Host and Enteroviral Proteases.

Unc93b is an endoplasmic reticulum (ER)-resident transmembrane protein that serves to bind and traffic toll-like receptors (TLRs) from the ER to their appropriate subcellular locations for ligand sensing. Because of its role in TLR trafficking, Unc93b is necessary for an effective innate immune response to coxsackievirus B3 (CVB), a positive-sense single stranded RNA virus belonging to the enterovirus family. Here, we show that Unc93b is cleaved by a CVB-encoded cysteine protease (3Cpro) during viral replication. Further, we define a role for Unc93b in the induction of apoptotic cell death and show that expression of wild-type Unc93b, but not a mutant incapable of binding TLRs or exiting the ER (H412R), induces apoptosis. Furthermore, we show that cellular caspases activated during apoptosis directly cleave Unc93b. Interestingly, we show that the 3Cpro- and caspase-mediated cleavage of Unc93b both occur within ten amino acids in the distal N-terminus of Unc93b. Mechanistically, neither caspase-mediated nor 3Cpro-mediated cleavage of Unc93b altered its trafficking function, inhibited its role in facilitating TLR3 or TLR8 signaling, or altered its apoptosis-inducing effects. Taken together, our studies show that Unc93b is targeted by both viral- and host cell-specific proteases and identify a function of Unc93b in the induction of apoptotic cell death.

RIP3 Regulates Autophagy and Promotes Coxsackievirus B3 Infection of Intestinal Epithelial Cells.

Receptor interacting protein kinase-3 (RIP3) is an essential kinase for necroptotic cell death signaling and has been implicated in antiviral cell death signaling upon DNA virus infection. Here, we performed high-throughput RNAi screening and identified RIP3 as a positive regulator of coxsackievirus B3 (CVB) replication in intestinal epithelial cells (IECs). RIP3 regulates autophagy, a process utilized by CVB for viral replication factory assembly, and depletion of RIP3 inhibits autophagic flux and leads to the accumulation of autophagosomes and amphisomes. Additionally, later in infection, RIP3 is cleaved by the CVB-encoded cysteine protease 3C(pro), which serves to abrogate RIP3-mediated necrotic signaling and induce a nonnecrotic form of cell death. Taken together, our results show that temporal targeting of RIP3 allows CVB to benefit from its roles in regulating autophagy while inhibiting the induction of necroptotic cell death.

Death waits for no man--does it wait for a virus? How enteroviruses induce and control cell death.

Enteroviruses (EVs) are the most common human viral pathogens. They cause a variety of pathologies, including myocarditis and meningoencephalopathies, and have been linked to the onset of type I diabetes. These pathologies result from the death of cells in the myocardium, central nervous system, and pancreas, respectively. Understanding the role of EVs in inducing cell death is crucial to understanding the etiologies of these diverse pathologies. EVs both induce and delay host cell death, and their exquisite control of this balance is crucial for their success as human viral pathogens. Thus, EVs are tightly involved with cell death signaling pathways and interact with host cell signaling at multiple points. Here, we review the literature detailing the mechanisms of EV-induced cell death. We discuss the mechanisms by which EVs induce cell death, the signaling pathways involved in these pathways, and the strategies by which EVs antagonize cell death pathways. We also discuss the role of cell death in both the resulting pathology in the host and in the facilitation of viral spread.

Focal adhesion kinase is a component of antiviral RIG-I-like receptor signaling.

Viruses modulate the actin cytoskeleton at almost every step of their cellular journey from entry to egress. Cellular sensing of these cytoskeletal changes may function in the recognition of viral infection. Here we show that focal adhesion kinase (FAK), a focal adhesion localized tyrosine kinase that transmits signals between the extracellular matrix and the cytoplasm, serves as a RIG-I-like receptor antiviral signaling component by directing mitochondrial antiviral signaling adaptor (MAVS) activation. Cells deficient in FAK are highly susceptible to RNA virus infection and attenuated in antiviral signaling. We show that FAK interacts with MAVS at the mitochondrial membrane in a virus infection-dependent manner and potentiates MAVS-mediated signaling via a kinase-independent mechanism. A cysteine protease encoded by enteroviruses cleaves FAK to suppress its role in innate immune signaling. These findings suggest that FAK serves as a link between cytoskeletal perturbations that occur during virus infection and activation of innate immune signaling.