C-Reactive Protein Levels Correlate with Measures of Dysglycemia and Gut Microbiome Profiles.

C-reactive protein (CRP) is a commonly used marker of low-grade inflammation as well as a marker of acute infection. CRP levels are elevated in those with diabetes and increased CRP concentrations are a risk factor for developing type 2 diabetes. Gut microbiome effects on metabolism and immune responses can impact chronic inflammation, including affecting CRP levels, that in turn can lead to the development and maintenance of dysglycemia. Using a high-sensitivity C-reactive protein (hsCRP) assay capable of detecting subtle changes in C-reactive protein, we show that higher hsCRP levels specifically correlate with worsening glycemia, reduced microbial richness and evenness, and with a reduction in the Firmicutes/Bacteroidota ratio. These data demonstrate a pivotal role for CRP not only in the context of worsening glycemia and changes to the gut microbiota, but also highlight CRP as a potential target for mitigating type 2 diabetes progression or as a therapeutic target that could be manipulated through the microbiome. Understanding these processes will provide insights into the etiology of type 2 diabetes in addition to opening doors leading to possible novel diagnostic strategies and therapeutics.

Impact of Diabetes on the Gut and Salivary IgA Microbiomes.

Mucosal surfaces like those present in the lung, gut, and mouth interface with distinct external environments. These mucosal gateways are not only portals of entry for potential pathogens but also homes to microbial communities that impact host health. Secretory immunoglobulin A (SIgA) is the single most abundant acquired immune component secreted onto mucosal surfaces and, via the process of immune exclusion, shapes the architecture of these microbiomes. Not all microorganisms at mucosal surfaces are targeted by SIgA; therefore, a better understanding of the SIgA-coated fraction may identify the microbial constituents that stimulate host immune responses in the context of health and disease. Chronic diseases like type 2 diabetes are associated with altered microbial communities (dysbiosis) that in turn affect immune-mediated homeostasis. 16S rRNA gene sequencing of SIgA-coated/uncoated bacteria (IgA-Biome) was conducted on stool and saliva samples of normoglycemic participants and individuals with prediabetes or diabetes (n = 8/group). These analyses demonstrated shifts in relative abundance in the IgA-Biome profiles between normoglycemic, prediabetic, or diabetic samples distinct from that of the overall microbiome. Differences in IgA-Biome alpha diversity were apparent for both stool and saliva, while overarching bacterial community differences (beta diversity) were also observed in saliva. These data suggest that IgA-Biome analyses can be used to identify novel microbial signatures associated with diabetes and support the need for further studies exploring these communities. Ultimately, an understanding of the IgA-Biome may promote the development of novel strategies to restructure the microbiome as a means of preventing or treating diseases associated with dysbiosis at mucosal surfaces.

Fecal microbiota transplantation in Parkinson's disease-A randomized repeat-dose, placebo-controlled clinical pilot study.

Background and purpose: The intestinal microbiome plays a primary role in the pathogenesis of neurodegenerative disorders and may provide an opportunity for disease modification. We performed a pilot clinical study looking at the safety of fecal microbiota transplantation (FMT), its effect on the microbiome, and improvement of symptoms in Parkinson's disease. Methods: This was a randomized, double-blind placebo-controlled pilot study, wherein orally administered lyophilized FMT product or matching placebo was given to 12 subjects with mild to moderate Parkinson's disease with constipation twice weekly for 12 weeks. Subjects were followed for safety and clinical improvement for 9 additional months (total study duration 12 months). Results: Fecal microbiota transplantation caused non-severe transient upper gastrointestinal symptoms. One subject receiving FMT was diagnosed with unrelated metastatic cancer and was removed from the trial. Beta diversity (taxa) of the microbiome, was similar comparing placebo and FMT groups at baseline, however, for subjects randomized to FMT, it increased significantly at 6 weeks (p = 0.008) and 13 weeks (p = 0.0008). After treatment with FMT, proportions of selective families within the phylum Firmicutes increased significantly, while proportion of microbiota belonging to Proteobacteria were significantly reduced. Objective motor findings showed only temporary improvement while subjective symptom improvements were reported compared to baseline in the group receiving FMT. Constipation, gut transient times (NS), and gut motility index (p = 0.0374) were improved in the FMT group. Conclusions: Subjects with Parkinson's disease tolerated multi-dose-FMT, and experienced increased diversity of the intestinal microbiome that was associated with reduction in constipation and improved gut transit and intestinal motility. Fecal microbiota transplantation administration improved subjective motor and non-motor symptoms. Clinical trial registration: ClinicalTrial.gov, identifier: NCT03671785.

IgA-Biome Profiles Correlate with Clinical Parkinson's Disease Subtypes.

BACKGROUND: Parkinson's disease is a heterogeneous neurodegenerative disorder with distinctive gut microbiome patterns suggesting that interventions targeting the gut microbiota may prevent, slow, or reverse disease progression and severity. OBJECTIVE: Because secretory IgA (SIgA) plays a key role in shaping the gut microbiota, characterization of the IgA-Biome of individuals classified into either the akinetic rigid (AR) or tremor dominant (TD) Parkinson's disease clinical subtypes was used to further define taxa unique to these distinct clinical phenotypes. METHODS: Flow cytometry was used to separate IgA-coated and -uncoated bacteria from stool samples obtained from AR and TD patients followed by amplification and sequencing of the V4 region of the 16 S rDNA gene on the MiSeq platform (Illumina). RESULTS: IgA-Biome analyses identified significant alpha and beta diversity differences between the Parkinson's disease phenotypes and the Firmicutes/Bacteroides ratio was significantly higher in those with TD compared to those with AR. In addition, discriminant taxa analyses identified a more pro-inflammatory bacterial profile in the IgA+ fraction of those with the AR clinical subclass compared to IgA-Biome analyses of those with the TD subclass and with the taxa identified in the unsorted control samples. CONCLUSION: IgA-Biome analyses underscores the importance of the host immune response in shaping the gut microbiome potentially affecting disease progression and presentation. In the present study, IgA-Biome analyses identified a unique proinflammatory microbial signature in the IgA+ fraction of those with AR that would have otherwise been undetected using conventional microbiome analysis approaches.

Worsening Glycemia Increases the Odds of Intermittent but Not Persistent Staphylococcus aureus Nasal Carriage in Two Cohorts of Mexican American Adults.

Numerous host and environmental factors contribute to persistent and intermittent nasal Staphylococcus aureus carriage in humans. The effects of worsening glycemia on the odds of S. aureus intermittent and persistent nasal carriage was established in two cohorts from an adult Mexican American population living in Starr County, Texas. The anterior nares were sampled at two time points and the presence of S. aureus determined by laboratory culture and spa-typing. Persistent carriers were defined by the presence of S. aureus of the same spa-type at both time points, intermittent carriers were S. aureus-positive for 1 of 2 swabs, and noncarriers were negative for S. aureus at both time points. Diabetes status was obtained through personal interview and physical examination that included a blood draw for the determination of percent glycated hemoglobin A1c (%HbA1c), fasting plasma glucose, and other blood chemistry values. Using logistic regression and general estimating equations, the odds of persistent and intermittent nasal carriage compared to noncarriers across the glycemic spectrum was determined controlling for covariates. Increasing fasting plasma glucose and %HbA1c in the primary and replication cohort, respectively, were significantly associated with increasing odds of S. aureus intermittent, but not persistent nasal carriage. These data suggest that increasing dysglycemia is a risk factor for intermittent S. aureus nasal carriage potentially placing those with poorly controlled diabetes at an increased risk of acquiring an S. aureus infection. IMPORTANCE Factors affecting nasal S. aureus colonization have been studied primarily in the context of persistent carriage. In contrast, few studies have examined factors affecting intermittent nasal carriage with this pathogen. This study demonstrates that the odds of intermittent but not persistent nasal carriage of S. aureus significantly increases with worsening measures of dysglycemia. This is important in the context of poorly controlled diabetes since the risk of becoming colonized with one of the primary organisms associated with diabetic foot infections can lead to increased morbidity and mortality.

Epidemiology of Antibiotic Use and Drivers of Cross-Border Procurement in a Mexican American Border Community.

Background: The U.S.-Mexico Border is an area of opportunity for improved health care access; however, gaps remain as to how and where U.S. border residents, particularly those who are underinsured, obtain care. Antibiotics are one of the most common reported drivers of cross-border healthcare access and a medication of particular concern since indiscriminate or inappropriate use is associated with antimicrobial resistance. In addition, many studies assessing preferences for Mexican pharmaceuticals and healthcare in U.S. border residents were done prior to 2010 when many prescription medications, including antibiotics, were available over the counter in Mexico. Methods: Data used in this study were collected during the baseline examination of an ongoing longitudinal cohort study in Starr Country, Texas, one of 14 counties on the Texas-Mexico border. Participants self-reported the name, date of use, and the source country of each antibiotic used in the past 12 months. Logistic regression was used to determine social, cultural, and clinical features associated with cross-border procurement of antibiotics. Results: Over 10% of the study cohort reported using antibiotics in the past 30 days with over 60% of all rounds used in the past 12 months sourced from Mexico. A lack of health insurance and generation score, a measure of acculturation, were the strongest predictors of cross-border procurement of antibiotics. Conclusions: Factors previously associated with cross-border acquisition of antibiotics are still present despite changes in 2010 to prescription drug regulations in Mexico. These results may be used to inform future public health initiatives to provide culturally sensitive education about responsible antibiotic stewardship and to address barriers to U.S. healthcare and pharmaceutical access in medically underserved, impoverished U.S.-Mexico border communities.

Original Antigenic Sin: the Downside of Immunological Memory and Implications for COVID-19.

The concept of original antigenic sin (OAS) was put forth many years ago to explain how humoral memory responses generated against one set of antigens can affect the nature of antibody responses elicited to challenge infections or vaccinations containing a similar but not identical array of antigens. Here, we highlight the link between OAS and the germinal center reaction (GCR), a process unique to activated B cells undergoing somatic hypermutation and class switch recombination. It is the powerful response of activated memory B cells and the accompanying GCR that establish the foundations of OAS. We apply these concepts to the current COVID-19 pandemic and put forth several possible scenarios whereby OAS may result in either beneficial or harmful outcomes depending, hypothetically, on prior exposure to antigens shared between SARS-CoV-2 and seasonal human coronaviruses (hCoVs) that include betacoronaviruses (e.g., HCoV-OC43 and HCoV-HKU1) and alphacoronaviruses (e.g., HCoV-NL63 and HCoV-HKU1) (E. M. Anderson, E. C. Goodwin, A. Verma, C. P. Arevalo, et al., medRxiv, 2020, https://doi.org/10.1101/2020.11.06.20227215; S. M. Kissler, C. Tedijanto, E. Goldstein, Y. H. Grad, and M. Lipsitch, Science 368:860-868, 2020, https://doi.org/10.1126/science.abb5793).

The impact of the Th17:Treg axis on the IgA-Biome across the glycemic spectrum.

Secretory IgA (SIgA) is released into mucosal surfaces where its function extends beyond that of host defense to include the shaping of resident microbial communities by mediating exclusion/inclusion of respective microbes and regulating bacterial gene expression. In this capacity, SIgA acts as the fulcrum on which host immunity and the health of the microbiota are balanced. We recently completed an analysis of the gut and salivary IgA-Biomes (16S rDNA sequencing of SIgA-coated/uncoated bacteria) in Mexican-American adults that identified IgA-Biome differences across the glycemic spectrum. As Th17:Treg ratio imbalances are associated with gut microbiome dysbiosis and chronic inflammatory conditions such as type 2 diabetes, the present study extends our prior work by examining the impact of Th17:Treg ratios (pro-inflammatory:anti-inflammatory T-cell ratios) and the SIgA response (Th17:Treg-SIgA axis) in shaping microbial communities. Examining the impact of Th17:Treg ratios (determined by epigenetic qPCR lymphocyte subset quantification) on the IgA-Biome across diabetes phenotypes identified a proportional relationship between Th17:Treg ratios and alpha diversity in the stool IgA-Biome of those with dysglycemia, significant changes in community composition of the stool and salivary microbiomes across glycemic profiles, and genera preferentially abundant by T-cell inflammatory phenotype. This is the first study to associate epigenetically quantified Th17:Treg ratios with both the larger and SIgA-fractionated microbiome, assess these associations in the context of a chronic inflammatory disease, and offers a novel frame through which to evaluate mucosal microbiomes in the context of host responses and inflammation.

Characterization of peripheral blood mononuclear cells gene expression profiles of pediatric Staphylococcus aureus persistent and non-carriers using a targeted assay.

Defects in innate immunity affect many different physiologic systems and several studies of patients with primary immunodeficiency disorders demonstrated the importance of innate immune system components in disease prevention or colonization of bacterial pathogens. To assess the role of the innate immune system on nasal colonization with Staphylococcus aureus, innate immune responses in pediatric S. aureus nasal persistent carriers (n = 14) and non-carriers (n = 15) were profiled by analyzing co-clustered gene sets (modules). We stimulated previously frozen peripheral blood mononuclear cells (PBMCs) from these subjects with i) a panel of TLR ligands, ii) live S. aureus (either a mixture of strains or stimulation with respective carriage isolates), or iii) heat-killed S. aureus. We found no difference in responses between carriers and non-carriers when PBMCs were stimulated with a panel of TLR ligands. However, PBMC gene expression profiles differed between persistent and non-S. aureus carriers following stimulation with either live or dead S. aureus. These observations suggest that individuals susceptible to persistent carriage with S. aureus may possess differences in their live/dead bacteria recognition pathway and that innate pathway signaling is different between persistent and non-carriers of S. aureus.

Mutations to Cysteine Residues in the Trypanosoma cruzi B-Cell Superantigen Tc24 Diminish Susceptibility to IgM-Mediated Hydrolysis.

B-cell superantigens (BC-SAgs) are immunoevasins that have evolved in response to innate catalytic IgM antibodies; germ-line encoded immunoglobulins present in the preimmune repertoire independent of prior antigen exposure. Catalysis is the result of a 2-step process that involves first the formation of a non-covalent bond between the BC-SAg and the immunoglobulin followed by covalent bond formation at the catalytic site resulting in target hydrolysis. Tc24 is a recently described Trypanosoma cruzi BC-SAg hypothesized to play a role in evading the humoral response early in the infection period. We previously demonstrated that exposure to Tc24 following immunization or infection resulted in the depletion of the catalytic IgM response, leaving a gap in the catalytic IgM repertoire. The present report compares the BC-SAg properties of wild-type Tc24 (Tc24-WT) to that of 2 recombinant Tc24 isoforms: Tc24-C2 (Cys to Ser mutations in the 2 most-proximal Cys residues) and Tc24-C4 (Cys to Ser mutations in all 4 Cys residues present). BC-SAg activity was assessed by immunizing mice with the respective isoforms and examining the ability of IgM purified from the respective groups to hydrolyze the 3 Tc24 isoforms. In addition, the ability of IgM purified from naive mice to hydrolyze the Tc24 isoforms was also assessed. Immunization with Tc24-WT, Tc24-C2, or Tc24-C4 resulted in loss of IgM-mediated hydrolysis of Tc24-WT. However, the ability of IgM purified from naive mice (previously shown to hydrolyze Tc24-WT) was less effective in hydrolyzing the 2 Tc24 isoforms. These data demonstrate that although the BC-SAg site in the mutants remained intact, their reduced susceptibility to IgM-mediated hydrolysis suggested that structural changes resulting from the Cys to Ser mutations altered accessibility to the catalytic site in the 2 isoforms.

Identification and Characterization of the Trypanosoma cruzi B-cell Superantigen Tc24.

Trypanosoma cruzi causes life-long disease after infection and leads to cardiac disease in 30% of infected individuals. After infection, the parasites are readily detectable in the blood during the first few days before disseminating to infect numerous cell types. Preliminary data suggested that the Tc24 protein that localizes to the T. cruzi membrane during all life stages possesses B-cell superantigenic properties. These antigens facilitate immune escape by interfering with antibody-mediated responses, particularly the avoidance of catalytic antibodies. These antibodies are an innate host defense mechanism present in the naive repertoire, and catalytic antibody-antigen binding results in hydrolysis of the target. We tested the B-cell superantigenic properties of Tc24 by comparing the degree of Tc24 hydrolysis by IgM purified from either Tc24 unexposed or exposed mice and humans. Respective samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, silver stained, and the degree of hydrolysis was measured. Data presented in this report suggest that the T. cruzi Tc24 is a B-cell superantigen based on the observations that 1) Tc24 was hydrolyzed by IgM present in serum of unexposed mice and humans and 2) exposure to Tc24 eliminated catalytic activity as early as 4 days after T. cruzi infection.

Genome-Wide Association Study of Staphylococcus aureus Carriage in a Community-Based Sample of Mexican-Americans in Starr County, Texas.

Staphylococcus aureus is the number one cause of hospital-acquired infections. Understanding host pathogen interactions is paramount to the development of more effective treatment and prevention strategies. Therefore, whole exome sequence and chip-based genotype data were used to conduct rare variant and genome-wide association analyses in a Mexican-American cohort from Starr County, Texas to identify genes and variants associated with S. aureus nasal carriage. Unlike most studies of S. aureus that are based on hospitalized populations, this study used a representative community sample. Two nasal swabs were collected from participants (n = 858) 11-17 days apart between October 2009 and December 2013, screened for the presence of S. aureus, and then classified as either persistent, intermittent, or non-carriers. The chip-based and exome sequence-based single variant association analyses identified 1 genome-wide significant region (KAT2B) for intermittent and 11 regions suggestively associated with persistent or intermittent S. aureus carriage. We also report top findings from gene-based burden analyses of rare functional variation. Notably, we observed marked differences between signals associated with persistent and intermittent carriage. In single variant analyses of persistent carriage, 7 of 9 genes in suggestively associated regions and all 5 top gene-based findings are associated with cell growth or tight junction integrity or are structural constituents of the cytoskeleton, suggesting that variation in genes associated with persistent carriage impact cellular integrity and morphology.