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Competing Interests: Conflicts of Interest: S.B.R. is scientific advisor for the Canadian Foundation for AIDS Research. D.H.S.T. is a site principal investigator for clinical trials sponsored by Glaxo Smith Kline.

Objective: To assess whether probiotic supplementation may reduce disease-linked systemic immune activation in people living with HIV with the immunologic nonresponder phenotype., Design: Phase 2b, randomized, double-blind, placebo-controlled pilot trial., Methods: HIV-positive individuals with blood CD4+ T-cell counts <350/mm3 despite viral suppression were randomized to 2:1 to receive De Simone Formulation Probiotic (DSFP; "Visbiome" commercially) or placebo for 48 weeks; target enrollment was 36 patients. The primary endpoint was the change in blood CD8+ T-cell coexpression of human leukocyte antigen-DR isotype and CD38 ("CD8 activation"). Secondary endpoints included biomarkers of inflammation, immune reconstitution, bacterial translocation, and gut permeability. Adjusted linear regression and linear mixed regression methods evaluated the differences between study arms from baseline to week 48. Study monitoring was performed by the CIHR Canadian HIV Trials Network Data Safety Monitoring Committee., Results: Nineteen patients received DSFP, whereas 10 received placebo. One probiotic arm patient withdrew early. Blood CD8 activation increased 0.82 percentage points (pp) in the probiotic arm (95% confidence interval: -1.23 to 2.87;) and decreased by 2.06 pp in the placebo arm (-4.81 to 0.70; between arms P = 0.097). CD4+ T-cell activation (%HLA-DR+) decreased in the placebo arm [-3.79 pp (-7.32 to -0.26)] but increased in the probiotic arm [1.64 (-0.98 to 4.26); between arms P = 0.018]. No differences were observed in plasma or urine biomarkers of inflammation or microbial translocation., Conclusions: Blood immune activation markers in immunologic nonresponder individuals on effective antiretroviral treatment were not reduced by supplementation with DSFP; CD4+ T-cell activation may have been increased., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Immunologic non-responders (INRs) are a subset of individuals living with HIV who have suboptimal blood CD4+ T cell recovery despite effective antiretroviral therapy (ART). They are at an increased risk of serious non-AIDS co-morbidities and death, and demonstrate enhanced systemic immune activation. In other populations diet has been correlated with markers of systemic inflammation through the Diet Inflammatory Index (DII), but this association has not been studied in persons living with HIV (PLWH). Blood was collected from 28 INR PLWH with a blood CD4+ T cell count <350/μL despite ≥2 years of effective ART. Participants completed a Canadian Diet History Questionnaire, and their responses were used to calculate the DII. Plasma inflammatory markers (IFNγ, TNF, IL-6, sVCAM, D-dimer, sCD14 and CRP) were assayed by ELISA, cellular immune activation (HLA-DR and CD38 on CD4+ and CD8+ T cells) was quantified using flow cytometry, and small bowel permeability assessed by calculation of the urine LacMan ratio after drinking a mix of lactulose and mannitol. Participants were a median age of 57 years, had been on effective ART for 15 years, and the median DII was -1.91 (range of -3.78 to +2.23). No correlation was observed between DII and plasma markers of inflammation, levels of T cell activation, gut permeability, or the biomarker of bacterial translocation sCD14. Self-reported alcohol intake, a potential confounder of the relationship between diet and inflammatory biomarkers, was also not associated with systemic inflammation or gut permeability. Our findings suggest that other mechanisms, rather than diet, are likely to be the major driver of systemic inflammation in INR individuals., Competing Interests: JH owns controlling interest in Connecting Health Innovations LLC (CHI), a company that has licensed the right to his invention of the dietary inflammatory index (DII®) from the University of South Carolina in order to develop computer and smart phone applications for patient counseling and dietary intervention in clinical settings. NS is an employee of CHI. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Malazogu, Rousseau, Shivappa, Huibner, Walmsley, Kovacs, Benko, Reinhard, Rosenes, Hebert and Kaul.)

Objective: Serious non-AIDS disease events (SNAE) are experienced disproportionately by immunologic non-responders (INRs), HIV-infected individuals who do not restore CD4 T cells in blood despite effective viral suppression. We aimed to characterize the inflammatory biomarker profile of the INR phenotype., Methods: Blinded cross-sectional cohort study comparing markers of immune activation and gut homing between INR and non-INR individuals. HIV-positive participants had HIV RNA suppression on antiretroviral therapy and were categorized as either INR (N = 36) or Clinical Responders ("CR"; CD4>350/mm3; N = 47). 18 HIV-negative comparator individuals were included. Cellular markers were assessed by flow cytometry, with soluble markers assessed by ELISA and LC/MS-MS. Multivariable linear regression models estimated the association between INR phenotype and markers, adjusting for age, sex, duration of ART, and recent infection/vaccination., Results: INR participants demonstrated a reduced CD4/CD8 ratio (p<0.001), 35% more CD8 activation (p = 0.02), 36% greater α4β7+ CD4 T cells (p<0.01), 54% more HLA-DR+ CD4 T cells (p<0.001), and 20% higher plasma VCAM (p<0.01) compared to CRs. The INR phenotype was not associated with levels of Kyn/Trp, CRP, TNF, IFNγ, IL-8, IL-6, sCD14, D-Dimer, I-FABP, MCP-1, ICAM or CD8%HLA-DR+., Conclusions: Peripheral CD4 non-recovery during long-term treated HIV infection is characterized by elevated CD8 activation and CD4 gut homing. Gut-focused interventions may be warranted in the INR context, and CD8 activation may serve as a surrogate endpoint for clinical interventions., Competing Interests: The authors have declared that no competing interests exist.

The ATP-binding cassette (ABC) transporter multidrug resistance protein 1 (MRP1/ABCC1) is responsible for the cellular export of a chemically diverse array of xenobiotics and endogenous compounds. Arsenic, a human carcinogen, is a high-affinity MRP1 substrate as arsenic triglutathione [As(GS)3]. In this study, marked differences in As(GS)3 transport kinetics were observed between MRP1-enriched membrane vesicles prepared from human embryonic kidney 293 (HEK) (Km 3.8 µM and Vmax 307 pmol/mg per minute) and HeLa (Km 0.32 µM and Vmax 42 pmol/mg per minute) cells. Mutant MRP1 lacking N-linked glycosylation [Asn19/23/1006Gln; sugar-free (SF)-MRP1] expressed in either HEK293 or HeLa cells had low Km and Vmax values for As(GS)3, similar to HeLa wild-type (WT) MRP1. When prepared in the presence of phosphatase inhibitors, both WT- and SF-MRP1-enriched membrane vesicles had a high Km value for As(GS)3 (3-6 µM), regardless of the cell line. Kinetic parameters of As(GS)3 for HEK-Asn19/23Gln-MRP1 were similar to those of HeLa/HEK-SF-MRP1 and HeLa-WT-MRP1, whereas those of single glycosylation mutants were like those of HEK-WT-MRP1. Mutation of 19 potential MRP1 phosphorylation sites revealed that HEK-Tyr920Phe/Ser921Ala-MRP1 transported As(GS)3 like HeLa-WT-MRP1, whereas individual HEK-Tyr920Phe- and -Ser921Ala-MRP1 mutants were similar to HEK-WT-MRP1. Together, these results suggest that Asn19/Asn23 glycosylation and Tyr920/Ser921 phosphorylation are responsible for altering the kinetics of MRP1-mediated As(GS)3 transport. The kinetics of As(GS)3 transport by HEK-Asn19/23Gln/Tyr920Glu/Ser921Glu were similar to HEK-WT-MRP1, indicating that the phosphorylation-mimicking substitutions abrogated the influence of Asn19/23Gln glycosylation. Overall, these data suggest that cross-talk between MRP1 glycosylation and phosphorylation occurs and that phosphorylation of Tyr920 and Ser921 can switch MRP1 to a lower-affinity, higher-capacity As(GS)3 transporter, allowing arsenic detoxification over a broad concentration range., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)