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The human lung is a complex organ that comprises diverse populations of epithelial, mesenchymal, vascular, and immune cells, which gains even greater complexity during disease states. To effectively study the lung at a single-cell level, a dissociation protocol that achieves the highest yield of viable cells of interest with minimal dissociation-associated protein or transcription changes is key. Here, we detail a rapid collagenase-based dissociation protocol (Col-Short) that provides a high-yield single-cell suspension that is suitable for a variety of downstream applications. Diseased human lung explants were obtained and dissociated through the Col-Short protocol and compared with four other dissociation protocols. Resulting single-cell suspensions were then assessed with flow cytometry, differential staining, and quantitative real-time PCR to identify major hematopoietic and nonhematopoietic cell populations, as well as their activation states. We observed that the Col-Short protocol provides the greatest number of cells per gram of lung tissue, with no reduction in viability when compared with previously described dissociation protocols. Col-Short had no observable surface protein marker cleavage as well as lower expression of protein activation markers and stress-related transcripts compared with four other protocols. The Col-Short dissociation protocol can be used as a rapid strategy to generate single cells for respiratory cell biology research. [ABSTRACT FROM AUTHOR]

Elite controllers (ECs) are people living with human immunodeficiency virus (HIV) who spontaneously control viral replication without antiretroviral therapy. We observed that elevated anti-cytomegalovirus (CMV) immunoglobulin G (IgG) levels correlated with annual CD4 T-cell count decay in ECs independently of age, sex, and human leukocyte antigen (HLA) type. Elevated anti-CMV titers may favor disease progression in ECs. [ABSTRACT FROM AUTHOR]

Background Cytomegalovirus (CMV) seropositivity and anti-CMV immunoglobulin G (IgG) levels are associated with adverse health outcomes in elderly populations. Among people living with human immunodeficiency virus (PLWH), CMV seropositivity has been associated with persistent CD8 T-cell elevation and increased risk of developing non-AIDS comorbidities despite long-term antiretroviral therapy (ART). Herein, we investigated whether CMV seropositivity and elevation of anti-CMV IgG levels were associated with increased epithelial gut damage, microbial translocation, and systemic inflammation. Methods A total of 150 PLWH (79 ART-naive and 71 ART-treated) were compared to 26 without human immunodeficiency virus (HIV) infection (uninfected controls). Plasma markers of HIV disease progression, epithelial gut damage, microbial translocation, nonspecific B-cell activation, anti-CMV and anti–Epstein-Barr virus (EBV) IgG levels, and proinflammatory cytokines were measured. Results CMV seropositivity and elevated anti-CMV IgG levels were associated with markers of epithelial gut damage, microbial translocation, and inflammation in PLWH and participants without HIV infection. In contrast, total nonspecific IgG, immunoglobulin M, immunoglobulin A, and anti-EBV IgG levels were not associated with these markers. CMV seropositivity was associated with markers of epithelial gut damage, microbial translocation, and inflammation independent of sociodemographic and behavioral characteristics of the study population. Conclusions CMV-seropositive people with and without HIV had increased epithelial gut damage, microbial translocation, and inflammation. Furthermore, anti-CMV IgG levels were independently associated with increased epithelial gut damage and microbial translocation. CMV coinfection may partially explain persistent gut damage, microbial translocation, and inflammation in ART-treated PLWH. [ABSTRACT FROM AUTHOR]

Background Microbial translocation from the gut to systemic circulation contributes to immune activation during human immunodeficiency virus (HIV) infection and is usually assessed by measuring plasma levels of bacterial lipopolysaccharide (LPS). Fungal colonization in the gut increases during HIV-infection and people living with HIV (PLWH) have increased plasma levels of fungal polysaccharide (1→3)-β-D-Glucan (βDG). We assessed the contribution of circulating DG to systemic immune activation in PLWH. Methods Cross-sectional and longitudinal assessments of plasma βDG levels were conducted along with markers of HIV disease progression, epithelial gut damage, bacterial translocation, proinflammatory cytokines, and βDG-specific receptor expression on monocytes and natural killer (NK) cells. Results Plasma βDG levels were elevated during early and chronic HIV infection and persisted despite long-term antiretroviral therapy (ART). βDG increased over 24 months without ART but remained unchanged after 24 months of treatment. βDG correlated negatively with CD4 T-cell count and positively with time to ART initiation, viral load, intestinal fatty acid–binding protein, LPS, and soluble LPS receptor soluble CD14 (sCD14). Elevated βDG correlated positively with indoleamine-2,3-dioxygenase-1 enzyme activity, regulatory T-cell frequency, activated CD38+Human Leukocyte Antigen - DR isotype (HLA-DR)+ CD4 and CD8 T cells and negatively with Dectin-1 and NKp30 expression on monocytes and NK cells, respectively. Conclusions PLWH have elevated plasma βDG in correlation with markers of disease progression, gut damage, bacterial translocation, and inflammation. Early ART initiation prevents further βDG increase. This fungal antigen contributes to immune activation and represents a potential therapeutic target to prevent non–acquired immunodeficiency syndrome events. [ABSTRACT FROM AUTHOR]

Background: Regenerating islet-derived protein 3α (REG3α) is an antimicrobial peptide secreted by intestinal Paneth cells. Circulating REG3α has been identified as a gut damage marker in inflammatory bowel diseases. People living with human immunodeficiency virus (PWH) on antiretroviral therapy (ART) present with an abnormal intestinal landscape leading to microbial translocation, persistent inflammation, and development of non-AIDS comorbidities. Herein, we assessed REG3α as a marker of gut damage in PWH.Methods: Plasma from 169 adult PWH, including 30 elite controllers (ECs), and 30 human immunodeficiency virus (HIV)-uninfected controls were assessed. REG3α plasma levels were compared with HIV disease progression, epithelial gut damage, microbial translocation, and immune activation markers.Results: Cross-sectionally, REG3α levels were elevated in untreated and ART-treated PWH compared with controls. ECs also had elevated REG3α levels compared to controls. Longitudinally, REG3α levels increased in PWH without ART and decreased in those who initiated ART. REG3α levels were inversely associated with CD4 T-cell count and CD4:CD8 ratio, while positively correlated with HIV viral load in untreated participants, and with fungal product translocation and inflammatory markers in all PWH.Conclusions: Plasma REG3α levels were elevated in PWH, including ECs. The gut inflammatory marker REG3α may be used to evaluate therapeutic interventions and predict non-AIDS comorbidity risks in PWH. [ABSTRACT FROM AUTHOR]