Highly Sensitive Analysis of Cervical Mucosal HIV-1 Infection Using Reporter Viruses Expressing Secreted Nanoluciferase.

Ex vivo cervical tissue explant models offer a physiologically relevant approach for studying virus-host interactions that underlie mucosal HIV-1 transmission to women. However, the utility of cervical explant tissue (CET) models has been limited for both practical and technical reasons. These include assay variation, inadequate sensitivity for assessing HIV-1 infection and replication in tissue, and constraints imposed by the requirement for using multiple replica samples of CET to test each experimental variable and assay parameter. Here, we describe an experimental approach that employs secreted nanoluciferase (sNLuc) and current HIV-1 reporter virus technologies to overcome certain limitations of earlier ex vivo CET models. This method augments application of the CET model for investigating important questions involving mucosal HIV-1 transmission.

Evaluation of Social Determinants of Health in Nursing Education through Service Learning.

Background: This manuscript describes an in-state nursing student global health-care experience. The 2021 Rio Grande Valley service learning team at Louise Herrington School of Nursing at Baylor University traveled from Dallas to McAllen, Texas to volunteer in a COVID vaccination clinic and refugee respite center on the U.S.-Mexican border. Method: A competency-based evaluation utilized the American Association of Colleges of Nursing's The Essentials: Core Competencies for Professional Nursing Education Featured Concepts, with a focus on social determinants of health, as a framework. Results: The evaluation of the service learning trip through the lens of social determinants of health and the Core Competencies can serve as a guideline for the design of future trips. Conclusion: The Rio Grande Valley service learning trip contributed to nursing students' self-reports of competency in global health education, in identifying the social determinants of health that characterized the immigrants and refugees, and in service and advocacy.

Assessing Diversity in Hospital Library Collections.

Public libraries have conducted collection diversity audits, but this is the first known report of a diversity audit in the hospital library community. A two-part questionnaire was sent to hospital librarians to determine their use of diversity audits in collection management and to provide a tool for a preliminary assessment of their collections' diversity. Results of the questionnaire indicate that developing diversity within hospital library collections is important to these respondents. These librarians also support diversity in their library personnel, open access, researching critical gaps, and programming.

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design.

Eliciting HIV-1-specific broadly neutralizing antibodies (bNAbs) remains a challenge for vaccine development, and the potential of passively delivered bNAbs for prophylaxis and therapeutics is being explored. We used neutralization data from four large virus panels to comprehensively map viral signatures associated with bNAb sensitivity, including amino acids, hypervariable region characteristics, and clade effects across four different classes of bNAbs. The bNAb signatures defined for the variable loop 2 (V2) epitope region of HIV-1 Env were then employed to inform immunogen design in a proof-of-concept exploration of signature-based epitope targeted (SET) vaccines. V2 bNAb signature-guided mutations were introduced into Env 459C to create a trivalent vaccine, and immunization of guinea pigs with V2-SET vaccines resulted in increased breadth of NAb responses compared with Env 459C alone. These data demonstrate that bNAb signatures can be utilized to engineer HIV-1 Env vaccine immunogens capable of eliciting antibody responses with greater neutralization breadth.

Mass Cytometric Analysis of HIV Entry, Replication, and Remodeling in Tissue CD4+ T Cells.

To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping, combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses, can be used as a discovery tool.

Mucosal stromal fibroblasts markedly enhance HIV infection of CD4+ T cells.

Understanding early events of HIV transmission within mucosal tissues is vital for developing effective prevention strategies. Here, we report that primary stromal fibroblasts isolated from endometrium, cervix, foreskin, male urethra, and intestines significantly increase HIV infection of CD4+ T cells-by up to 37-fold for R5-tropic HIV and 100-fold for X4-tropic HIV-without themselves becoming infected. Fibroblasts were more efficient than dendritic cells at trans-infection and mediate this response in the absence of the DC-SIGN and Siglec-1 receptors. In comparison, mucosal epithelial cells secrete antivirals and inhibit HIV infection. These data suggest that breaches in the epithelium allow external or luminal HIV to escape an antiviral environment to access the infection-favorable environment of the stromal fibroblasts, and suggest that resident fibroblasts have a central, but previously unrecognized, role in HIV acquisition at mucosal sites. Inhibiting fibroblast-mediated enhancement of HIV infection should be considered as a novel prevention strategy.

Optimized Replicating Renilla Luciferase Reporter HIV-1 Utilizing Novel Internal Ribosome Entry Site Elements for Native Nef Expression and Function.

We previously developed replication-competent reporter HIV-1 (referred to herein as LucR.T2A reporter viruses), utilizing a "ribosome skipping" T2A peptide strategy to link Renilla luciferase (LucR) with Nef expression. The demonstrated utility for HIV-1 vaccine and transmission study applications included measurement of neutralizing antibody (NAb) activity in vaccine sera, improved cell-mediated virus inhibition assays, such as T cell-mediated virus inhibition and antibody-dependent cell-mediated cytotoxicity (ADCC) assays, and humanized mouse models. Herein, we extend our prior work and introduce reporter virus technology for applications that require fully functional Nef. We demonstrate that in CD4(+) T cells productively infected with LucR.T2A reporter viruses, T2A peptide-driven Nef expression and function, such as down-regulation of surface CD4 and MHC-I, were impaired. We overcame this limitation of LucR.T2A reporter viruses and achieved physiological Nef expression and function by engineering novel LucR reporter HIV-1 comprising 11 different internal ribosome entry site (IRES) elements chosen for size and relative activity. A range of Nef expression was observed in 293T cells transfected with the different LucR.IRES reporter virus constructs. Iteratively, we identified IRES reporter genomes that expressed Nef closest to physiological levels and produced virus with infectivity, titers, and replication kinetics similar to nonreporter viruses. Our results demonstrated that LucR reporter activity was stable over multiple replication cycles in peripheral blood mononuclear cells (PBMCs). Furthermore, we analyzed Nef functionality, i.e., down-modulation of MHC-I and CD4, following infection of T cell lines and PBMCs. Unlike LucR.T2A reporter virus, one of the redesigned LucR.IRES reporter viruses [containing the modified encephalomyocarditis virus (EMCV) 6ATR IRES element, "6ATRi"] demonstrated Nef expression and function similar to parental "nonreporter" virus. In a previously validated (nef-independent) T cell-based NAb neutralization assay, LucR.6ATRi reporter virus performed indistinguishably from LucR.T2A reporter virus. In summary, reporter viruses comprising the "6ATRi" element promise to augment HIV-1 vaccine and transmission research approaches requiring a sensitive reporter readout combined with wild-type Nef function.

A Mediterranean-style low-glycemic-load diet increases plasma carotenoids and decreases LDL oxidation in women with metabolic syndrome.

Thirty-five women with metabolic syndrome and high plasma low-density lipoprotein (LDL) cholesterol (≥100 mg/dl) participated in a dietary intervention consisting of a Mediterranean-style low-glycemic-load diet for 12 weeks. Participants were randomly allocated to consume diet only (n=15) or diet plus a medical food containing soy protein and plant sterols (n=20). Plasma concentrations of carotenoids, lipoprotein subfractions and oxidized LDL (OxLDL) were measured. Independent of treatment, women had a significant increase in plasma lutein (P<.0001) and ß-carotene (P<.0001), while plasma lycopene was reduced (P<.05) after 12 weeks. Low-density lipoprotein cholesterol was reduced from 138±35 to 114±33 mg/dl (P<.0001). In addition, decreases were observed in the atherogenic subfractions: large very low-density lipoprotein (P<.05), small LDL (P<.00001) and medium high-density lipoprotein (P<.05). Oxidized LDL was significantly reduced by 12% in both groups (P<.01). Changes in OxLDL were inversely correlated with plasma lutein (r=-.478, P<.0001). The data indicate that women complied with the dietary regimen by increasing fruits and vegetable intake. Decreased consumption of high-glycemic foods frequently co-consumed with lycopene-rich tomato sauce such as pasta and pizza may be responsible for the lowering of this carotenoid in plasma after 12 weeks. These results also suggest that plasma lutein concentrations may protect against oxidative stress by reducing the concentrations of OxLDL.

Low HDL cholesterol is associated with increased atherogenic lipoproteins and insulin resistance in women classified with metabolic syndrome.

Both metabolic syndrome (MetS) and elevated LDL cholesterol (LDL-C) increase the risk for cardiovascular disease (CVD). We hypothesized that low HDL cholesterol (HDL-C) would further increase CVD risk in women having both conditions. To assess this, we recruited 89 women with MetS (25-72 y) and LDL-C ≥ 2.6 mmol/L. To determine whether plasma HDL-C concentrations were associated with dietary components, circulating atherogenic particles, and other risk factors for CVD, we divided the subjects into two groups: high HDL-C (H-HDL) (≥ 1.3 mmol/L, n = 32) and low HDL-C (L-HDL) (< 1.3 mmol/L, n = 57). Plasma lipids, insulin, adiponectin, apolipoproteins, oxidized LDL, Lipoprotein(a), and lipoprotein size and subfractions were measured, and 3-d dietary records were used to assess macronutrient intake. Women with L-HDL had higher sugar intake and glycemic load (P < 0.05), higher plasma insulin (P < 0.01), lower adiponectin (P < 0.05), and higher numbers of atherogenic lipoproteins such as large VLDL (P < 0.01) and small LDL (P < 0.001) than the H-HDL group. Women with L-HDL also had larger VLDL and both smaller LDL and HDL particle diameters (P < 0.001). HDL-C was positively correlated with LDL size (r = 0.691, P < 0.0001) and HDL size (r = 0.606, P < 0.001), and inversely correlated with VLDL size (r = -0.327, P < 0.01). We concluded that L-HDL could be used as a marker for increased numbers of circulating atherogenic lipoproteins as well as increased insulin resistance in women who are already at risk for CVD.

Biomarkers in Diagnosis of pancreatic carcinoma in fine-needle aspirates.

This study was undertaken to determine whether recently identified proteins could be translated to clinical practice as markers to distinguish pancreatic adenocarcinoma from chronic pancreatitis on fine-needle aspirate (FNA) samples. Resected pancreatic tissue sections (n = 40) and FNA samples (n = 65) were stained for clusterin-beta, MUC4, survivin, and mesothelin. For each biomarker, the staining patterns in adenocarcinoma and in reactive ductal epithelium were evaluated and compared. Clusterin-beta stained reactive ductal epithelium significantly more frequently than pancreatic adenocarcinoma (P < .001). In comparison, MUC4 and mesothelin were expressed more frequently in pancreatic adenocarcinoma on tissue sections. Positive staining for MUC4 (91% vs 0%; P < .001) and mesothelin (62% vs 0%; P = .01) and absence of staining for clusterin-beta (90% vs 7%; P < .001) were noted significantly more frequently in adenocarcinoma cells than in reactive cells in FNA samples. Clusterin-beta and MUC4 can help distinguish reactive ductal epithelial cells from the cells of pancreatic adenocarcinoma in FNA samples.