Pilot study of markers for high-grade anal dysplasia in a southern cohort from the Women's Interagency HIV Study (WIHS)

Background: Anal cancer rates have increased in HIV+ women. Factors associated with anal cancer precursor high-grade squamous intraepithelial lesions (HSIL) in HIV+ and at-risk-HIV- women were assessed. Methods: HIV+ and HIV- women from the Atlanta WIHS cohort were enrolled in a cross-sectional pilot study. All anal (AS) and cervical (CS) swab samples were analyzed for HPV-genotyping (Linear-Array® HPV-Genotyping Test, LA-HPVGT) and FAM19A4 and microRNA-124-2 promoter methylation. All women underwent high resolution anoscopy with biopsy of suspicious lesions and anal cytology (AC) collection. Logistic regression was conducted with anal HSIL histology (A-HSIL) as the dependent variable. Results: 75 women were enrolled: 52(69%) were HIV+ with 3/4 having undetectable viral load, 64(86%) Black, with mean age 49. 44(59%) AC samples were ASCUS/+hrHPV or higher, 38(51%) of all AS were +hrHPV by LA-HPVGT.13 anal biopsies were confirmed A-HSIL. 69(95%) AS and 19(26%) CS tested positive for hypermethylation, respectively. A-HSIL model included ASCUS/+hrHPV or greater on AC and cervical hypermethylation as covariates (Table 1). AS hypermethylation was not associated with A-HSIL. Conclusions: Our results suggest AC with hrHPV testing and/or cervical gene promoter hypermethylation measurements as promising non-invasive screening strategies for A-HSIL in both HIV+ and HIV- women. Lack of association between AS hypermethylation and A-HSIL may reflect characteristics of the anal milieu and warrants further investigation

Class-Based Antiretroviral Exposure and Cognition Among Women Living with HIV

Neurologic complications of the human immunodeficiency virus (HIV) are common in treated individuals, and toxicity of certain antiretroviral therapies (ART) may contribute to cognitive impairment. We investigated exposures to specific ART and cognition among women living with HIV (WLWH). Virologically suppressed (viral load <200 copies/mL during at least two semi-annual visits) WLWH and age/race matched HIV-seronegative controls enrolled in the Women's Interagency HIV Study who completed at least two biennial cognitive assessments were included. Analysis of WLWH was restricted to those with exposure to the drug class of interest and a nucleoside reverse transcriptase inhibitor (NRTI) backbone. Generalized estimating equations were used to evaluate repeated measures of cognition over time in association with ART class exposure. Among 1,242 eligible WLWH, 20% (n = 247) had isolated drug exposure to non-nucleoside reverse transcriptase inhibitors (NNRTI), 18% (n = 219) to protease inhibitors (PIs), and 6% (n = 79) to integrase inhibitors with a NRTI backbone. Cognitive assessments were performed at a median of 3 biennial visits {IQR 2-4 visits}. At the index assessment, 21% of WLWH demonstrated global cognitive impairment versus 29% at their last cognitive assessment. In multivariable analyses adjusted for hypertension, depression, diabetes mellitus, history of AIDS-defining illness, alcohol use, number of medications, and time on ART, WLWH exposed to NNRTIs demonstrated verbal learning improvements (mean T-score change 1.3, p = .020) compared to other treated women. Compared to HIV-seronegative women, WLWH exposed to PIs had worse verbal learning (mean T-score difference -2.62, p = .002) and verbal memory performance (mean T-score difference -1.74, p = .032) at baseline. Compared to HIV-seronegative women, WLWH exposed to PIs had improvements in verbal learning (mean T-score slope difference 0.36, p = .025) and verbal memory (mean T-score slope difference 0.32, p = .042). The index T-score and slope of change in the T-score were similar among other treated groups and the HIV-seronegative group. We noted emerging trends in cognition in WLWH exposed to specific drug classes. Ongoing study of this relatively young group is important to characterize long-term cognitive outcomes and effect of antiretrovirals as treatment guidelines evolve

Dysregulated B Cell Expression of RANKL and OPG Correlates with Loss of Bone Mineral Density in HIV Infection

<div><p>HIV infection is associated with high rates of osteopenia and osteoporosis, but the mechanisms involved are unclear. We recently reported that bone loss in the HIV transgenic rat model was associated with upregulation of B cell expression of the key osteoclastogenic cytokine receptor-activator of NF-κB ligand (RANKL), compounded by a simultaneous decline in expression of its physiological moderator, osteoprotegerin (OPG). To clinically translate these findings we performed cross-sectional immuno-skeletal profiling of HIV-uninfected and antiretroviral therapy-naïve HIV-infected individuals. Bone resorption and osteopenia were significantly higher in HIV-infected individuals. B cell expression of RANKL was significantly increased, while B cell expression of OPG was significantly diminished, conditions favoring osteoclastic bone resorption. The B cell RANKL/OPG ratio correlated significantly with total hip and femoral neck bone mineral density (BMD), T- and/or Z-scores in HIV infected subjects, but revealed no association at the lumbar spine. B cell subset analyses revealed significant HIV-related increases in RANKL-expressing naïve, resting memory and exhausted tissue-like memory B cells. By contrast, the net B cell OPG decrease in HIV-infected individuals resulted from a significant decline in resting memory B cells, a population containing a high frequency of OPG-expressing cells, concurrent with a significant increase in exhausted tissue-like memory B cells, a population with a lower frequency of OPG-expressing cells. These data validate our pre-clinical findings of an immuno-centric mechanism for accelerated HIV-induced bone loss, aligned with B cell dysfunction.</p></div

B cell subset RANKL and OPG expression in HIV-negative and HIV-positive individuals.

<p><b>A</b>) Total B cells (CD3⁻CD20⁺ cells) were identified in the lymphocyte gate. <b>B</b>) B cells were further divided into 4 distinct subsets: naïve (CD21^hiCD27⁻), resting memory (CD21^hiCD27⁺), activated memory (CD21⁻CD27⁺), and exhausted tissue-like memory (CD21⁻CD27⁻). <b>C</b>) B cell subset distribution in HIV-negative (N = 28) and seropositive (N = 24) individuals. Intracellular expression of <b>D</b>) OPG and <b>E</b>) RANKL in B cell subsets of HIV-negative and seropositive individuals combined. Graphs reflect individual individuals with bars at the mean (parametric data) or median [non-parametric data, Activated and tissue-like memory subset (<b>C</b>) and naïve and resting memory (<b>E</b>)]. Simple comparisons were done using Student's <i>t</i> test (for parametric data) or Wilcoxon rank sum test (for non-parametric data) for each of the subsets (<b>C</b>) and one-way ANOVA was used to compare multiple groups (<b>D</b>). Actual P values are reported for simple comparisons. For ANOVA *P<0.05 or ***P<0.001 or P =  not significant (ns).</p

Bone turnover outcomes.

<p>*Characteristics are compared between HIV serostatus groups with Wilcoxon rank-sum test for CTx and osteocalcin.</p>†<p>Multivariable analysis adjusted for age, gender, race, BMI, smoking, past 30 day alcohol consumption and fracture history</p><p>Bone turnover outcomes.</p

Increased frequency of RANKL-expressing B cells and decreased frequency of OPG-expressing B cells in HIV infection.

<p>Intracellular expression of: <b>A)</b> OPG and <b>B)</b> RANKL, by circulating peripheral blood B cells were quantified by flow cytometry in 56 HIV-negative and 57 HIV-infected individuals. Comparisons between HIV sero status were performed using Wilcoxon rank-sum test.</p

Association of bone mineral density and CTx with B cell OPG expression (%), B cell RANKL expression (%), RANKL/OPG ratio and Log RANKL/OPG ratio.

<p>r_s  =  Spearman rank correlation coefficient. Significant outcomes are in bold.</p><p>Association of bone mineral density and CTx with B cell OPG expression (%), B cell RANKL expression (%), RANKL/OPG ratio and Log RANKL/OPG ratio.</p

BMD and bone turnover outcomes.

<p>*Averaged across left and right sides.</p>†<p>Characteristics are compared between HIV serostatus groups with Wilcoxon rank-sum test for BMD. Chi-square test of proportions for normal, osteopenia and osteoporosis between HIV serostatus groups are significant for hip (p = 0.02) and any area (p = 0.05) therefore results for pairwise tests of proportion (normal osteopenia and normal-osteoporosis) are reported.</p><p>BMD and bone turnover outcomes.</p

Differential B cell subset RANKL and OPG expression in HIV-negative and HIV-positive individuals.

<p>Representative histograms (alternate top panels) of B cell subset staining for RANKL (<b>A</b>) and OPG (<b>B</b>) for HIV negative individuals (solid line) and HIV positive individuals (dashed line). Scatter plots (alternate bottom panels) represent cumulative data for B cell subset RANKL (<b>A</b>) or OPG (<b>B</b>) expression from individual HIV-negative (HIV⁻, N = 17) or seropositive (HIV⁺, N = 15) individuals with bars at the mean (parametric data) or median [non-parametric data, RANKL: tissue-like memory (HIV-); OPG: All HIV- subsets]. Comparisons were done using two-way ANOVA, followed by pairwise comparisons of individual B cell subsets using student's t-test (parametric data) or Wilcoxon rank sum test (non-parametric data).</p

Genetic basis for variation in plasma IL-18 levels in persons with chronic hepatitis C virus and human immunodeficiency virus-1 infections

Inflammasomes are multi-protein complexes integrating pathogen-triggered signaling leading to the generation of pro-inflammatory cytokines, including interleukin-18 (IL-18). Hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV) infections are associated with elevated IL-18, suggesting inflammasome activation. However, there is marked person-to-person variation in the inflammasome response to HCV and HIV. We hypothesized that host genetics may explain this variation. To test this, we analyzed the associations of plasma IL-18 levels and polymorphisms in 10 genes in the inflammasome cascade. 1538 participants with active HIV and/or HCV infection in 3 ancestry groups are included. Samples were genotyped using the Illumina Omni 1-quad and Omni 2.5 arrays. Linear regression analyses were performed to test the association of variants with logIL-18 including HCV and HIV infection status and HIV-RNA, in each ancestry group and then meta-analyzed. Eleven highly correlated SNPs (r2=0.98-1) in the IL18-BCO2 region were significantly associated with logIL-18; Each T allele of rs80011693 confers a decrease of 0.06 log pg/mL of IL-18 after adjusting for covariates (rs80011693; rs111311302 β=-0.06, P-value=2.7×10-4). In conclusion, genetic variation in IL18 is associated with IL-18 production in response to HIV and HCV infection and may explain variability in the inflammatory outcomes of chronic viral infections