Bradley E. Aouizerat

Abstract

Background: The effects of tobacco smoking on epigenome-wide methylation signatures in white blood cells (WBCs) collected from persons living with HIV may have important implications for their immune-related outcomes, including frailty and mortality. The application of a machine learning approach to the analysis of CpG methylation in the epigenome enables the selection of phenotypically relevant features from high-dimensional data. Using this approach, we now report that a set of smoking-associated DNA-methylated CpGs predicts HIV prognosis and mortality in an HIV-positive veteran population. Results: We first identified 137 epigenome-wide significant CpGs for smoking in WBCs from 1137 HIV-positive individuals (p < 1.70E-07). To examine whether smoking-associated CpGs were predictive of HIV frailty and mortality, we applied ensemble-based machine learning to build a model in a training sample employing 408,583 CpGs. A set of 698 CpGs was selected and predictive of high HIV frailty in a testing sample [(area under curve (AUC) = 0.73, 95%CI 0.63~0.83)] and was replicated in an independent sample [(AUC = 0.78, 95%CI 0.73~0.83)]. We further found an association of a DNA methylation index constructed from the 698 CpGs that were associated with a 5-year survival rate [HR = 1.46; 95%CI 1.06~2.02, p = 0.02]. Interestingly, the 698 CpGs located on 445 genes were enriched on the integrin signaling pathway (p = 9.55E-05, false discovery rate = 0.036), which is responsible for the regulation of the cell cycle, differentiation, and adhesion. Conclusion: We demonstrated that smoking-associated DNA methylation features in white blood cells predict HIV infection-related clinical outcomes in a population living with HIV.

Abstract

Sexual minority (i.e., non-heterosexual) individuals experience poorer mental and physical health, accounted for in part by the additional burden of sexual minority stress occurring from being situated in a culture favoring heteronormativity. Informed by previous research, the purpose of this study was to identify the relationship between sexual minority stress and leukocyte gene expression related to inflammation, cancer, immune function, and cardiovascular function. Sexual minority men living with HIV who were on anti-retroviral medication, had viral load < 200 copies/mL, and had biologically confirmed, recent methamphetamine use completed minority stress measures and submitted blood samples for RNA sequencing on leukocytes. Differential gene expression and pathway analyses were conducted comparing those with clinically elevated minority stress (n = 18) and those who did not meet the clinical cutoff (n = 20), covarying reactive urine toxicology results for very recent stimulant use. In total, 90 differentially expressed genes and 138 gene set pathways evidencing 2-directional perturbation were observed at false discovery rate (FDR) < 0.10. Of these, 41 of the differentially expressed genes and 35 of the 2-directionally perturbed pathways were identified as functionally related to hypothesized mechanisms of inflammation, cancer, immune function, and cardiovascular function. The neuroactive-ligand receptor pathway (implicated in cancer development) was identified using signaling pathway impact analysis. Our results suggest several potential biological pathways for future work investigating the relationship between sexual minority stress and health.

Abstract

Objective:Recent studies reported a lower human papillomavirus 16 (HPV16) prevalence in cervical precancer among African American than Caucasian women in the general population. We assessed this relationship in women with HIV.Design:Women living with or at risk for HIV in the Women's Interagency HIV Study were followed semi-annually with Pap tests, colposcopy/histology (if indicated), and collection of cervicovaginal lavage samples for HPV testing by PCR. Racial and ethnic groups were defined using genomic Ancestry Informative Markers (AIMs).Results:Among 175 cases of cervical intraepithelial neoplasia 3 or worse (CIN-3+), 154 were diagnosed in women with HIV. African American (27%) and Hispanic (37%) cases were significantly less likely than Caucasian (62%) women to test positive for HPV16 (P=0.01). In multivariate logistic regression models, these associations remained significant for African Americans (odds ratio=0.13; 95% confidence interval (CI) 0.04-0.44; P=0.001) but not Hispanics, after controlling for HIV status, CD4+ count, history of AIDS, age, smoking, and sexual behavior. Limiting the analysis to women with HIV did not change the findings.Conclusion:HPV16 prevalence is lower in African American compared with Caucasian women with HIV and cervical precancer, independent of immune status. Future studies to determine why these racial differences exist are warranted, and whether there are similar associations between race and invasive cervical cancer in women with HIV. Further, HPV types not covered by quadrivalent and bivalent vaccines may play an especially important role in cervical precancer among HIV-positive African American women, a possible advantage to using nonavalent HPV vaccine in this population.

Abstract

Stimulant use may accelerate HIV disease progression through biological and behavioral pathways. However, scant research with treated HIV-positive persons has examined stimulant-associated alterations in pathophysiologic processes relevant to HIV pathogenesis. In a sample of 55 HIV-positive, methamphetamine-using sexual minority men with a viral load less than 200 copies/mL, we conducted RNA sequencing to examine patterns of leukocyte gene expression in participants who had a urine sample that was reactive for stimulants (n = 27) as compared to those who tested non-reactive (n = 28). Results indicated differential expression of 32 genes and perturbation of 168 pathways in recent stimulant users. We observed statistically significant differential expression of single genes previously associated with HIV latency, cell cycle regulation, and immune activation in recent stimulant users (false discovery rate p < 0.10). Pathway analyses indicated enrichment for genes associated with inflammation, innate immune activation, neuroendocrine hormone regulation, and neurotransmitter synthesis. Recent stimulant users displayed concurrent elevations in plasma levels of tumor necrosis factor – alpha (TNF-α) but not interleukin 6 (IL-6). Further research is needed to examine the bio-behavioral mechanisms whereby stimulant use may contribute to HIV persistence and disease progression.

Abstract

Widespread pain and anxiety are commonly reported in cancer patients. We hypothesize that cancer is accompanied by attenuation of endogenous opioid-mediated inhibition, which subsequently causes widespread pain and anxiety. To test this hypothesis we used a mouse model of oral squamous cell carcinoma (SCC) in the tongue. We found that mice with tongue SCC exhibited widespread nociceptive behaviors in addition to behaviors associated with local nociception that we reported previously. Tongue SCC mice exhibited a pattern of reduced opioid receptor expression in the spinal cord; intrathecal administration of respective mu (MOR), delta (DOR), and kappa (KOR) opioid receptor agonists reduced widespread nociception in mice, except for the fail flick assay following administration of the MOR agonist. We infer from these findings that opioid receptors contribute to widespread nociception in oral cancer mice. Despite significant nociception, mice with tongue SCC did not differ from sham mice in anxiety-like behaviors as measured by the open field assay and elevated maze. No significant differences in c-Fos staining were found in anxiety-associated brain regions in cancer relative to control mice. No correlation was found between nociceptive and anxiety-like behaviors. Moreover, opioid receptor agonists did not yield a statistically significant effect on behaviors measured in the open field and elevated maze in cancer mice. Lastly, we used an acute cancer pain model (injection of cancer supernatant into the mouse tongue) to test whether adaptation to chronic pain is responsible for the absence of greater anxiety-like behavior in cancer mice. No changes in anxiety-like behavior were observed in mice with acute cancer pain.

Abstract

Objective: To assess variation in genes that regulate cholesterol metabolism in relation to the natural history of HIV infection. Design: Cross-sectional and longitudinal analysis of the Women's Interagency HIV Study. Methods: We examined 2050 single nucleotide polymorphisms (SNPs) in 19 genes known to regulate cholesterol metabolism in relation to HIV viral load and CD4 + T-cell levels in a multiracial cohort of 1066 antiretroviral therapy-naive women. Results: Six SNPs were associated with both HIV viral load and CD4 + T-cell levels at a false discovery rate of 0.01. Bioinformatics tools did not predict functional activity for five SNPs, located in introns of nuclear receptor corepressor 2, retinoid X receptor alpha (RXRA), and tetratricopeptide repeat domain 39B. Rs17111557 located in the 3′ untranslated region of proprotein convertase subtilisin/kexin type 9 (PCSK9) putatively affects binding of hsa-miR-548t-5p and hsa-miR-4796-3p, which could regulate PCSK9 expression levels. Interrogation of rs17111557 revealed stronger associations in the subset of women with HIV/hepatitis C virus (HCV) coinfection (n=408, 38% of women). Rs17111557 was also associated with low-density lipoprotein cholesterol levels in HIV/HCV coinfected (β: -10.4; 95% confidence interval: -17.9, -2.9; P=0.007), but not in HIV monoinfected (β:1.2; 95% confidence interval: -6.3, 8.6; P=0.76) women in adjusted analysis. Conclusion: PCSK9 polymorphism may affect HIV pathogenesis, particularly in HIV/HCV coinfected women. A likely mechanism for this effect is PCSK9-mediated regulation of cholesterol metabolism. Replication in independent cohorts is needed to clarify the generalizability of the observed associations.

Abstract

BACKGROUND: Reliable non-invasive markers to characterize inflammation, hepatocellular ballooning, and fibrosis in nonalcoholic fatty liver disease (NAFLD) are lacking. We investigated the relationship between plasma cytokine levels and features of NAFLD histology to gain insight into cellular pathways driving NASH and to identify potential non-invasive discriminators of NAFLD severity and pattern.METHODS: Cytokines were measured from plasma obtained at enrollment in pediatric participants in NASH Clinical Research Network studies with liver biopsy-proven NAFLD. Cytokines were chosen a priori as possible discriminators of NASH and its components. Minimization of Akaike Information Criterion (AIC) was used to determine cytokines retained in multivariable models. RESULTS: Of 235 subjects, 31% had "Definite NASH" on liver histology, 43% had "Borderline NASH", and 25% had NAFLD but not NASH. Total plasminogen activator inhibitor 1 (PAI1) and activated PAI1 levels were higher in pediatric participants with Definite NASH and with lobular inflammation. Interleukin-8 (IL-8) was higher in those with stage 3-4 fibrosis and lobular inflammation. sIL-2rα was higher in children with stage 3-4 fibrosis and portal inflammation. In multivariable analysis, PAI1 variables were discriminators of Borderline/Definite NASH, definite NASH, lobular inflammation and ballooning. IL-8 increased with steatosis and fibrosis severity; sIL-2rα increased with fibrosis severity and portal inflammation. IL-7 decreased with portal inflammation and fibrosis severity.CONCLUSIONS: Plasma cytokines associated with histology varied considerably among NASH features, suggesting promising avenues for investigation. Future, more targeted analysis is needed to identify the role of these markers in NAFLD and to evaluate their potential as non-invasive discriminators of disease severity.

Abstract

Persistent pain following breast cancer surgery is a significant problem. Both inherited and acquired mechanisms of inflammation appear to play a role in the development and maintenance of persistent pain. In this longitudinal study, growth mixture modeling was used to identify persistent breast pain phenotypes based on pain assessments obtained prior to and monthly for 6 months following breast cancer surgery. Associations between the “no pain” and “mild pain” phenotypes and single nucleotide polymorphisms (SNPs) spanning 15 cytokine genes were evaluated. The methylation status of the CpG sites found in the promoters of genes associated with pain group membership was determined using bisulfite sequencing. In the multivariate analysis, three SNPs (i.e., interleukin 6 (IL6) rs2069840, C-X-C motif chemokine ligand 8 (CXCL8) rs4073, tumor necrosis factor (TNF) rs1800610) and two TNF CpG sites (i.e., c.−350C, c.−344C) were associated with pain group membership. These findings suggest that variations in IL6, CXCL8, and TNF are associated with the development and maintenance of mild persistent breast pain. CpG methylation within the TNF promoter may provide an additional mechanism through which TNF alters the risk for mild persistent breast pain after breast cancer surgery. These genetic and epigenetic variations may help to identify individuals who are predisposed to the development of mild levels of persistent breast pain following breast cancer surgery.

Abstract

Objective/background Daytime napping longer than one hour has been associated with an increased risk for all-cause mortality. Associations between cytokine polymorphisms and daytime napping in chronic illnesses such as HIV, however, have not been well described. The purpose of this study was to examine cytokine polymorphisms associated with long daytime napping in adults living with HIV. Methods A cross-sectional analysis was conducted using a convenience sample of 257 adults living with HIV. Daytime napping was assessed with wrist actigraphy data collected over three days. Participants categorized as long nappers (≥60 min) were compared to short nappers and non-nappers (<60 min). Single nucleotide polymorphisms (SNPs) for 15 candidate genes involved in cytokine signaling were analyzed. Genes included: interferon-gamma (IFNG), IFNG receptor 1 (IFNGR1), interleukins (IL1B, IL1R, IL1R2, IL2, IL4, IL6, IL8, IL10, IL13, IL17A), nuclear factors of kappa light polypeptide gene enhancer in B cells (NFKB1 and NFKB2), and tumor necrosis factor alpha (TNFA). Results After adjusting for relevant demographic and clinical characteristics, long daytime napping was associated with 12 SNPs from seven genes: 1) IFNG rs2069728; 2) IL1B rs1143642, rs1143627, and rs16944; 3) IL2 rs2069763; 4) IL6 rs4719714, rs1554606, and rs2069845; 5) IL17A rs3819024 and rs8193036; 6) NFKB1 rs4648110; and 7) NFKB2 rs1056890. Conclusions Cytokine genetic variations may have a role in physiological regulation of daytime napping as well as nocturnal sleep. Cytokine polymorphisms associated with long daytime napping could help identify adults with HIV who may benefit from targeted therapeutic interventions.

Abstract

Context Adults with venous leg ulcers frequently experience multiple symptoms that may influence quality of life (QOL). Objectives The objective of this study was to identify patient subgroups based on their experience with a pain-depression-fatigue-sleep disturbance symptom cluster and to identify differences in patient characteristics and wound-healing and QOL outcomes between the subgroups. Methods Secondary data analysis from previous longitudinal studies of 247 patients with venous leg ulcers. Latent class analysis identified subgroups of patients with distinct experiences with the symptom cluster of pain, depression, fatigue, and sleep disturbance. Hierarchical regression analysis identified relationships between the subgroups and QOL outcomes. Survival analysis identified differences between the subgroups and ulcer healing. Results Latent class analysis found 67% of patients were in a mild symptom subgroup (i.e., experiencing no or mild pain, depressive symptoms, fatigue, or sleep disturbance). One-third of the samples were in a severe symptom subgroup, who reported moderate-to-severe levels of these symptoms. Compared with the mild subgroup, patients in the severe subgroup had poorer QOL scores (t = 8.06, P < 0.001). Symptom subgroup membership accounted for 19% of the variance (P < 0.001) within a hierarchical regression model that explained 42% of the variance in QOL (F(7,170) = 16.89, P < 0.001, R2 = 0.42). Cox proportional hazards regression found that at enrollment into the study, patients in the severe symptom subgroup were 1.5 times (95% confidence interval 1.02–2.08) less likely to heal in the following 24 weeks (P = 0.037). Conclusion Significant relationships were found between delayed ulcer healing, decreased QOL, and membership in the severe symptom subgroup. These findings suggest that comprehensive symptom assessment is needed to identify patients at higher risk for poor outcomes and enable early intervention.