Faculty Bibliography

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.

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.

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 6months 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.

Background: Latent class analysis is an exploratory analytic method that incorporates the associations between pre-specified independent variables without mandating consideration of an outcome, and has been successful in identifying clinically relevant subgroups in other heterogeneous diseases. Methods: Using a cross-sectional cohort of 648 adults from the NASH Clinical Research Network with biopsy proven NAFLD and clinical and biomarker data, we fit a series of latent class models to see if we could uncover distinct NAFLD classes. We included clinical factors, liver tests (ALT, AST, alkaline phosphatase and total bilirubin) and eleven plasma biomarkers previously demonstrated to have strong associations with NAFLD; adiponectin, activated plasminogen activator inhibitor 1 (PAI-1), haptoglobin, insulin-like growth factor 2, interleukin (IL)-8, monocyte chemoattractant protein-1, soluble IL-1 receptor 1, soluble IL-2 receptor <, tumor necrosis factor >and total PAI-1. Continuous measures were log-transformed and standardized to a common z-scale with a standard deviation scaled to 1. Results: We fitted models from 1 to 4 classes and evaluated fit using the Vuong-Lo-Mendell-Rubin test, Aikake Information Criterion, and Bayesian information criterion. A two-class model was more informative (p=0.04) than a one-class model and had mean probabilities of class assignment < 0.9 for both classes. Importantly in the two class model, one class had a relative elevation in levels of inflammatory biomarkers compared to the other class (Figure). Race and sex were not associated with either class. The "hyper"-inflammatory class was present in 24% (65/272) of patients with NAFLD without NASH and 54% (203/376) patients with definite NASH histology. Conclusions: We observed distinct clustering of plasma biomarkers in NAFLD which appear to co-localize with histological subgroups and, if validated, warrant evaluation in longitudinal studies as predictors of disease progression (Figure presented)

Background/UNASSIGNED: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/UNASSIGNED:as possible discriminators of NASH and its components. Minimization of Akaike Information Criterion (AIC) was used to determine cytokines retained in multivariable models. Results/UNASSIGNED: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/UNASSIGNED: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.

Cancer patients in pain require high doses of opioids and quickly become opioid-tolerant. Previous studies have shown that both chronic cancer pain and high dose opioid use lead to mu-opioid receptor down-regulation. In this study we explore down-regulation of OPRM1, the mu-opioid receptor gene, as a mechanism f,or opioid tolerance in the setting of opioid use for cancer pain. We demonstrate in a cohort of 84 cancer patients that high dose opioid use correlates with OPRM1 hypermethylation in peripheral leukocytes of these patients. We then reverse-translate our clinical findings by creating a mouse cancer pain model; we create opioid tolerance in the mouse cancer model to mimic opioid tolerance in the cancer patients. Using this model we determine the functional significance of OPRM1 methylation on cancer pain and opioid tolerance. We focus on two main cells within the cancer microenvironment: the cancer cell and the neuron. We show that targeted re-expression of mu-opioid receptor on cancer cells inhibits mechanical and thermal hypersensitivity, and prevents opioid tolerance, in the mouse model. The resultant analgesia and protection against opioid tolerance are likely due to preservation of mu-opioid receptor expression on the cancer-associated neurons.

OBJECTIVE: In the general population, lipoprotein(a) [Lp(a)] has been established as an independent causal risk factor for cardiovascular disease. Lp(a) levels are to a major extent regulated by a size polymorphism in the apolipoprotein(a) [apo(a)] gene. The roles of Lp(a)/apo(a) in human immunodeficiency virus (HIV)-related elevated cardiovascular disease risk remain unclear. APPROACH AND RESULTS: The associations between total plasma Lp(a) level, allele-specific apo(a) level, an Lp(a) level carried by individual apo(a) alleles, and common carotid artery intima-media thickness were assessed in 150 HIV-infected and 100 HIV-uninfected women in the WIHS (Women's Interagency HIV Study). Linear regression analyses with and without adjustments were used. The cohort was young (mean age, approximately 31 years), with the majority being Blacks ( approximately 70%). The prevalence of a small size apo(a) (/=30 mg/dL) was similar by HIV status. Total plasma Lp(a) level (P=0.029) and allele-specific apo(a) level carried by the smaller apo(a) sizes (P=0.022) were significantly associated with carotid artery intima-media thickness in the HIV-infected women only. After accounting for confounders (age, race, smoking, body mass index, blood pressure, hepatitis C virus coinfection, menopause, plasma lipids, treatment status, CD4+ T cell count, and HIV/RNA viral load), the association remained significant for both Lp(a) (P=0.035) and allele-specific apo(a) level carried by the smaller apo(a) sizes (P=0.010) in the HIV-infected women. Notably, none of the other lipids/lipoproteins was associated with carotid artery intima-media thickness. CONCLUSIONS: Lp(a) and allele-specific apo(a) levels predict carotid artery intima-media thickness in HIV-infected young women. Further research is needed to identify underlying mechanisms of an increased Lp(a) atherogenicity in HIV infection.

CONTEXT: Adults with venous leg ulcers frequently experience multiple symptoms which may influence quality of life (QOL). OBJECTIVES: To identify patient subgroups based on their experience with a pain-depression-fatigue-sleep disturbance (PDFS) symptom cluster and to identify differences in patient characteristics, as well as wound healing and quality of life outcomes between the subgroups. METHODS: Secondary data analysis from previous longitudinal studies of 247 patients with venous leg ulcers. Latent class analysis (LCA) 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: LCA 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 sample were in a severe symptom subgroup, who reported moderate to severe levels of these symptoms. Compared to 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=.42). Cox proportional hazards regression found that at enrolment into the study, patients in the severe symptom subgroup were 1.5 times (95%CI 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.

Some oncology outpatients experience a higher number of and more severe symptoms during chemotherapy (CTX). However, little is known about whether this high risk phenotype persists over time. Latent transition analysis (LTA) was used to examine the probability that patients remained in the same symptom class when assessed prior to the administration of and following their next dose of CTX. For the patients whose class membership remained consistent, differences in demographic and clinical characteristics, and quality of life (QOL) were evaluated. The Memorial Symptom Assessment Scale (MSAS) was used to evaluate symptom burden. LTA was used to identify subgroups of patients with distinct symptom experiences based on the occurrence of the MSAS symptoms. Of the 906 patients evaluated, 83.9% were classified in the same symptom occurrence class at both assessments. Of these 760 patients, 25.0% were classified as Low-Low, 44.1% as Moderate-Moderate and 30.9% as High-High. Compared to the Low-Low class, the other two classes were younger, more likely to be women and to report child care responsibilities, and had a lower functional status and a higher comorbidity scores. The two higher classes reported lower QOL scores. The use of LTA could assist clinicians to identify higher risk patients and initiate more aggressive interventions.