Metabolic Syndrome and Neurocognitive Function among older Hispanics/Latinos with HIV

Neurocognitive impairment is prevalent among persons with HIV (PWH), particularly among Hispanics/Latinos/as/x (henceforth Hispanics). We examined disparities in HIV-associated neurocognitive function between older Hispanic and non-Hispanic White PWH, and the potential role of metabolic syndrome (MetS) in explaining these disparities. Participants included 116 community-dwelling PWH ages 50-75, who were enrolled in a cohort study in southern California (58 Hispanic [53% Spanish-speaking] and 58 age-comparable non-Hispanic White; Overall group: Age: M=57.9, SD=5.7; Education: M=13, SD=3.4; 83% male, 58% AIDS, 94% on antiretroviral therapy [ART], 4% detectable plasma RNA). A global neurocognition score was derived from T-Scores on a comprehensive neurocognitive battery, with separate demographic adjustments for English and Spanish-speakers. MetS was ascertained via standard criteria that considered central obesity, elevated triglycerides, low high-density lipoprotein cholesterol, and elevated fasting glucose, as well as current medical treatment for these conditions. Covariates examined included sociodemographic, psychiatric, substance use and HIV-disease characteristics. Hispanics had higher rates of MetS (56%) than non-Hispanic Whites (37%; p<.05). A stepwise regression model on global neurocognition including ethnicity and covariates that differed between ethnic groups, selected only Hispanic ethnicity as a significant predictor (B=-3.82, SE=1.27, p<.01). A comparable model also including MetS showed that both Hispanic ethnicity (B=-3.39, SE=1.31, p=.01) and MetS (B=-2.73, SE=1.31, p=.04), were significantly associated with worse global neurocognition. Findings indicate that MetS does not fully explain disparities in neurocognitive function between Hispanic and non-Hispanic White older PWH, but rather is an independent predictor of neurocognitive function along with Hispanic ethnicity.

Therapeutic prediction of HIV-1 DNA decay: a multicenter longitudinal cohort study

Background Factors predicting peripheral blood total HIV-1 DNA size in chronically infected patients with successfully suppressed viremia remain unclear. Prognostic power of such factors are of clinical significance for making clinical decisions. Methods Two sets of study populations were included: 490 China AIDS Clinical Trial (CACT) participants (Training cohort, followed up for 144 to 288 weeks) and 117 outpatients from Peking Union Medical College Hospital (PUMCH) (Validation cohort, followed up for more than 96 weeks). All patients were chronically HIV-1-infected and achieved successful HIV-1 plasma RNA suppression within week 48. Total HIV-1 DNA in blood at baseline, 12, 24, 48, 96, 144 and 288 weeks after combined antiretroviral therapy (cART) initiation were quantified. Generalized estimating equations and logistic regression methods were used to derive and validate a predictive model of total HIV-1 DNA after 96 weeks of cART. Results The total HIV-1 DNA rapidly decreased from baseline [median = 3.00 log10 copies/106 peripheral blood mononuclear cells (PBMCs)] to week 24 (median = 2.55 log10 copies/106 PBMCs), and leveled off afterwards. Of the 490 patients who had successful HIV-1 plasma RNA suppression by 96 w post-cART, 92 (18.8%) had a low total HIV-1 DNA count (< 100 copies/106 PBMCs) at week 96. In the predictive model, lower baseline total HIV-1 DNA [risk ratio (RR) = 0.08, per 1 log10 copies/106 PBMCs, P < 0.001] and higher baseline CD4+ T cell count (RR = 1.72, per 100 cells/μL, P < 0.001) were significantly associated with a low total HIV-1 DNA count at week 96. In an independent cohort of 117 patients, this model achieved a sensitivity of 75.00% and specificity of 69.52%. Conclusions Baseline total HIV-1 DNA and CD4+ T cell count are two independent predictors of total HIV-1 DNA after treatment. The derived model based on these two baseline factors provides a useful prognostic tool in predicting HIV-1 DNA reservoir control during cART.

Optimization of Cell-free Plasma RNA Extraction for Downstream Application

The growing interest in biomedical studies has brought RNA from biofluids including plasma, as promising candidates for genetics profiling. The precision and reliability of an analysis in downstream application such as NanoString nCounter® MAX Analysis System (NanoString Technologies, Seattle, WA) ) depend on the RNA quality, purity and level. In this project, NanoString nCounter® miRNA panel was chosen due to rapid identification and ability to profile approximately 800 miRNAs per run which requires total RNAs from plasma with a minimum concentration of 33.3 ng/μL with 260/280 and 260/230 ratios of ≥1.8 for optimal results. Unlike tissues and cells, circulating RNAs in plasma are cell-free and are present in small sizes. However, the abundance of proteins and inhibitors in the plasma as possible contaminants could diminish the effectiveness of molecular isolation techniques and pose challenges in RNA isolation and quantification. This could skew data collection and elucidation. Therefore, the main objective is to determine the optimized plasma RNA isolation protocol to overcome problems in RNA quality and purity with regards NanoString nCounter® MAX Analysis System requirement. Several optimization steps were performed, including the addition of one chloroform extraction step with extra washing steps instead of conducting only once following the actual protocol. After conducting these steps, the average 260/280 ratio falls between 1.7 to 1.8, slightly increased compared to the results before optimization which was around 1.4 to 1.6 since these steps of optimization help to remove excess impurities including phenol and salt. Furthermore, increasing the incubation time in certain steps, for instance, after sample homogenization with Qiazol, during 95% ethanol precipitation and after RNase-free water addition have boosted the RNA recovery allowing RNA concentration of 15 ng/μL and above to be obtained. Hence, the optimized plasma RNA isolation protocol was determined since several issues related to plasma RNA concentration and purity were significantly improved by performing the additional steps in the protocol.

Impact of blood storage conditions on the transcript profile of plasma cell-free RNA

BACKGROUNDPlasma cell-free RNA (cfRNA) are potential biomarkers for disease prediction and diagnosis. However, pre-analysis factors, such as the delay in blood processing and storage may lead to unreliable results, though no study has systematically evaluated the effect of blood storage conditions on the whole transcriptome of plasma cfRNA yet.METHODSWe collected peripheral blood samples from four healthy subjects and allowed them to stand at room temperature or 4◻ for different time periods (0h, 2h, 6h and 24h) prior to plasma separation. Then, plasma cfRNA stability was evaluated by measuring expression changes of cell-free mRNA, lncRNA and miRNA using high throughput sequencing-based profiling. Finally, their paired leukocyte RNA data were integrated to depict the effect of leukocytes on plasma cfRNA during storage.RESULTSPlasma mRNA and lncRNA presented high correlations (Pearson R2 ≥ 0.8) and fewer variations when blood was stored at 4◻ for 6 hours or stored at RT for 2 hours. miRNA was more stable, with minimal R2 of 0.86 at 4◻ for at least 24 hours or at RT for 6 hours. Correlations of plasma RNA and leukocyte RNA increased with the incubation time, and the relative proportion of neutrophils in plasma grown from 14.3% to 61.2% at RT (P = 0.004), indicating leukocyte RNA contamination. Besides, the tissue enriched genes in plasma were down-regulated with the extension of storage time.CONCLUSIONSOur results characterized the effects of short-term storage of blood samples on plasma cfRNA, which will facilitate further researches or clinical applications to avoid bias resulting from sample processing.

Extracellular vesicles isolated by size-exclusion chromatography present suitability for RNomics analysis in plasma

Background Extracellular vesicles (EVs), known as cell-derived membranous structures harboring a variety of biomolecules, have been widely used in liquid biopsy. Due to the complex biological composition of plasma, plasma RNA omics analysis (RNomics) is easily affected, thus it is necessary to select an optimal strategy from exiting methods according to the performance for intended application. Methods In this study, four different strategies for EVs isolation were performed and compared (i.e. ultracentrifugation (UC), size exclusion chromatography (SEC), and two most frequently-used commercially available isolation kit (ExoQuick and exoEasy). We compared the yield, purity, PCR quantification of RNAs, miRNA-seq analyses and mRNA-seq analyses of RNAs from EVs isolated using four methods. Results The results showed that the lowest miRNA binding protein AGO2 (Argonaute-2) and the highest EVs-specific miRNA and lncRNA were observed in EVs obtained through SEC, meanwhile the content of the non-specific miRNA was the lowest. Further RNA-Seq data revealed that RNAs obtained via SEC presented more useful reads for both miRNA and mRNA. Furthermore, the mRNA delivered via SEC tended to have a concentration comparable to the ideal FPKM (Fragments Per Kilobase Million) value. Conclusions SEC shall be used as an optimal strategy for the isolation of EVs in plasma RNomics analysis.

Viral Reservoir in Early-Treated HIV-Infected Children and Markers for Sustained Viral Suppression

Background The impact of very early infant treatment on HIV reservoir, and markers for treatment success, require study. Methods The Early Infant Treatment Study (EIT) enrolled 40 children living with HIV started on antiretroviral treatment (ART) at &lt;7 days of age and 23 who had started treatment between 30-365 days to serve as controls. Quantitative HIV DNA was evaluated every 1-3 months in PBMCs. 84-week repeat qualitative whole blood DNA PCR and dual enzyme EIA were performed. Results Median quantitative cell-associated DNA after at least 84 weeks was significantly lower among the first 27 EIT children tested than among 10 controls (40.8 vs. 981.4 copies/million cells; p&lt;0.001) and correlated with pre-ART DNA. Median DNA after 84 weeks did not differ significantly by negative or positive serostatus at 84 weeks (p=0.94), and appeared unaffected by periods of unsuppressed plasma RNA from 24-84 weeks (p=0.70). However, negative 84-week serostatus was 67% predictive for sustained RNA suppression and positive serostatus was 100% predictive for viremia. Loss of qualitative DNA positivity at 84 weeks was 73% predictive for sustained suppression and persistent positivity was 77% predictive for viremia. Conclusions Lower viral reservoir was associated with starting ART at &lt;1 week. Negative serostatus and qualitative DNA were useful markers of sustained viral suppression from 24-84 weeks.