Objective: Early HIV infection is characterized by a dramatic depletion of CD4 T cells in the gastrointestinal tract and translocation of bacterial products from the gut into the blood. In this study, we evaluated if gut bacterial profiles were associated with immune status before and after starting antiretroviral therapy (ART). Design: We evaluated the gut microbiota of men recently infected with HIV (n = 13) who were participating in a randomized, double-blind controlled trial of combination ART and maraviroc versus placebo and who were followed for 48 weeks. Methods: To evaluate the gut microbiota of participants, we pyrosequenced the bacterial populations from anal swabs collected before and longitudinally after the initiation of ART. Associations of the gut flora with clinical variables (lymphocyte profiles and viral loads), activation and proliferation markers in peripheral blood mononuclear cells and gut biopsies (measured by flow cytometry) and markers of microbial translocation (lipopolysaccharide and soluble CD14) were performed by regression analyses using R statistical software. Results: Using pyrosequencing, we identified that higher proportions of Lactobacillales in the distal gut of recently HIV-infected individuals were associated with lower markers of microbial translocation, higher CD4% and lower viral loads before ART was started. Similarly, during ART, higher proportions of gut Lactobacillales were associated with higher CD4%, less microbial translocation, less systemic immune activation, less gut T lymphocyte proliferation, and higher CD4% in the gut. Conclusion: Shaping the gut microbiome, especially proportions of Lactobacillales, could help to preserve immune function during HIV infection.

Mitochondria are abundant organelles critical for energy metabolism and brain function. Mitochondrial DNA (mtDNA), released during cellular injury and as part of the innate immune response to viral pathogens, contains CpG motifs that act as TLR-9 ligands. We investigated relationships between cerebrospinal fluid (CSF) cell-free mtDNA levels and HIV viral load (VL), biomarkers of inflammation and iron transport, and neurocognitive (NC) function in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort. We quantified cell-free mtDNA in CSF by droplet digital PCR in 332 CHARTER participants who underwent comprehensive neuropsychiatric evaluation. NC performance was assessed using the global deficit score (GDS) as either a continuous or a binary measure (GDS ≥ 0.5, impaired vs. GDS < 0.5, unimpaired). CSF, clinical, and biomarker data from the earliest available time point were analyzed. Cell-free mtDNA associations with CSF inflammation and iron-related biomarkers [CXCL10, IL-6, IL-8, TNF-a, transferrin (TF), ceruloplasmin (CP), and vascular endothelial growth factor (VEGF)], VL, and GDS were evaluated by multivariable regression. CSF cell-free mtDNA levels were significantly lower in participants with undetectable (vs. detectable) VL in either plasma (p < 0.001) or CSF (p < 0.001) and in those on antiretroviral therapy (ART; p < 0.001). Participants on ART with undetectable VL in both CSF and plasma had lower mtDNA levels than those with detectable VL in both compartments (p = 0.001). Higher mtDNA levels were observed in participants in the highest vs. lowest tertile (T3 vs. T1) of CSF CXCL10 (T3 vs. T1, p < 0.001) and TNF-a (T3 vs. T1, p < 0.05) in unadjusted analyses. MtDNA levels also correlated with CSF leukocyte count. After adjusting for CSF leukocyte count and VL, mtDNA levels were also associated with other inflammation- and iron-related biomarkers in CSF, including TF (T3 vs. T1, p < 0.05) and CP (T3 vs. T1, p < 0.05). With additional correction for ART use, mtDNA was also negatively associated with CSF VEGF (p < 0.05) and IL-6 (p = 0.05). We observed no associations of CSF mtDNA levels with age or GDS-defined NC impairment. CSF cell-free mtDNA levels were associated with HIV RNA and ART status, as well as with biomarkers of iron transport and VEGF, a growth factor with known effects on mitochondrial integrity and autophagy. CSF mtDNA may be a biomarker of iron dysregulation and/or neuroinflammation during HIV infection.

Background: Similar to other resource-limited settings, cost restricts availability of viral load monitoring for most patients receiving antiretroviral therapy in Tijuana, Mexico. We evaluated if a pooling method could improve efficiency and reduce costs while maintaining accuracy. Methods: We evaluated 700 patient blood plasma specimens at a reference laboratory in Tijuana for detectable viremia, individually and in 10 × 10 matrix pools. Thresholds for virologic failure were set at ≥500, ≥1000 and ≥1500 HIV RNA copies per milliliter. Detectable pools were deconvoluted using pre-set algorithms. Accuracy and efficiency of the pooling method were compared with individual testing. Quality assurance (QA) measures were evaluated after 1 matrix demonstrated low efficiency relative to individual testing. Results: Twenty-two percent of the cohort had detectable HIV RNA (≥50 copies/mL). Pooling methods saved approximately one third of viral load assays over individual testing, while maintaining negative predictive values of >90% to detect samples with virologic failure (≥50 copies/mL). One matrix with low relative efficiency would have been detected earlier using the developed QA measures, but its exclusion would have only increased relative efficiency from 39% to 42%. These methods would have saved between $13,223 and $14,308 for monitoring this cohort. Conclusions: Despite limited clinical data, high prevalence of detectable viral loads and a contaminated matrix, pooling greatly improved efficiency of virologic monitoring while maintaining accuracy. By improving cost-effectiveness, these methods could provide sustainability of virologic monitoring in resource-limited settings, and incorporation of developed QA measures will most likely maximize pooling efficiency in future uses.

Despite initial responses to first-line treatment with platinum and taxane-based combination chemotherapy, most high-grade serous ovarian carcinoma (HGSOC) patients will relapse and eventually develop a cisplatin-resistant fatal disease. Due to the lethality of this disease, there is an urgent need to develop improved targeted therapies against HGSOC. Herein, we identified CASC10, a long noncoding RNA upregulated in cisplatin-resistant ovarian cancer cells and ovarian cancer patients. We performed RNA sequencing (RNA-seq) in total RNA isolated from the HGSOC cell lines OVCAR3 and OV-90 and their cisplatin-resistant counterparts. Thousands of RNA transcripts were differentially abundant in cisplatin-sensitive vs. cisplatin-resistant HGSOC cells. Further data filtering unveiled CASC10 as one of the top RNA transcripts significantly increased in cisplatin-resistant compared with cisplatin-sensitive cells. Thus, we focused our studies on CASC10, a gene not previously studied in ovarian cancer. SiRNA-mediated CASC10 knockdown significantly reduced cell proliferation and invasion; and sensitized cells to cisplatin treatment. SiRNA-mediated CASC10 knockdown also induced apoptosis, cell cycle arrest, and altered the expression of several CASC10 downstream effectors. Multiple injections of liposomal CASC10-siRNA reduced tumor growth and metastasis in an ovarian cancer mouse model. Our results demonstrated that CASC10 levels mediate the susceptibility of HGSOC cells to cisplatin treatment. Thus, combining siRNA-mediated CASC10 knockdown with cisplatin may represent a plausible therapeutic strategy against HGSOC.