MicroRNA Expression Profiles in Human Samples and Cell Lines Revealed Nine miRNAs Associated with Cisplatin Resistance in High-Grade Serous Ovarian Cancer.

Metastasis and drug resistance are major contributors to cancer-related fatalities worldwide. In ovarian cancer (OC), a staggering 70% develop resistance to the front-line therapy, cisplatin. Despite proposed mechanisms, the molecular events driving cisplatin resistance remain unclear. Dysregulated microRNAs (miRNAs) play a role in OC initiation, progression, and chemoresistance, yet few studies have compared miRNA expression in OC samples and cell lines. This study aimed to identify key miRNAs involved in the cisplatin resistance of high-grade-serous-ovarian-cancer (HGSOC), the most common gynecological malignancy. MiRNA expression profiles were conducted on RNA isolated from formalin-fixed-paraffin-embedded human ovarian tumor samples and HGSOC cell lines. Nine miRNAs were identified in both sample types. Targeting these with oligonucleotide miRNA inhibitors (OMIs) reduced proliferation by more than 50% for miR-203a, miR-96-5p, miR-10a-5p, miR-141-3p, miR-200c-3p, miR-182-5p, miR-183-5p, and miR-1206. OMIs significantly reduced migration for miR-183-5p, miR-203a, miR-296-5p, and miR-1206. Molecular pathway analysis revealed that the nine miRNAs regulate pathways associated with proliferation, invasion, and chemoresistance through PTEN, ZEB1, FOXO1, and SNAI2. High expression of miR-1206, miR-10a-5p, miR-141-3p, and miR-96-5p correlated with poor prognosis in OC patients according to the KM plotter database. These nine miRNAs could be used as targets for therapy and as markers of cisplatin response.

Oral microbiota and periodontitis severity among Hispanic adults.

Background: Periodontitis, one of the most common bacterial infections characterized by chronic inflammation, is also known to be a risk factor for chronic conditions, including cardiovascular disease and cancer. This inflammation is driven by an altered microbiota with an increase in pathogenic bacteria. We evaluated the association between oral microbiota and periodontitis severity in high-risk Hispanics. Method: This cross-sectional study recruited 134 sexually active participants aged 21 to 49 years old from STI Clinics in Puerto Rico. A periodontal examination, saliva collection, and an interviewer-administered questionnaire were performed. Periodontal severity was categorized as: having no disease, mild, and moderate/severe and BOP and tooth loos was noted. Saliva samples were collected for genomic DNA extraction, downstream 16S rDNA amplification sequencing, and bioinformatics analyses. Results: The structure, composition, and diversity of bacterial communities differed significantly according to periodontal severity. The richness and overall diversity also differed between participants without periodontitis and participants with some level of periodontal disease. A higher abundance of Prevotella, Veillonella, or Treponema was attributed to periodontal disease and Aggregatibacter to severe bleeding on probing, while Neisseria was found in higher abundance in healthy participants, decreasing its levels with drinking, smoking, and oral sex practices. Conclusions: Our findings indicate that dysbiosis occurs as periodontal disease progresses, and both alcohol consumption and smoking habits pose risk factors for oral dysbiosis. These results are of public health and clinical impact, as several bacteria identified could serve in the future as biomarkers for periodontitis and oral cancer risk.

Upregulation of the Long Noncoding RNA CASC10 Promotes Cisplatin Resistance in High-Grade Serous Ovarian Cancer.

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.

Sex and educational attainment differences in HPV knowledge and vaccination awareness among unvaccinated-sexually active adults in Puerto Rico.

Human papillomavirus (HPV) knowledge and HPV vaccination uptake remain suboptimal. We assessed sex and educational attainment differences in HPV knowledge and vaccine awareness. Data from a cross-sectional study (2018-2021) in Puerto Rico among adults aged 21-49 was analyzed (n = 278). Adequate knowledge was defined as a score of ≥70% of correct responses on a 13-item knowledge scale. Multivariable logistic regression was used to assess the association of sex (men vs. women) and education (high school or less vs. more than high school) categories with adequate HPV knowledge and vaccine awareness. Adequate HPV knowledge was higher among women (53%) and men (46%) with more than high school and was lower among women (46%) and men (27%) with high school or less. For HPV vaccine awareness, similar results were observed. Women (OR = 3.0 ; 95%CI = 1.4-6.2) and men (OR = 2.3 , 95%CI = 1.1-4.8) with more than high school and women with high school or less (OR = 2.3 , 95%CI = 1.0-5.2) were more likely to have adequate HPV vaccine knowledge than men with high-school or less education. Heightened HPV vaccine awareness was also seen among more educated women and men and women with similar lower education when compared to men with ≤ high school. Men and individuals with lower educational attainment were more likely to have inadequate HPV knowledge and vaccine awareness. HPV vaccine-oriented educational interventions should target these high-risk groups.

Association of Radiotherapy-Related Intestinal Injury and Cancer-related Fatigue: A Brief Review and Commentary.

Radiotherapy treatment-induced intestinal injury and gut microbial perturbation/dysbiosis have been implicated in the pathobiology of cancer-related fatigue. The objective of this brief review was to explore the available evidence of the relationship between intestinal injury and self-reported fatigue, especially among cancer patients. The scientific evidence-including our own-linking gut mucosal barrier dysfunction and gut microbial perturbation/dysbiosis induced by cancer treatment with worsening of cancer related fatigue (perhaps through the gut-brain axis) is limited but promising. Emerging data suggest that lifestyle interventions and the administration of specific probiotics may favorably modulate the gut microbiota and potentially mediate beneficial effects leading to improvements in fatigue.

The Role of Gut Microbiome Perturbation in Fatigue Induced by Repeated Stress from Chemoradiotherapy: A Proof of Concept Study.

OBJECTIVES: The objectives of this proof of concept study were to (a) examine the temporal changes in fatigue and diversity of the gut microbiome over the course of chemoradiotherapy (CRT) in adults with rectal cancers; (b) investigate whether there are differences in diversity of the gut microbiome between fatigued and nonfatigued participants at the middle and at the end of CRT; and (c) investigate whether there are differences in the relative abundance of fecal microbiota at the phylum and genus levels between fatigued and nonfatigued participants at the middle and at the end of CRT. METHODS: Stool samples and symptom ratings were collected prior to the inception of CRT, at the middle (after 12-16 treatments) and at the end (after 24-28 treatments) of the CRT. Descriptive statistics and Mann-Whitney U test were computed for fatigue. Gut microbiome data were analyzed using the QIIME2 software. RESULTS: Participants (N = 29) ranged in age from 37 to 80 years. The median fatigue score significantly changed at the end of CRT (median = 23.0) compared with the median score before the initiation of CRT for the total sample (median = 17.0; p ≤ 0.05). At the middle of CRT, the alpha diversity (abundance of Operational Taxonomic Units) was lower for fatigued participants (149.30 ± 53.1) than for nonfatigued participants (189.15 ± 44.18, t(23) = 2.08, p ≤ 0.05). At the middle of CRT, the alpha diversity (abundance of Operational Taxonomic Units) was lower for fatigued participants (149.30 ± 53.1) than for nonfatigued participants (189.15 ± 44.18, Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla for fatigued participants, and Escherichia, Bacteroides, Faecalibacterium, and Oscillospira were the most abundant genera for fatigued participants. CONCLUSION: CRT-associated perturbation of the gut microbiome composition may contribute to fatigue.

Gut microbiota perturbation is associated with acute sleep disturbance among rectal cancer patients.

Cancer treatment-associated gut microbial perturbation/dysbiosis has been implicated in the pathobiology of sleep disturbance; however, evidence is scarce. Eighteen newly diagnosed rectal cancer patients (ages 52-81 years; 10 males) completed a sleep disturbance questionnaire and provided stool samples for 16s RNA gene sequencing during chemo-radiotherapy. Descriptive statistics, Wilcoxon test and regression analyses were computed. Regression analyses showed the Shannon's diversity index to be a significant factor associated with sleep disturbance. This preliminary work suggests that the biological "gut-brain axis" mechanism may be associated with symptoms of sleep disturbance.

Hotspots of Sequence Variability in Gut Microbial Genes Encoding Pro-Inflammatory Factors Revealed by Oligotyping.

The gut microbiota has been implicated in a number of normal and disease biological processes. Recent studies have identified a subset of gut bacterial genes as potentially involved in inflammatory processes. In this work, we explore the sequence variability for some of these bacterial genes using a combination of deep sequencing and oligotyping, a data analysis application that identifies mutational hotspots in short stretches of DNA. The genes for pks island, tcpC and usp, all harbored by certain strains of E. coli and all implicated in inflammation, were amplified by PCR directly from stool samples and subjected to deep amplicon sequencing. For comparison, the same genes were amplified from individual bacterial clones. The amplicons for pks island and tcpC from stool samples showed minimal levels of heterogeneity comparable with the individual clones. The amplicons for usp from stool samples, by contrast, revealed the presence of five distinct oligotypes in two different regions. Of these, the oligotype GT was found to be present in the control uropathogenic clinical isolate and also detected in stool samples from individuals with colorectal cancer (CRC). Mutational hotspots were mapped onto the USP protein, revealing possible substitutions around Leu110, Glu114, and Arg115 in the middle of the pyocin domain (Gln110, Gln114, and Thr115 in most healthy samples), and also Arg218 in the middle of the nuclease domain (His218 in the uropathogenic strain). All of these results suggest that a level of variability within bacterial pro-inflammatory genes could explain differences in bacterial virulence and phenotype.

Mitochondrial biogenesis is altered in HIV+ brains exposed to ART: Implications for therapeutic targeting of astroglia.

The neuropathogenesis of HIV associated neurocognitive disorders (HAND) involves disruption of mitochondrial homeostasis and increased neuroinflammation. However, it is unknown if alterations in mitochondrial biogenesis in the brain underlie the neuropathogenesis of HAND. In this study, neuropathological and molecular analyses of mitochondrial biogenesis and inflammatory pathways were performed in brain specimens from a well-characterized cohort of HIV+ cases that were on antiretroviral regimens. In vitro investigations using primary human astroglia and neurons were used to probe the underlying mechanisms of mitochondrial alterations. In frontal cortices from HAND brains compared to cognitive normal brains, total levels of transcription factors that regulate mitochondrial biogenesis, peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and transcription factor A, mitochondrial (TFAM) were decreased. Immunohistochemical analyses revealed that TFAM was decreased in neurons and increased in astroglia. These changes were accompanied by decreased total mitochondrial DNA per cell and increased levels of messenger RNA for the proinflammatory cytokine interleukin (IL)-1ß. To determine how IL-1ß affects astroglial bioenergetic processes and mitochondrial activity, human astroglial cultures were exposed to recombinant IL-1ß. IL-1ß induced mitochondrial activity within 30 min of treatment, altered mitochondrial related gene expression, altered mitochondrial morphology, enhanced adenoside triphosphate (ATP) utilization and increased the expression of inflammatory cytokines. WIN55,212-2 (WIN), an aminoalkylindole derivative and cannabinoid receptor agonist, blocked IL-1ß-induced bioenergetic fluctuations and inflammatory gene expression in astroglia independent of cannabinoid receptor (CB)1 and peroxisome proliferator-activated receptor (PPAR) γ. A PPARα antagonist reversed the anti-inflammatory effects of WIN in human astroglia. These results show that mitochondrial biogenesis is differentially regulated in neurons and astroglia in HAND brains and that targeting astroglial bioenergetic processes may be a strategy to modulate neuroinflammation.

Cerebrospinal fluid cell-free mitochondrial DNA is associated with HIV replication, iron transport, and mild HIV-associated neurocognitive impairment.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Increased cell-free mitochondrial DNA is a marker of ongoing inflammation and better neurocognitive function in virologically suppressed HIV-infected individuals.

Cell-free mitochondrial DNA (mtDNA) is a highly immunogenic molecule that is associated with several inflammatory conditions and with neurocognitive impairment during untreated HIV infection. Here, we investigate how cell-free mtDNA in cerebrospinal fluid (CSF) is associated with inflammation, neuronal damage, and neurocognitive functioning in the context of long-term suppressive antiretroviral therapy (ART). We quantified the levels of cell-free mtDNA in the CSF from 41 HIV-infected individuals with completely suppressed HIV RNA levels in blood plasma (<50 copies/mL) by droplet digital PCR. We measured soluble CD14, soluble CD163, interferon γ-induced protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor-α (TNF-α), neopterin, and neurofilament light chain (NFL) by immunoassays in CSF supernatant or blood plasma. Higher levels of mtDNA in CSF were associated with higher levels of MCP-1 (r = 0.56, p < 0.01) in CSF and TNF-α (r = 0.43, p < 0.01) and IL-8 (r = 0.44, p < 0.01) in blood plasma. Subjects with a previous diagnosis of AIDS showed significantly higher levels of mtDNA (p < 0.01) than subjects without AIDS. The associations between mtDNA and MCP-1 in CSF and TNF-α in blood remained significant after adjusting for previous diagnosis of AIDS (p < 0.01). Additionally, higher levels of mtDNA were associated with a lower CD4 nadir (r = -0.41, p < 0.01) and lower current CD4% (r = -0.34, p = 0.03). Paradoxically, higher levels of mtDNA in CSF were significantly associated with better neurocognitive performance (r = 0.43, p = 0.02) and with less neuronal damage (i.e. lower NFL). Higher cell-free mtDNA is associated with inflammation during treated HIV infection, but the impact on neurocognitive functioning and neuronal damage remains unclear and may differ in the setting of suppressive ART.

(1→3)-ß-D-Glucan Levels Correlate With Neurocognitive Functioning in HIV-Infected Persons on Suppressive Antiretroviral Therapy: A Cohort Study.

Microbial translocation from the gut is associated with immune dysfunction, persistent inflammation, and likely plays a role in the pathogenesis of neurocognitive dysfunction during HIV infection. (1→3)-ß-D-Glucan (BDG) is a component of most fungal cell walls and might be a useful indicator of gut mucosal barrier impairment. The objective of this study was to evaluate whether higher blood BDG levels correlate with impaired neurocognitive functioning in a cohort of HIV-infected adults with suppressed levels of HIV RNA in blood plasma. In this cross-sectional cohort study, we measured levels of BDG in blood plasma and cerebrospinal fluid (CSF) supernatant samples in a cohort of adults with acute/early HIV infection, who initiated antiretroviral therapy (ART) during the earliest phase of infection and achieved suppressed levels of HIV RNA in blood plasma (<50 copies/mL) thereafter. We compared BDG with established biomarkers of microbial translocation, immune activation, and cognitive dysfunction (evaluated by global deficit score). We found that higher blood BDG levels were significantly related to higher global deficit scores, reflecting worse neurocognitive performance (Spearman r = 0.47; P = 0.042) among HIV-infected adults with suppressed viral loads who initiated ART early in infection. Two CSF samples presented elevated BDG levels. Interestingly, these 2 samples originated from the 2 subjects with the highest global deficit scores of the cohort. BDG may be a promising independent biomarker associated with neurocognitive functioning in virologically suppressed HIV-infected individuals.

Replication of Human Herpesviruses Is Associated with Higher HIV DNA Levels during Antiretroviral Therapy Started at Early Phases of HIV Infection.

UNLABELLED: Asymptomatic replication of human herpesviruses (HHV) is frequent in HIV-infected men and is associated with increased T-cell activation and HIV disease progression. We hypothesized that the presence of replication of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) (the most frequently detected HHV) might influence HIV DNA decay during antiretroviral therapy (ART). We investigated 607 peripheral blood mononuclear cell (PBMC) samples from 107 CMV-seropositive, HIV-infected men who have sex with men, who started ART within a median of 3 months from their estimated date of infection (EDI) and were monitored for a median of 19 months thereafter. Levels of HIV, CMV, and EBV DNA and cellular HIV RNA were measured by droplet digital PCR (ddPCR) for each time point. Using a general linear mixed-effect regression model, we evaluated associations between the presence of detectable CMV DNA and EBV DNA levels and HIV DNA decay and cellular HIV RNA levels, while adjusting for peak HIV RNA, nadir CD4(+)count, CD4/CD8 ratio, CMV IgG levels, time from EDI to ART initiation, time from ART initiation to virologic suppression, detectable CMV DNA pre-ART, and age. The presence of intermittent CMV DNA in PBMC during ART was significantly associated with slower decay of HIV DNA (P= 0.011) but not with increased cellular HIV RNA transcription or more detectable 2-long terminal repeat circles. Higher levels of EBV DNA were also associated with higher levels of HIV DNA (P< 0.001) and increased unspliced cellular HIV RNA transcription (P= 0.010). These observations suggest that replication of HHV may help maintain a larger HIV DNA reservoir, but the underlying mechanisms remain unclear. IMPORTANCE: Over three-fourths of HIV-infected men have at least one actively replicating human herpesvirus (HHV) in their mucosal secretions at any one time. Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) are the most common, and although it is often asymptomatic, such CMV and EBV replication is associated with higher levels of immune activation and HIV disease progression. We hypothesized that HHV-associated activation of HIV-infected CD4(+)T cells might lead to increased HIV DNA. This study found that detectable CMV in blood cells of HIV-infected men was associated with slower decay of HIV DNA even during antiretroviral therapy (ART) that was started during early HIV infection. Similarly, levels of EBV DNA were associated with higher levels of HIV DNA during ART. If this observation points to a causal pathway, interventions that control CMV and EBV replication may be able to reduce the HIV reservoir, which might be relevant to current HIV cure efforts.

Cell-free mitochondrial DNA in CSF is associated with early viral rebound, inflammation, and severity of neurocognitive deficits in HIV infection.

Cell-free mitochondiral DNA (mtDNA) is an immunogenic molecule associated with many inflammatory conditions. We evaluated the relationship between cell-free mtDNA in cerebrospinal fluid (CSF) and neurocognitive performance and inflammation during HIV infection. In a cross-sectional analysis, we evaluated the association of mtDNA levels with clinical assessments, inflammatory markers, and neurocognitive performance in 28 HIV-infected individuals. In CSF, we measured mtDNA levels by droplet digital PCR, and soluble CD14 and CD163, neurofilament light, and neopterin by ELISA. In blood and CSF, we measured soluble IP-10, MCP-1, TNF-α, and IL-6 by ELISA, and intracellular expression of IL-2, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells by flow cytometry. We also evaluated the relationship between CSF pleocytosis and mtDNA longitudinally in another set of five individuals participating in an antiretroviral treatment (ART) interruption study. Cell-free CSF mtDNA levels strongly correlated with neurocognitive performance among individuals with neurocognitive impairment (NCI) (r = 0.77, p = 0.001). CSF mtDNA also correlated with levels of IP-10 in CSF (r = 0.70, p = 0.007) and MCP-1 in blood plasma (r = 0.66, p = 0.01) in individuals with NCI. There were no significant associations between inflammatory markers and mtDNA in subjects without NCI, and levels of mtDNA did not differ between subjects with and without NCI. MtDNA levels preceded pleocytosis and HIV RNA following ART interruption. Cell-free mtDNA in CSF was strongly associated with the severity of neurocognitive dysfunction and inflammation only in individuals with NCI. Our findings suggest that within a subset of subjects cell-free CSF mtDNA is associated with inflammation and degree of NCI.

Cerebrospinal fluid (CSF) CD8+ T-cells that express interferon-gamma contribute to HIV associated neurocognitive disorders (HAND).

BACKGROUND: HIV associated neurocognitive disorders (HAND) continue to affect cognition and everyday functioning despite anti-retroviral treatment (ART). Previous studies focused on mechanisms related to monocyte/macrophage mediated inflammation. However, in the ART era, there is increasing evidence for the involvement of CD8+ T-cells in CNS pathogenesis. METHODS: To investigate the relationship between T-cell responses and neurocognitive impairment (NCI), cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T-cell intracellular cytokine (IFNγ, IL-2, TNFα) and lytic marker (CD107a) expression were assessed in HIV infected subjects who underwent comprehensive neurocognitive (NC) evaluation and either initiated or changed ART. RESULTS: Data were collected from 31 participants at 70 visits. The frequency of cytokine expressing T-cells in CSF was significantly higher than in peripheral blood for CD4+T-cells: TNFα, IL-2, IFNγ and CD8+T-cells: IL-2 and IFNγ. Analysis of T-cell activity and NCI as a function of CSF HIV RNA levels suggested a general association between NCI, high CSF CD8+ (but not CD4+T-cell) cytokine expression and CSF HIV RNA <103 copies/ml (p<0.0001). Specifically, CSF CD8+ T-cell IFNγ expression correlated with severity of NCI (r = 0.57, p = 0.004). Multivariable analyses indicated that CSF CD8+T-cell IFNγ and myeloid activation (CD163) contributed equally and independently to cognitive status and a composite variable produced the strongest correlation with NCI (r = 0.83, p = 0.0001). In contrast, CD8+ cytolytic activity (CD107a expression) was negatively correlated with NCI (p = 0.05) but was dependent on CD4 levels >400/µl and low CSF HIV RNA levels (<103 copies/ml). In our longitudinal analysis of 16 subjects, higher CSF CD8+IFNγ expression at baseline predicted NC decline at follow-up (p = 0.02). Severity of NCI at follow-up correlated with level of residual HIV RNA in CSF. CONCLUSIONS: Presence of IFNγ expressing CD8+ T-cells, absence of cytolytic CD8+ T-cells, high myeloid activation, and failure of ART to suppress HIV replication in CSF contribute to increased risk of HAND.

Correlation of (1→3)-ß-D-glucan with other inflammation markers in chronically HIV infected persons on suppressive antiretroviral therapy.

We evaluated associations between levels of BDG and other biomarkers of inflammation in blood from 41 virologically suppressed persons with chronic HIV-infection. We found a significant correlation between BDG and neopterin levels (r=0.68), and trends to significance for correlations with other inflammation markers (tumor-necrosis-factor-α: r=0.30; interleukin-8: r=0.30; interleukin-6: r=0.28). In conclusion, BDG levels correlated with inflammation markers in a cohort of virologically suppressed individuals with chronic HIV infection. Future studies are needed to evaluate whether BDG may be a marker for morbidity in chronic HIV infection.

Differential gene expression in HIV-infected individuals following ART.

Previous studies of the effect of ART on gene expression in HIV-infected individuals have identified small numbers of modulated genes. Since these studies were underpowered or cross-sectional in design, a paired analysis of peripheral blood mononuclear cells (PBMCs), isolated before and after ART, from a robust number of HIV-infected patients (N=32) was performed. Gene expression was assayed by microarray and 4157 differentially expressed genes (DEGs) were identified following ART using multivariate permutation tests. Pathways and gene ontology (GO) terms over-represented for DEGs reflected the transition from a period of active virus replication before ART to one of viral suppression (e.g., repression of JAK-STAT signaling) and possible prolonged drug exposure (e.g., oxidative phosphorylation pathway) following ART. CMYC was the DEG whose product made the greatest number of interactions at the protein level in protein interaction networks (PINs), which has implications for the increased incidence of Hodgkin's lymphoma (HL) in HIV-infected patients. The differential expression of multiple genes was confirmed by RT-qPCR including well-known drug metabolism genes (e.g., ALOX12 and CYP2S1). Targets not confirmed by RT-qPCR (i.e., GSTM2 and RPL5) were significantly confirmed by droplet digital (ddPCR), which may represent a superior method when confirming DEGs with low fold changes. In conclusion, a paired design revealed that the number of genes modulated following ART was an order of magnitude higher than previously recognized.

Pooled nucleic acid testing to detect antiretroviral treatment failure in Mexico.

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.

Peptide identification from mixture tandem mass spectra.

The success of high-throughput proteomics hinges on the ability of computational methods to identify peptides from tandem mass spectra (MS/MS). However, a common limitation of most peptide identification approaches is the nearly ubiquitous assumption that each MS/MS spectrum is generated from a single peptide. We propose a new computational approach for the identification of mixture spectra generated from more than one peptide. Capitalizing on the growing availability of large libraries of single-peptide spectra (spectral libraries), our quantitative approach is able to identify up to 98% of all mixture spectra from equally abundant peptides and automatically adjust to varying abundance ratios of up to 10:1. Furthermore, we show how theoretical bounds on spectral similarity avoid the need to compare each experimental spectrum against all possible combinations of candidate peptides (achieving speedups of over five orders of magnitude) and demonstrate that mixture-spectra can be identified in a matter of seconds against proteome-scale spectral libraries. Although our approach was developed for and is demonstrated on peptide spectra, we argue that the generality of the methods allows for their direct application to other types of spectral libraries and mixture spectra.