Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.

Neurotoxicity of HIV-1 Tat is attributed to its penetrating property.

We have recently engineered an exosomal Tat (Exo-Tat) which can activate latent HIV-1 in resting CD4+ T lymphocytes from antiretroviral treated HIV-1 infected patients. HIV-1 Tat protein can penetrate cell membrane freely and secrete into extracellular medium. Exo-Tat loses this penetrating property. HIV-1 Tat protein can damage the synaptic membranes contributing to the development of dementia in HIV-1 infected patients. To investigate whether the penetrating property attributes to synaptic damage in vivo, we have generated adeno-associated viruses AAV-Tat and AAV-Exo-Tat viruses. Vehicle control or AAV viruses (1 × 1012 GC/mouse in 200 µl PBS) were injected into Balb/cj mice via tail veins. The mRNA and protein expression levels in blood, brain, heart, intestine, kidney, liver, lung, muscle and spleen were determined on day 21. Intravenously injected AAV-Tat or AAV-Exo-Tat mainly infects liver and heart. Short-term expression of Tat or Exo-Tat doesn't change the expression levels of neuronal cytoskeletal marker ß3-tubulin and synaptic marker postsynaptic density 95 protein (PSD-95). Wild-type Tat, but not Exo-Tat, reduces the expression level of synaptic marker synaptophysin significantly in mice, indicating that penetrating property of HIV-1 Tat protein attributes to synaptic damage.

SARS-CoV-2 and HIV coinfection: clinical experience from Rhode Island, United States.

INTRODUCTION: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected >6 million people worldwide since December 2019. Global reports of HIV/SARS-CoV-2 coinfection are limited. To better understand the impact of the coronavirus disease 2019 (COVID-19) pandemic on persons with HIV and improve their care, we present an outpatient and inpatient clinical experience of HIV/SARS-CoV-2 coinfection from Rhode Island, US. METHODS: We describe outpatient and inpatient preparedness for the COVID-19 pandemic, and present a case series of all known patients with HIV/SARS-CoV-2 coinfection at The Miriam Hospital and Rhode Island Hospital, and The Miriam Hospital Infectious Diseases and Immunology Center, in Providence, Rhode Island, US. RESULTS AND DISCUSSION: The Infectious Diseases and Immunology Center rapidly prepared for outpatient and inpatient care of persons with HIV and SARS-CoV-2. Between 30 March and 20 May 2020, 27 patients with HIV were diagnosed with SARS-CoV-2. Twenty were male, six female and one transgender female; average age was 49 years; 13/27 were Hispanic and 6/27 were African American. All had HIV viral load <200 copies/mL and were on antiretroviral therapy with CD4 count range 87 to 1441 cells/µL. Twenty-six of the 27 had common COVID-19 symptoms for one to twenty-eight days and most had other co-morbidities and/or risk factors. Nine of the 27 were hospitalized for one to thirteen days; of those, three lived in a nursing home, six received remdesivir through a clinical trial or emergency use authorization and tolerated it well; eight recovered and one died. Overall, 17% of known Center people had HIV/SARS-CoV-2 coinfection, whereas the comparable state-wide prevalence was 9%. CONCLUSIONS: We highlight challenges of outpatient and inpatient HIV care in the setting of the COVID-19 pandemic and present the largest detailed case series to date from the United States on HIV/SARS-CoV-2 coinfection, adding to limited global reports. The aggregated clinical findings suggest that the clinical presentation and outcomes of COVID-19 appear consistent with those without HIV. Whether SARS-CoV-2 infection is more frequent among persons with HIV remains to be determined. More data are needed as we develop our understanding of how HIV and antiretroviral therapy are affected by or have an impact on this pandemic.

Depot medroxyprogesterone acetate administration increases cervical CCR5+CD4+ T cells and induces immunosuppressive milieu at the cervicovaginal mucosa.

OBJECTIVES: Depot medroxyprogesterone acetate (Depo-Provera) is the most commonly used injectable hormone contraceptive in Sub-Saharan Africa where HIV incidence is high. We determined the impact of Depo-Provera on cervical immune cells and mediators in healthy women. METHODS: In this longitudinal study, vaginal, endocervical, and rectal swabs were collected at baseline (visit 1), 1 month (visit 2), and 3 months (visit 3) after Depo-Provera injection. Cervical cells were collected by cytobrush and immune markers on cervical CD4 T cells were analyzed by multicolor flow cytometry at three different visits. The levels of immune mediators in cytobrush supernatants as well as vaginal, cervical, and rectal secretions from swabs were analyzed by multiplex assays and ELISA. RESULTS: Compared with baseline levels, we found a significant increase in the frequency of cervical CCR5CD4 T cells and a significant decrease in the frequency of cervical central memory CD4 T cells. Depo-Provera treatment had little effect on expression of immune mediators in rectal mucosa but significantly suppressed numerous immune mediators at cervicovaginal mucosa. Levels of MCP-1, G-CSF, IL-6, IL-10, GM-CSF, and IP-10 were significantly decreased in both vaginal and cervical secretions after Depo-Provera injection. In cervical samples collected by cytobrush, we found reduced levels of 22 of 25 immune mediators after Depo-Provera injection. Changes in immune mediators differed between vaginal and cervical mucosa, demonstrating compartment-specific responses. CONCLUSION: Depo-Provera altered immune profiles of cervical CD4 T cells and suppressed host immune response at cervicovaginal mucosa, suggesting its likely effect on transmission of sexually transmitted infections including HIV.

Inflammation-related gene expression profiles of salivary extracellular vesicles in patients with head trauma.

At present, there is no reliable biomarker for the diagnosis of traumatic brain injury (TBI). Studies have shown that extracellular vesicles released by damaged cells into biological fluids can be used as potential biomarkers for diagnosis of TBI and evaluation of TBI severity. We hypothesize that the genetic profile of salivary extracellular vesicles in patients with head trauma differs from that in uninjured subjects. Findings from this hypothesis would help investigate the severity of TBI. This study included 19 subjects, consisting of seven healthy controls who denied history of head trauma, six patients diagnosed with concussion injury from an outpatient concussion clinic, and six patients with TBI who received treatment in the emergency department within 24 hours after injury. Real-time PCR analysis of salivary extracellular vesicles in participants was performed using TaqMan Human Inflammation array. Gene expression analysis revealed nine upregulated genes in emergency department patients (LOX5, ANXA3, CASP1, IL2RG, ITGAM, ITGB2, LTA4H, MAPK14, and TNFRSF1A) and 13 upregulated genes in concussion clinic patients compared with healthy participants (ADRB1, ADRB2, BDKRB1, HRH1, HRH2, LTB4R2, LTB4R, PTAFR, CYSLTR1, CES1, KLK1, MC2R, and PTGER3). Each patient group had a unique profile. Comparison between groups showed that 15 inflammation-related genes had significant expression change. Our results indicate that inflammation biomarkers can be used for diagnosis of TBI and evaluation of disease severity. This study was approved by the Institutional Review Board on December 18, 2015 (approval No. 0078-12) and on June 9, 2016 (approval No. 4093-16).

Impact of exosomal HIV-1 Tat expression on the human cellular proteome.

HIV-1 exists in a latent form in all infected patients. When antiretroviral therapy is stopped, viral replication resumes. The HIV-1 Tat protein is a potent activator of viral transcription. Our previous work has demonstrated that exosomal formulations of Tat can reverse HIV-1 latency in primary CD4+ T lymphocytes isolated from long term antiretroviral treated individuals suggesting a potential role for Tat as a therapeutic HIV-1 Latency Reversal Agent (LRA). Here, we employed the label-free proteomic approach for profiling the proteomic changes associated with exosomal Tat production in human cell lines. Comparative proteomic analysis revealed that >30% peptides were differentially expressed in abundance in the Tat-expressing cell line compared with relevant controls. As expected, many of the known Tat-interactor proteins were upregulated. Tat expression also led to the upregulation of antioxidant proteins suggesting Tat-mediates an oxidative burst. Gene ontology and pathway analyses of these differentially expressed proteins showed enrichment of extracellular vesicular exosome and spliceosome localized proteins and proteins involved with transcriptional and translational mechanisms. Our work suggests that HIV-1 Tat expression leads to perturbations in cellular protein expression. In vivo administration of Tat using HIV/SIV animal models needs to be performed to assess the physiologic significance of Tat-induced proteomic changes prior to developing HIV-1 Tat as an LRA.

HIV Infection, HCV Coinfection, and Alcohol Use: Associations with Microbial Translocation and Immune Activation.

BACKGROUND: Human immunodeficiency virus (HIV) infection and heavy drinking independently promote microbial translocation and inflammation. However, it is not known how alcohol use may affect these processes in people living with HIV (PLWH). This study tested the hypothesis that alcohol exacerbates innate immune dysfunction in PLWH. METHODS: Participants were 75 PLWH and 34 uninfected controls. Groups were recruited to have similar proportions of nondrinkers, moderate drinkers, and heavy drinkers. Substance use data and plasma samples were collected at up to 3 visits over a 5-year study period. Recent alcohol use was assessed with the Timeline Followback Interview. Biomarkers of microbial translocation (lipopolysaccharide, LPS) and immune activation (lipopolysaccharide binding protein, LBP; soluble CD14, sCD14; soluble CD163, sCD163) were quantified using enzyme-linked immunosorbent assays. Analyses tested 2 hypotheses: (i) that biomarker levels would be significantly higher in PLWH than controls with comparable alcohol use and (ii) that current alcohol use would exacerbate biomarker elevations in PLWH. The second analysis included the interaction of alcohol use with hepatitis C virus (HCV) coinfection. RESULTS: Groups were matched on alcohol use, smoking, and other drug use. All biomarkers were significantly higher in PLWH relative to controls (LBP: p = 0.005; LPS: p = 0.014; sCD14: p < 0.001; sCD163: p < 0.001). In PLWH, alcohol use showed a significant, positive association with sCD163, but not with other biomarkers. However, the interaction of alcohol use with HCV coinfection was significant for all biomarkers (LBP: p = 0.002; LPS: p = 0.026; sCD14: p = 0.0004; sCD163: p = 0.001). In pairwise tests with sequential Bonferroni correction, HIV/HCV coinfected individuals who drank heavily had significantly higher sCD163 compared to coinfected nondrinkers and to HIV monoinfected nondrinkers, moderate drinkers, and heavy drinkers (ps < 0.005). Coinfected moderate drinkers had significantly higher sCD163 than each monoinfected group (ps < 0.003). In addition, sCD14 was significantly higher in coinfected moderate drinkers than coinfected nondrinkers (p = 0.027). CONCLUSIONS: As predicted, PLWH had higher levels of LBP, LPS, sCD14, and sCD163 than uninfected individuals with similar alcohol use. In PLWH, alcohol by itself was significantly associated only with higher sCD163. However, heavy or moderate alcohol use was associated with elevations in macrophage activation (sCD163) and monocyte activation (sCD14) in HIV/HCV coinfected individuals.

Potential biomarkers to detect traumatic brain injury by the profiling of salivary extracellular vesicles.

Traumatic brain injury (TBI) is a common cause of death and acquired disability in adults and children. Identifying biomarkers for mild TBI (mTBI) that can predict functional impairments on neuropsychiatric and neurocognitive testing after head trauma is yet to be firmly established. Extracellular vesicles (EVs) are known to traffic from the brain to the oral cavity and can be detected in saliva. We hypothesize the genetic profile of salivary EVs in patients who have suffered head trauma will differ from normal healthy controls, thus constituting a unique expression signature for mTBI. We enrolled a total of 54 subjects including for saliva sampling, 23 controls with no history of head traumas, 16 patients enrolled from an outpatient concussion clinic, and 15 patients from the emergency department who had sustained a head trauma within 24 hr. We performed real-time PCR of the salivary EVs of the 54 subjects profiling 96 genes from the TaqMan Human Alzheimer's disease array. Real-time PCR analysis revealed 57 (15 genes, p < 0.05) upregulated genes in emergency department patients and 56 (14 genes, p < 0.05) upregulated genes in concussion clinic patients when compared with controls. Three genes were upregulated in both the emergency department patients and concussion clinic patients: CDC2, CSNK1A1, and CTSD ( p < 0.05). Our results demonstrate that salivary EVs gene expression can serve as a viable source of biomarkers for mTBI. This study shows multiple Alzheimer's disease genes present after an mTBI.

Motivational interviewing with personalized feedback to reduce alcohol use in HIV-infected men who have sex with men: A randomized controlled trial.

OBJECTIVE: This randomized controlled trial tested the efficacy of motivational interviewing (MI) to reduce alcohol use among heavy drinking men who have sex with men (MSM) who are engaged in HIV care but not currently receiving addictions treatment. METHOD: One hundred eighty MSM living with HIV-recruited regardless of interest in changing drinking-were randomly assigned to MI or an assessment-only treatment as usual (TAU) control. MI comprised one in-person session followed by two brief phone calls and in-person booster sessions at 3 and 6 months. The Timeline Follow-Back Interview assessed past 30-day alcohol use and sexual behavior at 3, 6, and 12 months postbaseline, and serum samples and medical records assessed viral load, CD4 cell count, and liver function. RESULTS: At 6 and 12 months, MI compared to TAU resulted in significantly fewer drinks per week (6 months: b = -8.72, 95% confidence interval (CI) [-12.69, -4.76]; 12 months: b = -5.98, 95% CI [-9.77, -2.19]) and lower number of heavy drinking days (6 months: incidence rate ratio = 0.55, 95% CI [0.38, 0.79]; 12 months: incidence rate ratio = 0.50, 95% CI [0.33, 0.78]). Effects on viral load, CD4 cell count, and liver function were nonsignificant. Among those reporting condomless sex with nonsteady partners at baseline, MI resulted in significantly lower rates of this behavior at 3 and 12 months compared to TAU. CONCLUSIONS: In MSM living with HIV, MI shows substantial promise for reducing heavy drinking and for reducing condomless sex among those at risk. (PsycINFO Database Record

Exosomal Tat protein activates latent HIV-1 in primary, resting CD4+ T lymphocytes.

Replication competent HIV-1 persists in a subpopulation of CD4+ T lymphocytes despite prolonged antiretroviral treatment. This residual reservoir of infected cells harbors transcriptionally silent provirus capable of reigniting productive infection upon discontinuation of antiretroviral therapy. Certain classes of drugs can activate latent virus but not at levels that lead to reductions in HIV-1 reservoir size in vivo. Here, we show the utility of CD4+ receptor targeting exosomes as an HIV-1 latency reversal agent (LRA). We engineered human cellular exosomes to express HIV-1 Tat, a protein that is a potent transactivator of viral transcription. Preparations of exosomal Tat-activated HIV-1 in primary, resting CD4+ T lymphocytes isolated from antiretroviral-treated individuals with prolonged periods of viral suppression and led to the production of replication competent HIV-1. Furthermore, exosomal Tat increased the potency of selected LRA by over 30-fold in terms of HIV-1 mRNA expression, thereby establishing it as a potentially new class of biologic product with possible combinatorial utility in targeting latent HIV-1.

Proteomic Analysis of Exosomes and Its Application in HIV-1 Infection.

Exosomes are 30-100 nm extracellular vesicles secreted from late endosomes by various types of cells. Numerous studies have suggested that exosomes play significant roles in human immunodeficiency virus 1 (HIV-1) biogenesis. Proteomics coupled with exosome fractionation has been successfully used to identify various exosomal proteins and helped to uncover the interactions between exosomes and HIV-1. To inform the current progress in the intersection of exosome, proteomics, and HIV-1, this review is focused on: i) analyzing different exosome isolation, purification methods, and their implications in HIV-1 studies; ii) evaluating the roles of various proteomic techniques in defining exosomal contents; iii) discussing the research and clinical applications of proteomics and exosome in HIV-1 biology.

Markers of Microbial Translocation and Immune Activation Predict Cognitive Processing Speed in Heavy-Drinking Men Living with HIV.

HIV infection and alcohol use disorder are associated with deficits in neurocognitive function. Emerging evidence points to pro-inflammatory perturbations of the gut-brain axis as potentially contributing to neurocognitive impairment in the context of HIV and chronic heavy alcohol use. This study examined whether plasma markers of microbial translocation (LPS) from the gastrointestinal tract and related immune activation (sCD14, EndoCAb) were associated with neurocognition in 21 men living with HIV who were virally suppressed on antiretroviral therapy. All participants met federal criteria for heavy drinking and were enrolled in a randomized controlled trial (RCT) of a brief alcohol intervention. This secondary analysis utilized blood samples and cognitive scores (learning, memory, executive function, verbal fluency, and processing speed) obtained at baseline and three-month follow-up of the RCT. In generalized estimating equation models, LPS, sCD14, and EndoCAb individually were significant predictors of processing speed. In a model with all biomarkers, higher LPS and sCD14 both remained significant predictors of lower processing speed. These preliminary findings suggest that inflammation stemming from HIV and/or alcohol could have negative effects on the gut-brain axis, manifested as diminished processing speed. Associations of microbial translocation and immune activation with processing speed in heavy-drinking PLWH warrant further investigation in larger-scale studies.

National Institutes of Health Funding in Rhode Island.

OBJECTIVES: We present an overview of the National Institutes of Health (NIH) funding in Rhode Island through analysis of 935 NIH grants received during the fiscal years of 2012 to 2016. RESULTS: NIH funded over 2,600 grants from 2012 to 2016, of which approximately 900 were new grant awards, and the remainder were annual grant renewals. The most funded type of research in Rhode Island is mental health and substance abuse, followed by infectious disease, neurology, and public health. Research funding of cardiovascular diseases, on a per capita basis, are on par with the rest of the nation, while cancer research funding is less than one half the national average. The largest NIH institutional funding source is the National Institute of General Medical Sciences (NIGMS), followed by National Institute of Mental Health (NIMH) and National Institute on Alcohol Abuse and Alcoholism (NIAAA). While research grants (R01s) remain the predominant source of NIH funding, investigators in Rhode Island have secured additional funding through program project (P) grants with the aim of bolstering research resources and collaboration throughout the state. [Full article available at http://rimed.org/rimedicaljournal-2017-07.asp].

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells.

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Lactobacillus-derived extracellular vesicles enhance host immune responses against vancomycin-resistant enterococci.

BACKGROUND: Probiotic bacteria are known to modulate host immune responses against various pathogens. Recently, extracellular vesicles (EVs) have emerged as potentially important mediators of host-pathogen interactions. In this study, we explored the role of L. plantarum derived EVs in modulating host responses to vancomycin-resistant Enterococcus faecium (VRE) using both Caenorhabditis elegans and human cells. RESULTS: Our previous work has shown that probiotic conditioning C. elegans with L. acidophilus NCFM prolongs the survival of nematodes exposed to VRE. Similarly, L. plantarum WCFS1 derived extracellular vesicles (LDEVs) also significantly protected the worms against VRE infection. To dissect the molecular mechanisms of this EV-induced protection, we found that treatment of C. elegans with LDEVs significantly increased the transcription of host defense genes, cpr-1 and clec-60. Both cpr-1 and clec-60 have been previously reported to have protective roles against bacterial infections. Incubating human colon-derived Caco-2 cells with fluorescent dye-labeled LDEVs confirmed that LDEVs could be transported into the mammalian cells. Furthermore, LDEV uptake was associated with significant upregulation of CTSB, a human homologous gene of cpr-1, and REG3G, a human gene that has similar functions to clec-60. CONCLUSIONS: We have found that EVs produced from L. plantarum WCFS1 up-regulate the expression of host defense genes and provide protective effects on hosts. Using probiotic-derived EVs instead of probiotic bacteria themselves, this study provides a new direction to treat antimicrobial resistant pathogens, such as VRE.

Ultra-high performance liquid chromatography tandem mass-spectrometry for simple and simultaneous quantification of cannabinoids.

Cannabis is used widely in the United States, both recreationally and for medical purposes. Current methods for analysis of cannabinoids in human biological specimens rely on complex extraction process and lengthy analysis time. We established a rapid and simple assay for quantification of Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), 11-hydroxy Δ9-tetrahydrocannabinol (11-OH THC) and 11-nor-9-carboxy-Δ9-tetrahydrocannbinol (THCCOOH) in human plasma by U-HPLC-MS/MS usingΔ9-tetrahydrocannabinol-D3 (THC-D3) as the internal standard. Chromatographic separation was achieved on an Acquity BEH C18 column using a gradient comprising of water (0.1% formic acid) and methanol (0.1% formic acid) over a 6 min run-time. Analytes from 200µL plasma were extracted using acetonitrile (containing 1% formic acid and THC-D3). Mass spectrometry was performed in positive ionization mode, and total ion chromatogram was used for quantification of analytes. The assay was validated according to guidelines set forth by Food and Drug Administration of the United States. An eight-point calibration curve was fitted with quadratic regression (r2>0.99) from 1.56 to 100ngmL-1 and a lower limit of quantification (LLOQ) of 1.56ngmL-1 was achieved. Accuracy and precision calculated from six calibration curves was between 85-115% while the mean extraction recovery was >90% for all the analytes. Several plasma phospholipids eluted after the analytes thus did not interfere with the assay. Bench-top, freeze-thaw, auto-sampler and short-term stability ranged from 92.7 to 106.8% of nominal values. Application of the method was evaluated by quantification of analytes in human plasma from six subjects.

The Clinical Research Landscape in Rhode Island.

OBJECTIVES: To present an overview of clinical research activity and the state of medical research funding in Rhode Island. METHODS: We utilized clinicaltrials.gov registry to profile clinical studies between 2011 to 2016. NIH RePORT and other federal databases were used to extract information on levels of federal funding. Previously published hospital financial reports were reviewed for data on hospital-specific total external research funding. RESULTS: During 2011-2016, 1651 clinical studies were registered in clinicaltrials.gov. Nearly a third of all clinical studies were in oncology (21%) and cardiovascular diseases (10%). Alzheimer's dementia, breast cancer, HIV, and hepatitis C accounted for nearly 17% of all clinical trials. Seventy-five percent (75%) of clinical trials in RI were conducted in hospitals affiliated with Lifespan or Care New England. Financial support for clinical trials largely came from industry (60%) with 23% being supported by the National Institutes of Health (NIH). The rest are funded by nonprofit organizations, charitable foundations, educational institutions, and unlisted concerns. [Full article available at http://rimed.org/rimedicaljournal-2017-01.asp].

Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication.

A rare subset of HIV-1-infected individuals is able to maintain plasma viral load (VL) at low levels without antiretroviral treatment. Identifying the mechanisms underlying this atypical response to infection may lead to therapeutic advances for treating HIV-1. Here, we developed a proteomic analysis to compare peripheral blood cell proteomes in 20 HIV-1-infected individuals who maintained either high or low VL with the aim of identifying host factors that impact HIV-1 replication. We determined that the levels of multiple histone proteins were markedly decreased in cohorts of individuals with high VL. This reduction was correlated with lower levels of stem-loop binding protein (SLBP), which is known to control histone metabolism. Depletion of cellular SLBP increased promoter engagement with the chromatin structures of the host gene high mobility group protein A1 (HMGA1) and viral long terminal repeat (LTR), which led to higher levels of HIV-1 genomic integration and proviral transcription. Further, we determined that TNF-α regulates expression of SLBP and observed that plasma TNF-α levels in HIV-1-infected individuals correlated directly with VL levels and inversely with cellular SLBP levels. Our findings identify SLBP as a potentially important cellular regulator of HIV-1, thereby establishing a link between histone metabolism, inflammation, and HIV-1 infection.

The impact of pregnancy on anti-HIV activity of cervicovaginal secretions.

BACKGROUND: Mucosal immunity of the female genital tract plays a critical role in defense against sexually transmitted infections like HIV. Pregnancy is associated with both structural and immunologic alterations in the genital mucosa, but the impact of these changes on its ability to suppress HIV infection is unknown. Current epidemiologic data are conflicting as to whether pregnancy increases the risk of HIV acquisition. OBJECTIVE: The purpose of this study was to define the association between antimicrobial peptides and chemokines in cervicovaginal secretions and in vitro HIV infectivity among pregnant and nonpregnant women. STUDY DESIGN: Forty pregnant and 37 nonpregnant women were enrolled in a prospective longitudinal cohort study at a single tertiary care women's hospital in Providence, RI. Cervicovaginal lavage was performed at each study visit. For pregnant women, study visits occurred once per trimester, and there was an optional postpartum visit. For nonpregnant women, study visits occurred across a single cycle that was timed to occur in the proliferative, ovulatory, and secretory phases based on the presumption of a regular menstrual cycle. The impact of cervicovaginal lavage on HIV infectivity was evaluated using a TZM-bl assay and compared between pregnant and nonpregnant women for each visit. The previously validated TZM-bl assay, which uses a luciferase reporting gene to indicate HIV infection of TZM-bl cells, was measured with a luminometer with higher relative light units that indicate greater levels of in vitro HIV infection. Immune mediators were measured with a multiplex bead assay. HIV infectivity and median concentration of each mediator were compared between pregnant and nonpregnant groups with the Wilcoxon rank sum test. RESULTS: Cervicovaginal fluid from pregnant and nonpregnant women significantly decreased HIV infectivity in both groups compared with positive control (virus only; P<.01), but infectivity was not different between groups (P≥.44). During the second and third trimesters, pregnant women experienced suppression of several cervicovaginal immune mediators that included human beta defensin-2; lactoferrin; macrophage inflammatory protein-3α; regulated on activation, normally T-cell expressed and secreted; and stromal cell-derived factor-1 (all P≤.05). The antimicrobial peptide elafin was significantly correlated with HIV infectivity in both groups across all visits, except at the postpartum visit in the pregnant group (n=16). Secretory leukocyte protease inhibitor also was correlated significantly with infectivity across all visits, but in nonpregnant women only (P≤.03). CONCLUSION: Cervicovaginal secretions from both pregnant and nonpregnant women contain immune mediators that are associated with HIV infectivity in an in vitro assay; however, infectivity was not different between pregnant and nonpregnant groups. If pregnant women are at increased risk for HIV infection, it is unlikely to be mediated by alterations in the effectiveness of these protective secretions.