Chandravanu Dash

Cocaine Enhances HIV-1 Replication in CD4+ T Cells by Down-Regulating MiR-125b

The main objective of this study was to examine effects of cocaine on HIV-1 replication in primary CD4+ T cells. Cocaine a commonly used drug among HIV-1 positive individuals serves as a cofactor for HIV-1 infection and progression to acquired immunodeficiency syndrome (AIDS). Accumulating evidence suggest that cocaine increases HIV-1 replication in cell cultures, peripheral blood mononuclear cells (PBMCs) and animal models. Intriguingly, there are no studies on cocaine-induced alterations in HIV-1 replication in primary CD4+ T cells that serve as the main targets for HIV-1 replication in vivo. In this report, we demonstrate cocaine-induced enhancement of HIV-1 replication in primary CD4+ T cells isolated from human PBMCs. To decipher a potential mechanism, we examined whether cocaine targets the innate antiviral immunity of CD4+ T cells mediated by cellular microRNAs (miRNAs). This is because recently a network of anti-HIV miRNAs in CD4+ T cells is highlighted to suppress viral replication. Our genome wide miRNA expression analysis indicated downregulation of several anti-HIV miRNAs (miR-28, miR-125b, miR-150, miR-223, and miR-382) in cocaine treated CD4+ T cells. However, our real-time quantitative PCR analysis revealed significant downregulation of miR-125b only. Our results illustrated that miR-125b knockdown enhances HIV-1 replication, whereas overexpression of miR-125b decreases HIV-1 replication in these cells. Therefore, we believe miR-125b is a key player for the cocaine induced enhancement of HIV-1 replication in CD4+ T cells. Since, miR-125b targets the 3′ UTR regions of HIV-1 transcripts and inhibits viral protein translation, our data suggest modulation of post entry steps of HIV-1 by cocaine. Given that a plethora of studies suggest that cocaine regulates HIV entry, our results implicate a potentially novel mechanism by which cocaine can increase viral replication in CD4+ T cells.

Cocaine Enhances HIV-1–Induced CD4+ T-Cell Apoptosis: Implications in Disease Progression in Cocaine-Abusing HIV-1 Patients

Substance abuse is a major barrier in eradication of the HIV epidemic because it serves as a powerful cofactor for viral transmission, disease progression, and AIDS-related mortality. Cocaine, one of the commonly abused drugs among HIV-1 patients, has been suggested to accelerate HIV disease progression. However, the underlying mechanism remains largely unknown. Therefore, we tested whether cocaine augments HIV-1-associated CD4(+) T-cell decline, a predictor of HIV disease progression. We examined apoptosis of resting CD4(+) T cells from HIV-1-negative and HIV-1-positive donors in our study, because decline of uninfected cells plays a major role in HIV-1 disease progression. Treatment of resting CD4(+) T cells with cocaine (up to 100 μmol/L concentrations) did not induce apoptosis, but 200 to 1000 μmol/L cocaine induced apoptosis in a dose-dependent manner. Notably, treatment of CD4(+) T cells isolated from healthy donors with both HIV-1 virions and cocaine significantly increased apoptosis compared with the apoptosis induced by cocaine or virions alone. Most important, our biochemical data suggest that cocaine induces CD4(+) T-cell apoptosis by increasing intracellular reactive oxygen species levels and inducing mitochondrial depolarization. Collectively, our results provide evidence of a synergy between cocaine and HIV-1 on CD4(+) T-cell apoptosis that may, in part, explain the accelerated disease observed in HIV-1-infected drug abusers.

A prospective on drug abuse-associated epigenetics and HIV-1 replication.

Drugs of abuse serve as cofactors to susceptibility to HIV infection and disease progression. Although clinical reports indicate association between HIV/AIDS and drug use, the molecular mechanism of infection susceptibility and disease progression remains unclear. Drugs such as cocaine exert their addictive effects in part by epigenetic mechanisms. Given that epigenetic modifications play an important role in HIV-1 life cycle, it is essential to unravel whether drug abuse-associated epigenetic changes may contribute to HIV/AIDS. In this article we will provide a prospective on the impact of epigenetic mechanisms on HIV-1 life cycle.

XMRV accelerates cellular proliferation, transformational activity, and invasiveness of prostate cancer cells by downregulating p27(Kip1)

Xenotropic murine leukemia virus‐related retrovirus (XMRV) is a recently discovered gammaretrovirus that was originally detected in prostate tumors. However, a causal relationship between XMRV and prostate cancer remains controversial due to conflicting reports on its etiologic occurrence. Even though gammaretroviruses are known to induce cancer in animals, a mechanism for XMRV‐induced carcinogenesis remains unknown. Several mechanisms including insertional mutagenesis, proinflammatory effects, oncogenic viral proteins, immune suppression, and altered epithelial/stromal interactions have been proposed for a role of XMRV in prostate cancer. However, biochemical data supporting any of these mechanisms are lacking. Therefore, our aim was to evaluate a potential role of XMRV in prostate carcinogenesis.

A Novel Role of Proline Oxidase in HIV-1 Envelope Glycoprotein-induced Neuronal Autophagy

Background: Stress response autophagy is induced during HIV-1 glycoprotein “gp120”-mediated neurotoxicity. However, the underlying mechanisms are poorly understood. Results: HIV-1 gp120 induces proline oxidase that elicits ROS-mediated neuronal autophagy. Conclusion: Protective autophagy during HIV-1 gp120 neurotoxicity is partly dependent on proline oxidase-induced ROS. Significance: This is the first report that demonstrates the functional role of proline oxidase in HIV-1 gp120-mediated neuronal autophagy. Proline oxidase (POX) catalytically converts proline to pyrroline-5-carboxylate. This catabolic conversion generates reactive oxygen species (ROS) that triggers cellular signaling cascades including autophagy and apoptosis. This study for the first time demonstrates a role of POX in HIV-1 envelope glycoprotein (gp120)-induced neuronal autophagy. HIV-1 gp120 is a neurotoxic factor and is involved in HIV-1-associated neurological disorders. However, the mechanism of gp120-mediated neurotoxicity remains unclear. Using SH-SY5Y neuroblastoma cells as a model, this study demonstrates that gp120 treatment induced POX expression and catalytic activity. Concurrently, gp120 also increased intracellular ROS levels. However, increased ROS had a minimal effect on neuronal apoptosis. Further investigation indicated that the immediate cellular response to increased ROS paralleled with induction of autophagy markers, beclin-1 and LC3-II. These data lead to the hypothesis that neuronal autophagy is activated as a cellular protective response to the toxic effects of gp120. A direct and functional role of POX in gp120-mediated neuronal autophagy was examined by inhibition and overexpression studies. Inhibition of POX activity by a competitive inhibitor “dehydroproline” decreased ROS levels concomitant with reduced neuronal autophagy. Conversely, overexpression of POX in neuronal cells increased ROS levels and activated ROS-dependent autophagy. Mechanistic studies suggest that gp120 induces POX by targeting p53. Luciferase reporter assays confirm that p53 drives POX transcription. Furthermore, data demonstrate that gp120 induces p53 via binding to the CXCR4 co-receptor. Collectively, these results demonstrate a novel role of POX as a stress response metabolic regulator in HIV-1 gp120-associated neuronal autophagy.

Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients.

Epidemiologic studies suggest that cocaine abuse worsens HIV‐1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine‐abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4+ T cells that are the primary targets of HIV‐1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM–100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV‐1 integration in CD4+ T cells in a dose‐dependent manner. As integration can be modulated by several early postentry steps of HIV‐1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV‐1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4+ T cells and localize to the nucleus‐. In summary, our data provide strong evidence that cocaine can increase HIV‐1 integration in CD4+ T cells. Therefore, we hypothesize that increased HIV‐1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug‐abusing HIV‐1 patients.

Impact of cocaine abuse on HIV pathogenesis.

Over 1.2 million people in the United States are infected with the human immunodeficiency virus type 1 (HIV-1). Tremendous progress has been made over the past three decades on many fronts in the prevention and treatment of HIV-1 disease. However, HIV-1 infection is incurable and antiretroviral drugs continue to remain the only effective treatment option for HIV infected patients. Unfortunately, only three out of ten HIV-1 infected individuals in the US have the virus under control. Thus, majority of HIV-1 infected individuals in the US are either unaware of their infection status or not connected/retained to care or are non-adherent to antiretroviral therapy (ART). This national public health crisis, as well as the ongoing global HIV/AIDS pandemic, is further exacerbated by substance abuse, which serves as a powerful cofactor at every stage of HIV/AIDS including transmission, diagnosis, pathogenesis, and treatment. Clinical studies indicate that substance abuse may increase viral load, accelerate disease progression and worsen AIDS-related mortality even among ART-adherent patients. However, confirming a direct causal link between substance abuse and HIV/AIDS in human patients remains a highly challenging endeavor. In this review we will discuss the recent and past developments in clinical and basic science research on the effects of cocaine abuse on HIV-1 pathogenesis.

Effect of prolonged freezing of semen on exosome recovery and biologic activity

Exosomes are important vehicles of intercellular communication that shape host responses to physiologic, tumorigenic, and pathogenic conditions. The composition and function of exosomes are dynamic and depends on the state and condition of the cellular source. In prior work, we found that semen exosomes (SE) from healthy donors who do not use illicit drugs potently inhibit HIV-1. Following semen donation, specimens are either used immediately or frozen for use at a later time. It has been shown that short-term freezing of semen has no effect on SE-mediated HIV-1 inhibition. However, the effect of illicit drugs and prolonged freezing on SE bioactivity is unknown. Here, we show preservation of SE physical properties, (morphology, concentration, intensity/size) irrespective of illicit drug use or duration of semen freezing. Interestingly, illicit drugs and prolonged freezing decreased the levels of SE-bound CD63/CD9 and acetylcholinesterase activity respectively. Furthermore, we show differential effects of illicit drug use and prolonged freezing on SE-mediated HIV-1 inhibition. Our results highlight the importance of the source of SE and condition of semen storage on SE content and function. In-depth evaluation of donor drug-use and duration of semen storage on SE cargo and bioactivity will advance our understanding of SE composition and function.

Cocaine Enhances HIV-1 Transcription in Macrophages by Inducing p38 MAPK Phosphorylation.

Cocaine is a commonly used illicit drug among HIV-1 infected individuals and is known to increase HIV-1 replication in permissive cells including PBMCs, CD4+ T cells, and macrophages. Cocaine’s potentiating effects on HIV-1 replication in macrophages- the primary targets of the virus in the central nervous system, has been suggested to play an important role in HIV-1 neuro-pathogenesis. However, the mechanism by which cocaine enhances HIV-1 replication in macrophages remain poorly understood. Here, we report the identification of cocaine-induced signaling events that lead to enhanced HIV-1 transcription in macrophages. Treatment of physiologically relevant concentrations of cocaine enhanced HIV-1 transcription in a dose-dependent manner in infected THP-1 monocyte-derived macrophages (THP-1macs) and primary monocyte-derived macrophages (MDMs). Toward decoding the underlying mechanism, results presented in this report demonstrate that cocaine induces the phosphorylation of p38 mitogen activated protein kinase (p38 MAPK), a known activator of HIV-1 transcription. We also present data suggesting that the p38 MAPK-driven HIV-1 transcription is dependent on the induction of mitogen- and stress-activated protein kinase 1 (MSK1). Consequently, MSK1 mediates the phosphorylation of serine 10 residue of histone 3 (H3 Ser10), which is known to activate transcription of genes including that of HIV-1 in macrophages. Importantly, our results show that inhibition of p38 MAPK/MSK1 signaling by specific pharmacological inhibitors abrogated the positive effect of cocaine on HIV-1 transcription. These results validate the functional link between cocaine and p38 MAPK/MSK1 pathways. Together, our results demonstrate for the first time that the p38 MAPK/MSK1 signaling pathway plays a critical role in the cocaine-induced potentiating effects on HIV-1 infection, thus providing new insights into the interplay between cocaine abuse and HIV-1 neuro-pathogenesis.

Effect of oral contraceptives and doxycycline on endometrial MMP-2 and MMP-9 activity

OBJECTIVES To describe the effect of combined oral contraceptives (COCs) on matrix metalloproteinases MMP-2 and MMP-9 activity and compare MMP activity in women taking a COC with or without doxycycline. STUDY DESIGN Subjects (n=20) underwent endometrial biopsies (1) in the late luteal phase of a baseline cycle prior to initiating COCs, (2) on days 19-21 while taking COCs in a standard 28-day cycle (7-day hormone-free interval) and (3) on days 26-28 while taking active COCs continuously for a 28-day cycle. During the continuous COC cycle, they were randomized to receive daily subantimicrobial dose doxycycline 40mg or placebo. RESULTS Compared to baseline, COC treatment increased MMP-2 (p<.001) and MMP-9 (p<.001). MMP activity was lower in subjects taking a COC with doxycycline compared to those receiving placebo although only significantly lower for MMP-2 latent form (p=.002). CONCLUSIONS Unscheduled bleeding with COCs may be the result of increased endometrial MMPs. Sample size limitations prevent us from determining how doxycycline affects MMP activity in COC users.