cytochrome-c-t and HIV-Infections

Productive HIV infection of CD4(+) T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells.

Topics: Autophagy; Autophagy-Related Protein 5; CD4-Positive T-Lymphocytes; Cytochromes c; Female; Gene Expression Regulation; HIV; HIV Infections; Host-Pathogen Interactions; Humans; Intracellular Membranes; Lysosomes; Male; Membrane Proteins; Microtubule-Associated Proteins; Permeability; Tumor Suppressor Protein p53

Side effects and toxicity have posed a threat to the positive contribution of Antiretroviral Therapy (ART) in the management of human immunodeficiency virus (HIV) infection and Acquired Immune Deficiency Syndrome (AIDS). Symptoms of mitochondrial toxicity including myopathy, pancreatitis, hyperlipidaemia and lactic acidosis are found among HIVinfected patients on ART. To date, there is not a reliable biomarker for monitoring ART-related mitochondrial toxicity. Plasma level of Cytochrome c (Cyt-c) has been proposed as a potential biomarker for ART-related toxicity due to its strong association with apoptosis.. The present study assessed toxicity and level of plasma Cyt-c among HIV-infected patients receiving ART in Ghana.. A total of eighty (80) HIV patients were recruited into the study. Demographic data were obtained from personal interview and medical records. Plasma samples were screened for toxicity from sixty (60) participants due to limited resources, and plasma Cyt-c levels were determined using ELISA. Data were analyzed using Stata version 13.. Out of the 60 participants, 11 (18.3%) were found with symptoms of myopathy, 12 (20%) with pancreatitis, 21 (35%) with hyperlipidaemia and 36 (60%) with at least one of the symptoms. The concentration of plasma Cyt-c was higher (0.122 ng/ml) in patients with toxicity than in those without toxicity (0.05 ng/ml), though the difference was not statistically significant (p = 0.148). There was a weak correlation between plasma Cyt-c level and duration of ART (Spearman rho = 0.02, p = 0.89).. This study, therefore, demonstrated a high prevalence of ART-related toxicity and high levels of Cyt-c in HIV-infected patients in support of the argument that plasma Cyt-c levels are potential biomarkers for determining ART-related toxicity in HIV patients.

Topics: Adult; Antiretroviral Therapy, Highly Active; Cross-Sectional Studies; Cytochromes c; Drug-Related Side Effects and Adverse Reactions; Female; Ghana; HIV Infections; Humans; Hyperlipidemias; Male; Middle Aged; Muscular Diseases; Pancreatitis; Young Adult

A case-control study of the effect of antiretroviral therapy (ART) on apoptosis pathway genes comprising 16 cases (HIV infected with mitochondrial toxicity) and 16 controls (HIV uninfected) was conducted. A total of 26 of 84 genes of the apoptosis pathway were differentially expressed. Two of the upregulated genes, DFFA and TNFRSF1A, classified 75% of study participants correctly as either a case or control. Thus, apoptosis may be in the causal pathway of ART-associated mitochondrial toxicity. These two genes could be markers for detecting and monitoring ART-induced mitochondrial toxicity.

Topics: Adult; Aged; Anti-HIV Agents; Antiretroviral Therapy, Highly Active; Apoptosis; Apoptosis Regulatory Proteins; Case-Control Studies; Cytochromes c; DNA, Mitochondrial; Female; Gene Expression Regulation; HIV Infections; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Mitochondria; Principal Component Analysis; Receptors, Tumor Necrosis Factor, Type I

Endoplasmic reticulum (ER) stress triggered under hyperglycemic, hypoxic, and oxidative conditions has been implicated in cellular dysfunction through activation of the unfolded protein response (UPR). Recent clinical studies have documented that the release of soluble cellular and host factors following HIV infection in the central nervous system (CNS) results in induction of the ER stress response. Herein, we demonstrate that exposure of human brain microvascular endothelial cells (HBMECs) to HIV transactivator protein Tat101 resulted in early induction of several major ER stress regulators including ER chaperones Bip/GRP78 and ER stress sensors ATF6, p-PERK, and downstream mediators p-eIF2α and ATF4. Upregulation of the ER stress mediators was accompanied by decreased cell viability and increased apoptosis as evidenced by MTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays, respectively. Pretreatment of HBMECs with either ER inhibitor or knockdown of the effector C/EBP homologous protein (CHOP) resulted in increased cell viability and abrogation of apoptosis following Tat exposure. Notably, Tat-mediated activation of the UPR response involved reactive oxygen species. Furthermore, treatment of Tat also resulted in mitochondrial dysfunction, evidenced by decrease in Bcl2/Bax ratio, dysfunction of mitochondrial membrane potential, and release of cytochrome c, all of which could be partially reversed by the ER stress inhibitor. The current study demonstrates that exposure of HBMECs to Tat induces multiple stress responses, including ER stress and mitochondrial dysfunction which in turn lead to apoptosis.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Brain; Cell Survival; Cytochromes c; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Endothelial Cells; HIV Infections; Humans; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; tat Gene Products, Human Immunodeficiency Virus

Diagnosis of antiretroviral therapy (ART) toxicity is complicated. Apoptosis has been implicated in ART toxicity. Cytochrome c (Cyt-C) is a mitochondrial protein found in plasma during pro-apoptotic states. We conducted a study of HIV-infected individuals on ART with (cases, n=21) and without (controls, n=21) clinical evidence of toxicity to determine if elevated plasma Cyt-C is associated with ART toxicity. When corrected for CD4 count, viral load, and duration of HIV infection, cases are 7.86 times more likely than controls to have plasma Cyt-C>0.216 ng/mL. Cyt-C could be a useful clinical tool to guide treatment decisions in this population.

Topics: Adult; Aged; Anti-Retroviral Agents; Biomarkers; Case-Control Studies; CD4 Lymphocyte Count; Cytochromes c; Drug-Related Side Effects and Adverse Reactions; Female; HIV Infections; Humans; Male; Middle Aged; Mitochondria; Pilot Projects; Plasma; Viral Load

The emergence of drug resistant strains of Mycobacterium tuberculosis (M. tuberculosis) together with reports of co-infections with the human immunodeficiency virus (HIV) has renewed interest to better understand the intricate mechanisms prevalent during co-infections. In this study we report a synergistic effect of M. tuberculosis and HIV-1, and their antigens Rv3416 and Nef, respectively, in inhibiting apoptosis of macrophages. This inhibition involves the TLR2 pathway and second messengers that play complementing and contrasting roles in regulating apoptosis. Interestingly, the route of calcium influx into cells differentially regulates apoptosis during antigenic co-stimulation. While calcium released from intracellular stores was anti-apoptotic, calcium influx from the external milieu was pro-apoptotic. Further, molecular sensors of intracellular calcium release aid in antigen mediated inhibition of apoptosis. A cross-regulation between oxidative burst and differential routing of calcium influx governed apoptosis. Interestingly, the HIV-1 Nef supported anti-apoptotic responses in macrophages whereas Vpu had no significant effect. These results point to a synergistic liaison between M. tuberculosis and HIV-1 in regulating macrophage apoptosis.

Topics: Antigens; Apoptosis; Bacterial Proteins; Calcium; Cells, Cultured; Coinfection; Cytochromes c; Gene Expression Regulation; HEK293 Cells; HIV Infections; HIV-1; Homeostasis; Humans; Leukocytes, Mononuclear; Macrophages; Membrane Potential, Mitochondrial; Mycobacterium tuberculosis; nef Gene Products, Human Immunodeficiency Virus; Respiratory Burst; RNA, Small Interfering; Signal Transduction; Toll-Like Receptor 2; Tuberculosis

Topics: Antibodies, Monoclonal; Apoptosis; Caspase 8; Caspase 9; CD4-Positive T-Lymphocytes; Cell Death; Cytochromes c; Drug Resistance, Viral; Enzyme Activation; Epitopes; HIV Infections; HIV Protease; Humans; Prognosis; Protein Structure, Tertiary

HIV entry into the CNS is an early event after peripheral infection, resulting in neurologic dysfunction in a significant number of individuals despite successful anti-retroviral therapy. The mechanisms by which HIV mediates CNS dysfunction are not well understood. Our group recently demonstrated that HIV infection of astrocytes results in survival of HIV infected cells and apoptosis of surrounding uninfected astrocytes by the transmission of toxic intracellular signals through gap junctions. In the current report, we characterize the intracellular signaling responsible for this bystander apoptosis. Here, we demonstrate that HIV infection of astrocytes results in release of cytochrome C from the mitochondria into the cytoplasm, and dysregulation of inositol trisphosphate/intracellular calcium that leads to toxicity to neighboring uninfected astrocytes. Blocking these dysregulated pathways results in protection from bystander apoptosis. These secondary messengers that are toxic in uninfected cells are not toxic in HIV infected cells, suggesting that HIV protects these cells from apoptosis. Thus, our data provide novel mechanisms of HIV mediated toxicity and generation of HIV reservoirs. Our findings provide new potential therapeutic targets to reduce the CNS damage resulting from HIV infection and to eradicate the generation of viral reservoirs. We demonstrated that HIV infection of astrocytes protects infected cells from apoptosis but results in cell death of surrounding uninfected astrocytes by a mechanism that is dependent on gap junction channels, dysregulation of mitochondrial cytochrome C (CytC), and cell to cell diffusion of inositol trisphosphate (IP3 ) and calcium. Our data provide essential information about generation of brain reservoirs and the mechanism of toxicity mediated by the virus.

Topics: Apoptosis; Astrocytes; Bystander Effect; Calcium; Calcium Signaling; Cell Survival; Cerebral Cortex; Cytochromes c; Cytoplasm; Fetus; Gap Junctions; HIV Infections; Humans; Inositol 1,4,5-Trisphosphate Receptors; Microinjections; Mitochondria

Tenofovir disoproxil fumerate (TDF) is an effective nucleoside reverse transcriptase inhibitor for HIV infection but it is potentially nephrotoxic. A selective mithochondrial toxicity has been hypothesized. To assess early markers of renal toxicity, we evaluated a cohort of antiretroviral (ARV)-experienced HIV patients who had been switched from a thymidinic backbone to either a TDF/emtricitabine regimen (TDF; 73 patients) or an abacavir/lamivudine (ABV) regimen (28 patients). Markers of mitochondrial toxicity (cytochrome c, Cyc) or cytosolic (α-glutathione S transferase, α-GST) together with common indicators of renal damage were assessed at baseline (T0) and after 1 (T1), 3 (T2), 6 (T3), and 12 (T4) months of patient exposure to therapy. Clinical features of both groups were comparable at T0. There was no significant variation in estimated glomerular filtration rate (eGRF), median urine protein excretion, or microalbuminuria and serum phosphate levels in both groups during the study period. There was a significant increase in urinary excretion of phosphate in patients on TDF compared to those on ABV at T3 and T4. Fractional excretion of uric acid was also altered in the two treatment groups; there was no change in the ABV (constantly less than 0.10), but a progressive increase in TDF patients. Serum potassium levels were significantly lower in ABV than in TDF treated patients. Urine concentrations of α-GST showed a nonsignificant variation in both groups, while Cyc excretion was significantly higher at T1 and T3 in TDF-treated compared to ABV-treated patients. In conclusion, TDF may be associated with subclinical mitochondrial damage, inducing at a later stage increased urinary excretion of phosphate and uric acid, as markers of incipient tubular injury.

Topics: Adenine; Anti-HIV Agents; Biomarkers; Cohort Studies; Cytochromes c; Deoxycytidine; Dideoxynucleosides; Drug Combinations; Emtricitabine; Female; Glomerular Filtration Rate; Glutathione Transferase; HIV Infections; Humans; Kidney Diseases; Kidney Tubules, Proximal; Lamivudine; Male; Middle Aged; Mitochondria; Organophosphonates; Tenofovir; Time Factors

HCV related liver disease is one of the most important complications in persons with HIV, with accelerated fibrosis progression in coinfected persons compared to those with HCV alone. We hypothesized that HCV-HIV coinfection increases HCV related hepatocyte apoptosis and that HCV and HIV influence TRAIL signaling in hepatocytes.. We analyzed the effect of HIV in JFH1-infected Huh7.5.1 cells. Apoptosis was measured by Caspase-Glo 3/7 assay and Western blotting for cleaved PARP. TRAIL, TRAIL receptor 1 (DR4), and 2 (DR5) mRNA and protein levels were assessed by real-time PCR and Western blot, respectively. We also investigated activation of caspase pathways using caspase inhibitors and assessed expression of Bid and cytochrome C.. We found increased caspase 3/7 activity and cleaved PARP in JFH1 HCV-infected Huh7.5.1 cells in the presence of heat-inactivated HIV, compared to Huh7.5.1 cells infected with JFH1 or exposed to heat-inactivated HIV alone. Both DR4 and DR5 mRNA and protein expression were increased in JFH1-infected cells in the presence of inactivated HIV compared to Huh7.5.1 cells infected with JFH1 or exposed to heat-inactivated HIV alone. Pancaspase, caspase-8, and caspase-9 inhibition blocked apoptosis induced by HCV, inactivated HIV, and HCV plus inactivated HIV. A caspase-9 inhibitor blocked apoptosis induced by HCV, HIV, and HCV-HIV comparably to pancaspase and caspase-8 inhibitors. HCV induced the activation of Bid cleavage and cytochrome C release. The addition of HIV substantially augmented this induction.. Our findings indicate that hepatocyte apoptosis is increased in the presence of HCV and HIV compared to HCV or HIV alone, and that this increase is mediated by DR4 and DR5 up-regulation. These results provide an additional mechanism for the accelerated liver disease progression observed in HCV-HIV co-infection.

Topics: Apoptosis; Base Sequence; BH3 Interacting Domain Death Agonist Protein; Caspase 3; Caspase 7; Cell Line; Cytochromes c; DNA Primers; Hepatitis C, Chronic; Hepatocytes; HIV Infections; HIV-1; Humans; Poly(ADP-ribose) Polymerases; Receptors, TNF-Related Apoptosis-Inducing Ligand; RNA, Messenger; RNA, Small Interfering; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

The era of highly active antiretroviral therapy (HAART) has controlled AIDS and its related disorders considerably; however, the prevalence of HIV-1-associated neurocognitive disorders has been on the rise in the post-HAART era. In view of these developments, we investigated whether a HAART drug combination of 3'-azido-2',3'-deoxythymidine (AZT) and indinavir (IDV) can alter the functionality of the blood-brain barrier (BBB) endothelial cells, thereby exacerbating this condition. The viability of hCMEC/D3 cells (in vitro model of BBB) that were exposed to these drugs was significantly reduced after 72h treatment, in a dose-dependent manner. Reactive oxygen species were highly elevated after the exposure, indicating that mechanisms that induce oxidative stress were involved. Measures of oxidative stress parameters, such as glutathione and malondialdehyde, were altered in the treated groups. Loss of mitochondrial membrane potential, as assessed by fluorescence microscopy and decreased levels of ATP, indicated that cytotoxicity was mediated through mitochondrial dysfunction. Furthermore, AZT+IDV treatment caused apoptosis in endothelial cells, as assessed by the expression of cytochrome c and procaspase-3 proteins. Pretreatment with the thiol antioxidant N-acetylcysteine amide reversed some of the pro-oxidant effects of AZT+IDV. Results from our in vitro studies indicate that the AZT+IDV combination may affect the BBB in HIV-infected individuals treated with HAART drugs.

Topics: Acetylcysteine; Adenosine Triphosphate; Antiretroviral Therapy, Highly Active; Apoptosis; Blood-Brain Barrier; Caspase 3; Cell Line, Transformed; Cytochromes c; Endothelial Cells; Glutathione; HIV Infections; HIV Protease Inhibitors; HIV-1; Humans; Indinavir; Malondialdehyde; Membrane Potential, Mitochondrial; Mitochondria; Oxidative Stress; Reactive Oxygen Species; Zidovudine

Topics: Amino Acid Chloromethyl Ketones; Animals; Cysteine Proteinase Inhibitors; Cytochromes c; HIV Envelope Protein gp120; HIV Infections; HIV-1; Interleukin-1; Neocortex; Rats

Topics: Animals; Apoptosis; Apoptosis Inducing Factor; Cytochromes c; Flavoproteins; France; Gene Products, vpr; History, 20th Century; History, 21st Century; HIV Infections; Humans; Membrane Proteins; Mitochondria; Neoplasms; Physiology; vpr Gene Products, Human Immunodeficiency Virus