Substances of abuse and their effect on SAR-CoV-2 pathogenesis.

Following the emergence of SARS-CoV-2, various reports suggest that there has been a significant increase in substance abuse due to social distancing and related issues. Several reports have suggested the impact of chronic substance use on individuals' physiological and psychological health. Therefore, there is a need to know the impact of SARS-CoV-2 on persons with substance use disorders. Individuals with substance use disorders are the most vulnerable groups and are at a high risk of SARS-CoV-2 infection due to their already existing health issues associated with substance use. This review discusses some of the molecular and systemic/organic effects chronic substance use such as alcohol, nicotine, marijuana (cannabis), opioids, methamphetamine, and cocaine have on SARS-CoV-2 infectivity and its potential cause for worsened disease outcomes in persons with substance use disorder. This will provide healthcare providers, public health policies, and researchers with the needed knowledge to address some of the many challenges faced during the Covid-19 pandemic to facilitate treatment strategies for persons with substance use disorders.

Metformin Suppresses Monocyte Immunometabolic Activation by SARS-CoV-2 Spike Protein Subunit 1.

A hallmark of COVID-19 is a hyperinflammatory state associated with severity. Monocytes undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism could inhibit the inflammatory response. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism associated with production of pro-inflammatory cytokines. This response was dependent on hypoxia-inducible factor-1α, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production and metabolic reprogramming. Likewise, metformin pre-treatment blocked cytokine induction by SARS-CoV-2 strain WA1/2020 in direct infection experiments. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by metformin, and metformin likewise suppresses inflammatory responses to live SARS-CoV-2. This has potential implications for the treatment of hyperinflammation during COVID-19.

Suppression of HIV-1 Viral Replication by Inhibiting Drug Efflux Transporters in Activated Macrophages.

BACKGROUND: Ethanol has been shown to increase oxidative stress, drug efflux transporter expression, and promote HIV progression. Macrophages, which express drug efflux transporters, serve as an essential sanctuary site for HIV. The antiretroviral drug lopinavir, a protease inhibitor, is a substrate of the drug efflux transporters P-glycoprotein and multidrug resistance-associated protein 1. The NF-κB signaling pathway is associated with inflammation and drug efflux transporter expression. OBJECTIVE: To examine the effects of ethanol on drug efflux transporters and HIV replication of macrophages and develop strategies to increase the efficacy of the protease inhibitor. METHODS: The expression of PGP and MRP1 was examined with western blot. The NF- κB inhibition was assessed with nuclear western blot. LC-MS/MS and p24 ELISA were used to assess intracellular LPV and viral replication. RESULTS: Ethanol at 40mM slightly increased drug efflux transporter PGP and MRP1 expression in activated macrophages. IKK-16, an NF- κB inhibitor, counteracted the increased transporter expression caused by ethanol exposure. MK571, an MRP1 inhibitor, and IKK-16 significantly increased intracellular LPV concentration with or without ethanol treatment. MK571 significantly increased LPV efficacy in suppressing viral replication with or without ethanol treatment. A decreasing trend and a significant decrease were observed with IKK-16+LPV treatment compared with LPV alone in the no ethanol treatment and ethanol treatment groups, respectively. CONCLUSION: In activated macrophages, inhibiting drug efflux transporter MRP1 activity and reducing its expression may represent a promising approach to suppress viral replication by increasing intracellular antiretroviral concentrations. However, different strategies may be required for ethanolrelated vs. untreated groups.

Opioid Use Disorders in People Living with HIV/AIDS: A Review of Implications for Patient Outcomes, Drug Interactions, and Neurocognitive Disorders.

The opioid epidemic has had a significant, negative impact in the United States, and people living with HIV/AIDS (PLWHA) represent a vulnerable sub-population that is at risk for negative sequela from prolonged opioid use or opioid use disorder (OUD). PLWHA are known to suffer from HIV-related pain and are commonly treated with opioids, leading to subsequent addictive disorders. PLWHA and OUD are at an increased risk for attrition in the HIV care continuum, including suboptimal HIV laboratory testing, delayed entry into HIV care, and initiation or adherence to antiretroviral therapy. Barriers to OUD treatment, such as medication-assisted therapy, are also apparent for PLWHA with OUD, particularly those living in rural areas. Additionally, PLWHA and OUD are at a high risk for serious drug-drug interactions through antiretroviral-opioid metabolic pathway-related inhibition/induction, or via the human ether-a-go-go-related gene potassium ion channel pathways. HIV-associated neurocognitive disorders can also be potentiated by the off-target inflammatory effects of opioid use. PLWHA and OUD might require more intensive, individualized protocols to sustain treatment for the underlying opioid addiction, as well as to provide proactive social support to aid in improving patient outcomes. Advancements in the understanding and management of PLWHA and OUD are needed to improve patient care. This review describes the effects of prescription and non-prescription opioid use in PLWHA.

An update on drug-drug interactions between antiretroviral therapies and drugs of abuse in HIV systems.

INTRODUCTION: While considerable progress has been made in the fight against HIV/AIDS, to date there has not been a cure, and millions of people around the world are currently living with HIV/AIDS. People living with HIV/AIDS have substance abuse disorders at higher rates than non-infected individuals, which puts them at an increased risk of drug-drug interactions. AREAS COVERED: Potential drug-drug interactions are reviewed for a variety of potential drugs of abuse, both licit and illicit. These drugs include alcohol, cigarettes or other nicotine delivery systems, methamphetamine, cocaine, opioids, and marijuana. Potential interactions include decreased adherence, modulation of drug transporters, or modulation of metabolic enzymes. We also review the relative incidence of the use of these drugs of abuse in People living with HIV/AIDS. EXPERT OPINION: Despite considerable improvements in outcomes, disparities in outcomes between PLWHA who use drugs of abuse, vs those who do not still exist. It is of critical necessity to improve outcomes in these patients and to work with them to stop abusing drugs of abuse.

Differential packaging of inflammatory cytokines/ chemokines and oxidative stress modulators in U937 and U1 macrophages-derived extracellular vesicles upon exposure to tobacco constituents.

Smoking, which is highly prevalent in HIV-infected populations, has been shown to exacerbate HIV replication, in part via the cytochrome P450 (CYP)-induced oxidative stress pathway. Recently, we have shown that extracellular vesicles (EVs), derived from tobacco- and/or HIV-exposed macrophages, alter HIV replication in macrophages by cell-cell interactions. We hypothesize that cigarette smoke condensate (CSC) and/or HIV-exposed macrophage-derived EVs carry relatively high levels of pro-oxidant and pro-inflammatory cargos and/or low levels of antioxidant and anti-inflammatory cargos, which are key mediators for HIV pathogenesis. Therefore, in this study, we investigated differential packaging of pro- and anti-inflammatory cytokines/chemokines and pro- and anti-oxidant contents in EVs after CSC exposure to myeloid cells (uninfected U937 and HIV-infected U1 cells). Our results showed that relatively long to short exposures with CSC increased the expression of cytokines in EVs isolated from HIV-infected U1 macrophages. Importantly, pro-inflammatory cytokines, especially IL-6, were highly packaged in EVs isolated from HIV-infected U1 macrophages upon both long and short-term CSC exposures. In general, anti-inflammatory cytokines, particularly IL-10, had a lower packaging in EVs, while packaging of chemokines was mostly increased in EVs upon CSC exposure in both HIV-infected U1 and uninfected U937 macrophages. Moreover, we observed higher expression of CYPs (1A1 and 1B1) and lower expression of antioxidant enzymes (SOD-1 and catalase) in EVs from HIV-infected U1 macrophages than in uninfected U937 macrophages. Together, they are expected to increase oxidative stress factors in EVs derived from HIV-infected U1 cells. Taken together, our results suggest packaging of increased level of oxidative stress and inflammatory elements in the EVs upon exposure to tobacco constituents and/or HIV to myeloid cells, which would ultimately enhance HIV replication in macrophages via cell-cell interactions.

Evaluating emtricitabine + rilpivirine + tenofovir alafenamide in combination for the treatment of HIV-infection.

Introduction: Antiretroviral therapy (ART) is recommended for all people who are living with HIV to suppress viral load and to stop the progression and transmission of HIV-1. Fixed-dose combinations of antiretrovirals largely reduce pill burden.Areas covered: The authors first provide an overview of the use of non-nucleoside reverse transcriptase inhibitor (NNRTI) based therapy in HIV care. They then summarize the properties of each drug in the fixed-dose combination of tenofovir alafenamide/emtricitabine/rilpivirine/(TAF/FTC/RPV). The efficacy and safety of each component and the combination as a whole are reviewed: FTC is non-inferior to lamivudine (3TC) at assessed dosages; TAF was non-inferior to tenofovir disoproxil fumarate (TDF); the viral efficacy of RPV is non-inferior with EFV at the assessed dosage; TAF/FTC/RPV is non-inferior in efficacy but shows less of a decline in bone mineral density and renal function compared to TDF/FTC/RPV. Finally, adverse effects and drug-drug interaction data with FTC/RPV/TAF are discussed.Expert opinion: TAF/FTC/RPV can be used as an initial regimen for people living with HIV whose HIV RNA <100,000 copies/ml and CD4 cell count > 200 cells/mm3 when INSTI-based regimens are not a treatment option. Future antiretroviral therapy development may focus on dual therapy-based regimens containing RPV, particularly as long-acting formulations.

Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-κB Signaling Pathways.

Azithromycin is effective at controlling exaggerated inflammation and slowing the long-term decline of lung function in patients with cystic fibrosis. We previously demonstrated that the drug shifts macrophage polarization toward an alternative, anti-inflammatory phenotype. In this study we investigated the immunomodulatory mechanism of azithromycin through its alteration of signaling via the NF-κB and STAT1 pathways. J774 murine macrophages were plated, polarized (with IFN-γ, IL-4/-13, or with azithromycin plus IFN-γ) and stimulated with LPS. The effect of azithromycin on NF-κB and STAT1 signaling mediators was assessed by Western blot, homogeneous time-resolved fluorescence assay, nuclear translocation assay, and immunofluorescence. The drug's effect on gene and protein expression of arginase was evaluated as a marker of alternative macrophage activation. Azithromycin blocked NF-κB activation by decreasing p65 nuclear translocation, although blunting the degradation of IκBα was due, at least in part, to a decrease in IKKß kinase activity. A direct correlation was observed between increasing azithromycin concentrations and increased IKKß protein expression. Moreover, incubation with the IKKß inhibitor IKK16 decreased arginase expression and activity in azithromycin-treated cells but not in cells treated with IL-4 and IL-13. Importantly, azithromycin treatment also decreased STAT1 phosphorylation in a concentration-dependent manner, an effect that was reversed with IKK16 treatment. We conclude that azithromycin anti-inflammatory mechanisms involve inhibition of the STAT1 and NF-κB signaling pathways through the drug's effect on p65 nuclear translocation and IKKß.

Nanotechnology approaches for delivery of cytochrome P450 substrates in HIV treatment.

Introduction: Antiretroviral therapy (ART) has led to a significant reduction in HIV-1 morbidity and mortality. Many antiretroviral drugs (ARVs) are metabolized by cytochrome P450 (CYP) pathway, and the majority of these drugs are also either CYP inhibitors or inducers and few possess both activities. These CYP substrates, when used for HIV treatment in the conventional dosage form, have limitations such as low systemic bioavailability, potential drug-drug interactions, and short half-lives. Thus, an alternative mode of delivery is needed in contrast to conventional ARVs. Areas covered: In this review, we summarized the limitations of conventional ARVs in HIV treatment, especially for ARVs which are CYP substrates. We also discussed the preclinical and clinical studies using the nanotechnology strategy to overcome the limitations of these CYP substrates. The preclinical studies and clinical studies published from 2000 to February 2019 were discussed. Expert opinion: Since preclinical and clinical studies for prevention and treatment of HIV using nanotechnology approaches have shown considerable promise in recent years, nanotechnology could become an alternative strategy for daily oral therapy as a future treatment.

Pharmacokinetics and pharmacodynamics of cytochrome P450 inhibitors for HIV treatment.

INTRODUCTION: Drugs used in HIV treatment; all protease inhibitors, some non-nucleoside reverse transcriptase inhibitors, and pharmacoenhancers ritonavir and cobicistat can inhibit cytochrome P450 (CYP) enzymes. CYP inhibition can cause clinically significant drug-drug interactions (DDI), leading to increased drug exposure and potential toxicity. Areas covered: A complete understanding of pharmacodynamics and CYP-mediated DDI is crucial to prevent adverse side effects and to achieve optimal efficacy. We summarized the pharmacodynamics of all the CYP inhibitors used for HIV treatment, followed by a discussion of drug interactions between these CYP inhibitors and other drugs, and a discussion on the effect of CYP polymorphisms. We also discussed the potential advancements in improving the pharmacodynamics of these CYP inhibitors by using nanotechnology strategy. Expert opinion: The drug-interactions in HIV patients receiving ARV drugs are complicated, especially when patients are on CYP inhibitors-based ART regimens. Therefore, evaluation of CYP-mediated drug interactions is necessary prior to prescribing ARV drugs to HIV subjects. To improve the treatment efficacy and minimize DDI, novel approaches such as nanotechnology may be the potential alternative approach. However, further studies with large cohort need to be conducted to provide strong evidence for the use of nano-formulated ARVs to effectively treat HIV patients.

Mannose-decorated hybrid nanoparticles for enhanced macrophage targeting.

Our goal was to design nanocarriers that specifically target and deliver therapeutics to polarized macrophages. Mannose receptors are highly overexpressed on polarized macrophages. In this study, we constructed Pluronic® -F127 polymer and tannic acid (TA) based nanoparticles (F127-TA core nanoparticles) with varying mannose densities. The particle size of the optimized mannose-decorated F127-TA hybrid nanoparticles (MDNPs) was found to be ~ 265 nm with a negative zeta potential of ~ - 4.5 mV. No significant changes in the size and zeta potentials of nanoparticles were observed, which demonstrated structural integrity and stability of the nanoformulation. Physicochemical characteristics of MDNPs were evaluated by FTIR and TGA and demonstrated the presence of mannose units on surface nanoparticles. A mannose-dependent cellular targeting and uptake of MDNPs was found in U937 macrophages. The uptake process was found to vary directly with time and volume of MDNPs nanoparticles. The uptake pattern is higher in M2 than M1. This behavior was also evident from the instantaneous and superior binding profile of M2 macrophage lysate protein with MDNPs over that of M1 macrophage lysate protein. These results demonstrated that an appropriate mannose ligand density was confirmed, suggesting efficient targeting of M2. Altogether, these data support that the MDNPs formulation could serve as a targeted therapeutic guide in the generation of nanomedicine to treat various conditions as an anti-inflammation therapy.

The role of cytochrome P450 2E1 on ethanol-mediated oxidative stress and HIV replication in human monocyte-derived macrophages.

BACKGROUND: Alcohol consumption is considered to be a major health problem among people living with HIV/AIDS. Our previous reports have shown that ethanol reduced intracellular concentrations of antiretroviral drugs elvitegravir and darunavir in the HIV-1-infected U1 cell line. Ethanol also increased HIV-1 replication despite the presence of elvitegravir. Our previous finding has also shown that the levels of cytochrome P450 enzyme 2E1 (CYP2E1) and oxidative stress in blood monocytes were induced, while the concentration of alcohol in the plasma was reduced in HIV-1-infected alcohol users compared to uninfected alcohol users. However, the role of CYP2E1 in ethanol-enhanced oxidative stress and HIV-1 replication is still unclear. METHODS: This study examined the chronic effects (14 days) of ethanol on HIV viral load, oxidative DNA damage, expression of CYP2E1, expression of antioxidant enzymes (AOEs), expression of reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM). Further, to evaluate the role of CYP2E1 in mediating ethanol-induced viral replication, CYP2E1 siRNA and CYP2E1 selective inhibitor were used in the HIV-1-infected U1 cell line following ethanol treatment. RESULTS: Chronic ethanol exposure demonstrated an increase in oxidative DNA damage and CYP2E1 expression in both non-infected and HIV-1-infected MDM. Our results showed that ethanol chronic exposure increased HIV-1 replication by ~3-fold in HIV-1-infected MDM. This ethanol-enhanced HIV-1 replication was associated with an increased oxidative DNA damage, an increased expression of CYP2E1, and a decreased expression of antioxidant enzyme PRDX6. In HIV-1-infected U1 cell line, we observed a decreased viral replication (~30%) and a decreased DNA damage (~100%) after repression of CYP2E1 by siRNA, upon ethanol exposure. We also observed a decreased viral replication (~25%) after inhibition of CYP2E1 by using selective CYP2E1 inhibitor. CONCLUSIONS: The data suggest that chronic ethanol exposure increases HIV-1 replication in MDM, at least in part, through CYP2E1-mediated oxidative stress. These results are clinically relevant to potentially find effective treatment strategies for HIV-1-infected alcohol users.

The dawn of precision medicine in HIV: state of the art of pharmacotherapy.

INTRODUCTION: Combination antiretroviral therapy (ART) reduces viral load to under the limit of detection, successfully decreasing HIV-related morbidity and mortality. Due to viral mutations, complex drug combinations and different patient response, there is an increasing demand for individualized treatment options for patients. AREAS COVERED: This review first summarizes the pharmacokinetic and pharmacodynamic profile of clinical first-line drugs, which serves as guidance for antiretroviral precision medicine. Factors which have influential effects on drug efficacy and thus precision medicine are discussed: patients' pharmacogenetic information, virus mutations, comorbidities, and immune recovery. Furthermore, strategies to improve the application of precision medicine are discussed. EXPERT OPINION: Precision medicine for ART requires comprehensive information on the drug, virus, and clinical data from the patients. The clinically available genetic tests are a good starting point. To better apply precision medicine, deeper knowledge of drug concentrations, HIV reservoirs, and efficacy associated genes, such as polymorphisms of drug transporters and metabolizing enzymes, are required. With advanced computer-based prediction systems which integrate more comprehensive information on pharmacokinetics, pharmacodynamics, pharmacogenomics, and the clinically relevant information of the patients, precision medicine will lead to better treatment choices and improved disease outcomes.

Sofosbuvir + velpatasvir + voxilaprevir for the treatment of hepatitis C infection.

INTRODUCTION: Hepatitis C is a disease with a significant global impact. Over the last several years, the treatment of the disease has been revolutionized. Therapy has transformed over the last several years with the approval of second generation direct acting antivirals, and currently utilized medications for the treatment of hepatitis C are significantly more efficacious with better safety profiles than previously approved treatments. Treatment for individuals who have failed therapy on direct acting antivirals has, until recently, been complex and difficult to treat, but the approval of sofosbuvir/velpatasvir/voxilaprevir represents a new therapeutic option for these individuals. Areas covered: Sofosbuvir/velpatasvir/voxilaprevir is a recently approved therapeutic combination for the treatment of hepatitis C. This article reviews the studies leading to the approval of the combination, and its efficacy and safety profile. Expert opinion: Sofosbuvir/velpatasvir/voxilaprevir fills one of the previously unfilled niches for the treatment of hepatitis C, that of the treatment of individuals who have failed therapy with resistant virus. With the filling of this niche, there appears to be a general slowing of the development of new therapeutics. Although understandable, in the long term, there are considerable risks associated with the decreased development of new drugs to treat hepatitis C.

Polarized macrophage subsets differentially express the drug efflux transporters MRP1 and BCRP, resulting in altered HIV production.

Introduction Macrophages play an important role in HIV, where they are a cellular reservoir. Macrophages are polarized into two phenotypes: pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages, which may have altered expression of drug efflux transporters, including BCRP and MRP1. These differences may result in subtherapeutic concentrations of antiretrovirals inside of macrophages and viral replication. Methods U937 and U1 cells were polarized to the M1 or M2 phenotype via IFN-γ and LPS, or IL-4, IL-13, and LPS. Transporter expression was assessed via PCR and Western blotting, and transporter function was assessed via fluorescent dye assays. Transporter function was blocked with the inhibitors MK571 or KO143. Protein expression was confirmed in monocyte-derived macrophages. p24 production was assessed in U1 cells via enzyme-linked immunosorbent assay. Results mRNA and protein analysis demonstrated higher expression of MRP1 in M1 macrophages, while BCRP expression was downregulated in M1 macrophages. Treatment with inhibitors of transporter function decreased the difference in intracellular fluorescence between polarized macrophages. Differences in protein expression, which were observed with U937 cells, were confirmed in monocyte-derived macrophages. M1, but not M2 cells treated with MK571, showed decreased p24 production, consistent with reported MRP1 transporter expression. Conclusions These results support our hypothesis that there is differential expression of MRP1 and BCRP on M1 and M2 polarized macrophages and suggests that these differences may result in altered intracellular concentrations of antiretrovirals in macrophages and alter viral production in these cells. Targeting these differences may be a strategy to decrease viral replication in HIV-infected individuals.

Tobacco and Antiretrovirals Modulate Transporter, Metabolic Enzyme, and Antioxidant Enzyme Expression and Function in Polarized Macrophages.

BACKGROUND: Cigarette smoking increases systemic oxidative stress, inflammation, and viral replication in individuals with HIV. Macrophages are infected during HIV infection and serve as an important reservoir throughout the process. Macrophages exist in two phenotypes, the classically activated M1 macrophage and alternatively activated M2 macrophage. The expression of drug efflux transporters and metabolic enzymes, which have direct effects on intracellular drug concentrations, differ between the pro-inflammatory M1 macrophage and the anti-inflammatory M2 macrophage. OBJECTIVE: To further explain the role of tobacco use in worsened outcomes in the HIV + population receiving antiretroviral therapy. METHODS: Western blotting was used to examine macrophage polarization and expression of drug efflux transporters, CYP enzymes, and antioxidant enzymes. The arginase assay was used to measure arginase activity. Cytokine production was measured using the human multiplex inflammatory cytokine assay kit. The 8-OHdG DNA Damage Quantification Direct Kit was used to quantify DNA damage. Viral replication under the influence of tobacco and antiretroviral drug use was measured by p24 Elisa. RESULTS: We observed phenotypic shifts from M1 to M2 with both individual and combination treatments with cigarette smoke condensate and the protease inhibitor antiretroviral drug lopinavir. These shifts lead to changes in cytokine production, the expression of CYP enzymes, anti-oxidant enzymes, and drug efflux transporters, as well as changes in viral replication. CONCLUSION: This data suggest a mechanism by which tobacco use impairs HIV antiretroviral therapy to increase intracellular drug concentrations in this important cellular reservoir.

Influence of Ethanol on Darunavir Hepatic Clearance and Intracellular PK/PD in HIV-Infected Monocytes, and CYP3A4-Darunavir Interactions Using Inhibition and in Silico Binding Studies.

PURPOSE: Although the prevalence of alcohol consumption is higher in HIV+ people than general public, limited information is available on how alcohol affects the metabolism and bioavailability of darunavir (DRV). METHODS: DRV was quantified by using LC-MS/MS method. All in vitro experiments were performed using human liver microsomes and HIV-infected monocytic cells. CYP3A4 and DRV/Ritonavir (RTV) docking was performed using GOLD suite 5.8. RESULTS: Ethanol (20 mM) significantly decreased apparent half-life and increased degradation rate constant of RTV-boosted DRV but not for DRV alone. Similarly, ethanol exposure increased hepatic intrinsic clearance for RTV-boosted DRV with no significant influence on DRV alone. Ethanol showed a limited influence on intracellular total DRV exposure in the presence of RTV without altering maximum concentration (Cmax) values in HIV-infected monocytic cells. Ethanol alone elevated HIV replication but this effect was nullified with the addition of DRV or DRV + RTV. Additionally, inhibitory potency of DRV was significantly reduced in the presence of ethanol. Our docking results projected that ethanol increases the average distance between DRV and CYP3A4 heme, and alter the orientation of DRV-CYP3A4 binding. CONCLUSIONS: Collectively these findings suggest that DRV metabolism is primarily influenced by ethanol in the liver, but has minor effect in HIV-residing monocytes.

Pneumocystis infection alters the activation state of pulmonary macrophages.

Recent studies show a substantial incidence of Pneumocystis jirovecii colonization and infection in patients with chronic inflammatory lung conditions. However, little is known about the impact of Pneumocystis upon the regulation of pulmonary immunity. We demonstrate here that Pneumocystis polarizes macrophages towards an alternatively activated macrophage-like phenotype. Genetically engineered mice that lack the ability to signal through IL-4 and IL-13 were used to show that Pneumocystis alternative macrophage activation is dependent upon signaling through these cytokines. To determine whether Pneumocystis-induced macrophage polarization would impact subsequent immune responses, we infected mice with Pneumocystis and then challenged them with Pseudomonas aeruginosa 14 days later. In co-infected animals, a higher proportion of macrophages in the alveolar and interstitial spaces expressed both classical and alternatively activated markers and produced the regulatory cytokines TGFß and IL-10, as well as higher arginase levels than in mice infected with P. aeruginosa alone. Our results suggest that Pneumocystis reprograms the overall macrophage repertoire in the lung to that of a more alternatively-activated setpoint, thereby altering subsequent immune responses. These data may help to explain the association between Pneumocystis infection and decline in pulmonary function.

Effect of Polyaryl Hydrocarbons on Cytotoxicity in Monocytic Cells: Potential Role of Cytochromes P450 and Oxidative Stress Pathways.

BACKGROUND: Benzo(a)pyrene (BaP), naphthalene (NPh), phenanthrene (Phe), benzo(a)antharacene (BeA), and benzo(b)fluoranthene (BeF) are known carcinogenic polyaryl hydrocarbons (PAHs) present in cigarette smoke. This study was designed to examine the relative effect of these constituents on the cytotoxicity of monocytic cells and the possible mechanism of PAH-mediated cytotoxicity. METHODS: We examined the acute (6-24 hours) and chronic (7 days) effects of these PAHs on the expression of cytochromes P450 (CYPs), oxidative stress, and cytotoxicity. The treated cells were examined for mRNA and protein levels of CYPs (1A1 and 3A4) and antioxidants enzymes (AOEs) superoxide dismutase-1 (SOD1) and catalase. Further, we assessed the levels of reactive oxygen species (ROS), caspase-3 cleavage activity, and cell viability. We performed these experiments in U937 and/or primary monocytic cells. RESULTS: Of the five PAHs tested, after chronic treatment only BaP (100 nM) showed a significant increase in the expression of CYP1A1, AOEs (SOD1 and catalase), ROS generation, caspase-3 cleavage activity, and cytotoxicity. However, acute treatment with BaP showed only an increase in the mRNA expression of CYP1A1. CONCLUSIONS: These results suggest that of the five PAHs tested, BaP is the major contributor to the toxic effect of PAHs in monocytic cells, which is likely to occur through CYP and oxidative stress pathways.