Cytochrome P450 enzymes and oxidative stress in tobacco/nicotine mediated HIV pathogenesis

Title from PDF of title page, viewed on June 8, 2016Dissertation advisor: Santosh KumarVitaIncludes bibliographical references (pages 147-161)Thesis (Ph.D.)--School of Pharmacy. University of Missouri--Kansas City, 2016In the US, the prevalence of cigarette smoking in the HIV-infected population is 50-70% compared to 15-20% in the general population, which further increases the risk of smoking-related health problems in this group. For example, HIV-infected smokers show decreased immune responses, poorer responses to antiretroviral therapy (ART), and greater risk of virological rebound, compared to HIV-infected non-smokers. Several in vivo and in vitro studies have shown that smoking/nicotine is associated with decreased immune responses, increased inflammation, increased oxidative stress, and increased numbers of opportunistic infections. Furthermore, in vitro studies have shown that smoking/nicotine enhances HIV replication in alveolar macrophages, microglia, and T cells. However, the mechanism by which smoking or nicotine increases HIV replication is largely unknown. As an exception, a report suggests that iron and oxidative stress are possible mechanisms of enhanced production of HIV by alveolar macrophages in cigarette smokers. The role of CYP enzymes has not been studied in context with smoking/nicotine and HIV pathogenesis. However, there are several elegant studies that show the involvement of CYP2A6, CYP2A13, CYP1A1, and CYP1B1 in smoking/nicotine-mediated toxicity leading to various types of cancers and hepatic toxicity. The majority of tobacco constituents, including nicotine, are metabolized and/or activated by CYP enzymes to generate reactive oxygen species (ROS) and/or reactive metabolites, NNK. Based on the fact that CYP pathways play a critical role in smoking mediated cell/organ toxicity, there is a critical need to explore the involvement of CYP pathways in smoking/nicotine-mediated HIV pathogenesis. The present study was based on the central hypothesis that tobacco/nicotine, or its metabolites, enhance HIV replication in monocytes/macrophages through CYP pathway. The key findings of our studies presented in this thesis indicate that (a) CYP2A6-mediated metabolism of nicotine increased the generation of ROS in HIV cell model, SVGA astrocytes, (b) Cigarette smoke condensate caused significant induction of CYP1A1, CYP2A6, ROS production, and cytotoxicity in U937 monocytes as well as enhanced HIV replication in HIV-infected primary macrophages, (c) Mild-to-moderate smoking increased viral load in HIV-infected individuals. Furthermore, our study suggested that smoking and HIV independently increase oxidative stress in the plasma as well as in monocytes. (d) There was a decrease in the level of nicotine and subsequent increase in the level of nicotine metabolites, suggesting an increase in nicotine metabolism in HIV-infected smokers compared with uninfected smokers. In conclusion, our in vitro and ex vivo results are consistent with the hypothesis that CYP and CYP-mediated oxidative stress by tobacco/nicotine are associated with increased viral load by smoking/smoking constituents. This study has clinical implications in terms of targeting CYP and oxidative stress pathways to find potentially novel therapeutic interventions, as well as drug dose adjustment to treat HIV-infected smokers effectively.Tobacco smoking mediated oxidative stress in organ toxicities: role of cytochrome P450 systems -- Tobacco smoking effect on HIV pathogenesis: role of cytochrome P450 isozymes -- General material and methods -- An LC-MS method for concurrent determination of nicotine metabolites and the role of CYP2A6 in nicotine metabolite mediated oxidative stress in SVGA astrocytes -- Cigarette smoke consensate (CSC) mediated oxidative stress and cytoxicity in U937 monocytes -- Effect of mild-tomoderate smoking on viral load, cytokines, oxidative stress, and cytochrome P450 enzymes in HIV-infected individuals -- Enhanced nicotine metabolism in HIV-positive smokers compared to HIV-negative smokers: simultaneous determination of nicotine and its four metabolites in their plasma using a simple and sensitive ESI-LC-MS/MS technique -- Summary and future direction

Stelleninhaber geht – Wissen bleibt!

In Deutschland nimmt der Anteil älterer Arbeitnehmerinnen und Arbeitnehmer tendenziell zu. Deshalb muss sich die Bibliotheksleitung verstärkt auf das altersbedingte Ausscheiden älterer Arbeitnehmer einstellen. Eine langjährige Fachkraft verfügt über spezielles Erfahrungswissen im direkten Aufgabenfeld. Die Bibliotheksleitung muss den Transfer allen relevanten Wissens, dazu gehört das Erfahrungswissen, vom Stelleninhaber auf seinen Nachfolger ermöglichen und unterstützen. Am Beispiel der Universitätsbibliothek der Bergakademie Freiberg wird untersucht, wie das Wissensmanagement im Rahmen eines Stellenwechsels derzeit geregelt ist. Das geschieht mit Hilfe von Tiefeninterviews in verschiedenen Abteilungen. Die Auswertung der Interviews bildet die Basis für ein Konzept für das Wissensmanagement beim Stellenwechsel an der UB Freiberg. Das Konzept benennt u. a. Maßnahmen zur Identifikation des stellenbezogenen Wissens, Maßnahmen zur Dokumentation des relevanten Wissens und Instrumente zur Wissensweitergabe beim Stellenwechsel

Effects of Cigarette Smoke Condensate on Oxidative Stress, Apoptotic Cell Death, and HIV Replication in Human Monocytic Cells.

While cigarette smoking is prevalent amongst HIV-infected patients, the effects of cigarette smoke constituents in cells of myeloid lineage are poorly known. Recently, we have shown that nicotine induces oxidative stress through cytochrome P450 (CYP) 2A6-mediated pathway in U937 monocytic cells. The present study was designed to examine the effect of cigarette smoke condensate (CSC), which contains majority of tobacco constituents, on oxidative stress, cytotoxicity, expression of CYP1A1, and/or HIV-1 replication in HIV-infected (U1) and uninfected U937 cells. The effects of CSC on induction of CYP1 enzymes in HIV-infected primary macrophages were also analyzed. The results showed that the CSC-mediated increase in production of reactive oxygen species (ROS) in U937 cells is dose- and time-dependent. Moreover, CSC treatment was found to induce cytotoxicity in U937 cells through the apoptotic pathway via activation of caspase-3. Importantly, pretreatment with vitamin C blocked the CSC-mediated production of ROS and induction of caspase-3 activity. In U1 cells, acute treatment of CSC increased ROS production at 6H (>2-fold) and both ROS (>2 fold) and HIV-1 replication (>3-fold) after chronic treatment. The CSC mediated effects were associated with robust induction in the expression of CYP1A1 mRNA upon acute CSC treatment of U937 and U1 cells (>20-fold), and upon chronic CSC treatment to U1 cells (>30-fold). In addition, the CYP1A1 induction in U937 cells was mediated through the aromatic hydrocarbon receptor pathway. Lastly, CSC, which is known to increase viral replication in primary macrophages, was also found to induce CYP1 enzymes in HIV-infected primary macrophages. While mRNA levels of both CYP1A1 and CYP1B1 were elevated following CSC treatment, only CYP1B1 protein levels were increased in HIV-infected primary macrophages. In conclusion, these results suggest a possible association between oxidative stress, CYP1 expression, and viral replication in CSC-treated cells of myeloid lineage. This study warrants a closer examination of the role of CYP1B1 in smoking-mediated enhanced HIV replication

Advancing Cancer Therapy With Present And Emerging Immuno-Oncology Approaches

Immuno-oncology (I-O) is a young and growing field on the frontier of cancer therapy. Contrary to cancer therapies that directly target malignant cells, I-O therapies stimulate the body\u27s immune system to target and attack the tumor, which is otherwise invisible to, or inhibiting the immune response. To this end, several methods have been developed: First, passive therapies that enable T-cells to fight the tumor without direct manipulation, typically through binding and modifying the intracellular signaling of surface receptors. Checkpoint inhibitors, perhaps the most well known of I-O therapies; are an example of such. These are monoclonal antibodies that block binding of the tumor cell at receptors that inactivate the T-cell. A variety of small molecules can achieve the same effect by affecting metabolic or signaling pathways to boost the immune response or prevent its attenuation. Drugs originally formulated for unrelated disease states are now being used to treat cancer under the I-O approach. Second, active therapies which often involve direct manipulations that occur in vitro and once introduced to the patient will directly attack the tumor. Adoptive cell transfer is the oldest of these methods. It involves the removal of T-cells from the body, which are then expanded and genetically modified for specificity toward tumor-associated antigens (TAAs), and then reintroduced to the patient. A similar approach is taken with cancer vaccines, where TAAs are identified and reintroduced with adjuvants to stimulate an immune response, sometimes in the context of antigen-presenting cells or viral vectors. Oncolytic viruses are genetically modified natural viruses for selectivity toward tumor cells. The resulting cytotoxicity has the potential to elicit an immune response that furthers tumor cell killing. A final active approach is bi-specific T-cell engagers. These modified antibodies act to link a T-cell and tumor cell through surface receptors and thereby forcibly generate immune recognition. The therapies in each of these subfields are all still very new and ongoing clinical trials could provide even further additions. The full therapeutic potential of the aforementioned therapies, alone or in combination, has yet to be realized, but holds great promise for the future of cancer treatment

Advancing Cancer Therapy with Present and Emerging Immuno-Oncology Approaches

Immuno-oncology (I-O) is a young and growing field on the frontier of cancer therapy. Contrary to cancer therapies that directly target malignant cells, I-O therapies stimulate the body\u27s immune system to target and attack the tumor, which is otherwise invisible to, or inhibiting the immune response. To this end, several methods have been developed: First, passive therapies that enable T-cells to fight the tumor without direct manipulation, typically through binding and modifying the intracellular signaling of surface receptors. Checkpoint inhibitors, perhaps the most well known of I-O therapies; are an example of such. These are monoclonal antibodies that block binding of the tumor cell at receptors that inactivate the T-cell. A variety of small molecules can achieve the same effect by affecting metabolic or signaling pathways to boost the immune response or prevent its attenuation. Drugs originally formulated for unrelated disease states are now being used to treat cancer under the I-O approach. Second, active therapies which often involve direct manipulations that occur in vitro and once introduced to the patient will directly attack the tumor. Adoptive cell transfer is the oldest of these methods. It involves the removal of T-cells from the body, which are then expanded and genetically modified for specificity toward tumor-associated antigens (TAAs), and then reintroduced to the patient. A similar approach is taken with cancer vaccines, where TAAs are identified and reintroduced with adjuvants to stimulate an immune response, sometimes in the context of antigen-presenting cells or viral vectors. Oncolytic viruses are genetically modified natural viruses for selectivity toward tumor cells. The resulting cytotoxicity has the potential to elicit an immune response that furthers tumor cell killing. A final active approach is bi-specific T-cell engagers. These modified antibodies act to link a T-cell and tumor cell through surface receptors and thereby forcibly generate immune recognition. The therapies in each of these subfields are all still very new and ongoing clinical trials could provide even further additions. The full therapeutic potential of the aforementioned therapies, alone or in combination, has yet to be realized, but holds great promise for the future of cancer treatment

Utility of a Novel Three-Dimensional and Dynamic (3DD) Cell Culture System for PK/PD Studies: Evaluation of a Triple Combination Therapy at Overcoming Anti-HER2 Treatment Resistance in Breast Cancer

Background: Emergence of Human epidermal growth factor receptor 2 (HER2) therapy resistance in HER2-positive (HER2+) breast cancer (BC) poses a major clinical challenge. Mechanisms of resistance include the over-activation of the PI3K/mTOR and Src pathways. This work aims to investigate a novel combination therapy that employs paclitaxel (PAC), a mitotic inhibitor, with everolimus (EVE), an mTOR inhibitor, and dasatinib (DAS), an Src kinase inhibitor, as a modality to overcome resistance.Methods: Static (two dimensional, 2D) and three-dimensional dynamic (3DD) cell culture studies were conducted using JIMT-1 cells, a HER2+ BC cell line refractory to HER2 therapies. Cell viability and caspase-3 expression were examined after JIMT-1 cell exposure to agents as monotherapy or in combination using a 2D setting. A pharmacokinetic/pharmacodynamic (PK/PD) combination study with PAC+DAS+EVE was conducted over 3 weeks in a 3DD setting. PAC was administered into the system via a 3 h infusion followed by the addition of a continuous infusion of EVE+DAS 24 h post-PAC dosing. Cell counts and caspase-3 expression were quantified every 2 days. A semi-mechanistic PK/PD model was developed using the 2D data and scaled up to capture the 3DD data. The final model integrated active caspase-3 as a biomarker to bridge between drug exposures and cancer cell dynamics. Model fittings were performed using Monolix software.Results: The triple combination significantly induced caspase-3 activity in the 2D cell culture setting. In the 3DD cell culture setting, sequential dosing of PAC then EVE+DAS showed a 5-fold increase in caspase-3 activity and 8.5-fold decrease in the total cell number compared to the control. The semi-mechanistic PK/PD models fit the data well, capturing the time-course profiles of drug concentrations, caspase-3 expression, and cell counts in the 2D and 3DD settings.Conclusion: A novel, sequential triple combination therapeutic regimen was successfully evaluated in both 2D and 3DD in vitro cell culture systems. The efficacy of this combination at inhibiting the cellular proliferation and re-growth of HER2/mTOR resistant cell line, JIMT-1, is demonstrated. A biomarker-linked PK/PD model successfully captured all time-course data. The latter can be used as a modeling platform for a direct translation from 3DD in vitro settings to the clinic

Effect of acute and chronic CSC treatment in HIV-infected U1 cells.

<p>a) CSC treatment (50 μg/ml) for 6H resulted in significantly enhanced level of ROS in U1 cells, compared to control cells. b) Chronic (4-day) daily treatment with CSC (25 μg/ml) resulted in significantly higher ROS levels in U1 cells. c) PMA stimulation following 4-day CSC treatment was associated with statistically significant increase in HIV replication, as measured by HIV-1 p24 antigen level in cell supernatant, in CSC treated U1 cells compared to vehicle-treated control group. *p≤0.05; **p≤0.01.</p

Effect of CSC treatment on CYP1A1 gene transcription in monocytic cells.

<p>a) mRNA levels of CYP1A1 were found to be significantly higher in CSC treated monocytic cells compared to control cells. Acute (6H) treatment of U937 and U1 cells with CSC resulted in ~25 and ~15 fold higher CYP1A1 mRNA level respectively, as compared to DMSO-treated cells. Chronic treatment (4-day) of U1 cells with CSC was also associated with statistically significant upregulation in CYP1A1 mRNA level (~40 fold). b) The CSC-mediated upregulation in CYP1A1 gene transcription was found to be mediated via the aromatic hydrocarbon receptor (AHR). Pretreatment of U937 cells with the AHR antagonist, CH223191, significantly blocked the CSC-mediated induction of CYP1A1 mRNA level. *p≤0.05; **p≤0.01.</p