calcein-am and HIV-Infections

To evaluate the role of P-glycoprotein (P-gp) and multidrug-resistant-protein 1 (MRP1) on raltegravir intracellular drug disposition in CD4+ T cells, investigate the effect of HIV-1 infection on P-gp expression and correlate HIV-1 viraemia with P-gp activity in primary CD4+ T cell subsets.. The cellular accumulation ratio of [(3)H]raltegravir was quantified in CD4+ T cell lines overexpressing either P-gp (CEM-P-gp) or MRP1 (CEM-MRP1) and in primary CD3+CD4+ T cells with high (P-gp(high)) and low P-gp activity (P-gp(low)); inhibition of efflux transporters was confirmed by the intracellular retention of calcein-AM. The correlation of P-gp activity with HIV-1 viraemia was assessed in naive and memory T cell subsets from 21 HIV-1-infected treatment-naive subjects.. [(3)H]Raltegravir cellular accumulation ratio decreased in CEM-P-gp cells (P < 0.0001). XR9051 (a P-gp inhibitor) and HIV-1 PIs reversed this phenomenon. Primary CD4+P-gp(high) cells accumulated less raltegravir (38.4% ± 9.6%) than P-gp(low) cells, whereas XR9051 also reversed this effect. In vitro HIV-1 infection of PBMCs and stimulation of CD4+ T cells increased P-gp mRNA and P-gp activity, respectively, while primary CD4+P-gp(high) T cells sustained a higher HIV-1 replication than P-gp(low) cells. A significant correlation between HIV-1 viraemia and P-gp activity was found in different CD4+ T cell subsets, particularly memory CD4+ T cells (r = 0.792, P < 0.0001).. Raltegravir is a substrate of P-gp in CD4+ T cells. Primary CD4+P-gp(high) T cells eliminate intracellular raltegravir more readily than P-gp(low) cells and HIV-1 viraemia correlates with P-gp overall activity. Specific CD4+P-gp(high) T cell subsets could facilitate the persistence of viral replication in vivo and ultimately promote the appearance of drug resistance.

Topics: Anti-HIV Agents; ATP Binding Cassette Transporter, Subfamily B; Benzylidene Compounds; CD4-Positive T-Lymphocytes; Cell Line; Cells, Cultured; Fluoresceins; Healthy Volunteers; HIV Infections; HIV-1; Humans; Immunologic Memory; Raltegravir Potassium; Ritonavir; Tetrahydroisoquinolines; Viral Load; Viremia; Virus Replication

In developing countries, demand exists for a cost-effective method to evaluate human immunodeficiency virus patients' CD4(+) T-helper cell count. The TH (CD4) cell count is the current marker used to identify when an HIV patient has progressed to acquired immunodeficiency syndrome, which results when the immune system can no longer prevent certain opportunistic infections. A system to perform TH count that obviates the use of costly flow cytometry will enable physicians to more closely follow patients' disease progression and response to therapy in areas where such advanced equipment is unavailable. Our system of two serially-operated immiscible phase exclusion-based cell isolations coupled with a rapid fluorescent readout enables exclusion-based isolation and accurate counting of T-helper cells at lower cost and from a smaller volume of blood than previous methods. TH cell isolation via immiscible filtration assisted by surface tension (IFAST) compares well against the established Dynal T4 Quant Kit and is sensitive at CD4 counts representative of immunocompromised patients (less than 200 TH cells per microliter of blood). Our technique retains use of open, simple-to-operate devices that enable IFAST as a high-throughput, automatable sample preparation method, improving throughput over previous low-resource methods.

Topics: Acquired Immunodeficiency Syndrome; Automation, Laboratory; Biomarkers; CD4 Lymphocyte Count; CD4-Positive T-Lymphocytes; Cell Separation; Developing Countries; Fluoresceins; Fluorescent Dyes; Fluorometry; Health Care Costs; High-Throughput Screening Assays; HIV Infections; Humans; Microfluidics; Point-of-Care Testing; Reproducibility of Results

The ATP binding cassette (ABC)-transporters are energy dependent efflux pumps which regulate the pharmacokinetics of both anti-cancer chemotherapeutic agents, e.g. taxol, and of human immunodeficiency virus-1 (HIV-1) protease inhibitors (HPIs), e.g. saquinavir. Increased expression of several ABC-transporters, especially P-glycoprotein (P-gp) and multidrug resistance protein 2 (MRP2), are observed in multidrug resistant (MDR) tumor cells and on HIV-1 infected lymphocytes. In addition, due to their apical expression on vascular endothelial barriers, both P-gp and MRP2 are of crucial importance towards dictating drug access into sequestered tissues. However, although a number of P-gp inhibitors are currently in clinical trials, possible inhibitors of MRP2 are not being thoroughly investigated. The experimental leukotriene receptor antagonist (LTRA), MK-571 is known to be a potent inhibitor of MRP transporters. Using the MRP2 over-expressing Madin-Darby canine kidney cell line, MDCKII-MRP2, we evaluated whether the clinically approved LTRAs, e.g. montelukast (Singulair) and zafirlukast (Accolate), can similarly suppress MRP2-mediated efflux. We compared the efficacy of increasing concentrations (20-100 microM) of MK-571, montelukast, and zafirlukast, in suppressing the efflux of calcein-AM, a fluorescent MRP substrate, and the radiolabeled [(3)H-] drugs, taxol and saquinavir. Montelukast was the most potent inhibitor (p<0.01) of MRP2-mediated efflux of all three substrates. Montelukast also increased (p<0.01) the duration of intracellular retention of both taxol and saquinavir. More than 50% of the drugs were retained in cells even after 90 min post removal of montelukast from the medium. Our findings implicate that montelukast, a relatively safe anti-asthmatic agent, may be used as an adjunct therapy to suppress the efflux of taxol and saquinavir from MRP2 overexpressing cells.

Topics: Acetates; Animals; Anti-Asthmatic Agents; Antineoplastic Agents, Phytogenic; Biological Transport; Cell Line; Chemotherapy, Adjuvant; Cyclopropanes; Dogs; Drug Resistance, Neoplasm; Drug Resistance, Viral; Fluoresceins; HIV Infections; HIV Protease Inhibitors; Indoles; Leukotriene Antagonists; Multidrug Resistance-Associated Proteins; Neoplasms; Paclitaxel; Phenylcarbamates; Propionates; Quinolines; Saquinavir; Sulfides; Sulfonamides; Time Factors; Tosyl Compounds

Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95% +/- 3.56, p < 0.0001, for 24 hours and mean of 37.67% +/- 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.

Topics: Annexin A5; Apoptosis; Biomarkers; Cell Membrane Permeability; Ethidium; Flow Cytometry; Fluoresceins; Fluorescent Dyes; HIV Infections; Humans; Lymphocytes; Propidium; Sensitivity and Specificity; Time Factors