Bridget Maher

Tnf-α promoter region gene polymorphisms in Hiv-positive patients with lipodystrophy

Objective: The pathogenic mechanisms underlying lipodystrophy in HIV-positive patients are largely unknown. TNF-α has many actions that are consistent with the features of lipodystrophy; therefore, an analysis was carried out to determine whether functionally active polymorphisms in the promoter region of the TNF-α gene are associated with the development of lipodystrophy. Design: Genetic case–control association study. Methods: Individuals were genotyped for the −238 and −308 polymorphisms in the TNF-α gene using polymerase chain reaction–restriction fragment length polymorphism analysis. The genotype and allele frequencies for 61 HIV-positive patients with lipodystrophy were compared with (a) 35 HIV-positive patients with no evidence of lipodystrophy and (b) 239 healthy HIV-negative individuals. Results: The frequency of the variant rare −238 allele was significantly different (P = 0.01) in HIV-positive patients with lipodystrophy than in those without lipodystrophy. At the genotype level, a trend towards a difference between patients with and without lipodystrophy was observed (χ2 for linear trend 5.2, P = 0.02). For the −308 polymorphism, no difference was found in genotype and allele frequencies between HIV patients with and without lipodystrophy. Conclusions: The data suggest that the −238 (but not the −308) promoter region TNF-α gene polymorphism is a determinant in the development of HIV-related lipodystrophy. However, the results need to be confirmed in larger numbers of patients as well as in an ethnically diverse population.

Development of Enzymatic Assays for Quantification of Intracellular Lamivudine and Carbovir Triphosphate Levels in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus-Infected Patients

ABSTRACT In this paper, we describe the development and use of enzymatic assays to determine intracellular lamivudine triphosphate (3TCTP) and carbovir triphosphate (CBVTP) concentrations in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)-infected patients. The assays involve inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates into a synthetic template primer. For the 3TCTP assay, a preincubation procedure was added whereby 3TCTP becomes incorporated before [3H]dCTP. At a 1:400 template primer dilution, control product formation was reduced by 88.0% with 0.8 pmol of 3TCTP. Standard 3TCTP inhibition curves were performed using this procedure. For the CBVTP assay, 0.1 pmol of CBVTP inhibited control product formation with and without the use of a preincubation step, so inhibition curves were constructed using both procedures. However, reduced template primer stability with assays using preincubation steps led to a single-incubation procedure being adopted for future studies. The presence of PBMC extracts interfered with the 3TCTP assay. However, this was overcome by the addition of CuSO4. PBMC extracts did not interfere with the CBVTP assay. Intracellular 3TCTP and CBVTP concentrations were determined in PBMCs from HIV-infected patients over 24 h or greater. Peak concentrations were obtained 6 to 8 h after dosing, and the half-lives of the anabolites suggested the possibility of once-daily dosing. These assays are currently being used for determination of 3TCTP and CBVTP concentrations in clinical studies.

Influence of Prior Exposure to Zidovudine on Stavudine Phosphorylation In Vivo and Ex Vivo

ABSTRACT Intracellular phosphorylation of stavudine (d4T) and zidovudine (ZDV) was investigated in peripheral blood mononuclear cells (PBMCs) isolated from ZDV-naive and ZDV-experienced human immunodeficiency virus (HIV)-positive patients. An in vivo study measured the amount of d4T triphosphate (d4TTP), while an ex vivo study assessed the capacity of cells to phosphorylate added d4T. Endogenous dTTP was also measured. d4TTP and dTTP were determined in vivo using a reverse transcriptase chain termination assay. In ex vivo studies, d4T (1 μM) was incubated in resting and phytohemagglutinin-stimulated (10 μg ml−1; 72 h) PBMCs for 24 h. After washing and methanol extraction, radiolabeled anabolites were detected by high-performance liquid chromatography. d4TTP reached its highest level 2 to 4 h after dosing (0.21 ± 0.14 pmol/106cells; n = 27 [mean ± standard deviation]). Comparison of ZDV-naive and ZDV-experienced individuals showed no significant difference in levels of d4TTP (ZDV naive, 0.23 ± 0.17 pmol/106 cells [n = 7] versus ZDV experienced, 0.20 ± 0.14 pmol/106 cells [n = 20]; P = 0.473) or the d4TTP/dTTP ratio (0.14 ± 0.12 [n = 7] and 0.10 ± 0.08 [n = 20], respectively;p = 0.391). Ex vivo data demonstrated no significant difference in the formation of d4TTP or total d4T phosphates in naive and experienced patients (0.086 ± 0.055 pmol/106cells in ZDV-naive patients [n = 17] versus 0.081 ± 0.038 pmol/106 cells in ZDV-experienced patients [n = 22]; P = 0.767). The ability of HIV-infected patients to phosphorylate d4T in vivo and ex vivo was unchanged with increasing exposure to ZDV.