Tumor necrosis factor-alpha levels in HIV-1 seropositive injecting drug users.

TNF-alpha is a highly pleiotropic cytokine and plays an important role in regulating HIV-1 replication. It may compromise the integrity of the blood-brain-barrier and, thus, may contribute to the neurotoxicity of HIV-1-infection. Both intravenous drug abuse (IDU) and HIV infection can increase TNF-alpha activity, but little information is available on the effects of a combination of these factors on TNF-alpha. We investigated plasma TNF-alpha levels and mRNA in the peripheral monocytes of 166 men and women in three groups: HIV-1-positive IDUs, HIV-1-negative IDUs, and HIV-negative non-IDU control participants. HIV-1-positive IDUs had higher TNF-alpha levels than HIV-1-negative IDUs who, in turn, had higher levels than controls. TNF-alpha mRNA expression in peripheral monocytes was significantly increased in both HIV-1-positive and negative IDUs compared to controls. These findings show that the effects of HIV infection and intravenous drug use may be additive in increasing TNF-alpha levels. Given the multiple effects of TNF-alpha in HIV infection, additional investigation of its role is needed.

Human immunodeficiency virus type 1 RNA Levels in different regions of human brain: quantification using real-time reverse transcriptase-polymerase chain reaction.

Human immunodeficiency virus type 1 (HIV-1) enters the central nervous system shortly after the infection and becomes localized in different regions of the brain, leading to various neurological abnormalities including motor disorders and neurocognitive deficits. Although HIV-1-associated functional abnormalities of the central nervous system (CNS) can be evaluated during life by using various test batteries, HIV-1 virus concentration in different brain regions can be measured only after death. The tissues obtained at autopsy provide a valuable source for determining the role of various factors, including that of HIV-1 viral load in the CNS, that may contribute to the regional CNS neuropathogenesis. For this study, we obtained from the National Institutes of Health-sponsored National NeuroAIDS Tissue Consortium (NNTC) the tissues from different brain regions collected at autopsy of HIV-1-positive (N = 38) and HIV-negative (N = 11) individuals, with postmortem intervals of 2 to 29 h, and measured HIV-1 RNA concentration in the frontal cortex, frontal cortex area 4, frontal cortex area 6, basal ganglia, caudate nucleus, putamen, globus pallidus, substantia nigra, and cerebrospinal fluid. Because HIV-1+ individuals were infected with the virus for up to 21 years and the majority of them had used highly active antiretroviral therapy (HAART), we used highly sensitive real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay in order to detect a wide dynamic range of HIV-1 RNA with lower detection limit of a single copy. The primers and probes were from the long terminal repeat (LTR) region of HIV genome for achieving higher specificity and sensitivity of detection and amplification. Our results demonstrate a wide variation in the concentration of HIV-1 RNA in different brain regions (5.51 and 8,144,073; log(10) 0.74 and 6.91 copies/g tissue), and despite the high specificity and sensitivity of this method, viral RNA was not detected in 50% of all the samples, and in 30% to 64% of samples of each region of HIV-1+ individuals. However, the highest concentration of viral RNA was found in the caudate nucleus and the lowest concentration in the frontal cortex and cerebrospinal fluid. The viral RNA was undetectable in all samples of HIV-negative individuals.

Presenilin-1 detection in brain neurons and FOXp3 in peripheral blood mononuclear cells: normalizer gene selection for real time reverse transcriptase pcr using the DeltaDeltaCt method

Quantification of gene expression is important to confirm changes in levels of gene expression in disease. Prior quantification methods include standard curves, absolute quantification, and relative quantification. This paper describes an analytic method for the relative quantification of Presenilin-1 (PS-1) in neurons and Forkhead-box (FOX) p3 in PBMNCs using real-time PCR analytic techniques. A comparative Ct method (deltadelatCt) is described in which the quantity of target normalized to a normalizer gene reference is given by 2-deltadelatCt where deltadelatCt = [Ct of the gene of interest in the unknown specimen - Ct normalizer gene in the unknown specimen] - [Ct of the gene of interest in the calibrator specimen - Ct normalizer gene in the calibrator specimen]. The calibrator specimen is ideally from a non-treated control specimen and is analyzed on every assay plate with the unknown specimens of interest. The use of the deltadelatCt methodology allows for a higher throughput and a more economical approach to investigate gene expression. We applied this methodology to the quantification of PS-1 and FOXp3 genes and compare the levels of expression by normalizing to different normalizer genes using the deltadelatCt methodology. We find that use of GAPDH is the optimum normalizer gene for the genes analyzed in neurons from human brain and in PBMNCs.