Interferon gamma (IFN-γ) negative CD4+ and CD8+ T-cells can produce immune mediators in response to viral antigens.

Evaluation of antigen-specific T-cell responses to viral antigens is frequently performed on IFN-γ secreting cells. However, T-cells are capable of producing many more functions than just IFN-γ, some of which, like Perforin, are associated with immune protection in HIV-1 disease elite controllers. We evaluated the extent of missed T-cell functions when IFN-γ secretion is used as a surrogate marker for further evaluation of T-cell functions. Intracellular cytokine staining assay and flow cytometry were used to assess peripheral blood mononuclear cells (PBMCs) from 31 HIV-infected ART-naive individuals for the extent to which gated CD4+ and CD8+ IFN-γ producing and non-producing T-cells also secreted IL-2, Perforin, and TNF-α functions. Similarly, the extent of missed virus-specific responses in IFN-γ ELISpot assay negative T-cells from 5 HIV-1 uninfected individuals was evaluated. Cells from HIV-infected individuals were stimulated with pooled consensus group M (Con M) peptides; and those from healthy individuals were stimulated with pooled adenovirus (Ad) peptides. Overall, frequencies of virus-specific IFN-γ secreting CD4+ and CD8+ cells were low. Proportions of IFN-γ negative CD4+ expressing IL-2, Perforin, or TNF-α to Con M were significantly higher (5 of 7 functional profiles) than the corresponding IFN-γ positive CD4+ (0 of 7) T-cell phenotype, p = 0.02; Fisher's Exact test. Likewise, proportions of CD8+ T-cells expressing other functions were significantly higher in 4 of the 7 IFN-γ negative CD8+ T-cells. Notably, newly stimulated Perforin, identified as Perforin co-expression with IL-2 or TNF-α, was significantly higher in IFN-γ negative CD8+ T-cell than in the positive CD8+ T-cells. Using SEB, lower responses in IFN-γ positive cells were most associated with CD4+ than CD8+ T-cells. These findings suggest that studies evaluating immunogenicity in response to HIV and Adenovirus viral antigens should not only evaluate T-cell responsiveness among IFN-γ producing cells but also among those T-cells that do not express IFN-γ

BIOCHEMICAL STUDIES ON CHLORPROMAZINE: 1. THE EFFECT OF CHLORPROMAZINE ON RESPIRATORY ACTIVITY OF ISOLATED RAT BRAIN CORTEX

Chlorpromazine exerts a progressive inhibitory activity (at 0.3–0.6 mM) on the respiration of brain cortex in presence of either glucose, fructose, pyruvate, or L-glutamate. A similar progressive inhibition occurs with other phenothiazine derivatives such as methylene blue and phenergan. However, chlorpromazine does not inhibit oxygen uptake in the presence of succinate. Potassium-stimulated respiration is highly sensitive to chlorpromazine, as it is markedly diminished by 0.2 mM concentration of the drug, a concentration which does not affect the unstimulated respiration. The increased inhibition of potassium-stimulated respiration is only clearly seen during the early part of the experiment.Chlorpromazine is bound by tissue constituents. At a constant concentration of chlorpromazine (0.6 mM), its inhibitory effect on cortical respiration may be abolished by markedly increasing the amount of tissue present. The inhibitory effect of chlorpromazine may be diminished by addition of plasma proteins (αβ-globulin) or by addition of heated homogenized brain, liver, or kidney. No binding occurs with polyglutamic acid, ribonucleic, and deoxyribonucleic acids, but binding does occur with certain acid dyes such as trypan red. Trypan red may be used to immobilize free chlorpromazine. When the latter drug is absorbed, however, by the nervous tissue, the addition of trypan red has no effect on the metabolic inhibitions brought about by the absorbed chlorpromazine.It is concluded that chlorpromazine resembles a large variety of narcotics and anaesthetics in its marked inhibitory effects on potassium-stimulated respiration of the brain. Its action, in vitro, however, differs from that of the narcotics in bringing about progressive, apparently irreversible, inhibitions and in its high binding power with tissue proteins. Such apparently irreversible inhibition is consistent with the conclusion that the drug, after combination with the tissue, gradually diffuses into the cell bringing about metabolic inhibitions.</jats:p

BIOCHEMICAL STUDIES ON CHLORPROMAZINE: 2. EFFECTS OF CHLORPROMAZINE ON INCORPORATION INTO PROTEINS, AND BREAKDOWN OF GLYCINE-1-C¹⁴BY ISOLATED RAT BRAIN CORTEX

Glycine is decomposed in rat brain cortex to yield carbon dioxide. This process, in which C14O2is formed from glycine-1-C14, is markedly stimulated by the presence of 10 mM glucose, the rate of production of C14O2being increased at least threefold. The presence of succinate exercises a much smaller stimulation of C14O2formation. The addition of KCl (0.1 M) or of 2,4-dmitrophenol (0.025 mM), whilst stimulating the rate of oxygen uptake, does not increase the rate of C14O2formation from glycine-1-C14. The addition of K+tends to diminish the rate. The process of glycine-1-C14breakdown to C14O2is almost insensitive to chlorpromazine, under the given experimental conditions, until relatively high concentrations (e.g. 0.6 mM) are used. The presence of chlorpromazine, however, brings about an inhibition of the rate of glycine-1-C14incorporation into rat brain cortex proteins, an inhibition of 20% being recorded at a concentration of the drug (0.2 mM) that has little or no effect on the respiration of the brain or on the rate of breakdown of glycine-1-C14into C14O2. Glycine incorporation into brain cortex proteins is a process relatively sensitive to chlorpromazine, the magnitude of inhibition being of the same order as that brought about by amytal at similar concentrations. It is suggested that chlorpromazine brings about its effects by an uncoupling of phosphorylation from oxidation in brain cortex slices.</jats:p

BIOCHEMICAL STUDIES ON CHLORPROMAZINE: 1. THE EFFECT OF CHLORPROMAZINE ON RESPIRATORY ACTIVITY OF ISOLATED RAT BRAIN CORTEX

Chlorpromazine exerts a progressive inhibitory activity (at 0.3–0.6 mM) on the respiration of brain cortex in presence of either glucose, fructose, pyruvate, or L-glutamate. A similar progressive inhibition occurs with other phenothiazine derivatives such as methylene blue and phenergan. However, chlorpromazine does not inhibit oxygen uptake in the presence of succinate. Potassium-stimulated respiration is highly sensitive to chlorpromazine, as it is markedly diminished by 0.2 mM concentration of the drug, a concentration which does not affect the unstimulated respiration. The increased inhibition of potassium-stimulated respiration is only clearly seen during the early part of the experiment.Chlorpromazine is bound by tissue constituents. At a constant concentration of chlorpromazine (0.6 mM), its inhibitory effect on cortical respiration may be abolished by markedly increasing the amount of tissue present. The inhibitory effect of chlorpromazine may be diminished by addition of plasma proteins (αβ-globulin) or by addition of heated homogenized brain, liver, or kidney. No binding occurs with polyglutamic acid, ribonucleic, and deoxyribonucleic acids, but binding does occur with certain acid dyes such as trypan red. Trypan red may be used to immobilize free chlorpromazine. When the latter drug is absorbed, however, by the nervous tissue, the addition of trypan red has no effect on the metabolic inhibitions brought about by the absorbed chlorpromazine.It is concluded that chlorpromazine resembles a large variety of narcotics and anaesthetics in its marked inhibitory effects on potassium-stimulated respiration of the brain. Its action, in vitro, however, differs from that of the narcotics in bringing about progressive, apparently irreversible, inhibitions and in its high binding power with tissue proteins. Such apparently irreversible inhibition is consistent with the conclusion that the drug, after combination with the tissue, gradually diffuses into the cell bringing about metabolic inhibitions.</jats:p