Effect of polyamine on the regeneration of reduced glutathione (GSH) was analyzed in yeast. Spermine stimulated the reduction of oxidized glutathione in the presence of glucose 6-phosphate or isocitrate in permeabilized yeast cells. Stimulation of GSH formation was due to the enhancement of NADPH (reduced nicotinamide adenine dinucleotide phosphate) supply. Polyamine activated mitochondrial NADP (nicotinamide adenine dinucleotide phosphate)-isocitrate dehydrogenase (EC 1.1.1.42) and cytosolic glucose 6-phosphate dehydrogenase (EC 1.1.1.49) from yeast. Activating action of polyamine was largely the effect on the maximal velocity of these enzymes, and the order of effectiveness as the activators was spermine > spermidine > putrescine. The concentrations of spermine required for 50% activation of the enzymes were about 1—2 mM, values that were within the range of physiological concentrations of spermine. Polyamine can act as an antioxidant by stimulating the regeneration of reduced glutathione, the principal reactive oxygen scavenger, through the activation of cytosolic glucose 6-phosphate dehydrogenase and mitochondrial NADP-isocitrate dehydrogenase as the NADPH-generating enzymes in yeast.

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Enhanced T helper 1 (Th1)-type immunity was observed in patients with hepatitis C virus (HCV) infection during investigations on the efficiency of interferon (IFN) therapy. The mechanism for the shift to Th1-type immunity is, however, obscure in HCV infection. In this study, we examined the in vitro effect of IFN-α subtypes (IFN-α1, -α2, -α5, -α8 and -α10) on the Th1/Th2 balance in the peripheral blood mononuclear cells (PBMC) obtained from healthy control volunteers and HCV-infected patients. A two-day incubation without IFN stimulation did raise the Th1-type cell percentage but not the Th2-type cell percentage in the PBMC of the control group. The Th1-type cell percentage and Th1/Th2 ratio were significantly larger in the PBMC of patients when compared to controls both before and after treatment with the IFNs. IFN-α5 treatment induced an increase of the Th2-type cell percentage in both control and patient PBMC but did not show any significant changes in the Th1/Th2 ratio. Furthermore, IFN-α8 treatment slightly promoted an increase in the Th1/Th2 ratio only in patient PBMC. Statistical analysis revealed that effects of the IFN-α subtypes on the Th1/Th2 balance differed between two patient groups with severe liver damage (alanine aminotransferase; ALT: ≥ 80 IU/ml) and mild liver damage (ALT: ⟨ 80 IU/ml). IFN-α5 treatment lowered the Th1/Th2 ratio in patients with mild liver damage, whereas IFN- α 8 treatment raised the Th1/Th2 ratio in patients with severe liver damage without IFN-α5-induced decrease in the ratio. These findings imply that HCV infection and its disease status modify the effects of IFN-α subtypes on Th1 and Th2 immune balance in patients. Our findings should help to elucidate the mechanisms underlying IFN therapy for HCV infection.

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When gold beads coated with plasmid DNA encoding the β-galactosidase (β-gal) gene having a strong immunostimulating sequence (ISS) with CpG-motif were inoculated intradermally by gene gun, blue colored cells producing β-gal could be seen within regional lymph nodes and spleen. Electron microscopic analysis suggested that cells expressing the β-gal gene in the regional lymph nodes look like activated interdigitating dendritic cells (DC). In addition, multiple gold beads were observed in blue colored cells in the regional lymph nodes but not in the spleen where β-gal was actively produced. The DC expressing the β-gal gene may prime directly β-gal epitope peptide (TPHPARIGL)-specific, CD8⁺ cytotoxic T lymphocytes (CTL) without requiring Th1 type of help in this priming step. Moreover, HIV-1 gp120-specific CD8⁺ CTL could be primed when mice were immunized with gold beads coated with gp120 plasmid DNA together with the β-gal plasmid, though gold beads coated with HIV-1 gp120 plasmid alone or a mixture of two distinct gold beads coated with either β-gal or gp120 plasmid could not prime HIV-1-specific CTL. These results suggest that intradermal immature DC, like Langerhans cells, activated by ISS-containing plasmids encoding strong CpG-motif such as β-gal DNA, can efficiently prime CTL specific for not only original β-gal epitope but also the products of another plasmid without encoding ISS when both plasmids are captured simultaneously by the same dermal DC.

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C57BL/6 (B6) female mice were injected with 10⁷ splenocytes of B6 male mice to immunize male H-Y antigens. This stimulation induced a slight increase in the proportions of CD3^intIL-2Rβ⁺ cells, CD3^intNK1.1⁺ cells (i.e., NKT cells), and CD4⁺T cells in the liver and spleen. However, cytotoxic activity against male H-Y antigens was not detected in hepatic and splenic lymphocytes. When these in vivo primed hepatic or splenic lymphocytes were cultured in vitro with irradiated splenocytes of male mice (i.e., mixed lymphocyte reaction, MLR), potent cytotoxicity against male H-Y antigens was induced. Unprimed (in vivo) hepatic and splenic lymphocytes did not acquire such activity even by in vitro stimulation. If the primed mice were in vivo stimulated again with male lymphocytes, such cytotoxicity was not obtained. In other words, in vivo priming and in vitro culture stimulation were both required for the induction of cytotoxicity. Phenotypic characterization and cell-depletion study using normal B6 mice and NKT-deficient mice revealed that effector cells were both CD8⁺NK1.1⁻TCR^int and CD8⁺NK1.1⁺TCR^int cells. These results suggest that recognition of H-Y antigens and effector function against H-Y antigens may be events of innate immunity similar to the case of other self-related antigens.

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