The purpose of this study was to investigate the behavior of L929 fibroblasts on substrates of different pore sizes and to find an appropriate pore size that allows them to differentiate into type 1 collagen synthesizing cells. The behavior of cultured fibroblasts on Millipore filters of 4 different pore sizes (1.2 μm, 3.0 μm, 5.0 μm and 8.0 μm) was compared morphologically. The expression of type 1 collagen mRNA was also analyzed by RT-PCR. The cell length was the greatest in the control group (without a filter) compared to the experimental groups cultured on filters at 3 days, however there were no significant differences among any of the groups at 7 days. At the ultrastructural level, small and fine cell processes had invaded the pores of filter membranes with pore sizes of 1.2 μm or 3.0 μm, while long and thick cell processes containing many organelles (some of them degenerated) had invaded the pores of filter membranes with pore sizes of 5.0 μm or 8 μm. The expression of type 1 collagen mRNA was the highest in cells cultured on membranes with 1.2 μm pores at 3 days but was not significantly changed among any of the groups at 7 days. This suggests that Millipore filters with a 1.2 μm pore size are the most suitable to allow L929 fibroblasts to express the phenotype for type 1 collagen synthesis at early time periods.
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 18.104.22.168) and cytosolic glucose 6-phosphate dehydrogenase (EC 22.214.171.124) 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.
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.
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.
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 CD3intIL-2Rβ+ cells, CD3intNK1.1+ 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−TCRint and CD8+NK1.1+TCRint 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.
Using spectrophotometric assay, we have studied the distribution of α-D-mannosidase, β-D-galactosidase, and N-acetyl-β-D-glucosaminidase in hog serum, the aqueous humor, optic nerve, retina, and uvea (iris, ciliary body, choroid). N-acetyl-β-D-glucosaminidase was the most active gycosidase in all tissues that we studied. The highest activity of α-D-mannosidase was found in the extracts prepared from the iris, while β-D-galactosidase and N-acetyl-β-D-glucosaminidase were the most active in the extracts of the choroid and ciliary body, respectively. The aqueous humor and serum had several times lower specific activity of glycosidases than did the extracts of ocular tissues or optic nerve. There was no significant difference between the activity of these enzymes in the aqueous humor and serum. High activity of glycosidases in the optic nerve suggests their role for intrinsic axonal repair. Retinal extracts had the smallest glycosidase activity of all ocular extracts prepared. The physiological role of these findings is discussed.
The purpose of this study was to further evaluate the effects of diode laser irradiation on the surface of hydroxyapatite (HA). For laser irradiation of the HA plate, the distance from the HA plate to the tip of the probe was 50 mm, and the irradiation time was 60 seconds at a power output of 10 watts with a continuous wave. Laser-irradiated HA plates were smoother and the ridges were larger than non-laser irradiated HA. Laser irradiation has a tendency to change zeta potential, and has a possibility to control the adsorption of bacteria.