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Transcriptional expression of a gene or genes is absolutely required for induction of glucocorticoid-induced thymocyte apoptosis. We have previously shown that expression of T cell death-associated gene 8 (TDAG8) is quickly induced exclusively in the thymus after dexamethasone (DEX) treatment. Here, we present data that TDAG8 expression is induced prior to induction of DEX-mediated apoptosis. In contrast, TDAG8 expression in thymocytes was not induced in the process of gamma-irradiation-mediated apoptosis. TDAG8 expression accelerated only DEX-induced, but not TCR-mediated or gamma-irradiation-induced, thymocyte apoptosis in transgenic mice overexpressing TDAG8. Interestingly, these effects were specifically detected in CD4(+)CD8(+) double-positive thymocytes. Moreover, activation of caspase-3, -8 and -9 was enhanced in thymocytes of TDAG8 transgenic mice after DEX stimulation. In conclusion, TDAG8 expression is involved in glucocorticoid-induced signals to activate caspase-9, -8 and -3 for subsequent apoptosis induction in CD4(+)CD8(+) double-positive thymocytes.

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How melanosomal proteins such as enzymic proteins (tyrosinase and tyrosinase-related proteins, Tyrps) and structural protein (gp100) are transported from Golgi to melanosomal compartments is not yet fully understood. A number of small GTPases have been found to be associated with melanosomes and we have identified one of them, Rab7, a regulator of vesicular transport, organelle motility, phospholipid signaling and cytosolic degradative machinery, as being involved in the transport of Tyrp1 from Golgi to stage I melanosomes. This study further characterizes the role of Rab7 as a regulator of differential sorting of melanosomal proteins in this process. Murine melanocytes were transiently transfected with a plasmid encoding either wild-type (Rab7WT), constitutively active (Rab7Q67L) or dominant-negative (Rab7N125I and Rab7T22N) Rab7. Through immunocytostaining and confocal laser scanning microscopy, we quantitatively compared the bio-distribution of melanosomal proteins between Rab7WT-expressing cells and mutant Rab7-expressing cells. We also characterized their differential elimination from melanosomal compartments by Rab7 by utilizing a proteasome inhibitor, MG132. Our findings indicate that Rab7 plays an important role in differential sorting of tyrosinase, Tyrp1 and gp100 in early melanogenesis cascade, and that it is more specifically involved with Tyrp1 than tyrosinase and gp100 in the trafficking from Golgi to melanosomes and the specific exit from the degradative process.

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ABSTRACT. MRL/lpr mouse is an established animal model which develops autoimmune diseases including glomerulonephritis, sialoadenitis, hepatitis and inflammatory lung disease. Additionally, it has been reported that lpr strains uniquely accumulate CD3 + CD4 -CD8 -B220 + (double negative, DN) T cells in lymphoid organs leading to lymphadenopathy and splenomegaly. To investigate the role of CD28/ CTLA4-B7 pathway in the development of lymphadenopathy and splenomegaly, MRL/lpr mice were treated with soluble form of CTLA4 molecules, CTLA4IgG, which efficiently blocks this pathway. It was demonstrated that (i) the development of DN T cells was independent of the CD28/CTLA4-B7 pathway, (ii) the CD28/CTLA4-B7 pathway was required for the development of lymphadenopathy and splenomegaly, (iii) the CD28/CTLA4-B7 pathway was important for the accumulation of various cell populations in the lymph node and spleen, (iv) composition of the accumulating cell populations was not altered by CTLA4IgG treatment, and (v) activation of conventional T cells and IL-4 production from conventional T cells were the CD28/CTLA4-B7 pathway dependent. Thus, we concluded that the CD28/ CTLA4-B7 pathway was required for the development of full-blown lymphadenopathy and splenomegaly in MRL/lpr mice.-KEY WORDS: costimulatory signal, CTLA4IgG, IL-4, lymphadenopathy, MRL/lpr.

Serologic monitoring of infectious diseases is important for microbial control in colonies of laboratory mice. Rapid and simple tests that do not require killing animals are valuable for this purpose. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to mouse hepatitis virus (MHV), Sendai virus (also known as hemagglutinating virus of Japan [HVJ]), and Clostridium piliforme (The pathogen that causes Tyzzer disease), which are major infectious diseases in mice. For this assay, an ICA strip was put into a microtube containing 150 μL PBS and either 0.75 μL mouse serum or 1.5 μL whole blood. Binding antibodies were visualized by using protein A-conjugated colloidal gold. Under these conditions, multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. To evaluate the sensitivity and specificity of multiplex ICA, positive serum samples for each infectious disease were used. Sensitivities of the multiplex ICA test for MHV, HVJ, and C. piliforme were 100%, 100%, and 90%, respectively. No nonspecific reaction was observed in any of the 30 positive sera. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA test. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid, simple, and safe serologic testing of laboratory mice.