Site differences of Toll-like receptor expression in the mucous epithelium of rat small intestine.

Toll-like receptors (TLRs) are known to recognize pathogen-associated molecular patterns and might function as receptors to detect microbes. In this study, the distribution of TLR-2, -4 and -9 were immunohistochemically investigated in the rat small intestine. As a result, TLR-2 was detected in the striated borders of villous columnar epithelial cells throughout the small intestine, except for the apices of a small number of intestinal villi. TLR-4 and -9 were detected in the striated borders of the villous columnar epithelial cells only in the duodenum. TLR-4-immunopositive minute granules were found in the apical cytoplasms of epithelial cells, subepithelial spaces and blood capillary lumina. TLR-2 and -4 were detected in the striated borders of undifferentiated epithelial cells and in the luminal substances of the intestinal crypts throughout the small intestine, but TLR-9 was not detected in the crypts throughout the small intestine. Only TLR-4 was detected in the secretory granules of Paneth cells in both the jejunal and ileal intestinal crypts. These findings suggest that duodenal TLRs might monitor indigenous bacteria proliferation in the upper alimentary tract, that TLR-2 might also monitor the proliferation of colonized indigenous bacteria throughout the small intestine, that the lack of TLR-2 at the villous apices might contribute to the settlement of indigenous bacteria, and that TLR-2 and -4 are secreted from intestinal crypts.

Immunohistochemical study of the apoptosis process in epidermal epithelial cells of rats under a physiological condition.

Epidermal homeostasis is maintained by both epithelial proliferation in the stratum basale (SB) and the apoptosis of epithelial cells under physiological conditions. In this study, the induction and regulation mechanisms of epidermal apoptosis were immunohistochemically investigated in the epidermis from Wistar rat's palm and foot pad by using several apoptotic related proteins under a physiological condition. The results showed that Fas and Fas-L were expressed in cellular membranes of the stratum spinosum (SS), whereas TNF-R1 did not show any membranous expression in any epidermal layers. TNF-α was not observed in the epidermis. Caspase-10, cleaved caspase-3 and DNase-1 were found in the epithelial cytoplasms from the SS to stratum granulosum (SG), whereas caspase-8 was not detected in the epidermis. XIAP and Bak were found in the cytoplasm from the SS to SG, and the intensity of Bak-positivity was stronger in the SG than the SS, whereas Bid, Apaf-1 and cleaved caspase-9 were restricted in the SG. Homogenous cytoplasmic immunoreactivity of Bcl-2 was found in the SB and the intensity was gradually decreased from the SB to the SG. The granular-cytoplasmic immunopositivity of cytochrome C gradually altered into homogenous cytoplasmic expression in the upper half of the SG. Single-stranded DNA was rarely detected in the upper portion of the SG. These results suggest that epidermal apoptosis is induced by the interaction between Fas and Fas-L and the activation of caspase-10, and might initially proceed through a mitochondrial-independent pathway, and that a mitochondrial-dependent pathway finally accelerated under physiological conditions.

Tumor rejection antigens on BALB3T3 cells transformed by activated oncogenes.

The clonal expression of tumor rejection Ag (TRA) was analyzed on nine different clones derived from parental BALB3T3 cells that were transfected with activated H-ras, polyoma middle T (PyMT), c-myc, and v-src oncogenes. It was shown that Bras-h clone, which is an activated H-ras oncogene-induced transformant, expressed TRA as assessed in the transplantation study using syngeneic BALB/c mice. This TRA was not detected on parental BALB3T3 nontransformed cells, suggesting that TRA could be expressed in the BALB3T3 cell transformation. Furthermore, the cross-protection experiments indicated that this TRA was also conferred on other BALB3T3 transformants with high anchorage-independent growth potential such as an activated H-ras transformant Bras-d, and PyMT transformants BMT-f, BnMT-11, BnMT-20, except in the case of one H-ras transformant Bnr-12. In contrast, this TRA was not expressed on the transfectants with little or no anchorage independent growth potential such as a PyMT transfectant BnMT-4, a c-myc transfectant Bmyc-7, and a v-src transfectant Bsrc-7. We developed the mAb BRH19 that could react with TRA+ clones but not with TRA- clones. This mAb makes an immunoprecipitate, which is composed of a 50-kDa single polypeptide chain from Bras-h cell lysate. An injection to mice with this antigen could confer the protection against Bras-h challenge. These data indicate that the 50-kDa putative TRA molecule could be expressed in close association with the cell transformation, irrespective of the introduced oncogenes, and there may exist some regulatory mechanisms rather than individually distinct manners for the expression of TRA.

Development of monoclonal antibodies reacting against mycobacterial 65 kDa heat shock protein by using recombinant truncated products.

A mycobacterial 65 kDa molecule is a member of the GroEL heat shock protein family. We developed mAbs reacting against recombinant 65 kDa protein by using a gene (pTB12) which encodes this protein. Three mAbs (B20, B97 and B167) reacted selectively with 65 kDa proteins of Mycobacterium tuberculosis, BCG and Mycobacterium leprae, although B20 and B167 may weakly react with a 15 kDa molecule of mammalian cells. One (B108) was obviously cross-reactive between mycobacterial 65 kDa and the mammalian intracytoplasmic protein. We also developed deletion mutants of pTB12. The localization of these mAb-defined epitopes was determined by using truncated proteins of the Mycobacterium tuberculosis 65 kDa molecule produced in E. coli. Immunohistochemical analysis showed that B20, B97 and B167 mAbs could detect this antigen in experimental granulomas induced by injection of BCG in the subcutaneous tissue of rats. These mAbs should be useful for analyzing the immunobiologic roles of mycobacterial 65 kDa molecules.

HDL-C Concentrations in Weight-Trained, Endurance-Trained, and Sedentary Females.

In brief: Serum high-density lipoprotein cholesterol (HDL-C) levels and percent HDL-C were significantly higher in nine female endurance runners than in equal groups of female weight trainers and sedentary female controls. Weight trainers and controls showed no significant differences in HDL-C and percent HDL-C. Subjects who had higher HDL-C levels were more likely to be non-smokers who consumed little alcohol and did not use oral contraceptives. No significant dose-response relationships were found for either runners or weight trainers when daily training duration, weekly training frequency, and weekly mileage were correlated with HDL-C. It was concluded that HDL-C levels in females are associated with specific training methods.