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The functional relationship between fish and mammalian thrombocytes is relatively unknown. In this study, a panel of monoclonal antibodies (mAbs) was used to investigate the functional properties of rainbow trout thrombocytes. The mAbs recognize cell-surface molecules on thrombocytes with molecular weights ranging from 17 to 160 kDa. Flow cytometric and immuno-electron microscopic analyses demonstrate that these molecules are expressed at different levels and that surface expression increased upon activation with bovine collagen. Two of these cell-surface molecules (17 and 21 kDa) were directly involved in collagen-induced aggregation of thrombocytes since aggregation was blocked upon pre-treatment with mAbs that recognize the two surface markers. Interestingly, the percentage of thrombocytes in blood increased after stimulation using different antigens. The transcriptional profile of trout thrombocytes was then examined after immuno-magnetic enrichment using the described mAbs to assess potential roles of trout thrombocytes in immune functions. Trout thrombocytes express components of the MHC class Ia pathway, IL1beta, TNFalpha, TGFbeta, the interleukin receptor common gamma chain as well as CXC and CC chemokines. MHC class IIB and TNFalpha were expressed at low levels in resting thrombocytes. No evidence was found for the expression of TCRalphabeta, Ig heavy chain, CD8alpha or CK1 mRNA. Taken together, these results suggest that rainbow trout thrombocytes express molecules involved in activation, aggregation and genes encoding proteins, that are involved in antigen presentation and immune regulation.

A monoclonal antibody against carp intestinal T cells (WCL38; of IgM class) was produced by immunization of mice with isolated membrane molecules of carp intestinal intraepithelial lymphoid cells. Flow cytometric analysis showed that WCL38 reacted with 50-70% of the lymphoid cells isolated from intestine, gills or skin, with less than 6% of lymphoid cells isolated from thymus, head kidney or spleen and with a negligible number of PBL. WCL38+ cells were abundant in the intestinal epithelium and less numerous in the lamina propria. Immunogold labelling confirmed that WCL38 reacted with lymphoid cells; in gills and skin some of them have the morphology of large granular lymphoid cells. Immunochemical analysis showed that WCL38 reacted with dimeric membrane molecule on mucosal lymphoid cells with an Mr of 76 kDa, consisting of two 38 kDa subunits. WCL 38+ lymphoid cells are postulated to T cells, since WCL38 does not react with B cells, macrophages or non-specific cytotoxic cells. In conclusion, like higher vertebrates, carp seem to have a distinct (Putative) T cell population in their mucosal tissues.

To assess the efficacy of various forms of vitamin E in protection of skin from UV-light-induced oxidative stress, vitamin E (tocotrienol-rich fraction of palm oil, TRF) was applied to mouse skin and the contents of antioxidants before and after exposure to UV-light were measured. Four polypropylene plastic rings (1 cm2) were glued onto the animals' backs, and 20 microliters 5% TRF in polyethylene glycol-400 (PEG) was applied to the skin circumscribed by two rings and 20 microliters PEG to the other two rings. After 2 h, the skin was washed and half of the sites were exposed to UV-irradiation (2.8 mW/cm2 for 29 mi: 3 MED). TRF treatment (n = 19 mice) increased mouse skin alpha-tocopherol 28 +/- 16-fold, alpha-tocotrienol 80 +/- 50-fold, gamma-tocopherol 130 +/- 108-fold, and gamma-tocotrienol 51 +/- 36-fold. A significantly higher percentage of alpha-tocopherol was present in the skin as compared with that in the applied TRF. After UV-irradiation, all vitamin E forms decreased significantly (p < .01), while a larger proportion of the vitamin E remained in PEG-treated (approximately 80%) compared with TRF-treated (approximately 40%) skin. Nonetheless, vitamin E concentrations in irradiated TRF-treated skin were significantly higher than in the nonirradiated PEG-treated (control) skin (p < .01). Thus, UV-irradiation of skin destroys its antioxidants: however, prior application of TRF to mouse skin results in preservation of vitamin E.

A monoclonal antibody against thymocytes (WCL9; of IgG1 class) was produced by immunization of mice with isolated membrane molecules of carp thymocytes. Flow cytometric and fluorescence microscopic analysis showed that WCL9 was reactive with 30-50% of thymocytes and not with lymphoid cells from blood, pronephros, spleen and intestine. Cryo-sections of thymus showed a WCL9+ and WCL9- region in 3-month-old fish and only WCL9+ cells in 1-week-old fish. Because the WCL9- region is more medulla-like, the WCL9+ cells can be considered as cortical thymocytes. The majority of WCL9+ thymocytes appeared to have a higher density (1.06-1.07 g/mL) than the WCL9- cells (1.02-1.06 g/mL). Immunogold labelling or comparison of both density fractions did not show clear ultrastructural differences between WCL9+ and WCL9- thymocytes. The WCL9- fraction could be stimulated much better with PHA than the WCL9+ fraction. Removal of adherent cells or adding adherent accessory cells did not influence this result. Immunochemical analysis showed that WCL9 reacted with a protein determinant present on two molecules (M(r): 200 and 155 kDa) under reduced and non-reduced conditions. These results, together with the absence of WCL9+ cells in other lymphoid organs, strongly suggest that WCL9 is a specific marker for early thymocytes.

To evaluate skin susceptibility to ozone (O3) and to localize possible oxidative damage within the skin layers, hairless mice were exposed to 10 ppm O3 or air (0 ppm O3) for 2 h. The mice were euthanized, the skin removed and frozen. Three skin layers (upper epidermis, lower epidermis/papillary dermis, and dermis) were separated, antioxidant concentrations (alpha-tocopherol and ascorbic acid) and the lipid peroxidation product malondialdehyde (MDA) measured. In the upper epidermis, O3 significantly depleted alpha-tocopherol (22%; p < .05) and ascorbic acid (55%; p < .01). These antioxidants were unchanged by O3 in the lower skin layers. More remarkably, MDA increased ten-fold in the upper epidermis (p < .001) and two-fold in the lower epidermis/papillary epidermis (p < .05); it was unchanged in the dermis. Thus, exposure to O3 in vivo depletes ascorbic acid and alpha-tocopherol and strongly induces lipid peroxidation in skin. High MDA concentrations measured in the upper epidermis suggest that O3 reacts directly with fatty acids on the skin surface layers. These results further suggest that chronic exposure to lower O3 concentrations found in urban smog could potentially have implications for skin health.