Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane.

Matriptase and prostasin, acting as a tightly coupled proteolytic cascade, were reported to be required for epidermal barrier formation in mouse skin. Here we show that, in human skin, matriptase and prostasin are expressed with an inverse pattern over the course of differentiation. Matriptase was detected primarily in epidermal basal keratinocytes and the basaloid cells in the outer root sheath of hair follicles and the sebaceous gland, where prostasin was not detected. In contrast, prostasin was detected primarily in differentiated cells in the epidermal granular layer, the inner root sheath of hair follicles, and the sebaceous gland, where matriptase expression is negligible. While co-expressed in the middle stage of differentiation, prostasin was detected as polarized patches, and matriptase at intercellular junctions. Targeting to different subcellular localizations is also observed in HaCaT human keratinocytes, in which matriptase was detected primarily at intercellular junctions, and prostasin primarily on membrane protrusion. Furthermore, upon induction of zymogen activation, free active prostasin remains cell-associated and free active matriptase is rapidly shed into the extracellular milieu. Our data suggest that matriptase and prostasin likely function as independent entities in human skin rather than as a tightly coupled proteolytic cascade as observed in mouse skin.

[The effect of phosphoinositide-3-kinase and signal transducer and activator of transcription-6 on the proliferation of T lymphocytes in bronchial asthma].

OBJECTIVE: To investigate the effect of phosphoinositide-3-kinase (PI(3)K) and signal transducer and activator of transcription-6 (STAT(6)) on the proliferation of CD(4)(+) and CD(8)(+) T lymphocytes in bronchial asthma. METHODS: CD(4)(+) and CD(8)(+) T lymphocytes of 15 asthmatic patients or 15 healthy control subjects were cultured in vitro from August of 2004 to February of 2005. The T lymphocytes of asthmatic patients were divided into four groups and stimulated with or without 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) and IFN-gamma. The four groups included a control group (group A), the LY294002 group (group B), IFN-gamma group (group C) and LY294002 + IFN-gamma group (group D). The cell cycle phases and the expression of cell cycle proteins (P27kip1, Cyclin D, Cyclin E), PI(3)K and STAT(6) were analyzed by flow cytometry. RESULTS: (1) The percentage of G(0)/G(1) phase, the expression rate of P27kip1 in CD(4)(+) and CD(8)(+) T lymphocytes from asthmatic patients were 82.0%, 44.6%, 13.2%, 4.5%; and those from the control group were 99.0%, 100.0%, 47.2%, 46.3%, respectively; the difference was significant between the two groups (Z value were 3.54, 4.23, 3.09 and 2.51, all P < 0.05). The percentage of S phase, the expression rate of Cyclin E, PI(3)K and STAT(6) in CD(4)(+) and CD(8)(+) T lymphocytes from the asthmatic patients were 18.0%, 51.7%, 7.9%, 9.3%, 7.6%, 8.7%, 8.2%, 6.3%; and those from the control group were 0.2%, 0.0%, 3.7%, 3.5%, 3.3%, 3.4%, 1.9%, 2.4%, respectively; there were significant differences between the two groups (Z value were 2.88, 4.61, 1.95, 2.06, 2.51, 2.32, 4.38 and 2.22, all P < 0.05). (2) The percentage of G(0)/G(1) phase, S phase, the expression rate of Cyclin D, Cyclin E in CD(4)(+) T lymphocytes of B group were 95.6%, 1.9%, 13.3% and 3.1%; and those of A group were 82.0%, 18.0%, 35.0%, 7.9%; there were significant differences between the two groups (Z value were 2.04, 2.23, 2.78 and 1.99, all P < 0.05). The percentage of S phase, the expression rate of Cyclin E in CD(8)(+) T lymphocytes of B group were 1.0% and 4.1%; and those of A group were 51.7% and 9.3%; there were significant differences between the two groups (Z value were 3.06 and 2.56, all P < 0.05). The percentage of G(0)/G(1) phase, S phase, the expression rate of P27kip1 in CD(4)(+) T lymphocyte of C group were 94.0%, 1.5%, 46.1%; and those of A group were 82.0%, 18.0%, 13.2%; there were significant differences between the two groups (Z value were 2.17, 2.54 and 2.81, all P < 0.05). The percentage of S phase, the expression rate of P27kip1 in CD(8)(+) T lymphocyte of C group were 10.8% and 23.1%; and those of A group were 51.7% and 4.5%; there were significant differences between the two groups (Z value were 2.67 and 2.05, all P < 0.05). The percentage of G(0)/G(1) phase, S phase, the expression rate of P27kip1, Cyclin D in CD(4)(+) T lymphocytes of D group were 97.0%, 0.0%, 40.4%, 21.5%; and those of A group were 82.0%, 18.0%, 13.2%, 35.0%; there were significant differences between the two groups (Z value were 2.73, 2.79, 2.56 and 2.10, all P < 0.05). The percentage of S phase, the expression rate of P27kip1 in CD(8)(+) T lymphocytes of D group were 92.1% and 1.7%; and those of A group were 44.6% and 51.7%; there were significant differences between the two groups (Z were 2.22 and 3.12, all P < 0.05). CONCLUSION: PI(3)K and STAT(6) may be new therapeutic targets for the treatment of asthma. Further studies are necessary to explore the relationship between PI(3)K and JAK1-STAT(6) signal pathway during the enhancement of CD(4)(+) T or CD(8)(+) T lymphocyte proliferation in bronchial asthma.

[Application of immunomagnetic screening strategy for separation of CD4+ and CD8+ T cell subpopulations of peripheral blood].

To evaluate the separation of T lymphocyte subsets by immunomagnetic beads and to find optimization of strategy for specific binding of antibody-coated beads to cells, two strategies to isolate enriched T lymphocyte subpopulation CD4+ T cells and CD8+ T cells from small volumes (< 5 ml) of peripheral blood by using immunomagnetic beads or complement cytotoxicity method were compared. The purity and activity of CD4+ T cells and CD8+ T cells were measured by using flow cytometry, trypan-blue dye exclusion test, etc. The results showed that the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using immunomagnetic beads were (94.2 +/- 1.4)% and (93.8 +/- 3.0)% respectively, higher than those of control group and the group of using completement cytotoxicity method (P < 0.05). At the same time, the yields of CD4+ T lymphocytes and CD8+ T lymphocytes by using complement cytotoxicity method were (76.0 +/- 2.8)% and (77.0 +/- 3.0)% respectively, higher than those of unenriched group (P < 0.05). The trypan-blue dye exclusion test confirmed that there were no influences on activity of CD4+ T cells and CD8+ T cells when immunomagnetic beads were used for separation of these cells from peripheral blood. It is concluded that the immunomagnetic bead method has a higher efficiency for separation of CD4+ T cells and CD8+ T cells from peripheral blood than complement cytotoxic method, especially for small sample. This method has no influence on activity and proliferation of T lymphocyte subpopulations, and would be expected to establish conditions for research of biological characteristics of CD4+ T cells and CD8+ T cells in future.