C. Masuda

Involvement of Th1 cells and heat shock protein 60 in the pathogenesis of intestinal Behcet's disease.

Involvement of excessive Th1 cell functions and heat shock protein expression in the pathogenesis of Behçets disease (BD) has been reported. In this study we have characterized immune responses in intestinal lesions of BD. Peripheral blood lymphocytes (PBL) of BD and healthy controls (HC) and tissue specimens of intestinal Behçets disease (intestinal BD), Crohns disease (CD) and ulcerative colitis (UC) were analysed for mRNA and protein expression by reverse transcriptase‐polymerase chain reaction (PCR) and immunohistochemistry, respectively. PBL of BD patients expressed the Th1‐related chemokine receptor, CCR5 and CXCR3 preferentially compared with those of healthy controls. Intestinal lesions of BD expressed interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐12 mRNA, indicating Th1 skewed responses in vivo. mRNA of Txk, a Tec family tyrosine kinase specific to Th1 cells, was expressed in the lesions, suggesting its contribution to the Th1‐dominant responses. In the intestinal samples, CCR5 was detected in all the cases with BD, whereas Th2‐related CCR3 and CCR4 were detected randomly, mainly in the cases with inactive BD and those receiving large amounts of prednisolone, indicating the Th1‐dominant immune responses in the intestinal lesions. As the ligands of CCR5, MIP1α and MIP1β were detected, whereas RANTES was not. Heat shock protein (HSP) 60 was expressed in PBL and intestinal tissues of BD. Th1‐dominant immune responses and HSP60 expression may induce the inflammatory responses and thus be associated with the pathogenesis of intestinal BD.

Excessive expression of Txk, a member of the Tec family of tyrosine kinases, contributes to excessive Th1 cytokine production by T lymphocytes in patients with Behcet's disease

Excessive Th1 cell function is importantly involved in the pathogenesis of Behcets disease (BD). We previously found that Txk, a member of the Tec family of tyrosine kinases, acts as a Th1 cell specific transcription factor. To investigate immune aberration in the pathogenesis of BD, we studied the expression of Txk and Th1 cytokines in peripheral blood lymphocytes (PBL) and skin lesions in patients with BD. Cytokine production by the lymphocytes was assessed using ELISA. PBL produced excessive Th1 associated cytokines including IFN‐γ and IL‐12 spontaneously and in response to exogenous HSP60‐derived peptide stimulation, which was shown to induce proliferation of PBL, in patients with BD. Circulating CD4+ T cells expressed excessive Txk protein. A majority of cells infiltrating into skin lesions expressed IFN‐γ in the BD specimens. IL‐12 and IL‐18 were also expressed in the mononuclear cell aggregates. Lymphocytes accumulating in the skin lesion expressed higher levels of Txk as compared with atopic dermatitis lesions, a typical Th2 disease. IFN‐γ, IL‐18 and Il‐12 were detected in the BD skin lesions, which may induce preferential development of Th1 cells in patients with BD. The mononuclear cell aggregates contained Txk expressing cells in such skin lesions. Collectively, Txk expressing Th1 cells and the Th1 associated cytokines may play a critical role in the development of skin lesions in BD.

Transplantation of myocyte precursors derived from embryonic stem cells transfected with IGFII gene in a mouse model of muscle injury.

Background. Reconstruction of skeletal muscle tissue is hampered by the lack of availability of functional substitution of the tissue. Methods. Embryonic stem (ES) cells were transfected with the insulin-like growth factor (IGF) II gene and were selected with G418. The resultant cell clones were analyzed regarding their myogenic differentiation in vitro and in vivo. Results. The cells expressed early and late myogenic differentiation markers, including myoD, myogenin, and dystrophin in vitro. They had phosphorylated Akt within the cells, suggesting their activation by the secreted IGFII. Transplantation of the cells to injured anterior tibial muscle of mice significantly improved their motor functions compared to injured mice transplanted with undifferentiated ES cells and injured mice given vehicle alone. The transfected cells adapted to the injured muscle, formed myofibers positive for dystrophin and negative for MyoD and myogenin. Trichrome staining and toluidine blue staining support myofiber formation in vivo. The enzymatic activity of acetylcholine esterase suggested the functional activity of the regenerated motor units. The evoked electromyogram of anterior tibial muscle transplanted with the transfected cells showed significantly higher potentials compared to that transplanted with undifferentiated ES cells and that injected with phosphate-buffered saline (control injury). Electron microscopic examination confirmed the myofiber formation in the cells in vivo. Conclusions. Transfection of IGFII gene into ES cells may be applicable for transplantation therapy of muscle damage due to injury and myopathies.

Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells

Purpose: Corneal epithelial stem cells are deficient in cases of limbal disorders, leading to conjunctival epithelial ingrowth, vascularization, and eventually visual disturbance. We introduced the eye development-associated transcription factor pax6 to embryonic stem (ES) cells and tested whether pax6-transfected cells resembling purified corneal epithelial cells were applicable as a cell source for corneal transplantation. Methods: pax6 cDNA with green fluorescence protein was electrotransfected to ES cells and the cells were cultured with G418 for 14 days. They were characterized by reverse transcription-polymerase chain reaction and immunohistochemistry. The cells were transplanted onto experimentally damaged mouse corneas. Histologic reconstitution of the corneal epithelium was assessed. Results: pax6-transfected cells formed a monolayer of epithelium-like cells in vitro. They expressed cytokeratin12, a specific keratin of corneal epithelial cells, E-cadherin, and CD44, which are important adhesion molecules of corneal epithelial cells on the cell membrane. They accumulated to make a colony that gave a staining pattern of reticular configuration for cytokeratin 12, E-cadherin, and CD44. When the cells were transplanted onto damaged cornea, they have been kept alive on the cornea. Conclusions: The purified corneal epithelium-like cells derived from ES cells transfected with pax6 gene adapted to the injured cornea and were kept alive on it. These results suggested application of ES cell-derived corneal epithelial cells for treating corneal injuries.