Background-Although immunosuppressive therapy for myocarditis has attracted a great deal of attention, its effectiveness is controversial. Interleukin (IL)-10 has a variety of immunomodulatory properties. Among the nonviral techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive, and safe. Methods and Results-We examined the applicability of murine IL-10 (mIL-10) gene transfer to the treatment of rats with experimental autoimmune myocarditis. Nine-week-old Lewis rats were inoculated with pig myosin (day 0). A plasmid vector expressing mIL-10 cDNA (800 g per rat) was transferred into the tibialis anterior muscles by electroporation 3 times (5 days before immunization and at days 4 and 13); control rats received empty plasmid. Electroporation increased the serum mIL-10 levels to Ͼ250 pg/mL. The 21-day survival rate in rats treated with mIL-10 cDNA was higher (15 of 15; 100%) than that of the control group (9 of 15; 60% In a rat model in which myocarditis was induced by purified cardiac myosin, T cells were reported to play an important role in inducing myocarditis. [3][4][5] Immune dysfunction associated with autoimmune disease may be related to an imbalance between T helper type 1 and 2 cells. 6 The T helper type 2-associated cytokine interleukin (IL)-10 has a variety of immunomodulatory properties, including the inhibition of T helper type 1 cells and the production of proinflammatory cytokines. 7,8 Recent reports have suggested that the immunosuppressive effects associated with IL-10 are effective in suppressing the rejection of transplanted organs and immune complex diseases, and clinical trials of IL-10 have been performed in patients with these disorders. 9,10 Electroporation has been widely used to introduce DNA into various types of cells in vitro. Gene transfer by electroporation in vivo has been effective for introducing DNA into mouse skin, chick embryos, rat liver, and murine melanoma and muscle. [11][12][13] We previously showed that gene transfer into muscles by electroporation in vivo can be used to deliver cytokines systemically. 13,14 In the present study, we applied this method for the delivery of IL-10 in a rat model of autoimmune myocarditis. Methods AnimalsNine-week-old male Lewis rats were injected with the antigenadjuvant emulsion in their foot pads according to the procedure described previously. [3][4][5] The morbidity of experimental autoimmune myocarditis was 100% in rats immunized by this method. [3][4][5] Throughout the studies, all animals were treated in accordance with the guidelines for animal experiments of our institute. 5 Construction of Mouse IL-10 Expression VectorMouse IL-10 (mIL-10) cDNA cloned by polymerase chain reaction was inserted into the unique Xho I site between the cytomegalovirus immediate early enhancer-chicken ␤-actin hybrid promoter and rabbit ␤-globin poly A site of the pCAGGS expression plasmid. 13,14 The resulting plasmid, pCAGGS-IL-10, was grown in Escherichia coli DH 5␣ and prepared using plasmid purifica...

Production of blood cells is regulated by the interplay of various cytokines and bone marrow stromal cells. Recently, a ligand for the orphan receptor Mpl was identified as thrombopoietin (TPO), which specifically regulates megakaryocyte differentiation, and it was reported to be expressed mainly in liver and kidney. As it was found that thrombopoietin is also produced in bone marrow stromal cells, we studied further the roles of bone marrow stromal cells on megakaryocytopoiesis and platelet formation. The stromal cells stimulated growth and maturation of bone-marrow-derived megakaryocytes in the presence of thrombopoietin, and also supported growth of BaF3 cells expressing exogenous Mpl without thrombopoietin. Thrombopoietin induces drastic morphological change of megakaryocytes in bone marrow cells in vitro, ie, the formation of lengthy beaded cytoplasmic processes (proplatelet formation). However, when the purified megakaryocytes were cocultured with the stromal cells with or without thrombopoietin, most of the megakaryocytes adhered to the stromal cells and remained unchanged, while free megakaryocytes induced proplatelet formation. These observations indicated that the stromal cells in a hematopoietic microenvironment in bone marrow secrete thrombopoietin and stimulate proliferation and maturation of megakaryocytes, but the interaction of megakaryocytes with the stromal cells may suppress proplatelet formation.

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ABSTRACT-Recent progress in analyzing the structures and functions of G-protein coupled receptors (GPCRs) including > -adrenoceptors (>-ARs) has been made by pharmacological, physiological and molecular biological techniques. The three-dimensional (3D) structures, interaction sites with ligands and conformational changes of these receptor subtypes due to ligand binding are now better understood by the simulation of these receptors using computer-aided molecular modeling. Based on these techniques, numbers and conformations of amino acid sequences of each subtype (> 1-, >2-and >3-ARs) were defined and also interaction sites or modes of interaction between ligands and >-ARs could be analyzed three-dimensionally. In addition, simulation of 3D structures of > -ARs by molecular modeling could clearly determine the limited size, space or pocket for fitting with ligands. These studies will give some clues for the clarification of other GPCRs. Thus, this review summarizes current findings on chemical structures of ligands, amino acid sequences, 3D structures and important amino acids of >-AR subtypes for interacting with ligands obtained from mutagenesis, chimeric studies and molecular modeling techniques.

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Senescence-accelerated mouse (SAMP8) is known as a murine model of accelerated aging and memory dysfunction. The binding activity of [3H] 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK-11195) as a neurochemical marker of gliosis markedly increased with aging in the cerebral cortex and hippocampus of SAMP8. Immunoreactivity for glial fibrillary acidic protein (GFAP) was also enhanced. A beta-amyloid precursor protein (APP)-like immunoreactivity and 27-kDa-carboxyl terminal fragments of APP increased in SAMP8 brain. In addition, anti-APP antibody stained reactive astrocytes surrounding spongy degeneration in brain stern of SAMP8. These results suggest that astrocytosis and production of APP-derived fragments occur markedly in SAMP8 brains.