Background-A novel immunomodulator, KRP-203, the molecular structure of which has some similarity to FTY720, has been developed for use in organ transplantation. The present study was designed to investigate the potency and safety of KRP-203 on allograft survival against both acute and chronic rejection in rat skin and heart transplantation. Methods and Results-KRP-203 significantly prolonged skin or heart allograft survival of a minor histocompatibility complex (mHC)-disparate (LEW to F344) rat combination. Histopathological and immunohistochemical analysis at 100 days after mHC-disparate rat heart transplantation revealed that KRP-203 treatment significantly inhibited infiltration of inflammatory cells, including macrophages and T cells; expression of endothelin-1 and transforming growth factor-␤ 1 ; and IgG deposition and eventually attenuated neointimal formation and myocardial fibrosis. KRP-203 also prolonged heart allograft survival in a major histocompatibility complex (MHC)-incompatible (DA to LEW) rat combination, but the efficacy was not as significant. However, KRP-203 combined with a subtherapeutic dose of cyclosporin A synergistically prolonged the heart allograft survival. Flow cytometric analysis demonstrated that KRP-203 reduced the number of peripheral blood mononuclear cells (lymphocytes and monocytes) but not granulocytes and enhanced lymphocyte homing into peripheral lymph nodes. The influence of KRP-203 on heart rate changes in Hartley guinea pigs was examined. KRP-203 had less of a tendency to cause bradycardia than FTY720. Conclusions-These findings demonstrated that KRP-203 prolonged skin and heart allograft survival and significantly attenuated chronic rejection and bradycardia as an adverse effect. Therefore, KRP-203 offers considerable potential as a novel therapeutic immunosuppressant in patients with organ transplantation. (Circulation. 2005;111:222-229.)

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Current treatments for patients with Crohn's disease (CD) are based on recent advances in elucidating the pathophysiology of the disease. A satisfactory therapeutic strategy has not been well established. A new sphingosine 1-phosphate (S1P) receptor agonist, 2-amino-2-propanediol hydrochloride (KRP-203), has been developed for immunomodulation in autoimmune diseases and organ transplantation. We aimed to evaluate the efficacy and potency of KRP-203 on the treatment of chronic colitis in an interleukin (IL)-10 gene-deficient (IL-10

Mouse CD38 has been implicated in the regulation of both B-cell proliferation and protection of B cells from irradiation-induced apoptosis. CD38 ligation on B cells by CS/2, an anti-mouse CD38 monoclonal antibody, induced proliferation, IgM secretion, and tyrosine phosphorylation of Bruton tyrosine kinase in B cells from wild-type mice. B CD38 is a type II transmembrane glycoprotein and is an ectoenzyme that possesses both ADP-ribosyl cyclase and cADP-ribosyl hydrolase activities, which generate cADPribose and ADP-ribose from NADI (1-4). CD38 has been implicated in the regulation of both B-cell proliferation and rescue of B cells from apoptosis (5, 6). A role for CD38 in regulation of B-cell activation has been suggested by analysis using agonistic monoclonal antibody (mAb) to mouse CD38, which mitogenically stimulates resting B cells (5, 6) and induces tyrosine phosphorylation of cellular proteins (7). We have reported that ligation of CD38 by CS/2, an agonistic mAb against mouse CD38, also demonstrated mitogenic activity on resting B cells (8). At present, the molecular mechanisms underlying CD38-mediated activation of B cells are poorly understood.Interleukin 5 (IL-5) is a cytokine that stimulates proliferation and differentiation of B cells, eosinophils, and basophils (9, 10). IL-5 signals through the IL-5 receptor (IL-SR) complex, which is composed of an a chain (IL-5Ra) and a 13chain (11-13). IL-5Ra specifically binds IL-5 and forms with the ( chain, which cannot bind IL-5 by itself, a high-affinity receptor complex indispensable for IL-5 signal transduction (12-17).The (3 chain is shared among receptors for IL-5, IL-3, and granulocyte/macrophage colony-stimulating factor (GM-CSF) (14,18,19). IL-5 induces rapid tyrosine phosphorylation of IL-SR (3 chain, phosphatidylinositol 3-kinase, Shc, Vav, and HS1 and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.activates B-cell-specific nonreceptor-type Bruton tyrosine (Btk) and JAK2 kinases (20,21 . It is not clear whether aberrant expression of Btk in Xid mice is involved in this impaired B-cell responsiveness. In this study, we have examined the role of Btk activation in signaling through CD38 ligation and the synergistic effect of CD38 and IL-5 on IgM secretion. We show that Btk is tyrosine phosphorylated after CD38 cross-linking. Moreover, CD38 ligation with IL-5 costimulation enhances B-cell proliferation, Blimpl (B-lymphocyte-induced maturation protein) gene expression, and IgM production. MATERIALS AND METHODSReagents. Mouse recombinant IL-5 was prepared and purified using anti-mIL-5 mAb coupled beads as described (11). Rat anti-mouse CD38 mAb, CS/2, and anti-mouse IL-SRa mAb, H7, were prepared as described (8,32). Rat anti-mouse Btk polyclonal antibodies was prepared by immunizing rats with glutathione S-transferase-Btk fusion protein (33). Antiphosphotyrosine mAb, 4G10, was obtained...

CD38 is an ectoenzyme that possesses both ADP ribosyl cyclase and cADP ribosyl hydrolase activities, which generate cADP ribose and ADP ribose from NAD ϩ (1-4). A role for CD38 in regulation of B cell activation has been suggested by analysis using agonistic mAb to mouse CD38 (5-9). We have reported that CD38 ligation on B cells by anti-mouse CD38 mAb induced proliferation, IgM secretion, and tyrosine phosphorylation of Bruton tyrosine kinase (Btk) in B cells from wild-type mice. B cells from X chromosome-linked immunodeficient (Xid) mice did not respond at all to anti-CD38 mAb although CD38 expression on these B cells was comparable to that on wild-type B cells (8, 9). We infer from these results that Btk activity is involved in B cell triggering after cross-linkage of CD38.Interleukin 5 (IL-5) induces proliferation and differentiation of B cells, eosinophils, and basophils (10, 11). IL-5 signals through the IL-5 receptor (IL-5R) complex, which is comprised of an ␣ chain (IL-5R␣) and a ␤ chain (12-18). Analysis of the synergistic effects of various cytokines with CD38 ligation of B cell activation revealed that IL-5 showed the most potent effect on B cell proliferation, Blimp-1 gene expression, and IgM production (9). Although neither CD38 nor IL-5R carries the catalytic domain for protein tyrosine kinases, CD38 ligation induces activation of Btk (9) Lyn and Fyn are expressed in a broad range of cell types and tissues (27). Lyn has been shown to be physically associated with a number of hematopoietic cell surface receptors, including the B cell antigen receptor (28-30), CD40 (31), the lipopolysaccharide (LPS) receptor (32), the high affinity Fc RI complex (33), the granulocyte-macrophage colonystimulating factor receptor (34), and IL-5R (11, 35). Furthermore, Lyn and Fyn were shown to be associated with Btk (36). Lyn is most likely the predominant src family kinase involved in activation of Btk in B cells (37). It is not clear, however, whether Fyn and Lyn are involved in the signaling cascade of CD38 ligation. In this study, using fyn-deficient (Fyn Ϫ͞Ϫ ), lyn-deficient (Lyn Ϫ͞Ϫ ), and fyn͞lyn double-deficient (Fyn͞ Lyn Ϫ͞Ϫ ) mice, we have examined the role of Fyn and Lyn in signaling through CD38 ligation and in the synergistic effect of CD38 ligation and IL-5 on B cell triggering. We describe that CD38 ligation and IL-5 induce IgG1 secretion, which is impaired in B cells from Lyn Ϫ͞Ϫ and Fyn͞Lyn Ϫ͞Ϫ mice.