Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation model.

The effects of FK506, and TAK-779, antagonists of CCR5 and CXCR3, were investigated using a rat intestinal transplantation model. Small intestines from DA rats were heterotopically transplanted into LEW rats. The recipients were treated with FK506 (1mg/kg/day, day 0-5) and TAK-779 (10mg/kg/day, day 0-10). Graft survival and immunological responses to these materials were estimated by mixed lymphocyte reactions and IFN-γ production. The expression of chemokine receptors on lymphocytes was also examined. The average duration of survival was 7.0±0.3, 12.0±1.0, 9.8±0.5 and 18.0±1.5days in the allogeneic, FK506, TAK-779 and the two-drug combined groups, respectively. Cell proliferative responses and IFN-γ production were suppressed to a significant extent in the FK506 group compared with the TAK-779 group. In addition, the two-drug combination showed a tendency for stronger suppression than FK506 alone, correlated with in vivo and histopathological data. The numbers of both CD4(+) and CD8(+) cells were significantly suppressed in the blood of the recipients of both the FK506 and the TAK-779 groups, and in Peyer's patches of the graft of the TAK-779 group, but the FK506 group was not, as evidenced by FACS analysis. In addition, double-staining of graft-infiltrating lymphocytes showed a significant reduction in lymphocyte numbers, expressing CCR5 and CXCR3 in the TAK-779 group, but not evident in the FK506 group, compared to the allogeneic group. While FK506 suppresses cell proliferation and effecter function, it has less effect on the expression of CCR5 and CXCR3 in lymphocytes. Further exploration of the effects of a combined therapy with TAK-779 could represent a novel treatment for intestinal transplantation.

Effects of blocking the chemokine receptors, CCR5 and CXCR3, With TAK-779 in a rat small intestinal transplantation model.

BACKGROUND: The effect of blocking lymphocyte chemokine receptors with TAK-779 was investigated using a rat intestinal transplantation model. METHODS: Dark Agouti rat small intestines were heterotopically transplanted into Lewis rats. The recipients were treated with TAK-779 (10 mg/kg per day) by subcutaneous injection. Graft survival, histologic changes, mixed lymphocyte reaction, and antibody production were examined. Furthermore, expression of the chemokine receptors on the graft-infiltrating lymphocytes in the mesenteric lymph node (MLN) and Peyer's patches (PP) were measured using fluorescence-activated cell sorter analysis. RESULTS: Average duration of survival was greater for group T (with TAK-779: 9.8+/-1.4) than group A (without TAK-779: 7.0+/-0.6). Histologic findings and immunohistochemistry of the graft were consistent with the prolonged survival in group T. In the fluorescence-activated cell sorter analysis, several CD4+ and CD8+ cells were significantly suppressed in the blood, spleen, and MLN of the recipient and in the PP of the graft on postoperative day (POD) 6, but recovered in recipient spleen and MLN by POD 9. However, double-staining of graft-infiltrating lymphocytes in MLN and PP showed a significant reduction in the numbers of T cells expressing CCR5 and CXCR3 by POD 9. On the other hand, mixed lymphocyte reaction and cytokine production, and the antidonor antibody titer were suppressed on POD 6 but not on POD 9. CONCLUSION: TAK779 diminished not only the number of the graft-infiltrating cells and their expression of CCR5 and CXCR3, but also the total number of recipient T cells that play a role in graft rejection. Further exploration of these effects will provide the novel treatment of the intestinal transplantation.

Analysis of the serine protease function of porcine factor I produced by liver cells for xenotransplantation.

The use of a bioartificial liver with pig liver cells in the treatment of fulminant hepatic failure has already begun as a clinical trial in several countries. Therefore, studies on plasma complement regulatory proteins of the pig are necessary, because the liver produces them. Complement factor I is a serine protease that cleaves C3b and C4b. The DNA sequences of factor I have been reported in many species, with the noted exception of pigs. In this study, porcine factor I was cloned and an open reading frame of 1794 base pairs were identified. The predicted amino acid sequence maintained a relatively high homology compared to those of other mammals, especially in the serine protease (SP) region. The cell membrane-bound forms of the porcine and the human SP domain of factor I were constructed. Amelioration of complement-mediated cell lysis by these molecules was then tested, using several kinds of sera and Chinese hamster ovary (CHO) cell transfectants. Both molecules had a suppressing effect on pig, human and dog complements, indicating little species-specificity. Further studies of other plasma complement regulatory proteins produced from pig liver cells will need to be considered as the primary feature of the pig bioartificial liver.

Studies of monkey complement: measurement of cynomolgus monkey CH50, ACH50, C4, C2 and C3.

BACKGROUND: The cynomolgus monkey is commonly used as the recipient in transplantation experiments. However, study of the complement system of cynomolgus monkeys is lacking. In the present study, the complement system of cynomolgus monkeys was compared with that of humans, by checking hemolytic titers. METHODS: Hemolytic titers of complement from cynomolgus monkeys were calculated using the same methods as are used in humans. The complement regulatory function of human decay accelerating factor (DAF, CD55) in cynomolgus monkey serum was next studied using erythrocytes from human DAF-transgenic pigs. RESULTS: The results indicated relatively high values, except for C4: CH50: 211.19 +/- 42.78 units/ml, ACH50: 51.47 +/- 12.43 units/ml, C4: 30 170 +/- 14 300 SFU/ml C2: 33831 +/- 7442 SFU/ml and C3: 93612 +/- 30131 SFU/ml. Western blot experiments using antibodies for human complement components revealed similarities between the cynomolgus monkey and human complement systems. Human DAF inhibited pig erythrocyte lysis from approximately 60-70% to 17% in both human and cynomolgus monkey sera, indicating an almost identical complement regulatory function. CONCLUSION: The hemolytic titer of cynomolgus monkeys was greater than the titer measured in human serum. However, human DAF showed nearly the same complement regulatory function in both human and cynomolgus monkey sera.