Identification of two down-regulated genes in rat liver allografts by mRNA differential display

Total RNA differential display (DD) using random primers was performed for rat orthotopic liver transplantation (OLT) models. DA (RT1^a) donor livers were transplanted into DA, PVG (RT1^c), and LEW (RT1^l) recipients: (1) syngeneic OLT (DA-DA): no rejection occurs; (2) allogeneic OLT (DA-PVG): rejection occurs, but is naturally overcome without immunosuppression; (3) allogeneic OLT (DA-LEW): animals die of acute rejection within 14 days. cDNA was isolated from selected bands, re-amplified for sequencing, and confirmed by Northern blots. Two down-regulated genes were observed in day-7 allogeneic OLT livers (DA-PVG, DA-LEW), while they were consistently expressed in day-7 syngeneic OLT (DA-DA) livers. These two genes were identified as α-glutathione sulfotransferase (α-GST) Ya gene and estrogen sulfotransferase (EST), respectively. Northern blots confirmed that their expression was down-regulated in OLT (DA-PVG) livers on days 7–26 and gradually restored. The mRNA expression of GST and EST may be good markers to predict rejection or induction of tolerance. Received: 14 December 1999 Revised: 28 September 2000 Accepted: 16 March 2001     

Cyclosporin, toxicity and efficacy in rejection of liver allografts in the rat

52 orthotopic liver transplants were performed in DA to lewis rat strain combination, in order to appreciate cyclosporine toxicity, and efficacy at doses of 10 mg/kg day (G II) and 20 mg/kg/day (GIII) compared to liver allografts in DA/lewis rats. The first signs of cyclosporine hepatotoxicity are biological (increased plasma level of bilirubine and transaminase) that were noticed at the dose of 20 mg/kg/day. Histological signs (cells inclusion, hepatocytic necrosis) appeared late and were less constant as well as difficult to assert creatinine plasma level was the best reflect of cyclosporine nephrotoxicity. Renal toxicity was practically constant at the dose of 20 mg/kg/day. In spite of renal and hepatic toxicity, cyclosporin by itself, allows the abolition of the acute rejection of liver allografts in the rat.     

Role of natural killer cells in the rejection process of corneal allografts in rats

BACKGROUND: The exact mechanism of human corneal allograft rejection, which is the major cause of corneal transplant failure, remains unclear. We investigated the role of natural killer (NK) cells in rat corneal allograft rejection by examining the aqueous humor (AH) cell infiltrate on different postoperative days. METHODS: Flow cytometric analysis was performed on the AH and submandibular draining lymph node (DLN) cells before transplantation and at different time points thereafter. In addition, we performed functional cytotoxicity assays with cells present in the AH during corneal rejection. RESULTS: We demonstrated a gradual increase in the absolute cell number of different hematopoietic subpopulations in the AH after allogeneic cornea transplantation. CD3CD4 cells, mainly monocytes and macrophages, were the predominant subpopulation 2 days after transplantation, followed by a successive relative increase of CD4 T cells, CD8 T cells, CD161 T cells, and NK cells. NK and CD161 T cells were present at a 10- to 15-fold higher percentage than in the DLN, suggestive of local expansion of these cells. A higher percentage of NK cells were CD8-negative compared with DLN NK cells. AH cells specifically lysed allogeneic cells, and this cytotoxicity was mainly attributable to NK cells but not to CD4 or CD8 T lymphocytes. CONCLUSION: These results confirm the crucial role of CD4 cells in the allogeneic corneal graft rejection process and implicate NK cells as possible mediators of the rejection.     

Alloreactivity of natural killer cells in allogeneic liver transplantation

BACKGROUND: The cytolytic attack of natural killer (NK) cells is blocked by recognition of the idiotypic phenotype of certain polymorphisms in HLA class I molecules, specifically by HLA-C alleles (Asn77, Lys80 or Ser77, Asn80) or HLA-Bw4 allotypes. Because liver allograft rejection is associated closer with mismatch in HLA class I than class II, we investigated the role of NK cells in acute hepatic allograft rejection in vivo/in vitro. METHODS: The HLA pattern was typed with serological and polymerase chain reaction (PCR) techniques. In 31 liver transplantations, mononuclear cells from donor spleen and peripheral blood of recipients (before/after transplantation) were cultured in mixed lymphocyte cultures (MLC). MLC-derived effector cells were analyzed by flow cytometry and tested in 51Cr-release assays. RESULTS: Patients with NK allospecific constellations tended to have higher numbers of NK cells in peripheral blood during the first 4 weeks after transplantation, and patients' lymphocytes stimulated with donor cells had a significantly higher cytotoxic activity on days 14 and 21 compared with patients without NK allospecificity. However, acute rejection occurred with similar frequency in both groups (31% with allospecific constellations vs. 40% without). Moreover, acute rejection episodes were not associated with an increase in NK cells in vivo or enhanced cytotoxicity of NK cells to donor target cells. CONCLUSIONS: Under standard immunosuppressive therapy, NK allospecific constellations did not seem play a major role in acute hepatic allograft rejection. Strategies to prevent or treat NK allospecific constellations after liver transplantation are not likely to reduce the incidence or severity of acute allograft rejection.     

大鼠辅助肝移植病理改变

将Ｗｉｓｔａｒ大白鼠随机发Ａ、Ｂ、Ｃ三组，分别于辅助肝移植术后２、６和１２天处死。用光镜和电镜观察供肝病理变化。结果（１）Ａ组呈肝细胞增生明显，核大、核仁大，线粒体和粗面内质网增生；（２）Ｂ组可见肝细胞脂肪变性，线粒体肿胀以及血管周转有单核细胞，淋巴细胞浸润；（３）Ｃ组肝细胞丧失伴大量坏列经和纤维组织增生。     

Natural killer T-cells participate in rejection of islet allografts in the liver of mice

A role of natural killer T (NKT) cells in transplant rejection remains unknown. Here, we determined whether NKT cells participate in rejection of islet allografts, using NKT cell-deficient mice. Survival of islet allografts in streptozotocin-induced diabetic CD1d(-/-) mice or Valpha14 NKT cell(-/-) mice was significantly prolonged without immunosuppression when grafted into the liver, but not beneath the kidney capsule, compared with wild-type mice. Acceptance of intrahepatic islet allografts was achieved in CD1d(-/-) mice by a subtherapeutic dose of rapamycin, which was abrogated in conjunction with the transfer of hepatic mononuclear cells from wild-type, but not from CD1d(-/-), mice at islet transplantation. The second islet grafts from a donor-specific, but not from a third-party, strain in CD1d(-/-) mice bearing functional islet allografts were accepted without immunosuppression at 120 days after the initial transplantation. These findings demonstrate that NKT cells play a significant role in rejection of islet allografts in the liver of mice, but that NKT cells are not essential for induction of donor-specific unresponsiveness in this model. The current study indicates that NKT cells might be considered as a target for intervention to prevent islet allograft rejection when the liver is the site of transplantation.     

Expression of CD44 in rat liver allografts during rejection

As CD44 is believed to be a homing receptor involved in lymphoid trafficking and inflammatory responses, it is expected to be closely linked to transplant rejection. In this study, the expression of CD44 during liver transplant rejection was compared with the expression of lymphocyte-function associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1), which play an essential role in cell interactions and the initiation of immune responses. Male Brown Norway (BN) and Lewis (LEW) rats were used as donors and recipients, respectively. Orthotopic liver transplantation (OLTX) was done using the cuff technique of Kamada and Calne. Animals were killed on days 3, 5, and 7 after OLTX, and a piece of tissue from each of the liver grafts was obtained. Immunohistochemical staining was used to investigate the expression of CD44, ICAM-1, and LFA-1. CD44 was strongly expressed in portal areas of the rejected liver, and LFA-1 and ICAM-1 were expressed mainly on sinusoids and hepatocytes. These findings indicate that CD44 is closely involved in lymphocyte infiltration, which is dominant in portal areas, and that lymphocyte infiltration during the rejection process may involve a homing mechanism. Received for publication on Oct. 17, 1997; accepted on Oct. 21, 1997     

Identification and characterization of a beta-adrenergic receptor in hamster Sertoli cells

Sertoli cells cultured from immature hamsters contain a beta-adrenergic receptor which is coupled to the cAMP second messenger system. Thus, isoproterenol, epinephrine, and norepinephrine, which act via beta-adrenergic receptors, all stimulate cAMP accumulation in Sertoli cells cultured for 4-5 days. This cAMP response to isoproterenol is inhibited stereospecifically by the beta-receptor blocker, propranolol. It is also sensitive to inhibition by beta-adrenergic antagonists in this order of potency: nonspecific beta receptor antagonists, propranolol, timolol, hydroxypindolol greater than beta 1 selective antagonists, oxyprenolol, metoprolol much much greater than beta 2 selective antagonist, butoxamine. Butoxamine was at least 1000-fold less sensitive than either the nonspecific or the beta 1 selective antagonists at inhibiting the response of either isoproterenol (nonspecific), dobutamine (beta 1 selective) or zinterol (beta 2 selective). The hamster Sertoli cell beta receptor is, therefore, predominantly of the B1 subtype. This beta receptor mediated increase in cAMP is sensitive to homologous desensitization and is stimulated synergistically by forskolin. In addition, Seroli cells freshly isolated from immature hamsters contain an active beta receptor. However, this beta receptor mediated increase in cAMP is dependent on the type of trypsin used in the cell preparation. In agreement with Kierszenbaum et al (1985), freshly isolated Sertoli cells from immature rats never responded to the catecholamines regardless of the type of trypsin used; indicating an important physiologic difference between rat and hamster Sertoli cells.     

Sirolimus inhibits lymphangiogenesis in rat renal allografts,a novel mechanism to prevent chronic kidney allograft injury

Lymphangiogenesis occurs in renal allografts and it may be involved in the maintenance of the alloreactive immune response and thus participate in the development of chronic kidney allograft injury. Sirolimus (SRL) has been shown to inhibit lymphangiogenesis. The aim of this study was to describe lymphangiogenesis and its regulation during the development of chronic kidney allograft injury and to investigate the effect of SRL on allograft lymphangiogenesis and chronic kidney allograft injury. A rat renal transplantation model was used. Allografts treated with cyclosporine A or with SRL were analyzed in various time points. Syngenic transplantations were used as controls. Kidney function was followed with serum creatinine. Histology was analyzed by Chronic Allograft Damage Index (CADI). Immunohistochemistry was used to detect lymphatic vessels, VEGF‐C and VEGFR‐3. In cyclosporine‐treated allografts VEGF‐C/VEGFR‐3 pathway was strongly upregulated leading to extensive lymphangiogenesis 60 days after transplantation. Lymphangiogenesis correlated positively with the CADI score. Sirolimus efficiently inhibited lymphangiogenesis, improved graft function and attenuated the development of chronic kidney allograft injury when compared with cyclosporine. In conclusion, lymphangiogenesis is associated with chronic kidney allograft injury and SRL is a potent inhibitor of lymphangiogenesis in renal allografts. Inhibition of lymphatic proliferation could mediate the nephroprotective properties of SRL.