Antithrombin III treatment improves parameters of acute inflammation in a highly histoincompatible model of rat lung allograft rejection

BACKGROUND: Antithrombin III (AT-III) is an antithrombotic agent with known anti-inflammatory properties that is also known to attenuate acute inflammation, prevent ischemia-reperfusion injury, and disseminated intravascular coagulation (DIC) associated with sepsis and endotoxemia. Here, we examined the ability of AT-III to modify parameters of acute inflammation in a highly histoincompatible model of rat lung allograft rejection (AR). METHODS: After left single lung transplantations (BN-->Lew), recipient animals were treated i.v. with 50 U/kg of human AT-III (low dose group), 500 U/kg of human AT-III (high dose group), or normal saline (control group) on days 2 and 4 posttransplant. All animals were sacrificed on day 6, and several pathological categories of acute inflammation related to AR were scored (0-4). The effect of AT-III on concanavalin A (Con A)-stimulated rat spleen cell proliferation was also examined. RESULTS: The stage of AR, and the degrees of edema, hemorrhage, and necrosis were significantly reduced in the high dose group compared with the control group. AT-III significantly inhibited rat spleen cell proliferation in response to Con A, in a dose-dependent manner. Maximal inhibition was seen at 15 U/ml in culture. Identical inhibition of Con-A-stimulated cultures occurred in both serum free and serum-containing media, indicating that AT-III inhibition of Con-A-stimulated rat spleen cell proliferation is independent of its actions on thrombin. CONCLUSIONS: 1) AT-III treatment significantly improves parameters of acute inflammation seen in a highly histoincompatible model of rat lung AR. 2) AT-III inhibits in vitro T cell proliferation to the potent mitogen Con A, suggesting that protease inhibition may inhibit T cell activation in vitro. 3). The beneficial effects of AT-III on parameters of lung AR relate to the anti-coagulant, anti-inflammatory, and possibly immunoregulatory actions of AT-III.     

弓形虫感染对大鼠移植心脏存活时间的影响

目的探讨受者弓形虫感染对同种心脏移植物存活时间的影响。方法通过腹腔注射的方法建立弓形虫感染的大鼠模型,分别于感染后4~7d(急性感染组)或感染后27~32d(慢性感染组)进行同种异体颈部心脏移植,并设注射生理盐水的对照组和同系移植对照组。术后不用免疫抑制剂,观察移植心脏的存活时间以及移植物的病理变化。结果同系对照组的移植物存活时间为(135.3±30.4)d;慢性感染组移植心脏的存活时间为(73.6±49.3)d,明显长于生理盐水对照组和急性感染组(P<0.01),其移植心脏组织中的炎症细胞浸润也较对照组显著减轻,尤其是移植物存活时间超过100d者,无慢性移植物病变。结论受者的弓形虫感染能显著延长同种心脏移植物的存活时间,减轻移植物炎症反应。     

Prolongation of rat heart allograft survival with K(+)ATP-dependent channel modulators

Controversies exist regarding the immunoregulatory properties of K(+) ATP channel modulators. We investigated the effects of aprikalim, a K(+) ATP-dependent channels activator, and glibenclamide and gliclazide, two inhibitors of K(+) ATP-dependent channels, on the prolongation of heart allograft survival in the rat. Nine groups (n >/= 5) were involved in this study with the Brown-Norway to Lewis rat combination treated with aprikalim, glibenclamide, gliclazide, and/or cyclosporine. The results indicate that modulators of K(+) ATP-dependent channels can improve the survival of rat heart allograft without interfering with the immunosuppressive properties of cyclosporine.     

Prolongation of rat cardiac allograft survival by treatment with prostacyclin or aspirin during acute rejection

Hearts taken from DA (RT1a) rats were transplanted heterotopically to PVG (RT1c) rats of the same sex (day 0). On day 1 or on day 5 rats were treated with prostacyclin (PGI2), 250 ng/kg/min, by continuous infusion of alkaline solution into the inferior vena cava until the time of rejection. Controls received glycine buffer infusion alone, from day 1 or day 5. Cessation of palpable graft beat was taken as the end point of rejection. When PGI2 was infused from day 5 median graft survival time was prolonged from a control of 7.8 days to 9.3 days (P less than 0.05). When PGI2 was infused from day 1, graft survival time was prolonged from a control of 7.4 days to 8.6 days (P less than 0.05). Other groups of rats were treated from day 1 or from day 5 with aspirin (acetylsalicylic acid), 200 mg/kg/day, by 8-hourly subcutaneous injection in saline. Control groups received saline alone. When aspirin was given from day 5, graft survival time was prolonged from a control of 7.3 days to 9.5 days (P less than 0.05). When aspirin was given from day 1 graft survival time was prolonged from a control of 7.2 days to 14.9 days (P less than 0.01), and in two cases this led to very prolonged survival. Histological examination at the time of rejection showed lymphocyte and neutrophil infiltration to be much more prominent than vessel occlusion in all groups. These results imply that PGI2 and aspirin may be beneficial to graft survival in acute rejection, but this is not due to reduced occlusion of blood vessels by platelets.     

供者骨髓基质干细胞输注延长大鼠移植心存活时间的探讨

目的 探讨供者骨髓基质干细胞(MSC)输注在大鼠同种心脏移植术后的免疫调节及延长移植心存活时间的作用.方法 供者为近交系Wistar大鼠,受者为Fisher 344大鼠.处死供者后抽取其股骨和胫骨中骨髓,分离和培养MSC.通过混合淋巴细胞试验观察不同密度的MSC对异源性T淋巴细胞增殖反应的抑制作用.建立大鼠异位心脏移植模型,根据处理方式的不同,将受者分为MSC输注组和对照组,每组8只.Msc输注组:将含有2×106个MSC的林格氏液分别于术前1周、术中及术后连续3 d经尾静脉注入受者体内;对照组:用与MSC输注组相同的方法在相同时间点注入不含MSC的林格氏液.术后第5天,采用实时逆转录聚合酶链反应检测移植心组织中细胞因子的表达情况.结果 供者MSC可明显抑制异源性T淋巴细胞的增殖反应,且MsC密度越高抑制作用越强.MSC输注组Th1类细胞因子白细胞介素(IL)-1β和γ干扰素的表达要显著低于对照组;MSC输注组Th2类细胞冈子IL-4和IL-10呈高表达,而对照组基本不表达.MSC输注组移植心平均存活时间为(12.4±5.3)d,与对照组的(6.4±2.0)d比较,差异有统计学意义(P＜0.01).结论 输注供者MSC可通过改变Th1/Th2类细胞因子的平衡向Th2偏移诱导受者产生免疫调节作用,从而延长移植心存活时间.     

Viral IL-10 gene transfer prolongs rat islet allograft survival

Islet transplantation is a potential cure for diabetes. However, allotransplant rejection severely limits its clinical application. In this study, we sought to transfect rat islets with an adenoviral vector containing the viral IL-10 (vIL-10) gene and examine its efficacy in preventing graft rejection. The immunosuppressive effect of vIL-10 is reported but its efficacy is somehow debatable in transplantation model. vIL-10 transfected islets were transplanted into streptozotocin-induced diabetic rats. Blood glucose, serum vIL-10 concentration, graft histology, and graft cytokine expression were used to monitor graft function up to day 21 after transplantation. Transfected islets released a large amount of vIL-10 protein without affecting their viability and functional integrity. When we transplanted the transfected islets into allogeneic hosts, the survival of grafted islets was not significantly increased. However, the combined use of vIL-10 and subtherapeutic doses of CsA (cyclosporine) significantly prolonged graft survival beyond that achieved with either agent alone (p < 0.001). vIL-10 and CsA-treated rats contain high level of vIL-10 in serum, which is evidenced by the inhibition of allogeneic mixed lymphocyte reaction (MLR). Histological analysis additionally revealed the presence of viable islets up to 21 days. IL-10 mRNA expression in grafted liver was higher and IFN-gamma mRNA was lower in vIL-10 and CsA-treated animals, compared with other groups. The synergistic effect of this combination therapy is potentially correlated with the induction of inhibitory cytokine secretion and downregulation of proinflammatory cytokine secretion from host cells.     

Prolongation of heart allograft survival of rats treated by a Th2 inhibitor

In terms of Th1/Th2 balance in response to signals given during donor antigen presentation, induction of tolerance is more often correlated with Th2-type than with Th1-type reactions. However, in our study, heart allograft survival was prolonged by treatment of rats with a Th2 inhibitor. Suplatast tosilate (IPD; Taiho; Tokyo, Japan) is a novel immunoregulator that suppresses IgE production and eosinophil infiltration through selective inhibition of interleukin (IL)-4 and IL-5 synthesis by Th2-like cells but not IFN-gamma production in Th1 cells. Five LEW rats of DA heart grafts were treated with IPD (100 microg/day, p.o.) for 10 days. Heart allograft survival of all IPD-treated cases was prolonged more than 14 days while the beating of heart grafts in control groups was stopped within 9 days. In an in vitro study, the cell proliferation both in Con A blast and in mixed lymphocyte reaction assay was suppressed by IPD in dose-dependent manner. We could at least in part conclude that Th2 inhibition might temporarily suppress heart allograft rejection.     

Prolongation of renal allograft survival in the rat treated with amniotic fluid.

To assess the role of amniotic fluid (AMF) in the maintenance of pregnancy, immunosuppressive effects of AMF were studied in vivo, and the mechanisms of suppressor activity were analyzed immunologically in vitro in the rat. Female Lewis (LEW, RT-1l) rats mated with Brown-Norway (BN, RT-1n) rats for 14 days were sacrificed and cell-free AMF was obtained. AMF was diafiltered with PBS (PH 7.2) and reconstituted to 2 OD units measured at 280 nm. Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.2 days (n = 10). Five days of intravenous inoculation of AMF into LEW hosts remarkably enhanced BN graft survivals (MST = 20.3 +/- 4.4 days, n = 12) compared with controls (P less than 0.01), and slightly prolonged third-party DA (RT-1a) graft survivals (MST = 9.4 +/- 0.8 days, n = 7) compared with control LEW hosts engrafted with a DA kidney (MST = 7.6 +/- 0.2 days, n = 6). Five days of intravenous inoculation of pregnant sera into LEW hosts had no effect on BN graft survival. The AMF suppressed the proliferative response of LEW lymphocytes against not only irradiated BN stimulator cells but also irradiated third-party DA stimulators. The AMF also suppressed allokiller T cell generation of normal LEW lymphocytes against BN cells by 70.1% and 51.3%, and against DA cells by 64.9% and 38.9% at concentrations of 25% and 12.5%, respectively (P less than 0.01). To dissect the immunosuppressive activity of AMF, the effect of AMF on cytokine production and interleukin 2 (IL-2) receptor expression of concanavalin A-stimulated lymphocytes were investigated. AMF suppressed interferon and IL-2 production. Interestingly, however, AMF did not suppress interleukin 3 (IL-3) and interleukin 6 (IL-6) production, as well as IL-2 receptor expression. These results demonstrated that rat AMF displayed a strong immunosuppression in vivo as well as in vitro, and that AMF might play an important role in the maintenance of pregnancy.     

Presensitization accelerates chronic allograft rejection in a heterotopic rat tracheal allograft model with immunosuppression

BACKGROUND: In the field of organ transplantation, the effect of pretransplant humoral allosensitization on the organ transplant outcome has been highlighted. To clarify the correlation of presensitization with chronic rejection that can lead to a poor prognosis in transplant recipients, we examined humoral alloreactivity and allograft rejection in sensitized recipients by using a rat heterotopic tracheal transplant model. METHODS: An MHC fully incompatible combination strain was used in this study. Lewis (LEW) rats sensitized by transplantation with Brown Norway (BN) skin grafts received tracheal segments from BN rats in the dorsal subcutaneous pouch. Four allogenic groups (n=5) were investigated. Group 1, non-sensitized recipients without cyclosporine A (CsA) administration; group 2, non-sensitized recipients with CsA administration; group 3, sensitized recipients without CsA administration; and group 4, sensitized recipients with CsA administration. In the immunosuppressant groups (groups 2 and 4), the recipients were administered a subcutaneous injection of CsA (25 mg x kg(-1) x day(-1)) for 3 days from the day of operation. All recipients were sacrificed 21 days after transplantation. Tracheal segments were extracted from the recipients and histologically evaluated with regard to the following parameters: (1) airway lining epithelial loss, (2) lymphocyte/plasma cell infiltration, and (3) luminal obliteration due to granulation tissue formation and/or fibrosis. In order to analyze alloantibody (allo-Ab) responses, sera samples were tested by the flow cytometric cross-match (FCXM) technique. RESULTS: Histological findings revealed that the chronic rejection score in sensitized recipients treated with CsA was significantly higher than that in non-sensitized recipients treated with CsA (9.0+/-1.2 vs. 3.0+/-0.54, p<0.05). In other words, CsA therapy reduced the rejection score in non-sensitized recipients, but not in sensitized recipients. No significant differences were observed in the level of IgM Abs among the groups. However, donor-specific IgG Abs were induced after presensitization by donor skin grafting prior to tracheal transplantation. Heterotopic tracheal implantation also induced IgG Ab production. This elevation in the Ab levels was inhibited by CsA treatment in non-sensitized recipients. Conversely, the Ab levels were significantly higher in sensitized recipients than in non-sensitized recipients, regardless of CsA administration. CONCLUSIONS: Our data showed that presensitization accelerates chronic allograft rejection with a marked elevation in the level of donor specific IgG Abs. These results suggest that presensitization will be a significant risk factor for the lung transplant recipient, furthermore, the effect of immunosuppression might be insufficient in sensitized recipients.     

Interleukin 2 receptor in rat heart allograft rejection

Soluble interleukin 2 receptors (S-R-IL-2) of truncated Tac chain, produced in vitro during T lymphocyte activation, may represent an in vivo marker of an alloimmune reaction. We analyzed serum S-R-IL-2 production during acute heart allograft rejection and compared soluble and membranous Tac chain (blood lymphocytes and graft invading cells) regulation during rejection. Serum S-R-IL-2 was tested in an immunoradiometric assay, with a combination of two mouse IgG1 anti-IL2-R mAbs (ART18 and OX39). Membranous Tac chain was analyzed by immunochemistry in graft tissue, and by immunofluorescence on blood and spleen leukocytes. Four experimental groups were used: untreated allogeneic, untreated syngeneic, CsA-treated (10 mg/kg/day for 15 days) allogeneic and CsA-treated syngeneic graft recipients. In the untreated allogeneic group, S-R-IL-2, tested every day until rejection (9.14 +/- 1.6 days), increased as early as day 3 after transplantation, peaked at day 6, and plateaued thereafter. The allograft was infiltrated at day 5 by Tac chain-positive cells (10% of OX1 cells and 84% of OX19 cells). A small percentage of mononucleated cells was labeled in blood, but not in spleen, by ART18 and OX39 at day 7 only. In contrast, in untreated syngeneic and CsA-treated allogeneic combinations, there was no increase of baseline S-R-IL-2 level (P less than 0.001), and graft infiltrate did not contain IL-2-R positive cells. CsA treatment prolonged heart allograft survival (41.3 +/- 2.8 days). Baseline S-R-IL-2 levels during treatment were lower than those observed in untreated animals. In the CsA-treated allogeneic group, after CsA treatment interruption, S-R-IL-2 levels significantly increased, reaching a plateau at day 37. Results suggest that S-R-IL-2 measurement can be useful for clinical diagnosis of allograft rejection.