Anastomotic Healing in a Small Bowel Transplantation Model in the Rat

Anastomotic healing is impaired after intestinal surgery because of ischemia and reperfusion injury (IRI), which can result in intestinal leaks leading to increased mortality. The objective of this study was to determine the effects of transplant IRI and immune mechanisms on intestinal graft anastomotic healing. Orthotopic intestinal transplantations (OIT) were performed in rats. The experimental design consisted of six groups A–F (n = 5/group): A, allogeneic OIT treated with tacrolimus (1mg/kg/day); B, syngeneic OIT treated with tacrolimus; C, syngeneic OIT; D, allogeneic OIT; E, proximal and distal anastomoses performed in nontransplanted animals; F, same as in group E but treated with tacrolimus. Anastomotic bursting pressure (ABP), hydroxyproline content (HPC), and mucosal inflammatory infiltrate (MII) were determined at the anastomotic sites (proximal and distal) and compared between groups. ABP was significantly (p < 0.001) reduced in OIT groups A, B, C, and D compared to control groups E and F at both the proximal and distal anastomotic sites. HPC was 1 g/mg of tissue in groups A, B, C, and D, and 5g/mg of tissue in groups E and F. This demonstrates a significant (p < 0.001) reduction in HPC after OIT. MII was significantly (p < 0.001) increased in OIT groups when compared to nontransplanted control groups. MII was also significantly (p < 0.05) increased in allogeneic OIT groups A and D compared to syngeneic OIT groups B and C. Generally, ABP and HPC were inversely proportional to MII in both nontransplanted control and OIT groups. Reduced anastomotic strength was demonstrated in both syngeneic and allogeneic OIT anastomotic sites irrespective of immunosuppressive therapy, and is probably related to IRI.     

大网膜防止聚丙烯网片与腹腔脏器粘连的实验研究

目的:探讨大网膜防止聚丙烯网片与腹腔脏器粘连的作用及其丧失保护作用的原因。方法:1.动物实验:将雄性Wistar大鼠分为5组:(A)大网膜复盖组(n=15);(B)无大网膜复盖组(n=15);(C)大网膜切除组(n=15);(D)大网复盖 伤口葡萄球菌种植组(n=15);(E)大网膜切除组 伤口葡萄球菌种植组(n=15)。结果:D、E组动物术后死亡率明显高于A、B、C组(P<0.05)。术后1个月,D和E组粘连评分明显高于A、B、C组(P<0.05),术后2个月和3个月各组间粘连评分无显著性差别(P>0.05),但A和B组均为网膜与网片粘连,而D和E组则主要为肠管或肝脏参与粘连,C组为部分肠管或肝脏与网片粘连。结论:在无感染情况下大网膜具有防止腹腔脏器与网片粘连的作用,而感染后大网膜则失去保护作用,同样发生网片与腹腔脏器间的严重粘连。     

热休克蛋白70在大鼠胰腺移植后急性排斥反应诊断中的意义

目的　探讨热休克蛋白 (HSP) 70在大鼠胰腺移植急性排斥反应诊断中的意义。方法　建立大鼠全胰、十二指肠移植模型 ,用免疫组织化学及WesternBlotting法定量检测移植胰腺组织中HSP70的表达 ,并观察其与病理学检查的相关性。结果　同基因移植组移植胰腺HSP70的表达水平在术后无明显变化 (P >0 .0 5) ;异基因移植组移植胰腺HSP70的表达水平在术后第 3、5和 7d逐渐升高 (P <0 .0 1 ) ,而且与病理学评分之间存在着明显的正相关 (P <0 .0 1 ,r =0 .934)。结论　检测HSP70在移植胰中的表达对急性排斥反应的早期诊断有一定价值     

Patchy distribution of rejection changes in small intestinal transplantation.

The aim of this study is to determine the distribution of histological changes of rejection in small intestinal transplantation. Thirty-nine rats were randomized into two groups: group I (n = 15), syngeneic transplants and group II (n = 24), allogeneic transplants. Grafts were excised and examined on days 2, 4, and 8 after transplantation. All grafts of the syngeneic group showed normal mucosa by day 4. However, by this time, all grafts of the allogeneic group demonstrated rejection changes with a patchy distribution in the mucosa. Therefore, with a biopsy from the stoma site there is a risk of missing early rejection.     

Cryopreservation does not alter antigenic expression of aortic allografts

Cryopreserved aortic homografts are reportedly viable, but no cross-matching or immunosuppression is utilized. Alterations of the antigenic expression by cryo-preservation must be assumed. We designed a protocol to test this premise. Fisher 344 rats served as recipients in all cases. Lewis rats, a mildly disparate strain, were utilized as donors. Four cohorts of animals were utilized. Group I (N = 11) served as a "first set" control. All animals received a syngeneic skin graft. After 28 days an allogeneic skin graft was placed; rejection was seen at 10.3 +/- 0.5 days. Group II (N = 16) first received allogeneic skin grafts with a similar "first set" rejection pattern of 10.4 +/- 0.47 days. A second skin graft was placed and demonstrated an accelerated rejection response of 6.06 days +/- 0.25 days. Group III (N = 17) received two leaflets from a "fresh" Lewis heart valve inserted into a subcutaneous pouch. Allogeneic skin grafts in this group demonstrated a similar second set rejection at 7.05 +/- 0.82 days. Group IV (N = 22) also underwent implantation of heart valve leaflets, except "cryopreserved" Lewis leaflets were implanted into the subcutaneous pouch. An allogeneic skin graft was placed and demonstrated a second set rejection at 7.18 +/- 0.39 days. A one-way analysis of variance shows no significant difference in Groups III and IV, but a significant difference with respect to Group I (P less than 0.00001). Cryopreservation does not alter the antigenic expression in this model, and at present we strongly recommend that at least ABO compatibility be utilized in all patients undergoing aortic homograft implantation.     

Alteration in gastrointestinal peptide tissue levels in rejecting small bowel transplants

Gastrointestinal (GI) peptide tissue levels were measured following intestinal transplantation in rats and evaluated as a possible early marker of transplant rejection. Vascularized syngeneic and allogeneic jejunal transplants were performed in rats without immunosuppressive therapy. Serial tissue samples of transplanted intestine were obtained from each group of animals. Baseline levels of peptides were determined in nontransplanted jejunum of the same animals. Results were correlated with histology at all experimental time points. Tissue levels of gut peptides (somatostatin, vasoactive intestinal peptide and substance P) were determined by two methods--immunoperoxidase staining and radioimmunoassay. Normal levels of gut peptides in syngeneic bowel were maintained up to 1 year after transplantation. Allogeneic bowel showed a progressive decline in gut peptide concentrations simultaneously with (or preceding) histologic evidence of rejection. The monitoring of GI peptide tissue levels may prove to be a useful method of detecting small bowel transplant rejection.     

Mitomycin-C-treated peripheral blood mononuclear cells (PBMCs) prolong allograft survival in composite tissue allotransplantation

BackgroundComposite tissue allotransplantation (CTA) was introduced as a potential treatment for complex reconstructive procedures and has become a clinical reality. Hand and face transplantation, the most widely recognized forms of CTA, have intensified immunological research in this emerging field of transplantation. Mitomycin C (MMC) is an alkylating agent that suppresses allogeneic T-cell responses. MMC-treated dendritic cells/PBMCs have been shown to induce donor-specific tolerance in solid organ allograft transplantations.MethodsFully mismatched rats were used as hind limb donors [Lewis (RT1¹)] and recipients [Brown-Norway (RT1ⁿ)]. Fifty-five allogeneic hind limb transplantations were accomplished in six groups. Group A (n = 10) received donor-derived MMC-treated PBMCs on transplantation day. Group B (n = 10) rats received no immunosuppression, group C (n = 10) received FK506 and prednisolon, group D consisted in isograft transplantation without immunosuppression, group E (n = 10) received non-treated PBMCs, and group F (n = 5) received PBS without any donor-derived cells. Rejection was assessed clinically and histologically.ResultsIn group A, the survival times of the allografts were prolonged to an average of 8.0 d. Rejection was significantly delayed compared with the averages of the corresponding control groups B, E, and F (5.5, 5.9, and 5.8 d). No rejection was seen in control groups C and D.ConclusionThese results demonstrate that MMC-treated donor PBMCs significantly prolong allograft survival when administered systemically on the day of transplantation. However, the immunomodulatory effect is relatively modest with further research being required to clarify dose–effect relations, cell characteristics, and an optimized mechanism and timing for cell application.     

Short-course immunosuppression using FK506 for rat tracheal allografts

BACKGROUND: A minimizing immunosuppression after a tracheal allotransplantation is desirable. METHODS: We examined the usefulness of a short-course of immunosuppression after tracheal allotransplantation in rat. Each transplant consisting of a 5-ring segment was heterotopically implanted into the omentum. Four animals underwent a syngeneic transplantation and thus served as controls (Group A). Thirty animals underwent an allogeneic transplantation and were randomly classified into 4 groups as follows: No immunosuppression (Group B, n=6), treatment with 0.5 mg/kg of Tacrolims (FK506) (Group C, n=8), 1.0 mg/kg of FK506 (Group D, n=8), and 1.5 mg/kg of FK506 (Group E, n=8). Different doses of FK506 were administered intramuscularly for only three consecutive days after heterotopic tracheal allotransplantation. The serum levels of FK506 were then investigated 3, 7, 14, 21, and 28 days after transplantation in groups C, D, and E. All rats were killed 28 days after transplantation and then the implanted tracheae were harvested, and evaluated histologically. RESULTS: All animals survived for the protocol period. The graft morphology of Group E was significantly better than that of groups B, C, and D regarding both macro- and microscopy, and also showed the same findings as that of Group A, except for low-grade mononuclear cell infiltration. Only in Group E, the FK506 blood level was maintained at over 0.5 ng/ml, which is the lowest detectable limit in this assay, until 21 days after transplantation. CONCLUSIONS: We thus conclude that 1.5 mg/kg of FK506 which was administered for only three consecutive days after surgery may be used to maintain the morphology of tracheal allografts in rats for 28 days after transplantation.     

巨噬细胞在小肠移植急性排斥反应期的极化状态及意义

目的  探究小肠移植术后发生急性排斥反应（AR）时巨噬细胞极化状态的改变。 方法  将6只Brown Norway（BN）大鼠和24只Lewis大鼠分为假手术组（6只Lewis大鼠）、同基因组（Lewis→Lewis,供受体各6只）和异基因组（BN→Lewis,供受体各6只）。对各组大鼠术后7 d的移植肠组织进行苏木素-伊红（HE）染色和脱氧核糖核酸末端转移酶介导的 dUTP 缺口末端标记（TUNEL）法检测,观察其病理学表现和细胞凋亡情况;采用酶联免疫吸附试验（ELISA）检测血清中M1和M2型巨噬细胞极化相关细胞因子表达水平;利用免疫荧光技术检测各组移植肠组织中M1和M2型巨噬细胞表面标志物并进行共定位计数分析。 结果  HE染色和TUNEL检测结果显示假手术组与同基因组肠上皮形态结构正常,未见明显凋亡小体;异基因组大鼠术后7 d移植肠组织上皮层绒毛结构破坏严重,隐窝数量减少,凋亡小体增多,炎症细胞浸润肠壁全层,呈现中-重度AR。ELISA结果显示异基因组受体鼠血清中M1型巨噬细胞极化相关细胞因子肿瘤坏死因子（TNF）-α、干扰素（IFN）-γ和白细胞介素（IL）-12表达水平高于假手术组和同基因组,同基因组中M2型巨噬细胞极化相关细胞因子IL-10和转化生长因子（TGF）-β表达水平高于假手术组和异基因组,差异均有统计学意义（均为P<0.05）。免疫荧光结果显示异基因组移植肠组织中M1型巨噬细胞计数多于假手术组和同基因组,同基因组M2型巨噬细胞计数多于假手术组和异基因组,差异均有统计学意义（均为P<0.05）。 结论  小肠移植术后发生AR的移植物中,大量巨噬细胞浸润肠壁全层,以M1型为主并分泌大量促炎因子,调控巨噬细胞极化方向是治疗小肠移植术后AR的潜在方法。     

白细胞介素-15在心脏移植排斥反应中的表达及意义

目的 探讨白细胞介素-15（IL-15）在心脏移植排斥反应中的表达及其与排斥反应的关系。方法 采用小鼠颈部心脏移植模型，随机分为2组：同基因移植组，供、受体均为C57BL／6小鼠；异基因移植组，供、受体分别为BALB／C、C57BL／6小鼠。以pactin作内参照，分别于术后第1、3、5、7天取移植心脏，用逆转录-聚合酶链式反应（RT—PCR）法观察IL-15的表达情况。结果 随着术后天数的增加，异基因移植组IL-15表达逐渐升高，第5天达高峰（与同基因移植组相比，P〈0．01）。结论 IL-15的表达与心脏移植急性排斥反应的发生发展密切相关，可作为心脏移植急性排斥反应的监测指标，对急性排斥反应的早期诊断和移植物的预后估计具有重要的临床意义。     

Gemcitabine does not prevent acute rejection of the transplanted liver in rats

Abstract Our study was designed to determine effect of gemcitabine on acute rejection of liver in rats. Liver transplantation was performed in rats of the Dark Agouti (DA) and Lewis (LEW) strains. Recipients were divided into three groups: A, DA-to-LEW without immunosuppression; B, DA-to-LEW, treated with cyclosporine A; C, DA-to-LEW, treated with gemcitabine. Immunosuppressants were subcutaneously injected for seven consecutive days after transplantation. On day 7, blood samples and liver graft tissue specimens were harvested. Group A showed severe rejection changes (RAI 8/9); in group B no rejection changes were present (RAI 0/9), and in group C moderate rejection changes were observed (RAI 6/9). Differences were significant between B vs C and A vs C groups; P >0.05. Serum creatinine and urea levels in the gemcitabine group were significantly lower than those in the cyclosporine A group. We did not confirm gemcitabine ability to prevent liver allograft rejection.     

大鼠移植心脏的细胞凋亡及其与急性排斥反应的关系

目的 观察移植心脏的细胞凋亡现象及其与急性排斥反应的关系。方法 建立大鼠异位心脏移植模型,用ＨＥ梁色和原位末端标记（ＴＵＮＥＬ）技术检测移植心脏切片,进行排斥反应的病理分级,计算凋亡指数（ＡＩ）。结果 发生凋亡的细胞主要是心肌细胞,在各级排斥反应中均可见凋亡细胞存在,且ＡＩ与急性排斥发生的分级成正相关;各级的ＡＩ与０级（无排斥反应）比较,差异均有显著性。结论 细胞凋亡与移植心脏急性排斥反应的严重程     

Ultra‐short echo‐time magnetic resonance imaging distinguishes ischemia/reperfusion injury from acute rejection in a mouse lung transplantation model

To investigate whether lung tissue characterization by ultra‐short echo‐time (UTE) magnetic resonance imaging (MRI) allows ischemia/reperfusion injury to be distinguished from acute rejection in a mouse lung transplantation model. After orthotopic lung transplantation with 6 mice receiving syngeneic (C57Bl/6) lung transplants and 6 mice receiving allogeneic (BALB/c) transplants, they underwent postoperative imaging using three‐dimensional UTE‐MRI (echo times TE = 50–5000 μs) and conventional T2‐weighted fast spin‐echo imaging. Quantitative T2* values of lung transplant parenchyma and spin density (SD) were compared by region‐of‐interest analysis. All samples underwent histological and immunohistochemical workup. In the allogeneic group, alveolar infiltration resulting from acute organ rejection was visualized in the UTE sequences. This was reflected by the quantitative measurements of SD and T2* values with higher values in the allogeneic group compared with the syngeneic group and nontransplanted lung at the first time point (24 h postoperative: Tx allogeneic group SD: 2133.9 ± 516; Tx syngeneic group SD: 1648.61 ± 271; P = 0.004; Tx allogeneic group T2*: 1710.16 ± 644 μs, Tx syngeneic group T2*: 577.16 ± 263 μs; P = <0.001). Changes caused by acute rejection after lung transplantation can be visualized and characterized using a UTE sequence due to different relaxation properties compared with both syngeneic lung transplants and normal lung tissue.     

Increased expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in rat cardiac allografts

This study was designed to investigate the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) during chronic cardiac allograft rejection. Wistar rats were used as donors, and SD rats as recipients heterotopic cardiac transplants. Recipients pretreated with inoculation of donor splenocytes (SPC) followed by cyclophosphamide (CP) were divided into 4 groups: (A) untreated group (n = 18) without immunosuppression; (B) SPC plus CP-treated group (n = 18) that were euthanized at 15-120 days posttransplantation; (C) CsA-treated group (n = 18) euthanized at 2-3 months posttransplantation; and (D) tolerance group (n = 18) treated with SPC plus CP and monitored for at least 1 year posttransplantation. Cardiac allografts were harvested at various times for immunohistochemical studies performed to evaluate the expression of ICAM-1 and VCAM-1. Pretreatment of animals with SPC and CP induced long-term cardiac allograft survival. Immunohistochemical staining demonstrated a low level of ICAM-1 and VCAM-1 expression in cardiac allograft muscle and coronary arteries among Groups B and D. In contrast, the expressions of ICAM-1 and VCAM-1 in cardiac allografts of Groups A and C were significantly higher than those in Groups B and D. Our results suggested that the expression of ICAM-1 and VCAM-1 plays an important role during the development of chronic cardiac allograft rejection.     

The effect of selective inhibition of inducible nitric oxide synthase on cytochrome P450 after liver transplantation in a rat model

BACKGROUND: Activity levels of cytochrome P450 (CYP) provide markers for liver function and graft rejection episodes after orthotopic liver transplantation (OLT). Some in vitro studies have shown decreased CYP activation of inducible nitric oxide synthase (iNOS) in rejecting liver grafts. The aim of this study was to evaluate CYP isoenzyme activity changes in vivo and to examine histopathologic aspects during inhibition of iNOS after treatment with aminoguanidine (AG) using OLT in the rat. MATERIALS AND METHODS: Thirty DA-(RT1av1) rats that served as donors and LEWIS-(RT(1)) rats as recipients were divided into three groups: group I (controls, syngeneic rats; n = 6), group II (allogeneic rats without immunosupression; n = 11), and group III (allogeneic rats with AG treatment; n = 13). On postoperative days 5, 8, and 10 we performed laboratory investigations and liver biopsies for histopathologic investigations. RESULTS: On postoperative day 5, activities of CYP-1A1 and -3A4 were significantly lower (P = .022) in group III and the activity of CYP-1A2 higher (P < .05) compared with group II. At postoperative days 8 and 10, the activities of all CYP isoenzymes were significant higher in AG-treated rats (group III) in contrast with group II after allogeneic OLT without immunosuppression. Histopathologic findings revealed less distinct rejection signs in group III specimens after AG treatment compared with group II. CONCLUSION: Summarizing our results, we concluded that AG treatment led to increased CYP activity and less distinction of graft rejection after OLT in rats.     

Leakage of intraluminal low molecular weight polyethylene glycol as a marker of small bowel transplant rejection

To facilitate early detection of small bowel allograft rejection, we correlated transluminal leakage of low molecular weight polyethylene glycol (PEG) with the development of allograft rejection. Vascularized allogeneic and syngeneic jejunal transplants were performed in rats, without immunosuppression. A control group underwent creation of jejunal Thiry-Vella fistulas of similar length. Jejunal segments were perfused with a physiologic solution containing [3H]-PEG-900. At the end of an equilibrium period, an urinary bladder aspirate was collected and [3H]-PEG-900 measured by scintillation counting. Results are expressed as disintegrations per minute per 100 microL urine. Histologic examinations were performed at all experimental time points. Two days following transplantation, urinary PEG levels were elevated in both allogeneic and syngeneic groups (3943 +/- 935 and 4007 +/- 1164, respectively). Four days after the transplant, syngeneic urine PEG levels decreased to 581 +/- 159, and were not significantly different (P greater than .05) from Thiry-Vella controls (635 +/- 145). Syngeneic levels remained at this low level for the rest of the experiment. The allogeneic group continued to show significantly higher levels (P less than .05) compared with syngeneic and Thiry-Vella groups from day 4 until the end of the experiment. These elevated levels most likely represented the development of rejection, preceding the first significant histologic signs of rejection, which were found at six days post-transplant. Detection of transluminal leakage of low molecular weight PEG may be a useful adjunct in monitoring for small bowel transplant rejection.     

Intestinal transplantation with alemtuzumab (Campath-1H) induction for adult patients

BACKGROUND: Alemtuzumab (Campath-1H [C1H]) is a humanized monoclonal antibody directed against the CD 52 antigen that is present on the surface of T cells, B cells, natural killer cells and monocytes. We studied its application in intestinal transplantation. METHODS: This is a retrospective review of adult patients who underwent intestinal transplantation between December 1994 and May 2005. Group 1: non-C1H group (n = 39); group 2: C1H group (n = 37). C1H was administered as an induction immunosuppression in four doses (0.3 mg/kg), or in two doses (30 mg/kg). Tacrolimus levels were maintained at low level (5-10 ng/dL). No maintenance steroids were given. RESULTS: One-year survival of group 1 and group 2 patients were 57% and 70%, respectively. This difference is not statistically significant. Of 37 patients in group 2, 21 are alive. The incidence of rejection was lower in group 2 (P < .005). Average current tacrolimus level is 6.97 +/- 3.98 ng/dL. Seventeen patients (81%) are steroid free, and 15 (71%) are maintained solely on tacrolimus. There was no graft versus host disease in group 2. CONCLUSIONS: Our preliminary data suggest that C1H can provide effective immunosuppression for intestinal transplantation. Incidence of rejection was less with this regimen using low maintenance tacrolimus and minimal steroids.     

Gemcitabine does not prevent acute rejection of the transplanted liver in rats

Our study was designed to determine effect of gemcitabine on acute rejection of liver in rats. Liver transplantation was performed in rats of the Dark Agouti (DA) and Lewis (LEW) strains. Recipients were divided into three groups: A, DA-to-LEW without immunosuppression; B, DA-to-LEW, treated with cyclosporine A; C, DA-to-LEW, treated with gemcitabine. Immunosuppressants were subcutaneously injected for seven consecutive days after transplantation. On day 7, blood samples and liver graft tissue specimens were harvested. Group A showed severe rejection changes (RAI 8/9); in group B no rejection changes were present (RAI 0/9), and in group C moderate rejection changes were observed (RAI 6/9). Differences were significant between B vs C and A vs C groups; P<0.05. Serum creatinine and urea levels in the gemcitabine group were significantly lower than those in the cyclosporine A group. We did not confirm gemcitabine ability to prevent liver allograft rejection.     

不同免疫治疗方案诱导大鼠原位肝移植免疫耐受的实验研究

目的 观察抗CD-40L单抗加小剂量CsA联合免疫治疗对肝移植大鼠受体免疫耐受诱导的作用.方法 在建立稳定大鼠肝移植模型的基础上,将肝移植模型分为5组.A组为SD→SD对照组;B组为SD→Wistar对照组,A,B组术后不用任何治疗措施;C组为SD→Wistar,术后用CsA1～5 d;D组为SD→Wistar,术后用CsA 1～5 d加抗CD-40L(CD-154)单抗0～2d;E组为D组+术前供体特异性输血(DSBT).观察受体存活时间、移植肝病理改变以及术后外周血中细胞因子的变化.结果 A,D,E组受体大鼠存活时点(均>60 d)均明显长于B组和C组.D,E组移植肝急性排斥反应明显减轻.B组IL-2和IFN-γ的血清水平显著高于其余各组(P＜0.05).B,C,D,E 4组IL-4和IL-10较A组均有明显增加,尤其D,E组的IL-10表达较B组显著增高(P＜0.05). 结论 抗CD-40L单抗加小剂量CsA(伴或不伴DSBT)联合免疫治疗,可有效延长肝移植大鼠受体生存时间、减轻急性排斥反应并诱导Th2类细胞因子的高水平表达,有助于受体和移植肝的长期存活.