Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation: effect of donor-specific cell augmentation

INTRODUCTION: Immunomodulation by portal vein delivery of donor antigen reduces intestinal graft rejection. We investigated the impact of portal venous donor-specific cell augmentation (blood versus bone marrow) on cytokine expression in intestinal grafts versus native livers. METHODS: Ten groups of intestinal transplants (brown Norway male to Lewis female rats) varied by (1). the type of donor-specific cell augmentation and (2). the use and dose of tacrolimus-based immunosuppression. Tissue samples for histologic analysis and cytokine mRNA analysis were obtained at designated time points. RESULTS: Without immunosuppression, no type of cell augmentation reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood transfusion (versus bone marrow infusion). Irrespective of the type of cell augmentation, severe rejection caused strong intragraft expression of IL-1alpha, IL-1beta, IFN-gamma, and TNF-alpha; liver expression mainly involved TNF-alpha. Of note, nonimmunosuppressed, cell-augmented rats showed hardly any differences in cytokine expression in their grafts versus significant increases in their native livers. With immunosuppression, bone marrow infusion (versus blood transfusion) increased intragraft cytokine expression of IL-1alpha, IL-1beta, IFN-gamma, as well as TNF-alpha, and liver expression of IL-1beta. CONCLUSIONS: (1). Rejection and donor-specific cell augmentation independently caused differences in intragraft versus native liver cytokine expression after intestinal transplants. (2). Portal donor-specific blood transfusion (versus bone marrow infusion) lowered the incidence of rejection and diminished intragraft cytokine up-regulation. (3). In our study, TNF-alpha appeared to be the cytokine most strongly associated with rejection.     

Role of MLC serum inhibitory factors in high MLC-reactive kidney transplant recipients pretreated with donor-specific blood transfusion (DST)

In order to clarify the beneficial effect of donor-specific blood transfusions (DST) on kidney allograft survival, sera from 16 patients treated with DST were studied using the mixed lymphocyte culture (MLC) serum inhibition test. The results demonstrate that MLC inhibitory factors could be induced in the serum of the recipients after the completion of DST, and that these factors are directed against cells of the recipient but not against cell from the donor. Regarding the correlation with rejection episodes and clinical outcome, a significant improvement in renal transplant survival and reduction in rejection episodes was observed when MLC inhibitory factors were present in post-DST sera. These data suggest that such factors may contain antibodies directed against recognition sites on T lymphocytes, e.g., antiidiotypic antibodies, and the associated with prolonged graft survival of living-related, high MLC-reactive one-haplotype-mismatched kidney.     

The effect of donor-specific transfusion and cyclosporin A on small bowel transplantation in the rat

Pre-transplant blood transfusions are given as a means of desensitization to reduce the required dose of cyclosporin A (CsA). In this study, the effect of pretransplant blood transfusion on host survival and T-cell function against alloantigen were investigated. Male Lewis rats (RT1¹) were used as the recipients in all experiments, and male DA rats (RT1^a) were used as the blood and small bowel donors, and as a source of allogeneic stimulator cells. Male BUF rats (RT1^b) were used as donors of third party blood, and of allo-stimulator cells in a delayed-type hypersensitivity (DTH) response. In our experimental design, Lewis rats were divided into the following groups according to the type of administration: (1) a donor-specific blood transfusion (DST) 8 days preoperatively and a concurrent 5-day course of CsA at 10 mg/kg per day; (2) a nonspecific third party blood transfusion (NST) and CsA at 10 mg/kg per day from day 8 to day 4 preoperatively; (3) CsA alone from day 8 to day 4 preoperatively; (4) DST alone 8 days preoperatively; or (5) no treatment, being the control group. Postoperative treatment consisted of CsA at 2.5 mg/kg per day for 30 days. Rats conditioned with NST plus CsA, CsA alone, DST alone, and the untreated control rats survived for 7.2 ±1.2, 9.0 ± 2.2, 6.8 ± 0.4, and 7.4 ± 1.6 days, respectively. In contrast, the five rats conditioned with DST plus CsA survived for 100 days or more. This study demonstrates that long-term survival of a small bowel allograft can be achieved by host-conditioning with a combined treatment of DST and low-dose CsA.This paper was presented at the 10th Congress of the Asian Association of Pediatric Surgeons held in Seoul, Korea from March 26–29, 1990, and at the 90th Annual Meeting of the Japanese Surgical Society held in Sapporo, Japan in 1990.     

Use of donor-specific red blood cell transfusions for patients undergoing liver transplantation during the COVID-19 pandemic

BackgroundIncreased risks have been found for patients undergoing liver transplantation due to the blood supply shortage following the ongoing coronavirus disease 2019 (COVID-19) pandemic. Hence, exploring a method to alleviate this dilemma is urgent. This phase I, nonrandomized, prospective trial aimed to evaluate the safety and feasibility of using donor-specific red blood cell transfusion (DRBCT) as an urgent measurement to alleviate the blood supply shortage in deceased donor liver transplantation (DDLT).MethodsThe outcomes of 26 patients who received DRBCT and 37 patients in the control group who only received 3rd party packed red blood cells (pRBCs) transfusion between May 2020 and January 2021 were compared.ResultsPatients receiving DRBCT did not develop transfusion-related complications, and the incidence of postoperative infection was similar to that in the control group (23.1% vs. 18.9%, P=0.688). Because the patients received the red blood cells from organ donors, the median volume of intraoperative allogeneic red blood cell transfusion from blood bank was 4.0 U (IQR 1.1–8.0 U) in the DRBCT group, which is significantly lower than that (7.5 U, IQR 4.0–10.0 U) in the control group (P=0.018). The peak aspartate aminotransferase (AST) level was significantly lower in the DRBCT group than in the control group (P=0.008) and so were the AST levels in the first two days after the operation (P=0.006 and P=0.033).ConclusionsDRBCT is a safe and effective procedure to lower the need for blood supply and is associated with a reduction in AST levels after transplantation. DRBCT is beneficial to patients receiving life-saving transplantation without sufficient blood supply during the COVID-19 pandemic.     

Intestinal allograft survival in the rat following pretreatment with donor-specific UV-B-irradiated leukocytes and peritransplant immunosuppression with cyclosporine

Previous studies from our laboratory showed that pretreatment with ultraviolet-B-irradiated donor leukocytes (UV-B DL) combined with brief peritransplant cyclosporine (CyA) resulted in indefinite survival of Wistar/Furth rat cardiac allografts in Lewis recipients. This study was designed to examine the effect of pretransplant UV-B DL with or without peritransplant CyA on orthotopic intestinal allografts in the same rat strain combination. The results showed that while low-dose CyA treatment alone (10 mg/kg i.m. on days 0, +1, and +2) had no effect on intestinal allograft rejection, 20 mg/kg (on days 0, +1, and +2) CyA significantly (P0.001) prolonged graft survival, with 33% of the hosts surviving indenfinitely. The highest dose of CyA (30 mg on days 0, +1, and +2) abrogated rejection, but most transplant recipients succumbed to infection and functional ileus due to a toxic side effect of CyA. Pretreatment with UV-B DL on days -14 and-7 alone did not prolong intestinal allograft survival. Combination of a subtherapeutic CyA dose (20 or 10 mg/kg) given on days 0, +1, and +2 with pretransplant UV-B DL on days-14 and -7 did not alter the survival of intestinal allografts compared to treatment with CyA alone. This suggests that pretreatment with UV-B DL with or without peritransplant administration of CyA has no effect on intestinal allograft survival, in contrast to the effect of such combined treatment on cardiac allograft survival, where indefinite graft survival is observed. This difference in the effect of pretransplant UV-B DL on intestinal and cardiac allograft survival is most likely due to organ-specific immunogenicity, particularly to the relative density of class I and II histocompatibility antigens present in heart or intestine.     

Synergistic effect of donor-specific blood transfusion and a short course of deoxyspergualin in rat kidney transplantation

Deoxyspergualin (DSG), an analogue of spergualin produced by B. laterosporus, has a strong immunosuppressive effect in various transplantation models. We have investigated the mechanism of donor-specific prolongation of survival time in rat kidney grafting by donor-specific blood transfusion (DST) and a short course of DSG. Lewis (LEW) kidney allografts were transplanted into fully allogeneic BN rats. Fresh, whole LEW blood 1.0 ml, was injected i.v. into BN rats 2 days prior to transplantation. Then, DSG, 6 mg/kg per day, was administered by i.m. injection on days 0, 1, and 2 after transplantation. The recipients were divided into five groups: group 1 (n=6) no treatment: group 2 (n=6) DST only; group 3 (n=7) DSG only; group 4 (n=7) DST and DSG; and group 5 (n=6), third party (ACI rats) blood transfusion and DSG. Lymphocytes (cervical lymph nodes) and serum were harvested from BN recipients on day 7 postgrafting. For suppressor cell assays, lymphocytes from BN recipients in each group were added as a third cell to the mixed lymphocyte reaction (MLC) between nontransplanted BN lymphocytes (responder) and LEW or other third party (PVGC, ACI, WKA rats) lymphocytes (stimulator). Antidonor lymphocytotoxic antibody (ADLA) was checked by microcytotoxicity assays. Median survival times (MST) for each group were: group 1, 10 days; group 1, 10 days; group 3, 13 days; group 4, 75 days; and group 5, 13 days. Remarkable prolongation of MST was only noted in group 4. In the suppressor cell assay, group 4 showed significant suppression (40%; P<0.05); the other groups did not show any suppression. This suppressive activity in group 4 was effective only during the MLC between BN and LEW, not during the MLC of third party-BN combinations. Thus, suppressor cells from DST/DSG-treated BN recipients appear to be donor-specific. In the microcytotoxicity assay, the only group that showed any ADLA was group 2, which was not treated with DSG. These results clearly show that both induction of donor-specific suppressor cells and inhibition of ADLA production are associated with the remarkable donor-specific prolongation of kidney allograft survival in DST/DSG-treated recipients.     

Competition between native liver and graft in auxiliary liver transplantation in a rat model

The competition between the native and the grafted liver in heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) was investigated in a rat model. The experimental groups were: HALT with flow-regulated PVA and 70% resection of a native liver and graft (n = 32; group I) versus 70% liver resection (n = 32; group II). After HALT, the weight of the native liver increased until the sixth postoperative week (431% +/- 55% of the intraoperative weight), whereas, the graft weight was only 76% +/- 31% of the intraoperative weight at this time. In group II, liver weight increased continuously to 529% +/- 30% of the intraoperative weight after 6 weeks. On postoperative day 2, there was significantly increased proliferative hepatocellular activity in all groups. This was highest in the resected livers of group II, followed by the native livers of group I, and the grafts of group I (301 +/- 126 vs 262 +/- 97 vs 216 +/- 31 Ki-67-positive hepatocytes/10 visual fields). However, the differences between the groups were not significant. With regard to hepatocellular apoptosis, the livers were similar among all groups and at all time points, M30-positive hepatocyte counts were 相似文献   

大鼠肝移植小移植物模型的研究

目的 建立大鼠肝移植小移植物模型，探讨移植物体积对受体生存的影响。方法 48只雄性SD大鼠随机分为4组：70％肝切除组(A组)、全肝移植组(B组)、中等移植物组(C组)、小移植物组(D组)。观察各组大鼠生存率。于术后不同时间点，采取外周静脉血检测肝功能；行肝组织常规组织学及透射电镜检测。结果 D组第3天生存率为56％，显著低于A、B、C组(P＜0．01)；各时间点D组各生化指标均显著高于其他3组(P＜0．01)；A、C和D组肝脏组织学以肝细胞再生的表现为主，B组以淋巴细胞浸润为主。D组术后第3天光镜下和透射电镜检查可见分裂肝细胞；第21天移植肝体积已达正常水平。结论 大鼠肝移植小移植物模型供肝量应超过全肝重量的45％。影响小移植物成活的主要因素是缺血—再灌注损伤和手术技术。     

20%小体积的大鼠肝脏移植模型

目的 应用显微外科技术建立20%小体积移植物的大鼠原位肝脏移植模型.方法 原位移植建立20%小体积大鼠肝脏移植模型.雄性Lewis大鼠40只,供体20只,受体20只.供肝经门静脉用4℃ UW液灌注.肝上下腔静脉用端端吻合连续缝合的方法.肝下下腔静脉和门静脉分别用套管方法固定.套叠缝合法重建肝动脉.胆管重建采用内支架管端端连接的方法.观察移植物的存活率.免疫组化检测肝细胞摄取溴脱氧尿核苷的情况.结果 共施行肝脏移植手术20例,移植手术成功率为100%.20%小体积肝脏移植物的存活率为93.8%(＞14 d).组织学检查移植后的肝脏组织结构良好.移植术后72 h溴脱氧尿核苷染色阳性的肝细胞计数明显增多.结论 20%小体积大鼠肝脏移植物可启动完成移植后的肝脏再生.显微外科技术是移植模型成功的关键.该模型稳定性强,适合于部分肝脏移植领域的基础研究.     

胆汁细胞学及细胞因子基因表达诊断大鼠肝移植急性排斥反应的实验研究

目的 为肝移植术后急性排斥反应寻找可靠而又无创作的诊断方法。方法 应用袖套技术建立大鼠原位肝移植模型。分为同种异体移植组,同基因移植组和免疫抑制组,依据病理学变化诊断肝移植术后急性排斥反应,光学显微镜定量胆汁细胞学检查,应用逆转录多聚酶链反应技术检测术后1、2、3、5、7d胆汁中细胞因子IL-2、IFN-γ、IL-4和IL-6基因表达。结果 同种异体移植组胆汁中细胞数持续升高,并出现淋巴母细胞和淋巴细胞,同种异体移植组检测到IL-2和IFN-γmRNA转录,其特异性分别为70%和67%（7/10和8/12）,敏感性分别为39%（7/18）和44%（8/18）,3组胆汁中均检测到IL-4、IL-6基因表达。结论 胆汁细胞学检查和细胞因子IL-2、IFNγ基因表达检测是急性排斥反应有效而又无创伤的辅助诊断方法。     

大鼠原位和异位小肠移植慢性排斥反应模型的制作与比较

目的  比较大鼠原位和异位小肠移植慢性排斥反应模型的建模效果。方法  采用F344(RT1^1vr)大鼠作为供体, Lewis (RT1¹)大鼠作为受体, 构建异系异位和原位大鼠小肠移植模型(各8只), 术后0~14 d给予皮下注射环孢素。观察术后受体的体质量变化及存活时间。采用苏木素-伊红(HE)染色观察肠组织病理学变化, 酒精苏木素染色后观察肠组织胶原纤维和弹性纤维的变化。计算两组受体大鼠的成模率。结果  异位和原位小肠移植大鼠均能长期存活, 大部分超过90 d。原位小肠移植组大鼠术后第3日恢复正常饮食, 于术后14 d左右体质量可恢复至术前水平, 之后缓慢增长, 但大部分原位小肠移植大鼠在术后150 d出现持续的体质量下降, 且不能被环孢素逆转。异位小肠移植组大鼠术后第1日恢复进食, 于术后25~30 d才能恢复至术前的体质量水平, 术后30~90 d期间, 体质量逐渐上升并保持在较高水平。原位小肠移植组大鼠术后90 d的小肠组织未出现慢性排斥反应的病理学改变且未见明显纤维化, 术后163 d和术后200 d的小肠组织出现慢性排斥反应的病理学改变, 且出现移植小肠系膜纤维化。异位小肠移植组大鼠术后90 d和术后200 d的小肠组织均出现典型的慢性排斥反应的病理学改变和移植小肠系膜纤维化。异位小肠移植组全部出现特征性病理改变, 成模率为100%, 与原位小肠移植组成模率75%比较, 差异无统计学意义(P > 0.05)。结论  应用F344→Lewis大鼠组合建立原位和异位小肠移植模型, 术后给予小剂量环孢素, 均可在术后不同时间点出现慢性排斥反应。与原位大鼠小肠移植模型相比, 大鼠异位小肠移植模型建模操作简单, 慢性排斥反应成模时间较短, 病理改变程度相对一致, 更适合用于实验研究。     

小体积肝移植和辅助性原位小体积肝移植治疗猪急性肝功能衰竭的近期疗效观察

目的观察小体积肝移植和辅助性原位小体积肝移植治疗猪急性肝功能衰竭的近期疗效。方法急性肝功能衰竭猪随机分为3组接受肝移植治疗:A组行全肝移植(n=5);B组行小体积肝移植(n=5);C组行辅助性原位小体积肝移植(n=5)。各组动物开腹后即刻、切脾后即刻和再灌注后30 min分别监测门静脉压力,并观察术后生化指标变化、病理改变和1周生存率。结果A、B和C三组的移植肝重量与受体体重之比分别为(2．44±0．30)％、(0．76±0．02)％和(0．75±0．03)％。再灌注后30 min,B组移植肝门静脉压力显著高于其它两组(A:B:C=13．3:17．5:12．2 cmH2O, P<0．01),C组原肝门静脉压力显著高于移植肝门静脉压力(14．3:12．2 cmH2O,P<0．05)。A组和C组术后第2天起血清天冬氨酸转氨酶、总胆红素、凝血酶原时间、乳酸和血氨水平明显下降,术后第7天基本恢复至正常水平。B组术后上述生化指标一直维持在较高的水平,术后第2～4天明显高于其它两组(P<0．01)。A组、B组和C组1周生存率分别为100％、20％和80％,B组明显低于其它两组(P<0．05)。结论辅助性原位小体积肝移植治疗急性肝功能衰竭近期疗效优于小体积肝移植,术中不必干预原肝门静脉。     

Intraoperative measurement of graft blood flow - a necessity in liver transplantation

Abstract Portal venous and hepatic arterial flow was measured intraop-eratively in the 70 most recent patients undergoing liver transplantation in our institution. Impaired graft flow due to vascular abnormalities was detected in six patients. One patient suffered from arterial steal due to stenosis of the recipient celiac trunk with blood shunting from the hepatic to the splenic artery. Ligation of the recipient hepatic artery restored the arterial graft flow. In two patients we found reduced portal venous flow due to large portosystemic collaterals. The collaterals accountable for the impaired portal flow were identified and ligated, which restored portal venous graft flow. Excessive sensitivity of the portal venous flow to the position of the graft was found in a 6-month-old boy. Portal venous flow varied considerably, depending upon the position of the graft, and intraoperative flow measurement allowed the best position of the graft to be identified. Two patients developed arterial thrombosis in the early postoperative course. Immediate laparatomy with thrombectomy resulted in good, palpable pulsation in the graft artery in both patients. Intraoperative flow measurement demonstrated satisfactory arterial flow in one patient, whereas there was no net flow in the other patient's graft artery. Pulsation in this patient was caused by blood oscillating in and out of the liver. In conclusion, we find that causes of primary graft dysfunction due to technically flawed reperfusion of the graft can be identified and alleviated by intraoperative measurement of the flow in the graft vessels.     

Intraoperative measurement of graft blood flow — a necessity in liver transplantation

Portal venous and hepatic arterial flow was measured intraoperatively in the 70 most recent patients undergoing liver transplantation in our institution. Impaired graft flow due to vascular abnormalities was detected in six patients. One patient suffered from arterial steal due to stenosis of the recipient celiac trunk with blood shunting from the hepatic to the splenic artery. Ligation of the recipient hepatic artery restored the arterial graft flow. In two patients we found reduced portal venous flow due to large portosystemic collaterals. The collaterals accountable for the impaired portal flow were identified and ligated, which restored portal venous graft flow. Excessive sensitivity of the portal venous flow to the position of the graft was found in a 6-month-old boy. Portal venous flow varied considerably, depending upon the position of the graft, and intraoperative flow measurement allowed the best position of the graft to be identified. Two patients developed arterial thrombosis in the early postoperative course. Immediate laparatomy with thrombectomy resulted in good, palpable pulsation in the graft artery in both patients. Intraoperative flow measurement demonstrated satisfactory arterial flow in one patient, whereas there was no net flow in the other patient's graft artery. Pulsation in this patient was caused by blood oscillating in and out of the liver. In conclusion, we find that causes of primary graft dysfunction due to technically flawed reperfusion of the graft can be identified and alleviated by intraoperative measurement of the flow in the graft vessels.     

Lazaroid U-74389G预处理对大鼠心脏停搏供体肝移植缺血再灌注损伤的保护作用

目的探讨在心脏停搏供体肝移植中 ,LazaroidU 74 389G预处理对大鼠供肝热缺血损伤的保护作用。方法实验以U 74 389G预处理与否、以及供肝获取前经历的心脏停搏时间 4 5或6 0min分为 4组 ,分别为N 4 5组、N 6 0组、tN 4 5组和tN 6 0组 ,而后行大鼠原位肝移植 ,比较各组术后存活率、肝功能、MDA和肝脏病理学改变。结果N 4 5组、N 6 0组、tN 4 5组和tN 6 0组肝移植术后1周的存活率分别为 2 5 % (2 / 8)、0 (0 / 8)、5 8% (7/ 12 )和 33% (4/ 12 )。U 74 389G的预处理可以显著提高心脏停搏供体肝移植的存活率 ,并能够改善术后肝功能和减轻肝脏病理学改变 ,降低MDA的表达。结论在心脏停搏供体肝移植中 ,U 74 389G预处理可以减轻热缺血再灌注对供肝造成的损伤 ,提高肝移植存活率     

Intraoperative cell salvage with autologous transfusion in liver transplantation

Liver transplant(LT) is the primary treatment for patients with end-stage liver disease. About 25000 LTs are performed annually in the world. The potential for intraoperative bleeding is quite variable. However, massive bleeding is common and requires blood transfusion. Allogeneic blood transfusion has an immunosuppressive effect and an impact on recipient survival, in addition to the risk of transmission of viral infections and transfusion errors, among others.Techniques to prevent excessive bleeding or to use autologous blood have been proposed to minimize the negative effects of allogeneic blood transfusion.Intraoperative reinfusion of autologous blood is possible through previous selfdonation or blood collected during the operation. However, LT does not normally allow autologous transfusion by prior self-donation. Hence, using autologous blood collected intraoperatively is the most feasible option. The use of intraoperative blood salvage autotransfusion(IBSA) minimizes the perioperative use of allogeneic blood, preventing negative transfusion effects without negatively impacting other clinical outcomes. The use of IBSA in patients with cancer is still a matter of debate due to the theoretical risk of reinfusion of tumor cells. However, studies have demonstrated the safety of IBSA in several surgical procedures, including LT for hepatocellular carcinoma. Considering the literature available to date, we can state that IBSA should be routinely used in LT, both in patients with cancer and in patients with benign diseases.     

不同比例缩小体积肝移植的大动物研究

目的研究不同比例缩小体积肝移植的结果,确定猪能耐受的最小体积的肝移植。方法远系繁殖猪70头分为原位全肝移植作为对照组和3组不同比例缩小体积原位肝移植(按照缩小体积的移植肝占受体切除肝脏重量的百分比;1组:60%;2组:30%;3组:20%);实验采用原位经典肝移植方式(静脉转流)。术后第3天和第5天取肝标本。结果对照组和3组不同比例缩小体积肝移植的移植物与受体肝脏重量百分比(GIWRW)分别为87·4%±8·3%、59·9%±5·2%、33·6%±4·9%和22·1%±3·4%;移植物与受体体重百分比(GIWBW)分别2·4%±0·4%、1·43%±0·17%、0·81%±0·09%和0·53%±0·06%。对照组、1组和2组存活率达100%;3组存活率仅为53%。4个组的动物处死后肝脏移植物重量均有显著的增加。结论安全的缩小体积肝移植,应以移植物与受体肝脏重量百分比不小于33%,同时移植肝与受体重量百分比不小于0·8%为限。