Enteral supplementation with glycyl-glutamine improves intestinal barrier function after liver transplantation in rats

BACKGROUND:Most patients after liver transplantation (LT) suffer from intestinal barrier dysfunction.Glycyl-glutamine (Gly-Gln) by parenteral supplementation is hydrolyzed to release glutamine,which improves intestinal barrier function in intestinal injury.This study aimed to investigate the effect of GlyGln by enteral supplementation on intestinal barrier function in rats after allogenetic LT under immunosuppressive therapy.METHODS:Twelve inbred Lewis rats were selected randomly as donors,and 24 inbred Bro...     

Protective effect of probiotics on intestinal barrier function in malnourished rats after liver transplantation

BACKGROUND: Most patients waiting for liver transplantation have end-stage liver diseases with malnutrition, which is prone to induce intestinal barrier dysfunction after liver transplantation. We aimed to study the effect of probiotics on intestinal barrier function in malnourished rats following liver transplantation with long-term antibiotics. METHODS: Twelve Lewis rats were selected as donors. Twelve BN rats, which served as recipients, were subjected to malnutrition by semi-starvation for 4-5 weeks. Th...     

Effects of n－3 fatty acid, fructose－l,6－diphosphate and glutamine on mucosal cell proliferation and apoptosis of small bowel graft after transplantation in rats

AIM: To evaluate the effects of n-3 fatty acids (n-3FA), fructose-1,6-diphosphate (FDP) and glutamine (GLN) on mucosal cell proliferation and apoptosis of small bowel graft. METHODS: One hundred and ninety-six inbred strain Wistar rats were grouped as donors and recipients, and underwent heterotopic small bowel transplantation (SBT). n-3FA, FDP and GLN were administered via gastric tube as well as venous infusion for 10 days before and after surgery, respectively. The proliferation and apoptosis of mucosal cells were analyzed with flow cytometry and in situ cell death detection kits. RESULTS: Apparent apoptosis and minor proliferation of mucosal cells of small bowel graft after transplantation were observed. A higher mucosal cell proliferative index and lower apoptotic index were found in all small bowel grafts after supplying with n-3FA, FDP and GLN. CONCLUSION: Nutritional support with n-3FA, FDP and GLN promotes mucosal cell proliferation significantly, and prevents mucosal cell from undergoing apoptosis with different degrees. These regulatory effects on the apoptosis alter the structure and absorption function of transplanted small bowel favorably.     

Chronic bile duct hyperplasia is a chronic graft dysfunction following liver transplantation

AIM: To investigate pathological types and influential factors of chronic graft dysfunction (CGD) following liver transplantation (LT) in rats.METHODS: The whole experiment was divided into three groups: (1) normal group (n = 12): normal BN rats without any drug or operation; (2) syngeneic transplant group (SGT of BN-BN, n = 12): both donors and recipients were BN rats; and (3) allogeneic transplant group (AGT of LEW-BN, n = 12): Donors were Lewis and recipients were BN rats. In the AGT group, all recipients were subcutaneously injected by Cyclosporin A after LT. Survival time was observed for 1 year. All the dying rats were sampled, biliary tract tissues were performed bacterial culture and liver tissues for histological study. Twenty-one day after LT, 8 rats were selected randomly in each group for sampling. Blood samples from caudal veins were collected for measurements of plasma endotoxin, cytokines and metabonomic analysis, and faeces were analyzed for intestinal microflora.RESULTS: During the surgery of LT, no complications of blood vessels or bile duct happened, and all rats in each group were still alive in the next 2 wk. The long term observation revealed that a total of 8 rats in the SGT and AGT groups died of hepatic graft diseases, 5 rats in which died of chronic bile duct hyperplasia. Compared to the SGT and normal groups, survival ratio of rats significantly decreased in the AGT group (P < 0.01). Moreover, liver necrosis, liver infection, and severe chronic bile duct hyperplasia were observed in the AGT group by H and E stain. On 21 d after LT, compared with the normal group (25.38 ± 7.09 ng/L) and SGT group (33.12 ± 10.26 ng/L), plasma endotoxin in the AGT group was remarkably increased (142.86 ± 30.85 ng/L) (both P < 0.01). Plasma tumor necrosis factor-α and interleukin-6 were also significantly elevated in the AGT group (593.6 ± 171.67 pg/mL, 323.8 ± 68.30 pg/mL) vs the normal (225.5 ± 72.07 pg/mL, 114.6 ± 36.67 pg/mL) and SGT groups (321.3 ± 88.47 pg/mL, 205.2 ± 53.06 pg/mL) (P < 0.01). Furthermore, Bacterial cultures of bile duct tissues revealed that the rats close to death from the SGT and AGT groups were strongly positive, while those from the normal group were negative. The analysis of intestinal microflora was performed. Compared to the normal group (7.98 ± 0.92, 8.90 ± 1.44) and SGT group (8.51 ± 0.46, 9.43 ± 0.69), the numbers of Enterococcus and Enterobacteria in the AGT group (8.76 ± 1.93, 10.18 ± 1.64) were significantly increased (both P < 0.01). Meanwhile, compared to the normal group (9.62 ± 1.60, 9.93 ± 1.10) and SGT group (8.95 ± 0.04, 9.02 ± 1.14), the numbers of Bifidobacterium and Lactobacillus in the AGT group (7.83 ± 0.72, 8.87 ± 0.13) were remarkably reduced (both P < 0.01). In addition, metabonomics analysis showed that metabolic profiles of plasma in rats in the AGT group were severe deviated from the normal and SGT groups.CONCLUSION: Chronic bile duct hyperplasia is a pathological type of CGD following LT in rats. The mechanism of this kind of CGD is associated with the alterations of inflammation, intestinal barrier function and microflora as well as plasma metabolic profiles.     

Primary Sclerosing Cholangitis: The Emerging Role for Liver Transplantation

Primary sclerosing cholangitis is a progressive liver disease for which orthotopic liver transplantation is the only curative procedure. Questions exist regarding the role of temporizing procedures and the timing of transplantation. During the past 4 yr, we have performed liver transplants in 177 adult recipients. Twenty-six patients (14.6%) with primary sclerosing cholangitis received 30 transplants including 12 men and 14 women. The recipients were examined for a number of preoperative and postoperative variables. The 4-yr actuarial survival in patients with primary sclerosing cholangitis after transplantation was 88%. Patients were segregated according to preoperative risk variables. Twenty patients were low and medium risk, with one death (95% survival). Three patients were high risk, with two deaths (33% survival). In conclusion, orthotopic liver transplantation is safe and effective therapy for primary sclerosing cholangitis. Early referral for transplantation is recommended to reduce the mortality associated with this procedure in those with advanced hepatic failure.     

骨髓间充质干细胞移植治疗大鼠急性肝功能衰竭

目的探讨骨髓间充质干细胞（BMSCs）移植对急性肝功能衰竭的治疗作用。方法大鼠90%肝脏切除制备急性肝功能衰竭模型,实验组大鼠肝内移植2×106个BMSCs,对照组仅注射生理盐水。观察大鼠的生存情况,RT-PCR检测BMSCs在肝脏的植入,血清检测肝功能确认BMSCs移植对肝脏修复的作用。结果移植BM-SCs的实验组大鼠存活80%,对照组大鼠仅存活20%。RT-PCR显示,BMSCs移植后定植于大鼠肝脏内。肝功能检测显示,实验组大鼠的血清丙氨酸氨基转移酶水平、天冬氨酸氨基转移酶水平显著降低,血清白蛋白水平显著升高。结论 BMSCs移植可以显著提高急性功能衰竭大鼠的生存率,促进其肝功能的恢复,对急性肝功能衰竭的治疗具有积极作用。     

Change of intestinal mucosa barrier function in the progress of non-alcoholic steatohepatitis in rats

AIM： To explore the change of intestinal mucosa barrier function in the progress of non-alcoholic steatohepatitis （NASH） in rats. METHODS： Thirty-two Sprague-Dawley （SD） rats were randomly divided into control group and model group. Rats in the control group were given normal diet, and rats in the model group were given fat-rich diet. Eight rats in each group were killed at end of the 8th and 12th wk, respectively. The levels of endotoxin, D-xylose, TG, TC, ALT and AST, intestinal tissue SOD and MDA as well as intestinal mucus secretory IgA （sIgA） were measured. The pathology of liver was observed by HE staining. RESULTS： At end of the 8th wk, there was no marked difference in the levels of endotoxin, D-xylose and sIgA between the two groups. At end of the 12th wk, rats in the model group developed steatohepatitis and had a higher serum level of endotoxin （P = 0.01） and D-xylose （P = 0.00） and a lower serum level of sIgA （P = 0.007）. CONCLUSION： Intestinal mucosa barrier malfunction may exist in NASH rats and may be an important promoter of NASH in rats.     

中国肝移植受者选择与术前评估技术规范(2019版)

肝移植作为各种类型不可逆急、慢性肝病的有效治疗手段,已被广泛接受。经过几十年稳步持续的发展,肝移植技术逐渐成熟。随着肝移植技术的发展、新型免疫抑制剂的应用以及围手术期管理的进步,肝移植适应证和禁忌证也在发生变化。详细的术前检查和准备是保证肝移植预后的重要环节。为进一步规范我国肝移植受者选择以及术前评估和准备,中华医学会器官移植学分会组织肝移植专家,总结国内外相关研究最新进展,结合国际指南和临床实践,从肝移植适应证和禁忌证、受者术前检查、术前准备以及常见并发症处理等方面,制订《中国肝移植受者选择与术前评估技术规范(2019版)》。     

体外膜肺氧合技术对脑心双死亡器官捐献供肝的保护作用

目的探讨体外膜肺氧合(ECMO)技术在公民逝世器官捐献供肝保护中的应用,总结ECMO技术保护供肝的初步体会及经验。方法收集江西省人民医院2015年1月-2018年12月运用ECMO技术完成脑心双死亡器官捐献(DBCD)肝移植供者/受者及常规DBCD肝移植供者/受者的临床资料,对供肝的保护及移植效果进行对比分析。计量资料两组间比较采用t检验;计数资料两组间比较采用χ2检验。结果共纳入一般情况及肝功能接近的供者32例,根据采用的方法将其分为对照组(常规DBCD肝移植)和研究组(运用ECMO技术完成DBCD肝移植),每组各16例;32例肝移植受者分为对应的对照组(n=16)和研究组(n=16)。器官获取前供者对照组与供者研究组比较,心率、收缩压、舒张压、血氧分压、乳酸水平、中心静脉压、TBil、ALT、AST差异均有统计学意义(t值分别为14.121、-17.817、-19.187、-8.927、4.559、-3.495、3.357、4.111、3.553,P值均<0.05)。与受者对照组术后第7天肝功能相比,受者研究组肝移植术后肝功能恢复速度更快,两组TBil、DBil、ALT、AST、ALP、GGT比较,差异均有统计学意义(t值分别为9.309、4.783、5.067、2.203、4.774、5.257,P值均<0.05);受者研究组患者住院时间明显缩短[(12.65±2.86)d vs(20.87±4.98)d,t=5.756,P<0.001]。结论运用ECMO技术获取并实施肝移植临床效果较好,科学合理运用ECMO技术可以有效改善供肝质量,对我国公民逝世器官捐献工作有着积极的作用。     

Pretreatment with cyclosporine and anti-interleukin 2 receptor antibody abrogates the anti-idiotype response in rat recipients of cardiac allografts.

A 10-day course with ART-18, a mouse monoclonal antibody (mAb) directed against the rat interleukin 2 receptor (IL-2R), prolongs the survival of (LEW x BN)F1 cardiac allografts in LEW recipients to approximately 3 weeks (acute rejection = 8 days, P less than 0.001). We examined host responses against ART-18 idiotype (Id) and mouse immunoglobulin in recipients immunomodulated with ART-18 mAb. Treatment with ART-18 elicited high titers of anti-Id antibodies 14 days after transplantation. However, naive rats given ART-18 before transplantation showed strong anti-Id responses as early as day 4 after engraftment, coinciding with abrogation of the treatment effect (graft survival, approximately 10 days). Preimmunization with irrelevant mouse IgG, which elicited high titers of anti-IgG, did not influence the efficacy of ART-18 upon graft survival (17 days). The use of cyclosporin A (CsA) in conjunction with ART-18 prior to transplantation suppressed the anti-Id response and led to dramatic graft prolongation (greater than 58 days), with two of five grafts surviving indefinitely. This striking effect of CsA plus ART-18 pretreatment did not depend upon CsA per se, as grafts were rejected within 12 days in animals pretreated with CsA alone; in both groups CsA trough levels were comparable. Moreover, administration of CsA before transplantation in concert with control IgG (instead of ART-18) prompted rejection within 2-4 weeks. Thus, discrete interaction(s) between anti-IL-2R mAb and CsA prior to engraftment induces partial host unresponsiveness/tolerance to anti-IL-2R mAb treatment following transplantation and suppresses the neutralizing anti-Id responses, which results in long-term/permanent graft acceptance. This study provides a strategy to overcome the anti-Id response mounted by graft recipients that otherwise limits the efficacy of anti-IL-2R mAb treatment.     

Effect of cyclosporine on liver regeneration after orthotopic reduced-size hepatic transplantation in the rat

These experiments were undertaken to study the effects of cyclosporine A (CsA) on liver regeneration after an isogeneic orthotopic reduced-size hepatic transplantation (RSHT) in rats. Male Wistar rats were treated with or without a daily injection of CsA beginning 24 hr before surgery and were subjected to a 68% partial hepatectomy. A isogeneic orthotopic reduced-size hepatic transplantation was performed in recipient rats pretreated with or without CsA. A daily injection of CsA was continued until the recipient rats were sacrificed. Animals were sacrificed at various time points (12, 24, 36, 48, and 72 hr) postoperatively. The incorporation of bromodeoxyuridine (BrdU) into the DNA of the remnant hepatocytes was evaluated by immunohistochemical staining with a monoclonal antibody against BrdU. CsA (10 mg/kg/day) significantly augmented BrdU incorporation into hepatocytes after hepatectomy. The maximum labeling index (LI) was observed at 24 hr after hepatectomy. In contrast, the maximum LI in the recipient rats not receiving CsA was seen at 36 hr after RSHT, and 10 mg/kg/day of CsA decreased the LI at 36 hr after RSHT. A lower dose of CsA (3 mg/kg/day), however, significantly increased the LI in the recipient rats (P<0.01), and it reached a peak at 24 hr after RSHT when compared to the transplant recipients not receiving CsA. The time course of the increase in the LI in the transplant recipient rats receiving 3 mg/kg/day of CsA was similar to that observed in the rats after hepatectomy. This dosage improved the delay in the reduced-size hepatic transplant LI reaching its peak. These findings suggest that after RSHT the liver graft is more sensitive to both hepatotrophic and hepatotoxic effects of CsA.This work was supported in part by Mika Fund.     

Influence of estrogen and androgen on the outcome of liver transplantation

BACKGROUND/AIMS: To assess influence of sex hormone on outcome of orthotropic liver transplantation (OLT). METHODOLOGY: Adult female Wistar rats were used as donors and male Wistar rats as recipients. Two experimental series were established. The first series consisted of the orchectomized (ORC) group. 17beta-estradiol (E2) treated-ORC group and recipient Sham-ORC group; the second series consisted of the ovariectomized (OVX) group, dihydrotestosterone (DHT)-treated OVX group and donor Sham-OVX group. Recipients were sacrificed on postoperative day 7 (POD 7); the survival rate (SVR), histomorphological damage score of liver, graft-recipient weight ratio (GRWR) and the levels of alanine aminotransferase (ALT), alkaline phosphatase (AKP) and albumin (ALB) on POD 7 were detected. RESULTS: The histomorphological damage score and the level of ALT and AKP in the E2-treated ORC group, OVX group and DHT-treated OVX group was significantly lower compared with their respective sham group (P<0.05). CONCLUSIONS: Estrogen in recipient rats was responsible for the observed beneficial effects of liver transplantation, but at the same time, influence on liver of estrogen for a long time decreased the adaptability of graft to environment change. In contrary, androgen had less influence than estrogen.     

大鼠移植骨髓细胞向肝细胞转化的实验研究

目的 探讨体内骨髓细胞向肝细胞转化的可行性。方法 将雌性SD大鼠随机分为3组，每组15只。①R BMT(全身照射 骨髓移植)；②2—AAF R BMT；③2—AAF PH(部分肝切) BMT。进行交叉性别骨髓细胞移植，雄性骨髓植入雄性受体，分别于第5、10、20天处死雌鼠。以雄性性别决定基因sry作为细胞标记，用原位杂交和FISH作为检测方法对骨髓细胞的肝细胞转化进行分析。结果 PCR移植效果初步分析可见，R BMT组11例中有10例PCR阳性；2AAF PH BMT组11例中有7例阳性，2AAF B BMT组10例中有6例阳性。sry原位杂交染色发现，第5天各组雌性受体肝索中均未见sry阳性的肝细胞。第10天R BMT组可见1例sry阳性的细胞位于肝细胞索,FISH染色可见这一细胞白蛋白mRNA阳性。第20天各组PCR阳性各例均可在肝索中检测到sry阳性的细胞。FISH染色可见白蛋白mRNA阳性。经统计学分析第20天各组sry阳性细胞数无明显差异。结论 在B BMT、2—AAF PH BMT和2—AAF R BMT模型中移植的骨髓细胞均可以植入肝脏，并存在于肝细胞索。植入肝索的骨髓细胞最早可见于移植后第10天，并发生转分化，表达白蛋白mRNA。不经过全身照射的2—AAF PH BMT组，移植的骨髓细胞也可以进入肝脏发生转分化，因此全身照射并不一定是移植骨髓细胞活化、植入和转化的必须条件。     

Effect of lithium ingestion on digestive and absorptive function of rat intestine

The long-term effect of lithium treatment on the digestive and absorptive function has been investigated in male albino rats. The uptake of D-glucose, amino acids and activities of cellular and brush border enzymes were evaluated after every 3 months. Significantly increased uptake was observed in 6-month lithium-treated rats. The absorptive capacity (Vmax) for D-glucose increased significantly without alteration in the Michaelis constant. Activities of cellular, brush border membrane disaccharidase, leucine aminopeptidase and Na+,K+-ATPase enzymes were significantly augmented in 6-month lithium-treated animals. The elevation in sucrase activity may be due to induction of enzyme since only Vmax was increased in lithium-treated animals. The present biochemical alterations suggest that long-term lithium ingestion stimulates the small bowel digestive and absorptive functions.     

Anti-A of Donor Lymphocyte Origin in Three Recipients of Organs from the Same Donor

The development of iso- and alloantibodies reactive with recipient red blood cells subsequent to organ transplantation is an established phenomenon. However, development of self-reactive antibodies in multiple recipients of organs from a single donor source has only been reported in one instance involving the formation of anti-D after transplantation. We observed the development of a delayed hemolytic transfusion reaction 10 days after transplantation of a group O liver into a group A recipient. Serologic studies revealed a positive direct antiglobulin test due to coating of autologous A cells with anti-A. Close follow-up of the group A recipients of the kidneys transplanted from the same group O donor revealed development of hemolysis secondary to anti-A on day 11 in one recipient and hemolysis due to anti-A on day 13 in a second recipient. Significant anemia, a 2-3 g/dl drop in hemoglobin, occurred in both kidney recipients. These findings suggest that recipient of organs from donors whose transferred lymphocytes have produced antirecipient RBC antibodies in another recipient may be at risk for developing a similar self-limited hemolytic episode and should be followed accordingly.     

NF－κB activation and zinc finger protein A20 expression in mature dendritic cells derived from liver allografts undergoing acute rejection

AIM：To investigate the role of NF-κB activation and zinc finger protein A20 expression in the regulation of maturation of dendritic cells (DCs) derived from liver allografts undergoing acute acute rejection.METHODS:Sixty donor male SD rats and sixty recipient male lew rats weighing 220-300 g were randomly divided into whole liver transplantation group and partial liver transplantation group. Allogeneic (SD rat to LEW rat) whole and 50% partial liver transplantation were performed. DCs from liver grafts 0 hour and 4 days after transplantation were isolated and propagated in the presence of GM-CSF in vitro. Morphological characteristics and phenotypical features of DCs propagated for 10 days were analyzed by electron microscopy and flow cytometry,respectively. NF-κB binding activity, IL-1 2P70 Protein and zinc finger protein A20 expression on these DCs were measured by EMSA and Western blotting, respectively. Histological grading of rejection was determined.RESULTS: Allogeneic whole liver grafts showed no signs of rejection on day 4 after the transplantation. In contrast,allogeneic partial liver grafts demonstrated moderate to severe rejection on day 4 after the transplantation. After propagation for 10 days in the presence of GM-CSF in vitro,DCs from allogeneic whole liver grafts exhibited features of immature DC with absence of CD40 surface expression,these DCs were found to exhibit detectable but very low level of NF-κB activity, 1L-12 p70 protein and zinc finger protein A20 expression. Whereas, DCs from allogeneic partial liver graft 4 days after transplantation displayed features of mature DC, with high level of CD40 surface expression, and as a consequence, higher expression of lL-12p70 protein, higher activities of NF-κB and higher expression of zinc finger protein A20 compared with those of DCs from whole liver grafts (P&lt;0.001).CONCLUSION: These results suggest that A20 expression is up-regulated in response to NF-κB activation in mature DCs derived from allogeneic liver grafts undergoing acute rejection. Given the NF-κB inhibition function of this gene, it is suggested that their expression survives to limit NF-κB activation and maturation of DCs,and consequently inhibits the acute rejection and induces acceptance of liver graft.     

Immunotolerance of liver allotransplantation induced by intrathymic inoculation of donor soluble liver specific antigen

AIM: To study the effects of liver specific antigen (LSA) on the immunoreaction of liver allotransplantation and its significance.METHODS: Orthotopic liver transplantation was used in this study. Group Ⅰ: syngeneic control (Wistar-to-Wistar);Group Ⅱ: acute rejection (SD-to-Wistar). Group Ⅲ: acute rejection treated by intramuscular injection of cyclosporine A (CsA) (SD-to-Wistar+CsA). Group Ⅳ: Intrathymic inoculation of SD rat LSA one week before transplantation (LSA+SD-to-Wistar). The common situation and survival time, rejection grades, NF-κB activity of splenocytes and intragraft cytokine gene expression were observed to analyze the acute rejection severity and immune state of animals.RESULTS: The common situation of Wistar-to-Wistar group was very good after the transplantation and no signs of rejection were found. Recipients of SD-to-Wistar group lost body weight progressively. All died within 9 to 13 days after transplantation with the median survival time of 10.7±0.51days. It was an optimal control for acute rejection. The common situation of SD-to-Wistar+CsA group was bad during CsA medication but only with mild rejection. As for LSA+SD-to-Wistar group, 5 of 6 recipients survived for a long time and common situation was remarkably better than that of SD-to-Wistar group and SD-to-Wistar+CsA group.Its rejection grades were significantly lower than that of SD-to-Wistar group (P=0.026). Furthermore, no significant discrepancies of rejection were found between SD-to-Wistar group and LSA+SD-to-Wistar group at day7 and day12(P=0.067). NF-κB activity, IFN-γ and IL-2mRNA expression were significantly inhibited in LSA+SD-to-Wistar group compared with that of SD-to-Wistar group (P＜0.05).CONCLUSION: LSA is an important transplantation antigen which involves in the immunorejection of liver transplantation directly. We reported for the first time that intrathymic inoculation of LSA can induce immnotolerance of liver allotransplantation and grafts can survive for a long time thereby, thus leading to a novel way to liver transplantation immunotolerance.