Apoptosis in the human liver during allograft rejection and end-stage liver disease

The contribution of apoptosis (programmed cell death) to cellular damage in human liver disease is unknown. Using the in situ DNA end labelling method (ISEL), evidence was sought of programmed cell death (PCD) in liver tissue from patients with various liver diseases. In particular, the study aimed to determine whether PCD is involved in either the loss of interlobular bile ducts (vanishing bile duct syndrome—VBDS) or the perivenular hepatocyte drop-out, both of which are characteristic of irreversible graft rejection. Large numbers of apoptotic hepatocytes were found in pervenular areas in tissues taken from patients with chronic graft rejection. Significant hepatocyte apoptosis, was not seen in long-term stable allografts, primary biliary cirrhosis, cholestasis, paracetamol-induced fulminant hepatic failure, or fulminant hepatic failure of indeterminate origin (non-A, non-B, non-C hepatitis). Bile ducts rarely stained positively, but mononuclear cells present in the post-transplant tissues were frequently positive, showing nuclear or cytoplasmic staining. The presence of cytoplasmic staining suggested that some mononuclear cells had ingested apoptotic DNA from other cellular sources. PCD may thus contribute to the perivenular hepatocyte loss in chronic rejection. The absence of ductular epithelial cell staining suggests that PCD is not involved significantly in the bile duct loss of VBDS. Furthermore, apoptosis of monomuclear cells implies that PCD may be involved in regulating the inflammatory cell infiltration of graft rejection.     

带血管蒂肝圆韧带瓣转位修复胆管缺损的应用解剖

观察30例成人肝圆韧带标本,肝圆韧带呈纤维条索样,内含少许不规则残腔。肝圆韧带的血液供养主要来自肝右动脉的分支及伴行的附脐静脉。由于肝圆韧带近端紧邻胆管,远端易于游离,提出了带血管蒂的肝圆韧带瓣作为自体生物材料来修复肝外胆管的缺损,并讨论了该术式有关的应用解剖要点。     

调节性T细胞在大鼠小肠移植急性排斥反应中的作用

目的 分析调节性T细胞在大鼠急性排斥反应中的作用。方法 采用免疫组化SABC染色法，测定BN→LEW大鼠小肠移植急性排斥反应时，外周血及移植肠浸润淋巴细胞中调节性T细胞：CD4^ ,CD8^ ,CD25^ T淋巴细胞及相关细胞因子IL－4和IFN－γ的表达，并与同基因大鼠间小肠移植（BN→BN）作比较。结果 外周血淋巴细胞分析显示，大鼠小肠移植急性排斥反应时，以CD4^ ,CD25^ ,T细胞为主，CD8^ 淋巴细胞只占少部分；分泌IL－4的细胞在术后4，7，14d分别只占14．3％，16．2％和16．9％。移植肠基底浸润的淋巴细胞以CD4^ ,CD25^＋和分泌IFN－γ的淋巴细胞为主。结论 在大鼠同种小肠移植中，急性排斥反应与CD25^ ,CD4^ T细胞及Th1相关细胞因子IFN－γ的表达增加相关。而CD8^＋T淋巴细胞和Th2相关细胞因子IL－4可能具有保护作用。     

Contribution of Fas ligand to cardiac allograft rejection

Effector mechanisms for allograft injury remain unclear. Inthe present study, we verified the contribution of Fas and Fasligand (FasL) to cardiac allograft rejection by utilizing theFas-deficlent lpr or FasL-deficient gid mice as the donor orrecipient. Cardiac myocytes prepared from normal mice, but notthose from lpr mice, constitutively expressed Fas and were susceptibleto FasL-mediated lysis. Survival of cardiac allografts was substantiallyprolonged when gld or lpr mice were used as the recipient. Incontrast, cardiac allografts from ipr mice were normally rejectedwithout a delay. Histological examination of the grafts in thegld or lpr recipients demonstrated a lesser cellular infiltrationand much milder myocyte damage. Proliferative response and cytotoxicT lymphocyte induction against the donor-type alloantigens werenot impaired in the gld or lpr recipients. These results indicatea substaintial contribution of FasL to cardiac allograft rejection,independent of Fas in the grafts. This raises a possibilitythat FasL may be more generally involved in tissue damage associatedwith various diseases than expected from the expression of Fasin the target organs.     

Mast cells and c-Kit expression in liver allograft rejection

AIMS : The pathogenesis of rejection following liver transplantation is not fully understood. It has been postulated that mast cells may play a role in acute and chronic rejection of a number of other solid organ grafts. The aim of this study was to assess the possible role of mast cells and c-Kit+ cells in acute and chronic liver allograft rejection. METHODS AND RESULTS : Biopsy specimens from (i) 'time zero' grafts with a minimal degree of perfusion injury (controls), (ii) transplanted livers with different grades of acute rejection, and (iii) transplanted livers with end-stage chronic rejection, were stained immunohistochemically using monoclonal anti-mast cell tryptase and polyclonal anti-c-Kit antibodies. Tryptase- and c-Kit-positive cell densities were assessed by image analysis. Tryptase-positive mast cell densities (P<0.001) were strongly correlated with acute liver allograft rejection grades and chronic liver allograft rejection. Furthermore, a similarly strong relationship was found between c-Kit+ cell densities and increasing rejection grade (P<0.001). CONCLUSIONS : Tryptase- and c-Kit-positive mast cells form part of the inflammatory infiltrate in both acute and chronic liver allograft rejection, and may be important effector cells in these processes.     

Immunohistochemical identification of renal lysozyme during allograft rejection in man

An immunoperoxidase staining method was used to identify lysozyme in biopsy or transplantectomy specimens of human renal allografts during reversible and irreversible rejection of the grafts. Proximal tubules in apparently functioning nephrons showed lysozyme staining. In irreversibly rejected grafts, infiltrating mononuclear phagocytes in and near peritubular and glomerular capillaries also stained intensely for lysozyme. In acute necrotizing arteritis, lysozyme-positive cells (mononuclear phagocytes) infiltrated the blood vessel wall. The presence of infiltrating lysozyme-positive cells in the transplant was consistent with poor graft survival. The variation in lysozyme staining of proximal tubular cells apparently was a reflection of the differences in the reabsorption capacity of the tubular cells, attributable to the tubular dysfunction of renal allografts. The infiltrating lysozyme-positive cells probably contribute to the increased urinary excretion of lysozyme during acute rejection.     

Identification and characterization of cells infiltrating the graft and aqueous humour in rat corneal allograft rejection

In a rat model of corneal transplantation, Fischer 344 (RT1^lv1) rats received orthotopic corneal isografts or Wistar-Furth (RT1^u) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8⁺ than CD4⁺ cells was found at days 1–3 following rejection, whereas there was a higher proportion of CD4⁺ cells at days 5–8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1–2, 4 and 7–10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4⁺ > CD8⁺ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1–2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes—from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4⁺ cells was found in both aqueous and graft at later times.     

TLRs与同种异型移植排斥

Toll样受体(Toll-like receptors,TLRs)是一类在机体识别、清除入侵病原微生物免疫过程中起重要作用的受体.除此之外,新近的研究表明TLRs还参与同种异型移植过程中的排斥反应.本文就TLRs的这一进展进行了综述,内容包括TLRs的结构、功能和信号通路,TLRs与无菌性炎症,以及TLRs与皮肤、内脏移植排斥.     

Human interleukin-10 gene inhibits acute rejection by triggering apoptosis in allograft vascular transplantation

The aim this study is to explore effect of IL-10 on apoptosis of VSMCs in allograft arterial transplantation rats, and to investigate mechanism. SD rats were divied into three groups, including control group (CN, with physiological saline), blank vector group (BV, with blank adenovirus) and combined gene group (CG, with adenovirus carried IL-10 gene). The isolated donor vascular was transfected with the adenovirus carried hIL-10 gene for 30 minutes by immersing method. Forty-five days post transplantation, the grafts were harvested. The allografts pathologioc changes were observed and the size of vascular intima and middle layer of allografts were measured. The expression of hIL-10 was detected by RT-PCR, ELISA and immunohistochemistry, respectively. The repression of Fas/Fasl in artery allografts was also examined by immunohistochemistry method. The results indicated that 45 days after transplantation, the intimal and middle hyperplasia ratio in CG group was significantly lower than that in CN and BV group (P < 0.05). The transgene expression of human interleukin-10 was significantly enhanced in CG group compared to CN and BV group by ELISA, RT-PCR and immunohistochemistry (P < 0.05). The expression of Fas/FasL was higher in CG group compared with the other groups (P < 0.05). The level of apoptotic smooth muscle cells were significantly increased in CG group compared to CN and BV group (P < 0.05). In conclusion, adenovirus mediated IL-10 expression could up-regulate Fas/FasL expression, induce smooth muscle cell apoptosis and alleviate angiosclerosis process. The IL-10 gene transfer to allograft artery could inhibit acute rejection reaction of allograft vascular transplantation.     

Prognostic significance of fascin expression in extrahepatic bile duct carcinomas

Extrahepatic bile duct (EBD) carcinoma is an aggressive cancer with a poor prognosis. Fascin is an actin-bundling protein with roles in forming cell protrusions and mesenchymal and neuronal cell motility. Many human neoplasms up-regulate fascin. High fascin expression is thought to be a poor prognostic factor in some cancers; however, few data are available for the role of fascin in EBD carcinoma. We investigated fascin immunoreactivity in EBD carcinoma and tested it for correlations between fascin expression and clinicopathological parameters.Conventional tissue sections of 114 cases of EBD carcinomas were immunohistochemically analyzed for fascin expression.Fascin expression was tested by cytoplasmic staining. Negative, weak positive, and strong positive fascin staining was observed in 49 (43.0%), 32 (28.1%), and 33 cases (28.9%), respectively. Fascin expression in EBD carcinomas was significantly correlated with histological grade (P<0.0001), primary tumor (T) (P=0.002), TNM stage (P=0.036), lymphatic invasion (P=0.048), venous invasion (P=0.024), and adjacent organ invasion (P<0.0001). High fascin expression was a significant poor prognostic factor (P=0.0001) in EBD carcinoma. High fascin expression (P=0.004) and TNM stage (P=0.001) in EBD carcinoma were independent adverse prognostic factors.High fascin expression is significantly correlated with aggressive tumor phenotype in EBD carcinoma and is an independent poor prognostic factor in EBD carcinoma.     

慢性排斥肾肥大细胞形态与超微结构的研究

目的：了解肾移植慢性排斥反应时肥大细胞在排斥肾中的形态、超微结构特点及分布规律，以探讨肥大细胞是否在肾移植慢性排斥反应中所起的作用。方法：采用甲苯胺蓝、爱尔沙蓝特殊染色法及电子显微镜技术。结果：发现慢性排斥肾中，有较多的肥大细胞，主要分布于肾皮质区。特殊染色及超微结构的结果显示，排斥肾中至少存在幼稚与成熟两种类型的肥大细胞。在移植后３个时间段（〈１年，１年￣４年及〉４年），每个高倍视野中，肥大细胞     

大鼠肝移植排斥反应期一氧化氮合酶的动力学变化

目的 探讨供体特异性输血（DST）预处理后大鼠同种肝移植物一氧化氮合酶（NOS）的动力学变化。方法 观察同基因肝移植（Ⅰ组），同种肝移植（Ⅱ组），DST预处理的同种肝移植（Ⅲ组）术后NOS的表型及亚硝酸盐/硝酸盐，细胞因子的动态变化。结果 Ⅱ组血中亚硝酸盐/硝酸盐，INF-α，TNF-α浓度明显增高，免疫组化显示，Ⅱ组移植物中macNOS^ 及ED1^ ,ED2^ 细胞数明显增多，Northern blot分析，Ⅲ组移植物中IL-10和TGF-BmRNA呈高水平表达；Ⅱ组移植物的iNOS和IL-12mRNA水平明显高于其他组。结论 DST预处理的移植物处于免疫无应答状态，NOS被抑制与TH2及TH3类细胞因子高表达有关。     

TNF-alpha and TGF-beta1 gene polymorphisms and renal allograft rejection in Koreans

This study was performed in order to evaluate the association of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1) gene polymorphisms with renal allograft rejection in Koreans. Five TNF-alpha (-1031T/C, -863C/A, -857C/T, -308G/A and -238G/A) and two TGF-beta1 (codon 10 T/C and codon 25 G/C) single-nucleotide polymorphism (SNP) sites were studied by using polymerase chain reaction (PCR) single-strand conformation polymorphism and PCR restriction fragment length polymorphism methods in 100 controls and 164 patients. The patients underwent renal transplantation, having one or more Human leukocyte antigen (HLA)-A, HLA-B and HLA-DR antigens mismatched with their donors. For the TGF-beta1 gene, we also studied the polymorphism of donors. The allele frequencies of each SNP site in controls were not different from those of patients. The frequency of TNF-alpha high-producer genotype, -308GA, and TGF-beta1 lower (intermediate)-producer genotype, codon 10 CC and codon 25 GG, were significantly higher in patients with recurrent acute rejection episodes (REs), compared to those in patients with no or one RE. The highest risk group for developing recurrent REs showed the combination of TNF-alpha high- and TGF-beta1 lower-producer genotypes. Analysis of chronic renal allograft dysfunction (CRAD) revealed that TGF-beta1 high-producer genotype of donors, codon 10 TT/TC and codon 25 GG, is associated with CRAD especially in patients with recurrent REs. The highest risk group for developing CRAD showed the combination of recipient's TNF-alpha high- and donor's TGF-beta1 high-producer genotypes. These results would be useful for predicting high-risk group for acute rejection or CRAD in renal transplantation.     

The role of CC and CXC chemokines in cardiac allograft rejection in rats

Acute cellular rejection is due in part to an upregulation of chemokine genes, resulting in eventual cell-mediated cytotoxicity. The role of chemokines in acute cardiac allograft rejection is not fully characterized presently. These studies compared the patterns of expression for multiple chemokines in rodent cardiac allograft rejection. Allogeneic transplants were performed from Brown-Norway donors to Lewis recipients. Survival studies utilized daily administration of neutralizing antisera to MCP-1, CINC, and MIP-1alpha. Patterns of mRNA and protein expression were determined by Northern blots and immunohistochemistry. Allogeneic controls rejected at mean of 6.5 days. Neutralization of MCP-1 (10.8 days, P<0.001) and MIP-1alpha (7.5 days, P=0.004) function, but not CINC (6.2 days, P>0.05), significantly prolonged allograft survival. Message expression for the beta chemokines studied were increased by day 2 and continued to increase until day 6 just before rejection, while CINC levels did not change as dramatically after day 2. Chemokine protein levels mirrored mRNA patterns by IHC analysis. MCP-1 and MIP-1alpha appear to play regulatory roles in cardiac allograft rejection, while CINC is expressed, but not functional, in injury development. Beta chemokine activity should be studied further in hope of developing more targeted immunosuppression, or identifying specific chemokines that may be useful for immunosurveillance purposes.     

Histopathology of spleen allograft rejection in miniature swine

Spleen transplantation (SpTx) has established donor-specific tolerance in rodents, but not in large animals or humans. We report the histopathology of rejection in an established model of SpTx in major histocompatibility complex (MHC)-defined miniature swine. Of the 17 SpTx, rejection was observed in two grafts transplanted into untreated, MHC-matched, minor antigen-disparate recipients (group 1, n=4), but not in the two that received a 12-day course of cyclosporin A (CyA). Rejection also occurred in five grafts transplanted into fully MHC-disparate recipients (group 2, n=12), one of which was untreated and four of which received some form of immunosuppressive therapy. One recipient of an MHC class-I-mismatched spleen treated with 12 days of CyA did not show rejection. Following biopsy and/or necropsy, fixed allograft tissue sections were treated with multiple stains, immunohistochemical markers and TUNEL assay. Common features of rejection occurred in grafts from both groups, but with varying time courses. Necrosis developed as early as day 8 in group 2 and day 27 in group 1, ranging from focal fibrinoid necrosis of arteriolar walls and sinusoids to diffuse liquefactive necrosis, usually associated with haemorrhage. Other features of rejection included white pulp expansion by atypical cells and decreased staining of basement membranes and reticular fibres. A doubling of the baseline TUNEL index preceded histologically identifiable rejection. This study establishes histologic guidelines for diagnosing and, perhaps, in future studies, predicting acute rejection of splenic allografts transplanted across known histocompatibility barriers in a large-animal model.     

带血管蒂空肠瓣修复胆管狭窄的临床应用

目的：报道用于带血管蒂空肠瓣修复胆管狭窄的手术方法。方法：采用带血管蒂空肠瓣修复胆管狭窄17例,采用带血管蒂空肠段行肝胆管桥式吻合9例。结果：本组除3例术后并发胆瘘外行引流在2周内治愈外,其余23例均获满意效果。结论：本术式具有防止胆汁返流和逆行感染、促进持肝外胆管生理机能及保持胆汁的正常PH环境等优点。     

Factors producing bile infarction and bile duct proliferation in biliary obstruction

To clarify the factors producing bile infarction and bile duct proliferation in obstructive jaundice, the incidence of the hepatic lesions and the serum levels of the bile constituents were examined in three rat models. (1) Ligation of the common bile duct induced bile infarction, bile duct proliferation, retention of bile in the liver, and elevation of the serum levels of total bilirubin and total bile acids. (2) The rats treated by choledochotomy had bile in the abdominal cavity, but there was no retention of bile in the liver. The degree of development of bile infarction was similar to that of the common bile duct ligation group, but bile duct proliferation was not found: the serum levels of total bilirubin and total bile acids were elevated. (3) In the rats subjected to partial bile duct ligation, bile infarction and bile duct proliferation were seen only in the lobes with ligation of the hepatic ducts: only slight or no elevation of the serum levels of total bilirubin and total bile acids was found. These data suggest that bile infarction is caused by the toxic action of bile constituents other than bilirubin and bile acids, absorbed into the blood from the obstructed biliary system, and that bile duct proliferation is due to mechanical factors following bile retention or direct actions of retained bile in the liver.     

移植物细胞因子表达与小肠移植排斥反应相关性的实验和临床病例研究

目的结合移植物细胞因子表达的实验和临床病例研究，探索早期诊断小肠移植急性排斥反应的细胞因子相关的敏感指标。方法①两例短肠综合症患者接受活体小肠移植术。定期或病情变化时随时行内镜组织学检查并测定受体大鼠移植物sIL-2R、IL-4、IL-6和IFN-γ表达水平。②BN-LEW大鼠部分小肠移植，A组：SBT（n=20）；B组：SBT＋FK506（2．5mg／kg，n=20），术后第1、4、7、14和30天测定受体大鼠移植物sIL-2R、IL-4、IL-6和IFN-γ水平同时取移植肠黏膜行病理组织学检查。结果首例术后67d发生排斥反应，第2例于术后20d和80d分别发生强烈排斥反应。发生排斥反应相应时相均发生IL-2Rα、IFN-γ表达的显著升高，排斥反应控制后IL-2Rα迅速恢复，但IFN-γ仍在较高水平维持较长时间。A组大鼠术后第1天始即显示IL-2Rα、IFN-γ和IL-6表达的显著升高，于术后7d达到最高，移植后14d仍在高水平。B组仅术后第1天出现IL-2Rα、IFN-γ和IL-6表达的迅速升高，第4天已恢复至基本正常。结论移植物IL-2Rα、IFN-γ表达的升高与小肠移植急性排斥反应密切相关，有望成为早期诊断小肠移植急性排斥反应的敏感指标。