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Acute cellular rejection (ACR) remains a major concern after liver transplantation. Predicting and monitoring acute rejection by non-invasive methods are very important for guiding the use of immunosuppressive drugs. Many studies have shown that exosomes and their contents are potential biomarkers for various liver diseases. Here, we identify and validate the role of exosomes and galectin-9 in ACR after liver transplantation. Exosomes were isolated from three sets of paired patients, with and without ACR, and the proteins within the exosomes were isolated and identified. Candidate proteins were then validated using a tissue microarray containing resected liver samples from 73 ACR and 63 non-rejection patients. Finally, protein expression and clinical manifestations were included in Kaplan-Meier survival and Cox regression analyses. Circulating exosomes were isolated from ACR and non-rejection patients and characterized using transmission electron microscopy and western blotting for CD63/CD81. Western blotting experiments revealed higher levels of galectin-9 protein in circulating exosomes from ACR recipients. Immunohistochemical analysis of the tissue microarray showed that the expression of galectin-9 in resected liver was significantly higher in the ACR group than in the non-rejection group (P<0.05). Higher levels of galectin-9 expression in resected livers were associated with poorer prognosis (P<0.05). Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation.

Aim: To study the influence of different doses of tacrolimus (FK506) on gut microbiota after liver transplantation (LT) in rats., Methods: Specific pathogen-free Brown Norway (BN) rats and Lewis rats were separated into five groups: (1) Tolerance group (BN-BN LT, n = 8); (2) rejection group (Lewis-BN LT, n = 8); (3) high dosage FK506 (FK506-H) group (Lewis-BN LT, n = 8); (4) middle dosage FK506 (FK506-M) group (Lewis-BN LT, n = 8); and (5) low dosage FK506 (FK506-L) group (Lewis-BN LT, n = 8). FK506 was administered to recipients at a dose of 1.0 mg/kg, 0.5 mg/kg, and 0.1 mg/kg body weight for 29 d after LT to the FK506-H, FK506-M, and FK506-L groups, respectively. On the 30 th day after LT, all rats were sampled and euthanized. Blood samples were harvested for liver function and plasma endotoxin testing. Hepatic graft and ileocecal tissues were collected for histopathology observation. Ileocecal contents were used for DNA extraction, Real-time quantitative polymerase chain reaction (RT-PCR) and digital processing of denaturing gradient gel electrophoresis (DGGE) profiles and analysis., Results: Compared to the FK506-H and FK506-L groups, FK506-M was optimal for maintaining immunosuppression and inducing normal graft function; the FK506-M maintained gut barrier integrity and low plasma endotoxin levels; furthermore, DGGE results showed that FK506-M induced stable gut microbiota. Diversity analysis indicated that FK506-M increased species richness and rare species abundance, and cluster analysis confirmed the stable gut microbiota induced by FK506-M. Phylogenetic tree analysis identified crucial bacteria associated with FK506-M; seven of the nine bacteria that were decreased corresponded to Bacteroidetes , while increased bacteria were of the Bifidobacterium species. FK506-M increased Faecalibacterium prausnitzii and Bifidobacterium spp . and decreased Bacteroides-Prevotella and Enterobacteriaceae , as assessed by RT-PCR, which confirmed the crucial bacterial alterations identified through DGGE., Conclusion: Compared to the low or high dosage of FK506, an optimal dosage of FK506 induced immunosuppression, normal graft function and stable gut microbiota following LT in rats. The stable gut microbiota presented increased probiotics and decreased potential pathogenic endotoxin-producing bacteria. These findings provide a novel strategy based on gut microbiota for immunosuppressive dosage assessment for recipients following LT., Competing Interests: Conflict-of-interest statement: All authors declare no conflict of interest exists.

Objective: Acute rejection resulting from alloimmune responses is a major risk factor affecting patient survival following liver transplantation. Since interleukin (IL)-6 can mediate acute rejection, the association between IL-6 gene single nucleotide polymorphisms (SNPs) and incidence of acute rejection in liver transplant recipients was investigated., Methods: Patients who received liver transplant between January 2005 and December 2010 were typed for IL6-572C/G (rs1800796) polymorphisms using the snapshot technique. Association between genotype and acute rejection was analysed using the SNP Statistics website: http://bioinfo.iconcologia.net/snpstats/start.htm. Allelic and genotypic distributions for rs1800796 were compared among 335 patients with or without acute rejection within the first 6 months following liver transplant., Results: Incidence of acute rejection was 11.94%. A heterozygous CG genotype for IL6-572C/G was associated with a lower acute rejection rate compared with homozygous CC or GG genotypes., Conclusion: IL6-572 CG genotype may be related to protection from acute rejection following liver transplant in Han Chinese patients.

Copy number variation (CNV) in gene was a novel type of genetic diversity in human that affect the pathogenesis, progress, and prognosis of disease. The aim of this study was to determine the associations between CNV in CCL3L1 gene and the rejection risk in liver-transplant recipients. The 266 Han-Chinese patients and 135 volunteers were enrolled in this study. Genomic DNA and mRNA samples were obtained from the whole peripheral blood; a quantitative real-time PCR-based assay was carried out to determine the copy number of CCL3L1, and real-time PCR was carried out to calculate the CCL3L1/CCL3 mRNA ratio. The CNV distributions in patients and health controls had no significant differences. Copy number in acute rejection group (AR) shifted to higher number compared with non-acute rejection group (4.74±1.87 vs. 3.88±2.13, p=0.017). Moreover, patients with higher CCL3L1 copies (≥3 copies) had a significantly higher rejection risk than patients lower copies CCL3L1 (OR=3.85, 95% CI, 1.14-13.04; p=0.021). No association was found between CNV and rejection grades. In conclusion, liver transplant recipients with high copy number of CCL3L1 gene had a significant higher risk of AR. CNV might be a novel genetic diversity that correlated with allograft rejection., (© 2011 John Wiley & Sons A/S.)

Objectives: This study sought to investigate the associations between polymorphisms in the chemokine receptor 5 gene and acute rejection in liver transplant., Materials and Methods: A total of 266 patients who underwent a liver transplant between January 2006 and March 2009 were enrolled in this study. Genomic DNA was extracted from whole blood, and chemokine receptor 5Δ32 was detected by polymerase chain reaction. Eight nucleotide polymorphism loci in the chemokine receptor 5 gene were detected by Applied Biosystems SNaPShot and TaqMan technologies., Results: Chemokine receptor 5Δ32 mutation was not detected in all the individuals from China. There was no significant association between the single nucleotide polymorphism in chemokine receptor 5 gene and acute rejection., Conclusions: Single nucleotide polymorphisms in a single gene of the chemokine receptor 5 might not play a role in acute rejection after liver transplant.

T help cell 17 (Th17), recently identified as a new subset of CD4(+) T cells, has been implicated in autoimmune diseases, tumor immunity, and transplant rejection. To investigate the role of Th17 in acute hepatic rejection, a rat model of allogeneic liver transplantation (Dark Agouti (DA) to Brown Norway (BN)) was established and isogeneic liver transplantation (BN to BN) was used as controls in the study. The expression of Th17-related cytokines in the liver and peripheral blood was determined by immunohistochemistry, flow cytometry, enzyme-linked immunosorbent assay (ELISA), or real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Strong expression of interleukin-17A (IL-17A), IL-6, transforming growth factor-β (TGF-β), IL-8, and myeloperoxidase (MPO) was observed in liver allografts. The ratios of Th17 to CD4(+) lymphocytes in the liver and peripheral blood were dramatically increased in the allograft group compared with the control (P<0.01). Secreted IL-17 and IL-6 in liver homogenate and serum were significantly elevated in the allograft group, while secreted TGF-β was increased in liver homogenate and decreased in serum compared with the control (P<0.01). The messenger RNA (mRNA) levels of IL-17, IL-21, and IL-23 were enhanced in the allografts compared with the control (P<0.01). Correlation analysis showed significant correlations between IL-17 and IL-6 and TGF-β and between IL-17 and IL-21 and IL-23. The present study demonstrates that Th17 plays a role in promoting rat liver allograft rejection.

Background: Acute rejection (AR) after liver transplantation is a cell-mediated immune response that takes place within the allograft and results in graft dysfunction and failure, but the molecular mechanisms about hepatocyte dysfunction remain poorly understood. Here we characterized global protein expression changes in liver allograft during AR., Methods: The effect of an alloantigen-dependent immunological response was evaluated by syngeneic and allogeneic rat orthotopic liver transplantation (OLT). Using a combination of two-dimensional gel electrophoresis and mass spectrometry, we identified 18 differentially expressed proteins in AR allograft compared with matched tolerance allograft. Serum chemistry and allograft histology were determined., Results: Allogeneic OLT recipients exhibited elevated plasma levels of liver injury markers, progressive portal and venous inflammation and cellular infiltration in liver allograft compared with syngeneic OLT. 18 protein expressions altered by AR play important roles in metabolism, oxidative stress defense, signal transduction, biotransformation and transport. Decreased expression of protein disulfide isomerase in AR allograft was confirmed by Western blotting and immunohistochemistry., Conclusions: This study uncovered new mechanistic insights into graft dysfunction in AR of liver allograft. Several significantly altered protein expressions act coordinately in hepatocyte dysfunction by depressed energy, enhanced oxidative stress-induced molecular damage and restrained biotransformation. The present findings may open new avenues for the understanding and prevention of graft dysfunction and failure during AR., (Copyright 2009 S. Karger AG, Basel.)

Background: Acute rejection (AR) and hepatitis B virus (HBV) recurrence after liver transplantation (LT) are the two major complications leading to chronic graft dysfunction. Genomic polymorphisms in interleukin (IL)-10, tumor necrosis factor (TNF)alpha and transforming growth factor (TGF)beta1 genes have been found to affect the susceptibility to certain diseases. However, the relationship between cytokine gene polymorphisms and risk of AR as well as HBV recurrence after LT in Han Chinese has not been reported. The objective of the present study was to investigate the association of polymorphisms within these cytokine genes with the risk of AR as well as HBV recurrence., Methods: One hundred eighty six Chinese LT recipients in which 41 patients developed AR and 29 patients experienced HBV recurrence were enrolled; 151 age- and gender-matched healthy individuals were selected as controls. Single-nucleotide polymorphisms (SNPs) at loci of IL-10 -1082, -819, -592, and TNFalpha -308, -238, as well as TGFbeta1 -988, -800, -509, +869, and +915 were determined by using DNA sequencing and then confirmed by restriction fragment length polymorphism (PCR-RFLP). Analyses of linkage disequilibrium and haplotype frequency were performed using Haploview program., Results: The -819 and -592 polymorphisms in the IL-10 gene were in complete linkage (r(2) = 1). Another linkage was found at -509 and +869 in the TGFbeta1 gene (r(2) = 0.66). A significant difference was observed in the distribution of allelic frequencies at position -819 and -592 in the IL-10 gene between ARs and non-ARs (p = 0.036, OR = 1.134, 95% CI 0.999-1.287 and p = 0.036, OR = 1.134, 95% CI 0.999-1.287, respectively). After adjustment for a Bonferroni correction, there was no significant difference between the polymorphism and AR (p >0.05). Furthermore, the overall genotype distribution between HBV recurrence patients and non-HBV recurrence patients was also not significantly different (p >0.05)., Conclusions: Our study suggests that gene polymorphisms of IL10, TNFalpha, and TGFbeta1 do not have a major independent role in AR and HBV recurrence after LT and may not be risk factors of AR and HBV recurrence after LT in Chinese liver transplant recipients.

Toll-like receptors (TLRs) are germline-encoded receptors expressed on antigen-presenting cells (APCs) that identify a variety of microbial and endogenous ligands and activate the innate immune responses to the presence of danger. However, their role in the development of allograft rejection after liver transplantation remains unknown. In this study, we used flow cytometry to assess TLR-4 and TLR-2 expression among circulating CD14+ monocytes in 64 liver transplant patients and 24 healthy volunteers. We demonstrated significantly higher TLR-2 and TLR-4 expression on circulating monocytes among conditioned liver transplantation recipients with acute rejection compared with those in clinically stable with normal liver function. Steroid pulse therapy significantly reduced the expression of TLR-4 and TLR-2 on the monocytes of recipients with acute rejection. Based on these data, we have suggested that activation of innate immunity in liver transplant recipients through TLR-4 and TLR-2 contributes to the development of acute allograft rejection after liver transplantation. The reduced expression of TLR-4 and TLR-2 may be one of the mechanisms by which steroid pulse therapy inhibits the development of acute rejection. Estimation of TLR expression on APCs may be predictive of in acute rejection after liver transplantation.

Background and Aim: Rejection remains a problem in the transplantation field. The aim of this study was to establish acute and chronic rejection models in rats and to investigate the roles of non-interleukin (IL)-2 T cell growth factors such as IL-15, IL-7 and IL-13 during rejection., Methods: A liver transplant model was established using Dark Agouti and Brown Norway rats. The rats were divided into group A, left without treatment; group B, received cyclosporinee (1 mg/kg/day); and group C, cyclosporinee (4 mg/kg/day). Histopathological, reverse transcriptase-polymerase chain reaction and western blot were performed in liver specimens obtained from different time-points after transplantation in the three groups., Results: In group A, the livers showed irreversible acute cellular rejection with cell infiltration. In group B, chronic liver rejection was found, with graft infiltration, ductular damage or proliferation, obliterative arteriopathy and liver fibrosis. No apparent histological alterations were observed in group C. IL-15, IL-7 and IL-13 messenger RNA and their protein were all highly expressed in the liver specimens of groups A and B. Upregulated expression was found in IL-15 since the first day after transplantation and in IL-7 and IL-13 since day 6. The extent of IL-15 upregulation was more than that of IL-7 and IL-13., Conclusions: Liver transplantation in Dark Agouti to Brown Norway rats with low-dose immunosuppression can induce chronic rejection. In the process of acute and chronic allograft rejections, non-IL-2 T cell growth factors such as IL-15, IL-7 and IL-13 play roles. Strategies should pay more attention to regulating these cytokines after liver transplantation.

Objective: To study the effect and implication of nonmyeloablative donor specific bone marrow (DSBM) infusion on the immunoreaction of liver allotransplantation., Methods: Orthotopic liver transplantation model was used in this study. Groups were set as follows: Group I, syngeneic control (Wistar-to-Wistar); Group II, acute rejection (SD-to-Wistar); Group III, acute rejection treated with cyclosporine A (CsA) by intramuscular injection (SD-to-Wistar+CsA); Group IV, bone marrow infusion at 7 d pretransplantation followed by short-term CsA treatment (SD-to-Wistar+DSBM); Another group of short-term CsA treatment preoperatively without bone marrow infusion was also set as control. General characteristics and survival time were observed. Histological grades of rejection were determined by pathological examination. IL-2 and IFN-gamma level in peripheral blood and donor liver were detected respectively by Enzyme-Linked Immuno-Sorbent Assay (ELISA) and Western blot. Chimerism of donor cells was measured by PCR for a male-specific marker (Y-chromosome-specific sequence, Sry)., Results: No signs of rejection were found in Group I. Acute rejection occurred in both Group II and the short-term CsA treated group. All the recipients died at (9-15) d posttransplantation with a median survival time of (10.7+/-0.5) d and (11.2+/-2.4) d, respectively. Only mild rejection could be seen in Group III. In Group IV, 4 out of 6 recipients had long-term survival (>100 d), the histological grade of rejection was significantly lower than that of Group II, so did the expression level of IL-2 and IFN-gamma in both peripheral blood and grafted liver. Y-chromosome-specific sequence (Sry) of male SD rats could be detected in the bone marrow, spleen and thymus of female recipients at 15 d after bone marrow infusion., Conclusion: Mild preconditioning nonmyeloablative donor specific bone marrow infusion can enhance chimerism formation in recipients, alleviate the rejection of liver allotransplantation and prolong survival of liver allotransplantation.

Objective: To evaluate the effect of ciclosporin A (CsA) on the organogenesis and function of embryonic metanephroi allografted into adult rats., Methods: The whole metanephroi from the 15, 16 and 17 embryonic day-old (E15, E16, E17) embryos of Sprague-Dawley (SD) rat were allografted into the omenta of SD adult rats with their left kidneys resected, which were divided into 2 categories: 3 CsA-treated groups of 10 rats (E15CsASD, E16CsASD, and E17CsASD) and 3 non-CsA-treated groups of 10 rats (E15SD, E16SD, and E17SD). Thirty SD rats without kidney resection were divided into 6 equal groups and underwent allografting embryonic metanephroi in the same manner as mentioned above. The E15 metanephroi of Lewis rats were allografted into the omenta of Thirty adult Brown Norway (BN) rats with one kidney resected. Were divided into 6 equal groups, received allografting of embryonic metanephroi, and injected with CsA of normal saline in the manner as mentioned above (E15CsABN and E15BN). Two to 4 weeks after implantation, the metanephroi allografted in host rats were removed for histopathological examination or anastomosed for renal function measurement 4 weeks later., Results: (1) Four weeks after implantation, the E17SD and E16SD metanephroi showed signs of acute rejection as hypercellular glomeruli and mononuclear cell infiltration in the interstitium. The E16CsASD and E17CsASD metanephroi formed mature nephrons and collecting ducts with few lymphocytic infiltrates. After CsA was discontinued, the E16CsASD and E17CsASD metanephroi were rejected fully within 21 days. (2) 4 weeks after implantation, the E15SD metanephroi were enlarged, became vascularized, and developed mature tubules and glomeruli; however, they were rejected by 100 days after implantation. The E15 Lewis metanephroi were fully rejected within two weeks in the BN adult rats. With CsA administrated, the E15 Lewis metanephroi developed normal mature nephrons and collecting ducts within the adult BN rats. If CsA was discontinued, the E15CsABN metanephroi were rejected. (3) The E15CsASD metanephroi had significantly lower values of wet weight (P = 0.006) and higher values of creatinine clearances (P = 0.007) than the E15SD metanephros transplants, but were identical to those of the E16CsASD metanephroi (P = 0.948, P = 0.840). (4) The metanephroi did not grow or differentiate in the rats without host kidney resection., Conclusions: (1) Cyclosporin A may suppress graft rejection, thus normalizing the growth and function of fetal metanephroi in the omenta of host rats. (2) A variety of factors affect the growth and development of allografted metanephroi, whereas rejection remains the major one.