Galectin-9 contributes to the survival of fully allogeneic skin grafts by modulating effector T cells and Tregs imbalance.

Allogeneic skin transplantation is an important clinical treatment for many diseases. Although Galectin-9 demonstrates multifaceted roles in modulating immune cell homeostasis and inflammation, its precise impact on balancing effector T cells and Tregs during allogeneic skin transplantation remains uncertain. This work was performed to evaluate the modulation of the survival time of allogeneic skin grafts by Gal-9 and to explore the critical mechanism involved in this process. Skin graft transplantation was conducted using C57BL/6 and BALB/c mice. Additionally, the levels of IL-2, IFN-γ, IL-4, and IL-17A were measured. Hematoxylin and eosin staining assay was performed to analyze the pathological conditions of skin grafts of experiment mice. The results revealed that Gal-9 noticeably prolonged the survival of the allogeneic skin graft and ameliorated the damage caused by acute immune rejection. Furthermore, Gal-9 resulted in selective reduction of effectors T cells such as Th1, and Th17. Simultaneously, Gal-9 didn't attenuate the protective function for allograft, which alleviated the immune rejection caused by abnormal immune imbalance. Gal-9 exhibited a therapeutic effect on the allogeneic skin graft by selectively reducing CD4+TIM-3+ T effector cells and inducing a shift from a Th1 to an anti-inflammatory Th2 response. Furthermore, Gal-9 did not attenuate the protective function. Our present study may represent a novel therapeutic candidate for modulating effector T cells and Tregs imbalance-based therapy of allograft transplantation.

[To promote the kidney transplantation of sensitized recipients in China by multilateral cooperation].

Sensitized recipients have an increased risk of kidney transplantation due to the immune memory of human leucocyte antigen. They lag on the waiting list and have poorer outcomes of transplantation. The society should give every patient an equal opportunity for treatment. Facing this worldwide problem, Chinese transplant community has some practical difficulties under current status of transplantation development in our country. It requires the cooperation of transplant laboratories, organ procurement organizations, clinicians, and the national organ allocation system to explore a path of solution.

A prediction model of delayed graft function in deceased donor for renal transplant: a multi-center study from China.

BACKGROUND: Kidneys obtained from deceased donors increase the incidence of delayed graft function (DGF) after renal transplantation. Here we investigated the influence of the risk factors of donors with DGF, and developed a donor risk scoring system for DGF prediction. METHODS: This retrospective study was conducted in 1807 deceased kidney donors and 3599 recipients who received donor kidneys via transplants in 29 centers in China. We quantified DGF associations with donor clinical characteristics. A donor risk scoring system was developed and validated using an independent sample set. RESULTS: The incidence of DGF from donors was 19.0%. Six of the donor characteristics analyzed, i.e., age, cause of death, history of hypertension, terminal serum creatinine, persistence of hypotension, and cardiopulmonary resuscitation (CPR) time were risk factors for DGF. A 49-point scoring system of donor risk was established for DGF prediction and exhibited a superior degree of discrimination. External validation of DGF prediction revealed area under the receiver-operating characteristic (AUC) curves of 0.7552. CONCLUSIONS: Our study determined the deceased donor risk factors related to DGF after renal transplantation pertinent to the Chinese cohort. The scoring system developed here had superior diagnostic significance and consistency and can be used by clinicians to make evidence-based decisions on the quality of kidneys from deceased donors and guide renal transplantation therapy.

mTOR masters monocyte development in bone marrow by decreasing the inhibition of STAT5 on IRF8.

Monocytes and macrophages play a key role in defending pathogens, removing the dead cells or cell debris, and wound healing. The mammalian target of rapamycin (mTOR) inhibitor rapamycin (RPM) is widely used in clinics to treat patients with organ transplantation or tumors. The role of mTOR in monocyte/macrophage development remains to be clarified. Here we found that mTOR intrinsically controls monocyte/macrophage development, as evidenced by the decreased percentages and cell numbers of CD11b+F4/80+ cells resulting from mTOR inhibition in SCID mice, mTOR-deficient mice, and mixed chimera mice, and the in vitro colony formation and monocyte/macrophage induction assays. However, Lyzs-mTOR knockout mice displayed normal levels of monocytes/macrophages, indicating that mTOR is not essential for the survival and maturation of monocytes/macrophages. Further studies showed that mTOR deficiency significantly reduced macrophage colony-stimulating factor receptor CD115 expression at the transcriptional and translational levels. The molecular mechanism studies indicate that the impaired monocyte/macrophage development caused by mTOR deficiency is mainly a result of the overactivated STAT5 and subsequent downregulation of IRF8, but not the altered cell metabolism and autophagy. Therefore, our work identifies that mTOR is an intrinsic master for monocyte/macrophage development at the early stages through regulating STAT5-IRF8-dependent CD115-expressing pathway. Long-term usage of RPM may cause a defect of myeloid progenitors in bone marrow.

Outcomes in kidney transplantation with mycophenolate mofetil-based maintenance immunosuppression in China: a large-sample retrospective analysis of a national database.

There is no large data analysis reporting the outcome of Chinese kidney transplant patients using mycophenolate mofetil (MMF). This study analyzed 6719 patients from the Chinese Scientific Registry of Kidney Transplantation using MMF, which included 1153 from donation after cardiac death (DCD), 1271 from donation after brain and cardiac death (DBCD), and 4295 from living donor (LD). Compared with the transplants from deceased donor (DD), better outcomes including 3-year graft survival probabilities (LD = 95.8% vs. DD = 91.3%), incidence of delayed graft function (DGF, LD = 2.4% vs. DD = 17.7%), infection (LD = 10.7% vs. DD = 20.7%), graft loss (LD = 2.3% vs. DD = 6.3), and death (LD = 1.3% vs. DD = 3.2%) were shown in the LD group, with similar incidences of acute rejection (AR, LD = 3.7% vs. DD = 4.7%), hyperuricemia (LD = 21.7% vs. DD = 22.2%) within postoperative 1 year, and serum creatinine (Scr) >133 µmol/l at 1 year (LD = 18.8% vs. DD = 18.6%). Nonsignificant differences were found between the DCD and DBCD group. The 5-year survival of patient and graft in the LD group were 97.5% and 93.0%. Adjusted Cox model for graft loss showed significant associations with DGF [hazard ratio 3.7 (95% CI: 2.4, 5.8)], AR [2.8 (1.7, 4.6)], Scr >133 µmol/l at 1 year [2.6 (1.5, 4.2)], hyperuricemia [2.3 (1.6, 3.3)], and DD [1.6 (1.1, 2.4)].

Characterisation of Tertiary Lymphoid Organs in Explanted Rejected Donor Kidneys.

PURPOSE: Tertiary lymphoid organs (TLOs) have been described within organ allografts, but whether they promote destructive or beneficial alloimmune responses remains controversial. This study aimed to characterize TLO distribution in human chronically rejected renal allografts and to explore their functions. METHODS: A total of 29 explanted chronically rejected and 12 acutely rejected renal allografts were analyzed by immunohistochemistry. The distribution of TLOs, T cells, follicular dendritic cells, B cells, and follicular regulatory T (Tfr) cells, as well as Ki67, peripheral lymph node addressin (PNAd), podoplanin, AID, IL-17, IL-21, IL-10, and C4d expression were detected by immunohistochemistry. Correlations between lymphoid neogenesis and the expression of IL-17, IL-21, C4d, podoplanin, IL-10, and Foxp3 were evaluated. In addition, the duration of graft function was compared between allografts that harbored or lacked TLOs. RESULTS: TLOs were detected in 27.6% of chronically rejected renal grafts, but they rarely had germinal centers. Lymphoid neogenesis negatively correlated with CXCR5 expression, and almost completely correlated with IL-17 expression. Those grafts that harbored a TLO functioned for an average of 5.98 years and those without a TLO lasted only about half as long with an average of 2.91 years. However, in grafts that harbored a TLO, Foxp3(+) cells were comparitively less than those without a TLO. Foxp3(+)CXCR5(+) Tfr cells and IL-10(+) cells were rare in grafts, irrespective of the presence of a TLO. CONCLUSION: TLOs in chronically rejected kidney allografts may be an epiphenomenon of the inflammatory process that is related to graft duration.

Effectiveness and safety of calcineurin inhibitor withdrawal in kidney transplantation: a meta-analysis of randomized controlled trials.

BACKGROUND: The purpose was to compare the effectiveness and safety of calcineurin inhibitors (CNIs) withdrawal and continued therapies in kidney transplant recipients. METHODS: We searched the PubMed, MEDLINE, Springer, Elsevier Science Direct, Cochrane Library and Google Scholar up to May 2014. Risk ratio (RR) or weighted mean difference (WMD) and their 95 % confidence intervals (CIs) were calculated in fixed-effects model or random-effects model when appropriate. Besides, sensitivity analysis was performed based on the addition of sirolimus in initial immunosuppression protocols. RESULTS: Total seven studies with 1071 kidney transplant recipients received CNIs withdrawal therapy (experimental group) and 792 kidney transplant recipients received CNIs continued therapy (control group) were included in the meta-analysis. The overall estimates of acute rejection rate (RR = 1.64, 95 % CI: 1.19-2.27, P = 0.003), mean measured glomerular filtration rate (WMD = 9.50, 95 % CI = 2.96-16.03, P = 0.004), thrombocytopenia (RR = 3.39, 95 % CI: 2.27-5.05, P < 0.00001) and hypertension (RR = 0.56, 95 % CI: 0.40-0.78, P = 0.0006) showed that there were significant differences between the CNIs withdrawal and continued therapies in kidney transplant recipients, while no significant differences were found between groups in survival rate, graft survival rate, diabetes, hypercholesterolemia, hypertriglyceridemia and malignancies. In addition, two studies, in which sirolimus was not used in initial immunosuppression protocol, were excluded in sensitivity analysis and the results were still consistent with the overall analysis. CONCLUSIONS: CNIs withdrawal therapy in kidney transplant recipients could significantly decrease risk of hypertension and improve glomerular filtration rate, accompanying with increased risk of acute rejection and thrombocytopenia, compared with the CNIs continued therapy.

Ketanserin, a serotonin 2A receptor antagonist, alleviates ischemia-related biliary fibrosis following donation after cardiac death liver transplantation in rats.

Biliary fibrosis is a major complication after donation after cardiac death (DCD) liver transplantation. In this process, the roles of serotonin [5-hydroxytryptamine (5-HT)] and the 5-HT2A receptor subtype are still unknown. In this study, we analyzed markers of portal fibroblast (PF)/myofibroblast (MF) transdifferentiation such as transforming growth factor ß1 (TGF-ß1), phosphorylated smad2/3, α-smooth muscle actin (α-SMA), collagen I, and collagen III in a primary culture system of PFs after the administration of 5-HT or 5-HT plus ketanserin (a selective 5-HT2A receptor antagonist). A rat DCD transplant model was established with 30 minutes of warm ischemia and 4 hours of cold ischemia during organ procurement. Recipients were intraperitoneally injected with ketanserin (1 mg·kg(-1)·day(-1)) or normal saline. Grafts without in situ warm ischemia instead of minimal cold storage (30 minutes) served as controls. The serum biochemistry, the liver contents of 5-HT and hydroxyproline (HYP), and the expression of fibrosis-related genes (including TGF-ß1, matrix metalloproteinase 2, procollagen α1, and α-SMA messenger RNA) were determined. The extent of biliary fibrosis was also assessed histopathologically. The results indicated that ketanserin inhibited 5-HT-activated TGF-ß1-smad2/3 signaling in vitro and thereby depressed the MF conversion of PFs. Rats receiving DCD livers showed increased liver contents of 5-HT and HYP, impaired biliary function, up-regulation of fibrosis-related genes, and aggravated biliary fibrosis. However, these phenomena were alleviated by treatment with ketanserin. We concluded that the profibrotic activity of 5-HT occurred through the activation of TGF-ß1 signaling and the 5-HT2A receptor. Thus, these data suggest that the 5-HT2A receptor may be a potential therapeutic target for ischemia-related biliary fibrosis after DCD liver transplantation.

[Frequency analyses of human cytomegalovirus specific CD8(+) T cell in kidney transplantation].

OBJECTIVE: To establish a new detection method for cytomegalovirus (CMV) specific cytotoxic CD8(+)T cells and examine its proportion and significance in peripheral blood from kidney transplant recipients. METHODS: A total of 30 recipients of kidney transplantation from January 2009 to December 2010 for the first time were enrolled. And 10 healthy volunteers were selected as health control group. Tetramer technology was applied. The proportions of CMV antigen specific T cells expressed in each group were detected directly by flow cytometry. RESULTS: The positive rate of CMV antigen specific CTL, CMV-pp65 specific CD8(+)T cell was between 0.16%-7.21% in kidney transplant recipients (n = 30) and health control group (n = 10). The proportions of CMV antigen specific CTL were 2.95% ± 0.62% in CMV+ group. And it was significantly higher than that in CMV-group (1.17% ± 0.45%) and health control group (0.65% ± 0.17%) (P = 0.003,0.006). In CMV+ group, the proportion of CMV antigen specific CTL was 2.95% ± 0.62% in CMV-pp65 positive phase and decreased significantly to 1.50% ± 0.32% after turning into negative phase. In CMV+ group (n = 15), the proportion of CMV antigen specific CTL was positively related with the number of CMV-pp65 positive cells (Pearson test, r = 0.871, P < 0.01). CONCLUSIONS: The proportion of specific CTL is an important guide for evaluating and judging the prognosis of CMV infection. And it may provide rationales for future targeted therapy in kidney transplant recipients.

[Expression of T lymphocyte cytokines in peripheral blood of renal transplant recipients].

OBJECTIVE: To evaluate the clinical values of T-lymphocyte cytokines in renal transplant acute rejection. METHODS: A total of 31 recipients with renal transplantation and 15 healthy volunteers from January 2010 to January 2012 were enrolled and divided into acute rejection group (n = 11) and stable renal allograft function group (n = 20) according to the inclusion criteria. Peripheral blood was collected from the patients before transplantation, 1, 7, 14, 28 day after transplantation and acute rejection onset. Cytometric bead array (CBA) was used to monitor the levels of interleukin-17 (IL-17), interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin(IL)-10, IL-6, IL-4 and IL-2. The associations of the changes and levels of cytokines in 3 groups were examined with Pearson correlation analysis. RESULTS: The levels of IL-17A, TNF, IL-10 and IL-2 in recipients before transplantation were (3.40 ± 1.29) , (1.79 ± 0.53) , (2.73 ± 0.65) and (1.79 ± 1.02) ng/L respectively and decreased significantly compared to healthy volunteers ((4.52 ± 2.01), (3.36 ± 1.09) , (3.91 ± 0.42) , (3.12 ± 1.07) ng/L respectively, all P < 0.05). However the levels of IFN-γ, IL-6 and IL-4 showed no significant changes between two groups (all P > 0.05). In acute rejection group after transplantation, the levels of IL-17A, IFN-γ, IL-10 and IL-6 were (9.47 ± 4.75) , (5.01 ± 2.23) , (12.20 ± 5.79) , (6.55 ± 3.45) ng/L respectively and increased significantly compared to the renal allograft function group ((4.39 ± 1.26), (2.90 ± 0.87),(5.68 ± 2.25) and (2.10 ± 0.70) ng/L respectively, all P < 0.05); the level of IL-17A was correlated with those of IFN-γ and IL-4 (Pearson r = 0.519, 0.395, both P < 0.01), the level of IFN-γ was correlated with those of IL-4 and IL-2 (r = 0.276, 0.335, all P < 0.05) , the level of TNF was correlated with that of IL-4 (r = 0.423, P < 0.05) and the level of IL-10 was correlated with that of IL-6 (r = 0.361, P < 0.05). CONCLUSIONS: Cytokines play an important role in acute rejection of renal transplant. And further understanding of its mechanism may provide experimental and preventive rationales.

Combination of IL-1 receptor antagonist, IL-20 and CD40 ligand for the prediction of acute cellular renal allograft rejection.

PURPOSE: The outcome of renal transplantation is difficult to predict, even with an allograft biopsy. The aim of this study was to develop a sensitive, specific and noninvasive method for prediction of acute cellular rejection (ACR). METHODS: Luminex analysis was used to determine the levels of 95 cytokines/chemokines and their soluble receptors in sera from recipients with: ACR (in the first month post-transplantation, before and during rejection, and after rejection reversal); stable allograft function; delayed graft function (DGF); pulmonary infection. Evaluation of significant differential protein expression in ACR patients compared with stable allograft controls revealed a three-analyte combination as a marker of renal transplantation outcome. The predictive value of this combination was further validated in DGF and infection groups and in a blind binary code study of 24 additional serum samples. RESULTS: Significant differential expression was detected in 26 proteins expressed in patients during the period preceding an ACR episode compared with stable controls. A blood test for discrimination of such patients was developed based on the simultaneous quantification of three analytes (IL-1 receptor antagonist, IL-20 and sCD40 ligand). This test exhibited 90.9 % sensitivity, 96 % specificity, a positive predictive value (PPV) of 95.2 % and a negative predictive value (NPV) of 92.3 %. Moreover, this combination allowed discrimination between patients with ACR and DGF and pulmonary infection. CONCLUSIONS: With further development and validation, this blood test can be used to predict ACR and direct the treatment of transplant patients in the clinic.

Γ-aminobutyric acid B receptor improves carbon tetrachloride-induced liver fibrosis in rats.

BACKGROUND: It was well known that angiotension II can inhibit hepatic stellate cell activation. The GABAB receptor was upregulated when the hepatic stellate cell line was stimulated by angiotension II in our previous study. But the role of the GABAB receptor in liver fibrosis has never been reported. AIM: In the present study, we investigated the effects of this receptor on carbon tetrachloride-induced liver fibrosis in rats. METHODS: The rats were divided into four groups including GABAB receptor agonist, antangonist, model and control group. α-smooth muscle actin (α-SMA) and GABAB receptor expression levels were detected by immunohistochemistry and real-time polymerase chain reaction. Liver function tests were performed once blood samples was taken; Western blot analysis was used to detect protein expression level of α-SMA and TGF-ß1. RESULTS: We found baclofen ameliorated the CCl4-induced rats's liver fibrosis. The highest liver enzymes and α-SMA protein levels were found in the CGP35348 group. CONCLUSION: The GABAB receptor may have a protective role in the liver.

A comparison of hepatic segmental anatomy as revealed by cross-sections and MPR CT imaging.

To compare the areas of human liver horizontal sections with computed tomography (CT) images and to evaluate whether the subsegments determined by CT are consistent with the actual anatomy. Six human cadaver livers were made into horizontal slices with multislice spiral CT three-dimensional (3D) reconstruction was used during infusion process. Each liver segment was displayed using different color, and 3D images of the portal and hepatic vein were reconstructed. Each segmental area was measured on CT-reconstructed images, which were compared with the actual area on the sections of the same liver. The measurements were performed at four key levels namely: (1) the three hepatic veins, (2) the left, and (3) the right branch of portal vein (PV), and (4) caudal to the bifurcation of the PV. By dividing the sum of these areas by the total area of the liver, the authors got the percentage of the incorrectly determined subsegmental areas. In addition to these percentage values, the maximum distances of the radiologically determined intersegmental boundaries from the true anatomic boundaries were measured. On the four key levels, an average of 28.64 ± 10.26% of the hepatic area of CT images was attributed to an incorrect segment. The mean-maximum error between artificial segments on images and actual anatomical segments was 3.81 ± 1.37 cm. The correlation between radiological segmenting method and actual anatomy was poor. The hepatic segments being divided strictly according to the branching point of the PV could be more informative during liver segmental resection.

Crystallization and preliminary crystallographic study of a trypsin-resistant catalytic domain of human calcineurin.

Calcineurin, a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase, plays a key role in a number of cellular pathways, including T-cell activation, and is an important molecular target of the immunosuppressive drugs cyclosporin A and FK506. To understand the structural basis underlying the activation of calcineurin by calmodulin, X-ray crystallography was employed to solve the three-dimensional structure of the free calcineurin catalytic domain (residues 20-347 of the A subunit). To accomplish this, a bacterially expressed glutathione S-transferase (GST) fusion protein of the human calcineurin catalytic domain was first purified by GST-affinity chromatography. After limited digestion by trypsin, the catalytic domain (Cncat) was purified using anion-exchange and size-exclusion chromatography. Crystallization of Cncat was achieved by the hanging-drop vapour-diffusion method at pH 6.5 using PEG 6000 as precipitant. The diffraction results showed that the Cncat crystal belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 161.6, b = 87.4, c = 112.0 Å. There are four Cncat molecules in the asymmetric unit, with 49.5% solvent content. An X-ray diffraction data set was collected to 2.87 Šresolution and a clear molecular-replacement solution was obtained. The active site of Cncat is open to the solvent channels in the crystal packing.

LNCRNA XIST Inhibits miR-377-3p to Hinder Th17 Cell Differentiation through Upregulating ETS1.

Background: Th17 cell differentiation is involved in the development and progression of many diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Present study mainly focused on the role of LINC-XIST in Th17 cell differentiation. Methods: The naïve CD4+ T cells were isolated from human whole blood. Cells were cultured under Th17 cell-polarizing condition for 6 days. The expression of LINC-XIST and miR-153-3p was measured by qPCR. The relationship between LINC-XIST, miR-153-3p, and ETS1 was predicted by TargetScan website and authenticated by luciferase reporter assay. ELISA assays were conducted to evaluate the IL-17 concentration. Western blot was utilized to measure the protein expression of ETS1. Th17 cell frequency was examined by flow cytometry. Results: The expression of XIST markedly decreased and miR-153-3p expression markedly increased with Th17 cell differentiation. The mRNA expression of IL-17, IL-17 concentration, and Th17 cell frequency were observably decreased in overexpressed LINC-XIST group. Luciferase reporter assay authenticated that miR-153-5p was directly regulated by LINC-XIST. miR-153-3p inhibitor observably decreased IL-17 concentration, mRNA expression of IL-17, and Th17 cell frequency while si-XIST reversed this impact. ETS1 was confirmed to be regulated by miR-153-5p via luciferase reporter assay. In addition, ETS1 markedly decreased IL-17 mRNA expression, IL-17 concentration, and Th17 cell frequency while miR-153-5p mimic reversed this impact. Conclusion: LNCRNA XIST inhibited miR-377-3p to hinder Th17 cell differentiation through upregulating ETS1.

Serum hematopoietic growth factors as diagnostic and prognostic markers of acute renal allograft rejection: a potential role for serum stem cell factor.

OBJECTIVES: Hematopoietic growth factors (HGFs) are proliferative and chemotactic agents for hematopoietic stem cells, although their functions on renal transplantation are rarely reported. This study aimed to investigate the association of HGFs with acute T cell-mediated renal rejection. EXPERIMENTAL DESIGN: A cytokine bead array was used to analyze 10 HGFs in the sera of 32 patients with acute T cell-mediated renal allograft rejection before and during rejection and after rejection reversal. Stable renal allograft recipients (n=38) were also investigated. RESULTS: Serum levels of stem cell factor (SCF), IL-3, flt3 ligand, G-CSF, M-CSF and GM-CSF were elevated during episodes of rejection compared with before and after rejection. Serum concentrations of SCF, G-CSF, flt3 ligand and IL-3 were significantly higher than in the absence of rejection. Moreover, serum SCF levels were significantly higher in patients before rejection versus stable controls. CONCLUSIONS: HGFs are reported to be involved in acute T cell-mediated renal allograft rejection. A direct correlation was observed between SCF levels and the occurrence of acute renal allograft rejection which may represent an effective diagnostic and predictive biomarker for acute cellular renal allograft rejection.

Comparison of hepatitis B X gene mutation between patients with hepatocellular carcinoma and patients with chronic hepatitis B.

Hepatitis B virus (HBV), a major causative agent of hepatocellular carcinoma (HCC), encodes an oncogenic X protein (HBx) that transcriptionally activates multiple viral and cellular promoters. The present study aimed to identify the specific gene mutation related to the clinical outcome of HCC. Seventy-two HBV-infected patients (38 with chronic HBV infection and 34 with HCC) with well-characterized clinical profiles were enrolled. The HBx region was amplified from patient serum samples and analyzed by sequencing. Genotypes were determined using the National Center for Biotechnology Information genotype tool. All isolates were genotype B or C. Enhancer II nucleotide substitutions in the HCC group were significantly different from those in the chronic hepatitis B (CHB) group (Ρ < 0.05). HCC patients with genotype C had a higher risk of harboring the 1762/1764 double mutation than those with genotype B. The incidence of the 1762/1764 double mutation was higher in the high viral load group (>10(6) copies/ml) than in the low viral load group (≤10(6) copies/ml) (P = 0.03). The 1762/1764 double mutations may be related to the genotype and viral load. We found significantly more direct repeat sequence 1 (DR1) nucleotide substitutions in the HCC group (32.4%, 11/34) than in the CHB group (10.5%, 4/38) (Ρ < 0.05). Patients with higher viral load and genotype C had a higher incidence of 1762/1764 double mutations, which may not be related with development of HCC. Enhancer II and DR1 may play an important role in HCC development via nucleotide substitution.

[Role of rapamycin in cholangiocytes regeneration after liver transplantation].

OBJECTIVE: To investigate the role of IL-6/STAT3 pathway in the proliferation of cholangiocytes after liver transplantation and determine whether or not rapamycin (RPM) depresses the regeneration of cholangiocytes by blocking the activation of STAT3. METHODS: Rats were randomized into OLT-1 h and OLT-12 h groups (supplied livers preserved for 1 or 12 h), anti-sIL-6R group (rats of the OLT-12 h group injected intravenously with 16.7 µg/kg anti-rat sIL-6R antibody at 1 hour pre-operation and daily post-operation), RPM group (rats of the OLT-12 h group injected intraperitoneally with 0.05 mg/kg RPM for 3 days pre-operation and daily post-operation) and sham group (transverse laparotomy and closure without liver manipulation). At 1, 3, 7, 14 d post-operation, the IL-6 concentration in liver homogenate and cholangiocytes proliferation were detected by ELISA (enzyme linked immunosorbent assay) and histochemistry respectively. The expressions of IL-6 mRNA, phosphorylated-STAT3 and cyclin D1 protein in cholangiocytes were determined by real-time RT-PCR (reverse transcription-polymerase chain reaction) or Western blot. The DNA binding activity of STAT3 was determined by electrophoretic mobility shift assay. The serum concentrations of ALP (alkaline phosphatase) and GGT (γ-glutamyltransferase) were also measured. RESULTS: The minimal expressions of IL-6, p-STAT3, cyclin D1 and DNA binding activity of STAT3 were detected in OLT-1h group. And a slight increase of IOD (integral optical density) ratio (38 ± 10 and 22 ± 7) indicated a mild cholangiocytes proliferation. The concentrations of GGT were (69 ± 6) U/L, (34 ± 4) U/L and ALP (86 ± 9) U/L, (45 ± 3) U/L. The expression of IL-6 in liver homogenate were (273 ± 20) ng/g, (159 ± 18) ng/g and 0.40 ± 0.04, 0.23 ± 0.04 in cholangiocytes. The expressions of P-STAT3 were 0.420 ± 0.023 and 0.230 ± 0.040 in cholangiocytes and cyclin D1 0.580 ± 0.023 and 0.420 ± 0.015 respectively. Cholangiocytes responded to extended cold preservation with severe bile duct injures and marked increases in IL-6 secretion, p-STAT3 and cyclin D1 protein expression and DNA binding activity of STAT3, followed by compensatory cholangiocytes regeneration. Meanwhile biochemical index and morphology indicated that bile duct injury recovered at 14 d post-operation. The IOD ratios were 38 ± 10 and 22 ± 7 respectively. The expressions of IL-6 were (659 ± 28) and (446 ± 23) ng/g in liver homogenate and 0.73 ± 0.06 and 0.54 ± 0.04 in cholangiocytes. The expression of P-STAT3 were 0.72 ± 0.04 and 0.58 ± 0.06 in cholangiocytes and cyclin D1 0.88 ± 0.04 and 0.74 ± 0.07 respectively. However, anti-sIL-6R inhibited the cholangiocytes proliferation and reduced the expressions of IL-6, STAT3 and cyclin D1. The DNA binding activity of STAT3 with cellular injury and the increases of serum ALP or GGT were also abrogated by the administration of anti-sIL-6R. With similar results, the RPM treatment had insignificant effects on the expression of IL-6. CONCLUSION: The IL-6/STAT3 pathway initiates the cholangiocytes regeneration after liver transplantation so as to accelerate the biliary recovery. However RPM represses the cholangiocytes regeneration by inhibiting the STAT3 activation. It may have a negative effect on the healing and recovery of bile ducts in grafts with extended cold preservation.

[Identification and functional study of Tim-1(+)CD19(+) regulatory B cell in kidney transplantation recipients].

OBJECTIVE: To observe the ratio of Tim-1(+)CD19(+) B cell in the peripheral blood of kidney transplantation recipients and elucidate its functions. METHODS: From December 2009 to June 2010, a total of 35 pairs of kidney transplant recipients were selected and divided into 3 groups: healthy donors as control (n = 35), pre-transplantation (n = 35) and post-transplantation (n = 35). The profiles of Tim-1(+)CD19(+) B cell in kidney transplantation donors and recipients were analyzed and sorted by flow cytometry (FCM). Mixed lymphocyte culture (MLC) was carried out between kidney transplantation donors and recipients. After the additions of Tim-1(+)CD19(+) and Tim-1(-)CD19(+) B cells, there were 3 groups: Tim-1(+), Tim-1(-) and blank. Lymphocyte proliferation and inhibition status were evaluated by propidium iodide uptake and Annexin V binding. And the cytokine levels were detected by FCM. RESULTS: The absolute values of peripheral CD19(+)B cells were (170 ± 90), (202 ± 99), (155 ± 71) cells/µl in the pre-transplantation, post-transplantation and control groups respectively, post-transplantation group were higher than control group (P = 0.0300). The Tim-1(+)CD19(+) cell ratios were (2.20 ± 0.98)%, (35.46 ± 10.66)% and (1.95 ± 0.95)% in three groups. And the differences were statistically significant (both P < 0.01). Tim-1(+)CD19(+) B and Tim-1(-)CD19(+) B cells were added into MLC respectively. The early apoptotic cells of the Tim-1(+) group were higher than those in the Tim-1(-) group [(45.31 ± 12.37)% vs (10.92 ± 2.14)%, P < 0.05] and significantly higher than the blank group [(1.93 ± 0.26)%, P < 0.01]. Late apoptotic and dead cells of the Tim-1(+) group were higher than those in the Tim-1(-) group [(21.32 ± 5.67)% vs (2.32 ± 0.31)%, P < 0.01] and the blank group [(1.27 ± 0.19)%, P < 0.05]. The interleukin 10 levels in MLC supernatant of the Tim-1(+) group were significantly higher than those in the Tim-1(-) group [(5.32 ± 0.37) pg/ml vs (2.46 ± 0.25) pg/ml, P = 0.0001]. However, the interferon-γ levels were lower than those in the Tim-1(-) group [(1.51 ± 0.22) pg/ml vs (4.69 ± 0.32) pg/ml, P = 0.0015]. CONCLUSION: Present in the peripheral blood of kidney transplantation recipients, Tim-1(+)CD19(+) B cell has the capacity of promoting lymphocytic apoptosis. As a new regulatory subset of B cells, it plays important roles in the immune responses of transplantation.