Exosome-derived galectin-9 may be a novel predictor of rejection and prognosis after liver transplantation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

The study on the relationship between expression of B7-H1 on HBV transgenic mice and immune tolerance to HBV / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether there is an association between the expression of B7-H1 in HBV transgenic mice and the immune tolerance to HBV.</p><p><b>METHODS</b>T cells stimulatory capacity of DC was analyzed using mixed lymphocyte reaction. Expression of MHC-II, CD80, CD86, B7-H1 on DC was detected by Flow Cytometry. IL-2, IFNgamma, IL-10 production of T cells were determined by using ELISA. B7-H1 mRNA and protein expression in liver tissue were detected by RT-PCR and Western blotting respectively.</p><p><b>RESULTS</b>The ability of DC cells from HBV transgenic mice to stimulate T cell proliferation was significantly impaired compared with DC cells from control mice (t = 16.674, 19.674, 21.712, P less than 0.01). Expression of MHC-II, CD80 on DC was markedly decreased in transgenic mice (t = 7.910, 6.413, P less than 0.05). Meanwhile, the expression of CD86 and B7-H1 on DC cells in HBV transgenic mice were not significantly different from that in control mice. The levels of IL-2, IFNgamma, IL-10 in supernatant of T cells was significantly lower compared with controls (t = 18.712, 18.712 and 11.683, P less than 0.05). There was no significant difference in B7-H1 expression at mRNA and protein levels in liver tissue compared with controls.</p><p><b>CONCLUSIONS</b>Functional defect of DC, partly due to decreased expression of MHC-II, CD80, but not related to B7-H1 expression, is the cause for immune tolerance to HBV in HBV transgenic mice.</p>

Spectrum and risk factors for invasive candidiasis and non-Candida fungal infections after liver transplantation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Invasive fungal infections are an important cause of posttransplant mortality in solid-organ recipients. The current trend is that the incidence of invasive candidiasis decreases significantly and invasive aspergillosis occurs later in the liver posttransplant recipients. The understanding of epidemiology and its evolving trends in the particular locality is beneficial to prophylactic and empiric treatment for transplant recipients.</p><p><b>METHODS</b>A retrospective analysis was made of recorded data on the epidemiology, risk factors, and mortality of invasive fungal infections in 352 liver transplant recipients.</p><p><b>RESULTS</b>Forty-two (11.9%) patients suffered from invasive fungal infection. Candida species infections (53.3%) were the most common, followed by Aspergillus species (40.0%). There were 21 patients with a superficial fungal infection. The median time to onset of first invasive fungal infection was 13 days, first invasive Candida infection 9 days, and first invasive Aspergillus infection 21 days. Fifteen deaths were related to invasive fungal infection, 10 to Aspergillus infection, and 5 to Candida infection. Invasive Candida species infections were associated with encephalopathy (P = 0.009) and postoperative bacterial infection (P = 0.0003) as demonstrated by multivariate analysis. Three independent risk factors of invasive Aspergillus infection were posttransplant laparotomy (P = 0.004), renal dysfunction (P = 0.005) and hemodialysis (P = 0.001).</p><p><b>CONCLUSIONS</b>The leading etiologic species of invasive fungal infections are Candida and Aspergillus, which frequently occur in the first posttransplant month. Encephalopathy and postoperative bacterial infection predispose to invasive Candida infection. Posttransplant laparotomy and poor perioperative clinical status contribute to invasive Aspergillus infection. More studies are needed to determine the effect of prophylactic antifungal therapy in high risk patients.</p>

Expression and prognostic value of Polo-like kinase 1, E-cadherin in the patients with hepatocellular carcinoma / 中华外科杂志

<p><b>OBJECTIVES</b>To study the expression of Polo-like kinase 1 (PLK1) and E-cadherin in the tissues of hepatocellular carcinoma, and to discuss the relationship between them and clinical-pathological features, and to evaluate their prognostic value of hepatocellular carcinoma after liver transplantation.</p><p><b>METHODS</b>mRNA and protein expression of PLK1, E-cadherin were detected by RT-PCR and immunohistochemistry method respectively, the correlations of them with clinical-pathological data, tumor free time, recurrence rate were compared and analyzed.</p><p><b>RESULTS</b>The mRNA expression was observed in 90.0% for PLK1 and 96.0% for E-cadherin, and higher in cancerous' tissues than paracancerous' of all cases for PLK1 but no trend for E-cadherin. The positive and decreased expression rate for PLK1 and E-cadherin was observed in 60.0% and 50.0% respectively, the positive PLK1 expression was correlated with preoperative serum alpha-fetoprotein (AFP) only (chi2 = 4.433, P = 0.035), while E-cadherin expression was associated with none of the clinical-pathological features. There was a correlation between the positive PLK1 and decreased E-cadherin expression (chi2 = 5.333, P = 0.021). PLK1 (P = 0.006), E-cadherin (P = 0.019) and larger tumor (P = 0.019), portal vein tumor thrombi (P = 0.030), Edmondson grading (P = 0.019), preoperative serum AFP (P = 0.020) were all correlated with recurrence rate under Kaplan-Meier analysis, while only PLK1 (RR = 3.104, P = 0.009) had significant difference under Cox regression analysis.</p><p><b>CONCLUSIONS</b>The positive PLK1 expression and the decreased E-cadherin expression indicate higher recurrence rate of HCC after liver transplantation, and PLK1 is a independent risk factor.</p>

Efficacy of hepatitis B immunoglobulin in relation to the gene polymorphisms of human leukocyte Fcgamma receptor III (CD16) in Chinese liver transplant patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although the use of hepatitis B immunoglobulin (HBIG) may lead to a significant reduction in recurrent hepatitis B virus (HBV) infection and improve the survival of patients who have undergone liver transplantation (LT) for hepatitis B-related diseases, the recurrence of the disease still remains at a lower level. Different clinical curative effects were observed in patients with the same HBV-related diseases and the same therapy. This study was undertaken to investigate whether the efficacy of HBIG is associated with FCGR3A gene polymorphisms in Chinese liver transplant patients.</p><p><b>METHODS</b>Altogether 77 patients who had received liver transplantation for hepatitis B-related diseases with more than one-year survival after surgery were studied. The recurrence of HBV was characterized by the appearance of HBsAg in serum after the operation. The FCGR3A genotyping was performed using genomic DNA sequencing (ABI 3037). Single nucleotide polymorphism at nucleotide 559 was detected by Polyphred.</p><p><b>RESULTS</b>Of the 77 patients, 14 were complicated with HBV recurrence post-transplant. The FCGR3A at nucleotide 559 TT was observed in 35 (45.5%) subjects, whereas TG in 31 (40.3%) and GG in 11 (14.3%). In the 559G carrier group (n = 42, 54.5%), the risk of HBV recurrence was 9.5%, and 1- and 2-year recurrence-free survival rates were 95.2% and 88.7%, respectively. In the 559G noncarrier group (n = 35, 45.5%), the risk of HBV recurrence was 28.6%, and 1- and 2-year recurrence-free survival rates were 74.3% and 69.3%, respectively. The risk of HBV recurrence and the recurrence-free survival rate were both statistically different between the 559G carrier and noncarrier groups (P < 0.05).</p><p><b>CONCLUSIONS</b>A single nucleotide polymorphism (T/G) at position 559 of the FCGR3A gene was found in Chinese patients. The efficacy of HBIG in prophylaxis of HBV recurrence after LT is associated with the gene polymorphism, so detecting FCGR3A genotypes can be a clinical reference of the HBIG administration.</p>

Organogenesis of developing metanephroi allografted into nonimmunosuppressed adult rats / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To explore novel methods of possible donor organ supply and immunologic tolerance induction of organ transplantation.</p><p><b>METHODS</b>Whole metanephroi from d14-19 (E14-E19) embryos of pregnant rats were grouped and allografted into the omenta or near remnants of renal vessels of nonimmunosupressed adult rats. At the time of implantation, host rats underwent unilateral nephrectomy. Four weeks after implantation, allografted metanephroi in host rats were removed for gross, biochemical and histopathological examination.</p><p><b>RESULT</b>Four weeks post-implantation, (1) E19 and E18 metanephroi had enlarged,but were replaced by connective tissues. (2) E17 and E16 metanephroi showed the signs of acute rejection such as hypercellular glomeruli and lymphocyte infiltration in peritubular spaces. E16 grafted metanephroi underwent mild acute rejection of Banff schema, while E17 had moderate or severe acute rejection. When Cyclosporine A was administrated, E17 metanephroi formed mature nephrons and collecting ducts with few lymphocyte infiltration. (3) Metanephroi from E15 and E14 embryos allografted into the omentum or near remnants of renal vessels of uninephrectomized adult rats were enlarged and vascularized, and formed mature tubules and glomeruli. (4) The concentrations of urea nitrogen and creatinine in cyst fluid of E15 and E16 metanephroi were increased 40-fold and 50-fold, which were comparable to those in bladder urine. (5) In contrast, rat metanephroi did not grow or differentiate in rats without host kidney resection.</p><p><b>CONCLUSION</b>E14 and E15 metanephroi allografted into nonimmunosuppressed adult rats or E17 into cyclosporine-treated hosts undergo growth and differentiation and become vascularized. A variety of factors affect the growth and development of allografted metanephroi, while rejection is the main one.</p>

Infusion of nonmyeloablative bone marrow alleviates acute rejection reaction in liver allotransplantation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To study the effect and implication of nonmyeloablative donor specific bone marrow (DSBM) infusion on the immunoreaction of liver allotransplantation.</p><p><b>METHODS</b>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).</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>

Ciclosporin down-regulates interferon-gamma gene transcription via its inhibition of nuclear factor kappaB activity after liver transplantation / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the relation between the activity of nuclear factor kappaB (NF-kappaB) and the expression of interferon (IFN)-gamma gene transcription with or without ciclosporin treatment after liver transplantation.</p><p><b>METHODS</b>Orthotopic liver transplantation was performed in this study. Group I: syngeneic control (Wistar-to-Wistar); Group II: acute rejection (SD-to-Wistar); Group III: acute rejection treated with ciclosporin by intramuscular route (SD-to-Wistar + ciclosporin). Electrophoretic mobility shift assay and reverse transcriptase polymerase chain reaction were used to analyze NF-kappaB activity of splenocytes and IFN-gamma gene transcription expression of grafted liver with or without ciclosporin treatment after liver transplantation. Histopathological examination was also used in this study.</p><p><b>RESULTS</b>Low NF-kappaB activity was only detected at day 5 and day 7 in Wistar-to-Wistar group after transplantation, meanwhile low IFN-gamma mRNA expression was detected at any time in this group. In contrast, high NF-kappaB activity was detected in SD-to-Wistar group and high level IFN-gammamRNA expression was detected at all time points in this group. The activity of NF-kappaB and IFN-gammamRNA expression were significantly inhibited in SD-to-Wistar + ciclosporin group which was significantly lower than that of SD-to-Wistar group (P < 0.001). A good correlation was found between activity of NF-kappaB and IFN-gamma mRNA expression in this study (r=0.815, P <0.01).</p><p><b>CONCLUSIONS</b>The change of expression IFN-gamma mRNA is at least partially due to the activity change of NF-kappaB after orthotopic liver transplantation. CsA down-regulates NF-kappaB activity and further inhibit IFN-gamma gene transcription.</p>

Antitumor effect of immunizations with fusions of dendritic and hepatocellular carcinoma cells in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of immunization with fusions of dendritic cells and H22 cells on tumor-bearing mice and their possible mechanisms.</p><p><b>METHODS</b>Fusion cells of DC and H22 cells were prepared with polyethylene glycol (PEG). Expression of MHC and costimulatory molecules by dendritomas were determined by FACs. To study the antitumor immune preventative and therapeutic effects, fusions were subcutaneously injected into tumor-bearing mice. The cytotoxic T lymphocyte (CTL) activity was determined by LDH method, the expression of TNF-a and IFN-g in tumors were assayed by RT-PCR.</p><p><b>RESULTS</b>The data showed that the hybridomas of DC and H22 cells acquired both DC and H22 cell phenotypes. Immunization of BALB/C mice with DC/H22 fusions induced potent CTL activity (mean CTL activity=0.624+/-0.024, compared with DC + H22, DC, H22 groups, F = 65.46) and a protective immunity against a high dose of H22 tumor challenge. After treatment with hybridomas, the survival time of tumor-bearing mice was greatly extended (x2=18.45). The expression levels of TNF-a and IFN-g mRNA were remarkably increased (TNF-a, F = 47.84; IFN-g, F = 37.23).</p><p><b>CONCLUSIONS</b>The hybridomas of DC and H22 cells could induce effective antitumor immune responses and may have a useful potential in prevention and management of the recurrences and metastases of HCC.</p>

Influence of combined cyclosporine A and tacrolimus with 5-fluorouracil on hepatocellular carcinoma rats / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the effect of combined CsA and FK506 with 5-FU on hepatocellular carcinoma rats.</p><p><b>METHODS</b>A syngeneic rat model of hepatocellular carcinoma was used. Control group (A) underwent 4 ml 5% GS. Treatment group was divided into 3 groups namely, group B: only 5-FU and 5% GS; group C: 5-FU, CsA and 5% GS; group D: 5-FU, FK506 and 5%GS. Cell cycle, apoptosis, necrosis and mitochondrial transmembrane potential were measured by flow cytometry, laser scanning confocal microscopy, and electron transmission microscopy. Statistical analysis was performed by SPSS 10.0 for Windows software. Statistical comparisons were made with ANOVA followed by Dunnett's T3 or LSD test.</p><p><b>RESULTS</b>Compared to the control group, the percentage of apoptotic cells including trifle necrotic cells was significantly higher, and among the treatment group, group D was the highest, and group C was higher than group B. In the treatment group, cell cycle of hepatoma cells was mainly arrested at S phase, but in group D, G0/G1 phase cells were significantly decreased and S phase cells significantly increased. Compared to the control group, mitochondrial transmembrane potential was significantly decreased in the treatment group, among with, group B was the lowest, group C was higher than group D. Morphological changes demonstrated by electron microscopy included dispersed nuclear chromatin, loss of nucleoli, membrane bleeding, cell shrinkage, typical apoptotic bodies and marked swelling of mitochondria in the treatment group. In the control group, however, they were characterized by normal cell ultrastructure.</p><p><b>CONCLUSIONS</b>The present study reveals that 5-FU combined with CsA or FK506 demonstrated a synergistic effect on hepatocellular carcinoma rats. For FK506, the powerful mutual effect is related to the increase of tumor cell's quantity in S phase. Both CsA and FK506 can provide protection on mitochondrial transmembrane potential reduction against hepatoma cells damage from 5-FU.</p>

Alteration of P73 and P51 genes and its significance in human gastric carcinogenesis / 浙江大学学报·医学版

OBJECTIVE: To evaluate the relatinship between the expression of P51, P73 and the oncogenesis and development of human gastric carcinoma. METHODS: The expression of P73 mRNA were detected both in 32 human gastric carcinoma tissues and adjacent normal gastric tissues by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Overexpressions of P73 mRNA were found in 17/32 gastric carcinoma tissues,in 2/32 adjacent normal gastric tissues.The positive expression rate of P73 mRNA in gastric carcinooma tissues was significantly higher than in adjacent normal gastric tissues( P<0.01). However, a significant correlation was found between the positive expression rate of P73 mRNA in gastric carcinoma tissues and the TNM staging(P<0.05). THe low expressions of P51A mRNA and P51B were found in all gastric carcinoma tissues and adjacent normal gastric tissues. The expression of P51A in gastric carcinoma tissues were much higher than adjacent normal gastric tissues (P<0.05). The expression of P51B is no significant correlation was observed between gastirc carcinoma tissues and adjacent normal gastric tissues. CONCLUSION: The results suggest that there is an overexpression odf P73 and P51A mRNA in gastric cancer tissues, and their expressions is relationship with oncogenesis and developnment of gastric carcinoma.

The effect of cyclosporine A on hepatitis B virus replication in vitro / 中华传染病杂志

Objective To investigate the effect of cyclosporine A(CsA)on viral protein syn- thesis and hepatitis B virus(HBV)DNA replication in vitro.Methods The HBV DNA transfected cell line HepG2.2.15 was treated with different concentration of CsA(0.6-20.0?g/ml)for 4 days. Hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)in supernatant were detected by enzyme-linked immunosorbentassay(ELISA);intracellular hepatitis B core antigen(HBcAg)mR- NA and HBV DNA were analyzed by RT-PCR and slot blot hybridization,respectively;the phospho- rylation at tyrosine acid position 402 of PyK2 kinase(PyK2 Y402)was detected by Western blot.Re- sults CsA could suppress the expression of HBsAg and HBeAg,and inhibit the HBV DNA replica- tion in a dose-dependent manner.The suppression rate of HBsAg and HBeAg under the action of CsA at the concentration of 10.0?g/ml for 4 days was 49.7% and 34.3%,respective;similar effect was observed on HBV DNA replication,HBV DNA was only 34.9% of the control at the concentration of 10.0?g/ml of CsA.The phosphorylation level of PyK2 Y402 was declined under the action of CsA at the concentration of 2.0?g/ml.Conclusions CsA can inhibit the expression of HBsAg,HBeAg and HBV DNA replication in the HepG2.2.15 cell line in a dose-dependent manner.Suppression of the phosphorylation level of PyK2 Y402 maybe involved in the mechanism of the inhibitory activity of CsA on HBV replication.