Bile: miRNA pattern and protein-based biomarkers may predict acute cellular rejection after liver transplantation.

CONTEXT: Bile rather than blood depicts the local inflammation in the liver and may improve prediction and diagnosis of acute cellular rejection (ACR) after liver transplantation (OLT). METHODS: Secretome and miRNAs were analyzed during the first two weeks and on clinical suspicion of ACR in the bile of 45 OLT recipients. RESULTS: Levels of CD44, CXCL9, miR-122, miR-133a, miR-148a and miR-194 were significantly higher in bile of patients who developed ACR within the first 6 months after OLT and during ACR. CONCLUSION: Analysis of secretome and miRNA in bile could improve our understanding of the local inflammatory process during rejection.

Cytoreductive Surgery for Pancreatic Cancer Improves Overall Outcome of Gemcitabine-Based Chemotherapy.

OBJECTIVES: Pancreatoduodenectomy is feasible also in patients with locally advanced pancreatic adenocarcinoma (PA) nowadays. Data on risk and survival analysis of palliative pancreatic resections followed by gemcitabine-based chemotherapy (Cx) are limited. METHODS: Between 2000 and 2009, a total of 45 patients had primary cytoreductive surgery (cS) (pancreaticoduodenectomy or total pancreatectomy) followed by gemcitabine-based Cx (cS + Cx) for advanced PA. We matched 1:1 the cS + Cx group with 45 contemporaneous patients who primarily started palliative gemcitabine-based Cx for age, sex, performance status, and body mass index. Overall, survival was evaluated. RESULTS: Local R0 and R1 resection in metastatic patients was achieved in 27% and 27%, respectively. The R2 resection status without distant metastasis resulted in 33%, whereas 13% showed a local R2 status with additional metastasis (M1). Median overall survival was 10.4 months after cytoreductive pancreatic surgery and consecutive gemcitabine-based Cx versus 7.2 months after upfront gemcitabine-based Cx (P = 0.009). Median survival for R0/M1 patients was 14.4 months and 11.0 months for R2/M0 patients, whereas the median survival for R1/M1 and for R2/M1 patients was 7.3 months and 6.1 months, respectively. CONCLUSIONS: Individual patients with advanced PA had a significantly longer overall survival after palliative pancreaticoduodenectomy followed by Cx than patients in a matched control group who underwent primarily palliative Cx.

CD44 and CXCL9 serum protein levels predict the risk of clinically significant allograft rejection after liver transplantation.

The diagnosis of acute cellular rejection (ACR) after liver transplantation is based on histological analysis of biopsies because noninvasive biomarkers for allograft rejection are not yet established for clinical routines. CD31, CD44, and chemokine (C-X-C motif) ligand (CXCL) 9 have previously been described as biomarkers for cross-organ allograft rejection. Here, we assessed the predictive and diagnostic value of these proteins as serum biomarkers for clinically significant ACR in the first 6 months after liver transplantation in a prospective study. The protein levels were measured in 94 patients immediately before transplantation, at postoperative days (PODs) 1, 3, 7, and 14 and when biopsies were performed during episodes of biochemical graft dysfunction. The CD44 serum protein levels were significantly lower at POD 1 in patients who experienced histologically proven ACR in the follow-up compared with patients without ACR (P < 0.001). CXCL9 was significantly higher before transplantation (P = 0.049) and at POD 1 (P < 0.001) in these patients. Low CD44 values (cutoff, <200.5 ng/mL) or high CXCL9 values (cutoff, >2.7 ng/mL) at POD 1 differentiated between rejection and no rejection with a sensitivity of 88% or 60% and a specificity of 61% or 79%, respectively. The combination of both biomarker cutoffs at POD 1 had a positive predictive value of 91% and a negative predictive value of 67% for clinically significant ACR. Moreover, CD44 was significantly lower at the time of ACR (P < 0.001) and differentiated the rejection group from patients with graft dysfunction due to other reasons. Our results suggest that CD44 and CXCL9 may serve as predictive biomarkers to identify liver allograft recipients at risk for clinically significant ACR.

Hedgehog pathway as a potential treatment target in human cholangiocarcinoma.

BACKGROUND: Innovative treatment concepts targeting essential signaling pathways may offer new chances for patients suffering from cholangiocarcinoma (CCC). For that, we performed a systematic molecular genetic analysis concerning the Hedgehog activity in human CCC samples and analyzed the effect of Hh inhibition on CCC cells in vitro and in vivo. METHODS: Activation of the Hh pathway was analyzed in 50 human CCC samples using quantitative polymerase chain reaction (qPCR). The efficacy of Hh inhibition using cyclopamine and BMS-833923 was evaluated in vitro. In addition, the effect of BMS-833923, alone or in combination with gemcitabine, was analyzed in vivo in a murine subcutaneous xenograft model. RESULTS: Expression analysis revealed a significant activation of the Hh-signaling pathway in nearly 50% of CCCs. Hh inhibition resulted in a significant decrease in cell proliferation of CCC cells. Moreover, a distinct inhibition of tumor growth could be seen as a result of a combined therapy with BMS-833923 and gemcitabine in CCC xenografts. CONCLUSION: The results of our study suggest that the Hh pathway plays a relevant role at least in a subset of human CCC. Inhibition of this pathway may represent a possible treatment option for CCC patients in which the Hh pathway is activated.

YKL-40-gene polymorphism affects acute cellular rejection and fibrosis progression after transplantation for hepatitis C virus-induced liver disease.

BACKGROUND AND AIM: The development of end-stage graft disease is suspected to be partially determined by an individual genetic background. The aim of our study was to determine the prevalence of YKL-40-gene polymorphism in hepatitis C virus (HCV)-positive patients and its impact on the incidence of acute cellular rejection (ACR), graft fibrosis and antiviral treatment response. METHODS: A total of 149 patients, who underwent liver transplantation for HCV-induced liver disease, were genotyped for YKL-40 (rs4950928; G/C) by TaqMan Genotyping Assay. The results were correlated with 616 post-transplant graft biopsies regarding inflammation, fibrosis and evidence for ACR. RESULTS: No association of YKL-40-genotypes was observed regarding mean inflammation grade (P = 0.216) and antiviral treatment outcome (P = 0.733). However, the development of advanced fibrosis (F3-4) was significantly faster in patients with YKL-40-G-allele: t(CC) = 4.6 versus t(CG/GG) = 2.4 years; P = 0.006. Patients with lower fibrosis (F0-2) compared to advanced fibrosis (F3-4) received significantly more frequent dual immunosuppression (calcineurin inhibitors [CNIs]/mofetile mycophenolate [MMF] vs CNIs; P = 0.003). ACR-occurrence was associated with YKL-40-genotypes (ACR: CC = 60.4%, CG = 25.0% and GG = 14.6% vs non-ACR: CC = 74.2%, CG = 23.8% and GG = 2.0%; P = 0.009) and with gender compatibility between donor and recipient (P = 0.012). CONCLUSION: Fibrosis progression and ACR-incidence after transplantation for HCV-induced liver disease seem to be under genetic control. The negative impact of G-allele on post-transplant events observed in our study, deserves attention and should be verified in larger liver transplantation-cohorts.

Genetic variants of STAT-4 affect the development of graft fibrosis after liver transplantation for HCV-induced liver disease.

BACKGROUND: Hepatitis C virus (HCV) reinfection after liver transplantation may lead to recirrhosis and seems to be influenced by genetic factors. The aim of our study was to evaluate the role of STAT-4-polymorphisms in the development of HCV-related graft disease based on protocol biopsies. METHODS: One hundred sixty transplant patients with HCV recurrence were genotyped for STAT-4 (rs7574865) by polymerase chain reaction. Fibrosis stages were determined based on Desmet and Scheuer classification. SPSS was used for the statistical analysis of genotype distribution and of time to the development of advanced fibrosis among the genotypes. RESULTS: During a comparable observation period of 86.2 months (P=0.654), 65 patients (46.5%) developed advanced fibrosis. Advanced fibrosis was observed significantly more frequent in patients (n=34) with at least one T-allele (53.1 vs. 32.3%; P=0.013) compared with homozygotes for G-allele (n=31). Significant differences in the duration of advanced fibrosis development were detected between patients with at least one T-allele compared with G-allele (34.4 vs. 49.0 months; P=0.022). No impact was observed regarding the outcome of interferon-based antiviral treatment (P=0.297) and the occurrence of acute cellular rejection (P=0.365). CONCLUSION: Present results indicate a possible impact of genetic confounders in the recipient on graft fibrogenesis, thus explaining significantly different graft behavior observed after transplantation for HCV-associated liver disease. STAT-4-T-allele is identified as fibrogenic factor and seems to have a negative impact on HCV-induced fibrosis development.

Safety and efficacy of new integrated bipolar and ultrasonic scissors compared to conventional laparoscopic 5-mm sealing and cutting instruments.

BACKGROUND: Hemostasis is a central issue in laparoscopic surgery. Ultrasonic scissors and bipolar clamps are commonly used, with known advantages with each technique. METHODS: The prototype of new surgical scissors, delivering ultrasonically generated frictional heat energy and bipolar heat energy simultaneously (THUNDERBEAT(®) [TB]), was compared to ultrasonic scissors (Harmonic ACE(®) [HA]) and an advanced bipolar device (LigaSure(®) [LS]) using a pig model. As safety parameters, temperature profiles after single activation and after a defined cut were determined. As efficacy parameters, seal failures and the maximum burst pressure (BP) were measured after in vivo sealing of vessels of various types and diameters (categories 2-4 and 5-7 mm). Moreover, the vertical width of the tissue seal was measured on serial histological slices of selected arteries. The cutting speed was measured during division of isolated arteries and during dissection of a defined length of compound tissue (10 cm of mesentery). Burst-pressure measurement and histological analysis were performed by investigators blinded to the used sealing device. RESULTS: Using the TB, the burst pressure in larger arteries was significantly higher (734 ± 64 mmHg) than that of the HA (453 ± 50 mmHg). No differences in the rate of seal failures were observed. The cutting speed of the TB was significantly higher than that of all other devices. Safety evaluation revealed temperatures below 100 °C in the bipolar device. The maximum temperature of the HA and the TB was significantly higher. No relevant differences were observed between the HA and the TB. CONCLUSIONS: The ultrasonic and bipolar technique of the TB has the potential to surpass the dissection speed of ultrasonic devices with the sealing efficacy of bipolar clamps. However, heat production that is comparable to conventional ultrasonic scissors should be minded for clinical use.

Role of IL28B polymorphism in the development of hepatitis C virus-induced hepatocellular carcinoma, graft fibrosis, and posttransplant antiviral therapy.

BACKGROUND: The development of liver graft disease is partially determined by individual genetic background. Interleukin 28B (IL28B) is strongly suspected to be involved in susceptibility for hepatitis C virus (HCV) infection, inflammation, and antiviral treatment response before and after liver transplantation (LT). Currently, the role of IL28B polymorphism (rs12979860) in the development of hepatocellular carcinoma (HCC) is unclear, and only limited data are available on the course of HCV recurrence. METHODS: One hundred sixty-seven HCV-positive patients after LT were genotyped for IL28B (C→T; rs12979860). Sixty-one patients with histologically confirmed HCC in the explanted liver were compared with 106 patients without HCC regarding IL28B genotypes. Among patients with HCC, IL28B genotypes were correlated with tumor histology and pretransplant α-fetoprotein (AFP) levels. Furthermore, the role of IL28B polymorphism was evaluated regarding interferon-based treatment success and fibrosis progression after LT. RESULTS: The prevalence of HCC in explanted livers was significantly higher among patients with TT genotype, suggesting a protective role of the C allele in HCC development (P=0.041). Median AFP level was closely to significance higher in the presence of T allele (P=0.052). Significant differences in IL28B genotype distribution were detected between AFP-negative and AFP-positive HCCs (<15 µg/L vs. >15 µg/L; P=0.008). Although no impact could be observed regarding acute cellular rejection (P=0.940), T allele was significantly associated with antiviral therapy failure (P=0.028) and faster development of advanced fibrosis (P=0.017) after LT. CONCLUSION: IL28B polymorphism seems to be involved in the development of HCV-induced HCC and in the course of HCV recurrence after LT. T allele may be regarded as a genetic risk factor for HCV-related carcinogenesis, posttransplant fibrosis progression, and antiviral therapy failure.

Transforming growth factor-ß1-gene polymorphism in the development of kidney disease after liver transplantation.

BACKGROUND: The development of kidney dysfunction is one of the most important after liver transplantation (LT). Genetic variants of pathogenetically relevant cytokines may influence the development and course of the disease. The aim of our study was to evaluate the role of transforming growth factor-ß1 (TGF-ß1) polymorphism in this context. METHODS: Four hundred eighty-six liver graft recipients were genotyped for TGF-ß1 codon 25 (guanine → cytosine, G → C) by polymerase chain reaction. Renal function before and after LT was characterized by estimation of glomerular filtration rate (GFR) using four-parameter-modification of diet in renal disease formula on defined dates. GFR was compared among TGF-ß1-genotype groups of the entire cohort within the median observation period of 7 years. For the assessment of renal function recovery after LT, patients were divided into three groups by GFR difference (ΔGFR = ± 10 mL/min). RESULTS: Mean pretransplant GFR differed significantly among TGF-ß1-genotype groups (GG: 85.0 mL/min vs. GC/CC: 75.3 mL/min; P=0.016). The significance disappeared in the follow-up period. Although GG genotype demonstrated higher mean GFR levels, patients with GC/CC genotype tended to improve kidney function compared with GG genotype (P=0.013). Interestingly, lower mean GFR rates were observed among female compared with male recipients before (P=0.002), separately at all dates and cumulatively after LT (P<0.001). CONCLUSIONS: Genetic variants of one of the most important cytokine TGF-ß1 at codon 25 may have an impact on kidney function, suggesting an unfavorable effect of C allele in pretransplant setting and serve as marker for the recovery of renal function after LT. The identification of further confounders seems to be promising.

Combination of hedgehog signaling blockage and chemotherapy leads to tumor reduction in pancreatic adenocarcinomas.

OBJECTIVES: Activation of the hedgehog signal transduction pathway, triggered by hedgehog binding to the transmembrane receptor patched 1 (PTCH1) or by mutations in the PTCH1 gene, plays an important role in the development of various tumors. METHODS: To investigate whether the Hedgehog signaling pathway is also active in human pancreatic adenocarcinomas, we determined the expression levels of the known Hedgehog target genes PTCH1 and GLI-1 in pancreatic tumors. To determine whether alterations in the PTCH1 gene are responsible for this pathway activation, we screened pancreatic carcinomas for mutations in PTCH. To investigate the contribution of hedgehog signaling to the tumorigenicity of pancreatic tumor cells, we blocked the Hedgehog pathway in cultured tumor cells and xenografts using the steroidal alkaloid cyclopamine and the small-molecule Hedgehog inhibitor Hh-Antag. RESULTS: We identified single nucleotide polymorphisms (SNPs) within the PTCH1 gene but no somatic PTCH1 mutations. Pathway-blockage resulted in a significant dose-dependent reduction of tumor cell growth in vitro and in vivo. Moreover, combined treatment with cyclopamine and the conventional antimetabolite gemcitabine revealed a synergistic effect on the reduction of tumor growth in pancreatic adenocarcinoma xenografts. CONCLUSIONS: Inhibition of Hedgehog signaling could be a promising approach for the treatment of pancreatic adenocarcinomas.

Association of mannose-binding lectin-2 gene polymorphism with the development of hepatitis C-induced hepatocellular carcinoma.

BACKGROUND: Development of end-stage liver and graft disease is suspected to be partially determined by the individual genetic background. Mannose-binding lectin (MBL) is an important immunomodulatory factor, which is supposed to be involved in complement activation and oncogenesis. Genetic polymorphisms of MBL-2 alter MBL functionality. The aim of our study was to determine the prevalence of MBL-2 polymorphism (rs7096206) in hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC) based on histological analysis of explanted livers in patients undergoing liver transplantation (LT). METHODS: One hundred and seventy-seven patients, who underwent LT for HCV-induced liver disease, were genotyped for MBL-2 by TaqMan genotyping assay. Sixty-two patients with histologically confirmed HCC were compared with 115 patients without HCC. MBL-2 genotypes were corelated with the growth patern, tumour size and pretransplant α-fetoprotein (AFP) level of HCC patients. RESULTS: The prevalence of GG/GC genotypes was significantly higher among HCC patients compared with tumour-free explanted livers (P = 0.004; odds ratio 2.5; 1.3-4.8). GG/GC genotype group was significantly associated with the size of HCC (P = 0.022), higher pretransplant AFP level (P = 0.010) and bilobar tumour growth (P = 0.038). Furthermore, CC genotype was found to be significantly more frequent in AFP-negative HCCs (P = 0.002). CONCLUSION: Mannose-binding lectin-2 polymorphism seems to be involved in the development of pretransplant HCV-induced HCC and should be further investigated as potential risk factor for HCV-associated carcinogenesis.

Treatment of hepatitis C-virus-reinfection after liver transplant with silibinin in nonresponders to pegylated interferon-based therapy.

OBJECTIVES: Hepatitis C-virus-persistence after orthotopic liver transplant leads to reduced patient and graft survival compared to other indications. Current interferon-based antiviral therapy of hepatitis C-virus-infection posttransplant provides a sustained response rate of 30% to 40%. This study, performed in an hepatitis C-virus-reinfected liver transplant population, examines the antiviral effect of intravenously administered silibinin, recently reported to exhibit strong antiviral properties in the natural setting of hepatitis C-virus-related liver disease. PATIENTS AND METHODS: Four patients after orthotopic liver transplant with hepatitis C-virus-recurrence, previously having not responded to peg-interferon-ribavirin therapy, were treated with intravenous silibinin and additionally, after the 10th day, with standard interferon-based therapy. Aminotransferases and hepatitis C-virus-RNA were measured during treatment. RESULTS: All patients demonstrated normalization of liver enzymes and significant decline of hepatitis C-virus-RNA measured at day 10 (mean 2.8 logarithmic levels: 1.7, 2.3, 2.9, and 4.3) during silibinin monotherapy. One patient cleared hepatitis C-virus-RNA under silibinin monotherapy and another patient eliminated hepatitis C virus under subsequent interferon-based therapy. No adverse effects were observed during silibinin application. CONCLUSIONS: Intravenous silibinin is an effective therapeutic approach for treating hepatitis C-virus-reinfection after liver transplant and should be evaluated further.

Transforming growth factor ß1 polymorphisms and progression of graft fibrosis after liver transplantation for hepatitis C virus--induced liver disease.

Re-infection with the hepatitis C virus (HCV) is an important development after liver transplantation (LT); it can lead to graft fibrosis. The aim of this study was to assess the role of transforming growth factor ß1 (TGF-ß1) polymorphisms in the development of HCV-related graft disease by evaluating protocol liver biopsies. A total of 192 patients with a recurrence of HCV infection after LT were genotyped for TGF-ß1 codon 10 (C→T) and codon 25 (G→C) using the polymerase chain reaction. Histological evaluation of 614 protocol liver biopsies obtained from these patients was undertaken using the classification of Desmet and Scheuer to stage the degree of fibrosis. Mild stages of fibrosis (0-2) were compared to advanced stages of fibrosis (3-4) that developed during the period of infection with the virus. Correlations between the prevalence of TGF-ß1 genotypes and the different degrees of fibrosis that developed were determined. No statistically significant differences were found for genotype distributions (codons 10 and 25) with respect to recipient age, donor sex, occurrence of acute cellular rejection, and response to antiviral therapy. However, the C allele at codon 25 was significantly less frequent in the group with advanced fibrosis (P = 0.001). Furthermore, a positive association was found between progression of fibrosis and male recipient sex (P = 0.024), donor age (P = 0.041), and viral genotype 1b (P = 0.002). In conclusion, this study, in which the evolution of hepatic fibrosis was assessed histologically in a large cohort of patients with HCV re-infection after LT, has demonstrated that the C allele at codon 25 of the TGF-ß1 gene is a marker for the development of graft fibrosis.

Relationship between the interleukin-28b gene polymorphism and the histological severity of hepatitis C virus-induced graft inflammation and the response to antiviral therapy after liver transplantation.

Up to 30% of liver transplants will develop graft cirrhosis within 5 years after liver transplantation (LT) due to recurrent HCV-infection forwarding accelerated graft damage. Genetic variants of cytokines involved in the immune response may contribute to the degree of graft inflammation, fibrosis progression, and antiviral therapy outcome. The aim of our study was to analyze biochemical and histological inflammation extent based on protocol liver biopsies and to evaluate the role of genetic variants of IL-28b in HCV-related graft disease and antiviral treatment response. 183 patients, who underwent liver transplantation for HCV-induced liver disease, were genotyped for IL-28b (rs8099917, G ≥ T) by TaqMan Genotyping Assay. 56 of 159 patients have been successfully treated with interferon-based antiviral therapy. 605 protocol liver biopsies performed 0.5 to 10 and more than 10 years after transplantation were evaluated according to Desmet and Scheuer classification of inflammation and fibrosis. Prevalence of IL-28b-genotypes was correlated with histological severity of graft damage, levels of aminotransferases, occurrence of acute cellular rejection, pre-treatment viremia, and antiviral therapy outcome. Significant association of IL-28b-genotype distribution was observed to the median grade of inflammation (p < 0.001), mean levels of aminotransferases (ALT: p = 0.001, AST: p = 0.003), median pre-treatment viremia level within 1 year after LT (p = 0.046) and interferon-based antiviral therapy failure (p < 0.001). Among successfully treated patients, G-allele was significantly less frequent, and the genotype GG was not present at all. No differences were observed regarding acute cellular rejection (p = 0.798) and fibrosis stages (p = 0.586). IL-28b polymorphism seems to influence the degree of graft inflammation at biochemical and histological levels. G-allele might serve as a marker for graft inflammation and as a predictor for unfavorable antiviral therapy outcome in HCV-re-infected LT-population.

rHuEPo reduces ischemia-reperfusion injury and improves survival after transplantation of fatty livers in rats.

BACKGROUND: The scarcity of appropriate donor organs remains to be a major problem in transplantation surgery today. This has led to increased acceptance of so-called marginal grafts, incorporating the increased risk of poor posttransplant function. Erythropoietin has been shown to reduce ischemia-reperfusion injury in transplanted rat livers. We investigated whether these capacities may contribute to improve marginal organ function. METHODS: One hundred and forty Lewis rats were used. Fatty liver (>or=50% steatosis) was induced by a special diet in 70 donor animals. Seventy recipients received liver transplantation after donor organ treatment with 1000 IU rhuEpo or saline injection (controls) into portal veins (cold ischemia 6 hr, University of Wisconsin solution). Recipients were allocated to two groups which received 1000 IU rHuEpo at reperfusion or an equal amount of saline (control). Analysis of liver enzymes, histology (hematoxylin-eosin and periodic acid Schiff stain), immunostaining (terminal deoxynucleotide transferase- mediated dUTP nick-end labeling, hypoxyprobe, and tumor necrosis factor-alpha), and reverse transcriptase-polymerase chain reaction of cytokine messenger RNA (interleukin-1, interleukin-6, hypoxia induced factor-1 alpha, vascular endothelial growth factor, and hepatocyte growth factor) were performed at defined time points (2, 4.5, 24, 48 hr, and 7 days postoperatively). RESULTS: Alanine aminotransferase values were significantly reduced for epo-treated rats 48 hr after reperfusion; however, at all other time points enzyme levels were without significant differences. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling and hypoxyprobe analysis and necrotic index evaluation displayed significant reduction of apoptosis and hypoxic cells in rHuEpo-treated graft livers. Overall survival was significantly improved among epo-treated rats. CONCLUSION: Erythropoietin improves marginal graft function and recipient survival after transplantation of fatty livers in rats.

Erythropoietin reduces ischemia-reperfusion injury after liver transplantation in rats.

Human recombinant Erythropoietin (rHuEpo) has recently been shown to be a potent protector of ischemia- reperfusion injury in warm-liver ischemia. Significant enhancement of hepatic regeneration and survival after large volume partial hepatic resection has also been demonstrated. It was the aim of this study to evaluate the capacities of rHuEpo in the setting of rat liver transplantation. One-hundred-and-twenty Wistar rats were used: 60 recipients received liver transplantation following donor organ treatment (60 donors) with either 1000 IU rHuEpo or saline injection (controls) into portal veins (cold ischemia 18 h, University of Wisconsin (UW) solution). Recipients were allocated to two groups, which either received 1000 IU rHuEpo at reperfusion or an equal amount of saline (control). Animals were sacrificed at defined time-points (2, 4.5, 24, 48 h and 7 days postoperatively) for analysis of liver enzymes, histology [hematoxylin-eosin (HE) staining, periodic acid Schiff staining (PAS)], immunostaining [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), Hypoxyprobe] and real-time polymerase chain reaction (RT-PCR) of cytokine mRNA (IL-1, IL-6). Lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) values were significantly reduced among the epo-treated animals 24 and 48 h after liver transplantation (LT). The TUNEL and Hypoxyprobe analyses as well as necrotic index evaluation displayed significant reduction of apoptosis and necrosis in rHuEpo-treated graft livers. Erythropoietin reduces ischemia-reperfusion injury after orthotopic liver transplantation in rats.

Improvement of microsurgical techniques in orthotopic rat liver transplantation.

BACKGROUND: Because of the limited tolerance to portal venous clamping, the model of liver transplantation in rats represents a difficult task, which requires a great proportion of experience. Since techniques that include the introduction of an artificial stent increase the risk of thrombosis, it was our goal to modify the classical vascular hand-sewn venous anastomosis technique by using a modified end-to-end knotless procedure. MATERIALS AND METHODS: Seventy-two animals were randomly assigned to 1 of 2 experimental groups, which differed by the technique for the 3 venous anastomoses (supra- and infrahepatic vena cava, portal vein). Group 1 comprised the established suturing technique for rat liver transplantation, whereas all venous anastomosis of the second group were performed using our modified technique. RESULTS: With our method, average anhepatic time could be significantly reduced from 14 min 10 s (+/-100 s) to 11 min 40 s (+/-60 s) (P < 0.001). Kaplan-Meier survival rates demonstrated a better 7-d survival for the knotless (94%) compared to the classic technique (83%) (not significant, P = 0.137). Biliary complications were low in both groups but tended to be higher in the classical group. CONCLUSIONS: Our modified knotless anastomosis proves to be equally safe in regard to complications, improves timing, and provides excellent results in the model of orthotopic rat liver transplantation.

Erythropoietin promotes hepatic regeneration after extended liver resection in rats.

BACKGROUND AND AIM: It has been proven in various animal studies that recombinant human erythropoietin (rHuEPO) protects renal, cardiac and neuronal, as well as hepatic, tissue from ischemia, and promotes regeneration of adult central nervous system neurons. To date, no data are available as to whether rHuEPO has the ability to stimulate liver regeneration after liver resection. METHODS: Rats undergoing 70% or 90% hepatectomy received an intraportalvenous administration (i.p.) of rHuEPO prior to resection or a subcutaneous injection (s.c.) for 3 days postoperatively, control animals were treated with surgery and saline injection only. Regeneration capacity of remnant livers was studied over 7 days by histology and immunohistochemistry (Ki-67, proliferating cell nuclear antigen [PCNA]). Polymerase chain reaction was carried out to measure transforming growth factor beta (TGF-beta), hypoxia induced factor (HIF), signal transducing activator 3 and vascular endothelial growth factor. RESULTS: Ten-day survival in rats undergoing 90% hepatectomy significantly increased in i.p.-pretreated animals. After 70% hepatectomy the mitotic index was significantly increased in both rHuEPO-treated groups. These data were confirmed by PCNA and Ki-67 expression, which was significantly increased in the treated groups 24 h and 2 days after liver resection. TGF-beta and HIF mRNA both were upregulated in control animals 3 h after surgery. CONCLUSION: rHuEPO effectively increased liver regeneration in rats after 70% liver resection and enhanced survival after 90% hepatectomy. Thus, rHuEPO may increase the regenerative capacity after major hepatectomy.

Combination therapy of poly (ADP-ribose) polymerase inhibitor 3-aminobenzamide and gemcitabine shows strong antitumor activity in pancreatic cancer cells.

BACKGROUND AND AIM: Poly (ADP-ribose) polymerase (PARP) inhibitors such as 3-aminobenzamide (3-ABA) enhance the in vitro cytotoxicity of DNA mono-functional alkylating agents such as radiation or chemotherapeutic agents. The aim of this study was to test an approach combining the PARP inhibitor 3-ABA with standard gemcitabine therapy in human pancreatic cancer cells. METHODS: Cell viability was determined by proliferation assay (XTT). Cell-cycle analysis (FACS), ELISA (M30 Apoptosense), Western blot for caspase 8 and PARP, and electron microscopy were used to identify apoptosis. Tumor growth and survival was assessed in nude mice by subcutaneously injected Capan-1 cells. In addition, Ki67 staining was performed on tumors for cell proliferation and in vivo apoptosis induction was measured by TUNEL assay and ELISA. RESULTS: Combination therapy of gemcitabine and 3-ABA suppressed tumor cell growth more than gemcitabine alone in XTT, FACS and ELISA analysis. CONCLUSION: This in vivo study demonstrated a significantly reduced tumor weight and increased survival up to 40 days after cell inoculation with combination therapy compared to animals treated with PBS, gemcitabine or 3-ABA alone. Furthermore, TUNEL assay revealed a significant apoptosis induction and reduced proliferation in the combination group.