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There are two main enzymes that convert tryptophan (Trp) to kynurenine (Kyn): tryptophan-2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Kyn accumulation can promote immunosuppression in certain cancers. In this study, we investigated Trp degradation to Kyn by IDO and TDO in primary human hepatocytes (PHH) and tumoral HepG2 cells. To quantify Trp-degradation and Kyn-accumulation, using reversed-phase high-pressure liquid chromatography, the levels of Trp and Kyn were determined in the culture media of PHH and HepG2 cells. The role of IDO in Trp metabolism was investigated by activating IDO with IFN-γ and inhibiting IDO with 1-methyl-tryptophan (1-DL-MT). The role of TDO was investigated using one of two TDO inhibitors: 680C91 or LM10. Real-time PCR was used to measure TDO and IDO expression. Trp was degraded in both PHH and HepG2 cells, but degradation was higher in PHH cells. However, Kyn accumulation was higher in the supernatants of HepG2 cells. Stimulating IDO with IFN-γ did not significantly affect Trp degradation and Kyn accumulation, even though it strongly upregulated IDO expression. Inhibiting IDO with 1-DL-MT also had no effect on Trp degradation. In contrast, inhibiting TDO with 680C91 or LM10 significantly reduced Trp degradation. The expression of TDO but not of IDO correlated positively with Kyn accumulation in the HepG2 cell culture media. Furthermore, TDO degraded L-Trp but not D-Trp in HepG2 cells. Kyn is the main metabolite of Trp degradation by TDO in HepG2 cells. The accumulation of Kyn in HepG2 cells could be a key mechanism for tumor immune resistance. Two TDO inhibitors, 680C91 and LM10, could be useful in immunotherapy for liver cancers.

There are two main enzymes that convert tryptophan (Trp) to kynurenine (Kyn): tryptophan-2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Kyn accumulation can promote immunosuppression in certain cancers. In this study, we investigated Trp degradation to Kyn by IDO and TDO in primary human hepatocytes (PHH) and tumoral HepaRG cells. To quantify Trp-degradation and Kyn-accumulation, using reversed-phase high-pressure liquid chromatography, the levels of Trp and Kyn were determined in the culture media of PHH and HepaRG cells. The role of IDO in Trp metabolism was investigated by activating IDO with IFN-γ and inhibiting IDO with 1-methyl-tryptophan (1-DL-MT). The role of TDO was investigated using one of two TDO inhibitors: 680C91 or LM10. Real-time PCR was used to measure TDO and IDO expression. Trp was degraded in both PHH and HepaRG cells, but degradation was higher in PHH cells. However, Kyn accumulation was higher in the supernatants of HepaRG cells. Stimulating IDO with IFN-γ did not significantly affect Trp degradation and Kyn accumulation, even though it strongly upregulated IDO expression. Inhibiting IDO with 1-DL-MT also had no effect on Trp degradation. In contrast, inhibiting TDO with 680C91 or LM10 significantly reduced Trp degradation. The expression of TDO but not of IDO correlated positively with Kyn accumulation in the HepaRG cell culture media. Furthermore, TDO degraded L-Trp but not D-Trp in HepaRG cells. Kyn is the main metabolite of Trp degradation by TDO in HepaRG cells. The accumulation of Kyn in HepaRG cells could be a key mechanism for tumor immune resistance. Two TDO inhibitors, 680C91 and LM10, could be useful in immunotherapy for liver cancers.

BACKGROUND We evaluated whether effluent parameters prior to reperfusion correlate with post-transplant outcomes in liver transplant recipients. MATERIAL AND METHODS Concentrations of high mobility group box 1 protein (HMGB1), uncleaved cytokeratin-18 (M65), caspase-cleaved cytokeratin 18 fragment (M30), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), and alkaline phosphatase (ALP) were measured in effluent samples from 53 adult liver recipients (42 survived for 1 year and 11 did not survive). RESULTS Effluent concentrations of ALP (p=0.006), AST (p=0.050), and Ca++ (p=0.003) were higher in patients with bacteriemia in the first post-transplant year and ALP (p=0.015) was higher in patients with early graft dysfunction (EAD). Multivariate analysis of effluent parameters showed that Ca++ >0.30 mmol/l (p=0.012, odds ratio [OR]=7.12, confidence interval [CI]=1.56-32.58), and ALP ≥27 IU/l (p=0.033, OR=5.31, CI=1.14-27.74) were significantly associated with 1-year post-transplant bacteriemia, whereas ALP ≥27 IU/l (p=0.020, OR=5.56, CI=1.32-23.46) was significantly associated with EAD. HMGB1 >54 pg/ml (p=0.008, OR=6.05, CI=1.59-23.00) was significantly associated with the donor body mass index (p=0.008, OR=6.05, CI=1.59-23.00) and fatty liver (p=0.005, OR=11.68, CI=2.10-64.01). CONCLUSIONS Effluent parameters are indicators of liver quality and predict the outcome of liver transplantation. High effluent Ca++ and ALP are risk factors of post-transplant bacteriemia. In addition, high ALP is a risk factor of EAD, and high HMGB1 is an indicator of liver quality.

BACKGROUND The Model for End-Stage Liver Disease (MELD) score is a well-established tool for assessing hepatic failure. The present retrospective study investigated whether serum keratin 18 (M65) and caspase-cleaved cytokeratin (M30) were associated with liver dysfunction and post-transplant graft failure. MATERIAL AND METHODS A total of 147 patients with liver cirrhosis were categorized into 2 groups according to their baseline MELD score (group I: MELD score <20, n=87, and group II: MELD score ≥20, n=60). Serum M65 and M30 levels were measured by ELISA. RESULTS Cirrhotic patients had significantly higher serum M65 and M30 levels than healthy controls (p<0.0001). Serum M65 was correlated with the MELD score and serum bilirubin (p≤0.007) and serum M30 was correlated with the MELD score, international normalized ratio, and serum bilirubin (p≤0.001). Group II had significantly higher serum M65 and M30 levels than group I (M65, p=0.025 and M30, p<0.001). Patients who lost the allograft during the first post-transplant year had significantly higher serum M30 levels than patients with a graft survival of >1 year (p=0.004). In the regression analysis, serum M30 was associated with the MELD score (odds ratio [OR]=2.545, p=0.005), serum bilirubin (OR=2.605, p=0.005) and 1-year graft loss (OR=3.61, p=0.006). CONCLUSIONS Our data indicate that serum M30 levels reflect the degree of liver dysfunction and can predict 1-year graft loss.

Early allograft dysfunction (EAD) after liver transplantation is mostly a reversible event caused by factors related to ischemiaireperfusion (I/R) injury. EAD represents a hepatic injury associated with pre- and early post-transplant inflammatory cytokine responses. Aim of the present study was to evaluate the prognostic and diagnostic value of CRP in liver transplant recipients with EAD.Materials and methods: Forty-seven patients with EAD were compared with 115 non-EAD patients. Pre- and post-transplant parameters were analyzed. EAD was defined based on postoperative liver function tests such as INR, bilirubin and liver enzymes. Statistical analysis was performed using SPSS version 18.0.Results: Pre-transplant liver enzyme were not significantly different in the two groups. At day 3, 5 and 10 post-transplant CRP was significantly higher in patients with EAD than in non-EAD patients (p <= 0.001 for all investigations) and remained consistently high in patients with EAD and low in non-EAD patients. EAD patients with high CRP at post-transplant days 3 and 5 showed lower survival at 6-month and 12-month post-transplant than patients with low CRP.Conclusion: Our results indicate a prognostic and diagnostic value of CRP in patients with early graft dysfunction and predict 6-month and 12-month mortality in liver transplant recipients. (C) 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Cytomegalovirus seropositivity is an independent risk factor for atherosclerosis in patients with ESRD. Donor CMV seropositivity is associated with higher graft loss. Dendritic cells, macrophages and Th17 lymphocytes are defined as producers of IL-23. IL-23 is thought to be involved in the promotion of Th17 cell polarization. Latent CMV-induced Th17 might be involved in the pathogenesis of CMV infection in patients with ESRD. We aimed to evaluate associations of Th17-dependent cytokines with ESRD, CMV status and post-transplant outcome in kidney transplantation.

Objective: To investigate if early pregnancy serum neopterin concentrations (EPSN) could predict spontaneous preterm birth (SPB).Methods: EPSN was measured in 92 sera collected from 46 pregnant women with birth at term and 40 sera from 20 pregnant women with preterm birth. Two sera were collected for each case: in the first and early second trimester.Results: EPSN concentrations correlate with gestational age (rho=0.275, P=0.001), a correlation which was present in both groups: term and preterm birth. EPSN were higher in pregnancies with SPB compared with normal pregnancies (6.27 +/- 1.03 vs. 6.04 +/- 0.15, P=0.039). Patients with SPB showed a considerable increase of EPSN in the second trimester compared with patients with birth at term (7.30 +/- 1.53 vs. 6.16 +/- 0.23, P=0.043). A sharper increase was found in the group with SPB before 32 weeks of pregnancy (wp) (9.83 +/- 4.36 vs. 6.16 +/- 0.23, P=0.016). Pregnant women with an early second trimester serum neopterin value of above 8 nmol/L are associated with a risk of SPB before 32 wp (odds ratio=14.4, P=0.01) and of SPB before 34 wp (odds ratio=3.6, P=0.05), respectively.Conclusions: EPSN increases with the gestational age and predicts SPB in asymptomatic pregnant women.

Background: Most hepatocellular carcinomas (HCCs) are diagnosed at an advanced stage. The prognostic value of serum tumour markers alpha-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP) is limited. The aim of our study is to evaluate the diagnostic value of serum growth factors, apoptotic and inflammatory mediators of cirrhotic patients with and without HCC.Methods: Serum samples were collected from cirrhotic potential liver transplant patients (LTx) with (n = 61) and without HCC (n = 78) as well as from healthy controls (HCs; n = 39). Serum concentrations of CRP, neopterin and IL-6 as markers of inflammation and thrombopoietin (TPO), GCSF, FGF basic and VEGF, HMGB1, CK-18 (M65) and CK18 fragment (M30) and a panel of proinflammatory chemokines (CCL2, CCL3, CCL4, CCL5, CXCL5 and IL-8) were measured. Chi square, Fisher exact, Mann-Whitney U-tests, ROC curve analysis and forward stepwise logistic regression analyses were applied.Results: Patients with HCC had higher serum TPO and chemokines (P<0.001 for TPO, CCL4, CCL5 and CXCL5) and lower CCL2 (P = 0.008) levels than cirrhotic patients without HCC. Multivariate forward stepwise regression analysis for significant parameters showed that among the studied parameters CCL4 and CCL5 (P = 0.001) are diagnostic markers of HCC. Serum levels of TPO and chemokines were lower, whereas M30 was significantly higher in cirrhotic patients than in HCs.Conclusions: High serum levels of inflammatory chemokines such as CCL4 and CCL5 in the serum of cirrhotic patients indicate the presence of HCC.

Bacterial infections are the most common complications, and the major cause of mortality after liver transplantation (Tx). Neopterin, a marker of immune activation, is produced in monocyte/macrophages in response to inflammation. The aim of our study was to investigate whether early post-operation serum levels of neopterin were associated with post-transplant bacteremia and mortality in liver transplant recipients. We studied 162 of 262 liver Tx patients between January 2008 and February 2011 of whom pre- and early post-Tx sera samples were available. Pre- and early post-operative risk factors of infection and mortality were evaluated in 45 bacteremic patients and 117 non-bacteremic patients. During one-year follow-up, 28 of 262 patients died because of graft failure, septicemia and other diseases. Post-Tx serum neopterin on day 10 (p<0.001) were significantly higher in bacteriemic patients than in patients without bacteremia. Logistic regression analyses showed that day 10 post-Tx neopterin serum level ⩾40 nmol/l has a predictive value (OR=6.86: p<0.001) for bacteremia and mortality (OR=3.47: p=0.021). Our results suggest that early post-Tx neopterin serum levels are very sensitive predictive markers of one-year post-Tx bacteremia and mortality in liver Tx recipients.

Hantaviruses are emerging zoonotic pathogens which cause hemorrhagic fever with renal syndrome, an immune-mediated pathogenesis is discussed. The aim of the present study was to investigate the role of TGF-β expression in acute hantavirus infection.

PURPOSE Human corneal endothelial cells (HCEC) are a potential target of immune attack after corneal transplantation. The aim of this in vitro study was to investigate the role of HCEC during the alloimmune response of T-cells by examining cytokine profiles, function of the immunosuppressive enzyme indoleamine 2,3-dioxigenase (IDO), major histocompatibility complex (MHC-I/-II), T-cell proliferation, and the induction of cell death. METHODS Real-time PCR and RP-HPLC were used to determine IDO expression and activity. Multiplex assay was performed for quantification of cytokine levels. T-cell proliferation was assessed by thymidine incorporation, and HCEC cell death was measured by flow cytometry. RESULTS Human corneal endothelial cells induce strong proliferation of allogeneic T-cells and an increase of proinflammatory cytokines such as interleukin-1α (IL-1α), IL-1β, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). Tumor necrosis factor-alpha (and to a lesser extent IFN-γ) induces apoptosis. Moreover, IFN-γ strongly upregulates MHC-II molecules and IDO activity in HCEC as reflected by high kynurenine (Kyn) concentrations. Interestingly, the T-cell response was not affected by increased IDO activity, since blocking of IDO did not affect the proliferation rate. Indoleamine 2,3-dioxigenase-induced Kyn levels did not exceed concentrations of 175 ± 20 μM. Concentrations of ≥400 μM Kyn were required to suppress T-cell proliferation. CONCLUSIONS Our data show that T-cell attack on HCEC leads to increased concentrations of proinflammatory cytokines. Inflammatory cytokines induce apoptosis and upregulate MHC-II molecules and IDO in HCEC. Although increased IDO activity does not influence the T-cell response, it constitutes an inflammatory marker of the alloimmune response toward HCEC.

Background: Bacterial infections are not only one of the most common complications, but also the major cause of mortality and morbidity after liver transplantation. Prediction of liver transplant recipients at risk of post-transplant blood stream infection (BSI) is very important. The aim of this study was to determine the pre- and early post-transplant risk factors of BSI in patients underwent liver transplantation Material and Methods: We studied 151 patients who underwent liver transplantation between January 2008 and February 2011. Patients who experienced at least an episode of infection and lost the graft during 1 year post-transplant and patients who died because of sepsis included in the study. Pre- and early post-operative risk factors of infection and immune parameters in 44 patients with BSI, 52 patients with non-bacteriemic infection and 65 bacterial-negative patients were evaluated. Because the non-bacteriemic patients and bacterial-negative patients had no significant demographic and characteristic differences, we considered the two groups as patients without BSI. Results: Pre-transplant serum sIL-1RA (p=0.002) and post-transplant serum CRP on day 3 (p=0.002), day 5 (p=0.003) and day 10 (p=0.004) and serum neopterin on day 10 (p<0.001) were significantly higher in BSI-positive patients than in patients without BSI. Categorical analyses defined post-transplant day 3 CRP and day 10 neopterin as risk factors for BSI during 1 year post-transplant. Forward stepwise logistic regression analyses showed that only day 10 post-transplant serum neopterin > 45 nmol/l has the predictive value (relative risk= 7.88: p=0.005) for BSI during 1 year post-transplant. Conclusion: we concluded that early post-transplant neopterin serum levels are very sensitive predictive marker of one year post-transplant BSI in liver transplant recipients.