The biliary microbiome in ischaemic-type biliary lesions can be shaped by stenting but is resilient to antibiotic treatment.

This study aims to characterize the biliary microbiome as neglected factor in patients with ischaemic-type biliary lesions (ITBL) after liver transplantation. Therefore, the V1-V2 region of the 16S rRNA gene was sequenced in 175 bile samples. Samples from patients with anastomotic strictures (AS) served as controls. Multivariate analysis and in silico metagenomics were applied cross-sectionally and longitudinally. The microbial community differed significantly between ITBL and AS in terms of alpha and beta diversity. Both, antibiotic treatment and stenting were associated independently with differences in the microbial community structure. In contrast to AS, in ITBL stenting was associated with pronounced differences in the biliary microbiome, whereas no differences associated with antibiotic treatment could be observed in ITBL contrasting the pronounced differences found in AS. Bacterial pathways involved in the production of antibacterial metabolites were increased in ITBL with antibiotic treatment. After liver transplantation, the biliary tract harbours a complex microbial community with significant differences between ITBL and AS. Fundamental changes in the microbial community in ITBL can be achieved with biliary stenting. However, the effect of antibiotic treatment in ITBL was minimal. Therefore, antibiotics should be administered wisely in order to reduce emerging resistance of the biliary microbiome towards external antibiotics.

[Endoscopic Management of Biliary Complications Following Hepatobiliary Surgery]. / Endoskopisches Management von Gallengangskomplikationen nach Leberchirurgie.

Biliary complications are frequent after hepatic surgery and may greatly influence postoperative morbidity and mortality. Most of these are leaks or strictures to the bile duct, most frequently leaks after cholecystectomy and liver resection. Strictures are an important problem after liver transplantation. Patients after orthotopic liver transplantation are particularly vulnerable, as they are different from biliary complications after liver resection and must be treated very sensitively and carefully. Endoscopic retrograde cholangiography is an excellent procedure for treating these complications. The therapeutic properties include endoscopic sphincterotomy, insertion of prostheses and dilatations. These procedures can give success rates of up to 90% of cases. For hepatico-jejunostomies, there are alternative possibilities of intervention, such as balloon- or motor-supported antegrade enteroscopy, percutaneous transhepatic cholangio drainage or the increasing use of endosonographically supported procedures.

Endoscopic vacuum assisted closure (E-VAC) of upper gastrointestinal leakages.

OBJECTIVES: Endoscopic vacuum-assisted closure (E-VAC) of leaks of the upper gastrointestinal tract is an increasingly applied endoscopic technique. Data on indication, clinical success, complications and prognostic factors are still sparse. METHODS: Patients treated with E-VAC between 2012 and 2019 at a tertiary referral center have been retrospectively analyzed. RESULTS: Overall, 116 patients treated with E-VAC were identified. Indication for E-VAC placement was postoperative leakage in 94/116 (81%), iatrogenic perforations 7/116 (6%) and others 15/116 (13%). In 92/116 (79%) of the patients E-VAC therapy showed successful wound closure. The first E-VAC after detection of insufficiency was significantly more often placed intracavitary in patients with E-VAC failure (p = .031). There was a trend for longer intensive care unit treatment for patients with E-VAC failure (p = .069). Complications occurred significantly more often in patients with E-VAC failure (p = .009). Platelet count was significantly higher in patients with E-VAC success at day of insufficiency detection (257/Thsd/µL (interquartile range [IQR], 185-362) vs. 195 (IQR, 117-309); p = .039). Platelet count (375 Thsd/µL (IQR, 256-484) vs. 190 (IQR, 129-292)), hemoglobin (9.5 g/dL (IQR, 8.8-10.1) vs. 8.7 g/dL (IQR, 8.15-9.35)) and C-reactive protein level (79 mg/L (IQR, 39.7-121.9) vs. 152 mg/L (IQR, 73.7-231)) at day 14 differed significantly. The 30 days mortality rate was 33.3% (8/24) in E-VAC failure compared with 2.2% in patients with E-VAC success (p = .001). CONCLUSIONS: E-VAC is an emerging highly effective interventional endoscopic technique for gastrointestinal wound closure even in highly selected patients.

Bile and urine peptide marker profiles: access keys to molecular pathways and biological processes in cholangiocarcinoma.

BACKGROUND: Detection of cholangiocarcinoma (CCA) remains a diagnostic challenge. We established diagnostic peptide biomarkers in bile and urine based on capillary electrophoresis coupled to mass spectrometry (CE-MS) to detect both local and systemic changes during CCA progression. In a prospective cohort study we recently demonstrated that combined bile and urine proteome analysis could further improve diagnostic accuracy of CCA diagnosis in patients with unknown biliary strictures. As a continuation of these investigations, the aim of the present study was to investigate the pathophysiological mechanisms behind the molecular determinants reflected by bile and urine peptide biomarkers. METHODS: Protease mapping and gene ontology cluster analysis were performed for the previously defined CE-MS based biomarkers in bile and urine. For that purpose, bile and urine peptide profiles (from samples both collected at the date of endoscopy) were investigated from a representative cohort of patients with benign (n = 76) or CCA-associated (n = 52) biliary strictures (verified during clinical follow-up). This was supplemented with a literature search for the association of the individual biomarkers included in the proteomic patterns with CCA or cancer progression. RESULTS: For most of the peptide markers, association to CCA has been described in literature. Protease mapping revealed ADAMTS4 activity in cleavage of both bile and urine CCA peptide biomarkers. Furthermore, increased chymase activity in bile points to mast cell activation at the tumor site. Gene ontology cluster analysis indicates cellular response to chemical stimuli and stress response as local and extracellular matrix reorganization by tissue destruction and repair as systemic events. The analysis further supports that the mapped proteases are drivers of local and systemic events. CONCLUSIONS: The study supports connection of the CCA-associated peptide biomarkers to the molecular pathophysiology and indicates an involvement in epithelial-to-mesenchymal transition, generation of cancer-associated fibroblasts and activation of residual immune cells. Proteases, extracellular matrix components, inflammatory cytokines, proangiogenic, growth and vasoactive factors released from the tumor microenvironment are drivers of systemic early events during CCA progression.

Endoscopic decompression of acute intestinal distension is associated with reduced mortality in critically ill patients.

BACKGROUND: Endoscopic placement of intestinal decompression tubes is a feasible technique for treatment of acute intestinal dilation. Given the heterogeneity of the underlying diseases leading to intestinal obstruction data on the significance of endoscopic procedures for treatment of these conditions are sparse. METHODS: In the study period from 2008 to 2019 all patients receiving a decompression tube were identified by retrospective chart review and analyzed. RESULTS: A total of 59 decompression tubes were placed in 50 patients. Technical success was achieved in 98% (58/59 tubes). As major complication one small bowel perforation occurred (1/59; 1.7%). Causes for impaired intestinal transit comprised tumor stenoses 22% (11/50), infections 18% (9/50), post-operative paralysis 14% (7/50), neurological diseases 8% (4/50), trauma 2% (1/50) and others 36% (18/50). Most patients (74%; 37/50) were critically ill and treated on intensive care unit. Treatment response after tube insertion was documented in 76% of patients (38/50) whereas 24% (12/50) did not fulfill response criteria. Patients with treatment response showed a significantly better outcome compared to non-responders. Responders had a median survival of 113 days (95% CI 41-186) compared to 15 days (95% CI 6-24) in non-responders (p = 0.002). Analysis of laboratory parameters after stratification in responders and non-responders to endoscopic therapy showed that non-responders had significantly higher levels of CRP and lower platelet count at baseline (CRP 262 mg/L (IQR 101-307) vs. 94 mg/L (IQR 26-153): p = 0.027; platelets 69 thsd/µL (IQR 33-161) vs. 199 thsd/µL (IQR 138-289): p = 0.009). CONCLUSIONS: Endoscopic decompression is a safe procedure for acute management of impaired intestinal transit even in critically ill patients. Response to therapy is associated with improved outcome and markers of inflammation and organ function such as CRP, platelet count and serum lactate have to be taken into account for therapy monitoring and evaluation of prognosis.

Treatment with metformin is associated with a prolonged survival in patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal cancers. Nutrition- and life style-associated risk factors are increasingly prevalent. Metformin, the mainstay of type 2 diabetes mellitus (T2DM)-treatment, reduces the risk of hepatocarcinogenesis. However, its influence on the prognosis of patients with HCC has not been investigated on a large scale, yet. METHODS: Five thousand and ninety-three patients treated for HCC between 2000 and 2016 at three referral centres were included in this retrospective multicentre study. The aim of this study was to assess whether treatment with metformin for T2DM is associated with a prolonged overall survival (OS) in patients diagnosed with HCC. RESULTS: Among 5093 patients with HCC, 1917 patients (37.6%) were diagnosed with T2DM, of which 338 (17.6%) received treatment with metformin. Compared to diabetic patients not treated with metformin, patients on metformin had a significantly better hepatic function (Child-Pugh-Score A: 69.2% vs 47.4%, P < 0.001) and underwent significantly more often tumour resection (22.1% vs 16.5%, P = 0.024). Patients on metformin had a significantly longer median OS (mOS) compared to diabetic patients not treated with metformin (22 vs 15 months, P = 0.019). The prolongation of survival was most significant in patients treated with surgery. Using a propensity score match (PSM), patients were adjusted for hepatic function and initial therapy. In the matched cohorts, mOS remained significantly longer in metformin-treated patients (22 vs 16 months, P = 0.021). Co-treatment of metformin and sorafenib was associated with a survival disadvantage. CONCLUSION: Treatment with metformin was associated with an improved survival in patients with T2DM and HCC. This effect was most pronounced in patients at potentially curative tumour stages.

Endoscopic biliary drainage in patients with cholangiocarcinoma - self-expanding metal versus polyethylene stents.

Background: Endoscopic biliary drainage is the standard of care for patients with cholangiocarcinoma (CCA)-induced, obstructive jaundice. Self-expanding metal stents are supposed to be superior to polyethylene stents in terms of reduction of interventions and costs. So far, there are only few real-life data with respect to stent selection and survival in this patient cohort. Methods: In this study, we retrospectively analyzed patients with CCA treated with endoscopic biliary drainage from 2000 to 2015 at Hannover Medical School, Germany. The aim of this study was to analyze whether metal stenting reduces the frequency of interventions and influences survival in a large, real-life cohort. Results: Overall, 422 patients with CCA were included in this study. Indication for endoscopic biliary drainage was most often obstructive jaundice (n = 397; 94.1%). Among these patients, 20 patients (5%) were initially treated with a metal stent and 38 (9.6%) received a metal stent in the subsequent course. Median number of interventions per month was 2.4-fold reduced following metal stenting. Patients first treated with a metal stent had a more advanced tumor stage and a significantly shorter median overall survival (mOS) compared to patients who received a metal stent subsequently (7.5 months vs. 15.2 months; p=.019). There was no difference in mOS for metal vs. polyethylene stenting following a propensity score match for the confounders curative resection and chemotherapy (13.2 vs. 13.7 months, p=.555). Conclusions: Our data confirm that metal stenting reduces the frequency of interventions, but does not influence OS. Metal stenting should be considered specifically in younger patients who are suitable for chemotherapy.

Transjugular diagnostics in acute liver failure including measurements of hepatocentral venous biomarker gradients.

AIM: Acute liver failure (ALF) is a syndrome of severe liver injury that may need urgent liver transplantation and is associated with significant risk of death. Early outcome prediction and further possibilities to increase accuracy of prognosis scores are important. METHODS: We examined 30 patients with ALF, according to the novel criteria of the Intractable Hepato-Biliary Diseases Study Group, who underwent transjugular liver biopsy (TJLB) and investigated the relevance of histology for correct diagnosis and etiology. We assessed the suitability of necrosis (%), hepatic venous pressure gradients (HVPG), and hepatocentral venous gradients of serum biomarkers for outcome prediction. For this purpose, we calculated the difference of biomarker levels between hepatic vein (HV) and superior vena cava (SVC) blood samples. RESULTS: Histology of TJLB specimens contributed to finding the etiology in 83%. Necrosis (%) and HVPGs were not significantly different between outcome groups. In gradient measurements, caspase 3/7 activity and total cytokeratin 18 (CK-18) (M65) had significant and relevant levels different from zero. Although they were not accurate for outcome prediction, differences between outcome groups were detected in caspase activation: levels of caspase 3/7 activity in the HV and caspase-cleaved CK-18 (M30) in the SVC were significantly higher in spontaneously recovered patients. CONCLUSIONS: Our results underline the role of caspase activation in spontaneous recovery from ALF. Furthermore, the calculation of hepatocentral venous biomarker gradients could represent a new diagnostic tool whose clinical potential needs to be further investigated.

Retrograde portal vein flow and transarterial chemoembolization in patients with hepatocellular carcinoma - a case-control study.

BACKGROUND AND AIMS: Transarterial chemoembolization (TACE) is the most common treatment for hepatocellular carcinoma (HCC). In case of portal vein (PV) flow diversion, outcome may be compromised due to a decompensation of hepatic perfusion following arterial embolization. The aim of this study was to determine whether TACE in patients with retrograde PV flow results in a stronger deterioration of liver function and a poorer survival compared to patients with orthograde PV flow. METHODS: A database of 606 patients treated with TACE between 2000 and 2015 at Hannover Medical School was screened for Doppler ultrasound (US) findings on PV flow prior to TACE. A total of 407 patients were identified, among which 32 patients had retrograde PV flow. RESULTS: Patients with retrograde PV flow had significantly more often liver cirrhosis with advanced hepatic dysfunction (93.5% vs. 72.7%, p < .05). Median overall survival (OS) was 12 and 19 months in patients with retro- and orthograde PV flow, respectively (HR 1.27, p > .05). Patients with retrograde PV flow showed a trend for a shorter OS when matched for cirrhosis (12 vs. 21months, HR 1.51), Child-Pugh score/albumin-bilirubin grade (12 vs. 15 months). There was no difference in the deterioration of liver function after repeated treatments between both groups as assessed by increase of CP points and ALBI grade. CONCLUSIONS: Retrograde PV flow alone was not a significant prognostic marker, but patients with retrograde PV flow and advanced liver cirrhosis treated with TACE had a very short survival. Assessment of PV flow prior TACE may be helpful in borderline cases considered for TACE.

Experience from a real-life cohort: outcome of 606 patients with hepatocellular carcinoma following transarterial chemoembolization.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal cancers. Transarterial chemoembolization (TACE) has been accepted as the standard care for intermediate stage disease. METHODS: In this study, we characterized 606 with HCC patients from Hannover Medical School treated with TACE. RESULTS: 606 with HCC patients treated with TACE were identified between 2000 and 2015. Most patients (59.8%) were at intermediate stage. Following TACE, most patients subsequently received systemic therapy or best supportive care (BSC), whereas 227 (37.5%) patients were bridged to potentially curative local treatments. Depending on subsequent therapies, median post-TACE survival ranged from 7 to 162 months. Ascites, cholinesterase, c-reactive and alpha-feto protein and tumor size were identified as prognostic factors. These factors as well as the HAP, mHAP-II and STATE score also determined post-TACE survival independent of subsequent therapies. Hepatic function progressively deteriorated with repeated TACE sessions. Despite that, post-TACE survival was not shortened in frequently treated patients (≥5 times) as compared to patients treated 4 times or less (p = not significant [n.s.]). Patients treated ≥5 times with TACE received significantly more often systemic therapy following TACE (37.3%) as compared to patients with 3-4 (30.1%), 2 (27.4%) and 1 (21.8%) sessions (p < .05). CONCLUSION: TACE is performed in a heterogeneous population as bridging therapy to other local treatments and palliative therapy. The long-term survival following TACE is determined by baseline tumor, patient-related factors and by subsequent therapies. Post-TACE survival is not shorter in patients with frequent treatments (≥5), and the rate of subsequent systemic treatments is higher compared to less frequently treated patients.

Patterns and challenges of treatment sequencing in patients with hepatocellular carcinoma: Experience from a German referral center.

BACKGROUND AND AIM: Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers. Several local and systemic therapies are available for patients with HCC depending on the stage of the disease. In clinical practice, treatment decision-making, and sequencing may be very heterogeneous. METHODS: In this study, we retrospectively analyzed treatment algorithms in 2101 patients with HCC treated from 2000 to 2015 at Hannover Medical School, Germany. RESULTS: Transarterial chemoembolization was the most common initial treatment (n = 545; 25.9%), followed by resection (n = 435, 20.7%), local-ablative procedures (n = 283, 13.5%), systemic therapies (n = 275, 13.1%), and liver transplantation (n = 52; 2.5%). Most patients were treated only once (n = 960; 59.6%). A total of 433 (26.9%) and 160 (9.9%) patients received a second line and third line treatment after recurrent or progressive disease. Patients with more than one treatment line were diagnosed at significantly earlier disease stages (P < 0.001). Using binary logistic regression, AFP ≤ 200 µg/L, albumin > 36 g/L, and small tumor size (≤50 mm) were identified as predictors of achieving more than one treatment line. Subsequent treatment stage migration to a therapy suggested for the next advanced stage occurred only in 56.9%, whereas 43.1% received treatments suggested for earlier disease stages. Only 16% of all treated patients received systemic therapy in the salvage setting. CONCLUSION: Most patients were treated only once, and only a minority of patients received systemic treatment. The high dropout rate for subsequent therapies needs to be considered within therapy decision-making. There is an urgent need for prospective studies to define the best time point when to switch patients from local to systemic therapies.

Liver transplantation for critically Ill patients with secondary sclerosing cholangitis: Outcome and complications.

Secondary sclerosing cholangitis in critically ill patients (SSC-CIP) is a destructive cholangiopathy with a poor prognosis. Liver transplantation (LT) is an established therapeutic option in end-stage liver disease but is insufficiently evaluated in patients with SSC-CIP. Our aim was the retrospective analysis of the outcome and complications of patients with SSC-CIP undergoing LT between 2002 and 2012. Demographic characteristics, laboratory, transplantation, and follow-up data were compared to sex- and age-matched patients undergoing LT because of other reasons. Quality of life (QoL) before and after LT was assessed in a retrospective telephone interview. LT was performed in 21 patients with SSC-CIP. The main causes for intensive care unit admission comprised cardiothoracic surgery interventions (10/21, 48%), polytrauma (6/21, 29%), and pneumonia (3/21, 14%). Median follow-up period after LT was 82 months (interquartile range [IQR], 37-129) for patients with SSC-CIP and 83 months (IQR, 55-104) for control patients. Biopsy-proven rejection episodes in patients with SSC-CIP (4/21, 19%) were similar compared to control patients (12/60, 20%; P = 0.93). Cytomegalovirus infections were equal in both groups (10/21, 48% versus 25/60, 42%; P = 0.64). The 1-, 3-, and 5-year survival rates of patients with SSC-CIP versus control patients were 100% versus 98%, 86% versus 92%, and 76% versus 87%, respectively (P > 0.05). The QoL improved significantly after LT in SSC-CIP. In conclusion, LT is a valid option for patients with SSC-CIP with excellent long-term outcome and improvement of QoL.

Increased T-helper 2 cytokines in bile from patients with IgG4-related cholangitis disrupt the tight junction-associated biliary epithelial cell barrier.

BACKGROUND & AIMS: IgG4-related cholangitis is a chronic inflammatory biliary disease that involves different parts of the pancreatobiliary system, but little is known about its mechanisms of pathogenesis. A T-helper (Th) 2 cell cytokine profile predominates in liver tissues from these patients. We investigated whether Th2 cytokines disrupt the barrier function of biliary epithelial cells (BECs) in patients with IgG4-related cholangitis. METHODS: We assessed the Th2 cytokine profile in bile samples and brush cytology samples from 16 patients with IgG4-related cholangitis and respective controls, and evaluated transcription of tight junction (TJ)-associated proteins in primary BECs from these patients. The effect of Th2 cytokines on TJ-mediated BEC barrier function and wound closure was examined by immunoblot, transepithelial resistance, charge-selective Na(+)/Cl(-) permeability, and 4-kDa dextran flux analyses. RESULTS: Bile samples from patients with IgG4-related cholangitis had significant increases in levels of Th2 cytokines, interleukin (IL)-4, and IL-5. IL-13 was not detected in bile samples, but polymerase chain reaction analysis of whole-brush cytology samples from patients with IgG4-related cholangitis revealed increased levels of IL-13 mRNA, compared with controls. BECs isolated from the brush cytology samples revealed decreased levels of claudin-1 and increased levels of claudin-2 mRNAs. In vitro, IL-4 and IL-13 significantly reduced TJ-associated BEC barrier function by activating claudin-2-mediated paracellular pore pathways. Th2 cytokines also impaired wound closure in BEC monolayers. CONCLUSIONS: Th2 cytokines predominate in bile samples from patients with IgG4-related cholangitis and disrupt the TJ-mediated BEC barrier in vitro. Subsequent increases in biliary leaks might contribute to the pathogenesis of chronic biliary inflammation in these patients.

MicroRNAs in the bile of patients with biliary strictures after liver transplantation.

Biliary complications after liver transplantation remain a major cause of morbidity and reduced graft survival. Ischemic-type biliary lesions (ITBLs) are common and difficult to treat. The pathophysiology of ITBLs remains unclear, and diagnostic markers are still missing. The analysis of microRNA (miRNA) profiles is an evolving field in hepatology. Our aim was to identify specific miRNA patterns in the bile of patients with ITBLs after liver transplantation. Liver transplant patients with biliary complications were included in a cross-sectional study. Patients with ITBLs (n = 37), anastomotic strictures (ASs; n = 39), and bile duct stones (BDSs; n = 12) were compared. Patients with ITBLs were categorized by disease severity. The miRNA concentrations in bile were determined with global miRNA profiling and subsequent miRNA-specific polymerase chain reaction-mediated validation. The concentrations of microRNA 517a (miR-517a), miR-892a, and miR-106a* in bile were increased for patients with ITBLs versus patients with ASs or BDSs (P < 0.05). Categorization by ITBL severity showed higher median concentrations in patients with intrahepatic and extrahepatic strictures (P > 0.05). miR-210, miR-337-5p, miR-577, and miR-329 displayed no statistical differences. In conclusion, miR-517a, miR-892a, and miR-106a* are increased in the bile fluid of patients with ITBLs versus patients with ASs or BDSs. An analysis of miRNA profiles may be useful in the diagnosis and management of patients with ITBLs. Future studies are needed to prove the potential prognostic value of these miRNAs.

Safety and performance of liver biopsies in liver transplant recipients.

BACKGROUND: Liver biopsy in patients after liver transplantation (OLT) serves as a diagnostic tool to establish the cause of liver pathology. However, liver biopsy may cause life-threatening complications. Very limited information is available about complications and success rates of liver biopsies in patients after OLT. Our aim was to investigate biopsy-related complications and quality of specimen obtained by liver biopsy after OLT and to evaluate risks and benefits of this procedure. METHODS: Retrospective analysis of patients after OLT presenting for liver biopsy between January 2000 and October 2012. All patients were observed for 24 h after intervention. Twelve or more portal tracts were required for liver biopsy specimens to be considered as adequate. RESULTS: Of 703 liver biopsies were performed in 409 patients. Thirteen (1.9%) liver biopsies did not have an adequate number of portal tracts. Only 10 (1.4%) liver biopsies caused complications. Five patients suffered from pain, three patients developed post-procedural fever, and three patients had subcapsular/intercostal bleeding. One patient suffered from a vasovagal reaction. Pain was treated by analgesics; none of the patients required blood transfusion or surgery. CONCLUSIONS: Liver biopsy is a safe and adequate diagnostic tool in patients after OLT.

Calprotectin in bile: a disease severity marker in patients with primary sclerosing cholangitis.

GOALS: Our aim was to evaluate the diagnostic potential of calprotectin in serum and bile of patients with primary sclerosing cholangitis (PSC). BACKGROUND: PSC is a chronic cholestatic liver disease of unknown etiology. It is characterized by progressive inflammation and fibrosis of the bile ducts leading to biliary cirrhosis and eventually liver failure. Reliable markers for disease activity and severity are still lacking. Subunits of calprotectin, a fecal marker of inflammation in inflammatory bowel disease, have been recently identified in bile. STUDY: Calprotectin was measured in patients with PSC (n=56), cholangiocarcinoma (CC) complicating PSC (CC/PSC) (n=13), CC (n=30), and bile duct stones in bile (n=38) and serum (n=73) by enzyme-linked immunosorbent assay in a cross-sectional study. PSC patients were categorized by the Mayo risk score (MRS) to characterize the disease severity. RESULTS: Calprotectin is present in bile, and the median concentration was significantly higher in PSC patients (P<0.05). Stratification of PSC patients by MRS showed significantly elevated calprotectin levels in bile in the MRS-high group (P<0.05). Calprotectin and MRS correlated significantly (P<0.05). The presence or absence of inflammatory bowel disease in PSC patients did not alter calprotectin levels in bile. Serum AP and calprotectin in bile correlated significantly (P=0.013). No significant correlation was found for other liver-related parameters. In contrast, serum calprotectin levels were significantly higher in patients with CC, but there was no association with PSC or disease activity/severity. CONCLUSIONS: Calprotectin in bile is a promising disease marker in patients with PSC with a potential prognostic value.

Human CCR4+ CCR6+ Th17 cells suppress autologous CD8+ T cell responses.

The role of Th17 cells in cancer patients remains unclear and controversial. In this study, we have analyzed the phenotype of in vitro primed Th17 cells and further characterized their function on the basis of CCR4 and CCR6 expression. We show a novel function for a subset of IL-17-secreting CD4(+) T cells, namely, CCR4(+)CCR6(+)Th17 cells. When cultured together, CCR4(+)CCR6(+)Th17 cells suppressed the lytic function, proliferation, and cytokine secretion of both Ag-specific and CD3/CD28/CD2-stimulated autologous CD8(+) T cells. In contrast, CCR4(-)CCR6(+) CD4(+) T cells, which also secrete IL-17, did not affect the CD8(+) T cells. Suppression of CD8(+) T cells by CCR4(+)CCR6(+)Th17 cells was partially dependent on TGF-ß, because neutralization of TGF-ß in cocultures reversed their suppressor function. In addition, we also found an increase in the frequency of CCR4(+)CCR6(+), but not CCR4(-)CCR6(+) Th17 cells in peripheral blood of hepatocellular carcinoma patients. Our study not only underlies the importance of analysis of subsets within Th17 cells to understand their function, but also suggests Th17 cells as yet another immune evasion mechanism in hepatocellular carcinoma. This has important implications when studying the mechanisms of carcinogenesis, as well as designing effective immunotherapy protocols for patients with cancer.

Next Generation Sequencing Outperforms Cultivation-Based Methods for Detection of Bacterial Genera in Bile After Liver Transplantation.

Background and aims: Bacterial cholangitis is a common complication in patients with ischemic type biliary lesions and/or anastomotic strictures after liver transplantation (LTX). Patients frequently need antibiotics and endoscopic retrograde cholangiography (ERC) to improve the bile flow. Antibiotic treatment is based on findings in standard microbiological cultivation (SMC) of bile. However, the cultivation techniques are limited to a subset of bacteria easy-to-cultivate. Therefore, the aim of our study was to evaluate the value of next generation sequencing as an additional diagnostic tool to SMC in ischemic type biliary lesions and/or anastomotic strictures. Methods: We sequenced the V1-V2 region of the 16S rRNA gene in 242 stored bile samples in patients after LTX and compared the results with findings of SMC. SMC was performed in n = 135 (56%) fresh bile samples in addition to NGS. SMC was part of the clinical routine in these patients. Results: NGS detected bacterial genera in bile samples more often than SMC (P = 5.42 × 10-74). SMC showed insufficient discovery of bacterial genera compared to NGS with better performance in patients receiving antibiotics prior to ERC. SMC missed many bacterial genera detected by NGS. Conclusions: NGS was more sensitive in detecting bacteria in bile than SMC, no clinical parameters could be used to improve discovery rates in SMC and many genera were missed by SMC. Therefore, NGS should be used in a combined approach with SMC for improved diagnostics to achieve more specific and targeted antibiotic treatments.

Microbiologic analysis of peri-pancreatic fluid collected during EUS in patients with pancreatitis: impact on antibiotic therapy.

BACKGROUND: Pancreatitis is a potentially life-threatening condition frequently accompanied by peri-pancreatic fluid collections (PPFC), such as pseudocysts or pancreatic necrosis. Aspiration of PPFCs during EUS interventions for microbiologic analysis is still rarely performed in clinical routine. OBJECTIVE: To evaluate the role of routine microbiologic analysis of PPFCs and its impact on antibiotic therapy in patients with pancreatitis. DESIGN: Prospective, observational, multicenter study. SETTING: Four treatment centers. PATIENTS: A total of 44 consecutive patients who presented for endoscopic treatment of PPFCs were included. INTERVENTION: Concomitantly, PPFC during intervention and concomitant blood cultures were obtained. MAIN OUTCOME MEASUREMENTS: Microbiologic examination of PPFCs and blood samples. RESULTS: Colonization of PPFCs was found in 59% of PPFC cultures, whereas all but 2 concomitant blood cultures showed no microbial growth. Risk factors for a colonization were the presence of necrosis (P = .006), acute pancreatitis (P = .033), leukocytosis (P = .001), elevated C-reactive protein levels (P = .003), fever (P = .02), turbid material (P = .031), and longer hospital stay (P = .003). In 23 patients with fluid colonization despite empiric antibiotic therapy, the treatment had to be adjusted in 18 patients (78%) according to the observed antibiotic susceptibility profile. LIMITATIONS: Contamination cannot be totally excluded. CONCLUSION: The microbiologic colonization of PPFCs in patients with pancreatitis is common. Only the direct microbiologic analysis of PPFCs, but not of blood cultures, is useful to optimize an effective antibiotic therapy in patients with pancreatitis.

Biliary cast syndrome post-liver transplantation: risk factors and outcome.

BACKGROUND: Biliary cast syndrome (BCS) is characterized by the retention of lithogenic material leading to obstructive cholangitis and subsequent liver damage. BCS after orthotopic liver transplantation (OLT) can lead to retransplantation or death. AIM: Evaluation of aetiology, risk factors and outcome of BCS after OLT. METHODS: In a retrospective single centre analysis between 2002 and 2011, all OLT patients with BCS diagnosed by endoscopic retrograde cholangiography were identified and compared with a matched control group at a 2:1 ratio. RESULTS: Thirty patients with BCS after OLT were identified (30/887, 3.4%). Seventy per cent of those patients (21/30) underwent transplantation in the Model for Endstage Liver Disease (MELD) score era. Median time to diagnosis after OLT was 255 days (IQR 107-621). Intensive care unit treatment after OLT was significantly longer in BCS patients [16 days (IQR 8-42) vs. 9 (IQR 7-17) days; P = 0.039]. In a multivariate analysis, hepatic artery stenosis (P = 0.04), biliary strictures (P = 0.032) and need for renal replacement therapy (P = 0.002) were significantly associated with BCS. Immunosuppressant regimen, operation time, cold or warm ischaemia time, graft size, acute cellular rejection and cytomegalovirus infections were not significantly different between both groups. Retransplantation rate and 12-month mortality were significantly higher with BCS (9/30, 30% vs. 4/60, 7%, P = 0.003). CONCLUSIONS: BCS is a rare, but severe complication after OLT. Patients with hepatic artery stenosis, biliary strictures or renal replacement therapy have the highest risk to develop BCS and should therefore be monitored carefully.