Hanno Ehlken

c-FLIP Maintains Tissue Homeostasis by Preventing Apoptosis and Programmed Necrosis

The antiapoptotic protein c-FLIP blocks multiple cell death pathways in mice. FLIPping Multiple Death Signals Off The gene c-Flip, which encodes the antiapoptotic protein c-FLIP, is expressed in response to nuclear factor κB (NF-κB) activation. NF-κB–mediated protection of the intestine and liver from proapoptotic signaling is important for tissue maintenance (homeostasis). Avoiding the embryonic lethality caused by complete knockout of c-Flip in mice, Piao et al. selectively deleted c-Flip in intestinal epithelial cells (IECs) or hepatocytes. Whereas c-FLIP–deficient IECs exhibited tumor necrosis factor (TNF)–dependent apoptosis and programmed necrosis, a cell death process morphologically and mechanistically distinct from that of apoptosis, leading to perinatal death of the mice, c-FLIP–deficient hepatocytes exhibited apoptosis and programmed necrosis, and mice died in a TNF-independent manner. Induced loss of c-FLIP in hepatocytes in adult mice led to lethal hepatitis, which was prevented by blocking multiple proinflammatory factors that trigger apoptosis. Together, these data show that c-FLIP blocks both apoptosis and programmed necrosis to maintain tissue homeostasis and suggest that targeting both cell death pathways may be effective in treating certain viral infections in which c-FLIP abundance is reduced. As a catalytically inactive homolog of caspase-8, a proapoptotic initiator caspase, c-FLIP blocks apoptosis by binding to and inhibiting caspase-8. The transcription factor nuclear factor κB (NF-κB) plays a pivotal role in maintaining the homeostasis of the intestine and the liver by preventing death receptor–induced apoptosis, and c-FLIP plays a role in the NF-κB–dependent protection of cells from death receptor signaling. Because c-Flip–deficient mice die in utero, we generated conditional c-Flip–deficient mice to investigate the contribution of c-FLIP to homeostasis of the intestine and the liver at developmental and postnatal stages. Intestinal epithelial cell (IEC)– or hepatocyte-specific deletion of c-Flip resulted in perinatal lethality as a result of the enhanced apoptosis and programmed necrosis of the IECs and the hepatocytes. Deficiency in the gene encoding tumor necrosis factor–α (TNF-α) receptor 1 (Tnfr1) partially rescued perinatal lethality and the development of colitis in IEC-specific c-Flip–deficient mice but did not rescue perinatal lethality in hepatocyte-specific c-Flip–deficient mice. Moreover, adult mice with interferon (IFN)–inducible deficiency in c-Flip died from hepatitis soon after depletion of c-FLIP. Pretreatment of IFN-inducible c-Flip–deficient mice with a mixture of neutralizing antibodies against TNF-α, Fas ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL) prevented hepatitis. Together, these results suggest that c-FLIP controls the homeostasis of IECs and hepatocytes by preventing cell death induced by TNF-α, FasL, and TRAIL.

Transient elastography in autoimmune hepatitis: Timing determines the impact of inflammation and fibrosis.

BACKGROUND & AIMS There is an unmet need for the non-invasive monitoring of fibrosis progression in patients with autoimmune hepatitis (AIH). The aim of this study was to assess the diagnostic performance of transient elastography in patients with AIH and to investigate the impact of disease activity on its diagnostic accuracy. METHODS Optimal cut-offs were defined in a prospective pilot study (n=34) and the diagnostic performance of transient elastography validated in an independent second cohort (n=60). To explore the impact of disease activity on liver stiffness, patients were stratified according to biochemical response and the time interval between start of immunosuppression and transient elastography. RESULTS Liver stiffness strongly correlated with histological fibrosis stage (pilot study: ρ=0.611, p<0.001; validation cohort: ρ=0.777, p<0.0001). ROC curves defined an area under the receiver operating curve of 0.95 for diagnosing cirrhosis at the optimal cut-off of 16kPa. The performance of transient elastography was impaired when patients were analysed in whom transient elastography was performed within 3months from start of treatment. In this setting, liver stiffness correlated with histological grading (ρ=0.558, p=0.001), but not with staging. In contrast, using the cut-off of 16kPa, the accuracy for diagnosing cirrhosis was excellent in patients treated for 6months or longer (area under the receiver operating curve 1.0). CONCLUSIONS Liver inflammation has a major impact on liver stiffness in the first months of AIH treatment. However, transient elastography has an excellent diagnostic accuracy for separating severe from non-severe fibrosis after 6months of immunosuppressive treatment. LAY SUMMARY Transient elastography is a special ultrasound scan, which assesses liver stiffness as a surrogate marker for liver fibrosis/scarring. Transient elastography has been shown to be a reliable non-invasive method to assess liver fibrosis in various chronic liver diseases, it takes less than 5min and has a high patient acceptance. The current study validated for the first time this technique in a large cohort of patients with autoimmune hepatitis (AIH) and demonstrates that it is a reliable tool to detect liver fibrosis in treated AIH. For the monitoring of potential disease progression under treatment, the validation of liver stiffness as non-invasive marker of liver fibrosis will greatly improve patient care in autoimmune hepatitis.

Transient Elastography in Primary Sclerosing Cholangitis—the Value as a Prognostic Factor and Limitations

Reply. Thanks to Li et al for their interesting comments. The authors raise some important points regarding retrospective database studies on pancreatic cancer incidence among patients with chronic pancreatitis (CP). The key question is whether patients with pancreatic cancer in our cohort actually suffered from CP or may have been misdiagnosed with CP during examinations that finally demonstrated a pancreatic cancer. This is a general concern in retrospective studies using databases and the method to account for that uncertainty is to disregard cases of pancreatic cancer that are diagnosedwithin a certain period of time after the diagnose of CP. The preferable latency period has been 2 years, which to our knowledge was introduced by Lowenfels et al and has been used in later studies as well. The background for selection of 2 years of latency period seems not to be evidence based and more likely arbitrary. Furthermore, we do not know whether the excluded patients in these studies actually did also suffer from CP. It should also be remembered that CP patients may go undiagnosed for years, which could shorten the observed latency period between CP and pancreatic cancer diagnosis. According to Danish data, approximately 1000 persons are diagnosed with pancreatic cancer annually and the 1-year survival is 26% (www.cancer.dk). In our material the median survival time with pancreatic cancer without surgery was 86 days (Q1-Q3, 35–235). The observed survival time was independent of time since CP diagnoses: 0–1 year, 104 (39–280); 1–2 years, 67 (19–168); and >2 years, 117 (34–362) days. Hence, it is less likely that patients survive pancreatic cancer for >1 year. We think these considerations justify our approach to exclude the patients with pancreatic cancer within only 1 year of the diagnosis of CP. Furthermore, the results from retrospective studies should be interpreted with precaution; however, post hoc exclusion of patients based on assumptions could be hazardous. To address the request from Li et al, we have excluded the 338 patients with pancreatic cancer within 1 year of CP, and recalculated the hazard ratio (HR) of pancreatic cancer among patients with CP compared with our controls. We then found that 1.48% of the patients with CP and 0.21% of our controls developed pancreatic cancer yielding a HR of 4.5 (95% CI, 3.6–5.7). For other types of cancer, only small changes in the results were observed, which did not change the overall conclusion (not reported). Finally, we want to draw attention to the 388 patients in our study who were diagnosed with pancreatic cancer within 1 year of CP diagnosis. Whether CP was truly present or not should not change the conclusion that diagnostic attention should be given especially in the early phase of CP because 2.9% of patients with newly diagnosis of CP are diagnosed with a pancreatic cancer within 1 year.

Efficacy and Limitations of Budesonide as a Second-Line Treatment for Patients With Autoimmune Hepatitis

BACKGROUND & AIMS: Many patients with autoimmune hepatitis (AIH) develop steroid‐specific side effects or require doses of steroids that are unacceptable for long‐term treatment. We investigated the efficacy of budesonide as an alternative steroid for patients previously treated with prednisolone who developed side effects or were unable to reduce their dose of prednisolone below acceptable levels. We also report the effects of more than 12 months of budesonide treatment in a large cohort of patients with AIH. METHODS: We performed a retrospective analysis of data from 60 patients (51 female) with AIH who were treated initially with prednisolone (mean time, 47 mo) but then switched to budesonide, managed at a single center in Germany from 2001 through June 2016. Patients were evaluated after 6 months, 12 months, 24 months, 36 months, and at the last follow‐up evaluation; response to treatment with budesonide was assessed based on normal serum levels of aminotransferases and IgG (biochemical response). RESULTS: Thirty patients were switched to budesonide therapy because of prednisolone‐induced side effects and 30 patients switched because of prednisolone dependency. Overall, a biochemical response was detected in 55% of patients after 6 months of budesonide treatment, in 70% after 12 months, and in 67% after 24 months. At the last follow‐up evaluation (mean time, 63 mo) 23 patients (38%) still were receiving budesonide treatment. Fifteen patients (25%) had switched back to prednisolone therapy because of insufficient response to budesonide or its side effects. Fifteen patients with osteopenia at the beginning of budesonide treatment were followed up and evaluated by dual‐energy X‐ray absorptiometry. After a median of 24 months of budesonide treatment, bone mineral density had improved in 6 patients, remained stable in 8 patients, and worsened in 1 patient. CONCLUSIONS: We performed a retrospective analysis of patients with AIH that confirmed the therapeutic value of budesonide beyond 12 months of treatment in patients who are intolerant to or dependent on prednisolone. Although budesonide‐induced side effects appear to be mild in real life, effectiveness was limited in a considerable proportion of patients; close monitoring is advised.

Validation of Transient Elastography and Comparison with Spleen Length Measurement for Staging of Fibrosis and Clinical Prognosis in Primary Sclerosing Cholangitis

Background Patients with primary sclerosing cholangitis (PSC) develop progressive liver fibrosis and end-stage liver disease. Non-invasive and widely available parameters are urgently needed to assess disease stage and the risk of clinical progression. Transient elastography (TE) has been reported to predict fibrosis stage and disease progression. However, these results have not been confirmed in an independent cohort and comparison of TE measurement to other non-invasive means is missing. Methods In a retrospective study we collected data from consecutive PSC patients receiving TE measurements from 2006 to 2014 (n = 139). Data from 62 patients who also underwent a liver biopsy were used to assess the performance of TE and spleen length (SL) measurement for the staging of liver fibrosis. Follow-up data from this cohort (n = 130, Hamburg) and another independent cohort (n = 80, Paris) was used to compare TE and SL as predictors of clinical outcome applying Harrel’s C calculations. Results TE measurement had a very good performance for the diagnosis and exclusion of higher fibrosis stages (≥F3: AUROC 0.95) and an excellent performance for the diagnosis and exclusion of cirrhosis (F4 vs. < F4: AUROC 0.98). Single-point TE measurement had very similar predictive power for patient outcome as previously published. In a combined cohort of PSC patients (n = 210), SL measurements had a similar performance as TE for the prediction of patient outcome (5 x cross-validated Harrel’s C 0.76 and 0.72 for SL and TE, respectively). Conclusions Baseline TE measurement has an excellent performance to diagnose higher fibrosis stages in PSC. Baseline measurements of SL and TE have similar usefulness as predictive markers for disease progression in patients with PSC.

Spleen size for the prediction of clinical outcome in patients with primary sclerosing cholangitis

Dear Sir, We read with interest the work of van der Meer et al 1 who propose a risk score for patients with chronic Hepatitis C. The authors demonstrate that the assessment of readily available and objective parameters can stratify patients according to the risk of disease progression. Patients with primary sclerosing cholangitis (PSC) usually develop progressive liver fibrosis and end-stage liver disease within 10–20 years.2 Simple and non-invasive means for disease stratification and prediction of prognosis are urgently needed. Indeed, the International PSC Study Group recently declared the research on surrogate end-point markers as a high-priority task,3 since several clinical studies investigating novel treatment strategies …

Primary sclerosing cholangitis and cholangiocarcinoma: pathogenesis and modes of diagnostics.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease caused by progressive inflammation of the intra- and extrahepatic bile duct system. PSC patients have an increased risk to develop hepatobiliary as well as extrahepatic malignancies. The goal of a surveillance strategy for hepatobiliary malignancy in these patients is the detection of early cancer which will allow a potentially curative therapy. Here, we focus on a conceptual review of the pathogenesis of cholangiocellular carcinoma and gallbladder cancer and we will discuss a rational approach for the surveillance of these malignancies in PSC patients.

Opposing role of tumor necrosis factor receptor 1 signaling in T cell–mediated hepatitis and bacterial infection in mice

Death receptor (DR) ligands such as tumor necrosis factor (TNF) have been identified as fundamental mediators of liver damage both in mouse models and in humans. While the essential site of function of DR signaling is conceivably the hepatocyte, a systematic analysis is missing. Using mice with conditional gene ablation, we analyzed the tissue‐specific function of DR signaling in T cell–dependent (concanavalin A) and independent (lipopolysaccharide/galactosamine) hepatitis and in models of bacterial infection (Listeria monocytogenes, lipopolysaccharide). We report that lipopolysaccharide/galactosamine‐induced liver injury depends on hepatocyte‐intrinsic TNF receptor 1 (p55, TNFR1). In contrast, we show that T cell–induced hepatitis was independent of TNFR1 signaling in hepatocytes, T cells, or endothelial cells. Moreover, T cell–induced hepatitis was independent of hepatocyte‐intrinsic Fas‐associated protein with death domain, TNF‐related apoptosis‐inducing ligand receptor, or Fas signaling. Instead, concanavalin A–induced hepatitis was completely prevented in mice with myeloid‐derived cell (MDC)–specific deletion of TNFR1. Significantly, however, mice lacking TNFR1 in MDCs succumbed to listeria infection, although they displayed similar sensitivity toward endotoxin‐induced septic shock when compared to control mice. These results suggest that TNFR1 signaling in MDCs is a critical mediator of both the detrimental and the protective functions of TNF in T cell–induced hepatitis and bacterial infection, respectively. Conclusion: The critical site of action of DRs is completely dependent on the nature of hepatitis; the data specify MDCs as the essential cell type of TNFR1 function in T cell–mediated hepatitis and in the response to listeria, thereby identifying the opposing role of MDC TNFR1 in autoimmunity and bacterial infection. (Hepatology 2016;64:508‐521)

Risk of cholangiocarcinoma in patients with primary sclerosing cholangitis: diagnosis and surveillance

Purpose of review Primary sclerosing cholangitis (PSC) is associated with an increased risk of hepatobiliary and extrahepatic malignancy. Particularly the risk of cholangiocarcinoma (CCA) is greatly increased. To provide potentially curative treatments for affected patients an early diagnosis of CCA is crucial. We here review the current advances with respect to CCA diagnosis and surveillance and discuss a rational approach on how to perform surveillance of CCA in PSC patients. Recent findings Given the shortcomings of the current modalities for the surveillance and diagnosis of CCA in PSC, recent studies have focused on novel biomarkers for CCA. These include serum biomarkers (e.g., circulating angiopoeitin-2, cytokeratin-19 fragments, and antiglycoprotein 2 IgA autoantibodies, microRNA) as well as proteomics obtained from urine and/or bile. Novel approaches that may enhance the diagnostic value of brush cytology in future include the optimization of fluorescence in situ hybridization probes and the assessment of genetic aberrations. In addition, studies on advanced techniques (e.g., single-operator cholangioscopy and probe-based confocal laser endomicroscopy) have shown promising results with respect to CCA detection. Summary Despite recent advances in the diagnosis of CCA in PSC, the detection of early-stage CCA remains difficult. A better understanding of CCA pathogenesis and large prospective studies on novel biomarkers and techniques are required to timely diagnose CCA in the future.

How Should Cancer Surveillance in Primary Sclerosing Cholangitis Be Performed

Background: Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease affecting the intra- and extrahepatic bile duct system that can ultimately cause liver cirrhosis. Apart from the risk of progression to end-stage liver disease the prognosis of PSC is primarily determined by the risk to develop hepatobiliary or extrahepatic malignancies. A reasonable surveillance strategy for PSC patients must allow the detection of early cancer that will permit a potentially curative therapy. Methods: Current guidelines on malignancy within the context of PSC as well as the primary literature were reviewed for this article. Results: Here, we focus on a concise review of the three tumors most commonly associated with PSC: cholangiocellular carcinoma (CCA), gallbladder cancer, and colorectal carcinoma. For cancer surveillance in this patient group, endoscopy, cholangiography, cross-sectional imaging, and the use of serum tumor markers are principally available. Furthermore, for the diagnosis of CCA novel approaches were recently suggested to improve sensitivity and specificity to detect this malignancy. Conclusion: We review different aspects of cancer surveillance in patients with PSC. Since prospective data on the surveillance of malignant tumors is unavailable, we discuss a rational approach on how to perform cancer surveillance in patients with PSC.