Peter R. Galle

BackgroundNo effective systemic therapy exists for patients with advanced hepatocellular carcinoma.A preliminary study suggested that sorafenib, an oral multikinase inhibitor of the vascular endothelial growth factor receptor, the platelet-derived growth factor receptor, and Raf may be effective in hepatocellular carcinoma. MethodsIn this multicenter, phase 3, double-blind, placebo-controlled trial, we randomly assigned 602 patients with advanced hepatocellular carcinoma who had not received previous systemic treatment to receive either sorafenib (at a dose of 400 mg twice daily) or placebo.Primary outcomes were overall survival and the time to symptomatic progression.Secondary outcomes included the time to radiologic progression and safety. ResultsAt the second planned interim analysis, 321 deaths had occurred, and the study was stopped.Median overall survival was 10.7 months in the sorafenib group and 7.9 months in the placebo group (hazard ratio in the sorafenib group, 0.69; 95% confidence interval, 0.55 to 0.87; P<0.001).There was no significant difference between the two groups in the median time to symptomatic progression (4.1 months vs. 4.9 months, respectively, P = 0.77).The median time to radiologic progression was 5.5 months in the sorafenib group and 2.8 months in the placebo group (P<0.001).Seven patients in the sorafenib group (2%) and two patients in the placebo group (1%) had a partial response; no patients had a complete response.Diarrhea, weight loss, hand-foot skin reaction, and hypophosphatemia were more frequent in the sorafenib group. ConclusionsIn patients with advanced hepatocellular carcinoma, median survival and the time to radiologic progression were nearly 3 months longer for patients treated with sorafenib than for those given placebo.(ClinicalTrials.govnumber, NCT00105443.)

Liver cancer is the fifth most common cancer and the second most frequent cause of cancer-related death globally.Hepatocellular carcinoma represents about 90% of primary liver cancers and constitutes a major global health problem.The following Clinical Practice Guidelines will give up-to-date advice for the clinical management of patients with hepatocellular carcinoma, as well as providing an in-depth review of all the relevant data leading to the conclusions herein.

There have been major advances in the armamentarium for hepatocellular carcinoma (HCC) since the last official update of the Barcelona Clinic Liver Cancer prognosis and treatment strategy published in 2018. Whilst there have been advances in all areas, we will focus on those that have led to a change in strategy and we will discuss why, despite being encouraging, data for select interventions are still too immature for them to be incorporated into an evidence-based model for clinicians and researchers. Finally, we describe the critical insight and expert knowledge that are required to make clinical decisions for individual patients, considering all of the parameters that must be considered to deliver personalised clinical management.

Diagnosis of autoimmune hepatitis (AIH) may be challenging. However, early diagnosis is important because immunosuppression is life-saving. Diagnostic criteria of the International Autoimmune Hepatitis Group (IAIHG) were complex and purely meant for scientific purposes. This study of the IAIHG aims to define simplified diagnostic criteria for routine clinical practice. Candidate criteria included sex, age, autoantibodies, immunoglobulins, absence of viral hepatitis, and histology. The training set included 250 AIH patients and 193 controls from 11 centers worldwide. Scores were built from variables showing predictive ability in univariate analysis. Diagnostic value of each score was assessed by the area under the receiver operating characteristic (ROC) curve. The best score was validated using data of an additional 109 AIH patients and 284 controls. This score included autoantibodies, immunoglobulin G, histology, and exclusion of viral hepatitis. The area under the curve for prediction of AIH was 0.946 in the training set and 0.91 in the validation set. Based on the ROC curves, two cutoff points were chosen. The score was found to have 88% sensitivity and 97% specificity (cutoff > or =6) and 81% sensitivity and 99% specificity (cutoff > or =7) in the validation set.A reliable diagnosis of AIH can be made using a very simple diagnostic score. We propose the diagnosis of probable AIH at a cutoff point greater than 6 points and definite AIH 7 points or higher.

Patients with advanced hepatocellular carcinoma and increased α-fetoprotein concentrations have poor prognosis. We aimed to establish the efficacy of ramucirumab in patients with advanced hepatocellular carcinoma and α-fetoprotein concentrations of 400 ng/mL or higher.REACH-2 was a randomised, double-blind, placebo-controlled, phase 3 trial done at 92 hospitals, clinics, and medical centres in 20 countries. Eligible patients were aged 18 years or older and had histologically or cytologically confirmed hepatocellular carcinoma, or diagnosed cirrhosis and hepatocellular carcinoma, Barcelona Clinic Liver Cancer stage B or C disease, Child-Pugh class A liver disease, Eastern Cooperative Oncology Group (ECOG) performance statuses of 0 or 1, α-fetoprotein concentrations of 400 ng/mL or greater, and had previously received first-line sorafenib. Participants were randomly assigned (2:1) via an interactive web response system with a computer-generated random sequence to 8 mg/kg intravenous ramucirumab every 2 weeks or placebo. All patients received best supportive care. The primary endpoint was overall survival. Secondary endpoints were progression-free survival, proportion of patients achieving an objective response, time to radiographic progression, safety, time to deterioration in scores on the Functional Assessment of Cancer Therapy Hepatobiliary Symptom Index 8 (FHSI-8), and time to deterioration in ECOG performance status. We also pooled individual patient data from REACH-2 with data from REACH (NCT01140347) for patients with α-fetoprotein concentrations of 400 ng/mL or greater. Efficacy analyses were by intention to treat, whereas safety analyses were done in all patients who received at least one dose of study drug. This trial is registered with ClinicalTrials.gov, number NCT02435433.Between July 26, 2015, and Aug 30, 2017, 292 patients were randomly assigned, 197 to the ramucirumab group and 95 to the placebo group. At a median follow-up of 7·6 months (IQR 4·0-12·5), median overall survival (8·5 months [95% CI 7·0-10·6] vs 7·3 months [5·4-9·1]; hazard ratio [HR] 0·710 [95% CI 0·531-0·949]; p=0·0199) and progression-free survival (2·8 months [2·8-4·1] vs 1·6 months [1·5-2·7]; 0·452 [0·339-0·603]; p<0·0001) were significantly improved in the ramucirumab group compared with the placebo group. The proportion of patients with an objective response did not differ significantly between groups (nine [5%] of 197 vs one [1%] of 95; p=0·1697). Median time to deterioration in FHSI-8 total scores (3·7 months [95% CI 2·8-4·4] vs 2·8 months [1·6-2·9]; HR 0·799 [95% CI 0·545-1·171]; p=0·238) and ECOG performance statuses (HR 1·082 [95% CI 0·639-1·832]; p=0·77) did not differ between groups. Grade 3 or worse treatment-emergent adverse events that occurred in at least 5% of patients in either group were hypertension (25 [13%] in the ramucirumab group vs five [5%] in the placebo group), hyponatraemia (11 [6%] vs 0) and increased aspartate aminotransferase (six [3%] vs five [5%]). Serious adverse events of any grade and cause occurred in 68 (35%) patients in the ramucirumab group and 28 (29%) patients in the placebo group. Three patients in the ramucirumab group died from treatment-emergent adverse events that were judged to be related to study treatment (one had acute kidney injury, one had hepatorenal syndrome, and one had renal failure).REACH-2 met its primary endpoint, showing improved overall survival for ramucirumab compared with placebo in patients with hepatocellular carcinoma and α-fetoprotein concentrations of at least 400 ng/mL who had previously received sorafenib. Ramucirumab was well tolerated, with a manageable safety profile. To our knowledge, REACH-2 is the first positive phase 3 trial done in a biomarker-selected patient population with hepatocellular carcinoma.Eli Lilly.

Cholangiocarcinoma (CCA) comprises a heterogeneous group of cancers with pathologic features of biliary tract differentiation, and is presumed to arise from the intra- or extrahepatic biliary tract. Two recent papers suggest these cancers may also arise directly from transdifferentiation of hepatocytes [1,2]. Gallbladder cancer is distinct from cholangiocarcinoma in epidemiology, pathobiology, clinical presentation and management, and should be considered a different form of biliary tract cancer [3].

CD4(+)CD25(+) regulatory cells are a subpopulation of T lymphocytes of thymic origin. However, recent data suggest an alternative commitment of regulatory T cells in the periphery, although the precise mechanism is unknown. In the present work, we demonstrate that TGF-beta is able to induce Foxp3 expression and subsequently a regulatory phenotype in CD4(+)CD25(-) peripheral murine T cells. Similarly, TGF-beta induced Foxp3 in human CD4(+)CD25(-) T cells. Moreover, we show that the inhibitory Smad7 protein that is normally induced by TGF-beta and limits TGF-beta signaling, is strongly down-regulated by Foxp3 at the transcriptional level. Foxp3-mediated down-regulation of Smad7 subsequently rendered CD4(+)CD25(-) T cells highly susceptible to the morphogenic and regulatory effects of TGF-beta signaling via Smad3/4. In summary, we demonstrate that TGF-beta induces a regulatory phenotype in CD4(+)CD25(-) T cells through the induction of Foxp3 and a positive autoregulatory loop of TGF-beta signaling due to the absence of Smad7.

Every year, more than 945000 people develop colorectal cancer worldwide, and around 492000 patients die. This form of cancer develops sporadically, in the setting of hereditary cancer syndromes, or on the basis of inflammatory bowel diseases. Screening and prevention programmes are available for all these causes and should be more widely publicised. The adenoma-carcinoma sequence is the basis for development of colorectal cancer, and the underlying molecular changes have largely been identified. Prognosis depends on factors related to the patient, treatment, and tumour, and the expertise of the treatment team is one of the major determinants of outcome. New information on the molecular basis of this cancer have led to the development of targeted therapeutic options, which are being tested in clinical trials. Further clinical progress will largely depend on the broader implementation of multidisciplinary treatment strategies following the principles of evidence-based medicine.

Chemotherapeutic drugs cause DNA damage and kill cancer cells mainly by apoptosis. p53 mediates apoptosis after DNA damage. To explore the pathway of p53-dependent cell death, we investigated if p53-dependent apoptosis after DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated hepatoma, gastric cancer, colon cancer, and breast cancer cell lines upon treatment with different anticancer agents known to act via p53 accumulation. Cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin at concentrations present in the sera of patients during therapy led to an upregulation of both CD95 receptor and CD95 ligand. Induction of the CD95 ligand occurred in p53 wild-type (wt), p53 mutant (mt), and p53 deficient (p53−/−) cell lines and at wt and mt conformation of temperature-sensitive p53 mutants. In contrast, upregulation of the CD95 receptor was observed only in cells with wt p53, not in cells with mt or without any p53. Restitution of inducible wt p53 function restored the ability of p53−/− Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. This rendered the cells sensitive to CD95-mediated apoptosis. In an attempt to understand how CD95 expression is regulated by p53, we identified a p53-responsive element within the first intron of the CD95 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53-responsive elements in the promoter of the CD95 gene. wt p53 bound to and transactivated the CD95 gene, whereas mt p53 failed to induce apoptosis via activation of the CD95 gene. These observations provide a mechanistic explanation for the ability of p53 to contribute to tumor progression and to resistance of cancer cells to chemotherapy.

Background & Aims: Aconfocal laser endoscopy system has recently been developed that may allow subsurface imaging of living cells in colonic tissue in vivo. The aim of the present study was to assess its potential for prediction of histology during screening colonoscopy for colorectal cancer. Methods: Twenty-seven patients underwent colonoscopy with the confocal endoscope using acriflavine hydrochloride or fluorescein sodium with blue laser illumination. Furthermore, 42 patients underwent colonoscopy with this system using fluorescein sodium. Standardized locations and circumscript lesions were examined by confocal imaging before taking biopsy specimens. Confocal images were graded according to cellular and vascular changes and correlated with conventional histology in a prospective and blinded fashion. Results: Acriflavine hydrochloride and fluorescein sodium both yielded high-quality images. Whereas acriflavine hydrochloride strongly labeled the superficial epithelial cells, fluorescein sodium offered deeper imaging into the lamina propria. Fluorescein sodium was thus used for the prospective component of the study in which 13,020 confocal images from 390 different locations were compared with histologic data from 1038 biopsy specimens. Subsurface analysis during confocal laser endoscopy allowed detailed analysis of cellular structures. The presence of neoplastic changes could be predicted with high accuracy (sensitivity, 97.4%; specificity, 99.4%; accuracy, 99.2%). Conclusions: Confocal laser endoscopy is a novel diagnostic tool to analyze living cells during colonoscopy, thereby enabling virtual histology of neoplastic changes with high accuracy. These newly discovered diagnostic possibilities may be of crucial importance in clinical practice and lead to an optimized rapid diagnosis of neoplastic changes during ongoing colonoscopy. Background & Aims: Aconfocal laser endoscopy system has recently been developed that may allow subsurface imaging of living cells in colonic tissue in vivo. The aim of the present study was to assess its potential for prediction of histology during screening colonoscopy for colorectal cancer. Methods: Twenty-seven patients underwent colonoscopy with the confocal endoscope using acriflavine hydrochloride or fluorescein sodium with blue laser illumination. Furthermore, 42 patients underwent colonoscopy with this system using fluorescein sodium. Standardized locations and circumscript lesions were examined by confocal imaging before taking biopsy specimens. Confocal images were graded according to cellular and vascular changes and correlated with conventional histology in a prospective and blinded fashion. Results: Acriflavine hydrochloride and fluorescein sodium both yielded high-quality images. Whereas acriflavine hydrochloride strongly labeled the superficial epithelial cells, fluorescein sodium offered deeper imaging into the lamina propria. Fluorescein sodium was thus used for the prospective component of the study in which 13,020 confocal images from 390 different locations were compared with histologic data from 1038 biopsy specimens. Subsurface analysis during confocal laser endoscopy allowed detailed analysis of cellular structures. The presence of neoplastic changes could be predicted with high accuracy (sensitivity, 97.4%; specificity, 99.4%; accuracy, 99.2%). Conclusions: Confocal laser endoscopy is a novel diagnostic tool to analyze living cells during colonoscopy, thereby enabling virtual histology of neoplastic changes with high accuracy. These newly discovered diagnostic possibilities may be of crucial importance in clinical practice and lead to an optimized rapid diagnosis of neoplastic changes during ongoing colonoscopy. Colorectal cancer remains one of the leading causes of cancer death in the western world and is estimated to have affected 148,000 people in 2002 in the United States alone.1Grady W.M. Genetic testing for high-risk colon cancer patients.Gastroenterology. 2003; 124: 1574-1594Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 2Burt R.W. Colon cancer screening.Gastroenterology. 2000; 119: 837-853Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar It develops in about 5%–6% of the adult population, and almost one half will die as a consequence of the disease.3Gatta G. Ciccolallo L. Capocaccia R. Coleman M.P. Differences in colorectal cancer survival between European and US populations the importance of sub-site and morphology.Eur J Cancer. 2003; 39: 2214-2222Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 4Weir H.K. Thun M.J. Hankey B.F. Ries L.A. Howe H.L. Wingo P.A. Jemal A. Ward E. Anderson R.N. Edwards B.K. Annual report to the nation on the status of cancer, 1975–2000, featuring the uses of surveillance data for cancer prevention and control.J Natl Cancer Inst. 2003; 95: 1276-1299Crossref PubMed Scopus (768) Google Scholar The risk of developing colorectal cancer is influenced by hereditary and lifestyle factors. Based on familial clustering studies, it is estimated that 20%–30% of all colon cancer cases have a potentially definable inherited cause, whereas 3%–5% of colon cancers occur in genetically defined high-risk colon cancer family syndromes.1Grady W.M. Genetic testing for high-risk colon cancer patients.Gastroenterology. 2003; 124: 1574-1594Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar Many of the genetic mutations responsible for the development of colon cancer have already been determined and include mutations in oncogenes (e.g., K-RAS, SRC), tumor suppressor genes (e.g., APC, SMAD2/4), and DNA mismatch repair genes (e.g., hMLH1, hMSH2).5Chung D.C. The genetic basis of colorectal cancer insights into critical pathways of tumorigenesis.Gastroenterology. 2000; 119: 854-865Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar Screening colonoscopy is the widely accepted gold standard for early diagnosis of colorectal cancer and should be offered every 10 years to persons older than 50 years.6Winawer S. Fletcher R. Rex D. Bond J. Burt R. Ferrucci J. Ganiats T. Levin T. Woolf S. Johnson D. Kirk L. Litin S. Simmang C. Colorectal cancer screening and surveillance clinical guidelines and rationale—update based on new evidence.Gastroenterology. 2003; 124: 544-560Abstract Full Text PDF PubMed Scopus (1960) Google Scholar There is direct evidence that screening sigmoidoscopy reduces mortality related to colorectal cancer.7Selby J.V. Friedman G.D. Quesenberry C.P. Weiss N.S. A case control study of screening sigmoidoscopy and mortality from colorectal cancer.N Engl J Med. 1992; 326: 653-657Crossref PubMed Scopus (1560) Google Scholar Furthermore, colonoscopy has been shown to reduce the incidence of colorectal cancer in patients with adenomatous polyps.8Winawer S.J. Zauber A.G. Ho M.N. O’Brien M.J. Gottlieb L.S. Sternberg S.S. Waye J.D. Schapiro M. Bond J.H. et al.Prevention of colorectal cancer by colonoscopic polypectomy.N Engl J Med. 1993; 329: 1977-1981Crossref PubMed Scopus (3830) Google Scholar These data suggest the effectiveness of screening colonoscopy for the detection of premalignant changes. However, the prognosis of patients with colonic malignancies is strictly dependent on early detection of preneoplastic and neoplastic changes, because only intraepithelial neoplasias and early colorectal cancers can be cured by endoscopic resection.9Ortner M.A. Dorta G. Blum A.L. Michetti P. Endoscopic interventions for preneoplastic and neoplastic lesions mucosectomy, argon plasma coagulation, and photodynamic therapy.Dig Dis. 2002; 20: 167-172Crossref PubMed Scopus (18) Google Scholar For screening colonoscopy, modern video endoscopes using white light have been recommended.6Winawer S. Fletcher R. Rex D. Bond J. Burt R. Ferrucci J. Ganiats T. Levin T. Woolf S. Johnson D. Kirk L. Litin S. Simmang C. Colorectal cancer screening and surveillance clinical guidelines and rationale—update based on new evidence.Gastroenterology. 2003; 124: 544-560Abstract Full Text PDF PubMed Scopus (1960) Google Scholar When performing colonoscopy, it is important to differentiate between neoplastic (intraepithelial neoplasia, cancer) and nonneoplastic tissue (e.g., hyperplastic polyps), because only neoplastic tissue requires resection. This is particularly relevant for patients with large numbers of hyperplastic polyps due to genetic predisposition or inflammatory bowel diseases. Histologic analysis of biopsy material remains the gold standard for the final diagnosis of colorectal lesions. However, such workup and evaluation require time. This may limit the ability of the endoscopist to immediately determine the necessity for resection during ongoing colonoscopy, resulting in the need for repeat colonoscopies. Furthermore, overtreatment (resection of benign lesions) or undertreatment (biopsy instead of resection for neoplastic tissue) can lead to unnecessary risks (e.g., bleeding) for the patients. Recently, a confocal laser endomicroscope has been developed that is integrated in the distal tip of a conventional video colonoscope. This confocal laser microscope was designed to enable subsurface imaging of living tissue during colonoscopy and may offer an instant and reliable diagnostic tool for in vivo histology. The aim of the present study was to assess the suitability of this novel system for use in diagnosing intraepithelial neoplasias and colon cancer during ongoing colonoscopy. Laser scanning confocal microscopy is an adaptation of light microscopy whereby focal laser illumination is combined with pinhole limited detection to geometrically reject out-of-focus light. In single-point scanning confocal microscopes, the point is typically scanned in a raster pattern and measurement of light returning to the detector from successive points is digitized so that an image of the scanned region can be constructed. Importantly, each resultant image is an “optical section” representing approximately one focal plane within the specimen.10Pawley J.B. Limitations on optical sectioning in live-cell confocal microscopy.Scanning. 2002; 24: 241-246Crossref PubMed Scopus (44) Google Scholar The device assessed in the present study used a confocal microscope in which a single optical fiber acts as both the illumination point source and the detection pinhole, allowing the miniaturization required for endoscopy.11Delaney P.M. Harris M.R. Fiberoptics in confocal microscopy.in: Pawley J.B. Handbook of biological confocal microscopy. Plenum, New York, NY1995: 515-523Crossref Google Scholar The components of the confocal laser colonoscope are based on integration of a confocal laser microscope in the distal tip of a conventional video colonoscope (EC3870K; Pentax, Tokyo, Japan), which enables confocal microscopy in addition to standard videoendoscopy. The diameter of the distal tip and the insertion tube were 12.8 mm. The distal tip contained an air and water jet nozzle, 2 light guides, an auxiliary water jet channel (used for topical application of the contrast agent), and a 2.8-mm working channel (see Figure 1). Actuation of imaging plane depth was controlled using 2 remote control buttons on the handpiece. During laser endoscopy, an argon ion laser delivered an excitation wavelength of 488 nm and the maximum laser power output was ≤1 mW at the surface of the tissue. Confocal images were collected at a scan rate of 0.8 frames/second (1024 × 1024 pixels) or 1.6 frames/second (1024 × 512 pixels). The optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. The range of the z-axis was 0–250 μm below the surface layer. Confocal images could be generated simultaneously with endoscopic images. Patients on the normal endoscopy list were recruited for examination with the prototype instruments. The study protocols were reviewed and approved by the Human Research Ethics Committee of Cabrini Hospital (no. 08–14–10–02). Written informed consent was obtained from each patient before examination. All patients were prepared for routine colonoscopy by ingesting a commonly prescribed oral electrolyte lavage solution. Patient exclusion criteria were as follows: women of childbearing age, patients younger than 18 years of age, patients with an allergy or adverse reaction to acriflavine hydrochloride or fluorescein sodium, and those unable to give informed consent. Confocal images were collected following either the topical application of acriflavine hydrochloride (0.05% in saline; Sigma Pharmaceuticals, Australia; n = 9) or the intravenous administration of fluorescein sodium (5–10 mL of a 10% solution; Pharmalab, NSW, Australia; n = 18) during colonoscopy. Standard methods of conscious sedation (e.g., midazolam hydrochloride, fentanyl citrate, and propofol) and cardiopulmonary monitoring were used during each procedure. In asymptomatic patients with macroscopically normal colon, a series of images was collected from 4 to 5 randomly selected sites throughout the length of the large bowel from the rectum and sigmoid colon through to the cecum and in some instances the terminal ileum. Patients with an indication for screening or surveillance colonoscopy (after prior polypectomy) were recruited from the outpatient clinic of the First Department of Medicine at the University of Mainz (Mainz, Germany). Suitable patients were identified using inclusion and exclusion criteria. The inclusion criteria were indication for screening colonoscopy according to Winawer et al.6Winawer S. Fletcher R. Rex D. Bond J. Burt R. Ferrucci J. Ganiats T. Levin T. Woolf S. Johnson D. Kirk L. Litin S. Simmang C. Colorectal cancer screening and surveillance clinical guidelines and rationale—update based on new evidence.Gastroenterology. 2003; 124: 544-560Abstract Full Text PDF PubMed Scopus (1960) Google Scholar or indication for follow-up endoscopy after polypectomy or surgery according to Winawer et al.6Winawer S. Fletcher R. Rex D. Bond J. Burt R. Ferrucci J. Ganiats T. Levin T. Woolf S. Johnson D. Kirk L. Litin S. Simmang C. Colorectal cancer screening and surveillance clinical guidelines and rationale—update based on new evidence.Gastroenterology. 2003; 124: 544-560Abstract Full Text PDF PubMed Scopus (1960) Google Scholar Exclusion criteria were hereditary nonpolyposis colorectal cancer or familial adenomatous polyposis, known intraepithelial neoplasia or colorectal cancer or any other malignancy, acute gastrointestinal bleeding, coagulopathy (prothrombin time <50% of control; partial thromboplastin time >50 seconds), impaired renal function (creatinine level >1.2 mg/dL), pregnancy or breast-feeding, inability to provide informed consent, and known allergy to methylene blue or fluorescein sodium. The identified patients and their primary care physicians were invited to participate in the study, and informed consent was obtained. The study was approved by the local ethical committee in Rheinland-Pfalz, Germany (no. 837.321.03). Confocal colonoscopy was performed by 2 experienced endoscopists (R.K. and M.F.N.). Each had performed 5 confocal procedures before including the first patient in the current study. The confocal laser endoscope was first introduced into the terminal ileum or cecum. A total of 5 mL fluorescein (5–10 mL of a 10% solution; Alcon Laboratories, Inc.) sodium and 2 mL butylscopolamine (Buscopan; Boehringer Ingelheim, Germany) was then applied intravenously, because peristalsis may lead to artifacts. On withdrawal, all parts of the colon were evaluated as specified below. Standardized locations (every 10 cm in the colon and the distal part of the ileum) and every macroscopically visible lesion were examined with the help of the confocal laser imaging system. Every flat or suspected lesion was stained before the confocal examination in a targeted fashion by using methylene blue at a final concentration of 0.1% to clarify the borders of the lesions. In addition, the distal 20 cm of the colon was stained in an untargeted fashion with methylene blue as previously described12Kiesslich R. Bergh M.V. Hahn M. Hermann G. Jung M. Chromoendoscopy with indigocarmine improves the detection of adenomatous and nonadenomatous lesions in the colon.Endoscopy. 2001; 33: 1001-1006Crossref PubMed Scopus (252) Google Scholar to unmask circumscript lesions with the help of chromoendoscopy. Examinations before this study showed that such methylene blue staining does not cause an interference with the laser scanning system of the endoscope. No relevant interfering autofluorescence of the colon wall was noted. Every macroscopically visible, circumscript lesion was graded according to size (in centimeters), morphology (flat or depressed, sessile, polypoid), and location (distance in centimeters from the anal verge). The surface staining pattern was graded according to the modified pit pattern classification.13Kudo S. Tamura S. Nakajima T. Yamano H. Kusaka H. Watanabe H. Diagnosis of colorectal tumorous lesions by magnifying endoscopy.Gastrointest Endosc. 1996; 44: 8-14Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, 14Kiesslich R. Fritsch J. Holtmann M. Koehler H.H. Stolte M. Kanzler S. Nafe B. Jung M. Galle P.R. Neurath M.R. Methylene blue-aided chromoendoscopy for the detection of intraepithelial neoplasia and colon cancer in ulcerative colitis.Gastroenterology. 2003; 124: 880-888Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar, 15Kiesslich R. Neurath M.R. Surveillance colonoscopy in ulcerative colitis magnifying chromoendoscopy in the spotlight.Gut. 2004; 53: 165-167Crossref PubMed Scopus (84) Google Scholar The distal tip of the endoscope was placed in gentle contact with the mucosa and the position of the focal plane within the specimen adjusted using the buttons on the endoscope control body. Images were collected with the help of a foot pedal and stored as digital files. The confocal images of the different sides were analyzed for the presence of neoplastic changes by using a newly developed confocal pattern classification (see Table 1). The confocal pattern classification was developed based on experience from animal research16McLaren W. Anikijenko P. Barkla D. Delaney T.P. King R. In vivo detection of experimental ulcerative colitis in rats using fiberoptic confocal imaging (FOCI).Dig Dis Sci. 2001; 46: 2263-2276Crossref PubMed Scopus (42) Google Scholar, 17McLaren W.J. Anikijenko P. Thomas S.G. Delaney P.M. King R.G. In vivo detection of morphological and microvascular changes of the colon in association with colitis using fiberoptic confocal imaging (FOCI).Dig Dis Sci. 2002; 47: 2424-2433Crossref PubMed Scopus (52) Google Scholar and pathologic criteria18Schlemper R.J. Riddell R.H. Kato Y. Borchard F. Cooper H.S. Dawsey S.M. Dixon M.F. Fenoglio-Preiser C.M. Flejou J.F. Geboes K. Hattori T. Hirota T. Itabashi M. Iwafuchi M. Iwashita A. Kim Y.I. Kirchner T. Klimpfinger M. Koike M. Lauwers G.Y. Lewin K.J. Oberhuber G. Offner F. Price A.B. Rubio C.A. Shimizu M. Shimoda T. Sipponen P. Solcia E. Stolte M. Watanabe H. Yamabe H. The Vienna classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1696) Google Scholar and our own previous unpublished experience. The classification graduates neoplastic and nonneoplastic changes in 3 types (normal tissue, regeneration, and neoplasia). The confocal pattern classification and the macroscopic appearance of the lesions and the mucosal surface were taken together to predict histopathology. The endoscopic prediction was made immediately after the confocal imaging. However, after the procedure, all collected images were reevaluated and the quality was assessed. In the lower gastrointestinal tract, more than 70% of images were of satisfactory, good, or very good quality. In the remaining cases, moving artifacts were the most common cause of disturbance.Table 1Confocal Pattern Classification to Predict Colorectal PathologyGradingVessel architectureCrypt architectureNormalHexagonal, honeycomb appearance that presents a network of capillaries outlining the stroma surrounding the luminal openings of the cryptsRegular luminal openings and distribution of the crypts covered by a homogeneous layer of epithelial cells, including goblet cellsRegenerationHexagonal, honeycomb appearance with no or mild increase in the number of capillariesStar-shaped luminal crypt openings or focal aggregation of regular-shaped crypts with a regular or reduced amount of goblet cellsNeoplasiaDilated and distorted vessels with elevated leakage; irregular architecture with little or no orientation to adjunct tissueRidged-lined irregular epithelial layer with loss of crypts and goblet cells; irregular cell architecture with little or no mucin Open table in a new tab The endoscopic prediction of the different sites was compared prospectively with histology of biopsy specimens (H&E-stained serial sections of tissue cut in 2 different planes). The biopsy specimens, which were taken from the examined areas, were judged by 2 independent experienced pathologists in a blinded fashion and graded according to the modified Vienna classification.18Schlemper R.J. Riddell R.H. Kato Y. Borchard F. Cooper H.S. Dawsey S.M. Dixon M.F. Fenoglio-Preiser C.M. Flejou J.F. Geboes K. Hattori T. Hirota T. Itabashi M. Iwafuchi M. Iwashita A. Kim Y.I. Kirchner T. Klimpfinger M. Koike M. Lauwers G.Y. Lewin K.J. Oberhuber G. Offner F. Price A.B. Rubio C.A. Shimizu M. Shimoda T. Sipponen P. Solcia E. Stolte M. Watanabe H. Yamabe H. The Vienna classification of gastrointestinal epithelial neoplasia.Gut. 2000; 47: 251-255Crossref PubMed Scopus (1696) Google Scholar Mucosal specimens were obtained using standard biopsy forceps. The fixed biopsy specimens were orientated with a microscope for reverse light, embedded in paraffin, and sectioned vertically and transversely to facilitate the comparison between histology and confocal images. Afterward, the serial sections (4 μm) were stained with H&E for histopathologic examination. A computerized database was designed using Microsoft Excel 2000 (Microsoft Corp., Redmond, WA), and data were entered and stored in this program. Statistical analysis was performed by computer using the statistical software package SAS (release 6.08; SAS Institute, Inc., Cary, NC). Sensitivity, specificity, positive and negative predictive values, and accuracy rate were calculated for the prediction of neoplastic changes. McNemar’s test was used to compare paired proportions (histopathologic diagnosis and diagnosis by confocal laser endoscopy). The P value was calculated with McNemar’s test using the continuity correction. P values of ≤0.05 were considered significantly different. Due to the exploratory character of the study, no multiple adjusting was performed. To evaluate the potential of contrast agents for confocal laser endoscopy, 27 patients undergoing routine surveillance screening colonoscopy at the Cabrini Hospital, Australia, between January and September 2003 were recruited for examination. The indications for screening included a personal history of polyps, tumors, or diverticular disease or a family history of colorectal cancer. In all patients examined with the prototype instrument (16 men and 11 women; median age, 63 years; range, 37–77 years), the device was advanced to the cecum or terminal ileum in a time comparable to conventional colonoscopy (≤6 minutes). On average, the architecture of 6–12 crypts could be examined in a single field of view. Using either topical acriflavine hydrochloride or intravenously administered fluorescein sodium as a fluorescent contrast agent, the histologic correlates of the mucin-containing goblet cells and the columnar epithelial cells (including those vertically oriented across the surface in contact with the confocal imaging window and those radially oriented within crypts) were readily identifiable within the surface of the mucosa (Figure 2). However, when exploiting the subsurface optical sectioning capability of the device, acriflavine hydrochloride did not yield contrast beneath the epithelium and there was no observed temporal change in distribution after initial staining. Fluorescein sodium, however, distributed throughout the entire mucosa within seconds of intravenous administration. This resulted in strong contrast within the connective tissue matrix of the lamina propria and the microvasculature of the colon as well as permeation among the epithelial sheet. Fluorescein sodium also produced strong contrast in the epithelium, lamina propria, and internal microvasculature of the villi in the terminal ileum. A total of 138 consecutive patients from the First Department of Medicine at the University of Mainz were screened for possible inclusion in the present study, and 93 were excluded. A total of 45 patients fulfilled all inclusion criteria and were enrolled in this study, which was conducted between July and November 2003. Three patients had to be excluded from further analysis because of insufficient bowel preparation (possibly preventing the identification of relevant lesions) and 42 patients completed the study protocol (23 men and 19 women; mean age, 64.2 ± 12.1 years), including colonoscopy plus confocal laser endomicroscopy. The mean duration of the examination was 57 minutes (range, 37–82 minutes), and the mean time for reaching the cecum was 9 minutes (range, 3–16 minutes). After undergoing confocal laser endoscopy, all patients developed a slight yellow coloration of the skin (a side effect of fluorescein sodium), which disappeared in all cases within 60 minutes. No severe side effects were noted. Confocal laser endomicroscopy was performed on standardized locations (every 10 cm in the colon and terminal ileum) and all visible lesions on methylene blue staining. Confocal imaging of the normal terminal ileum during colonoscopy showed columnar epithelium of the villi, interspersed goblet cells, mononuclear cells, and capillaries filled with erythrocytes in high resolution (Figure 3). Similarly, histopathologic analysis of the same area showed normal appearance of ileal villi, goblet cells, intraepithelial lymphocytes, and subepithelial stroma tissue containing lymphocytes, plasma cells, and capillaries (Figure 3). However, whereas the nuclei of the intestinal epithelial cells were not readily visible during confocal endoscopy because of the pharmacologic properties of fluorescein sodium, confocal laser endoscopy showed a more prismatic appearance of intestinal epithelial cells in vivo as compared with formalin-fixed biopsy tissue, probably caused by the lack of fixation artifacts. In the normal colon, confocal laser endoscopy showed regular distribution of round-shaped crypts with round or oval crypt openings and a normal number of goblet cells (Figure 4). In addition, mononuclear cells surrounding the crypts were noted in the mucosa. Analysis of corresponding biopsy specimens from the same areas showed similar morphology of crypt and cell structure (Figure 4). However, whereas nuclei of intestinal epithelial cells were again not readily visible by confocal laser endoscopy, the superficial honeycomb lining of the crypts in the bowel wall could be easily analyzed by this method.Figure 4Confocal laser endomicroscopy of normal rectum mucosa. (A) Round-shaped regular colonic crypts with black mucin visible within goblet cells. (B) Corresponding histologic specimen shows the nuclei within the epithelial layer. 1, goblet cells; 2, crypt lumen; 3, stroma; 4, nuclei.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In addition, 134 circumscript lesions could be identified during colonoscopy after staining with methylene blue (94 flat, 13 sessile, and 27 polypoid) that were further analyzed by confocal laser endoscopy. The mean size of the lesions was 4 mm (flat lesions, 3 mm; sessile lesions, 5 mm; polypoid lesions, 9 mm). In 23 cases, confocal laser analysis showed aberrant crypt foci (Figure 5) with aggregated star-shaped crypts and clear demarcation against adjacent normal tissue. In contrast, intraepithelial neoplasias and colon cancers showed tubular, villous, or irregular architecture with a reduced number or loss of goblet cells, as determined by confocal laser endoscopy (Figure 6, Figure 7). A distinct gap between the cells was observed (Figure 6) in 11 of 38 neoplastic lesions but not in benign tissue. We postulate that this gap means “loss of cellular junction” due to loss of tight junctions as a potential sign of early malignancy. Furthermore, vessels with irregular structure could be identified in the tumor tissue. However, due to the lacking visibility of nuclei, a differentiation between low- and high-grade intraepithelial neoplasia was not possible by the use of confocal laser endoscopy.Figure 6Adenoma with high-grade intraepithelial neoplasia. (A) Videoendoscopy shows a large polyp. (B) Confocal laser endomicroscopy shows tubular-shaped crypts with a reduced amount of goblet cells and loss of cellular junctions. (C) Corresponding histologic specimen. 1, branched crypt structure in the area of intraepithelial neoplasia; 2, reduced amount of goblet cells; 3, loss of cellular junctions; 4, normal-shaped crypts.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Colorectal cancer. (A) Confocal laser endomicroscopy shows irregular cell architecture with total loss of goblet cells. (B) Corresponding histologic specimen.View Large

Chemotherapeutic drugs are cytotoxic by induction of apoptosis in drug-sensitive cells.We investigated the mechanism of bleomycin-induced cytotoxicity in hepatoma cells.At concentrations present in the sera of patients during therapy, bleomycin induced transient accumulation of nuclear wild-type (wt) p53 and upregulated expression of cell surface CD95 (APO-1/Fas) receptor in hepatoma cells carrying wt p53 (HepG2).Bleomycin did not increase CD95 in hepatoma cells with mutated p53 (Huh7) or in hepatoma cells which were p53 Ϫ / Ϫ (Hep3B).In addition, sensitivity towards CD95-mediated apoptosis was also increased in wt p53 positive HepG2 cells.Microinjection of wt p53 cDNA into HepG2 cells had the same effect.In contrast, bleomycin did not enhance susceptibility towards CD95-mediated apoptosis in Huh7 and in Hep3B cells.Furthermore, bleomycin treatment of HepG2 cells increased CD95 ligand (CD95L) mRNA expression.Most notably, bleomycin-induced apoptosis in HepG2 cells was almost completely inhibited by antibodies which interfere with CD95 receptor/ligand interaction.These data suggest that apoptosis induced by bleomycin is mediated, at least in part, by p53-dependent stimulation of the CD95 receptor/ligand system.The same applies to other anti-cancer drugs such as cisplatin and methotrexate.These data may have major consequences for drug treatment of cancer and the explanation of drug sensitivity and resistance.( J. Clin.Invest.

Conventional cytogenetic analysis in B-cell chronic lymphocytic leukemia (B-CLL) has been very difficult, and the prognostic significance of specific chromosome aberrations is under discussion. Recent improvements in fluorescence in situ hybridization (ISH) techniques have provided an alternative approach for the detection of chromosome aberrations. Here, an interphase cytogenetic study was performed to analyze the incidence and prognostic significance of a p53 gene deletion in B-CLL and related disorders. We studied mononuclear cells from 100 patients with chronic B-cell leukemias [B-CLL, 90 patients; B-prolymphocytic leukemia (B-PLL), 7; Waldenstrom's macroglobulinemia (WM), 3] by fluorescence ISH with a genomic p53 DNA probe. In a subset of patients, additional G-banding analysis and single strand conformation polymorphism (SSCP) analysis was performed. Seventeen of the 100 patients [17%; B-CLL, 11 of 90 (12%); WM, 1 of 3; B-PLL, 5 of 7] exhibited a monoallelic p53 gene deletion by ISH. G- banding analysis demonstrated abnormalities of chromosome 17 in 13 of these 17 patients, all leading to loss of band 17p13. SSCP analysis showed aberrant bands in 9 of 14 patients with a p53 gene deletion. None of 12 patients with a p53 gene deletion compared with 20 of 36 patients (56%) without a deletion responded to therapy with fludarabine or pentostatin (P &amp;lt; .001). The difference in survival probabilities from the time of diagnosis and from the start of treatment with purine analogs between the two groups was highly significant (P &amp;lt; .001). In multivariate analysis, p53 gene deletion was the strongest prognostic factor for survival. In conclusion, p53 gene deletion predicts for non- response to therapy with purine analogs and for poor survival in chronic B-cell leukemias.

Alterations of TGF-β signaling have been described in colorectal cancer, although the molecular consequences are largely unknown. By using transgenic mice overexpressing TGF-β or a dominant-negative TGF-βRII, we demonstrate that TGF-β signaling in tumor infiltrating T lymphocytes controls the growth of dysplastic epithelial cells in experimental colorectal cancer, as determined by histology and a novel system for high-resolution chromoendoscopy. At the molecular level, TGF-β signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell-derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-β-dependent IL-6 trans-signaling prevented tumor progression in vivo. Taken together, our data provide novel insights into TGF-β signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on inhibition of TGF-β-dependent IL-6 trans-signaling.

Apoptosis occurs in the normal liver and in various forms of liver disease. The CD95 (APO-1/Fas) (CD95) receptor mediates apoptosis, and liver cells in animal models are acutely sensitive to apoptosis initiated by this receptor. We have used primary human hepatocytes as a model system to investigate CD95-mediated apoptotic liver damage. Treatment of fresh human hepatocytes with low concentrations of agonistic antibodies against CD95 resulted in apoptosis of > 95% of the cultured liver cells within 4 and 7.5 h. Immunohistology of a panel of explanted liver tissues revealed that hepatocytes in normal livers (n = 5) and in alcoholic cirrhosis (n = 13) expressed low constitutive levels of CD95. CD95 receptor expression was highly elevated in hepatocytes in hepatitis B virus-related cirrhosis (n = 9) and in acute liver failure (n = 8). By in situ hybridization CD95 ligand messenger RNA expression was absent in normal liver but detected at high levels in livers with ongoing liver damage. In cases of hepatitis B virus-related cirrhosis and acute hepatic failure, ligand expression was found primarily in areas with lymphocytic infiltration. In contrast, in patients with alcoholic liver damage, high CD95 ligand messenger RNA expression was found in hepatocytes. These findings suggest that liver destruction in hepatitis B may primarily involve killing of hepatocytes by T lymphocytes using the CD95 receptor-ligand system. In alcoholic liver damage, death of hepatocytes might occur by fratricide and paracrine or autocrine mechanisms mediated by the hepatocytes themselves.

IMbrave150 demonstrated that atezolizumab plus bevacizumab led to significantly improved overall survival (OS) and progression-free survival (PFS) compared with sorafenib in patients with unresectable hepatocellular carcinoma at the primary analysis (after a median 8.6 months of follow-up). We present updated data after 12 months of additional follow-up.Patients with systemic treatment-naive, unresectable hepatocellular carcinoma were randomized 2:1 to receive 1,200 mg atezolizumab plus 15 mg/kg bevacizumab intravenously every 3 weeks or 400 mg sorafenib orally twice daily in this open-label, phase III study. Co-primary endpoints were OS and PFS by independently assessed RECIST 1.1 in the intention-to-treat population. Secondary efficacy endpoints included objective response rates and exploratory subgroup efficacy analyses. This is a post hoc updated analysis of efficacy and safety.From March 15, 2018, to January 30, 2019, 501 patients (intention-to-treat population) were randomly allocated to receive atezolizumab plus bevacizumab (n = 336) or sorafenib (n = 165). On August 31, 2020, after a median 15.6 (range, 0-28.6) months of follow-up, the median OS was 19.2 months (95% CI 17.0-23.7) with atezolizumab plus bevacizumab and 13.4 months (95% CI 11.4-16.9) with sorafenib (hazard ratio [HR] 0.66; 95% CI 0.52-0.85; descriptive p <0.001). The median PFS was 6.9 (95% CI 5.7-8.6) and 4.3 (95% CI 4.0-5.6) months in the respective treatment groups (HR 0.65; 95% CI 0.53-0.81; descriptive p < 0.001). Treatment-related grade 3/4 adverse events occurred in 143 (43%) of 329 and 72 (46%) of 156 safety-evaluable patients in the respective groups, and treatment-related grade 5 events occurred in 6 (2%) and 1 (<1%) patients.After longer follow-up, atezolizumab plus bevacizumab maintained clinically meaningful survival benefits over sorafenib and had a safety profile consistent with the primary analysis.NCT03434379.The primary analysis of IMbrave150 showed that atezolizumab plus bevacizumab had significantly greater benefits than sorafenib in patients with advanced hepatocellular carcinoma, but survival data were not yet mature. At this updated analysis done 12 months later, median overall survival was 5.8 months longer with atezolizumab plus bevacizumab than sorafenib, and the severity profile of treatment-related side effects remained similar. These updated results confirm atezolizumab plus bevacizumab as the first-line standard of care for advanced hepatocellular carcinoma.

Human asthma is associated with airway infiltration by T helper 2 (TH2) lymphocytes. We observed reduced expression of the TH1 transcription factor, T-bet, in T cells from airways of patients with asthma compared with that in T cells from airways of nonasthmatic patients, suggesting that loss of T-bet might be associated with asthma. Mice with a targeted deletion of the T-bet gene and severe combined immunodeficient mice receiving CD4+ cells from T-bet knockout mice spontaneously demonstrated multiple physiological and inflammatory features characteristic of asthma. Thus, T-bet deficiency, in the absence of allergen exposure, induces a murine phenotype reminiscent of both acute and chronic human asthma.

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-γ/interleukin (IL)-4 and transforming growth factor (TGF)-β activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L+ CD4+ T cells exacerbated colitis in reconstituted SCID mice, T-bet–deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet–deficient CD62L− CD4+ T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-β signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-γ/IL-4 and TGF-β responses and the development of chronic intestinal inflammation in T cell–mediated colitis. Furthermore, TGF-β was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-β and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell–mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.

Confocal laser endomicroscopy allows subsurface analysis of the intestinal mucosa and in vivo histology during ongoing endoscopy. Here, we have applied this technique to the in vivo diagnosis of Barrett's epithelium and associated neoplasia.Fluorescein-aided endomicroscopy was performed by applying the endomicroscope over the whole columnar-lined lower esophagus. Images obtained within 1 cm of the columnar-lined lower esophagus were stored digitally and a targeted biopsy examination or endoscopic mucosal resection of the examined areas was performed. In vivo histology was compared with the histologic specimens. All digitally stored images were re-assessed by a blinded investigator by the confocal Barrett classification system to predict histology. Intraobserver and interobserver variations of the involved endoscopists were evaluated by using kappa statistics.Endomicroscopy allowed distinguishing between different types of epithelial cells and detected cellular and vascular changes in Barrett's epithelium at high resolution during ongoing endoscopy in 63 patients. Barrett's esophagus and associated neoplasia could be predicted with a sensitivity of 98.1% and 92.9% and a specificity of 94.1% and 98.4%, respectively (accuracy, 96.8% and 97.4%). The mean kappa value for interobserver agreement for the prediction of histopathological diagnosis was .843, whereas the intraobserver agreement showed a mean kappa value of .892.Fluorescence-aided endomicroscopy of Barrett's esophagus allows in vivo histology of the mucosal layer during ongoing endoscopy. Gastric and Barrett's epithelium and Barrett's-associated neoplastic changes can be diagnosed with high accuracy. Thus, endomicroscopy may be helpful in the management of patients with Barrett's esophagus.

The intermediate stage of hepatocellular carcinoma (HCC) comprises a highly heterogeneous patient population and therefore poses unique challenges for therapeutic management, different from the early and advanced stages. Patients classified as having intermediate HCC by the Barcelona Clinic Liver Cancer (BCLC) staging system present with varying tumor burden and liver function. Transarterial chemoembolization (TACE) is currently recommended as the standard of care in this setting, but there is considerable variation in the clinical benefit patients derive from this treatment. In April 2012, a panel of experts convened to discuss unresolved issues surrounding the application of current guidelines when managing patients with intermediate HCC. The meeting explored the applicability of a subclassification system for intermediate HCC patients to tailor therapeutic interventions based on the evidence available to date and expert opinion. The present report summarizes the proposal of the expert panel: four substages of intermediate HCC patients, B1 to B4.

BACKGROUND A single, high priming dose of tremelimumab (anti-cytotoxic T lymphocyte-associated antigen 4) plus durvalumab (anti-programmed cell death ligand-1), an infusion regimen termed STRIDE (Single Tremelimumab Regular Interval Durvalumab), showed encouraging clinical activity and safety in a phase 2 trial of unresectable hepatocellular carcinoma. METHODSIn this global, open-label, phase 3 trial, the majority of the patients we enrolled with unresectable hepatocellular carcinoma and no previous systemic treatment were randomly assigned to receive one of three regimens: tremelimumab (300 mg, one dose) plus durvalumab (1500 mg every 4 weeks; STRIDE), durvalumab (1500 mg every 4 weeks), or sorafenib (400 mg twice daily).The primary objective was overall survival for STRIDE versus sorafenib.Noninferiority for overall survival for durvalumab versus sorafenib was a secondary objective. RESULTSIn total, 1171 patients were randomly assigned to STRIDE (n5393), durvalumab (n5389), or sorafenib (n5389).The median overall survival was 16.43 months (95% confidence interval [CI], 14.16 to 19.58) with STRIDE, 16.56 months (95% CI, 14.06 to 19.12) with durvalumab, and 13.77 months (95% CI, 12.25 to 16.13) with sorafenib.Overall survival at 36 months was 30.7%, 24.7%, and 20.2%, respectively.The overall survival hazard ratio for STRIDE versus sorafenib was 0.78 (96.02% CI, 0.65 to 0.93; P50.0035).Overall survival with durvalumab monotherapy was noninferior to sorafenib (hazard ratio, 0.86; 95.67% CI, 0.73 to 1.03; noninferiority margin, 1.08).Median progression-free

Abstract Data regarding the role of TGF-β for the in vivo function of regulatory CD4+CD25+ T cells (Treg) are controversial. A transgenic mouse model with impaired TGF-β signaling specifically in T cells was used to assess the role of endogenous TGF-β for the in vivo function of CD4+CD25+ Treg in a murine model of colitis induced by dextran sulfate. Transfer of wild-type, but not transgenic CD4+CD25+ Treg was found to suppress colitis in wild-type mice. In addition, by transferring CFSE-labeled CD4+CD25+ Treg we could demonstrate that endogenous TGF-β promotes the expansion of CD4+CD25+ Treg in vivo. Transgenic mice themselves developed reduced numbers of peripheral CD4+CD25+ Treg and were more susceptible to the induction of colitis, which could be prevented by the transfer of wild-type Treg. These data indicate that TGF-β signaling in CD4+CD25+ Treg is required for their in vivo expansion and suppressive capacity.

Abstract Hepatocellular carcinoma (HCC) is one of the most common causes of cancer‐related deaths globally due, in part, to the majority of patients being diagnosed with intermediate or advanced stage disease. Our increased understanding of the heterogeneous molecular pathogenesis of HCC has led to significant developments in novel targeted therapies. Despite these advances, there remains a high unmet need for new treatment options. HCC is a complex disease with multiple pathogenic mechanisms caused by a variety of risk factors, making it difficult to characterize with a single biomarker. In fact, numerous biomarkers have been studied in HCC, but alpha‐fetoprotein (AFP) remains the most widely used and accepted serum marker since its discovery over 60 years ago. This review summarizes the most relevant studies associated with the regulation of AFP at the gene and protein levels; the pathophysiology of AFP as a pro‐proliferative protein; and the correlation of AFP with molecular HCC subclasses, the vascular endothelial growth factor pathway and angiogenesis. Also described are the historical and current uses of AFP for screening and surveillance, diagnosis, its utility as a prognostic and predictive biomarker and its role as a tumour antigen in HCC. Taken together, these data demonstrate the relevance of AFP for patients with HCC and identify several remaining questions that will benefit from future research.

Loss of intestinal barrier function plays an important role in the pathogenesis of inflammatory bowel disease (IBD). Shedding of intestinal epithelial cells is a potential cause of barrier loss during inflammation. The objectives of the study were (1) to determine whether cell shedding and barrier loss in humans can be detected by confocal endomicroscopy and (2) whether these parameters predict relapse of IBD.Confocal endomicroscopy was performed in IBD and control patients using intravenous fluorescein to determine the relationship between cell shedding and local barrier dysfunction. A grading system based on appearances at confocal endomicroscopy in humans was devised and used to predict relapse in a prospective pilot study of 47 patients with ulcerative colitis and 11 patients with Crohn's disease.Confocal endomicroscopy in humans detected shedding epithelial cells and local barrier defects as plumes of fluorescein effluxing through the epithelium. Mouse experiments demonstrated inward flow through some leakage-associated shedding events, which was increased when luminal osmolarity was decreased. In IBD patients in clinical remission, increased cell shedding with fluorescein leakage was associated with subsequent relapse within 12 months after endomicroscopic examination (p<0.001). The sensitivity, specificity and accuracy for the grading system to predict a flare were 62.5% (95% CI 40.8% to 80.4%), 91.2% (95% CI 75.2 to 97.7) and 79% (95% CI 57.7 to 95.5), respectively.Cell shedding and barrier loss detected by confocal endomicroscopy predicts relapse of IBD and has potential as a diagnostic tool for the management of the disease.

The cancer stem cells (CSCs) have important therapeutic implications for multi-resistant cancers including hepatocellular carcinoma (HCC). Among the key pathways frequently activated in liver CSCs is NF-κB signaling.We evaluated the CSCs-depleting potential of NF-κB inhibition in liver cancer achieved by the IKK inhibitor curcumin, RNAi and specific peptide SN50. The effects on CSCs were assessed by analysis of side population (SP), sphere formation and tumorigenicity. Molecular changes were determined by RT-qPCR, global gene expression microarray, EMSA, and Western blotting.HCC cell lines exposed to curcumin exhibited differential responses to curcumin and were classified as sensitive and resistant. In sensitive lines, curcumin-mediated induction of cell death was directly related to the extent of NF-κB inhibition. The treatment also led to a selective CSC-depletion as evidenced by a reduced SP size, decreased sphere formation, down-regulation of CSC markers and suppressed tumorigenicity. Similarly, NF-κB inhibition by SN50 and siRNA against p65 suppressed tumor cell growth. In contrast, curcumin-resistant cells displayed a paradoxical increase in proliferation and expression of CSC markers. Mechanistically, an important component of the CSC-depleting activity of curcumin could be attributed to a NF-κB-mediated HDAC inhibition. Co-administration of the class I/II HDAC inhibitor trichostatine sensitized resistant cells to curcumin. Further, integration of a predictive signature of curcumin sensitivity with human HCC database indicated that HCCs with poor prognosis and progenitor features are most likely to benefit from NF-κB inhibition.These results demonstrate that blocking NF-κB can specifically target CSC populations and suggest a potential for combined inhibition of NF-κB and HDAC signaling for treatment of liver cancer patients with poor prognosis.

The last 5 years have witnessed relevant advances in the systemic treatment of hepatocellular carcinoma. New data have emerged since the development of the EASL Clinical Practice Guidelines on the management of hepatocellular carcinoma in 2018. Drugs licensed in some countries now include 4 oral multi-tyrosine kinase inhibitors (sorafenib, lenvatinib, regorafenib and cabozantinib), 1 anti-angiogenic antibody (ramucirumab) and 4 immune checkpoint inhibitors, alone or in combination (atezolizumab in combination with bevacizumab, ipilimumab in combination with nivolumab, nivolumab and pembrolizumab in monotherapy). Prolonged survival in excess of 2 years can be expected in most patients with sensitive tumours and well-preserved liver function that renders them fit for sequential therapies. With different choices available in any given setting, the robustness of the evidence of efficacy and a correct matching of the safety profile of a given agent with patient characteristics and preferences are key in making sound therapeutic decisions. The recommendations in this document amend the previous EASL Clinical Practice Guidelines and aim to help clinicians provide the best possible care for patients today. In view of several ongoing and promising trials, further advances in systemic therapy of hepatocellular carcinoma are foreseen in the near future and these recommendations will have to be updated regularly.

Summary Background Understanding patients' experience of cancer treatment is important. We aimed to evaluate patient-reported outcomes (PROs) with atezolizumab plus bevacizumab versus sorafenib in patients with advanced hepatocellular carcinoma in the IMbrave150 trial, which has already shown significant overall survival and progression-free survival benefits with this combination therapy. Methods We did an open-label, randomised, phase 3 trial in 111 hospitals and cancer centres across 17 countries or regions. We included patients aged 18 years or older with systemic, treatment-naive, histologically, cytologically, or clinically confirmed unresectable hepatocellular carcinoma and an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, with disease that was not amenable to curative surgical or locoregional therapies, or progressive disease after surgical or locoregional therapies. Participants were randomly assigned (2:1; using permuted block randomisation [blocks of six], stratified by geographical region; macrovascular invasion, extrahepatic spread, or both; baseline alpha-fetoprotein concentration; and ECOG performance status) to receive 1200 mg atezolizumab plus 15 mg/kg bevacizumab intravenously once every 3 weeks or 400 mg sorafenib orally twice a day, until loss of clinical benefit or unacceptable toxicity. The independent review facility for tumour assessment was masked to the treatment allocation. Previously reported coprimary endpoints were overall survival and independently assessed progression-free survival per Response Evaluation Criteria in Solid Tumors 1.1. Prespecified secondary and exploratory analyses descriptively evaluated treatment effects on patient-reported quality of life, functioning, and disease symptoms per the European Organisation for Research and Treatment of Cancer (EORTC) quality-of-life questionnaire for cancer (QLQ-C30) and quality-of-life questionnaire for hepatocellular carcinoma (QLQ-HCC18). Time to confirmed deterioration of PROs was analysed in the intention-to-treat population; all other analyses were done in the PRO-evaluable population (patients who had a baseline PRO assessment and at least one assessment after baseline). The trial is ongoing; enrolment is closed. This trial is registered with ClinicalTrials.gov , NCT03434379 . Findings Between March 15, 2018, and Jan 30, 2019, 725 patients were screened and 501 patients were enrolled and randomly assigned to atezolizumab plus bevacizumab (n=336) or sorafenib (n=165). 309 patients in the atezolizumab plus bevacizumab group and 145 patients in the sorafenib group were included in the PRO-evaluable population. At data cutoff (Aug 29, 2019) the median follow-up was 8·6 months (IQR 6·2–10·8). EORTC QLQ-C30 completion rates were 90% or greater for 23 of 24 treatment cycles in both groups (range 88–100% in the atezolizumab plus bevacizumab group and 80–100% in the sorafenib group). EORTC QLQ-HCC18 completion rates were 90% or greater for 20 of 24 cycles in the atezolizumab plus bevacizumab group (range 88–100%) and 21 of 24 cycles in the sorafenib group (range 89–100%). Compared with sorafenib, atezolizumab plus bevacizumab reduced the risk of deterioration on all EORTC QLQ-C30 generic cancer symptom scales that were prespecified for analysis (appetite loss [hazard ratio (HR) 0·57, 95% CI 0·40–0·81], diarrhoea [0·23, 0·16–0·34], fatigue [0·61, 0·46–0·81], pain [0·46, 0·34–0·62]), and two of three EORTC QLQ-HCC18 disease-specific symptom scales that were prespecified for analysis (fatigue [0·60, 0·45–0·80] and pain [0·65, 0·46–0·92], but not jaundice [0·76, 0·55–1·07]). At day 1 of treatment cycle five (after which attrition in the sorafenib group was more than 50%), the mean EORTC QLQ-C30 score changes from baseline in the atezolizumab plus bevacizumab versus sorafenib groups were: –3·29 (SD 17·56) versus –5·83 (20·63) for quality of life, –4·02 (19·42) versus –9·76 (21·33) for role functioning, and –3·77 (12·82) versus –7·60 (15·54) for physical functioning. Interpretation Prespecified analyses of PRO data showed clinically meaningful benefits in terms of patient-reported quality of life, functioning, and disease symptoms with atezolizumab plus bevacizumab compared with sorafenib, strengthening the combination therapy's positive benefit–risk profile versus that of sorafenib in patients with unresectable hepatocellular carcinoma. Funding F Hoffmann–La Roche and Genentech.

Treatment of hepatocellular carcinoma (HCC) is dependent on the stage of the disease. Intermediate stage HCC encompasses the largest subgroup of patients with the disease, and is characterized by substantial heterogeneity. The standard therapeutic approach, transarterial chemoembolization (TACE), is probably over-used and may not be appropriate for all patients with intermediate stage HCC. In patients with extensive tumour bulk, multi-nodular spread or impaired liver function, TACE may not be optimal and other treatments can be considered as a first-line treatment. These include surgery, percutaneous ablation, radioembolization or systemic treatment. In addition, patients who do not achieve complete or partial necrosis (TACE failure) and patients with early recurrence after TACE, should be managed individually, considering systemic treatments usually reserved for advanced disease. In selected cases and in patients who achieve downstaging, radical approaches such as hepatic resection or even liver transplantation can be considered. In this review, we evaluate the current literature for the treatment strategies for patients with intermediate Barcelona Clinic Liver Cancer (BCLC) B stage HCC.

Abstract Background and Aims Atezolizumab plus bevacizumab (AtezoBev) is the standard of care for first‐line treatment of unresectable HCC. No evidence exists as to its use in routine clinical practice in patients with impaired liver function. Approach and Results In 216 patients with HCC who were consecutively treated with AtezoBev across 11 tertiary centers, we retrospectively evaluated treatment‐related adverse events (trAEs) graded (G) according to Common Terminology Criteria for Adverse Events v5.0, including in the analysis all patients treated according to label ( n = 202, 94%). We also assessed overall survival (OS), progression‐free survival (PFS), overall response (ORR), and disease control rates (DCR) defined by Response Evaluation Criteria in Solid Tumors v1.1. Disease was mostly secondary to viral hepatitis, namely hepatitis C ( n = 72; 36%) and hepatitis B infection ( n = 35, 17%). Liver function was graded as Child‐Pugh (CP)‐A in 154 patients (76%) and CP‐B in 48 (24%). Any grade trAEs were reported by 143 patients (71%), of which 53 (26%) were G3 and 3 (2%) G4. Compared with CP‐A, patients with CP‐B showed comparable rates of trAEs. Presence and grade of varices at pretreatment esophagogastroduodenoscopy did not correlate with bleeding events. After a median follow‐up of 9.0 months (95% CI, 7.8–10.1), median OS was 14.9 months (95% CI, 13.6–16.3), whereas median PFS was 6.8 months (95% CI, 5.2–8.5). ORR and DCR were respectively 25% and 73%, with no difference across CP classes. Conclusions This study confirms reproducible safety and efficacy of AtezoBev in routine practice. Patients with CP‐B reported similar tolerability compared with CP‐A, warranting prospective evaluation of AtezoBev in this treatment‐deprived population.

In light of a global rise in obesity and type 2 diabetes, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) represent an increasingly important underlying aetiology of hepatocellular carcinoma (HCC). HCCs arising from lipotoxicity-mediated chronic inflammation are characterised by several unique features: in contrast to virally driven HCC, up to 50% of NAFLD-HCC occurs in patients without cirrhosis and annual HCC incidence is comparatively low, complicating current surveillance strategies. On average, patients are older and are more frequently diagnosed at an advanced stage. While locoregional treatments are probably equally effective regardless of HCC aetiology, the picture is less clear for systemic therapy. Tyrosine kinase inhibitors are probably equally effective, while there have been initial signals that immune checkpoint inhibitors may be less effective in NAFLD-HCC than in viral HCC. Current international clinical practice guidelines for HCC do not consider aetiology, as there are insufficient data to draw specific conclusions or to recommend aetiology-specific modifications to the current management of patients with HCC. However, in light of the growing relevance of NAFLD-HCC, future clinical trials should assess whether HCC aetiology - and NAFLD/NASH in particular - influence the safety and efficacy of a given treatment.

HCC is one of the most common cancers worldwide, and the third leading cause of cancer-related death globally. HCC comprises nearly 90% of all cases of primary liver cancer. Approximately half of all patients with HCC receive systemic therapy during their disease course, particularly in the advanced stages of disease. Immuno-oncology has been paradigm shifting for the treatment of human cancers, with strong and durable antitumor activity in a subset of patients across a variety of malignancies including HCC. Immune checkpoint inhibition with atezolizumab and bevacizumab, an antivascular endothelial growth factor neutralizing antibody, has become first-line therapy for patients with advanced HCC. Beyond immune checkpoint inhibition, immunotherapeutic strategies such as oncolytic viroimmunotherapy and adoptive T-cell transfer are currently under investigation. The tumor immune microenvironment of HCC has significant immunosuppressive elements that may affect response to immunotherapy. Major unmet challenges include defining the role of immunotherapy in earlier stages of HCC, evaluating combinatorial strategies that use targeting of the immune microenvironment plus immune checkpoint inhibition, and identifying treatment strategies for patients who do not respond to the currently available immunotherapies. Herein, we review the rationale, mechanistic basis and supporting preclinical evidence, and available clinical evidence for immunotherapies in HCC as well as ongoing clinical trials of immunotherapy.

Fibrosis-4 (FIB-4) and the nonalcoholic fatty liver disease fibrosis score (NFS) are the 2 most popular noninvasive blood-based serum tests proposed for widespread fibrosis screening. We therefore aimed to describe the accuracy of FIB-4 and NFS to detect elevated liver stiffness as an indicator of hepatic fibrosis in low-prevalence populations.This study included a total of 5129 patients with concomitant measurement of FIB-4, NFS, and liver stiffness measurement (LSM) by Fibroscan (Echosens, France) from 5 independent population-based cohorts from Spain, Hong Kong, Denmark, England, and France; 3979 participants from the general population and 1150 from at-risk cohorts due to alcohol, diabetes, or obesity. We correlated LSM with FIB-4 and NFS, and calculated pre- and post-test predictive values of FIB-4 and NFS to detect elevated LSM at 8 kPa and 12 kPa cutoffs. The mean age was 53 ± 12 years, the mean body mass index was 27 ± 5 kg/m2, and 2439 (57%) were women. One in 10 patients (552; 11%) had liver stiffness ≥8 kPa, but 239 of those (43%) had a normal FIB-4, and 171 (31%) had normal NFS. The proportion of false-negatives was higher in at-risk patients than the general population. FIB-4 was false-negative in 11% of diabetic subjects, compared with 2.5% false-negatives with NFS. Waist circumference outperformed FIB-4 and NFS for detecting LSM ≥8 kPa in the general population. Almost one-third (28%-29%) of elevated FIB-4/NFS were false-positive in both the general population and at-risk cohorts.FIB-4 and NFS are suboptimal for screening purposes due to a high risk of overdiagnosis and a non-negligible percentage of false-negatives, especially in patients with risk factors for chronic liver disease. Waist circumference emerged as a potential first step to identify patients at risk for liver fibrosis in the general population.

Based on positive results from small single center studies, granulocyte-colony stimulating factor (G-CSF) is being widely used for the treatment of patients with acute-on-chronic liver failure (ACLF). Herein, we aimed to evaluate the safety and efficacy of G-CSF in patients with ACLF.In this multicenter, prospective, controlled, open-label phase II study, 176 patients with ACLF (EASL-CLIF criteria) were randomized to receive G-CSF (5 μg/kg daily for the first 5 days and every third day thereafter until day 26) plus standard medical therapy (SMT) (n = 88) or SMT alone. The primary efficacy endpoint was 90-day transplant-free survival analyzed by Cox regression modeling. The key secondary endpoints were overall and transplant-free survival after 360 days, the development of ACLF-related complications, and the course of liver function scores during the entire observation period.Patients treated with G-CSF had a 90-day transplant-free survival rate of 34.1% compared to 37.5% in the SMT group (hazard ratio [HR] 1.05; 95% CI 0.711-1.551; p = 0.805). Transplant-free and overall survival at 360 days did not differ between the 2 arms (HR 0.998; 95% CI 0.697-1.430; p = 0.992 and HR 1.058; 95% CI 0.727-1.548; p = 0.768, respectively). G-CSF did not improve liver function scores, the occurrence of infections, or survival in subgroups of patients without infections, with alcohol-related ACLF, or with ACLF defined by the APASL criteria. Sixty-one serious adverse events were reported in the G-CSF+SMT group and 57 were reported in the SMT group. In total, 7 drug-related serious adverse reactions occurred in the G-CSF group. The study was prematurely terminated due to futility after conditional power calculation.In contrast to previous findings, G-CSF had no significant beneficial effect on patients with ACLF in this multicenter controlled trial, which suggests that it should not be used as a standard treatment for ACLF. CLINICALTRIALS.NCT02669680 LAY SUMMARY: Granulocyte-colony stimulating factor was considered as a novel treatment for acute-on-chronic liver failure (ACLF). We performed the first randomized, multicenter, controlled phase II trial, which showed that G-CSF did not improve survival or other clinical endpoints in patients with ACLF. Therefore, G-CSF should not be used to treat liver disease outside clinical studies.

Single-agent anti-PD1 checkpoint inhibitors convey outstanding clinical benefits in a small fraction (∼20%) of patients with advanced hepatocellular carcinoma (aHCC) but the molecular mechanisms determining response are unknown. To fill this gap, we herein analyze the molecular and immune traits of aHCC in patients treated with anti-PD1.Overall, 111 tumor samples from patients with aHCC were obtained from 13 centers before systemic therapies. We performed molecular analysis and immune deconvolution using whole-genome expression data (n = 83), mutational analysis (n = 72), and histologic evaluation with an endpoint of objective response.Among 83 patients with transcriptomic data, 28 were treated in frontline, whereas 55 patients were treated after tyrosine kinase inhibitors (TKI) either in second or third line. Responders treated in frontline showed upregulated interferon-γ signaling and major histocompatibility complex II-related antigen presentation. We generated an 11-gene signature (IFNAP), capturing these molecular features, which predicts response and survival in patients treated with anti-PD1 in frontline. The signature was validated in a separate cohort of aHCC and >240 patients with other solid cancer types where it also predicted response and survival. Of note, the same signature was unable to predict response in archival tissue of patients treated with frontline TKIs, highlighting the need for fresh biopsies before immunotherapy.Interferon signaling and major histocompatibility complex-related genes are key molecular features of HCCs responding to anti-PD1. A novel 11-gene signature predicts response in frontline aHCC, but not in patients pretreated with TKIs. These results must be confirmed in prospective studies and highlights the need for biopsies before immunotherapy to identify biomarkers of response.

IMbrave150 has established the superiority of atezolizumab plus bevacizumab over sorafenib in patients with unresectable hepatocellular carcinoma (HCC).We generated a prospectively maintained database including patients treated with atezolizumab plus bevacizumab for unresectable HCC across Europe, Asia and USA. Clinico-pathologic characteristics were assessed for their prognostic influence on overall survival (OS) and progression-free survival (PFS) in univariable and multivariate analyses. Overall response rate by RECIST v1.1 and treatment-related adverse events (TRAEs) per CTCAE v.5.0 were reported.Out of 433 patients, 296 Child-Pugh A and ECOG performance status01 patients received atezolizumab plus bevacizumab in first line and were included. Patients were mostly male (82.7%), cirrhotic (75%) with history of viral hepatitis (65.9%). Overall, 68.9% had Barcelona Clinic Liver Cancer C-stage HCC with portal vein tumour thrombosis (PVTT, 35%) and extrahepatic spread (EHS, 51.7%). After a median follow-up of 10.0 months (95% confidence interval (CI): 9.4-10.4), median OS and PFS were 15.7 (95% CI: 14.5-NE) and 6.9 months (95% CI: 6.1-8.3), respectively. In the response-evaluable patients (n = 273), overall response rate was 30.8%. Overall, 221 patients (74.6%) developed TRAEs, with 70 (23.6%) reporting grade 3 or higher TRAEs; 25 (8.4%) patients had bleeding events. OS was independently associated with baseline Albumin-bilirubin (ALBI) grade and PVTT. Shorter PFS was associated with AFP≥ 400 ng/ml, worse ALBI and presence of EHS.This global observational study confirms the reproducible safety and efficacy of atezolizumab plus bevacizumab in routine clinical practice. Within Child-Pugh-A criteria, the presence of PVTT and higher ALBI grade identify patients with poorer survival.

<h2>Abstract</h2><h3>Background&Aims</h3> Immune-related liver injury(irLI) is commonly observed in patients with cancer treated with immune checkpoint inhibitors(ICIs). We aimed to compare incidence, clinical characteristics and outcomes of irLI between patients receiving ICIs for hepatocellular carcinoma(HCC) versus other solid tumours. <h3>Methods</h3> Two separate cohorts were included: 375 patients with advanced/unresectable HCC, Child-Pugh A class treated with first-line Atezolizumab+Bevacizumab from AB-real study and a non-HCC cohort, including 459 patients treated with first-line ICI therapy from INVIDIa-2 multicentre study. IrLI was defined as treatment‐related increase of transaminases levels after exclusion of alternative aetiologies of liver injury. Incidence of irLI was adjusted for the duration of treatment exposure. <h3>Results</h3> In HCC patients, incidence of any-grade irLI was 11.4% over a median treatment exposure of 4.4 months(95%CI 3.7-5.2), compared to 2.6% in INVIDIa-2 cohort over a median treatment exposure of 12.4 months(95%CI 11.1-14.0). Exposure-adjusted-incidence of any-grade irLI was 22.1 per 100-Patient-years(PY) in HCC patients and 2.1 per 100-PY in non-HCC patients(p<0.001), with median time to irLI of 1.4 and 4.7 months, respectively. Among patients who developed irLI, systemic corticosteroids were administered in 16.3% of HCC and 75.0% of non-HCC patients(p<0.001) and irLI resolution was observed in 72.1% and 58.3%, respectively(p=0.362). In HCC patients, rates of hepatic decompensation and treatment discontinuation due to irLI were 7%. Grade 1-2 irLI was associated with improved overall survival in HCC patients only(HR 0.53, 95%CI 0.29-0.96). <h3>Conclusions</h3> Despite higher incidence and earlier onset in patients with HCC, IrLI is characterised by high rates of remission, low requirement for corticosteroid therapy and low risk of decompensation compared to other solid tumours. Hepatotoxicity leads to discontinuation in 7% of patients with HCC and does not negatively affect oncological outcomes. <h3>Impact and implications</h3> Immune-related liver injury (irLI) is common in patients with cancer receiving immune checkpoint inhibitors (ICI), but whether irLI is more frequent or it is associated with a worse clinical course in patients with hepatocellular carcinoma (HCC), compared to other tumours, is not known. Herein, we compared characteristics and outcomes of irLI in two prospective cohorts including patients treated with ICIs for HCC or for other oncological indications. irLI is significantly more common and it occurs earlier in patients with HCC, also after adjustment for duration of treatment exposure. However, outcomes of patients with HCC who developed irLI are not negatively affected in terms of requirement of corticosteroid therapy, hepatic decompensation, treatment discontinuation and overall survival.

<h2>Abstract</h2><h3>Background</h3> In the phase III HIMALAYA study (NCT03298451) in unresectable hepatocellular carcinoma (uHCC), STRIDE (Single Tremelimumab Regular Interval Durvalumab) significantly improved overall survival (OS) versus sorafenib; durvalumab monotherapy was noninferior to sorafenib for OS. Results reported herein are from a four-year updated OS analysis of HIMALAYA. <h3>Patients and methods</h3> Participants with uHCC and no previous systemic treatment were randomized to STRIDE (<i>n</i>=393), durvalumab (<i>n</i>=389), or sorafenib (<i>n</i>=389). The updated data cut-off was January 23, 2023. OS and serious adverse events (AEs) were assessed. Additionally, baseline characteristics and subsequent therapies were analyzed in long-term survivors (≥36 months beyond randomization). <h3>Results</h3> For STRIDE, durvalumab, and sorafenib, median (95% CI) follow-up was 49.12 (46.95-50.17), 48.46 (46.82-49.81), and 47.31 (45.08-49.15) months, respectively. OS HR (95% CI) for STRIDE versus sorafenib was 0.78 (0.67-0.92). The 36-month OS rate for STRIDE was 30.7% versus 19.8% for sorafenib. The 48-month OS rate remained higher for STRIDE at 25.2%, versus 15.1% for sorafenib. The long-term OS benefit of STRIDE was observed across clinically relevant subgroups and was further improved in participants who achieved disease control. Long-term survivors with STRIDE (<i>n</i>=103) included participants across clinically relevant subgroups, and 57.3% (59/103) had no reported subsequent anticancer therapy. No new serious treatment-related AEs occurred with STRIDE from the primary analysis (17.5%; 68/388). Durvalumab maintained OS noninferiority to sorafenib and no late onset safety signals were identified. <h3>Conclusions</h3> These data represent the longest follow-up to date in phase III studies in uHCC. The unprecedented three- and four-year OS rates reinforce the sustained long-term OS benefit of STRIDE versus sorafenib. STRIDE maintained a tolerable yet differentiated safety profile from other current uHCC therapies. Results continue to support the long-term benefits of STRIDE in a diverse population, reflective of uHCC globally.

IL-12 p40-related cytokines such as IL-12 p35/p40 heterodimer and IL-23 (p19/p40) are potent regulators of adaptive immune responses.Little is known, however, about the transcriptional regulation of the p40 gene in vivo.In an attempt toward this goal, we have generated transgenic mice expressing firefly luciferase under the control of the IL-12 p40 promoter.High constitutive transgene expression was found in the small intestine only, whereas little reporter gene activity was observed in other tissues.Within the small bowel, constitutive promoter activity was restricted to the terminal ileum and associated with high expression of p40 mRNA as well as p40 and IL-23 p19/p40 proteins.The cells constitutively producing IL-12 p40 were identified as CD8α and CD11b double-negative CD11c + lamina propria dendritic cells (LPDCs) that represent a major cell population in the lamina propria of the small intestine, but not in the colon.FISH directly demonstrated the uptake of bacteria by a subset of LPDCs in the terminal ileum that was associated with p40 expression.Furthermore, little or no p40 protein expression in LPDCs was found in the terminal ileum of germfree mice, indicating a key role of the intestinal flora for constitutive p40 expression.In addition, analysis of transgenic mice with a mutated NF-κB target site in the p40 promoter showed a critical role of NF-κB for constitutive transgene expression.Our data reveal important functional differences between the mucosal immune systems of the small and large bowel in healthy mice and suggest that the high bacterial load in the terminal ileum activates p40 gene transcription in LPDCs through NF-κB.These data suggest a predisposition of the terminal ileum to develop chronic inflammatory responses through IL-23 and thus may provide a molecular explanation for the preferential clinical manifestation of Crohn disease in this part of the gut.

Helicobacter pylori infection is associated with chronic gastritis, peptic ulceration, and gastric carcinoma. The potential role of CD95-mediated apoptosis was investigated in a panel of gastric biopsies obtained from patients with H. pylori-associated chronic gastritis (n = 29) and with noninfected normal mucosa (n = 10). Immunohistochemistry revealed increased CD95 receptor expression in epithelial and lamina propria cells in chronic gastritis. By in situ hybridization, CD95 ligand mRNA was absent or low in normal mucosa but expressed at high levels in lamina propria lymphocytes and, unexpectedly, in epithelial cells in chronic gastritis. Apoptotic cells were rare in normal mucosa but were observed regularly in chronic gastritis in close proximity to CD95 ligand mRNA expression throughout the epithelial and lamina propria cells. In a functional analysis gastric epithelial cell lines were incubated with supernatants of H. pylori. Treatment with the cytotoxic isolate H. pylori 60190 but not with the noncytotoxic isolate Tx30a upregulated CD95 in up to 50% of gastric epithelial cells and induced apoptosis in these cells. H. pylori-induced apoptosis was partially prevented by blocking CD95, demonstrating the functional role of the CD95 system. These findings suggest that H. pylori-associated chronic gastritis involves apoptosis of gastric epithelial cells by activation of the CD95 receptor and ligand system.

Primary human hepatocytes were used to study bile salt hepatotoxicity and the hepatoprotective potential of ursodeoxycholate in vitro. Hepatocytes were obtained by collagenase perfusion of healthy human liver tissue and were treated with glycochenodeoxycholate for 24 hr 1 day after plating. Clear signs of cytotoxicity were observed at concentrations of about 100 μmol/L glycochenodeoxycholate. Toxicity was determined by release of alkaline phosphatase, γ-glutamyl transferase, AST, ALT or lactate dehydrogenase into the culture medium, by measuring DNA synthesis of the cultured liver cells and by testing the viability of the hepatocytes using trypan-blue dye exclusion. Addition of ursodeoxycholate, which by itself proved to be of little toxicity, significantly reduced the hepatotoxic effects of glycochenodeoxycholate: 72% ± 6% of the cells survived treatment with 500 μmol/L glycocheno-deoxycholate alone, but addition of 100 μmol/L ursodeoxycholate increased the survival rate to 87% ± 4% (p < 0.05). Moreover, all enzymes tested were secreted at a significantly lower level when ursodeoxycholate was present. Similarly, the cellular DNA synthesis was maintained at significantly higher levels as a result of ursodeoxycholate treatment. We conclude that (a) primary human hepatocytes are a suitable model for studying hepatotoxicity of bile salts in vitro, (b) ursodeoxycholate reduces hepatotoxicity of other bile salts and (c) ursodeoxycholate can act hepatoprotectively by itself (i.e., alteration of the metabolism of other bile salts is not necessarily required). (HEPATOLOGY 1990;12:486–491).

Hemochromatosis and Wilson disease (WD), characterized by the excess hepatic deposition of iron and copper, respectively, produce oxidative stress and increase the risk of liver cancer. Because the frequency of p53 mutated alleles in nontumorous human tissue may be a biomarker of oxyradical damage and identify individuals at increased cancer risk, we have determined the frequency of p53 mutated alleles in nontumorous liver tissue from WD and hemochromatosis patients. When compared with the liver samples from normal controls, higher frequencies of G:C to T:A transversions at codon 249 (P < 0.001) and C:G to A:T transversions and C:G to T:A transitions at codon 250 (P < 0.001 and P < 0.005) were found in liver tissue from WD cases, and a higher frequency of G:C to T:A transversions at codon 249 (P < 0.05) also was found in liver tissue from hemochromatosis cases. Sixty percent of the WD and 28% of hemochromatosis cases also showed a higher expression of inducible nitric oxide synthase in the liver, which suggests nitric oxide as a source of increased oxidative stress. A high level of etheno-DNA adducts, formed from oxyradical-induced lipid peroxidation, in liver from WD and hemochromatosis patients has been reported previously. Therefore, we exposed a wild-type p53 TK-6 lymphoblastoid cell line to 4-hydroxynonenal, an unsaturated aldehyde involved in lipid peroxidation, and observed an increase in G to T transversions at p53 codon 249 (AGG to AGT). These results are consistent with the hypothesis that the generation of oxygen/nitrogen species and unsaturated aldehydes from iron and copper overload in hemochromatosis and WD causes mutations in the p53 tumor suppressor gene.

Genes encoded on chromosome 6 within the major histocompatibility complex region are thought to play an important role in the pathogenesis of psoriasis. A potential candidate gene is tumor necrosis factor α. The tumor necrosis factor α promoter contains several polymorphisms including two G→A transitions at position -308 and -238, which are the most common in Caucasian populations. The TNF238.2 (-238A) allele has been strongly associated with psoriasis. We have investigated the effect of the -238 and -308 variants on transcription of the tumor necrosis factor α gene in luciferase reporter gene assays. In addition, peripheral blood mononuclear cells of 47 patients with psoriasis and 43 controls were stimulated with different antigens and mitogens (streptococcal sonicate and superantigen, lipopolysaccharide, phorbol-12-myristate, phytohemagglutinin, CD3 antibodies) and tumor necrosis factor α production was measured in supernatants by enzyme-linked immunosorbent assay. The psoriasis-associated tumor necrosis factor α promoter allele TNF238.2 showed a significantly decreased transcriptional activity. Peripheral blood mononuclear cells carrying this allele produced significantly less tumor necrosis factor α after stimulation with T cell mitogens and streptococcal antigens in comparison to controls. The promoter allele TNF238.2 seems to influence tumor necrosis factor α production; a possible role in the pathogenesis of psoriasis has to be further evaluated. Genes encoded on chromosome 6 within the major histocompatibility complex region are thought to play an important role in the pathogenesis of psoriasis. A potential candidate gene is tumor necrosis factor α. The tumor necrosis factor α promoter contains several polymorphisms including two G→A transitions at position -308 and -238, which are the most common in Caucasian populations. The TNF238.2 (-238A) allele has been strongly associated with psoriasis. We have investigated the effect of the -238 and -308 variants on transcription of the tumor necrosis factor α gene in luciferase reporter gene assays. In addition, peripheral blood mononuclear cells of 47 patients with psoriasis and 43 controls were stimulated with different antigens and mitogens (streptococcal sonicate and superantigen, lipopolysaccharide, phorbol-12-myristate, phytohemagglutinin, CD3 antibodies) and tumor necrosis factor α production was measured in supernatants by enzyme-linked immunosorbent assay. The psoriasis-associated tumor necrosis factor α promoter allele TNF238.2 showed a significantly decreased transcriptional activity. Peripheral blood mononuclear cells carrying this allele produced significantly less tumor necrosis factor α after stimulation with T cell mitogens and streptococcal antigens in comparison to controls. The promoter allele TNF238.2 seems to influence tumor necrosis factor α production; a possible role in the pathogenesis of psoriasis has to be further evaluated. peripheral blood mononuclear cells Psoriasis is a polygenic, T-cell-mediated skin disease with a heterogeneous genetic background (Bos and de Rie, 1999Bos J.D. de Rie M.A. The pathogenesis of psoriasis: immunological facts and speculations.Immunol Today. 1999; 20: 40-46https://doi.org/10.1016/s0167-5699(98)01381-4Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). One of the most consistent associations exists with genes of the major histocompatibility complex (MHC) on the short arm of chromosome 6 (Elder et al., 1994Elder J.T. Henseler T. Christophers E. Voorhees J.J. Nair R.P. The genetics of psoriasis.Arch Dermatol. 1994; 130: 216-224Crossref PubMed Scopus (228) Google Scholar). Although recent studies have located a potential candidate gene between human leukocyte antigen B (HLA-B) and HLA-C (Allen et al., 1999Allen M.H. Veal C. Faasesen A. Powis S.H. Vaughan R.W. Trembath R.C. Barker J.N.W. A non-HLA gene within the MHC in psoriasis.Lancet. 1999; 353: 1589-1590https://doi.org/10.1016/s0140-6736(99)01618-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), the considerable genetic heterogeneity of psoriasis suggests that other nearby encoded genes could act as disease modifiers. Tumor necrosis factor α (TNF-α) is encoded centromerically to HLA-B within the class III region. TNF-α is an important inflammatory mediator that is enriched in early psoriatic lesions (Ettehadi et al., 1994Ettehadi P. Greaves M.W. Wallach D. Aderka K. Camp R.D. Elevated tumor necrosis factor-alpha biological activity in psoriasis lesions.Clin Exp Immunol. 1994; 96: 145-151Google Scholar). Several polymorphisms in the TNF-α region have been described, including five microsatellites and several point mutations particularly in the promoter region (Jongeneel et al., 1991Jongeneel C.V. Briant L. Udalova I.A. Sevin A. Nedospasov S.A. Cambon-Thomsen A. Extensive genetic polymorphism in the human tumor necrosis factor region and relation to extended HLA haplotypes.Proc Natl Sci USA. 1991; 88: 9717-9721Crossref PubMed Scopus (177) Google Scholar;Nedospasov et al., 1991Nedospasov S.A. Udalova I.A. Kuprash D.Y. Tretskya R.L. D.N.A. sequence polymorphism at the human tumor necrosis factor locus.J Immunol. 1991; 147: 1053-1059PubMed Google Scholar;Skoog et al., 1999Skoog T. van't Hooft F.M. Kallin B. et al.A common functional polymorphism (C to A substitution -863) in the promoter region of the TNF-α gene associated with reduced circulating levels of TNF-α.Hum Mol Genet. 1999; 8: 1443-1449Crossref PubMed Scopus (307) Google Scholar). In Caucasian populations the most common exchanges are two G→A transitions in the promoter at positions -308 (308A/TNF308.2) and -238 (238A/TNF238.2) (Wilson et al., 1992Wilson A.G. Di Giovine F.S. Blakemore A.I.F. Duff G.W. Single base change in the human tumour necrosis factor alpha gene detectable by Nco I restriction of the PCR product.Hum Mol Genet. 1992; 1: 353Crossref PubMed Scopus (843) Google Scholar;D'alfonso and Richiardi, 1994D'alfonso S. Richiardi P.M. A polymorphic variation in a putative regulation box of the TNFA promoter region.Immunogenetics. 1994; 39: 150-154Crossref PubMed Scopus (368) Google Scholar). A rare polymorphism at promoter position -376 occurs strictly linked to the TNF238.2 allele (Hamann et al., 1995Hamann A. Mantzoros C. Vidal-Puig A. Flier J.S. Genetic variability in the TNF-α promoter is not associated with type II diabetes mellitus (NIDDM).Biochem Biophys Res Commun. 1995; 211: 833-839https://doi.org/10.1006/bbrc.1995.1887Crossref PubMed Scopus (136) Google Scholar;Knight et al., 1999Knight J.C. Udalova I. Hill A.V.S. Greenwood B.M. Peshu N. Marsh K. Kwiatkowski D. A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria.Nature Genet. 1999; 22: 145-150Crossref PubMed Scopus (422) Google Scholar). We and colleagues have found a strong association of the polymorphism at position -238 with psoriasis and psoriatic arthritis (Arias et al., 1997Arias A.I. Giles B. Eiermann T.H. Sterry W. Pandey J.P. Tumor necrosis factor-alpha gene polymorphism in psoriasis.Exp Clin Immunogenet. 1997; 14: 118-122PubMed Google Scholar;Hoehler et al., 1997Hoehler T. Kruger A. Schneider P.M. et al.A TNF-alpha-promoter polymorphism is associated with juvenile onset psoriasis and psoriatic arthritis.J Invest Dermatol. 1997; 109 (556): 562Crossref PubMed Scopus (175) Google Scholar). The functional consequences for the TNF238.2 allele are not yet clear (Pociot et al., 1995Pociot F. D'alfonso S. Compasso S. Scorza R. Richiardi P.M. Functional analysis of a new polymorphism in the human TNF-α gene promoter.Scand J Immunol. 1995; 42: 501-504Crossref PubMed Scopus (148) Google Scholar). Contradictory findings exist for the TNF308 polymorphism (Stuber et al., 1996Stuber F. Udalova I.A. Book M. et al.-308 TNF polymorphism is not associated with survival in severe sepsis and is unrelated to LPS inducibility of the huamn TNF promoter.J Inflammation. 1996; 46: 42-50Google Scholar;Kroeger et al., 1997Kroeger K.M. Carville K.S. Abraham L.J. The -308 tumor necrosis factor alpha promoter polymorphism effects transcription.Mol Immunol. 1997; 34: 391-399https://doi.org/10.1016/s0161-5890(97)00052-7Crossref PubMed Scopus (0) Google Scholar;Wilson et al., 1997Wilson A.G. Symons J.A. McDowell T.L. Di Giovine F.S. Duff G.W. Effects of a TNF-alpha promoter base transition on transcriptional activity.Proc Natl Acad Sci USA. 1997; 94: 3195-3199Crossref PubMed Scopus (2046) Google Scholar). In this study we have investigated the effect of the -238 polymorphism on transcriptional activity of the TNF-α promoter in comparison with another polymorphism at position -308. In addition we have studied the influence of these TNF-α promoter polymorphisms on TNF-α production of peripheral blood mononuclear cells (PBMC) in psoriasis patients after stimulation with different mitogens and streptococcal antigens. Forty-seven patients with type I psoriasis (15 with psoriatic arthritis) from the Department of Dermatology and the First Medical Department (Rheumatology), University Clinic of Mainz, Germany, were recruited for this study. Psoriatic arthritis was defined as a seronegative inflammatory arthritis, associated with psoriasis (Gladman, 1994Gladman D.D. Natural history of psoriatic disease.Baillere's Clin Rheum. 1994; 8: 379-394Abstract Full Text PDF PubMed Scopus (143) Google Scholar). All patients were seen by a rheumatologist (EMH). Thirty-nine male and eight female patients were included; mean age was 48.8 y (range 20–83 y) and 59.1 y (range 46–82 y), respectively. Female patients were only included after menopause. At the time of recruitment, none of the patients was receiving immunosuppressive treatment like corticosteroids, methotrexate, cyclosporine A or ultraviolet A therapy. Patients did not suffer from any other relevant disease nor did they show any signs of streptococcal infections at the time of recruitment. The healthy control population consisted of 43 Caucasoid male persons, mean age 38.3 y (range 23–91 y). The polymorphisms at positions -238 and -308 were studied as previously described (Wilson et al., 1992Wilson A.G. Di Giovine F.S. Blakemore A.I.F. Duff G.W. Single base change in the human tumour necrosis factor alpha gene detectable by Nco I restriction of the PCR product.Hum Mol Genet. 1992; 1: 353Crossref PubMed Scopus (843) Google Scholar;Gallagher et al., 1997Gallagher G. Eskdale J. Oh H.H. Richards S.D. Campbell D.A. Field M. Polymorphisms in the TNF gene cluster and MHC serotypes in the West of Scotland.Immunogenetics. 1997; 45: 188-194https://doi.org/10.1007/s002510050188Crossref PubMed Scopus (58) Google Scholar). In addition samples carrying the -238A exchange were sequenced for the detection of the -376 polymorphism. PBMC were isolated from freshly taken heparinized blood by standard Ficoll-Hypaque gradient centrifugation. All experiments were performed in duplicates in 96 well microtiter plates (Nunc, Roskilde, Denmark) with 105 mononuclear cells in 100 μl assay medium per well. The following mitogens and antigens were added: 10 ng per ml lipopolysaccharide (LPS; Sigma, Deisenhofen, Germany), 10 ng per ml phorbol-12-myristate (PMA; Sigma) (both stimulators of B cell and monocyte/macrophage proliferation); T cell mitogens – 1 μg per ml anti-CD3 monoclonal antibodies (CD3), 1 μg per ml phytohemagglutinin (Difco Labs, Detroit, MI), 0.1 μg per ml Streptococcus pyogenes sonicate, and 0.25 μg per ml streptococcal superantigen SPEC. Cells were incubated for 24 h at 37°C and 5% CO2 in a humified atmosphere. TNF-α production was measured in supernatants using a PharminGen TNF-α test kit (Pharmin-Gen, Hamburg, Germany). Human T cell derived Jurkat and B-lymphoblastoid Raji cell lines were maintained in culture medium consisting of RPMI-1640 supplemented with 2 mM L-glutamine, 100 U per ml of penicillin, 100 μg per ml of streptomycin and 5% fetal bovine serum, at 37°C with 5% CO2. The three naturally occurring allelic forms of the TNF-α promoter (wild-type, TNF308G/238G; TNF308.2, TNF308A/238G; TNF238.2, TNF308G/238A) were cloned into the luciferase reporter gene construct (Figure 1). A TNF308A/238A allele has never been reported as mutations have occurred independently on different haplotypes. A 691 bp fragment (position -585 to +106) of the TNF-α gene was amplified by polymerase chain reaction (Wilson et al., 1997Wilson A.G. Symons J.A. McDowell T.L. Di Giovine F.S. Duff G.W. Effects of a TNF-alpha promoter base transition on transcriptional activity.Proc Natl Acad Sci USA. 1997; 94: 3195-3199Crossref PubMed Scopus (2046) Google Scholar), cloned into the pGL3.basic-vector (Promega, Madison, WI), digested with XhoI (New England Biolabs, Schwalbach, Germany) and HindIII (New England Biolabs), and used to transform Escherichia coli (strain INVαF′, Invitrogen, Groningen, The Netherlands). Prior to transfection experiments plasmids were sequenced by the dideoxy chain terminator method and analyzed on an ABI 310 DNA sequencer (Applied Biosystems, Weiterstadt, Germany). Jurkat and Raji cells were transfected during their log growth phase by DEAE transfection with plasmid DNA prepared and purified using Qiagen Maxi prep-250 Kit (Qiagen, Hilden, Germany). A total of 107 cells were transfected with 10 μg of construct DNA plus 2.5 μg pcDNA3.1/HisB/lacZ control plasmid (Invitrogen). After transfection cells were distributed into two wells and incubated for 16 h (Jurkat) or 26 h (Raji). Cells were either left unstimulated or were induced for 8 h (Jurkat) or 15 h (Raji) with Ionomycin (1 μg per ml) and PMA (50 ng per ml) to achieve maximal stimulation. Luciferase and β-galactosidase activity of cell lysates was measured according to the manufacturers' instructions (Promega, Mannheim, Germany; Tropix, Bedford, OH). All transfections were performed five to six times. Luciferase activity was normalized against β-galactosidase activity. Wilcoxon and Kruskal-Wallis tests were used for statisitical analysis of enzyme-linked immunosorbent assay data. The results of the luciferase assays were analyzed by variance analysis. Distribution of the TNF-α promoter alleles is shown in Table 1. As expected from earlier studies the TNF238.2 allele was significantly enriched among psoriasis patients (p < 0.02). In only two of our probands carrying the TNF238.2 allele the rare -376G→A polymorphism was found.Table 1TNF-α promoter genotype distribution in groups of patientsGenotypePatients (n = 47)Controls (n = 43)-308-238n%n%G/GG/G2859.52856.1A/GG/G510.61023.2G/GA/G14 ap < 0.02.29.8511.6a p < 0.02. Open table in a new tab Results of the luciferase reporter gene assays are shown in Figure 2 The TNF308.2 construct had a transcriptional activity comparable to the wild-type construct, whereas the TNF238.2 variant showed a significantly decreased promoter activity after stimulation compared to these two constructs (p < 0.02). In the Raji cell line this decreased expression also became obvious in unstimulated cells (p < 0.04). As shown in Figure 3, PBMC from wild-type homozygous psoriasis patients produced more TNF-α than wild-type homozygous controls and other genotypes. Among psoriasis patients, PBMC carrying the TNF238.2 variant produced significantly less TNF-α after stimulation with CD3 (p < 0.04) and streptococcal antigens (p < 0.05) than wild-type homozygous patients. Controls heterozygous for the TNF238.2 allele had lower TNF-α secretion than either wild-type homozygous or TNF308.2 heterozygous controls, although results did not reach statistical significance because of the small numbers of TNF238.2 heterozygotes. Stimulation of PBMC with the B cell mitogens LPS and PMA did not induce significant differences (data not shown). Genomewide searches have established a strong association of psoriasis with the MHC and in particular with the ancestral haplotype 57.1 (Bhalero and Bowcock, 1998Bhalero J. Bowcock A.M. The genetics of psoriasis: a complex disorder of the skin and immune system.Hum Mol Genet. 1998; 7: 1537-1545Crossref PubMed Scopus (230) Google Scholar;Jenisch et al., 1998Jenisch S. Henseler T. Nair R.P. et al.Linkage analysis of human leukocyte antigen (HLA) markers in familial psoriasis: strong disequilibrium effects provide evidence for a major determinant in the HLA-B/-C region.Am J Hum Genet. 1998; 63: 191-199Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar,Jenisch et al., 1999Jenisch S. Westphal E. Nair R.P. et al.Linkage disequilibrium analysis of familial psoriasis: identification of multiple disease-associated MHC haplotypes.Tissue Antigens. 1999; 53: 135-146Crossref PubMed Scopus (50) Google Scholar). The exact role of the MHC in the pathogenesis of psoriasis is not yet clear. We now show that the TNF-α promoter allele TNF238.2, which is part of the B57.1 haplotype and found with increased frequency in psoriatic patients, is associated with a significantly decreased transcriptional activity in reporter gene assays. In addition PBMC of TNF238.2 heterozygous individuals show a significantly lower secretion of TNF-α after stimulation with CD3 antibodies and streptococcal sonicates. TNF-α secretion is predominantly regulated at the transcriptional level (Raabe et al., 1998Raabe T. Bukrinsky M. Currie R.A. Relative contribution of transcription and translation of the induction of tumor necrosis factos alpha by lipopolysaccharide.J Biol Chem. 1998; 273: 974-980Crossref PubMed Scopus (158) Google Scholar). Interindividual differences in TNF-α secretion are mostly genetically determined (Westendorp et al., 1997Westendorp R.G. Langermans J.A.M. Huizinga T.W.J. Elouali A.H. Verweij C.L. Boomsma D.I. Vandenbrouke J.P. Genetic influence on cytokine production and fatal meningococcal disease.Lancet. 1997; 349: 170-173https://doi.org/10.1016/s0140-6736(96)06413-6Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar) and have been associated with HLA-DR haplotypes and polymorphisms in the TNF-α promoter. Particular interest has been focused on the common G→A transition at position -308, but conflicting results have been reported. Increased transcriptional activity of the TNF308.2 allele (Wilson et al., 1997Wilson A.G. Symons J.A. McDowell T.L. Di Giovine F.S. Duff G.W. Effects of a TNF-alpha promoter base transition on transcriptional activity.Proc Natl Acad Sci USA. 1997; 94: 3195-3199Crossref PubMed Scopus (2046) Google Scholar) and increased TNF-α production of PBMC carrying this allele have been described (Bouma et al., 1996Bouma G. Crusius J.B.A. Oudkerk Pool M. et al.Secretion of TNF-alpha and LTα in relation to polymorphisms in the TNF genes and HLA-DR alleles. Relevance for inflammatory bowel disease.Scand J Immunol. 1996; 43: 456-463Crossref PubMed Scopus (376) Google Scholar;Louis et al., 1998Louis E. Franchimont D. Piron A. et al.TNF gene polymorphism influences TNF-α-production in LPS-stimulated whole blood cell cultures in healthy humans.Clin Exp Immunol. 1998; 113: 401-406Crossref PubMed Scopus (539) Google Scholar). Findings of other groups including our own, however, suggest that there is no significant effect of this allele on TNF production (Turner et al., 1995Turner D.M. Grant S.C. Lamb W.R. Brenchley P.E. Dyer P.A. Sinnott P.J. Hutchinson I.V. A genetic marker of high TNF-alpha production in heart transplant recipients.Transplantation. 1995; 60: 1113-1117Crossref PubMed Scopus (108) Google Scholar;Stuber et al., 1996Stuber F. Udalova I.A. Book M. et al.-308 TNF polymorphism is not associated with survival in severe sepsis and is unrelated to LPS inducibility of the huamn TNF promoter.J Inflammation. 1996; 46: 42-50Google Scholar;Kroeger et al., 1997Kroeger K.M. Carville K.S. Abraham L.J. The -308 tumor necrosis factor alpha promoter polymorphism effects transcription.Mol Immunol. 1997; 34: 391-399https://doi.org/10.1016/s0161-5890(97)00052-7Crossref PubMed Scopus (0) Google Scholar). In contrast, the TNF238.2 allele caused a significant decrease in promoter activity compared to the wild-type and the TNF308.2 allele.Fong et al., 1994Fong C.W. Siddiqui A.H. Mark D.F. Identification and characterisation of a novel repressor site in the human tumor necrosis factor alpha gene.Nucl Acid Res. 1994; 22: 1108-1114Crossref PubMed Scopus (83) Google Scholar have localized a repressor site (TRS) to a 25 bp stretch between positions -254 and -230 in the promoter. It is tempting to speculate that the -238A exchange could lead to an increased transcriptional repression. The findings in reporter gene assays are further strengthened by the results of our PBMC studies. TNF-α production of PBMC from patients heterozygous for the TNF238.2 allele showed a decreased TNF-α production after stimulation with different mitogens and antigens. In particular, stimulation with CD3 antibodies, streptococcal sonicates, and streptococcal superantigens led to a significantly decreased TNF-α secretion into supernatants. This effect was observed in heterozygous psoriasis patients as well as in heterozygous control subjects. Psoriasis is probably due to a disturbed interaction of T cells and keratinocytes that may be triggered and maintained by streptococcal infections (Valdimarsson et al., 1995Valdimarsson H. Baker B.S. Jonsdottir I. Powles A. Fry L. Psoriasis: a T-cell mediated autoimmune disease induced by streptococcal superantigens?.Immunol Today. 1995; 16 (114): 145Abstract Full Text PDF PubMed Scopus (329) Google Scholar;Fry, 1998Fry L. Psoriasis.Br J Dermatol. 1998; 119: 445-461Crossref Scopus (152) Google Scholar;Bos and de Rie, 1999Bos J.D. de Rie M.A. The pathogenesis of psoriasis: immunological facts and speculations.Immunol Today. 1999; 20: 40-46https://doi.org/10.1016/s0167-5699(98)01381-4Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). TNF-α is secreted mainly by monocytes, macrophages, T cells, and B cells and is found in early psoriatic lesions (Ettehadi et al., 1994Ettehadi P. Greaves M.W. Wallach D. Aderka K. Camp R.D. Elevated tumor necrosis factor-alpha biological activity in psoriasis lesions.Clin Exp Immunol. 1994; 96: 145-151Google Scholar). It induces the expression of HLA-DR, the production of cell adhesion molecules like intercellular adhesion molecule 1 (Barker et al., 1990Barker J.N. Sarma V. Mitra R.S. Dixit V.M. Nickoloff B.J. Marked synergism between tumor necrosis factor-alpha and interferon-gamma in regulation of keratinocyte-derived adhesion molecules and chemotactic factors.J Clin Invest. 1990; 85: 605-608Crossref PubMed Scopus (217) Google Scholar), and the production of chemoattractants like interleukin-8, which lead to the attraction of inflammatory cells like T lymphocytes and neutrophils (Valdimarsson et al., 1995Valdimarsson H. Baker B.S. Jonsdottir I. Powles A. Fry L. Psoriasis: a T-cell mediated autoimmune disease induced by streptococcal superantigens?.Immunol Today. 1995; 16 (114): 145Abstract Full Text PDF PubMed Scopus (329) Google Scholar). The pathogenetic consequences of our findings remain to be defined. Decreased TNF-α secretion could characterize psoriasis patients with a particular disease course or be related to an impaired clearance of skin infections with candida or streptococci, and thus predispose individuals to the development of the disease. This work was supported by the Deutsche Forschungsgemeinschaft, DFG grant HO 1309/2. The work contains major parts of the thesis of Sabine Grossmann; publication has been approved by the Dean of the Faculty of Medicine, University of Mainz.

Although IL-12 and IL-23 share the common p40 subunit, IL-23, rather than IL-12, seems to drive the pathogenesis of experimental autoimmune encephalomyelitis and arthritis, because IL-23/p19 knockout mice are protected from disease. In contrast, we describe in this study that newly created LacZ knockin mice deficient for IL-23 p19 were highly susceptible for the development of experimental T cell-mediated TNBS colitis and showed even more severe colitis than wild-type mice by endoscopic and histologic criteria. Subsequent studies revealed that dendritic cells from p19-deficient mice produce elevated levels of IL-12, and that IL-23 down-regulates IL-12 expression upon TLR ligation. Finally, in vivo blockade of IL-12 p40 in IL-23-deficient mice rescued mice from lethal colitis. Taken together, our data identify cross-regulation of IL-12 expression by IL-23 as novel key regulatory pathway during initiation of T cell dependent colitis.

ABSTRACT The vacuolating cytotoxin and the cytotoxin-associated protein, encoded by vacA and cagA , respectively, are important virulence determinants of Helicobacter pylori . Sixty-five H. pylori strains were isolated from dyspeptic patients (19 with peptic ulcer disease, 43 with chronic gastritis, and 3 with gastric cancer) and studied for differences in the vacA and cagA genes and their relationship to VacA and CagA expression, cytotoxin activity, and the clinical outcome of infection. By PCR, fifty-four (83.1%) of 65 strains had the vacA signal sequence genotype s1 and only 10 (15.4%) had the type s2. After primer modification, the vacA middle-region types m1 and m2 were detected in 24 (36.9%) and 41 (63.1%) strains, respectively. The combinations s1-m2 (31 [47.7%]) and s1-m1 (23 [35.4%]) occurred more frequently than s2-m2 (10 [15.4%]) ( P = 0.01). No strain with the combination s2-m1 was found. All 19 patients with peptic ulcers harbored type s1 strains, in contrast to 32 (74.4%) of 43 patients with gastritis ( P = 0.02). The vacA genotype s1 was associated with the presence of cagA ( P &lt; 0.0001), VacA expression ( P &lt; 0.0001), and cytotoxin activity ( P = 0.003). The cagA gene was detectable in 48 (73.8%) of 65 isolates and present in 16 (84.2%) of 19 ulcer patients and 29 (67.4%) of 43 patients with gastritis ( P = 0.17). The vacA genotypes of German H. pylori isolates are identical to those previously reported. H. pylori strains of vacA type s1 are associated with the occurrence of peptic ulceration and the presence of cagA , cytotoxin activity, and VacA expression.

OBJECTIVE Autoimmune hepatitis (AIH) may influence pregnancy outcome and pregnancy may affect AIH. We aimed at analyzing the disease course in pregnant AIH patients and at identifying disease-related risk factors for adverse pregnancy outcome. PATIENTS AND METHODS AIH patients with at least one pregnancy were identified at four liver units. The patients' records and the data obtained by detailed questionnaires were analyzed retrospectively. Forty-two pregnancies of 22 AIH patients were included. RESULTS The rate of adverse pregnancy outcome was 26%; a medical explanation could be elucidated in only 4 of 11 pregnancies with adverse outcome. Of note, the 7 unexplained adverse pregnancy outcomes were highly associated with the presence of antibodies to SLA/LP (odds ratio 51; p < 0.003) and Ro/SSA (odds ratio 27; p < 0.02). Of 35 live births, 30 children developed normally over a mean observation period of nearly 5 yr. Eleven of these had been exposed to azathioprine in utero. The rate of serious maternal complications was 9% and a high rate (52%) of postpartum flares was noted. CONCLUSIONS The presence of autoantibodies may be a risk factor for adverse pregnancy outcome in AIH patients. Close monitoring of both mother and fetus seems advisable due to a significant rate of maternal and fetal complications.

Background Although the pathomechanisms of autoimmune diseases in various organs remain unresolved, an accumulation of autoimmune diseases in individual patients has been observed. An overlap of autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) or primary sclerosing cirrhosis has been well documented. However, the overlap of autoimmune diseases other than PBC or PSC has not yet been investigated in a large cohort. Goal The goal of our analysis was to investigate the incidence of concurrent autoimmune diseases in patients with AIH. Study We analyzed our cohort of 278 patients with AIH for concurrent autoimmune diseases. Results A total of 111 patients (40%) were diagnosed with additional autoimmune diseases. Besides overlap syndromes for PBC and PSC, autoimmune thyroiditis was the most common concurrent disease (28 patients, 10%). Other concurrent autoimmune diseases comprised vitiligo (5 patients), rheumatoid arthritis (5 patients), Sjogren syndrome (4 patients), ulcerative colitis (4 patients), conjunctivitis (4 patients), celiac disease (3 patients), systemic lupus erythematodes (2 patients), type I diabetes (2 patients), multiple sclerosis (2 patients), polymyalgia rheumatica (2 patients), and urticaria (2 patients). One patient each was diagnosed with Crohn's disease, autoimmune gastritis, collagenous colitis, hypophysitis, and sarcoidosis. Investigating 100 patients with polyglandular syndrome and autoimmune thyroid disease for the occurrence of autoantibodies associated with AIH, we identified AIH-associated antibodies only in 1 patient. Conclusions Concurrent autoimmune diseases are common in patients with AIH and mirror the full range of known autoimmune diseases. Therefore, an extended diagnostic screening for accumulating autoimmune diseases, especially autoimmune thyroiditis, seems reasonable in patients with AIH.

To analyze the relevance of the microRNA miR-196a for colorectal oncogenesis.The impact of miR-196a on the restriction targets HoxA7, HoxB8, HoxC8 and HoxD8 was analyzed by reverse transcription polymerase chain reaction (RT-PCR) after transient transfection of SW480 cancer cells. The miR-196a transcription profile in colorectal cancer samples, mucosa samples and diverse cancer cell lines was quantified by RT-PCR. Transiently miR-196a-transfected colorectal cancer cells were used for diverse functional assays in vitro and for a xenograft lung metastasis model in vivo.HoxA7, HoxB8, HoxC8 and HoxD8 were restricted by miR-196a in a dose-dependent and gene-specific manner. High levels of miR-196a activated the AKT signaling pathway as indicated by increased phosphorylation of AKT. In addition, high levels of miR-196a promoted cancer cell detachment, migration, invasion and chemosensitivity towards platin derivatives but did not impact on proliferation or apoptosis. Furthermore, miR-196a increased the development of lung metastases in mice after tail vein injection.miR-196a exerts a pro-oncogenic influence in colorectal cancer.

Detection of disease activity in Crohn's disease (CD) is of crucial importance for diagnosis and management of the disease. Noninvasive methods for monitoring are desirable and comprise hydromagnetic resonance imaging (hydro-MRI) and leukocyte scintigraphy. In addition, a recent case report indicated the potential of 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) to assess CD activity. However, comparative prospective studies are lacking.Between February, 1999 and August, 2000, 59 patients with CD were enrolled in a prospective study to assess disease activity by FDG-PET, hydro-MRI, and immunoscintigraphy with anti-nonspecific cross-reacting antigen 95 antigranulocyte antibodies. In 28 of these patients, colonoscopy could be performed. Twelve patients with irritable bowel syndrome and 20 tumor patients without gut inflammation served as controls. Results were compared by statistical analysis.FDG-PET detected 127 pathological findings (average maximum standardized uptake value = 4.4 +/- 1.1) in the terminal/neoterminal ileum (37), small bowel (24), and colon (66) of 54 patients with CD, whereas no pathological findings were seen in five patients with CD, the control patients with irritable bowel syndrome, and the tumor patients without gut inflammation. In contrast, examination with hydro-MRI or granulocyte antibodies detected less pathological findings in CD patients. Forty-five of the detected foci were accessible to endoscopic verification. The correlation of the foci with endoscopic findings showed a high specificity (>89%) of all three methods to detect inflamed areas in the terminal ileum and colon of patients with CD, although analyses by hydro-MRI and granulocyte antibody scan had strikingly lower sensitivities (40.9% and 66.7%) than FDG-PET analysis (85.4%).FDG-PET appears to be a reliable noninvasive tool for simultaneous detection of inflamed areas in the small and large bowel of patients with CD. FDG-PET can be used to detect disease activity in the terminal ileum and colon of CD patients with high sensitivity and specificity.

In patients with autoimmune hepatitis, efficient immunosuppressive therapy is essential to avoid progression to cirrhosis. There is no established second line therapy for patients failing standard therapy with steroids and azathioprine. The aim of this study was to examine the possible role of mycophenolate mofetil (MMF) as second line treatment of autoimmune hepatitis (AIH).We were able to identify 37 patients (29 women, 8 men) with AIH proven according to International AIH Group criteria who failed standard therapy. One patient on MMF was excluded due to non-compliance. A total of 28 of 36 patients had experienced side effects necessitating stop of treatment. One patient stopped azathioprine due to pregnancy. A total of nine patients did not respond sufficiently to azathioprine. A total of four patients with a treatment duration of 3 months or less because of severe side effects were considered as intolerant to MMF. Remission was defined as aspartate transaminase (ASP) < twice upper normal limit (UNL).Of 36 patients on MMF included in the analysis, 14 patients (39%) experienced remission. A total of 22 patients (61%) did not respond sufficiently to MMF. The response rate to MMF was dependent on the cause of treatment cessation of azathioprine. Of eight patients with prior nonresponse to azathioprine, six (75%) did not respond to MMF and only two (25%) reached biochemical remission. Of 28 patients with azathioprine intolerance in 16 (57%) patients, the response to MMF was insufficient and in 12 patients (43%) remission was reached. The difference did not reach statistical significance due to the relatively small numbers included.In the light of its good tolerability, MMF seems to be an alternative for patients who could not tolerate azathioprine previously. However, our data suggest that a majority of patients fail MMF particularly if they are switched because of an insufficient response to azathioprine.

Oxidative stress has been associated with the induction of programmed cell death. The CD95 ligand/receptor system is a specific mediator of apoptosis. We have used the model of drug-induced apoptosis to assess whether the CD95 ligand mRNA is induced by reactive oxygen intermediates. Treatment of HepG2 hepatoma cells with bleomycin induced the production of reactive oxygen intermediates and, as an additional parameter of oxidative stress, resulted in glutathione (GSH) depletion. In parallel, CD95 ligand mRNA expression was induced. In a similar fashion CD95 ligand mRNA expression increased after treatment with H2O2. Additional treatment with the antioxidant and GSH precursor N-acetylcysteine resulted in partial restoration of intracellular GSH levels and in reduced induction of CD95 ligand mRNA. Induction of CD95 ligand mRNA by bleomycin was further reduced by combined treatment withN-acetylcysteine and deferoxamine. These data suggest a direct role of oxygen radicals in the induction of the CD95 ligand. Oxidative stress has been associated with the induction of programmed cell death. The CD95 ligand/receptor system is a specific mediator of apoptosis. We have used the model of drug-induced apoptosis to assess whether the CD95 ligand mRNA is induced by reactive oxygen intermediates. Treatment of HepG2 hepatoma cells with bleomycin induced the production of reactive oxygen intermediates and, as an additional parameter of oxidative stress, resulted in glutathione (GSH) depletion. In parallel, CD95 ligand mRNA expression was induced. In a similar fashion CD95 ligand mRNA expression increased after treatment with H2O2. Additional treatment with the antioxidant and GSH precursor N-acetylcysteine resulted in partial restoration of intracellular GSH levels and in reduced induction of CD95 ligand mRNA. Induction of CD95 ligand mRNA by bleomycin was further reduced by combined treatment withN-acetylcysteine and deferoxamine. These data suggest a direct role of oxygen radicals in the induction of the CD95 ligand. CD95 (APO-1/Fas) is a 45-kDa glycosylated transmembrane protein belonging to the tumor necrosis factor receptor family of type I membrane proteins (1Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3972) Google Scholar, 2Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4543) Google Scholar). The CD95 ligand (CD95L) is a 40-kDa Type II transmembrane protein and a member of the tumor necrosis factor family of cytokines (1Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3972) Google Scholar, 3Suda T. Okazaki T. Naito Y. Yokota N. Arai S. Ozaki K. Nakao K. Nagata S. J. Immunol. 1995; 154: 3806-3813PubMed Google Scholar). 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Med. 1995; 182: 1223-1230Crossref PubMed Scopus (676) Google Scholar). Thus, CD95L expression seems to be induced by different mechanisms of cellular injury and might be an important tool for the organism to eliminate damaged cells. Little is known about the exact mechanism of induction of CD95L. The CD95L promoter has been described to contain NF-κB binding sites (17Takahashi T. Tanaka M. Inazawa J. Abe T. Suda T. Nagata S. Int. Immunol. 1994; 6: 1567-1574Crossref PubMed Scopus (416) Google Scholar). Therefore, induction of CD95L mRNA might involve reactive oxygen species (ROS). 1The abbreviations used are: ROS, reactive oxygen species; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline. 1The abbreviations used are: ROS, reactive oxygen species; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate-buffered saline. In line with this assumption is the observation of apoptotic cell death in different cell lines after oxidative stress (18Jacobson M.D. Trends Biochem. Sci. 1996; 21: 83-86Abstract Full Text PDF PubMed Scopus (728) Google Scholar, 19Kroemer G. Zamzami N. Susin S.A. Immunol. Today. 1997; 18: 44-51Abstract Full Text PDF PubMed Scopus (1382) Google Scholar). In the present study we have used the model of bleomycin-induced apoptosis to investigate the possible role of ROS in the induction of CD95L mRNA. Bleomycin has been described as a potent inducer of apoptosis, involving up-regulation of CD95 receptor and ligand expression (15Müller M. Strand S. Hug H. Heinemann E.-A. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (716) Google Scholar). Whereas up-regulation of the CD95 receptor in response to cell damage apparently involves activity of the p53 tumor suppressor gene product, the mechanism of CD95L induction is unclear to date. Because bleomycin treatment induces oxidative stress (20Kilinc C. Ozcan O. Karaoz E. Sunguroglu K. Kutluay T. Karaca L. J. Basic Clin. Physiol. Pharmacol. 1993; 4: 249-269Crossref PubMed Scopus (35) Google Scholar, 21An J. Hsie A.W. Mutat. Res. 1992; 270: 167-175Crossref PubMed Scopus (25) Google Scholar, 22An J. Hsie A.W. Mutat. Res. 1993; 289: 215-222Crossref PubMed Scopus (26) Google Scholar) it provides a suitable model for the investigation of the potential association between induction of ROS and CD95L. In this study we demonstrate that CD95L mRNA induction indeed involves the action of ROS which can be blocked by the antioxidants N-acetylcysteine and deferoxamine. HepG2 cells, a human hepatoblastoma cell line, were cultured in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum. Bleomycin (Cell Pharm, Hannover, Germany) was used at 3 mg/ml. N-Acetylcysteine (Sigma) was used at 50 μm, deferoxamine (desferrioxamine mesylate; Sigma, Deisenhofen, Germany) at 50 mm. At these concentrations N-acetylcysteine and deferoxamine proved to be nontoxic for HepG2 cells as demonstrated by viability assays, lactate dehydrogenase release, and morphological analysis (data not shown). Poly(A)+-RNA was purified from about 5 × 105 HepG2 cells treated with bleomycin using the Oligotex Direct mRNA kit (Qiagen, Hilden, Germany) according to the protocol of the manufacturer. RNA was eluted with 50 μl of H2O. RT-PCR was performed using the Gen-Amp RNA-PCR kit from Perkin-Elmer. Reverse transcription was done with oligo(dT)16 and 3 μl of the poly(A)+-RNA under the conditions recommended by the manufacturer. The primers used for amplification of the CD95L mRNA have been described recently (23Peter M.E. Dhein J. Ehret A. Hellbardt S. Walczak H. Moldenhauer G. Krammer P.H. Int. Immunol. 1995; 7: 1873-1877Crossref PubMed Scopus (57) Google Scholar) and were used at a final concentration of 0.2 μm. Human β-actin primers were from Stratagene (Catalog No. 302010) and used at a final concentration of 0.1 μm. 35 PCR cycles were performed at 94 °C for 30 s, at 56 °C for 30 s, and at 72 °C for 2 min in a volume of 100 μl. 10 μl of the PCR sample were analyzed on 1.5% agarose gels. In all cases at least three independent sets of experiments were performed. Floating cells from the tissue culture supernatant were collected by centrifugation at 200 ×g. Adherent cells were harvested by incubation with 1% trypsin. HepG2 cells were collected by centrifugation at 200 ×g, washed with PBS, and fixed in 70% ethanol. This was followed by staining with propidium iodide (50 μg/ml PBS). DNA fluorescence was measured in a Becton Dickinson FACScan according to the method of Nicoletti et al. (24Nicoletti I. Migliorati G. Paggliacci M.C. Grignani F. Riccardi C.A J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4415) Google Scholar). A minimum of 10,000 events was measured per sample. Data analysis was performed with Lysis II software. HepG2 cells were maintained on 35-mm plates. After bleomycin treatment cells were harvested with a cell scraper, washed in PBS, and finally taken up in 300 μl of 2.5% trichloroacetic acid and analyzed for intracellular glutathione and glutathione disulfide as described (25Eck H.-P. Gmünder H. Hartmann M. Petzhold D. Biol. Chem. Hoppe-Seyler. 1989; 370: 101-108Crossref PubMed Scopus (315) Google Scholar). O·̄2 generation was detected by a chemiluminescence reaction as described previously (26Galle J. Bengen J. Schollmeyer P. Wanner C. Circulation. 1995; 92: 1582-1589Crossref PubMed Scopus (131) Google Scholar). HepG2 cells were subconfluently seeded in sterile scintillation vials and treated with 3 mg/ml bleomycin for 24 h. Thereafter, medium was discarded and replaced by the scintillation solution containing 0.25 mmol/L lucigenin (Sigma) dissolved in 2 ml of Krebs-HEPES buffer. Counts were obtained at 1-min intervals at room temperature. To determine the specificity of the reaction the O·̄2 scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron, 10 mmol/L, Sigma) was added. Treatment with bleomycin in concentrations between 10 μg/ml and 3 mg/ml induces apoptosis as demonstrated by the appearance of a sub-G1 fraction of fragmented nuclei using propidium iodide staining and FACS analysis according to Nicolettiet al. (24Nicoletti I. Migliorati G. Paggliacci M.C. Grignani F. Riccardi C.A J. Immunol. Methods. 1991; 139: 271-279Crossref PubMed Scopus (4415) Google Scholar) (Fig.1 A; see also Ref. 15Müller M. Strand S. Hug H. Heinemann E.-A. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (716) Google Scholar) and by morphological and DNA fragmentation analysis (data not shown). This is accompanied by an induction of CD95 mRNA (15Müller M. Strand S. Hug H. Heinemann E.-A. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (716) Google Scholar) and also of CD95L mRNA (Fig. 1 B). The functional relevance of this observation has been demonstrated by blocking access of CD95L to the CD95 receptor using F(ab′)2 antagonistic antibody fragments which largely inhibited induction of apoptosis (15Müller M. Strand S. Hug H. Heinemann E.-A. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (716) Google Scholar). Here, HepG2 cells were treated with 3 mg/ml bleomycin for 0, 5, 10, 24, 32, and 48 h, and expression of CD95L mRNA was assessed by RT-PCR. CD95L mRNA expression was detectable after 5 h, showed its highest level between 24 and 32 h, and decreased around 48 h (Fig.1 B). Recently we demonstrated dependence of CD95 induction on the presence of the tumor suppressor gene product p53 (15Müller M. Strand S. Hug H. Heinemann E.-A. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (716) Google Scholar). CD95 expression took place only in hepatoma cell lines with p53 wild type configuration (HepG2) but not with p53 mutant configuration (Huh7) or in the absence of p53 (Hep3b). In contrast, CD95L mRNA was found to be inducible independently of the presence of p53 wild type and thus, seems to be regulated in a different manner (15Müller M. Strand S. Hug H. Heinemann E.-A. Walczak H. Hofmann W.J. Stremmel W. Krammer P.H. Galle P. J. Clin. Invest. 1997; 99: 403-413Crossref PubMed Scopus (716) Google Scholar). Since bleomycin treatment has been described to result in oxidative stress (21An J. Hsie A.W. Mutat. Res. 1992; 270: 167-175Crossref PubMed Scopus (25) Google Scholar, 22An J. Hsie A.W. Mutat. Res. 1993; 289: 215-222Crossref PubMed Scopus (26) Google Scholar), we posed the question whether CD95L mRNA activation is correlated to the generation of ROS in response to bleomycin treatment. In initial experiments we investigated the induction of oxidative stress following bleomycin treatment in hepatoma cells. As a parameter of oxidative stress intracellular glutathione (GSH) levels were assessed. Bleomycin treatment resulted in a rapid depletion of total GSH from initial values of 18 nmol/mg of protein in untreated controls to 0.7 nmol/mg of protein after 48 h (Fig.2 A) indicating disturbances in the cellular redox status. Additional treatment withN-acetylcysteine partially prevented GSH depletion of bleomycin-treated cells, with GSH levels of 14.8 nmol/mg of protein after 48 h. As a direct measure of induction of reactive oxygen species in bleomycin-treated cells we investigated generation of superoxide (O·̄2) using a chemiluminescence assay (26Galle J. Bengen J. Schollmeyer P. Wanner C. Circulation. 1995; 92: 1582-1589Crossref PubMed Scopus (131) Google Scholar). We observed a strong induction in chemiluminescence following treatment with 3 mg/ml bleomycin for 24 h. This signal could be completely blunted by addition of the O·̄2 scavenger Tiron, demonstrating specificity of the reaction. To establish a causative relationship between the observed induction of CD95L mRNA and presence of reactive oxygen species we treated HepG2 cells with H2O2. In a manner similar to bleomycin treatment H2O2 at concentrations between 0.1 and 10 μm induced expression of CD95L mRNA (Fig.3 A). Higher concentrations proved to be cytotoxic as demonstrated by a decrease in cell number and a consecutive decrease in β-actin and CD95L mRNA expression (data not shown). Further evidence for ROS-mediated CD95L mRNA expression was obtained from experiments with the antioxidants deferoxamine andN-acetylcysteine. Deferoxamine is an iron chelator. It prevents the formation of the hydroxyl radical from hydrogen peroxide via the Fenton reaction (27Yu B.P. Physiol. Rev. 1994; 74: 139-162Crossref PubMed Scopus (2202) Google Scholar).N-Acetylcysteine interferes with the generation of ROS and is a glutathione precursor. A direct effect of glutathione depletion on the induction of apoptosis seems unlikely, because glutathione depletion alone (using buthionine sulfoximine treatment of human leukocytes) failed to induce apoptosis (28Sato N. Iwata S. Nakamura K. Hori T. Mori K. Yodoi J. J. Immunol. 1995; 154: 3194-3203PubMed Google Scholar). HepG2 cells were treated with bleomycin (3 mg/ml) for up to 48 h in the absence or presence of antioxidants as shown in Fig. 3 B. At ∼32 h maximum expression of CD95L mRNA was reached. This expression was reduced in the presence of N-acetylcysteine. A further reduction to almost undetectable levels is observed in the presence ofN-acetylcysteine and deferoxamine (Fig. 3 B,lower panel). Deferoxamine alone did not result in a reproducible decrease of CD95L mRNA expression (data not shown). In a similar fashion, H2O2-induced CD95L expression was reduced in the presence of N-acetylcysteine and deferoxamine (data not shown). Taken together the above data point to an important role of ROS in the transcriptional regulation of CD95L expression. Interestingly and in agreement with our experimental evidence in hepatoma cells, a positive correlation between the intracellular levels of ROS and CD95L expression has been observed recently in activation-induced death of mature T lymphocytes and hybridomas (29Williams M.S. Henkart P.A. J. Immunol. 1996; 157: 2395-2402PubMed Google Scholar). The exact mechanism of CD95L mRNA induction via ROS remains to be clarified. Potentially involved regulatory proteins include redox-dependent transcription factors such as NF-κB or AP-1. Our data suggest a coordinated activation of the CD95 system in induction of apoptosis and thus elimination of injured cells, which involves p53-mediated expression of the CD95 receptor in response to DNA damage and ROS-mediated expression of the CD95 ligand. This might result in autocrine suicide of damaged cells and might add to the concept of maintenance of genomic integrity as it has been suggested as an important functional role of p53. Any alteration of the cellular capability to respond to cytostatic agents both on the level of CD95 or CD95L might result in a decreased sensitivity toward chemotherapeutic drug action and could thus add to primary or secondary resistance to anti-cancer therapy. The expert technical assistance of Martina Seyferth is gratefully acknowledged. We are indebted to Heinz Schaller for generous support.

Confocal laser endomicroscopy is a newly introduced endoscopic tool that makes it possible to carry out confocal microscopic examination of the mucosal layer during ongoing endoscopy. Different types of tissue and diseases can be diagnosed immediately, facilitating early diagnosis of gastrointestinal cancer. Analysis of the in vivo microarchitecture is helpful in targeting biopsies to relevant areas. In addition, subsurface imaging can unmask microscopic diseases - (microscopic colitis) or bacterial infection ( HELICOBACTER PYLORI), for example. Molecular imaging is becoming feasible, and this will shortly open the door to new indications in gastrointestinal endoscopy (e.g., in vivo receptor analysis).

The immune response to bacterial infections must be tightly controlled to guarantee pathogen elimination while preventing tissue damage by uncontrolled inflammation. Here, we demonstrate a key role of interleukin (IL)-27 in regulating this critical balance. IL-27 was rapidly induced during murine experimental peritonitis induced by cecal ligation and puncture (CLP). Furthermore, mice deficient for the EBI3 subunit of IL-27 were resistant to CLP-induced septic peritonitis as compared with wild-type controls, and this effect could be suppressed by injection of recombinant single-chain IL-27. EBI3-/- mice displayed significantly enhanced neutrophil migration and oxidative burst capacity during CLP, resulting in enhanced bacterial clearance and local control of infection. Subsequent studies demonstrated that IL-27 directly suppresses endotoxin-induced production of reactive oxygen intermediates by isolated primary granulocytes and macrophages. Finally, in vivo blockade of IL-27 function using a newly designed soluble IL-27 receptor fusion protein led to significantly increased survival after CLP as compared with control-treated mice. Collectively, these data identify IL-27 as a key negative regulator of innate immune cell function in septic peritonitis. Furthermore, in vivo blockade of IL-27 is a novel potential therapeutic target for treatment of sepsis.

Epstein–Barr virus-induced gene 3 (EBI3) is a widely expressed IL-12p40-related protein that associates as a heterodimer with either IL-12p35 or an IL-12p35 homologue, p28, to create a new cytokine (IL-27). To define the function of EBI3 in vivo , we generated knockout mice in which the ebi3 gene was targeted by homologous recombination. EBI3 −/− mice exhibited normal numbers of both naive and mature CD4 + and CD8 + T cells and B cells, but markedly decreased numbers of invariant natural killer T cells (iNKT) as defined by staining with an α-galactosylceramide (αGalCer)-loaded CD1d-tetramer. iNKT cells from EBI3 −/− mice exhibited decreased IL-4 and, to a lesser extent, IFN-γ production after αGalCer stimulation in vitro . A sustained decrease in IL-4 production was also observed in EBI3 −/− mice after αGalCer stimulation in vivo in contrast to IFN-γ production, which was only transiently decreased under such stimulation. Notably, EBI3 −/− mice were resistant to the induction of immunopathology associated with oxazolone-induced colitis, a colitis model mediated primarily by T helper (Th) 2-type cytokine production by iNKT cells. In contrast, trinitrobenzene sulfonic acid-induced colitis, a predominantly Th1-mediated colitis model, was unaffected. Thus, EBI3 plays a critical regulatory role in the induction of Th2-type immune responses and the development of Th2-mediated tissue inflammation in vivo , which may be mediated through the control of iNKT cell function.

Antibodies to soluble liver antigen/liver pancreas (SLA/LP) are specific markers of autoimmune hepatitis. Their target antigen has recently been cloned.To establish standardised immunoassays using the recombinant antigen, and to assess the frequency and significance of seropositivity in patients from different countries.An enzyme linked immunoassay was developed using purified recombinant antigen and validated by testing sera from 200 healthy blood donors and 1026 patients with various liver and non-liver diseases. The assay was then applied to 454 sera from 419 patients with autoimmune hepatitis from different countries. All sera were also tested by inhibition immunoassay and western blot.Antibodies were reliably detected by the recombinant immunoassay and occurred exclusively in patients with autoimmune liver disease. Twenty three of 149 patients from the USA (15%), 23/132 from Brazil (17%), 21/108 from Germany (19%), and 2/30 from Japan (7%) were seropositive. Clinical features at presentation were similar between seropositive and seronegative patients. However, relapse after corticosteroid withdrawal or during maintenance therapy occurred more commonly in seropositive patients.Antibodies to SLA/LP can be reliably detected by these standardised immunoassays based on recombinant antigen. Antibodies to SLA/LP occur with similar frequencies in different geographical regions, races, and age groups, and are of exquisite diagnostic specificity. Whether SLA/LP positive patients represent a clinically distinct subgroup remains to be determined; relapse during treatment reduction appeared to be more common in the SLA/LP group.

Colonoscopy is the accepted gold standard for the detection of colorectal cancer. The aim of the current study was to prospectively compare high definition plus (HD+) colonoscopy with I-Scan functionality (electronic staining) vs. standard video colonoscopy. The primary endpoint was the detection of patients having colon cancer or at least one adenoma.A total of 220 patients due to undergo screening colonoscopy, postpolypectomy surveillance or with a positive occult blood test were randomized in a 1 : 1 ratio to undergo HD+ colonoscopy in conjunction with I-Scan surface enhancement (90i series, Pentax, Tokyo, Japan) or standard video colonoscopy (EC-3870FZK, Pentax). Detected colorectal lesions were judged according to type, location, and size. Lesions were characterized in the HD+ group by using further I-Scan functionality (p- and v-modes) to analyze pattern and vessel architecture. Histology was predicted and biopsies or resections were performed on all identified lesions.HD+ colonoscopy with I-Scan functionality detected significantly more patients with colorectal neoplasia (38 %) compared with standard resolution endoscopy (13 %) (200 patients finally analyzed; 100 per arm). Significantly more neoplastic (adenomatous and cancerous) lesions and more flat adenomas could be detected using high definition endoscopy with surface enhancement. Final histology could be predicted with high accuracy (98.6 %) within the HD+ group.HD+ colonoscopy with I-Scan is superior to standard video colonoscopy in detecting patients with colorectal neoplasia based on this prospective, randomized, controlled trial.

Despite great progress in diagnosis and management of hepatocellular carcinoma (HCC), the exact biology of the tumor remains poorly understood overall limiting the patients' outcome. Detailed analysis and characterization of the molecular mechanisms and subsequently individual prediction of corresponding prognostic traits would revolutionize both diagnosis and treatment of HCC and is the key goal of modern personalized medicine. Over the recent years systematic approaches for the analysis of whole tumor genomes and transcriptomes as well as epigenomes became affordable tools in translational research. This includes simultaneous analyses of thousands of molecular targets using microarray-based technologies as well as next-generation sequencing. Although currently diagnosis and classification of hepatocellular cancers still rely on histological examination of tumor sections, these technologies show great promise to advance the current knowledge of hepatocarcinogenesis, complement diagnostic classification in a setting of microarray-aided pathology, and rationalize the individual drug selection. This review aims to summarize recent progress of system biological approaches in hepatocarcinogenesis and outline potential areas for translational application in a clinical setting. Further, we give an update about known signaling pathways active in HCC, summarize the historical application of whole genomic approaches in liver cancer and indicate ongoing experimental research utilizing novel technologies in diagnosis and treatment of this deadly disease. This will also include the discussion and characterization of new molecular and cellular targets such as Cancer Stem Cells (CSCs).

Abstract The EBV-induced gene 3 (EBI3) is expressed in dendritic cells (DCs) and part of the cytokine IL-27 that controls Th cell development. However, its regulated expression in DCs is poorly understood. In the present study we demonstrate that EBI3 is expressed in splenic CD8−, CD8+, and plasmacytoid DC subsets and is induced upon TLR signaling. Cloning and functional analysis of the EBI3 promoter using in vivo footprinting and mutagenesis showed that stimulation via TLR2, TLR4, and TLR9 transactivated the promoter in primary DCs via NF-κB and Ets binding sites at −90 and −73 bp upstream of the transcriptional start site, respectively. Furthermore, we observed that NF-κB p50/p65 and PU.1 were sufficient to transactivate the EBI3 promoter in EBI3-deficient 293 cells. Finally, induced EBI3 gene expression in DCs was reduced or abrogated in TLR-2/TLR4, TLR9, and MyD88 knockout mice, whereas both basal and inducible EBI3 mRNA levels in DCs were strongly suppressed in NF-κB p50-deficient mice. In summary, these data suggest that EBI3 expression in DCs is transcriptionally regulated by TLR signaling via MyD88 and NF-κB. Thus, EBI3 gene transcription in DCs is induced rapidly by TLR signaling during innate immune responses preceding cytokine driven Th cell development.

Goals and Background The multikinase inhibitor sorafenib provides survival benefit for patients with advanced hepatocellular carcinoma (HCC) and liver cirrhosis (LCI) Child-Pugh A. We report our experiences with sorafenib in advanced HCC, particularly in patients with LCI Child-Pugh B/C, where only limited data are available in regard to safety and efficacy of sorafenib. Methods Thirty-four patients with advanced HCC were treated with sorafenib regardless of liver function and prior anticancer therapy. Adverse events (AEs) were graded using Common Toxicity Criteria version 3.0, tumor response was assessed according to Response Evaluation Criteria in Solid Tumors. Results Fifteen patients presented without LCI or with LCI Child-Pugh A, 15/4 patients had LCI Child-Pugh B/C. Barcelona Clinic Liver Cancer stage was B/C/D in 4/22/8 patients. During treatment period (median 2.2 mo), therapy was discontinued in 61.8% of patients due to tumor progression (32.3%), death (17.6%), AEs (8.8%), or noncompliance (2.9%). Most common grade 3/4 AEs included liver dysfunction (23.5%), diarrhea (14.7%), increased lipase (8.8%), fatigue (8.8%), and hand-foot skin reaction (5.9%). Worsening liver dysfunction/failure was more frequent (P=0.036) in patients with LCI Child-Pugh B/C compared with patients with maintained liver function (no LCI/LCI Child-Pugh A). Median overall survival was 7.2 months for patients with maintained liver function versus 3.3/3.4 months for patients with LCI Child-Pugh B/C. Conclusions These data do not support the use of sorafenib in patients with LCI Child-Pugh C, and patients with LCI Child-Pugh B should be treated with caution until larger trials provide more safety data and a clinically relevant survival benefit under sorafenib therapy.

To investigate the pathophysiological effect of interleukin 6 (IL-6) on lupus nephritis in MRL-Fas(lpr) mice.We generated IL-6-deficient MRL-Fas(lpr) mice using a backcross/intercross breeding scheme. Renal pathology was evaluated using immunohistochemistry detection for macrophages, lymphocytes, vascular cell adhesion molecule-1 (VCAM-1), and TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) for apoptotic cells, and renal IgG and C3 deposition by immunofluorescence staining. Expression of inflammatory markers in the spleen was analyzed by quantitative real-time reverse transcription-polymerase chain reaction. Serum cytokine concentrations were detected by FACS analysis.IL-6 deficiency was highly effective in prolonging survival and ameliorating the clinical, immunological, and histological indicators of murine systemic lupus erythematosus. During the study period of 6 months, MRL-Fas(lpr) IL-6 -/- mice showed delayed onset of proteinuria and hematuria compared to IL-6-intact control mice. Survival rate was 100% in IL-6-deficient MRL-Fas(lpr) mice and 25% in the control group at 6 months of age. The absence of IL-6 resulted in significant reduction of infiltrating macrophages in the kidney (p < 0.05), a decrease in renal IgG and C3 deposition, and a reduction of CD4+ and CD8+ lymphocytes. The parenchymal adhesion molecule VCAM-1 was found to be downregulated in kidneys of MRL-Fas(lpr) IL-6 -/- compared to IL-6-intact mice. We found elevated serum levels of IL-10 and interferon-gamma in IL-6-deficient mice, while splenic mRNA showed an overall downregulation of immunoregulatory genes.IL-6 is a strong promoter of lupus nephritis and may be a promising new therapeutic target in the treatment of human lupus nephritis.

Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 protein family. It interacts with proapoptotic Bcl-2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl-1 is essential for embryonal development and the maintenance of B cells, T cells, and hematopoietic stem cells. We have recently shown that induction of Mcl-1 by growth factors rescues primary human hepatocytes from CD95-mediated apoptosis. This prompted us to further analyze the relevance of Mcl-1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte-specific Mcl-1 knockout mouse (Mcl-1flox/flox-AlbCre). Deletion of Mcl-1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl-1flox/flox-AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl-1. Importantly, hepatic pericellular fibrosis occurs in Mcl-1 negative livers in response to chronic liver damage. Furthermore, Mcl-1flox/flox-AlbCre mice are more susceptible to hepatocellular damage induced by agonistic anti-CD95 antibodies or concanavalin A. Conclusion: The present study provides in vivo evidence that Mcl-1 is a crucial antiapoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction. (HEPATOLOGY 2009.)

The epidemic occurrence of obesity has led to a rapid increase in the incidence of non-alcoholic fatty liver disease (NAFLD) in industrial countries. The disease spectrum includes hepatic steatosis, lobular inflammation with steatohepatitis (NASH) and varying degrees of liver fibrosis, which can progress to cirrhosis. Hepatocellular carcinoma can develop in patients with NASH, even in the absence of cirrhosis. The majority of patients with primary NASH exhibit risk factors that define the metabolic syndrome including insulin resistance and visceral obesity. However, only a minority of patients with NAFLD progress to end-stage liver disease and, so far, predictors to identify these patients are not available. The course of disease progression appears to be slow and develops progressively over years, modulated by genetic susceptibility, nutritional misbehavior and environmental factors. Although risk factors have been identified in epidemiological studies, little is known about disease initiation and progression. This review summarizes the existing animal models of NAFLD, focusing on genetic and dietary models, and discusses their applicability in studying signaling events involved in steatohepatitis. Despite the shortcomings inherent to all experimental models, research in this field has helped to identify potential therapeutic targets and, thus, contributed significantly to our understanding of this disease. The validation and search for new in vivo and in vitro models will propagate the understanding of NASH and help clinicians to develop new treatment modalities.

The hepatocellular carcinoma (HCC) treatment landscape changed a decade ago, with sorafenib demonstrating survival benefit in the first-line setting and becoming the first systemic therapy to be approved for HCC. More recently, regorafenib and nivolumab have received approval in the second-line setting after sorafenib, with further positive phase 3 studies emerging in the first line (lenvatinib non-inferior to sorafenib) and second line versus placebo (cabozantinib and ramucirumab). A key recommendation in the management of patients receiving sorafenib is to promote close communication between the patient and the physician so that adverse events (AEs) are detected early and severe AEs can be prevented. Sorafenib-related AEs have been identified as clinical biomarkers for sorafenib efficacy. Healthcare professionals have become more efficient in managing AEs, identifying patients who are likely to benefit from treatment, and assessing response to treatment, resulting in a trend towards increased overall survival in the sorafenib arms of clinical studies. The rapidly changing treatment landscape due to the emergence of new treatment options (sorafenib and lenvatinib equally effective in first line; regorafenib, cabozantinib, and ramucirumab showing OS benefit in second line with nivolumab approved by the FDA based on response rate) underscores the importance of re-assessing the role of the first approved systemic agent in HCC, sorafenib.

Goals/Background Disease activity and response to treatment in autoimmune hepatitis is assessed best by liver biopsy, which does not suit for regular disease monitoring. It is frequent clinical practice to follow disease by assessment of serologic markers. Here, we assessed the diagnostic fidelity of this clinical practice. Study One hundred thirty-one biopsies from 82 patients with autoimmune hepatitis were analyzed for histologic activity. Serum samples, taken at the time of biopsy, were analyzed for aminotransferases [alanine aminotransferase (ALT), aspartate aminotransferase], IgG, and γ-globulin levels and compared with histology. Results All serum parameters were significantly associated with histologic activity (P<0.0075); ALT and IgG were most complementary. Presence of both elevated ALT and IgG were associated with high inflammatory activity (histologic activity scores ≥6) with 99% sensitivity. Elevation of either IgG or ALT was associated with residual inflammatory activity in almost all patients. Histologic remission is reliably indicated by normalization of both serum parameters, but about half of the patients with normal serum parameters still showed residual histologic activity of histologic activity index (HAI) 4 or 5. However, our patients with HAI scores 4 or 5 were at significantly lower risk of fibrosis progression than patients with scores ≥6 (P<0.02; odds ratio 14.2). Conclusions Histologic activity seems to be reliably indicated by elevated serum parameters. Normalization of serum parameters is not a reliable marker for complete histologic remission (HAI 1 to 3); however, normalized serum parameters identified patients at low risk of fibrosis progression. Thus, the common clinical practice of disease monitoring by serum markers seems to be suitable for regular follow-up.

<h3>Background</h3> Vascular endothelial growth factor (VEGF) is a therapeutic target in gastrointestinal cancer (GiC). However, its in vivo visualisation could not be achieved to date with endoscopic techniques. Confocal laser endomicroscopy (CLE) is a novel imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. The aim of the study was to evaluate CLE for in vivo molecular imaging of VEGF in GiC. <h3>Methods</h3> Molecular imaging of tumours in APCmin mice, in xenograft models and in surgical specimens of patients with colorectal cancer (CRC) was achieved after application of labelled antibodies. The tumour sites were scanned with the probe for the strongest specific fluorescent signal. From all tumour sites examined with CLE in vivo, targeted specimens were obtained for histology, immunohistochemistry (IHC) and fluorescence microscopy. <h3>Results</h3> A VEGF-specific signal was visualised in vivo in 13/15 APCmin mice and in 9/10 xenograft tumours. CLE enabled the cytoplasmatic distribution of VEGF to be displayed due to its subcellular resolution. In human tissue, a VEGF-specific signal was observed in 12/13 malignant specimens and in 10/11 samples from healthy mucosa from the patients (p&lt;0.03). CLE findings correlated well with ex vivo microscopy. <h3>Conclusion</h3> In vivo molecular imaging with specific targeting of VEGF is possible in murine tumours, human xenografts and tissue specimens using CLE. CLE with similar probes can be performed in human colonoscopy. Therefore—from a technical point of view—in vivo molecular imaging is transferable to stratification of patients with CRC during endoscopy even today. CLE could contribute to the identification of lesions at risk and potentially predict response to targeted treatment.

Since sorafenib has shown activity in different tumour types and gemcitabine regimens improved the outcome for biliary tract cancer (BTC) patients, we evaluated first-line gemcitabine plus sorafenib in a double-blind phase II study.102 unresectable or metastatic BTC patients with histologically proven adenocarcinoma of gallbladder or intrahepatic bile ducts, Eastern Cooperative Oncology Group (ECOG) 0-2 were randomised to gemcitabine (1000 mg/m2 once weekly, first 7-weeks+1-week rest followed by once 3-weeks+1-week rest) plus sorafenib (400 mg twice daily) or placebo. Treatment continued until progression or unacceptable toxicity. Tumour samples were prospectively stained for sorafenib targets and potential biomarkers. Serum samples (first two cycles) were measured for vascular endothelial growth factors (VEGFs), vascular endothelial growth factor receptor 2 (VEGFR-2) and stromal cell-derived factor 1 (SDF1)α by enzyme-linked immunosorbent assay (ELISA).Gemcitabine plus sorafenib was generally well tolerated. Four and three patients achieved partial responses in the sorafenib and placebo groups, respectively. There was no difference in the primary end-point, median progression-free survival (PFS) for gemcitabine plus sorafenib versus gemcitabine plus placebo (3.0 versus 4.9 months, P=0.859), and no difference for median overall survival (OS) (8.4 versus 11.2 months, P=0.775). Patients with liver metastasis after resection of primary BTC survived longer with sorafenib (P=0.019) compared to placebo. Patients who developed hand-foot syndrome (HFS) showed longer PFS and OS than patients without HFS. Two sorafenib targets, VEGFR-2 and c-kit, were not expressed in BTC samples. VEGFR-3 and Hif1α were associated with lymph node metastases and T stage. Absence of PDGFRβ expression correlated with longer PFS.The addition of sorafenib to gemcitabine did not demonstrate improved efficacy in advanced BTC patients. Biomarker subgroup analysis suggested that some patients might benefit from combined treatment.

•Optimal liver stiffness thresholds for community-based screening of at-risk patients are 9.1–9.5 kPa for fibrosis (stages ≥F2). •Transient elastography is a cost-effective intervention for identifying patients with liver fibrosis in primary care. •Between 2,500 to 6,500 PPP-adjusted euros are needed to gain an extra year of life, adjusted for quality of life. •The survival effect of screening is most pronounced for the identification of significant (≥F2) fibrosis. Background & Aims Non-alcoholic fatty liver disease and alcohol-related liver disease pose an important challenge to current clinical healthcare pathways because of the large number of at-risk patients. Therefore, we aimed to explore the cost-effectiveness of transient elastography (TE) as a screening method to detect liver fibrosis in a primary care pathway. Methods Cost-effectiveness analysis was performed using real-life individual patient data from 6 independent prospective cohorts (5 from Europe and 1 from Asia). A diagnostic algorithm with conditional inference trees was developed to explore the relationships between liver stiffness, socio-demographics, comorbidities, and hepatic fibrosis, the latter assessed by fibrosis scores (FIB-4, NFS) and liver biopsies in a subset of 352 patients. We compared the incremental cost-effectiveness of a screening strategy against standard of care alongside the numbers needed to screen to diagnose a patient with fibrosis stage ≥F2. Results The data set encompassed 6,295 participants (mean age 55 ± 12 years, BMI 27 ± 5 kg/m2, liver stiffness 5.6 ± 5.0 kPa). A 9.1 kPa TE cut-off provided the best accuracy for the diagnosis of significant fibrosis (≥F2) in general population settings, whereas a threshold of 9.5 kPa was optimal for populations at-risk of alcohol-related liver disease. TE with the proposed cut-offs outperformed fibrosis scores in terms of accuracy. Screening with TE was cost-effective with mean incremental cost-effectiveness ratios ranging from 2,570 €/QALY (95% CI 2,456–2,683) for a population at-risk of alcohol-related liver disease (age ≥45 years) to 6,217 €/QALY (95% CI 5,832–6,601) in the general population. Overall, there was a 12% chance of TE screening being cost saving across countries and populations. Conclusions Screening for liver fibrosis with TE in primary care is a cost-effective intervention for European and Asian populations and may even be cost saving. Lay summary The lack of optimized public health screening strategies for the detection of liver fibrosis in adults without known liver disease presents a major healthcare challenge. Analyses from 6 independent international cohorts, with transient elastography measurements, show that a community-based risk-stratification strategy for alcohol-related and non-alcoholic fatty liver diseases is cost-effective and potentially cost saving for our healthcare systems, as it leads to earlier identification of patients. Non-alcoholic fatty liver disease and alcohol-related liver disease pose an important challenge to current clinical healthcare pathways because of the large number of at-risk patients. Therefore, we aimed to explore the cost-effectiveness of transient elastography (TE) as a screening method to detect liver fibrosis in a primary care pathway. Cost-effectiveness analysis was performed using real-life individual patient data from 6 independent prospective cohorts (5 from Europe and 1 from Asia). A diagnostic algorithm with conditional inference trees was developed to explore the relationships between liver stiffness, socio-demographics, comorbidities, and hepatic fibrosis, the latter assessed by fibrosis scores (FIB-4, NFS) and liver biopsies in a subset of 352 patients. We compared the incremental cost-effectiveness of a screening strategy against standard of care alongside the numbers needed to screen to diagnose a patient with fibrosis stage ≥F2. The data set encompassed 6,295 participants (mean age 55 ± 12 years, BMI 27 ± 5 kg/m2, liver stiffness 5.6 ± 5.0 kPa). A 9.1 kPa TE cut-off provided the best accuracy for the diagnosis of significant fibrosis (≥F2) in general population settings, whereas a threshold of 9.5 kPa was optimal for populations at-risk of alcohol-related liver disease. TE with the proposed cut-offs outperformed fibrosis scores in terms of accuracy. Screening with TE was cost-effective with mean incremental cost-effectiveness ratios ranging from 2,570 €/QALY (95% CI 2,456–2,683) for a population at-risk of alcohol-related liver disease (age ≥45 years) to 6,217 €/QALY (95% CI 5,832–6,601) in the general population. Overall, there was a 12% chance of TE screening being cost saving across countries and populations. Screening for liver fibrosis with TE in primary care is a cost-effective intervention for European and Asian populations and may even be cost saving.

Hepatocellular carcinomas (HCCs) are characterised by considerable phenotypic and molecular heterogeneity. Treating HCC and designing clinical trials are particularly challenging because co-existing liver disease, present in most patients, limits aggressive therapeutic options. Positive results in recent phase III clinical trials have confirmed the high value of anti-angiogenic therapies for HCC in both first (sorafenib and lenvatinib) and second line (regorafenib and cabozantinib) treatment modalities. However, failure of several large randomised controlled clinical trials over the last 10 years underlines the necessity for innovative treatment strategies and implementation of translational findings to overcome the unmet clinical need. Furthermore, the promising results from novel immunotherapies are likely to complement the landscape of active compounds for HCC and will require a completely different approach to patients, as well as the development of prognostic/predictive biomarkers. Given our increasing understanding of the most abundant molecular alterations in HCC, effective enrichment of patients based on clinical and molecular biomarkers, as well as adaptive clinical trials, are now feasible and should be implemented. Herein, we aim to review important aspects of precision medicine approaches in HCC that might contribute to improving the molecular subclassification of patients in a clinical trial setting and pave the way for novel therapeutic strategies.

The interdisciplinary guidelines at the S3 level on the diagnosis of and therapy for hepatocellular carcinoma (HCC) constitute an evidence- and consensus-based instrument that is aimed at improving the diagnosis of and therapy for HCC since these are very challenging tasks. The purpose of the guidelines is to offer the patient (with suspected or confirmed HCC) adequate, scientifically based and up-to-date procedures in diagnosis, therapy and rehabilitation. This holds not only for locally limited or focally advanced disease but also for the existence of recurrences or distant metastases. Besides making a contribution to an appropriate health-care service, the guidelines should also provide the foundation for an individually adapted, high-quality therapy. The explanatory background texts should also enable non-specialist but responsible colleagues to give sound advice to their patients concerning specialist procedures, side effects and results. In the medium and long-term this should reduce the morbidity and mortality of patients with HCC and improve their quality of life.

To prospectively compare SIRT and DEB-TACE for treating hepatocellular carcinoma (HCC).From 04/2010-07/2012, 24 patients with histologically proven unresectable N0, M0 HCCs were randomized 1:1 to receive SIRT or DEB-TACE. SIRT could be repeated once in case of recurrence; while, TACE was repeated every 6 weeks until no viable tumor tissue was detected by MRI or contraindications prohibited further treatment. Patients were followed-up by MRI every 3 months; the final evaluation was 05/2013.Both groups were comparable in demographics (SIRT: 8males/4females, mean age 72 ± 7 years; TACE: 10males/2females, mean age 71 ± 9 years), initial tumor load (1 patient ≥25 % in each group), and BCLC (Barcelona Clinic Liver Cancer) stage (SIRT: 12×B; TACE 1×A, 11×B). Median progression-free survival (PFS) was 180 days for SIRT versus 216 days for TACE patients (p = 0.6193) with a median TTP of 371 days versus 336 days, respectively (p = 0.5764). Median OS was 592 days for SIRT versus 788 days for TACE patients (p = 0.9271). Seven patients died in each group. Causes of death were liver failure (n = 4 SIRT group), tumor progression (n = 4 TACE group), cardiovascular events, and inconclusive (n = 1 in each group).No significant differences were found in median PFS, OS, and TTP. The lower rate of tumor progression in the SIRT group was nullified by a greater incidence of liver failure. This pilot study is the first prospective randomized trial comparing SIRT and TACE for treating HCC, and results can be used for sample size calculations of future studies.

Advanced fibrosis has been established as the most important predictor of overall mortality in patients with non-alcoholic fatty liver disease (NAFLD). In contrast to cirrhosis, advanced, non-cirrhotic NAFLD is difficult to identify and data from Germany are lacking.To identify clinical factors associated with advanced, non-cirrhotic fibrosis.Patients were recruited in the prospectively enrolling European NAFLD Registry. Clinical characteristics and the performance of non-invasive surrogate scores compared with vibration-controlled transient elastography are reported.Two hundred and sixty-one patients with non-cirrhotic NAFLD on liver biopsy (mean age 51 years, equal sex distribution) were included. The prevalence of stage 3 fibrosis on liver biopsy was 15.7%. These patients were significantly older (57 vs 50 years, P < 0.01), had a higher body mass index (32.3 vs 30.5, P < 0.05), and more frequent arterial hypertension (78% vs 50%, P = 0.001) and type 2 diabetes (61% vs 24.1%, P < 0.001). On multivariate logistic regression, diabetes (OR = 4.68, 95% CI 2.17-10.10) and hypertension (OR = 2.91, 95% CI 1.12-7.18) were independent predictors of advanced fibrosis. Comedication included metformin in 50% and insulin in 33% of patients with diabetes. Despite the presence of cardiovascular risk factors, the use of statins was low. Liver stiffness measurement identified advanced fibrosis with an AUROC of 0.81 (95% CI 0.72-0.91). The performance of NAFLD fibrosis score, Fibrosis-4, and AST to platelet ratio index were lower with AUCs of 0.74, 0.71, and 0.67, respectively.The prevalence of metabolic comorbidities in a German population with non-cirrhotic biopsy-proven NAFLD is high. While the examined scores exhibit an acceptable specificity, liver stiffness measurement appeared to be superior to blood-based non-invasive surrogate scores in ruling out advanced fibrosis.

Human hepatocarcinogenesis is as a multi-step process starting from dysplastic lesions to early carcinomas (eHCC) that ultimately progress to HCC (pHCC). However, the sequential molecular alterations driving malignant transformation of the pre-neoplastic lesions are not clearly defined. This lack of information represents a major challenge in the clinical management of patients at risk.We applied next-generation transcriptome sequencing to tumor-free surrounding liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, eHCC (n = 5) and pHCC (n = 3) from 8 HCC patients with hepatitis B infection. Integrative analyses of genetic and transcriptomic changes were performed to characterize the genomic alterations during hepatocarcinogenesis.We report that changes in transcriptomes of early lesions including eHCC were modest and surprisingly homogenous. Extensive genetic alterations and subsequent activation of prognostic adverse signaling pathways occurred only late during hepatocarcinogenesis and were centered on TGFβ, WNT, NOTCH, and EMT-related genes highlighting the molecular diversity of pHCC. We further identify IGFALS as a key genetic determinant preferentially down-regulated in pHCC.Our results define new hallmarks in molecular stratification and therapy options for patients at risk for HCC, and merit larger prospective investigations to develop a modified clinical-decision making algorithm based on the individualized next-generation sequencing analyses.

Goals: The aim of this study was to analyze clinical presentation, course of disease, and management of patients with hepatocellular carcinoma (HCC) in a German referral center between 1998 and 2009. Background: HCC is a rare tumor in Germany, but its incidence has increased over the last 30 years. New therapies such as chemoembolization with drug-eluting beads, selective internal radiotherapy, and sorafenib were introduced recently; however, the impact on clinical management and overall survival (OS) is unclear. Study: In this retrospective analysis, 1066 patients with HCC, separated into two 6-year periods (n=385; 1998 to 2003 and n=681; 2004 to 2009) were evaluated. Results: The number of patients presenting each year (64 vs. 114 per year), with an age over 80 years or with nonalcoholic steatohepatitis increased significantly between periods. The main risk factors were alcoholic liver disease in 51.7%, chronic hepatitis C virus in 28.2%, and chronic hepatitis B virus in 13.4% of patients with liver cirrhosis and HCC. Patients presented with more advanced tumor stages and with worse liver function in period 2. The majority (61.6%) of patients received local treatment over a spectrum of Barcelona Clinic Liver-Cancer (BCLC) stages, whereas systemic therapy was offered to a minority (8.8%) and limited to BCLC stage C patients only. OS decreased in BCLC stage A and D and improved in BCLC stage B and C and decreased for all patients from 16.5 to 15.3 months between periods. Conclusions: No improvement of OS was observed when comparing time periods, partly because of the more advanced stage of HCC and because of the increasing age in the second time period. Improved and new therapeutic options and the intensification of surveillance programs are likely to increase survival of HCC patients in the future.

HepatologyVolume 73, Issue S1 p. 137-149 Review Local and Regional Therapies for Hepatocellular Carcinoma Roman Kloeckner, Roman Kloeckner orcid.org/0000-0001-5492-4792 Department of Diagnostic and Interventional Radiology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, GermanySearch for more papers by this authorPeter Robert Galle, Peter Robert Galle orcid.org/0000-0001-8294-0992 Department of Internal Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, GermanySearch for more papers by this authorJordi Bruix, Corresponding Author Jordi Bruix jbruix@clinic.cat orcid.org/0000-0002-9826-0753 Barcelona Clinic Liver Cancer Group, Liver Unit, Hospital Clínic, IDIBAPS, University of Barcelona, CIBEREHD Barcelona, Barcelona, Spain Address Correspondence and Reprint Requests to: Jordi Bruix, M.D, Ph.D. Barcelona Clinic Liver Cancer Group, Liver Unit, Hospital Clínic, IDIBAPS, University of Barcelona, CIBEREHD Villarroel 170 Barcelona 08036, Spain E-mail: jbruix@clinic.cat Tel.: 34932279803Search for more papers by this author Roman Kloeckner, Roman Kloeckner orcid.org/0000-0001-5492-4792 Department of Diagnostic and Interventional Radiology, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, GermanySearch for more papers by this authorPeter Robert Galle, Peter Robert Galle orcid.org/0000-0001-8294-0992 Department of Internal Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, GermanySearch for more papers by this authorJordi Bruix, Corresponding Author Jordi Bruix jbruix@clinic.cat orcid.org/0000-0002-9826-0753 Barcelona Clinic Liver Cancer Group, Liver Unit, Hospital Clínic, IDIBAPS, University of Barcelona, CIBEREHD Barcelona, Barcelona, Spain Address Correspondence and Reprint Requests to: Jordi Bruix, M.D, Ph.D. Barcelona Clinic Liver Cancer Group, Liver Unit, Hospital Clínic, IDIBAPS, University of Barcelona, CIBEREHD Villarroel 170 Barcelona 08036, Spain E-mail: jbruix@clinic.cat Tel.: 34932279803Search for more papers by this author First published: 18 June 2020 https://doi.org/10.1002/hep.31424Citations: 36 Supported by the Carlos III Health Institute (PI18/00768), Albert Einstein Cancer Center (PI044031), and World Cancer Research Fund/American Institute for Cancer Research (16-0026). Potential conflict of interest: Dr. Galle consults, advises, is on the speakers' bureau, and received grants from Bayer. He consults, advises, and is on the speakers' bureau for Bristol-Myers Squibb, MSD, Sirtex, Lilly, Ipsen, AstraZeneca, and Roche. Dr. Bruix consults and received grants from BTG and Ipsen. He consults for AbbVie, AstraZeneca, Arqule, Roche, Bristol-Myers Squibb, AdaptImmune, Basilea, Eisai, Gilead, Sirtex, Sanofi, Terumo, and Novartis. Dr. Kloeckner consults and advises for Boston Scientific, Bristol-Myers Squibb, Guerbet, Roche, and SIRTEX and is on the speakers' bureau for BTG, Eisai, Guerbet, Ipsen, MSD Sharp & Dohme, and SIRTEX. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume73, IssueS1Special Issue: Cancers of the Liver - Biology, Diagnosis and TreatmentJanuary 2021Pages 137-149 RelatedInformation

Nonalcoholic fatty liver disease (NAFLD), depression, and anxiety disorders are frequent diseases, and data on mutual influence are inconsistent. The aim of this study was to explore the incidence of depression and anxiety in a large primary care cohort in Germany and to study the impact of NAFLD over a 10‐year time frame. Patients with NAFLD diagnosed between 2010 and 2015 were matched to a cohort without NAFLD controlling for age, sex, physician, index year, and Charlson comorbidity index. The primary outcome of the study was the incidence of depression, anxiety, and first prescription of antidepressant drugs. We compared 19,871 patients with NAFLD to 19,871 matched controls. Within 10 years of the index date, 21.2% of patients with NAFLD and 18.2% of controls were diagnosed with depression ( P &lt; 0.001). On regression analysis, the hazard ratio (HR) for incidence of depression was 1.21 ( P &lt; 0.001). This association was similar for the endpoint of the first prescription of antidepressant drugs (HR, 1.21; P &lt; 0.001). Anxiety disorders were diagnosed in 7.9% of patients with NAFLD and 6.5% of controls during the observation time ( P = 0.003). The HR for incidence of anxiety was 1.23 ( P &lt; 0.001). This association remained significant in women ( P &lt; 0.001), while there was only a trend in men (HR, 1.15; 95% confidence interval, 0.99‐1.34; P &lt; 0.067). The risk of developing anxiety disorders was higher in younger patients. Conclusion: NAFLD constitutes an independent risk factor for emerging depression and anxiety even after controlling for confounding comorbidities.

4003 Background: Patients (pts) with advanced HCC and elevated AFP have a poorer prognosis compared to the general HCC population, and need effective, well tolerated treatment options. Increased VEGF and VEGFR2 expression is associated with high AFP expression in HCC tumors. Ramucirumab (RAM), a human IgG1 mAb, inhibits activation of VEGFR2. REACH-2 was designed to confirm the benefit of RAM treatment observed in the REACH study in pts with baseline AFP ≥400 ng/mL. Methods: Eligible pts were ≥18 yrs, had HCC with BCLC stage C or B disease refractory or not amenable to locoregional therapy, baseline AFP ≥400 ng/mL, Child-Pugh A, ECOG PS 0 or 1, adequate hematologic and biochemical parameters, had progressed on or following, or were intolerant to sorafenib. Pts were randomized (2:1) to receive RAM 8 mg/kg iv or placebo (PL) Q2W plus best supportive care, until disease progression or unacceptable toxicity. Primary endpoint was overall survival (OS). Secondary objectives included progression-free survival (PFS), objective response rate (ORR) per RECIST v1.1 and safety. Results: 292 pts were randomized to RAM (197) or PL (95). Baseline characteristics were generally balanced between arms. RAM treatment significantly improved OS (median OS 8.5 mo vs 7.3 mo PL; HR 0.710; 95% CI 0.531, 0.949; p=.0199). RAM significantly improved PFS (median PFS 2.8 mo vs 1.6 mo PL; HR 0.452; 95% CI 0.339, 0.603; p<.0001). ORR was 4.6% RAM vs 1.1% PL (p=.1156) and disease control rate (ORR + stable disease) was 59.9% RAM vs 38.9% PL (p=.0006). Grade ≥ 3 adverse events occurring in ≥ 5% pts in the RAM arm were hypertension (12.2% RAM, 5.3% PL) and hyponatremia (5.6%, 0%). Conclusions: REACH-2 met its primary endpoint showing a significant survival benefit, with RAM treatment reducing the risk of death (29%) in pts with HCC and AFP ≥ 400 ng/mL who progressed on or were intolerant to sorafenib. Treatment was well tolerated, with a safety profile consistent with the established profile for single agent RAM. REACH-2 is the first positive phase 3 study conducted in a biomarker-selected pt population with HCC. Clinical trial information: NCT02435433.

Lifestyle modifications remain the cornerstone of treatment in non-alcoholic fatty liver disease (NAFLD). However, they requently fail related to the inability of patients to implement lasting changes.To evaluate the effects of a short, web-based, individualised exercise program on non-invasive markers of hepatic steatosis, inflammation and fibrosis.Patients with histologically confirmed NAFLD underwent an 8-week, web-based, individualised exercise program that contained bidirectional feedback.Forty-four patients entered the study and 41 completed the assigned training goal (93.2%). In the completer population, 8 weeks of individualised exercise increased the VO2peak by 12.2% compared to baseline (P < .001). ALT and AST decreased by 14.3% (P = .002) and 18.2% (P < .001) and remained at this level until follow-up 12 weeks after the intervention. Markers of inflammation including hsCRP, ferritin, and M30 decreased. In parallel, gut microbiota exhibited increased metagenomic richness (P < .05) and at the taxonomic levels Bacteroidetes and Euryarchaeota increased whereas Actinobacteria phylum decreased. Surrogate scores of steatosis and fibrosis including the fatty liver index (FLI), FiB-4, APRI and transient elastography showed significant reductions. In parallel, a marker of procollagen-3 turnover (PRO-C3) decreased while C4M2, reflecting type IV collagen, degradation increased suggesting beneficial hepatic fibrosis remodelling from exercise. Also, an enhancement in health-related quality of life was reported.The current study underlines the plausibility and potential of an 8 week individualised web-based exercise program in NAFLD. Clinical trial number: NCT02526732.

Patients with advanced hepatocellular carcinoma (HCC) have historically had few options and faced extremely poor prognoses if their disease progressed after standard-of-care tyrosine kinase inhibitors (TKIs). Recently, the standard of care for HCC has been transformed as a combination of the immune checkpoint inhibitor (ICI) atezolizumab plus the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab was shown to offer improved overall survival in the first-line setting. Immunotherapy has demonstrated safety and efficacy in later lines of therapy as well, and ongoing trials are investigating novel combinations of ICIs and TKIs, in addition to interventions earlier in the course of disease or in combination with liver-directed therapies. Because HCC usually develops against a background of cirrhosis, immunotherapy for liver tumors is complex and oncologists need to account for both immunological and hepatological considerations when developing a treatment plan for their patients. To provide guidance to the oncology community on important concerns for the immunotherapeutic care of HCC, the Society for Immunotherapy of Cancer (SITC) convened a multidisciplinary panel of experts to develop a clinical practice guideline (CPG). The expert panel drew on the published literature as well as their clinical experience to develop recommendations for healthcare professionals on these important aspects of immunotherapeutic treatment for HCC, including diagnosis and staging, treatment planning, immune-related adverse events (irAEs), and patient quality of life (QOL) considerations. The evidence- and consensus-based recommendations in this CPG are intended to give guidance to cancer care providers treating patients with HCC.

•The detection of patients with early cirrhosis is important to improve prognosis. •The SEAL algorithm evaluates the utility of population-based screening to detect cirrhosis as early as possible. •Excluding patients with decompensated cirrhosis at initial diagnosis, SEAL was associated with a 60% higher chance of early cirrhosis detection. •Only 50% of the patients identified by SEAL in primary care utilized specialist assessment. Background & Aims Detection of patients with early cirrhosis is of importance to prevent the occurrence of complications and improve prognosis. The SEAL program aimed at evaluating the usefulness of a structured screening procedure to detect cirrhosis as early as possible. Methods SEAL was a prospective cohort study with a control cohort from routine care data. Individuals participating in the general German health check-up after the age of 35 (“Check-up 35”) at their primary care physicians were offered a questionnaire, liver function tests (aspartate and alanine aminotransferase [AST and ALT]), and follow-up. If AST/ALT levels were elevated, the AST-to-platelet ratio index (APRI) score was calculated, and patients with a score >0.5 were referred to a liver expert in secondary and/or tertiary care. Results A total of 11,859 participants were enrolled and available for final analysis. The control group comprised 349,570 participants of the regular Check-up 35. SEAL detected 488 individuals with elevated APRI scores (4.12%) and 45 incident cases of advanced fibrosis/cirrhosis. The standardized incidence of advanced fibrosis/cirrhosis in the screening program was slightly higher than in controls (3.83‰ vs. 3.36‰). The comparison of the chance of fibrosis/cirrhosis diagnosis in SEAL vs. in standard care was inconclusive (marginal odds ratio 1.141, one-sided 95% CI 0.801, +Inf). Of note, when patients with decompensated cirrhosis at initial diagnosis were excluded from both cohorts in a post hoc analysis, SEAL was associated with a 59% higher chance of early cirrhosis detection on average than routine care (marginal odds ratio 1.590, one-sided 95% CI 1.080, +Inf; SEAL 3.51‰, controls: 2.21‰). Conclusions The implementation of a structured screening program may increase the early detection rate of cirrhosis in the general population. In this context, the SEAL pathway represents a feasible and potentially cost-effective screening program. Registration DRKS00013460 Lay summary Detection of patients with early liver cirrhosis is of importance to prevent the occurrence of complications and improve prognosis. This study demonstrates that the implementation of a structured screening program using easily obtainable measures of liver function may increase the early detection rate of cirrhosis in the general population. In this context, the ‘SEAL’ pathway represents a feasible and potentially cost-effective screening program. Detection of patients with early cirrhosis is of importance to prevent the occurrence of complications and improve prognosis. The SEAL program aimed at evaluating the usefulness of a structured screening procedure to detect cirrhosis as early as possible. SEAL was a prospective cohort study with a control cohort from routine care data. Individuals participating in the general German health check-up after the age of 35 (“Check-up 35”) at their primary care physicians were offered a questionnaire, liver function tests (aspartate and alanine aminotransferase [AST and ALT]), and follow-up. If AST/ALT levels were elevated, the AST-to-platelet ratio index (APRI) score was calculated, and patients with a score >0.5 were referred to a liver expert in secondary and/or tertiary care. A total of 11,859 participants were enrolled and available for final analysis. The control group comprised 349,570 participants of the regular Check-up 35. SEAL detected 488 individuals with elevated APRI scores (4.12%) and 45 incident cases of advanced fibrosis/cirrhosis. The standardized incidence of advanced fibrosis/cirrhosis in the screening program was slightly higher than in controls (3.83‰ vs. 3.36‰). The comparison of the chance of fibrosis/cirrhosis diagnosis in SEAL vs. in standard care was inconclusive (marginal odds ratio 1.141, one-sided 95% CI 0.801, +Inf). Of note, when patients with decompensated cirrhosis at initial diagnosis were excluded from both cohorts in a post hoc analysis, SEAL was associated with a 59% higher chance of early cirrhosis detection on average than routine care (marginal odds ratio 1.590, one-sided 95% CI 1.080, +Inf; SEAL 3.51‰, controls: 2.21‰). The implementation of a structured screening program may increase the early detection rate of cirrhosis in the general population. In this context, the SEAL pathway represents a feasible and potentially cost-effective screening program.

The prevalence of liver disease, and especially of advanced liver fibrosis, in the German population is poorly defined. The aim of the study was to explore liver enzymes and surrogate scores of hepatic steatosis and advanced hepatic fibrosis in a population-based cohort study in Germany. In the cross-sectional population-based Gutenberg Health study, data of 14,950 participants enrolled between 2007 and 2012 were captured and analyzed. The distribution of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), fatty liver index (FLI), and Fibrosis-4 (FIB-4) score, as well as the underlying risk factors, were assessed by regression models. Elevated liver enzymes in this population-based sample were seen in 19.9% for ALT, 12.8% for AST, and 14% for GGT. Risk factors for liver disease included alcohol use and the presence of the metabolic syndrome, which were both risk factors associated with increased liver enzymes. The FLI suggested that 37.5% of the population exhibited hepatic steatosis and 1.1% of patients exhibited a FIB-4 above the upper cutoff, while 19.2% were in the intermediate range. Interestingly, advanced fibrosis was significantly more frequent in men compared with women (FIB-4: 1.5% vs. 0.6% [P < 0.0001]; NFS: 3.6% vs. 1.9% [P < 0.0001]). In addition, age was a relevant risk factor for exhibiting a noninvasive surrogate score suggestive of advanced fibrosis in the current study population. Conclusion: Elevated liver enzymes were seen in almost a fifth of the German population. At the population-based level, the prevalence of advanced fibrosis was estimated at 1% in Germany.

Abstract Background and Aims Different approaches are available after the progression of disease (PD) to immune checkpoint inhibitors (ICIs) for hepatocellular carcinoma (HCC), including the continuation of ICI, treatment switching to tyrosine kinase inhibitors (TKIs) and cessation of anticancer therapy. We sought to characterise the relationship between radiological patterns of progression and survival post‐ICI, also appraising treatment strategies. Methods We screened 604 HCC patients treated with ICIs, including only those who experienced PD by data cut‐off. We evaluated post‐progression survival (PPS) according to the treatment strategy at PD and verified its relationship with radiological patterns of progression: intrahepatic growth (IHG), new intrahepatic lesion (NIH), extrahepatic growth (EHG), new extrahepatic lesion (NEH) and new vascular invasion (nVI). Results Of 604 patients, 364 (60.3%) experienced PD during observation. Median PPS was 5.3 months (95% CI: 4.4–6.9; 271 events). At the data cut‐off, 165 patients (45%) received no post‐progression anticancer therapy; 64 patients (17.6%) continued ICI beyond PD. IHG (HR 1.64 [95% CI: 1.21–2.22]; p = .0013) and nVI (HR 2.15 [95% CI: 1.38–3.35]; p = .0007) were associated with shorter PPS. Multivariate models adjusted for progression patterns, treatment line and albumin‐bilirubin grade and Eastern Cooperative Oncology Group performance status at PD confirmed receipt of ICI beyond PD with (HR 0.17, 95% CI: 0.09–0.32; p &lt; .0001) or without subsequent TKI (HR 0.39, 95% CI: 0.26–0.58; p &lt; .0001) as predictors of prolonged PPS versus no anticancer therapy. Conclusions ICI‐TKI sequencing is a consolidated option in advanced HCC. nVI and IHG predict a poorer prognosis. Despite lack of recommendation, the continuation of ICI beyond progression in HCC is adopted clinically: future efforts should appraise which patients benefit from this approach.

The prevalence of minimal hepatic encephalopathy (MHE), in particular in different subgroups, remains unknown. This study aimed to analyze the prevalence of MHE in different subgroups to identify patients at high risk and to pave the way for personalized screening approaches.In this study, data of patients recruited at 10 centers across Europe and the United States were analyzed. Only patients without clinical signs of hepatic encephalopathy were included. MHE was detected using the Psychometric Hepatic Encephalopathy Score (PHES, cut-off < or ≤-4 depending on local norms). Clinical and demographic characteristics of the patients were assessed and analyzed.In total, 1,868 patients with cirrhosis with a median model for end-stage liver disease (MELD) of 11 were analyzed (Child-Pugh [CP] stages: A 46%, B 42%, and C 12%). In the total cohort, MHE was detected by PHES in 650 patients (35%). After excluding patients with a history of overt hepatic encephalopathy, the prevalence of MHE was 29%. In subgroup analyses, the prevalence of MHE in patients with CP A was low (25%), whereas it was high in CP B or C (42% and 52%). In patients with a MELD score <10, the prevalence of MHE was only 25%, but it was 48% in patients with a MELD score ≥20. Standardized ammonia levels (ammonia level/upper limit of normal of each center) correlated significantly, albeit weakly with PHES (Spearman ρ = -0.16, P < 0.001).The prevalence of MHE in patients with cirrhosis was high but varied substantially between diseases stages. These data may pave the way for more individualized MHE screening approaches.

Atezolizumab plus bevacizumab (Atezo/Bev) is first line-treatment for unresectable hepatocellular carcinoma (HCC). Body mass index (BMI) has demonstrated predictive value for response to immunotherapy in non-HCC cancer types. Our study investigated the effect of BMI on safety and efficacy of real-life use of Atezo/Bev for unresectable HCC.191 consecutive patients from seven centres receiving Atezo/Bev were included in the retrospective study. Overall survival (OS), progression-free survival (PFS), overall response rate (ORR) and disease control rate (DCR) defined by RECIST v1.1 were measured in overweight (BMI ≥ 25) and non-overweight (BMI < 25) patients. Treatment-related adverse events (trAEs) were evaluated.Patients in the overweight cohort (n = 94) had higher rates of non-alcoholic fatty liver disease (NAFLD) and lower rates of Hepatitis B compared to non-overweight cohort (n = 97). Baseline Child-Pugh class and Barcelona Clinic Liver Cancer stage were similar between cohorts, with lower rates of extrahepatic spread in the overweight group. Overweight patients had similar OS compared to non-overweight (median OS 15.1 vs. 14.9 months; p = 0.99). BMI did not influence median PFS (7.1 vs. 6.1 months; p = 0.42), ORR (27.2% vs. 22.0%; p = 0.44) and DCR (74.1% vs. 71.9%; p = 0.46). There were higher rates of atezolizumab-related fatigue (22.3% vs. 10.3%; p = 0.02) and bevacizumab-related thrombosis (8.5% vs. 2.1%; p = 0.045) in the overweight patients, but overall trAEs and treatment discontinuation were comparable between cohorts.Atezo/Bev has comparable efficacy in overweight HCC patients, with an increase in treatment-related fatigue and thrombosis. Combination therapy is safe and efficacious to use in overweight patients, including those with underlying NAFLD.

Type 2 diabetes mellitus (T2DM) is a major risk factor for advanced liver disease. The aim of this prospective cohort study was to assess the prevalence and associated risk factors of liver fibrosis and cirrhosis in primary care centers participating in the diabetes disease management program (DMP) in Germany.A total of 175 participants with the diagnosis of T2DM were enrolled in two primary care centers. Steatotic liver disease (SLD; hepatic steatosis, ≥275 dB/m), fibrosis (≥8 kPa), and cirrhosis (≥15 kPa) were assessed non-invasively using vibration-controlled transient elastography. Multivariable logistic regression analysis was performed to identify clinical predictors of fibrosis and cirrhosis. The AUDIT questionnaire was used to screen for alcohol consumption, and a score ≥8 was considered harmful alcohol consumption.The majority of participants were male (62%), and the median age was 66 years (interquartile range 59; 71). The median body mass index was 31.1 kg/m2 , with 58.9% of the participants being obese. Harmful alcohol consumption was prevalent in 8.0% and 20.0% of the entire cohort and in those with cirrhosis, respectively. The prevalence of SLD, fibrosis, and cirrhosis was 77.1%, 42.3%, and 12.0%, respectively. In multivariable logistic regression analysis, obesity, and harmful alcohol consumption were associated with the highest odds of fibrosis (odds ratio [OR] 5.198, 95% confidence interval [CI] 2.269-11.908) and cirrhosis (OR 5.615, 95% CI 1.274-24.756), respectively.The prevalence of fibrosis and cirrhosis in patients seen in the diabetes DMP in Germany is high. Obesity and harmful alcohol consumption increase the risk of fibrosis and cirrhosis in people with T2DM. Screening for advanced liver disease and associated risk factors within the DMP program may reduce the liver disease burden in this high-risk population.

The vaccination route may influence the success of immunization against pathogens. The conventional intramuscular (i.m.) application of a vaccine containing the hepatitis B virus (HBV) surface antigen (HBsAg) led to protective anti-HBs antibody levels in the majority of vaccine recipients. In this study, we vaccinated healthy volunteers and a group of i.m. vaccine nonresponders via the intradermal (i.d.) route and analyzed the HBV-specific B-cell response as well as class-II- and class-I-restricted T-cell responses by (3)H-thymidine uptake, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT). The results were then compared with i.m. vaccinated controls. I.d. vaccinations were well tolerated and induced neutralizing anti-HBs antibodies in all naive vaccine recipients and, importantly, all but one former i.m. nonresponder developed protective anti-HBs serum antibody levels after 2 or 3 i.d. immunizations. On the cellular level, i.d. vaccine recipients showed significantly higher anti-HBs producing B-cell frequencies and more vigorous class-II-restricted T-helper (Th) cell responses than i.m. controls. However, although the HBsAg-specific T cells were characterized by their cytokine release as Th1-like cells in both groups, human leukocyte antigen (HLA)-A2+ individuals who received the soluble HBsAg via the i.d. route developed higher peptide-specific cytotoxic CD8+ T cell precursor (CTLp) frequencies. In conclusion, i.d. HBsAg vaccination is more effective even in former i.m. vaccine nonresponders with respect to antibody induction and specific B- and T-cell responses. The induction of virus-specific CTLp may provide the rationale to study the i.d. HBsAg vaccine in the treatment of chronic hepatitis B.

Regulation of the homeostatic balance between cell proliferation and programmed cell death, apoptosis, is essential for development and maintenance of multicellular organisms. Apoptosis is a genetically and evolutionarily highly conserved process. Analysis of the molecular mechanisms of apoptosis has led to a better understanding of many human diseases. Notably in cancer, but also in infectious or autoimmune disease, a deficiency in apoptosis is one of the key events in pathophysiology. On the other hand, overefficient apoptosis, as observed in fulminant liver failure, may be equally harmful for the organism indicating that a tight regulation of the apoptotic machinery is essential for survival. The execution of apoptosis may be initiated by many different signals, either from within or outside the cell involving ligand-receptor interactions, as has been shown for Fas/Fas-ligand, TNF-alpha/TNF-receptor or TGF-beta/TGF-receptor, or potentially by more unspecific signals such as ceramide or DNA damage. During the modulation phase of apoptosis many different genes such as p53, c-myc or Bcl-2/Bax have been shown to able to shift the balance either to cell survival or cell death.

All human hepatitis B viruses characterized so far express three envelope proteins, pre-S1, pre-S2, and HBs, which are believed to function as binding proteins for the cellular receptor, as targets for immune-mediated virus elimination, and in virion morphogenesis and secretion. Here we report the characterization of infectious HBV variant genomes that are unable to express a pre-S2 protein and which were derived from serum of a highly viremic chronic carrier. Direct sequencing of the amplified pre-S region and sequencing of 50 cloned amplified pre-S DNA fragments revealed that in all molecules, in addition to numerous nucleotide changes, there were deletions of the pre-S2 translation initiation codon and three codons 54 nucleotides downstream thereof. No pre-S2 protein and altered pre-S1 proteins were found in the serum of the patient. Cloned infectious HBV DNA genomes having the pre-S region substituted by the variant pre-S region were replication competent in cultured hepatoblastoma cells. Morphologically normal virions were efficiently secreted and were infectious for primary human hepatocyte cultures. These data demonstrate that HBV devoid of pre-S2 protein can occur in vivo as a dominant or exclusive virus population and that expression of the pre-S2 protein is not essential for HBV replication, virion morphogenesis, secretion, or in vitro infectivity.

Background/Aims: Classification of autoimmune hepatitis (AIH) into different subgroups according to autoantibody status has been proposed: type I (ANA/SMA), type II (LKM-1) and type III (anti-SLA). However, whether type III AIH forms a clinically distinct disease entity remains controversial. The aim of this study was to evaluate the subclassification of AIH into ANA/SMA and anti-SLA positive patients with regard to clinical, biochemical and histologic differences. Methods: Ninety-seven consecutive patients with a well-documented long-term course of AIH with ANA/SMA and/or anti-SLA autoantibodies were studied. Clinical, biochemical and histological features of patients with ANA/SMA and/or anti-SLA autoantibodies were compared in a secondary analysis of data acquired prospectively. Results: Anti-SLA autoantibodies were found in 21.6% of patients. Anti-SLA-positive patients tended to have lower transaminases (mean: 153 vs. 247 IU/I), γ-globulins (25 vs. 31%) and bilirubin (1.8 vs. 3.3 mg/dl) in comparison to ANA/SMA positive patients, but there was a large overlap. HLA-type A1 B8 was more frequent in anti-SLA positive patients, while there was no difference in HLA DR3 and DR4 allotype. Response to immunosuppressive therapy was excellent, but relapse occurred frequently. Diagnosis of anti-SLA positive AIH was often delayed (mean: 68 months from first elevation of transaminases) since testing for anti-SLA autoantibodies is currently not generally available. Conclusions: ANA/SMA and anti-SLA positive patients share most clinical, biochemical, histologic and prognostic features. Distinction between type I and type III AIH is therefore clinically not helpful. However, testing for anti-SLA autoantibodies helps in the diagnosis of AIH in many patients who may otherwise be misdiagnosed.

The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, we evaluated tumour tissues from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription and compared our findings with the clinical data from each of these patients. We found that Hugl-1 was lost in 75% of tumour samples and these losses were associated with advanced stage and particularly with lymph node metastases. Reduced Hugl-1 expression during the adenoma-carcinoma sequence occurring as early as in colorectal adenomas was detected by both immunohistochemical and reverse transcription-polymerase chain reaction analysis. Functional assays with ecdysone-inducible cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Our studies thus indicate that downregulation of Hugl-1 contributes to CRC progression.

Recent studies have shown that IL-18, a pleiotropic cytokine that augments IFN-gamma production, is produced by intestinal epithelial cells and lamina propria cells from patients with Crohn's disease. In this study, we show that IL-18 is strongly expressed by intestinal epithelial cells in a murine model of Crohn's disease induced by transfer of CD62L+ CD4+ T cells into SCID mice. To specifically down-regulate IL-18 expression in this model, we constructed an E1/E3-deleted adenovirus expressing IL-18 antisense mRNA, denoted Ad-asIL-18, and demonstrated the capacity of such a vector to down-regulate IL-18 expression in colon-derived DLD-1 cells and RAW264.7 macrophages. Local administration of the Ad-asIL-18 vector to SCID mice with established colitis led to transduction of epithelial cells and caused a significant suppression of colitis activity, as assessed by a newly developed endoscopic analysis system for colitis. Furthermore, treatment with Ad-asIL-18 induced a significant suppression of histologic colitis activity and caused suppression of mucosal IFN-gamma production, whereas IFN-gamma production by spleen T cells was unaffected. Taken together, these data indicate an important role for IL-18 in the effector phase of a T cell-dependent murine model of colitis and suggest that strategies targeting IL-18 expression may be used for the treatment of patients with Crohn's disease.

Atherogenic lipoproteins cause injury to the vascular wall in the early phase of atherogenesis. We assessed the effects of native (nLDL) and oxidized (oxLDL) low-density lipoprotein (LDL) and lipoprotein (a) [Lp(a)] on O2- formation and cell death in cultured human umbilical vein endothelial cells (HUVECs) and rabbit aorta (RA).O2- formation of HUVECs and RA segments was not influenced by nLDL, but was dose dependently increased by oxLDL and was moderately increased by nLp(a). oxLp(a) was the most potent stimulus for O2- formation, increasing it in HUVECs by 356% at 5 micrograms/ml and in RA by 294% at 100 micrograms/ml. Apoptosis was detected by DNA fragmentation and Annexin assay in HUVECs and by TUNEL staining in RA. Incubation of HUVECs and RA with oxLDL, but not nLDL, dose and time dependently induced apoptosis with only a minimal effect on necrosis. nLp(a) elicited a small but significant effect on apoptosis, whereas oxLp(a) induced apoptosis more potently than oxLDL in HUVECs and RA and caused necrotic cell death in HUVECs. Induction of apoptosis by oxLDL and oxLp(a) in RA was enhanced by the superoxide dismutase (SOD) inhibitor, diethyl-dithio-carbamate, and was blunted by SOD and catalase in HUVECs and RA, suggesting that O2- formation was involved. The concentration of lysophosphatidylcholine, a lipoprotein oxidation product and stimulus for O2- formation, was significantly enhanced by factor 5 in oxLDL and by factor 7 in oxLp(a) compared with native lipoproteins.Atherogenic lipoproteins stimulate O2- formation and induction of apoptosis in HUVECs and RA, and may thereby influence the pathogenesis of atherosclerosis.

IL-12 p40-related cytokines such as IL-12 p35/p40 heterodimer and IL-23 (p19/p40) are potent regulators of adaptive immune responses. Little is known, however, about the transcriptional regulation of the p40 gene in vivo. In an attempt toward this goal, we have generated transgenic mice expressing firefly luciferase under the control of the IL-12 p40 promoter. High constitutive transgene expression was found in the small intestine only, whereas little reporter gene activity was observed in other tissues. Within the small bowel, constitutive promoter activity was restricted to the terminal ileum and associated with high expression of p40 mRNA as well as p40 and IL-23 p19/p40 proteins. The cells constitutively producing IL-12 p40 were identified as CD8alpha and CD11b double-negative CD11c+ lamina propria dendritic cells (LPDCs) that represent a major cell population in the lamina propria of the small intestine, but not in the colon. FISH directly demonstrated the uptake of bacteria by a subset of LPDCs in the terminal ileum that was associated with p40 expression. Furthermore, little or no p40 protein expression in LPDCs was found in the terminal ileum of germfree mice, indicating a key role of the intestinal flora for constitutive p40 expression. In addition, analysis of transgenic mice with a mutated NF-kappaB target site in the p40 promoter showed a critical role of NF-kappaB for constitutive transgene expression. Our data reveal important functional differences between the mucosal immune systems of the small and large bowel in healthy mice and suggest that the high bacterial load in the terminal ileum activates p40 gene transcription in LPDCs through NF-kappaB. These data suggest a predisposition of the terminal ileum to develop chronic inflammatory responses through IL-23 and thus may provide a molecular explanation for the preferential clinical manifestation of Crohn disease in this part of the gut.

The CD95 receptor is a death receptor capable of transducing apoptotic cell death upon ligation with the CD95 ligand (CD95L). The CD95/CD95L system plays a physiological role in apoptosis of lymphocytes and liver cells. In addition, the striking finding of acute hepatic failure in mice upon CD95 triggering has stimulated general interest in the involvement of CD95 mediated apoptosis in human liver disease. The currently available data point to a deregulated CD95 system in viral hepatitis, alcoholic hepatitis, acute hepatic failure of different etiology, diseases of the bile ducts, and hepatocellular carcinoma. Animal experiments suggest a causative relationship between CD95 activation and liver cell death, which, however, still has to be proven for liver disease in man. This review summarizes our present knowledge on CD95 mediated human liver disease.

Studies on the interaction of hepatitis B virus (HBV) with its host cell require a suitable tissue culture system. This study used primary adult hepatocytes from healthy human liver tissue to establish productive infection in vitro.Hepatocytes were inoculated overnight with HBV. Production of viral proteins was assessed by radioimmunoassay and by [35S]methionine labeling, and production of viral DNA was assessed by Southern blotting and endogenous polymerase assay.Secretion of high levels of hepatitis B surface antigen (HBsAg) and low levels of hepatitis B virus e antigen (HBeAg) into the medium was detectable 6 days after infection and reached maximum values after 12 days. Metabolic labeling showed production of viral proteins to be a result of de novo synthesis. The appearance of single-stranded HBV DNA in the cytoplasm of infected cells, typically present in immature cores, indicated viral replication. HBV DNA containing particles possessing an active viral DNA polymerase could be immunoprecipitated from the medium 12 days after infection. An antiserum specific for the preS1 region of the viral envelope was capable to block infection. Presence of dimethyl sulfoxide in the medium greatly improved the yield of viral proteins.Primary adult human liver cells are competent for infection with HBV.

Drosophila lethal giant larvae: (lgl), discs large (dlg) and scribble (scrib) are tumour suppressor genes acting in a common pathway, whose loss of function leads to disruption of cell polarity and tissue architecture, uncontrolled proliferation and growth of neoplastic lesions. Mammalian homologues of these genes are highly conserved and evidence is emerging concerning their role in cell proliferation control and tumorigenesis in humans. Here we investigate the functional conservation between Drosophila lethal giant larvae and its human homologue Hugl-1(Llgl1). We first show that Hugl-1 is lost in human solid malignancies, supporting its role as a tumour suppressor in humans. Hugl-1 expression in homozygous lgl Drosophila mutants is able to rescue larval lethality; imaginal tissues do not show any neoplastic features, with Dlg and Scrib exhibiting the correct localization; animals undergo a complete metamorphosis and hatch as viable adults. These data demonstrate that Hugl-1 can act as a tumour suppressor in Drosophila and thus is the functional homologue of lgl. Furthermore, our data suggest that the genetic pathway including the tumour suppressors lgl, dlg and scrib may be conserved in mammals, since human scrib and mammalian dlg can also rescue their respective Drosophila mutations. Our results highlight the usefulness of fruit fly as a model system for investigating in vivo the mechanisms linking loss of cell polarity and cell proliferation control in human cancers.