Iron as a therapeutic target in chronic liver disease

It was clearly realized more than 50 years ago that iron deposition in the liver may be a critical factor in the development and progression of liver disease.The recent clarification of ferroptosis as a specific form of regulated hepatocyte death different from apoptosis and the description of ferritinophagy as a specific variation of autophagy prompted detailed investigations on the association of iron and the liver.In this review,we will present a brief discussion of iron absorption and handling by the liver with emphasis on the role of liver macrophages and the significance of the iron regulators hepcidin,transferrin,and ferritin in iron homeostasis.The regulation of ferroptosis by endogenous and exogenous modulators will be examined.Furthermore,the involvement of iron and ferroptosis in various liver diseases including alcoholic and non-alcoholic liver disease,chronic hepatitis B and C,liver fibrosis,and hepatocellular carcinoma(HCC)will be analyzed.Finally,experimental and clinical results following interventions to reduce iron deposition and the promising manipulation of ferroptosis will be presented.Most liver diseases will be benefited by ferroptosis inhibition using exogenous inhibitors with the notable exception of HCC,where induction of ferroptosis is the desired effect.Current evidence mostly stems from in vitro and in vivo experimental studies and the need for well-designed future clinical trials is warranted.

Autophagy in liver diseases

Autophagy is the liver cell energy recycling system regulating a variety of homeostatic mechanisms.Damaged organelles,lipids and proteins are degraded in the lysosomes and their elements are re-used by the cell.Investigations on autophagy have led to the award of two Nobel Prizes and a health of important reports.In this review we describe the fundamental functions of autophagy in the liver including new data on the regulation of autophagy.Moreover we emphasize the fact that autophagy acts like a two edge sword in many occasions with the most prominent paradigm being its involvement in the initiation and progress of hepatocellular carcinoma.We also focused to the implication of autophagy and its specialized forms of lipophagy and mitophagy in the pathogenesis of various liver diseases.We analyzed autophagy not only in well studied diseases,like alcoholic and nonalcoholic fatty liver and liver fibrosis but also in viral hepatitis,biliary diseases,autoimmune hepatitis and rare diseases including inherited metabolic diseases and also acetaminophene hepatotoxicity.We also stressed the different consequences that activation or impairment of autophagy may have in hepatocytes as opposed to Kupffer cells,sinusoidal endothelial cells or hepatic stellate cells.Finally,we analyzed the limited clinical data compared to the extensive experimental evidence and the possible future therapeutic interventions based on autophagy manipulation.

Presence of disease specific autoantibodies against liver sinusoidal cells in primary biliary cirrhosis

AIM: To investigate the presence of autoantibodies directed against liver sinusoidal cells in primary biliary cirrhosis(PBC).METHODS: Liver biopsies from 21 PBC patients were studied and compared with 12 liver biopsies from disease controls [3 patients with hepatitis B(HBV) virus,3 patients with hepatitis C virus(HCV), 3 patients with non-alcoholic steatohepatitis and 3 patients with acute alcoholic hepatitis(AAH)]. As healthy controls, we used tissue specimens adjacent to metastatic liver adenocarcinoma. Normal serum was taken from staff members of the unit. The determination of the cell type targetedby autoantibodies present in the patients sera was performed by indirect immunofluorescence(IIF) analysis using paraffin-embedded liver sections as a substrate.Sera from homologous or heterologous PBC patients or sera from the disease control group were used as primary antibodies. The presence of autoantibodies was identified using confocal microscopy.RESULTS: In total, 18/21(85.7%) PBC patients exhibited positive staining in the sinusoidal cells, 10/21(47.6%) in lymphocytes, 8/21(38%) in cholangiocytes and 7/21(33.3%) in hepatocytes, when homologous serum and fluorescein isothiocyanate-conjugated immunoglobulin type G(IgG) secondary antibody were used. PBC sections incubated with heterologous PBC serum showed reduced staining(20% for sinusoidal cells, 20% for lymphocytes, 20% for cholangiocytes and 13.3% for hepatocytes). When IgM immunoglobulin, instead of IgG, was used as secondary antibody,positive staining was observed in 75% of lymphocytes,62.5% of cholangiocytes, 37.5% of hepatocytes and50% of the sinusoidal cells with a much stronger staining intensity. No staining was observed when either normal or PBC sera were used as a primary antibody on liver sections from the disease control group. When PBC sera were incubated with healthy control sections,weak positive staining of cholangiocytes was observed in 3/21(14.3%) PBC serum samples. Steatohepatitis serum on PBC sections gave a positive staining of some hepatocytes and lymphocytes but no staining on viral hepatitis sections. Incubation with HBV sera stained some hepatocytes, cholangiocytes and intra-sinusoidal or portal lymphocytes of PBC, HBV and AAH patients but not HCV patients.CONCLUSION: In this study, for the first time in diseased liver tissue, we have demonstrated that a large proportion of PBC patients have disease specific autoantibodies against liver sinusoidal cells.

A novel animal model for in vivo study of liver cancer metastasis

AIM:To establish an animal model with human hepatocyte-repopulated liver for the study of liver cancer metastasis.METHODS:Cell transplantation into mouse livers was conducted using alpha-fetoprotein(AFP)-producing hu-man gastric cancer cells(h-GCCs) and h-hepatocytes as donor cells in a transgenic mouse line expressing urokinase-type plasminogen activator(uPA) driven by the albumin enhancer/promoter crossed with a severe combined immunodeficient(SCID) mouse line(uPA/SCID mice).Host mice were divided into two groups(A and B).Group A mice were transplanted with h-GCCs alone,and group B mice were transplanted with h-GCCs and h-hepatocytes together.The replacement index(RI),which is the ratio of transplanted h-GCCs and h-hepatocytes that occupy the examined area of a histological section,was estimated by measuring h-AFP and h-albumin concentrations in sera,respectively,as well as by immunohistochemical analyses of h-AFP and human cytokeratin 18 in histological sections.RESULTS:The h-GCCs successfully engrafted,repopulated,and colonized the livers of mice in group A(RI = 22.0% ± 2.6%).These mice had moderately differentiated adenocarcinomatous lesions with disrupted glandular structures,which is a characteristics feature of gastric cancers.The serum h-AFP level reached 211.0 ± 142.2 g/mL(range,7.1-324.2 g/mL).In group B mice,the h-GCCs and h-hepatocytes independently engrafted,repopulated the host liver,and developed colonies(RI = 12.0% ± 6.8% and 66.0% ± 12.3%,respectively).h-GCC colonies also showed typical adenocarcinomatous glandular structures around the h-hepatocyte-colonies.These mice survived for the full 56 day-study and did not exhibit any metastasis of h-GCCs in the extrahepatic regions during the observational period.The mice with an h-hepatocyte-repopulated liver possessed metastasized h-GCCs and therefore could be a useful humanized liver animal model for studying liver cancer metastasis in vivo.CONCLUSION:A novel animal model of human liver cancer metastasis was established using the uPA/SCID mouse line.This model could be useful for in vivo testing of anti-cancer drugs and for studying the mechanisms of human liver cancer metastasis.

Peri-nuclear antibodies correlate with survival in Greek primary biliary cirrhosis patients

AIM:To investigate possible associations of anti-nuclear envelope antibody(ANEA)with disease severity and survival in Greek primary biliary cirrhosis(PBC)patients.METHODS:Serum samples were collected at diagnosis from 147 PBC patients(85%female),who were followed-up for a median 89.5 mo(range 1-240).ANEA were detected with indirect immunofluorescence on 1% formaldehyde fixed Hep2 cells,and anti-gp210 antibodies were detected using an enzyme linked immunosorbent assay.Findings were correlated with clinical data,histology,and survival.RESULTS:ANEA were detected in 69/147(46.9%) patients and 31/147(21%)were also anti-gp210 positive.The ANEA positive patients were at a more advanced histological stage(Ⅰ-Ⅱ/Ⅲ-Ⅳ56.5%/43.5% vs 74.4%/25.6%,P=0.005)compared to the ANEA negative ones.They had a higher antimitochondrial antibodies(AMA)titer(≤1:160/>1:160 50.7%/49.3%vs 71.8%/28.2%,P=0.001)and a lower survival time(91.7 ±50.7 mo vs 101.8±55 mo,P=0.043).Moreover,they had more advanced fibrosis,portal inflammation,interface hepatitis,and proliferation of bile ductules(P =0.008,P=0.008,P=0.019,and P=0.027,respectively).They also died more frequently of hepatic failure and/or hepatocellular carcinoma(P=0.016).ANEA positive,anti-gp210 positive patients had a difference in stage(Ⅰ-Ⅱ/Ⅲ-Ⅳ54.8%/45.2%vs 74.4%/25.6%,P= 0.006),AMA titer(≤1:160/>1:160 51.6%/48.4%vs 71.8%/28.2%,P=0.009),survival(91.1±52.9 mo vs 101.8±55 mo,P=0.009),and Mayo risk score(5.5 ±1.9 vs 5.04±1.3,P=0.04)compared to the ANEA negative patients.ANEA positive,anti-gp210 negative patients had a difference in AMA titer(≤1:160/>1:160 50%/50%vs 71.8%/28.2%,P=0.002),stage(Ⅰ-Ⅱ/Ⅲ -Ⅳ57.9%/42.1%vs 74.4%/25.6%,P=0.033),fibrosis(P=0.009),portal inflammation(P=0.018),interface hepatitis(P=0.032),and proliferation of bile ductules(P=0.031).Anti-gp210 positive patients had a worse Mayo risk score(5.5±1.9 vs 4.9±1.7,P=0.038)than the anti-gp210 negative ones.CONCLUSION:The presence of ANEA and anti-gp210 identifies a subgroup of PBC patients with advanced disease severity and poor prognosis.

Octreotide induces caspase activation and apoptosis in human hepatoma HepG2 cells

AIM: To investigate the role of octreotide on cellular proliferation and apoptosis of human hepatoma (HepG2) cells. METHODS: We studied cellular proliferation, apoptosis and the possible internal caspase-mediated apoptosis pathway involved, after treatment of HepG2 carcinoma cells with octreotide in comparison with the apoptosis caused by tumor necrosis factor-α (TNF-α). Activities of caspase-3, caspase-9, caspase-8 and caspase-2 were studied, while apoptosis was investigated through detection of DNA fragmentation and through identification of apoptotic cells with the annexin-V/propidium iodide flow cytometric method. RESULTS: After an initial increase in HepG2 cellular proliferation, a significant inhibition was observed with 10-8 mol/L octreotide, while TNF-α dose-dependently decreased proliferation. Early and late apoptosis was significantly increased with both substances. Octreotide significantly increased caspase-3, caspase-8 and caspase-2 activity. TNF-α signifi cantly increased only caspase-2. Cellular proliferation was decreased after treatment with octreotide or TNF-α alone but, in contrast to TNF-α, octreotide decreased proliferation only at concentrations of 10-8 mol/L, while lower concentrations increased proliferation. CONCLUSION: Our findings are suggestive of caspasemediated signaling pathways of octreotide antitumor activity in HepG2 cells, and indicate that measurements of serum octreotide levels may be important, at least in clinical trials, to verify optimal therapeutic drug concentrations.

Isolated right posterior bile duct injury following cholecystectomy:Report of two cases

Anatomic variations of the right biliary system are one of the most common risk factors for sectoral bile duct injury(BDI)during cholecystectomy.Isolated right posterior BDI may in particular be a challenge for both diagnosis and management.Herein we describe two cases of isolated right posterior sectoral BDI that took place during laparoscopic cholecystectomy.Despite effective external biliary drainage from the liver hilum in both cases,there was a persistent biliary leak observed which was not visible on endoscopic retrogradecholangiogram.Careful evaluation of images from both endoscopic and magnetic resonance cholangiograms revealed the diagnosis of an isolated right posterior sectoral BDI.These were treated with a delayed bisegmental(segments 6 and 7)liver resection and a Roux-en-Y hepaticojejunostomy respectively with good outcomes at 24 and 4 mo of follow-up.This paper discusses strategies for prevention of such injuries along with the diagnostic and therapeutic challenges it offers.

Increased ΤGF-β3 in primary biliary cirrhosis: An abnormality related to pathogenesis?

AIM: To investigate the transforming growth factor-β (TGF-β) isoforms in the peripheral and hepatic venous blood of primary biliary cirrhosis (PBC) patients. METHODS: We examined TGF-β1, TGF-β2 and TGF-β3 (enzyme-linked immunosorbent assay), in 27 stage Ⅳ PBC patients (27 peripheral and 15 hepatic vein sera), 35 early (Ⅰ-Ⅱ) PBC and 60 healthy controls. As disease controls 28 hepatitis C virus (HCV) cirrhosis (28 peripheral and 17 hepatic vein serum), 44 chronic HCV hepatitis and 38 HCV-related hepatocellular carcinomas were included. We also tested liver tissue by immunohistochemistry to identify localization of TGF isoforms. RESULTS: TGF-β1 was significantly decreased in all cirrhotics (PBC Ⅲ-Ⅳ: median 13.4 ng/mL; range, 7.4-26.2, HCV cirrhosis: 11.6 ng/mL; range, 5.0-33.8), compared to controls (30.9 ng/mL; range, 20.9-37.8). TGF-β2 was increased in viral cirrhosis but not in PBC and chronic hepatitis. TGF-β3 (47.2 pg/mL; range, 27.0-79.7 in healthy controls) was increased in early and late PBC (Ⅰ-Ⅱ: 94.3 pg/mL; range, 41.5-358.6; Ⅲ-Ⅳ: 152.8 pg/mL; range, 60.4-361.2; P < 0.001) and decreased in viral cirrhosis (37.4 pg/mL; range, 13.3-84.0; P < 0.05). Hepatic vein TGF-β levels were analogous to those in peripheral blood. Immunohistochemistry identified all isoforms in portal tract lymphocytes, sinusoidal cells and cholangiocytes. TGF-β3 was additionally overexpressed in hepatocytes in PBC patients. CONCLUSION: The serum profile of TGF-β isoforms is different in cirrhotics. Increased TGF-β3 is characteristic of PBC. These findings may be related to the immunological abnormalities of PBC.

Serrated adenoma of the stomach:Case report and literature review

Gastric serrated adenomas are histologically characterized by protruding glands with lateral saw tooth-like indentations lined with stratified dysplastic cells containing abundant eosinophilic cytoplasm.Since the first case of gastric serrated adenoma found in 2001,18 additional cases have been reported.Gastric serrated adenomas have a particular proclivity to progress to invasive carcinoma;75% or 15 of the 20 cases now in record-including the present one-exhibited invasive carcinoma.The 20 th case of gastric serrated adenoma reported here differs from the preceding ones in as much as it evolved in a patient with Lynch syndrome,implying that this adenoma phenotype may develop not only sporadically but also in patients with hereditary traits.

Carcinoma in gut-associated lymphoid tissue in ulcerative colitis: Case report and review of literature

The colorectal mucosa includes two quantitatively, structurally and functionally dissimilar areas: one, built with columnar and goblet cells, covers the vast majority of the mucosa, and the other consists of scattered minute gut-associated lymphoid tissue (GALT). The overwhelming majority of colorectal carcinomas evolve in GALT-free mucosal areas and very rarely in GALT aggregates. Remarkably, the colonic mucosa in patients with ulcerative colitis (UC) displays a high number of newly formed GALT-aggregates. The patient here described is a 68-year-old female with a history of UC since 1984. At surveillance colonoscopy in 2012, one of two detected polyps was a tubular adenoma with high-grade dysplasia. Beneath this adenoma, a well-circumscribed GALT sheltering a carcinoma was found. Serial sections revealed no connection between the villous adenomaand the GALT-carcinoma. The GALT-carcinoma here reported seems to have evolved in a newly formed, UC- dependent, GALT complex. This notion is substantiated by the fact that 27% or 4 out of the 15 cases of GALT- carcinomas in the colon reported in the literature (including the present case) evolved in patients with UC.

增生性结肠息肉综合征和结肠直肠癌

Background:Patients with hyperplastic polyposis coli syndrome(HPCS)have a propensity to develop colorectal carcinoma(CRC).Patients and Methods:Details were retrieved from the files of patients attending our hospital between 1988 and 2004 who fulfilled the World Health Organization criteria for HPCS.Results:Over a period of 16 years,10 cases of HPCS were identified at our hospital(0.625 cases/year or one case every 1.6 years).A mean of 40.3 hyperplastic polyps per patient were found(range 6-159).Other colorectal lesions were found as follows:two patients each had one mixed polyp;there were 15 serrated adenomas in eight patients;and there were 30 tubular,tubulovillous,or villous adenomas in eight patients.Among the 10 patients with HPCS,seven developed a CRC.Of the four villous adenomas,three were associated with a CRC,but only one of the 15 serrated adenomas was associated with a CRC.The pathway of cancer evolution in HPCS patients remains unresolved.Conclusions:Similarly to our results,a review of the literature indicates a high incidence of CRCs in HPCS patients.These patients are at a high risk of developing a CRC and should therefore receive regular colonoscopic surveillance.