Bruno Turlin

A New Mouse Liver-specific Gene, Encoding a Protein Homologous to Human Antimicrobial Peptide Hepcidin, Is Overexpressed during Iron Overload

Considering that the development of hepatic lesions related to iron overload diseases might be a result of abnormally expressed hepatic genes, we searched for new genes up-regulated under the condition of iron excess. By suppressive subtractive hybridization performed between livers from carbonyl iron-overloaded and control mice, we isolated a 225-base pair cDNA. By Northern blot analysis, the corresponding mRNA was confirmed to be overexpressed in livers of experimentally (carbonyl iron and iron-dextran-treated mice) and spontaneously (β2-microglobulin knockout mice) iron-overloaded mice. In addition, β2-microglobulin knockout mice fed with a low iron content diet exhibited a decrease of hepatic mRNA expression. The murine full-length cDNA was isolated and was found to encode an 83-amino acid protein presenting a strong homology in its C-terminal region to the human antimicrobial peptide hepcidin. In addition, we cloned the corresponding rat and human orthologue cDNAs. Both mouse and human genes named HEPC are constituted of 3 exons and 2 introns and are located on chromosome 7 and 19, respectively, in close proximity toUSF2 gene. In mouse and human, HEPC mRNA was predominantly expressed in the liver. During both in vivo and in vitro studies, HEPC mRNA expression was enhanced in mouse hepatocytes under the effect of lipopolysaccharide. Finally, to analyze the intracellular localization of the predicted protein, we used the green fluorescent protein chimera expression vectors. The murine green fluorescent protein-prohepcidin protein was exclusively localized in the nucleus. When the putative nuclear localization signal was deleted, the resulting protein was addressed to the cytoplasm. Taken together, our data strongly suggest that the product of the new liver-specific gene HEPCmight play a specific role during iron overload and exhibit additional functions distinct from its antimicrobial activity.

Insulin resistance–associated hepatic iron overload

BACKGROUND & AIMS Hepatic iron overload has been reported in various metabolic conditions, including the insulin-resistance syndrome (IRS) and nonalcoholic steatohepatitis (NASH). The aim of this study was to show that such hepatic iron overload is part of a unique and unrecognized entity. METHODS A total of 161 non-C282Y-homozygous patients with unexplained hepatic iron overload were included. We determined the age; sex; presence of IRS (1 or more of the following: body mass index of >25, diabetes, or hyperlipidemia); serum iron tests and liver iron concentration (LIC; reference value, <36 micromol/g); liver function test results; C282Y and H63D HFE mutations; and liver histological status. RESULTS Patients were predominantly male and middle-aged. Most (94%) had IRS. Transferrin saturation was increased in 35% (median, 42%; range, 13%-94%). LIC ranged from 38 to 332 micromol/g (median, 90 micromol/g), and LIC/age ratio ranged from 0.5 to 4.8 (median, 1.8). Allelic frequencies of both HFE mutations were significantly increased compared with values in normal controls (C282Y, 20% vs. 9%; H63D, 30% vs. 17%), only because of a higher prevalence of compound heterozygotes. Patients with no HFE mutations had similar degrees of iron overload as those with other genotypes, except for compound heterozygotes, who had slightly more iron burden. Steatosis was present in 25% of patients and NASH in 27%. Portal fibrosis (grades 0-3) was present in 62% of patients (grade 2 or 3 in 12%) in association with steatosis, inflammation, and increased age. Sex ratio, IRS, transferrin saturation, and LIC did not vary with liver damage. Serum ferritin concentration, liver function test results, and fibrosis grade were more elevated in patients with steatosis and NASH than in others, but LIC and allelic frequencies of HFE mutations were similar. CONCLUSIONS This study shows that patients with unexplained hepatic iron overload are characterized by a mild to moderate iron burden and the nearly constant association of an IRS irrespective of liver damage.

Noninvasive prediction of fibrosis in C282Y homozygous hemochromatosis

BACKGROUND & AIMS The diagnosis of hemochromatosis is now possible for C282Y homozygous patients using noninvasive molecular genetic tests. The aim of this study was to define noninvasive factors predictive of severe fibrosis (bridging fibrosis or cirrhosis) to avoid unnecessary liver biopsies in such patients. METHODS Clinical and biological data were recorded at the time of diagnosis in 197 French C282Y homozygous patients, 52 (26%) of whom had severe fibrosis. Variables significantly linked to severe fibrosis using univariate analysis were entered into a multivariate stepwise analysis. These variables were combined to obtain a simple index allowing for prediction of severe fibrosis. RESULTS Serum ferritin, hepatomegaly, and serum aspartate aminotransferase were selected using multivariate analysis. Their combination applied to the 96 patients with ferritin level of </=1000 microgram/L, normal aspartate aminotransferase values, and absence of hepatomegaly showed that no severe fibrosis was encountered in this subgroup of patients. The results were validated in 113 C282Y homozygous patients in Canada with a good reproducibility of negative prediction but a poor reproducibility of the positive prediction of severe fibrosis. CONCLUSIONS In C282Y homozygous patients, the diagnosis of severe fibrosis relies on liver biopsy, but absence of severe fibrosis can be accurately predicted in most patients on the basis of simple clinical and biochemical variables.

Transdifferentiation of hepatocyte‐like cells from the human hepatoma HepaRG cell line through bipotent progenitor

Hepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte‐like and biliary‐like cells at confluence. If hepatocyte‐like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21CIP1 and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of β‐catenin localization and HNF3β expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. Conclusion: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors. (HEPATOLOGY 2007;45:957–967.)

Iron and hepatocellular carcinoma

Abstract The high prevalence of hepatocellular carcinoma (HCC) in genetic hemochromatosis (GH) and the association between increased body iron stores and occurrence of HCC in subjects with iron overload unrelated to GH, and the experimental evidence of a co‐carcinogenic role of iron strongly support that iron is involved in the development of HCC.

Primary liver cancer in genetic hemochromatosis: A clinical, pathological, and pathogenetic study of 54 cases

BACKGROUND Although liver cancer arises frequently in the course of genetic hemochromatosis (GH), it has not been previously studied in a large series of patients with well-defined GH. METHODS The bioclinical and pathological data from 1 cholangiocarcinoma and 53 hepatocellular carcinomas (HCCs) complicating GH in 32 untreated and 22 de-ironed patients are reported. RESULTS This study (1) adds three new well-documented cases of HCC in noncirrhotic but only fibrotic hemochromatotic liver, (2) shows the high prevalence (83%) of proliferative and often dysplastic (70%) iron-free foci in the nontumorous liver of untreated patients, and (3) emphasizes the significant increase of cirrhosis (81% vs. 28%) and of associated noniron-related risk factors, mainly chronic alcoholism (48% vs. 25%) and tobacco smoking (50% vs. 18%) in patients with HCC compared with matched hemochromatotic patients without HCC. CONCLUSIONS These data (1) suggest that iron-free foci may be markers of an early stage of HCC in GH and (2) supply the basis for defining a cost-effective policy for the screening of HCC in GH patients.

Reversibility of hepatic fibrosis in treated genetic hemochromatosis: A study of 36 cases

The current study was undertaken to assess whether fibrosis could regress under venesection therapy in patients with C282Y homozygous genetic hemochromatosis. The 36 patients studied were recruited from a subfile of our database consisting of 125 C282Y homozygotes with either severe fibrosis or cirrhosis (F3 or F4 fibrosis stage, respectively, according to the METAVIR grading system). The second liver biopsy was performed for management of liver cancer, extrahepatic surgery, or assessment of liver fibrosis. All paired biopsies were reviewed by two pathologists without knowledge of clinical data. Among the 13 patients who had F3 fibrosis on their initial liver biopsy, 3 had F0, 6 had F1, and 2 had F2 on their second liver biopsy. Among the 23 patients with cirrhosis on their initial liver biopsy, 1 had F0, 4 had F1, 3 had F2, and 2 had F3 on their second liver biopsy. When defining regression of fibrosis as a decrease of at least 2 METAVIR units, fibrosis regressed in 9 of 13 (69%) F3 and in 8 of 23 (35%) F4. When the ratio of gammaglobulins (g/L) to (platelets [n/mm3] × prothrombin activity [%]) was greater than 7.5, fibrosis never regressed. In conclusion, these data extend the concept of regression of fibrosis to patients with treated genetic hemochromatosis and suggest that some simple biochemical tests would be predictive of further regression of fibrosis as a result of venesection therapy. If confirmed on larger series, this could modify the ultrasound screening policy of hepatocellular carcinoma in genetic hemochromatosis. (HEPATOLOGY 2006;44:472–477.)

Liver fibrosis in genetic hemochromatosis: Respective roles of iron and non-iron-related factors in 127 homozygous patients

A retrospective study of 127 patients with untreated homozygous genetic hemochromatosis (HGH) was conducted to evaluate the respective roles of iron overload and non-iron-related factors in the development of hepatic fibrosis in HGH. Twenty-seven percent of the patients had cirrhosis, 21% had liver fibrosis and 52% had no fibrosis (prefibrotic group). The mean value of liver iron concentration was increased significantly (p < 0.001) in cirrhotic (378 +/- 144 mumol/g dry wt.) and in fibrotic (331 +/- 168) subjects compared to prefibrotic (237 +/- 108) patients. Of 13 patients with liver iron concentration > or = 500, 12 had liver fibrosis or cirrhosis, versus 48/134 with liver iron concentration < 500. Chronic alcoholic men exhibited hepatic fibrosis or cirrhosis more frequently than non-alcoholic men (p < 0.001). Non-alcoholic men had hepatic fibrosis or cirrhosis more often than non-alcoholic women (p < 0.05). Cirrhotic and fibrotic patients were significantly older than prefibrotic patients whilst a significant correlation between age and liver iron concentration was found in younger patients only. These results suggest that the iron overload threshold necessary to induce fibrosis is modulated by non-iron-related factors such as alcoholism, sex and age. The development of fibrosis in HGH with liver iron concentration < 500 mumol/g is frequent and must lead to a search for associated non-iron-related fibrogenic factors.

Histologic Features of the Liver in Insulin Resistance-Associated Iron Overload A Study of 139 Patients

The aim of the present study was to describe histologic features of the liver in insulin resistance-associated hepatic iron overload (IR-HIO), defined as the association of metabolic disorders and hepatic iron overload. We included 139 patients in the study on the basis of one or more metabolic disorders and liver iron overload unrelated to usual causes. Liver biopsy specimens were reviewed, and histologic data were compared with those of a previously published, well-defined population with genetic hemochromatosis. Iron overload was characterized by a mixed pattern with iron deposits in hepatocytes and sinusoidal cells. Steatosis was present in 59.7% of patients with inflammation in 32.4% of cases. Periportal fibrosis was found in 67.4% of patients. These patients were older, had higher sinusoidal iron scores, and had a higher prevalence of steatosis and inflammation than patients without fibrosis. Iron overload in IR-HIO was histologically different from that in genetic hemochromatosis.

Value of immunohistochemical Ki-67 and p53 determinations as predictive factors of outcome in renal cell carcinoma☆

OBJECTIVES Nuclear grade and tumor stage are important prognostic factors in renal cell carcinoma, but tumors of similar stage and grade can exhibit a wide variation in biologic behavior and clinical outcome. In this retrospective study, we evaluated the immunologic markers, Ki-67 (MIB1) and p53, in 73 cases of conventional (clear cell) renal cell carcinoma and compared these markers with the accepted prognostic features of grade, stage, and tumor size in predicting outcome. METHODS Specimens of 73 renal cell carcinomas of different nuclear grade (20 Furhman I/II, 32 Fuhrman III, and 21 Fuhrman IV) and different stage (10 pT1, 23 pT2, 36 pT3, and 4 pT4) were immunostained with monoclonal antibodies against Ki-67 and p53. RESULTS Univariate statistical analysis showed that tumor size (P <0. 001), nuclear grade (P <0.01), tumor stage (P <0.01), Ki-67 index (P <0.001), and p53 immunostaining (P <0.03) correlated significantly with a poor prognosis. A Ki-67 index of 20% was a powerful predictor of survival in all patients (P <0.00001), with strong predictive values. On multivariate analysis, the Ki-67 index and metastases were significant independent prognostic factors (P <0.02 and <0.01, respectively). CONCLUSIONS Ki-67 immunostaining appeared to be an additional prognostic indicator of biologic aggressiveness in renal cell carcinoma. Immunohistochemical assessment of tumor antigens could be used to identify patients at high risk of tumor progression in addition to conventional prognostic factors.