H. P. Dienes

Simplified criteria for the diagnosis of autoimmune hepatitis

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 ≥6) and 81% sensitivity and 99% specificity (cutoff ≥7) in the validation set. Conclusion: 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. (HEPATOLOGY 2008.)

MicroRNA Gene Expression Profile of Hepatitis C Virus-Associated Hepatocellular Carcinoma

MicroRNAs are small noncoding RNAs that regulate gene expression by targeting messenger RNAs (mRNAs) through translational repression or RNA degradation. Many fundamental biological processes are modulated by microRNAs, and an important role for microRNAs in carcinogenesis is emerging. Because understanding the pathogenesis of viral‐associated hepatocellular carcinomas is important in developing effective means of classification, prognosis, and therapy, we examined the microRNA expression profiles in a large set of 52 human primary liver tumors consisting of premalignant dysplastic liver nodules and hepatocellular carcinomas by quantitative real‐time polymerase chain reaction. All patients were infected with hepatitis C, and most had liver cirrhosis. Initially, the accessibility of microRNAs from formalin‐fixed paraffin‐embedded archival liver tissue by real‐time polymerase chain reaction assays was shown. Subsequently, target parenchyma from routinely processed tissue was macrodissected, RNA was extracted, and reverse transcription followed by quantitative real‐time polymerase chain reaction was performed. Relative quantification was performed by the 2−ΔΔCt method with normal livers as a calibrator. In order to obtain a comprehensive microRNA gene expression profile, 80 microRNAs were examined in a subset of tumors, which yielded 10 up‐regulated and 19 down‐regulated microRNAs compared to normal liver. Subsequently, five microRNAs (miR‐122, miR‐100, miR‐10a, miR‐198, and miR‐145) were selected on the basis of the initial results and further examined in an extended tumor sample set of 43 hepatocellular carcinomas and 9 dysplastic nodules. miR‐122, miR‐100, and miR‐10a were overexpressed whereas miR‐198 and miR‐145 were up to 5‐fold down‐regulated in hepatic tumors compared to normal liver parenchyma. Conclusion: A subset of microRNAs are aberrantly expressed in primary liver tumors, serving both as putative tumor suppressors and as oncogenic regulators. (HEPATOLOGY 2008.)

Expression of the gene of the α-smooth muscle-actin isoform in rat liver and in rat fat-storing (ITO) cells

SummaryFat storing cells (FSCs) in the liver represent the main site of vitamin A deposition in the body. These cells are considered to play an important role during scar formation and fibrogenesis in the liver. The putative descent of FSCs from the fibroblastic or from the myofibroblastic system have not been determined yet by morphological or immunohistochemical studies. To further define the origin of these liver cells, we analysed the pattern of expression of three structural proteins: vimentin, desmin and the α-smooth muscle (SM)-actin isoform in FSCs of the rat liver, in smooth muscle cells (SMCs) from the aorta and in rat skin fibroblasts. FSCs were studied by immunohistochemical methods immediately after isolation, at days 3 and 7 after plating. FSC-geneexpression was also analysed by Northern blot analysis of total RNA extracted from cells in culture at days 3 and 7 after isolation. Arterial SMCs and skin fibroblasts were studied in a similar way. For comparison, isolated rat hepatocytes and Küpffer cells (Kc) were studied. Of freshly isolated FSCs, 100% were vimentin-positive, 50% were desmin-positive, but all were α-SM-actin negative. Three days after isolation, FSCs were clearly positive for vimentin and desmin and weakly α-SM-actinpositive, as demonstrated by indirect immunofluorescence as well as by the immunoperoxidase technique. Desmin, α-SM-actin and vimentin staining was further increased at day 7 after isolation, and α-actin specific transcripts in FSC-RNA were clearly detectable at day 7 after isolation. Passaged arterial SMCs were vimentin-and α-SM-actin-positive, but desmin-negative and fibroblasts were only vimentin-positive. α-SM-actin isoform gene expression was strongly increased in the rat liver after acute or chronic damage induced by treatment with carbon tetrachloride. Positive staining was detected in cells which were also desmin-positive. As FSCs are vimentin-, desmin-and α-SM-actin-positive, we suggest that they are smooth muscle-related myo-fibroblasts with special functions.

Atorvastatin attenuates hepatic fibrosis in rats after bile duct ligation via decreased turnover of hepatic stellate cells

BACKGROUND & AIMS Activation of hepatic stellate cells (HSC) and transdifferentiation to myofibroblasts following liver injury is the main culprit for hepatic fibrosis. Myofibroblasts show increased proliferation, migration, contraction, and production of extracellular matrix (ECM). In vitro, HMG-CoA reductase inhibitors (statins) inhibit proliferation and induce apoptosis of myofibroblastic HSC. To investigate the antifibrotic effects of atorvastatin in vivo we used bile duct ligated rats (BDL). METHODS BDL rats were treated with atorvastatin (15 mg/kg/d) immediately after ligation (prophylactically) or in on-going fibrosis (therapeutically). Fibrosis was assessed by hydroxyproline content and Sirius-red staining. The activation of HSC was investigated by analysis of alphaSMA expression. mRNA levels of cytokines and procollagen were analyzed by RT-PCR, and MMP-2 activity by zymography. Proliferation was assessed by expression of cathepsins (B and D), proliferating cell nuclear antigen (PCNA), and Ki67-staining. Apoptosis was characterized by caspase-3 activity, cleavage of PARP-1, and TUNEL assay. Hepatic inflammation was investigated by serum parameters and liver histology. RESULTS Prophylactic and early therapy with atorvastatin significantly attenuated fibrosis and HSC activation. Later therapy lacked significant effects on fibrosis but reduced profibrotic cytokine expression and led to a more quiescent state of HSC with less proliferation and apoptosis, while hepatic inflammation did not change. CONCLUSIONS This study shows that very early atorvastatin treatment inhibits HSC activation and fibrosis in the BDL model in vivo, while late treatment reduces HSC turnover and activity. Our findings underline that long-term studies in humans are warranted.

Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue levels of transforming growth factor-beta.

Although many patients with chronic viral hepatitis C infection suffer from progressive liver disease, the rate of fibrosis progression is highly variable and some patients do not show any measurable progression. However, our ability to predict which patients progress is very limited. Since transforming growth factor‐β (TGF‐β) is a key mediator of liver fibrogenesis, we assessed the predictive role of TGF‐β for fibrogenesis in chronic hepatitis C. We studied 39 patients with chronic hepatitis C in whom two liver biopsies were taken at least 12 months apart, and who did not receive therapy during this period. TGF‐β was measured by bioassay and by ELISA in serum samples taken at the time of the first biopsies, and TGF‐β was determined semiquantitatively by immunostaining of liver biopsy sections. Fibrosis was scored blinded in the biopsy samples by two pathologists independently. There was a close correlation between TGF‐β serum levels and the rate of fibrosis progression. Patients with no progression of fibrosis had significantly lower (59 ng/mL ± 22) TGF‐β serum levels than patients with progressive disease (115 ng/mL ± 20), and a TGF‐β level below 75 ng/mL was predictive for stable disease. Immunohistology for TGF‐β in biopsy samples was also predictive for progressive liver disease with fibrosis progression found in those patients displaying staining of hepatocytes and sinusoidal cells. No such correlation was found with other markers such as procollagen III peptide, viral load or transaminase levels. These results further support the role of TGF‐β in liver fibrogenesis, and offer an opportunity to predict clinical disease progression, which may help in selecting patients who are in need of therapeutic interventions.

Beta-catenin accumulation in the progression of human hepatocarcinogenesis correlates with loss of E-cadherin and accumulation of p53, but not with expression of conventional WNT-1 target genes

Beta‐catenin integrates intracellular WNT signalling and the intercellular E‐cadherin–catenin adhesion system. To date, little is known about the role of β‐catenin activation and nuclear accumulation in hepatocarcinogenesis. This study has analysed β‐catenin expression patterns in human dysplastic nodules (DNs), as well as in hepatocellular carcinomas (HCCs) in comparison with proliferation, expression of WNT‐1 target genes, E‐cadherin, and p53. One hundred and seventy HCCs and 25 DNs were categorized according to established criteria and analysed for the expression pattern of β‐catenin. Analysis of the proliferative activity and expression of E‐cadherin, cyclin D1, MMP‐7, c‐myc, and p53 was performed on a representative subgroup of cases. All DNs lacked nuclear β‐catenin, while 36% of all HCCs were positive, with the number of nuclear stained cells ranging from less than 1% to more than 90%. Increasing nuclear accumulation of β‐catenin correlated with reduced membranous E‐cadherin expression and nuclear p53 but not with proliferation. Cyclin D1, MMP‐7, and c‐myc expression was detected in 54%, 26%, and 65% of HCCs, respectively, but did not correlate with nuclear β‐catenin, proliferation, or grading. Sequence analysis of the β‐catenin gene revealed no detectable mutations in DNs, but mutations in the GSK‐3β binding site were present in 14.3% of the HCCs. In conclusion, this study has demonstrated that nuclear accumulation of β‐catenin is a frequent progression event in human hepatocarcinogenesis which correlates with nuclear p53 accumulation and loss of membranous E‐cadherin, but not with the expression pattern of established WNT‐1 target genes. It is hypothesized that the role of β‐catenin in human HCC differs significantly from its established function in colon carcinogenesis. Copyright

Hepatic and serum levels of miR-122 after chronic HCV-induced fibrosis

BACKGROUND & AIMS The progression of liver fibrosis in patients with chronic hepatitis C (CHC) is important to decide on the treatment of the virus. As liver biopsy and liver stiffness measurement for staging of fibrosis present limitations, circulating levels of miR-122 have been suggested as a novel biomarker to predict the extent of liver injury. We evaluated the potential of miR-122 as an indicator of fibrosis progression in CHC infection and performed, for the first time, a comprehensive analysis of hepatic and circulating miR-122 levels in patients with CHC. METHODS Patients with well-documented CHC infection were selected from the database of HepNet, the German-Competence-Network on Viral Hepatitis. All patients underwent blood sampling and liver biopsy with grading of inflammation and staging of fibrosis. RNA was extracted from 84 liver biopsies and 164 serum samples of CHC patients. miR-122 levels in liver and serum samples were quantified by real-time PCR normalized to RNU6 or spiked-in RNA, respectively. RESULTS Hepatic levels of miR-122 decreased significantly with the severity of fibrosis (p = 0.001). In addition, circulating miR-122 levels correlated negatively with increasing stages of fibrosis, although the inverse correlation was moderate due to a two-phase miR-122 pattern during fibrosis progression. Thus, circulating miR-122 levels decreased in patients with severe fibrosis (F3, F4), while at early stages with distinct fibrotic structures (F2) and high inflammatory activity, miR-122 serum levels were elevated. CONCLUSIONS We conclude that during progression of fibrosis less miR-122 is released into the blood stream due to the loss of liver cells and the decrease of hepatic miR-122 levels. Although the release of circulating miR-122 possibly mirrors acute liver injury, in chronic liver disease and fibrosis, the loss of liver cells and the decreased hepatocellular miR-122 expression render miR-122 an inappropriate marker, when exclusively used for interpretation of fibrosis progression.

Expression of MMP-2 is associated with progression and lymph node metastasis of gastric carcinoma.

Expression of MMP‐2 is associated with progression and lymph node metastasis of gastric carcinoma

Hepatic granulomas: histological and molecular pathological approach to differential diagnosis–a study of 442 cases

Background/Aims: The incidence of hepatic granulomas is reported in 2–15% of liver biopsies. This study was carried out to evaluate the incidence and aetiology of hepatic granulomas in a German Institute of Pathology with specialization in liver diseases.

Fat storing cells (FSC) of rat liver synthesize and secrete fibronectin: Comparison with hepatocytes

Fat storing cells (FSC) of rat liver were isolated and kept in culture for up to 2 weeks. Freshly isolated cells and cells in culture were characterized functionally (vitamin A content) and morphologically. Their synthetic capacity for fibronectin was studied quantitatively by ELISA-measurement and qualitatively by biosynthetic labeling, immunoprecipitation and SDS-PAGE analysis. The synthesis product and the kinetics of the synthesis and secretion were compared with those obtained from culture of hepatocytes. FSC were shown to synthesize and secrete fibronectin under the culture conditions used. No fibronectin synthesis was detected during the first 3 days of culture. Thereafter the synthesis increased continuously up to 2600 ng/24 h/100 micrograms cell protein. The molecular weight of fibronectin synthesized by fat storing cells seems to be the same as the one synthesized by hepatocytes, but the secretion proceeds faster in hepatocytes than in FSC. No differences between the two molecules were observed when both cell populations were treated with tunicamycin (TM), an antibiotic which inhibits the N-glycosylation of the primary translation product.