Frozen Liver Biopsy Research Articles

Clinical significance of CDT1 mRNA expression in chronic hepatitis C or liver cirrhosis.

We have previously reported that chromatin licensing and DNA replication factor 1 (CDT1) is associated with the postoperative recurrence of hepatocellular carcinoma (HCC). Based on this fact, we verified whether CDT1 mRNA expression is also associated with HCC development from chronic hepatitis C (CHC) and liver cirrhosis (LC). There were 142 cases with CHC or LC who underwent liver biopsy. Detection of CDT1 mRNA in liver was performed by RT-qPCR using frozen liver biopsy tissues. We examined the association between the CDT1 mRNA expression and clinical conditions and long-term outcome. We then examined the association between serum cytokine/chemokine levels and CDT1 mRNA expression in 58 cases. The cumulative incidence rates of HCC development in cases with CDT1 mRNA in the low expression group showed significantly lower than those in the high expression group (p = 0.0391). A significant correlation was found between CDT1 mRNA expression and the extent of proliferation of atypical hepatocytes in hematoxylin and eosin-stained sections (p<0.0001). CDT1 mRNA expression has been associated with cytokines involved in tumorigenesis in experimental and human cancers. We found that cases with high CDT1 mRNA expression were at risk for developing HCC, even if they were CHC or LC.

The comparative performance of a custom Canine NanoString® panel on FFPE and snap frozen liver biopsies

Formalin-Fixed Paraffin Embedded (FFPE) biopsies would provide a critical mass of cases to allow investigation of canine liver disease, however their use is often limited by challenges typically associated with transcriptomic analysis. This study evaluates the capability of NanoString® to measure the expression of a broad panel of genes in FFPE liver samples. RNA was isolated from matched histopathologically normal liver samples using FFPE (n = 6) and snap frozen in liquid nitrogen (n = 6) and measured using a custom NanoString® panel. Out of the 40 targets on the panel, 27 and 23 targets were above threshold for non-diseased snap frozen and FFPE tissue respectively. The binding density and total counts were significantly reduced in the FFPE samples relative to the snap frozen samples (p = 0.005, p = 0.01, respectively), confirming a reduction in sensitivity. The concordance between the snap frozen and FFPE samples was high, with correlations (R) ranging between 0.88 and 0.99 between the paired samples. An additional 14 immune-related targets, undetectable the non-diseased FFPE liver, were above threshold when the technique was applied to a series of diseased samples, further supporting their inclusion on this panel. This use of NanoString® based analysis opens up huge opportunity for retrospective evaluation of gene signatures in larger caseloads through harnessing the capacity of archived FFPE samples This information used alongside clinical and histological data will not only afford a way to explore disease etiopathogenesis, it may also offer insight into sub-types of liver disease in dogs, which cannot be discerned using more traditional diagnostic methods.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B

Background & AimsHBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms.MethodsDNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.ResultsHBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.ConclusionsTargeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.Lay summaryFresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

Characterization of the liver immune microenvironment in liver biopsies from patients with chronic HBV infection

Background & AimsWe aim to describe the liver immune microenvironment by analyzing liver biopsies from patients with chronic HBV infection (CHB). Host immune cell signatures and their corresponding localization were characterized by analyzing the intrahepatic transcriptome in combination with a custom multiplex immunofluorescence panel.MethodMatching FFPE and fresh frozen liver biopsies were collected from immune active patients within the open-label phase IV study GS-US-174-0149. RNA-Seq was conducted on 53 CHB liver biopsies from 46 patients. Twenty-eight of the 53 samples had matched FFPE biopsies and were stained with a 12-plex panel including cell segmentation, immune and viral biomarkers. Corresponding serum samples were screened using the MSD Human V-plex Screen Service to identify peripheral correlates for the immune microenvironment.ResultsUsing unsupervised clustering of the transcriptome, we reveal two unique liver immune signatures classified as immune high and immune low based on the quantification of the liver infiltrate gene signatures. Multiplex immunofluorescence analysis demonstrated large periportal lymphoid aggregates in immune high samples consisting of CD4 and CD8 T cells, B cells and macrophages. Differentiation of the high and low immune microenvironments was independent of HBeAg status and peripheral viral antigen levels. In addition, longitudinal analysis indicates that treatment and normalization of ALT correlates with a decrease in liver immune infiltrate and inflammation. Finally, we screened a panel of peripheral biomarkers and identified ICAM-1 and CXCL10 as biomarkers that strongly correlate with these unique immune microenvironments.ConclusionThese data provide a description of immune phenotypes in patients with CHB and show that immune responses are downregulated in the liver following nucleotide analogue treatment. This may have important implications for both the safety and efficacy of immune modulator programs aimed at HBV cure.Lay summaryLiver biopsies from patients with chronic hepatitis B were submitted to RNA-Seq and multiplex immunofluorescence and identified two different liver immune microenvironments: immune high and immune low. Immune high patients showed elevated immune pathways, including interferon signaling pathways, and increase presence of immune cells. Longitudinal analysis of biopsies from treatment experienced patients showed that treatment correlates with a marked decrease in inflammation and these findings may have important implications for both safety and efficacy of immune modulator programs for HBV cure.

Spatial Systems Lipidomics Reveals Nonalcoholic Fatty Liver Disease Heterogeneity.

Hepatocellular lipid accumulation characterizes nonalcoholic fatty liver disease (NAFLD). However, the types of lipids associated with disease progression are debated, as is the impact of their localization. Traditional lipidomics analysis using liver homogenates or plasma dilutes and averages lipid concentrations, and does not provide spatial information about lipid distribution. We aimed to characterize the distribution of specific lipid species related to NAFLD severity by performing label-free molecular analysis by mass spectrometry imaging (MSI). Fresh frozen liver biopsies from obese subjects undergoing bariatric surgery (n = 23) with various degrees of NAFLD were cryosectioned and analyzed by matrix-assisted laser desorption/ionization (MALDI)-MSI. Molecular identification was verified by tandem MS. Tissue sections were histopathologically stained, annotated according to the Kleiner classification, and coregistered with the MSI data set. Lipid pathway analysis was performed and linked to local proteome networks. Spatially resolved lipid profiles showed pronounced differences between nonsteatotic and steatotic tissues. Lipid identification and network analyses revealed phosphatidylinositols and arachidonic acid metabolism in nonsteatotic regions, whereas low–density lipoprotein (LDL) and very low–density lipoprotein (VLDL) metabolism was associated with steatotic tissue. Supervised and unsupervised discriminant analysis using lipid based classifiers outperformed simulated analysis of liver tissue homogenates in predicting steatosis severity. We conclude that lipid composition of steatotic and nonsteatotic tissue is highly distinct, implying that spatial context is important for understanding the mechanisms of lipid accumulation in NAFLD. MSI combined with principal component–linear discriminant analysis linking lipid and protein pathways represents a novel tool enabling detailed, comprehensive studies of the heterogeneity of NAFLD.

Interferon Lambda 4 Gene (IFNL4) Linked to Hepatitis C Virus Clearance, Treatment

Background: AdesignatedIFNL4 gene, encoding the interferon-λ4 protein (IFNL4), which is moderately similar to IFNL3, is more strongly associated with HCV clearance in individuals of African ancestry, whereas it provides comparable information in Europeans and Asians. Aim of the work: The study was attempted for the identification of interferon Lambda 4 (IFNL4) gene expression in the liver biopsy and the recombinant IFNL4 protein in the serum of CHCV patients. Patients and methods: Eighty five patients with chronic hepatitis C virus infection (CHCV), whose age ranged between 19 and 57 years, were selected from the National Hepatology and Tropical Medicine Research Institute were included in this study, before chronic HCV therapy, during the preparation of patients, and ten healthy individuals were included to serve as controls. All the patients and controls were subjected to the following: history, clinical examination, abdominal ultrasonography and collection of blood samples for routine laboratory investigations, CBCs. Liver biopsy was done to all patients and controls. Patients revealed mild fibrosis (Metavir fibrosis from F1 to F3). Using freshly frozen liver biopsies to identify gene (IFNL4) by real time-PCR and the detection of its serum protein levels by ELISA. Results: Patientswith CHCVhave higher hepatic expression of IFNL4 before treatment and also recombinant IFNL4 protein expression was detectable in serum with high levels. Conclusion: An inducible human protein-coding gene IFNL4, which is related to, known IFNs have been identified in genotype 4 CHCV patients. Recommendations: Thetherapeutic inhibition of IFNL4 might represent a novel biological target for the treatment of HCV and HBV infection and possibly other diseases.

Identification of core gene networks and hub genes associated with progression of non-alcoholic fatty liver disease by RNA sequencing.

Non-alcoholic fatty liver disease (NAFLD) progresses because of the interaction between numerous genes. Thus, we carried out a weighted gene coexpression network analysis to identify core gene networks and key genes associated with NAFLD progression. We enrolled 39 patients with mild NAFLD (fibrosis stages 0-2) and 21 with advanced NAFLD (fibrosis stages 3-4). Total RNA was extracted from frozen liver biopsies, and sequenced to capture a large dynamic range of expression levels. A total of 1777 genes differentially expressed between mild and advanced NAFLD (q-value <0.05) clustered into four modules. One module was enriched for genes that encode cell surface or extracellular matrix proteins, and are involved in cell adhesion, proliferation, and signaling. This module formed a scale-free network containing four hub genes (PAPLN, LBH, DPYSL3, and JAG1) overexpressed in advanced NAFLD. PAPLN is a component of the extracellular matrix, LBH and DPYSL3 are reported to be tumor suppressors, and JAG1 is tumorigenic. Another module formed a random network, and was enriched for genes that accumulate in the mitochondria. These genes were downregulated in advanced NAFLD, reflecting impaired mitochondrial function. However, the other two modules did not form unambiguous networks. KEGG analysis indicated that 71 differentially expressed genes were involved in "pathways in cancer". Strikingly, expression of half of all differentially expressed genes was inversely correlated with methylation of CpG sites (q-value <0.05). Among clinical parameters, serum type IV collagen 7s was most strongly associated with the epigenetic status in NAFLD. Newly identified core gene networks suggest that the NAFLD liver undergoes mitochondrial dysfunction and fibrosis, and acquires tumorigenic potential epigenetically. Our data provide novel insights into the pathology and etiology of NAFLD progression, and identify potential targets for diagnosis and treatment.

The Fundamental Role Played by Cell Cycle Proteins in Controlling Cell Proliferation in Chronic Hepatitis C Virus Infection.

Background: examining the alteration of cell cycle genes in early hepatitis C virus (HCV) found that altered expression of mitotic checkpoint genes, MAD2L1, KNTC1, CDC16 and CDC34, KNTC1 known as “rough deal protein” (ROD) is part of a complex involved in elaborating an inhibitory signal due to improper chromosomal aligment during cell division. Aim of the work: attempt for the identification of proteins (genes), which act as predictive factors to identify patients with high risk of cell transformation and HCC development. Patients and methods: fifty three patients with chronic HCV infection, age ranged between 18 and 58 years, time of assessment was before starting therapy of hepatitis C at the National Hepatology and Tropical Medicine Research Institute. Ten healthy individuals were included to serve as controls. All the patients and controls were subjected to the following: history, clinical examination, abdominal ultrasonography, and collection of blood samples for routine laboratory investigation; CBCs. Liver biopsy was done to all patients and controls, patients revealed mild fibrosis (Metavir fibrosis scores from F1 to F3). Also, we used freshly frozen liver biopsies mRNA levels with perspective protein levels of four genes: P27, P15, KNTC1, MAD2L1. Results: significant association of P27, P15, KNTC1 and MAD2L-1 with the progression of liver fibrosis in chronic HCV liver biopsy was found. Conclusion: there is altered gene expression in HCV-associated liver disease. Recommendations: the emerging interest of hepatologists in the influence of genetic factors in HCV. Evaluation of the expression of key proteins related to the cell cycle and apoptosis in chronically infected patients with HCV would be of significance to understand disease pathogenesis, and will help in identifying novel prognostic indicators.

Mallory-Denk bodies: Correlation with steatosis, severity, zonal distribution, and identification with ubiquitin.

Intermediate filament proteins contain few aggregates as their main component. Among those, Mallory-Denk bodies (MDBs) are by far the best recognized component. To identify the presence of MDBs in individuals having chronic liver disease and to evaluate the correlation among MDBs and steatosis as well as the severity and zonal distribution of hepatocyte balloon degeneration. Tertiary reference hospital. Three hundred consecutive liver specimens derived from our patients with chronic liver disease were included in the current study. Immunohistochemistry analysis was conducted on frozen liver biopsies fixed at room temperature with acetone anti-rabbit antibody to ubiquitin. In addition, histological activity was evaluated by the routine staining of liver biopsy sections with hematoxylin-eosin and periodic staining by acid-Schiff stain, reticulin, Masson trichrome, and iron. The presence of MDBs, steatosis, severity, and the zonal distribution of hepatocyte balloon degeneration were evaluated in every patient. Histopathologic diagnosis were chronic hepatitis B (n=219), alcoholic steatohepatitis (n=23), non-alcoholic steatohepatitis (n=20), chronic hepatitis C (n=20), overlap syndrome (n=10), and primary biliary cirrhosis (n=8). The distribution of MDBs stained positive for ubiquitin was 80% in the overlap syndrome, 86% in chronic hepatitis B, and 100% in alcoholic steatohepatitis, NASH, chronic hepatitis C, and primary biliary cirrhosis. There was a correlation between the severity of steatosis and ubiquitin positivity, particularly in zone 2. A conspicuous correlation existed between the severity of hepatocyte balloon degeneration and ubiquitin positivity. These findings have demonstrated that the observation of MDB together with ubiquitin positivity will be helpful in the evaluation of the models of diagnosis, staging, and therapy in patients with chronic liver disease.

Relationship Between Methylome and Transcriptome in Patients With Nonalcoholic Fatty Liver Disease

Cirrhosis and liver cancer are potential outcomes of advanced nonalcoholic fatty liver disease (NAFLD). It is not clear what factors determine whether patients will develop advanced or mild NAFLD, limiting noninvasive diagnosis and treatment before clinical sequelae emerge. We investigated whether DNA methylation profiles can distinguish patients with mild disease from those with advanced NAFLD, and how these patterns are functionally related to hepatic gene expression. We collected frozen liver biopsies and clinical data from patients with biopsy-proven NAFLD (56 in the discovery cohort and 34 in the replication cohort). Samples were divided into groups based on histologic severity of fibrosis: F0-1 (mild) and F3-4 (advanced). DNA methylation profiles were determined and coupled with gene expression data from the same biopsies; differential methylation was validated in subsets of the discovery and replication cohorts. We then analyzed interactions between the methylome and transcriptome. Clinical features did not differ between patients known to have mild or advanced fibrosis based on biopsy analysis. There were 69,247 differentially methylated CpG sites (76% hypomethylated, 24% hypermethylated) in patients with advanced vs mild NAFLD (P < .05). Methylation at fibroblast growth factor receptor 2, methionine adenosyl methyltransferase 1A, and caspase 1 was validated by bisulfite pyrosequencing and the findings were reproduced in the replication cohort. Methylation correlated with gene transcript levels for 7% of differentially methylated CpG sites, indicating that differential methylation contributes to differences in expression. In samples with advanced NAFLD, many tissue repair genes were hypomethylated and overexpressed, and genes in certain metabolic pathways, including 1-carbon metabolism, were hypermethylated and underexpressed. Functionally relevant differences in methylation can distinguish patients with advanced vs mild NAFLD. Altered methylation of genes that regulate processes such as steatohepatitis, fibrosis, and carcinogenesis indicate the role of DNA methylation in progression of NAFLD.

Reply to the Sannier et al. letter entitled “MDR3 immunostaining on frozen liver biopsy is not a sensitive diagnosis tool for the detection of heterozygous MDR3/ABCB4 gene mutation”

Sir or Madam, We read with great interest the letter by Sannier et al. The data presented are consistent with our publication [1]. The authors studied a new series of adult patients with unexplained cholestasis and heterozygous MDR3/ABCB4 mutations. In five cases, a MDR3 immunostaining was performed on liver frozen sections and showed an intense canalicular staining similar to a normal staining. These results are in contradiction with their initial report that showed a faint MDR3 immunostaining in four patients with heterozygous MDR3/ABCB4 mutations [2]. This discrepancy may be explained by technical hazards or by the type of MDR3/ABCB4 mutations (indeed, the mutations were different in the two series). It could be interesting to perform MDR3 immunostainings on the formalin-fixed paraffin-embedded liver tissues in these two series that would enable a comparison with our published data which were obtained with this technique. To conclude, it seems that MDR3/ABCB4 heterozygous mutation diagnosis requires gene sequencing.

MDR3 immunostaining on frozen liver biopsy samples is not a sensitive diagnostic tool for the detection of heterozygous MDR3/ABCB4 gene mutations

Dear Editor, We read with great interest the recent article of Wendum et al. [1] describing histological lesions observed in the liver of 13 adult patients with heterozygous MDR3/ABCB4 gene mutations. They performed multidrug resistance Pglycoprotein 3 (MDR3) immunostaining on formalin-fixed paraffin-embedded sections of 11 patients and reported that the staining had a diffuse and strong canalicular pattern in patients and controls, except in one case who disclosed a faint staining. This finding suggested a lack of sensitivity of this technique for the diagnosis of MDR3/ABCB4 heterozygous mutations. Since we previously suggested that MDR3 immunostaining on frozen liver sections could be useful for the diagnosis of unexplained anicteric cholestasis in adult patients [2], we would like to contribute by providing our experience of MDR3 immunostaining realized on frozen liver biopsy sections of a new series of adult patients with MDR3/ABCB4 mutations. Between April 2007 and March 2011, 32 adult patients with unexplained anicteric cholestasis had ABCB4 gene sequencing, using polymerase chain reaction amplification and DNA sequencing of exons 2 to 28 and all splice junctions. Among these 32 patients, ten carried six different ABCB4 mutations. These ten patients (six females, 17– 72 years of age) displayed various clinical characteristics: three fulfilled the criteria for low phospholipid-associated cholelithiasis syndrome [3]; one had an anteriority of intrahepatic cholestasis of pregnancy; and six had an unexplained anicteric cholestasis—two of them with associated elevated aminotransferase level. Histopathological analysis using Picrosirius red and hematoxylin–eosin-stained liver biopsy sections showed that nine patients had portal fibrosis (three F1, four F2, one F3, and one F4 according to Ishak fibrosis score); six of them displaying a jigsaw pattern of biliary-type fibrosis. Bile duct lesions corresponding to mild cholangitis could be observed in four patients. Ductopenia, defined as a loss of more than 50 % of bile ducts, was seen in two patients. Lastly, six cases showed a mild to moderate ductular reaction. The clinicopathological features are detailed in Table 1. Frozen liver biopsies were available for 14 patients (five cases and nine controls). Immunohistochemistry was performed on 4-μm frozen sections by using MDR3 monoclonal antibody (clone P3II-26) and MRP2 antibody (clone M2III-6) as a control to check tissue immune reactivity. As shown in Fig. 1, we observed a diffuse and intense canalicular staining with MDR3 antibody, in the available frozen liver biopsies of the five patients with heterozygous ABCB4 mutations and in the nine controls. Positive canalicular staining was also observed with MRP2 antibody, confirming the immunoreactivity of our samples. These results are in contradiction with our previous report [2] in which no or faint MDR3 immunostaining was observed in frozen liver biopsies from four patients with A. Sannier (*) :M. Tepper :M. Ziol Service d’Anatomie Pathologique, GHU Paris-Seine-Saint-Denis, Hopital Jean Verdier, AP-HP, Avenue du 14 Juillet, 93143 Bondy Cedex, France e-mail: aureliesannier@free.fr

Role of hepatic HCV–RNA level on the severity of chronic hepatitis C and response to antiviral therapy

BackgroundCorrelation between hepatic HCV–RNA and serum HCV–RNA, severity of liver disease and response to therapy is poorly known. ObjectivesTo assess the influence of hepatic HCV–RNA level on severity of liver disease and response to therapy in a large cohort of chronic hepatitis C (CHC) patients. Study DesignHCV–RNA was measured in frozen liver biopsies and serum samples from 130 CHC patients the day of liver biopsy prior to treatment. Liver fibrosis was assessed by Ishaq scoring. A Sustained Virological Response (SVR) was observed in 52% of the patients, non-response (NR) in 34%. ResultsMean±standard deviation hepatic HCV–RNA level was 7.69±0.67 log10 copies/mg of liver. Mean serum HCV–RNA level was 6.21±0.72 log10 copies/ml. There was a correlation between hepatic and serum HCV–RNA in genotype 1 and 4 (p=0.008 and p=0.03) and age (p=0.006). Mean hepatic HCV–RNA was 7.70±0.69 vs 7.67±0.68 log10 copies/mg of liver, in patients with significant fibrosis vs those with mild fibrosis, respectively (p=0.7); 8.04±0.68; 7.44±0.47; 7.43±0.49 and 7.44±0.71 log10 copies/mg of liver in genotypes 1, 2, 3 and 4, respectively (p=0.0001); higher in women than in men (p=0.04); 7.60±0.63, 7.71±0.54 and 7.96±0.73 log10 copies/mg in SVR, relapsers and NR, respectively (p=0.1). Multivariate analysis showed that high hepatic HCV–RNA level was independently associated with genotype and response to therapy was associated with genotype independently from hepatic HCV–RNA level. ConclusionsHepatic HCV–RNA level was not associated with severity of liver disease. High level was strongly associated with HCV genotype independently from response to therapy.

Utility of formalin‐fixed, paraffin‐embedded liver biopsy specimens for global proteomic analysis in nonalcoholic steatohepatitis

To compare the proteomic profiles of formalin-fixed, paraffin-embedded (FFPE) liver biopsy material and matched frozen liver tissue from patients with nonalcoholic steatohepatitis (NASH). A label-free mass spectrometry-based approach was used to profile global protein expression in FFPE and frozen liver biopsy specimens from five patients with NASH. Eight hundred and sixty proteins were identified with >75% confidence: 225 common proteins were identified in both the FFPE and frozen tissues, and an additional 142 and 493 proteins were identified in the FFPE and frozen tissues, respectively. Functional analyses revealed a general, nonspecific reduction in the number of proteins identified in FFPE tissue compared with frozen tissue. No bias toward proteins located in any specific subcellular compartments or implicated in any particular biological functions was observed. The relative abundance of several proteins with functions relating to the pathogenesis of NASH (peroxiredoxin-1, fatty acid binding protein 1, fatty acid synthase, vimentin, catalase, and glutathione S-transferase A1) was similar in FFPE and frozen liver tissues. FFPE liver biopsy material from NASH patients can be used for global proteomic analysis and biomarker identification studies, although a universal reduction in the number of identified proteins compared with frozen tissue is likely.

Modulations of cell cycle checkpoints during HCV associated disease

BackgroundImpaired proliferation of hepatocytes has been reported in chronic Hepatitis C virus infection. Considering the fundamental role played by cell cycle proteins in controlling cell proliferation, altered regulation of these proteins could significantly contribute to HCV disease progression and subsequent hepatocellular carcinoma (HCC). This study aimed to identify the alterations in cell cycle genes expression with respect to early and advanced disease of chronic HCV infection.MethodsUsing freshly frozen liver biopsies, mRNA levels of 84 cell cycle genes in pooled RNA samples from patients with early or advanced fibrosis of chronic HCV infection were studied. To associate mRNA levels with respective protein levels, four genes (p27, p15, KNTC1 and MAD2L1) with significant changes in mRNA levels (> 2-fold, p-value < 0.05) were selected, and their protein expressions were examined in the liver biopsies of 38 chronic hepatitis C patients.ResultsIn the early fibrosis group, increased mRNA levels of cell proliferation genes as well as cell cycle inhibitor genes were observed. In the advanced fibrosis group, DNA damage response genes were up-regulated while those associated with chromosomal stability were down-regulated. Increased expression of CDK inhibitor protein p27 was consistent with its mRNA level detected in early group while the same was found to be negatively associated with liver fibrosis. CDK inhibitor protein p15 was highly expressed in both early and advanced group, but showed no correlation with fibrosis. Among the mitotic checkpoint regulators, expression of KNTC1 was significantly reduced in advanced group while MAD2L1 showed a non-significant decrease.ConclusionCollectively these results are suggestive of a disrupted cell cycle regulation in HCV-infected liver. The information presented here highlights the potential of identified proteins as predictive factors to identify patients with high risk of cell transformation and HCC development.

HCV liver infection and liver steatosis: evidence for indirect mechanisms in genotype 3?

Hepatitis C virus (HCV) infection is associated with the appearance of liver steatosis. To search for a correlation between the number of HCV infected hepatocytes and the presence, amount and distribution of steatosis. A total of 124 frozen liver biopsies from HCV patients (genotype 3 = 21) were studied. HCV-antigens were detected on frozen liver sections using a four steps immunoperoxidase technique. Steatosis was graded by haematoxilin-eosin counterstaining on a serial section. Steatosis was detected in 82 of 124 (66.1%) patients without differences between different genotypes. Uric acid, body mass index, gammaGT levels significantly correlated with steatosis in non-3 (P < 0.01, P < 0.05, P < 0.01, respectively) but not in genotype 3 patients. HCV-antigens were detected in 95 of 124 (76.6%) cases. A positive correlation between steatosis and the number of infected hepatocytes was observed only in genotype 3 patients (P = 0.06). In most cases the number of cells with steatosis greatly outnumbered that of HCV infected cells. We confirm a possible role of the virus in the genesis of steatosis in HCV genotype 3 infected patients; however, as steatosis do not appear to be directly related to the presence of HCV-antigens within single hepatocytes, an indirect, possibly cytokine mediated, mechanism might be operative.

Evidence of angiogenesis in primary biliary cirrhosis: an immunohistochemical descriptive study

The intrahepatic inflammatory process occurring during primary biliary cirrhosis contributes to bile duct destruction, but the cellular and molecular pathways involved are largely unknown. Furthermore, additional pathogenetic mechanisms may exist. We aimed at evaluating the cellular infiltrate phenotype; the expression of lymphocyte activation, antigen recognition and cell-adhesion molecules; the occurrence of hepatic angiogenesis and the molecules involved. Immunohistochemical investigations were performed in frozen liver biopsy sections from primary biliary cirrhosis patients. CD8+ and CD69+ T cells were predominant in inflammatory infiltrates around damaged cholangiocytes; beta2-microglobulin conformational epitope and intercellular adhesion molecule-1 expression were enhanced in bile ducts and hepatocytes. Inflamed portal areas showed vascular cell adhesion molecule-1 up-regulation; formation of tubule-like structures (neovessels) by endothelial cells expressing vascular endothelial-cadherin and CD-31; vascular endothelial growth factor expression in surrounding sinusoidal endothelial cells; and enhanced expression of angiopoietins 1 and 2, their receptor Tie-2 and endoglin, suggesting their involvement in new vascular structure formation. The inflammatory infiltrate in primary biliary cirrhosis shows an increased reactivity for lymphocyte activation, antigen recognition and cell- and vascular-adhesion molecules. Additionally, intrahepatic angiogenesis occurs, involving vascular endothelial growth factor, angiopoietins 1 and 2, Tie-2 and endoglin in neovessel formation.

Detection of recipient's cells in liver graft using antibodies to mismatched HLA class I antigens

Engraftment by recipient's (R) cells has been already demonstrated in gender mismatched liver grafts using fluorescence in situ hybridization (FISH), with contrasting results concerning epithelial cells. Mismatch for human leukocyte antigen (HLA) class I (HLA-I) is quite common in patients with orthotopic liver transplantation (OLT). We thus aimed to assess whether monoclonal antibodies (MoAbs), currently employed in the HLA typing process, could be used to study the dynamics of R cells in liver grafts. A total of 50 frozen liver biopsies from 37 patients receiving a HLA mismatch liver were tested. Biopsies were obtained from 3 days to more than 360 days after OLT. Frozen sections of graft biopsies were stained using an immunoperoxidase technique with the proper MoAbs. In selected cases, a double immunofluorescence was also performed. Circulating R blood cells and sinusoidal cells were occasionally observed in liver biopsies obtained within 10 days after OLT and were commonly detected after 1 month. The number of sinusoidal cells continued to increase up to 6 months, as shown on serial biopsies. On the whole, R blood cells and R sinusoidal cells were detected in 86% and 82% of the biopsies, respectively. R hepatocytes and biliary cells were detected after 40 and 60 days after OLT, respectively, in 14% (hepatocytes), 8% (bile ducts), and 12% (proliferating bile ducts) of the biopsies. R hepatocytes presented as single cells or groups of few cells; their number was lower than 1% and apparently did not increase with time after OLT. In conclusion, it is possible to detect R cells in liver graft using MoAbs to specific mismatched HLA-I alleles. R sinusoidal cells start to appear after 10 days and are commonly observed after 1 month; bile duct cells and hepatocytes appear later and their number does not increase with time. Engraftment by R epithelial cells seems to be less important than previously reported.

THE HEPATIC EXTRACELLULAR MATRIX DURING COLD ISCHEMIA/WARM REPERFUSION OF THE RAT LIVER: ROLE OF MATRIX METALLOPROTEINASES IN ITS ALTERATION.

A310 Background: Poor initial graft function remains a major clinical problem in human liver transplantation. Its incidence (15 to 25% of transplanted patients) appears to depend on the length of cold storage and has thus been ascribed to injury from harvesting, cold storage and warm reperfusion after blood vessel reconnection; i.e. cold ischemia/warm re-perfusion (CI-WR) injury. The underlying mechanism of CI-WR injury has been studied extensively over the last decade, but it remains poorly understood. Recently, it has been demonstrated that different time courses and mechanisms of cell death occur in rat livers after CI-WR, with early sinusoidal endothelial cell necrosis followed by delayed hepatocyte apoptosis. These processes did not appear to be of major importance in the mechanism of graft failure since both were similar under non-lethal and lethal conditions. By contrast, prolonged CI was associated with an alteration of the extracellular matrix (ECM), leading to extensive coagulative necrosis of hepatocytes as found in conditions ending in graft failure. Aims: Since it is known that warm ischemia/WR of livers induces an increased local synthesis of metalloproteinases (MMPs), proteases implicated in the degradation of the hepatic ECM, we looked if the same phenomenon was occurring during prolonged CI/WR of rat livers ensuing in ECM disappearance responsible for the graft malfunction. Methods: Rat livers were preserved for 0, 24 (viable livers) and 42 h (non-viable livers) in the UW solution (4°C), then reperfused for 0, 10, 20, 30, 40, 60, 90, and 120 minutes with a krebs buffer solution (37°C) containing 20% erythrocytes. The first 15 mLs of the liver rinsing solution were taken during early WR and frozen liver biopsies were obtained at the end of each period (n=4 in all groups). Total liver RNAs and proteins were extracted, then MMP and their natural inhibitor (TIMP) expression was analysed by Northern Blot quantitative analysis and MMP activity was nalyzed using a fluorescent substrate (MCA-MAT) or by zymography. Results: although most of these genes are not expressed in the normal rat liver, there was an induction of MMP-13 and 14 expression as well as of TIMP-1 and 2 expression in all livers, independently from CI or WR periods. There was also an increased MMP activity in all livers, as measured ding a in presence or absence of an inhibitor (BB94), whatever the experimental condition. An increased activity of MMPs (particularly gelatinases A and B) was found in the initial flush solution. Comments: These data are quite different from those observed following warm ischemia/WR, particularly when considering that, following CI, the ECM rapidly deteriorates with WR. However since this phenomenon occurs very early during WR, it might be due to the liberation of MMPs already present in sinusoidal endothelial cells and released by their necrosis during prolonged CI, as suggested by our data and others. Conclusion: Induction of hepatic MMP synthesis does not occurs following prolonged CI/WR of rat livers and thus has no major role in the alteration of the hepatic extracellular matrix observed under these conditions. This phenomenon might result from the local passive release of preformed MMPs from dead hepatic cells.

HCV VIRAL RECURRENCE AND LIVER MITOCHONDRIAL DAMAGE AFTER LIVER TRANSPLANTATION (LT) IN HIV-HCV COINFECTED PATIENTS

O30 End stage liver disease requiring LT become an emerging problem in HIV-HCV co-infected patients. Aims: We need to assess the incidence and severity of HCV recurrence and highly active antiretroviral therapy (HAART) toxicity. Patients and Methods: Nineteen HIV-HCV co-infected patients were transplanted between December 1999 and March 2004 for HCV cirrhosis. All patients were treated by HAART. Liver biopsies were performed 1 month, 6 months, and every year post-LT. Recurrent hepatitis C was assessed by the METAVIR score. HAART-related toxicity was asssessed on (1) the presence of microvesicular steatosis, (2) activity of the four complexes of the four complexes of the respiratory chain was analyzed byspectrophotometric analysis on homogenate from frozen liver biopsy specimens. Quantification of the mitochondrial (mt) DNA copy number was performed using real-time PCR amplication. Results: In March 2004, the median follow-up was 10 months (range, 1–51). Three patients died, 4, 11 and 22 months after LT due mt toxicity (n=2), and pancreatic carcinoma (n=1). At the last biopsy, METAVIR score was staged F4 in two patients, F3 in two and F1 in three. One patient, showed a virological sustained response to anti-HCV treatment. Four other patients showed a partial (n=3) or a complete biochemical response (n=1) with anti-HCV therapy. Microvesicular steatosis was observed in 8 patients (42%). A significant enzymatic defect of complex IV was observed in 7 of the 10 patients analyzed. A low level of mtDNA was detected in 2 of 5 patients studied. Conclusions: Mitochondrial toxicity and severe HCV recurrence occur after LT in HIV-HCV coinfected patients.