An automated pattern recognition system for the quantification of inflammatory cells in hepatitis-C-infected liver biopsies

This paper presents an automated system for the quantification of inflammatory cells in hepatitis-C-infected liver biopsies. Initially, features are extracted from colour-corrected biopsy images at positions of interest identified by adaptive thresholding and clump decomposition. A sequential floating search method and principal component analysis are used to reduce dimensionality. Manually annotated training images allow supervised training. The performance of Gaussian parametric and mixture models is compared when used to classify regions as either inflammatory or healthy. The system is optimized using a response surface method that maximises the area under the receiver operating characteristic curve. This system is then tested on images previously ranked by a number of observers with varying levels of expertise. These results are compared to the automated system using Spearman rank correlation. Results show that this system can rank 15 test images, with varying degrees of inflammation, in strong agreement with five expert pathologists

An Automated Pattern Recognition System for the Quantification of Inflammatory Cells in Hepatitis C Infected Liver Biopsies.

Hepatitis C is a common viral infection of the liver. The degree of inflammation associated with the infection is normally estimated manually from a liver biopsy, by considering the quantity and nature of inflammatory cells. This paper presents an automated pattern recognition system for the quantification of inflammatory cells in liver biopsies. Initially, images are corrected for colour variation. Features are then extracted for from colour biopsy images at positions of interest identified by adaptive thresholding and clump decomposition. A sequential floating search method and principal component analysis are used to reduce the dimensionality of the feature vector. Manually annotated training images allow supervised training by providing the class membership for each position of interest. Gaussian parametric and Gaussian mixture model density estimation methods are compared and are used to classify cells as either inflammatory or healthy via Bayes' theorem. The system is optimised using a response surface method, where the response or system performance is derived from the area under the receiver operating characteristic curve. The optimised system is then tested on test images previously ranked by a number of observers with varing levels of pathology experience. The observers results are compared to the automated system using Spearman rank correlation. Results show that this system can rank 15 test images, with varying degrees of inflammation, in strong agreement with five expert pathologists

Identification of woodchuck toll-like receptors and their expression during the course of hepadnaviral infection in the woodchuck model of hepatitis B

The woodchuck hepatitis virus (WHV) is closely related to human hepatitis B virus (HBV), the prototypic member of the Hepanaviridae family. Toll-like receptors (TLRs) may play an important role in the pathogenesis of hepadnaviral hepatitis, however, little is known about their expression during the course of hepadnaviral infection. In this study, woodchuck TLRs1-10 gene exon fragments were identified and their transcriptional profiles investigated in livers, hepatocytes isolated from these livers, and peripheral blood mononuclear cells (PBMCs) from healthy woodchucks and animals with different stages of experimental WHV infection. Overall expression analysis revealed that livers from woodchucks with acute hepatitis (AH) and chronic hepatitis (CH) had significantly upregulated expression of TLRs2-10 when compared to the livers of healthy animals and those with self-limited acute hepatitis (SLAH) and primary occult infection (POI). This was likely due to intrahepatic immune cell infiltration. In contrast, a significant downregulation of TLR3, TLR5, TLR7, TLR8, and TLR10 expression was identified in hepatocytes of woodchucks with CH when compared to hepatocytes from healthy animals and those with pre-acute hepatitis (PreAH), SLAH and POI. This may suggest WHV active suppression of the innate immune response in these cells. Upregulated transcription of the majority of TLRs was found in PBMCs during CH but not in other stages of infection. In summary, this study uncovered that TLR expression is significantly modulated depending on the stage of WHV infection and form of hepatitis. Treatments designed to restore hepatocyte TLR expression may allow for better control of the virus through activation of a stronger intrahepatocyte immune response during CH

Liver Biopsy

Liver biopsy is recommended as the gold standard method to determine diagnosis, fibrosis staging, prognosis and therapeutic indications in patients with chronic liver disease. However, liver biopsy is an invasive procedure with a risk of complications which can be serious. This book provides the management of the complications in liver biopsy. Additionally, this book provides also the references for the new technology of liver biopsy including the non-invasive elastography, imaging methods and blood panels which could be the alternatives to liver biopsy. The non-invasive methods, especially the elastography, which is the new procedure in hot topics, which were frequently reported in these years. In this book, the professionals of elastography show the mechanism, availability and how to use this technology in a clinical field of elastography. The comprehension of elastography could be a great help for better dealing and for understanding of liver biopsy

Spatio-temporal differences in presentation of CD8 T cell epitopes during HBV infection

Distinct populations of hepatocytes infected with HBV or only harboring HBV-DNA integrations coexist within an HBV chronically infected liver. These hepatocytes express HBV antigens at different levels and with different intracellular localizations but it is not known whether this heterogeneity of viral antigen expression could result in an uneven hepatic presentation of distinct HBV epitopes/HLA class-I complexes triggering different level of activation of HBV-specific CD8+ T cells.Using antibodies specific to two distinct HLA-A*02:01/HBV epitope complexes of HBV nucleocapsid and envelope proteins, we mapped their topological distribution in liver biopsies of two anti-HBe+ chronic HBV (CHB) patients. We demonstrated that the core and envelope CD8+T cell epitopes were not uniformly distributed in the liver parenchyma but preferentially located in distinct and sometimes mutually exclusive hepatic zones. The efficiency of HBV epitope presentation was then tested in vitro utilizing HLA-A*02:01/HBV epitope-specific antibodies and the corresponding CD8+ T cells, in primary human hepatocyte and hepatoma cell lines either infected with HBV or harboring HBV-DNA integration. We confirmed the existence of a marked variability in the efficiency of HLA-class I/HBV epitope presentation among the different targets that was influenced by presence of IFN-γ and availability of newly-translated viral antigens. In conclusion, HBV antigen presentation can be heterogeneous within an HBV-infected liver. As a consequence, CD8+ T cells of different HBV specificities might have different antiviral efficacy.Importance The inability of patients with chronic HBV infection to clear HBV is associated with defective HBV-specific CD8+ T cells. Hence, the majority of immunotherapy developments focus on HBV specific T cell function restoration. However, knowledge of whether distinct HBV-specific T cells can equally target all the HBV-infected hepatocytes of a chronically infected liver are lacking. In this work, analysis of CHB patient liver parenchyma and in vitro HBV infection models shows a non-uniform distribution of HBV CD8+ T cells epitopes that is influenced by presence of IFN-γ and availability of newly-translated viral antigens. These results suggest that CD8+ T cells recognizing different HBV epitopes can be necessary for efficient immune therapeutic control of chronic HBV infection

Interferon signaling in chronic hepatitis C : mechanisms and implications for therapy

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide and can lead to liver cirrhosis and hepatocellular carcinoma. The current standard therapy of chronic hepatitis C (CHC) consists of a combination of pegylated interferon alpha (pegIFNα) and ribavirin. However, sustained viral clearance is achieved in only 50-60% of patients. The underlying mechanism of failure of pegIFNα based therapy remains unknown and no molecular or genetic markers have been identified that could predict the treatment outcome. The overall aim of the study described in this thesis is to understand the molecular basis for failure of IFNα based therapies in patients with CHC. The study has focused on the IFNinduced Jak-STAT (janus kinase-signal transducer and activator of transcription) signaling pathway. To address the molecular basis of treatment response to IFN therapy, three experimental approaches have been employed. The first approach involved the analysis of IFNα signaling and expression of interferon stimulated genes (ISGs) in liver biopsies and peripheral blood mononuclear cells (PBMCs) of HCV patients undergoing pegIFNα treatment. Paired liver biopsies and PBMCs from 16 patients were collected before andhours after the first injection of pegIFNα, and were subjected to analysis of global gene expression using Affymetrix arrays. Further, activation of the IFN-induced Jak-STAT signaling pathway was analyzed by immunoblotting, immunohistochemistry and gel shift assays. The correlation of these biochemical and molecular data with the clinical response to treatment demonstrated that in the liver of patients with a rapid response pegIFNα induced a strong upregulation of ISGs, whereas in patients that did not respond to therapy, induction of IFN-dependent gene expression was impaired. Surprisingly, the non-responders had maximally induced ISG expression already before treatment with pegIFNα. Furthermore, the analyses of STAT1 phosphorylation, nuclear localization and DNA binding confirmed that the endogenous IFN signaling pathway in non-responders is pre-activated and refractory to further stimulation. In contrast to liver samples, ISG expression in PBMCs was stimulated by pegIFNα in both responders and nonresponders, indicating that PBMCs are not a good surrogate marker for IFNα responses in the liver and that chronic HCV infection has strong local effects on the IFN system in liver. Our findings support an interesting concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection, but may also impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway in the liver. In the second approach we addressed the mechanisms underlying the pre-activation of the endogenous IFN system in a defined group of HCV patients (future non-responders). For this purpose, we analyzed ISG expression by quantitative RT-PCR and nuclear localization of STAT1 by immunohistochemistry in a cohort of 112 patients with CHC. By subdividing this cohort according to the HCV genotype (GT), we discovered that patients infected with HCV GT 1 and 4 more often show hepatic ISG preactivation than GT 2 and 3 patients, thus providing an explanation for the poor response to IFN therapy seen in GT 1/4 patients. We analyzed the possible involvement of viral sensory pathways in type I IFN production and ISG upregulation. Previously, the viral HCV NS3-4A protease was shown to interfere with viral sensory pathways by cleaving and thereby inactivating an important adaptor molecule, Cardif. We therefore assessed Cardif cleavage in liver biopsies of HCV patients and found that cleavage more often occurred in patients infected with HCV GTs 2 and 3. Our findings support a concept that the success of the virus in preventing the induction of the endogenous IFN system in the livers of these patients would, however, come at the cost of being more susceptible to IFNα therapies as is the case with GT 2/3 patients. In the third approach we designed an experimental model to study the molecular basis of refractoriness of IFN signaling in vivo. Previously, cell culture experiments demonstrated a long lasting desensitization period, which followed the initial activation of the IFNα signaling pathway. In the approach used here, we established a mouse model in which continuous presence of IFNα in vivo was achieved by multiple subcutaneous injections, mimicking the constitutively high serum levels achieved by pegIFNα in patients. Interestingly, this resulted in refractoriness of IFNα signaling. Activation of STAT1 and STAT2, but not STAT3, in the mouse liver was desensitized by continuous IFNα stimulation. To elucidate the mechanism of this refractoriness, the role of negative regulators of the Jak- STAT signaling pathway was investigated. IFN signaling remained refractory in mice deficient in suppressor of cytokine signaling (SOCS) 3 and persisting refractoriness was also observed in mice deficient in IL-10, a strong inducer of SOCS3. Ubiquitin specific peptidase 18 (USP18/UBP43) was recently identified as novel negative regulator of IFNα signal transduction. Interestingly, refractoriness could be overcome in USP18/UBP43 knockout mice. These data strongly indicate that UBP43 is the decisive factor in inducing a refractory state in the IFNα signaling pathway in vivo