Structural and Functional Aspects of the Hepatic Sinusoids

The hepatic sinusoids were long regarded as simple, inert vascular channels lined by phagocytic and non-phagocytic cells derived from a common progenitor cell. This thesis describes studies dealing with various structural and functional aspects of the mammalian sinusoids. The aims of the work included can be broadly summarized as follows: (i) To investigate the role of sinusoidal cells in the synthesis of complement proteins. (ii) To characterize the composition of the extracellular matrix of the space of Disse in normal liver and to examine alterations to this in liver disease. (iii) To identify the cellular origin of such extracellular matrix proteins with particular attention to the role of sinusoidal cells. (iv) Having identified fat-storing cells as a major source of extracellular matrix proteins in (iii), to study the response of these cells in vivo to acute and chronic liver injury. (v) To identify and characterize intra-sinusoidal nerve fibres and to examine alterations to sinusoidal innervation in liver disease. The recently developed methods of sinusoidal cell isolation and culture were used to investigate the capacity of Kupffer cells, sinusoidal endothelial cells and fat-storing cells to synthesize C3, the most abundant protein of the complement cascade pathway. C3 was detected in Kupffer cell supernatants using a sensitive ELISA. Release of C3 could be reversibly inhibited by addition of the protein synthesis inhibitor cycloheximide, indicating active synthesis of the protein by these cells. Secretion of C3 could be enhanced by the prior exposure of the cells to bacterial endotoxin. Smaller amounts of C3 were also detected in fat-storing cell cultures after 5 days in culture but were undetectable in sinusoidal endothelial cell cultures. This study was the first to demonstrate C3 synthesis and secretion by sinusoidal cells. Although it was calculated that Kupffer cells are likely to contribute less than 1% of total hepatic C3 production, the observation of enhanced secretion following exposure to endotoxin suggests that C3 release by these cells may play an important role in the local intrahepatic host response to bacterial toxins. In order to characterize the extracellular matrix of the space of Disse in human liver, a panel of affinity-purified polyclonal antibodies to a wide range of matrix components was used for immunolocalization of the proteins by light microscopy and an ultracryomicrotomy/ immuno-gold labelling method was applied for their identification at the ultrastructural level in liver biopsy specimens. The normal space of Disse was found to contain several types of interstitial collagen (types I, III and V), a "minor" collagen (type VI), fibronectin, undulin and vitronectin. In spite of the paucity of a recognisable basement membrane at this site, the basement membrane components collagen type IV and laminin could also be identified. Increased immunolabelling for fibronectin was noted in the space of Disse in material from patients with acute liver disease. By contrast, in various forms of chronic (fibrotic) liver disease increased labelling was noted for interstitial collagens (types I and III) and the basement membrane proteins collagen type IV and laminin. Such changes in the extracellular matrix of the space of Disse may adversely affect parenchymal cell function by altering normal cell-matrix interactions and may perpetuate liver injury by interfering with the transport of nutrients between the sinusoidal blood flow and parenchymal cells. Two methods were used to examine the role of sinusoidal cells in the synthesis of extracellular matrix proteins. First, immunohistochemistry was used to identify intracellular proteins by light microscopy and at the ultrastructural level. Second, in vitro biosynthesis of matrix proteins by isolated rat liver sinusoidal cells was studied using [3H]-proline incorporation and SDS-PAGE analysis. Evidence is presented which suggests that fat-storing cells are a major source of such proteins; these cells may therefore be important in hepatic fibrogenesis. In order to study the response of these cells in vivo to various forms of experimental liver injury, a method for their immunolocalization in rat liver using monoclonal antibodies to the intermediate filament protein desmin was developed. This method was used to monitor the response of fat-storing cells to two forms of experimental injury: acute carbon tetrachloride-induced damage and chronic cholestatic injury (common bile duct ligation). In both models, desmin-positive fat-storing cells accumulated in areas of parenchymal cell damage. A novel double-labelling method was used to demonstrate that expansion of the desmin-positive fat-storing cell population was, at least in part, due to local cell proliferation. Furthermore, in both models evidence of phenotypic modulation in these cells towards that of myofibroblasts was found by demonstration of expression of the alpha-(smooth muscle) isoform of actin. (Abstract shortened by ProQuest.)

Multi-agent planning using an abductive : event calculus

Temporal reasoning within distributed Artificial Intelligence Systems is faced with the problem of concurrent streams of action. Well known, logic-based systems using the SITUATION CALCULUS solve the frame problem in a purely linear manner. Recent research, however, has revealed that the EVENT CALCULUS under the abduction principle is capable of nonlinear planning. In this report, we present a planning service module which incorporates this approach into a constraint logic framework and even allows a notion of strong nonlinearity. The work includes the axiomatisation of appropriate versions of the EVENT CALCULUS, the development of a suitably sound and complete proof procedure that supports abduction and the implementation of both of these layers on the constraint platform OZ. We demonstrate prototypically how this module, EVE, can be integrated into an existing multi-agent architecture and evaluate the behaviour of such agents within an application domain, the loading dock scenario

A PDCD1 Role in the Genetic Predisposition to NAFLD-HCC?

Obesity and non-alcoholic fatty liver disease (NAFLD) are contributing to the global rise in deaths from hepatocellular carcinoma (HCC). The pathogenesis of NAFLD-HCC is not well understood. The severity of hepatic steatosis, steatohepatitis and fibrosis are key pathogenic mechanisms, but animal studies suggest altered immune responses are also involved. Genetic studies have so far highlighted a major role of gene variants promoting fat deposition in the liver (PNPLA3 rs738409; TM6SF2 rs58542926). Here, we have considered single-nucleotide polymorphisms (SNPs) in candidate immunoregulatory genes (MICA rs2596542; CD44 rs187115; PDCD1 rs7421861 and rs10204525), in 594 patients with NAFLD and 391 with NAFLD-HCC, from three European centres. Associations between age, body mass index, diabetes, cirrhosis and SNPs with HCC development were explored. PNPLA3 and TM6SF2 SNPs were associated with both progression to cirrhosis and NAFLD-HCC development, while PDCD1 SNPs were specifically associated with NAFLD-HCC risk, regardless of cirrhosis. PDCD1 rs7421861 was independently associated with NAFLD-HCC development, while PDCD1 rs10204525 acquired significance after adjusting for other risks, being most notable in the smaller numbers of women with NAFLD-HCC. The study highlights the potential impact of inter individual variation in immune tolerance induction in patients with NAFLD, both in the presence and absence of cirrhosis

Genome-wide association study of non-alcoholic fatty liver and steatohepatitis in a histologically-characterised cohort

Correction: Volume: 74 Issue: 5 Pages: 1274-1275 DOI: 10.1016/j.jhep.2021.02.003 Correction: Volume78, Issue5 Page: 1085-1086 DOI: 10.1016/j.jhep.2023.02.028 Published MAY 2023Background and Aims Genetic factors associated with non-alcoholic fatty liver disease (NAFLD) remain incompletely understood. To date, most GWAS studies have adopted radiologically assessed hepatic triglyceride content as reference phenotype and so cannot address steatohepatitis or fibrosis. We describe a genome-wide association study (GWAS) encompassing the full spectrum of histologically characterized NAFLD. Methods The GWAS involved 1483 European NAFLD cases and 17781 genetically-matched population controls. A replication cohort of 559 NAFLD cases and 945 controls was genotyped to confirm signals showing genome-wide or close to genome-wide significance. Results Case-control analysis identified signals showing p-values ≤ 5 x 10-8 at four locations (chromosome (chr) 2 GCKR/C2ORF16; chr4 HSD17B13; chr19 TM6SF2; chr22 PNPLA3) together with two other signals with pPeer reviewe

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis

Background & Aims: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. Methods: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting–purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. Results: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor β was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB–IL34/CCL8 signaling that promotes macrophage chemotaxis. Conclusions: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC

The European NAFLD Registry: A real-world longitudinal cohort study of nonalcoholic fatty liver disease.

Non-Alcoholic Fatty Liver Disease (NAFLD), a progressive liver disease that is closely associated with obesity, type 2 diabetes, hypertension and dyslipidaemia, represents an increasing global public health challenge. There is significant variability in the disease course: the majority exhibit only fat accumulation in the liver but a significant minority develop a necroinflammatory form of the disease (non-alcoholic steatohepatitis, NASH) that may progress to cirrhosis and hepatocellular carcinoma. At present our understanding of pathogenesis, disease natural history and long-term outcomes remain incomplete. There is a need for large, well characterised patient cohorts that may be used to address these knowledge gaps and to support the development of better biomarkers and novel therapies. The European NAFLD Registry is an international, prospectively recruited observational cohort study that aims to establish a large, highly-phenotyped patient cohort and linked bioresource. Here we describe the infrastructure, data management and monitoring plans, and the standard operating procedures implemented to ensure the timely and systematic collection of high-quality data and samples. Already recruiting subjects at secondary/tertiary care centres across Europe, the Registry is supporting the European Union IMI2-funded LITMUS 'Liver Investigation: Testing Marker Utility in Steatohepatitis' consortium, which is a major international effort to robustly validate biomarkers that diagnose, risk stratify and/or monitor NAFLD progression and liver fibrosis stage. The European NAFLD Registry has the demonstrable capacity to support research and biomarker development at scale and pace

A liver fibrosis cocktail? Psoriasis, methotrexate and genetic hemochromatosis

BACKGROUND: Pathologists are often faced with the dilemma of whether to recommend continuation of methotrexate therapy for psoriasis within the context of an existing pro-fibrogenic risk factor, in this instance, patients with genetic hemochromatosis. CASE PRESENTATIONS: We describe our experience with two male psoriatic patients (A and B) on long term methotrexate therapy (cumulative dose A = 1.56 gms and B = 7.88 gms) with hetero- (A) and homozygous (B) genetic hemochromatosis. These patients liver function were monitored with routine biochemical profiling; apart from mild perivenular fibrosis in one patient (B), significant liver fibrosis was not identified in either patient with multiple interval percutaneous liver biopsies; in the latter instance this patient (B) had an additional risk factor of partiality to alcohol. CONCLUSION: We conclude that methotrexate therapy is relatively safe in patients with genetic hemochromatosis, with no other risk factor, but caution that the risk of fibrosis be monitored, preferably by non-invasive techniques, or by liver biopsy