s of the 29 th National Congress of Digestive Diseases / Digestive and Liver Disease 55S2 (2023) S77-S209 with an increase in body mass index and waist-to-hip ratio (p<0.0001 for all).Low/high-density lipoprotein, glucose, insulin, homeostatic model assessment for insulin resistance, and transaminases worsened at T2 (insulin: p=0.0003, glucose: p=0.0007; p<0.0001 for the others).The non-invasive tools assessments revealed a NAFLD fibrosis score, liver stiffness measurement, and controlled attenuation parameter impairment at T2 (p<0.0001all).The BIA evidenced an increase in Fat Mass (FM) and a reduction of Free Fat Mass (FFM), Body Cell Mass (BCM), and Extracellular Mass (ECM) at T2 in comparison to the other study time points.During the confinement, the overall HCC and Milan-out staged HCC occurrence revealed HR:2.521, p=0.01, and HR:13.78,p=0.0009 respectively.Contrariwise to the modifications of the biochemical parameters, a significant association between body composition parameters modification during the study evaluations with HCC overall and HCC Milan-out criteria occurrence was highlighted.

OBJECTIVE:Diagnosis of coeliac disease is based on the presence of villous atrophy which recovers following a gluten-free diet. The presence of circulating antiendomysial antibodies as well as their disappearance after a gluten-free diet supports the diagnosis. It has also been demonstrated that antiendomysial antibodies are detectable in supernatants of cultured intestinal biopsies from patients with coeliac disease. The objective of this study was to compare the histology and antiendomysial antibodies in culture supernatants of intestinal biopsies to validate the in vitro organ culture system as a future diagnostic tool for coeliac disease.MATERIAL AND METHODS:Seventy-five antiendomysial serum-positive patients on a gluten-containing diet were evaluated. Patients underwent endoscopy with 5 biopsy fragments: 3 for histology, 1 cultured with and the other without gliadin-peptide activator. Antiendomysial antibodies were evaluated in all culture supernatants.RESULTS:Sixty-eight patients had evidence of villous atrophy, while 73 out of 75 were positive to the organ culture system. The agreement rate between organ culture and histology results was 94%.CONCLUSIONS:As all the centres participating in the study obtained good agreement between organ culture and histology results, the new system could be considered a reliable tool for the diagnosis of coeliac disease. Nevertheless, it is possible to highlight cases with an organ culture-positive and -negative histology. This feature could be of considerable interest because, as the sensitivity of organ culture seems to be greater than the initial histology, the new system might be useful in uncertain cases where the risk of missing the diagnosis of coeliac disease is high.

Experimental evidence indicates that reactive oxygen species (ROS) are involved in the development of hepatic fibrosis; they induce hepatic stellate cells (HSC) proliferation and collagen synthesis. To address the role of matrix metalloproteinase (MMP)-2 in promoting HSC proliferation during hepatic injury, we investigated whether oxidative stress modulates the growth and invasiveness of HSC by influencing MMP-2 activation. Cell invasiveness and proliferation, which were studied using Boyden chambers and by counting cells under a microscope, were evaluated after treatment with a superoxide-producing system, xanthine plus xanthine oxidase (X/XO), in the presence or absence of antioxidants and MMP inhibitors. Expression and activation of MMP-2 were evaluated via gel zymography, immunoassay, and ribonuclease protection assay. The addition of X/XO induced proliferation and invasiveness of human HSC in a dose-dependent manner. The addition of antioxidants as well as MMP-2-specific inhibitors impaired these phenomena. X/XO treatment increased MMP-2 expression and secretion appreciably and significantly induced members of its activation complex, specifically membrane-type 1 MMP and tissue inhibitor metalloproteinase 2. To study the intracellular signaling pathways involved in X/XO-induced MMP-2 expression, we evaluated the effects of different kinase inhibitors. The inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphatidyl inositol 3-kinase (PI3K) abrogated X/XO-elicited MMP-2 upregulation and completely prevented X/XO-induced growth and invasiveness of HSC. In conclusion, our findings suggest that MMP-2 is required for the mitogenic and proinvasive effects of ROS on HSC and demonstrate that ERK1/2 and PI3K are the main signals involved in ROS-mediated MMP-2 expression.

Thiazolidinediones (TZDs) are a new class of antidiabetic drugs that have also been shown to possess antitumoral properties in different human cancers. TZDs bind and activate the peroxisome proliferator-activated receptor (PPAR)-gamma, which is a nuclear receptor acting as a transcription factor in several tissues. In the present study, we evaluated PPARgamma mRNA and protein expression in tissue samples of human adrenocortical carcinomas (ACCs), normal adrenal glands, and the human ACC cell line H295R. PPARgamma mRNA was expressed in six of eight ACC, two of three normal adrenal glands and the H295R cells. These results were confirmed by immunohistochemistry. PPARgamma transcriptional activity in H295R cells, monitored by a reporter gene assay, was induced 2- to 3-fold by TZDs, such as rosiglitazone (RGZ) and pioglitazone, whereas in PPARgamma-transfected cells RGZ alone or RGZ plus 9-cis retinoic acid further increased reporter activity. TZDs inhibited both the proliferation and invasiveness of H295R cells in a dose-dependent manner. Thymidine incorporation was reduced by about 60% by 20 mum of both TZDs. Cotreatment with the retinoic X receptor ligand 9-cis retinoic acid had an additive effect. TZDs increased the number of cells in the G(0)/G(1) phase and decreased them in the S phase. Western blot analysis showed that TZDs increased the expression of the cell cycle inhibitors p21 and p27 and reduced the expression of cyclin D1. Twenty micromoles of RGZ and pioglitazone reduced H295R invasiveness through Matrigel by about 85%. Zymography and ELISA tests showed that TZD inhibited metalloproteinase-2 secretion by H295R cells in a dose-dependent manner. These data suggest that TZDs reduce the malignant potential of the H295R ACC cell line and, therefore, might potentially constitute a novel tool in the medical treatment of human ACCs.

Background/Aim: Controlled proteolysis is a prerequisite for cell migration, angiogenesis, and matrix remodelling during gastric ulcer healing. We studied the temporal and spatial expression of three matrix metalloproteinases, gelatinase A (MMP-2), interstitial collagenase (MMP-13), stromelysin (MMP-3), and their major inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) during experimental gastric ulcer healing induced in rats by acetic acid injection. Methods: Gastric tissue specimens were hybridized with antisense 35S-labelled RNA probes and the autoradiographic signal was analyzed by a computer aided image system. Gelatinase activity was analyzed by in situ and gel zymography. Results: During gastric ulcer healing, MMP-2, MMP-3, and MMP-13 RNA expression was increased in stromal cells of the gastric mucosa bordering the ulcer, suggesting a prevalent role of non-epithelial cells in pericellular proteolysis. Gelatinolytic activity was increased during ulcer healing and it was associated with extracellular matrix of the healing mucosa and newly formed vessels. In contrast to MMP-2 RNA, which was homogeneously distributed in all layers of the ulcer bed, MMP-3 and MMP-13 RNAs were confined to the upper layers of the granulation tissue. TIMP-1 RNA was detected in both epithelial and stromal cells of the gastric mucosa adjacent to the ulcer, as well as in the granulation tissue of the ulcer bed. Both MMP and TIMP-1 expression returned to basal levels during the late stages of tissue remodeling. Conclusion: Gastric ulcer repair is associated with a transient expression of specific metalloproteinases and their inhibitors in a distinct anatomical pattern pointing to complex cellular and cell/matrix interactions in the various layers of the healing mucosa.

BACKGROUND/AIMS:Thiazolidinediones (TZD) are a new class of oral antidiabetic drugs that have been shown to inhibit growth of some epithelial cancer cells. Although TZD were found to be ligands for peroxisome proliferators activated receptor gamma (PPARgamma) the mechanism by which TZD exert their anticancer effect is currently unclear. Furthermore, the effect of TZD on local motility and metastatic potential of cancer cells is unknown. The authors analysed the effects of two TZD, rosiglitazone and pioglitazone, on invasiveness of human pancreatic carcinoma cell lines in order to evaluate the potential therapeutic use of these drugs in pancreatic adenocarcinoma.METHODS:Expression of PPARgamma in human pancreatic adenocarcinomas and pancreatic carcinoma cell lines was measured by reverse transcription polymerase chain reaction and confirmed by western blot analysis. PPARgamma activity was evaluated by transient reporter gene assay. Invasion assay was performed in modified Boyden chambers. Gelatinolytic and fibrinolytic activity were evaluated by gel zymography.RESULTS:TZD inhibited pancreatic cancer cells' invasiveness, affecting gelatinolytic and fibrinolytic activity with a mechanism independent of PPARgamma activation and involving MMP-2 and PAI-1 expression.CONCLUSION:TZD treatment in pancreatic cancer cells has potent inhibitory effects on growth and invasiveness suggesting that these drugs may have application for prevention and treatment of pancreatic cancer in humans.

Background/Aims: Little is known about the role of fractalkine (CX3CL1) in the liver. The aim of this study was to investigate the expression patterns of fractalkine and its receptor CX3CR1 in normal human liver and in conditions of injury.Methods: Distribution and expression of fractalkine and its receptor were investigated using immunohistochemistry, in situ hybridization, flow cytometry and reverse transcriptase-polymerase chain reaction. In vitro experiments were conducted in HepG2 cells.Results: Both fractalkine and CX3CR1 were up-regulated during chronic injury, in areas of portal and lobular inflammation. In severe acute hepatitis, fractalkine and CX3CR1 were expressed at high levels not only in areas of inflammation but also in regenerating epithelial cells within bile duct-like structures, which showed co-expression of fractalkine and cytokeratin-7 or CX3CR1. The human hepatocarcinoma cell line HepG2 expressed fractalkine at the gene and protein level, and HepG2-conditioned medium was chemotactic for cells overexpressing CX3CR1. Transcripts for CX3CR1 were detected in HepG2, and exposure of these cells to recombinant fractalkine induced cell migration.Conclusions: This study shows that the fractalkine system is up-regulated during liver damage, and suggests that fractalkine may play a role in the recruitment and adhesion of inflammatory cells and in the biology of liver epithelial cells. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

The activation of hepatic stellate cells (HSC) during liver fibrogenesis has been shown to be mediated by paracrine and autocrine loops involving transforming growth factor-beta1 (TGF-beta1) as the main fibrogenic mediator secreted by activated macrophages, endothelial cells and liberated by disintegrated platelets. The cell-associated plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We have studied whether TGF-beta1 could modulate the plasminogen activation system in human HSC and the role of such protease system in the activity of TGF-beta1 on HSC. Urokinase plasminogen activator receptors (u-PAR), u-PA and plasminogen activator inhibitor type 1 (PAI-1) were determined by immunoassay and RNase protection assay. Cell migration, evaluated either as chemotaxis or as chemoinvasion, was studied in Boyden chambers after addition of TGF-beta1, and inhibition with anti-u-PA and anti-u-PAR antagonists [antibodies against u-PA and u-PAR and antisense oligonucleotides (aODN) against u-PAR mRNA]. We have shown that TGF-beta1 is not mitogenic for HSC, while it is a powerful motogen either in chemotaxis or chemoinvasion assays. TGF-beta1 up-regulates the synthesis and expression of PAI-1, as well as u-PAR expression and exposure at the cell membrane, while it does not affect u-PA levels. TGF-beta1-dependent chemoinvasion of reconstituted basement membrane exploits the cell-associated plasminogen activation system, since it is blocked by monoclonal antibodies against u-PA and against various u-PAR domains, as well as by anti-u-PAR aODN. We have also observed a cumulative effect of TGF-beta1, b-FGF and PDGF in the invasion assay of HSC: in the presence of low amounts of TGF-beta1 the chemoinvasive activity of PDGF and bFGF is dramatically increased. Also this cooperation requires u-PAR and is inhibited by monoclonal antibodies against u-PAR domains I, II and III.

Chronic alcoholic pancreatitis (CAP) is characterized by progressive pancreatic fibrosis and loss of the acinar cell mass, but the pathogenesis of pancreatic fibrosis in the human is poorly understood. It has been recently suggested that lipid peroxidation-derived aldehydes such as 4-hydroxynonenal (HNE) are involved in tissue damage and fibrosis in other organs. The aim of this study was to evaluate the role of oxidative stress in the development of alcohol-induced pancreatic fibrosis in humans, and to assess the contribution of pancreatic periacinar stellate cells (PSC) in the in vivo synthesis of extracellular matrix components during CAP. Lipid peroxidation was evaluated in tissue specimens obtained from patients with CAP who underwent surgical procedures, by immunohistochemistry using a monoclonal antibody directed against HNE-protein adducts. Immunohistochemical determination of collagen type I, alpha-smooth muscle actin (alpha-SMA), and the beta subunit of human platelet-derived growth factor (PDGF-Rbeta) was also performed. In addition, the tissue mRNA expression of procollagen I, PDGF-Rbeta, and transforming growth factor-beta1 (TGF-beta1) was evaluated by in situ hybridization. In CAP, increased formation of HNE-protein adducts was evident in acinar cells adjacent to the interlobular connective tissue that stained positively for collagen type I. HNE staining was absent in normal pancreas. Several non-parenchymal periacinar cells (PSC) underlay the HNE-stained acinar cells. Those PSC stained positively for alpha-SMA and PDGF-Rbeta and showed active synthesis of procollagen type I by in situ expression of the specific mRNAs. The pattern of expression of PDGF-Rbeta mRNA reflected that observed in immunostaining, showing increased amounts of transcripts in PSC. TGF-beta1 mRNA expression was increased in CAP, but transcripts were found in several cell types including PSC, acinar, and ductal cells. These results indicate that significant lipid peroxidation phenomena occur in CAP and that they are associated with active synthesis of collagen by PSC.

During liver fibrogenesis, hepatic stellate cells (HSC) proliferate and migrate under the influence of growth factors, including platelet-derived growth factor (PDGF) and basic-fibroblast growth factor (b-FGF). The plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We evaluated the expression and biological functions of the plasminogen activation system in human HSC and its interaction with PDGF and b-FGF. Urokinase-plasminogen activator receptors (u-PAR) were measured by radioligand binding, cell cross-linking, immunoassay, and RNAse protection assay. u-PA and plasminogen activator inhibitors (PAIs) expression and activities were analyzed by zymography, immunoassay, and RNase protection assay. Cell migration and proliferation, studied in Boyden chambers and by microscopic counting, were evaluated after the addition of PDGF, b-FGF, and blockade with anti-u-PA, anti-u-PAR antibodies, and antisense oligodeoxynucleotides (aODN) against u-PAR mRNA. We have shown that HSC produce u-PAR, u-PA, and PAI-1. PDGF and b-FGF up-regulate u-PA and u-PAR, but not PAI-1, and exogenous addition of u-PA stimulates HSC proliferation, chemotaxis, and chemoinvasion. Inhibition of u-PA/u-PAR with antibodies against u-PA or u-PAR and with u-PAR aODN inhibit the proliferative, chemotactic, and chemoinvasive activity of PDGF and b-FGF. These findings indicate that u-PA and u-PAR are required for the mitogenic and chemoinvasive activity of PDGF and b-FGF on HSC.

Increased expression of monocyte chemotactic protein-1 (MCP-1) has been indicated as a mechanism underlying leukocyte recruitment after liver injury. In this study we examined the temporal relationship between MCP-1 expression and the appearance of monocyte infiltration during acute liver injury. In addition, we tested the effects of vitamin E, a well known antioxidant, on these parameters. Rats were intoxicated with a single intragastric administration of CCl4 with or without pretreatment with vitamin E (atocopherol).Monocyte chemotactic protein-1 expression was analyzed by northern blotting and in situ hybridization and monocyte infiltration was determined by ED-1 immunostaining. The results were quantitated by computerized image analysis. Expression of MCP-1 mRNA was significantly increased as early as 12 hours following injury, and progressively increased thereafter. In contrast, a significant increase in the number of ED-1 positive cells, an index of monocyte infiltration, was observed only 24 and 48 hours after injury.Vitamin E markedly reduced MCP-1 expression at the mRNA and protein levels, and caused a significant reduction in the number of monocyte/macrophages, indicating a role for oxidative stress in the induction of MCP-1 expression in vivo. Accordingly, in cultured hepatic stellate cells, different oxidative stress-related molecules increased MCP-1 mRNA.These data suggest the existence of a direct relationship between MCP-1 expression and monocyte infiltration after acute liver injury, and that preventing the generation of oxidative stress-related molecules results in decreased expression and release of this chemokine.

Background/Aims: Chronic cholestasis stimulates a fibroductular reaction which may progress to secondary biliary fibrosis and cirrhosis, Since platelet-derived growth factor has been indicated as a major fibrogenic factor in chronic liver disease, we analyzed its expression and that of its receptor beta subunit in a rat model of chronic cholestasis.Methods: Liver tissue samples collected at 7, 10, 21, and 28 days after induction of cholestasis obtained by bile duct ligation, were analyzed by immunohistochemistry, in situ hybridization and RNase protection assay for the expression of platelet-derived growth factor (PDGF)-B chain and; receptor beta subunit, Furthermore, the expression of PDGF-B chain mRNA was analyzed in highly purified cholangiocytes from normal and cholestatic rat liver.Results: In cholestatic liver, platelet-derived growth factor-BE and B chain mRNA expression increased up to 4 weeks in epithelial cells of proliferating bile ducts, and periductular mesenchymal cells. The increased expression of PDGF-B chain mRNA was confirmed in highly purified cholangiocytes obtained from normal and cholestatic rat liver. The expression of the receptor beta subunit progressively increased after induction of cholestasis and was mainly localized to desmin-positive periductular hepatic stellate cells.Conclusions: These data suggest that platelet-derived growth factor-B chain can be synthesized by cholangiocytes during chronic cholestasis. The presence of its receptor on periductular hepatic stellate cells raises the possibility that, in this experimental setting, this cytokine might contribute to fibrogenesis in vivo.

Thrombin is generated during tissue damage in several organs, including the liver, and participates in the process of tissue repair through proteolytic activation of a specific thrombin receptor (TR). The aim of this study was to investigate TR expression in human liver by immunohistochemistry and in situ hybridization. In normal liver, immunostaining for TR was present in the endothelial lining of the hepatic sinusoids. During chronic hepatitis, several cells expressing the TR were detected in the inflammatory infiltrate of portal tracts. In cirrhosis with chronic active hepatitis, expression of the TR was also present in mesenchymal cells of fibrous septa. TR expression was markedly up-regulated during fulminant hepatitis, with the highest expression in mesenchymal cells in areas of regeneration. Up-regulation of TR expression was associated with increased levels of TR messenger RNA (mRNA), as assessed by in situ hybridization and RNAse protection assay of liver RNA. Immunostaining of serial sections using specific cellular markers showed that different nonparenchymal cells contribute to TR expression during liver injury. TR expression was also shown in cultured human hepatic stellate cells, with increasing signal comparing activated versus quiescent cells. Because thrombin is rapidly generated after tissue damage, regulated TR expression may be involved in tissue remodeling and/or scarring during liver damage.