Emanuele Bendia

Effect of pirfenidone on rat hepatic stellate cell proliferation and collagen production

BACKGROUND/AIMS Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC). METHODS HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70(S6K) activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and alpha1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively. RESULTS Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 microM, with a maximal effect at 1000 microM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70(S6K) activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na(+)/H(+) exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 microM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 microM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor(beta1) by 54% and 92%, respectively, as well as TGF(beta1)-induced alpha1(I) procollagen mRNA expression. RESULTS Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases.

Intracellular pathways mediating Na+/H+ exchange activation by platelet-derived growth factor in rat hepatic stellate cells

BACKGROUND & AIMS The Na+/H+ exchanger is the main intracellular pH regulator in hepatic stellate cells (HSCs), and its activity is increased by platelet-derived growth factor (PDGF). Amiloride, an Na+/H+ exchange inhibitor, reduces PDGF-induced HSC proliferation, suggesting that the Na+/H+ exchanger plays a role in regulating HSC proliferative response. The aim of this study was to characterize the intracellular pathways mediating activation of the Na+/H+ exchanger by PDGF in HSCs. METHODS The activity of the Na+/H+ exchanger and HSC proliferation rate were evaluated under control condition and after incubation with PDGF in the absence or presence of specific inhibitors of the main intracellular pathways of signal transduction. Na+/H+ exchange protein expression was evaluated by means of Western blot. RESULTS PDGF induced a significant increase in the activity of the Na+/H+ exchanger without modifying protein expression. Inhibition of the calcium/calmodulin- and protein kinase C-dependent pathways resulted in a significant inhibition of both Na+/H+ exchange activity and of PDGF-induced HSC proliferation. The involvement of the two pathways was confirmed by showing that incubation of HSCs with both phorbol-12-myristate-13-acetate, a potent protein kinase C activator, and thapsigargin, which increases intracellular calcium levels, significantly increased both the Na+/H+ exchanger activity and HSC proliferation rate. Inhibition of the protein kinase A pathway did not modify either PDGF-induced Na+/H+ exchange activation or PDGF-induced HSC proliferation. On the contrary, inhibition of the mitogen-activated protein kinase- and of phosphatidylinositol 3-kinase-dependent pathways significantly reduced PDGF-induced HSC proliferation without affecting the activity of the Na+/H+ exchanger. CONCLUSIONS Activation of the Na+/H+ exchanger by PDGF in HSCs is mediated by calcium/calmodulin- and protein kinase C-dependent pathways. PDGF-induced HSC proliferation is mediated by Na+/H+ exchange-dependent and -independent pathways.

Intracellular pH regulation and Na+/H+ exchange activity in human hepatic stellate cells: effect of platelet-derived growth factor, insulin-like growth factor 1 and insulin

BACKGROUND/AIMS The Na+/H+ exchanger is involved in rat hepatic stellate cell (HSC) proliferation induced by platelet-derived growth factor (PDGF). We therefore evaluated in human HSC: (1) the mechanisms of intracellular pH regulation; (2) the relationship between Na+/H+ exchange activation and cell proliferation induced by PDGF, insulin-like growth factor 1 (IGF-1) and insulin. METHODS/RESULTS pH(i) regulation was mainly dependent on the activity of the Na+/H+ exchanger, which was evaluated by measuring pH(i) recovery from an acute acid load. PDGF (25 ng/ml) gradually increased the activity of the Na+/H+ exchanger which peaked at 18 h and remained stable until the 24th h. IGF-1 (10 nmol/l), but not insulin (100 nmol/l), slightly but significantly increased the activity of the Na+/H+ exchanger. Amiloride (100 micromol/l) and 20 micromol/l 5-N-ethyl-N-isopropyl-amiloride completely inhibited HSC proliferation (evaluated by measurement of bromodeoxyuridine incorporation) induced by PDGF and IGF-1, but did not affect proliferation of HSC induced by insulin. Finally, IGF-1 did not modify the activity of the Na+/Ca2+ exchanger. CONCLUSIONS The Na+/H+ exchanger is involved in HSC proliferation induced by PDGF and IGF-1, whereas the proliferative effect of insulin is mediated by intracellular pathways which are Na+/H+ exchange-independent.

Rearrangement of the cytoskeletal network induced by platelet-derived growth factor in rat hepatic stellate cells: role of different intracellular signalling pathways.

BACKGROUND/AIMS Cytoskeletal reorganization plays an important role in the regulation of different cell functions, such as proliferation and migration. Since platelet-derived growth factor (PDGF) stimulates both proliferation and chemotaxis of hepatic stellate cells (HSC), we investigated the effects of this cytokine on cytoskeletal components of cultured rat HSC. METHODS/RESULTS Exposure of HSC to PDGF induced the formation of stress fibres and of a ruffled configuration of the plasma membrane, evaluated by both fluorescence and electron microscopy. These modifications were also induced by exposure to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) and abolished by pretreatment with the PKC inhibitor calphostin C, with the Rho inhibitor C3 exoenzyme and with the intracellular calcium chelator MAPTAM, but not with the PI-3 kinase inhibitor wortmannin or with the mitogen-activated protein kinase kinase inhibitor PD 98059. PDGF induced a translocation of Rho from the cytosol to the membrane which was inhibited by C3 exoenzyme and by calpostin C, and which was also induced by PMA. Moreover, PDGF induced a rearrangement of vinculin which was prevented by C3 exoenzyme and calphostin C. CONCLUSIONS PDGF-induced cytoskeletal reorganization in HSC is dependent on PKC and Rho, thus suggesting that these two pathways may play an important role in the response of liver to injury.

Role of Small Intestinal Bacterial Overgrowth and Helicobacter pylori Infection in Chronic Spontaneous Urticaria: A Prospective Analysis

The aim of this study is to assess the associations between chronic spontaneous urticaria (CSU), Helicobacter pylori infection and small intestinal bacterial overgrowth. Forty- eight patients with CSU were studied by scoring the urticaria activity and assesing the quality of life. Patients with H. pylori infection (n=11) or small intestinal bacterial overgrowth (n=13) were specifically treated for one week and clinically evaluated both before and 4 weeks after the eradication therapy. Eradication of H. pylori infection led to a significant improvement in CSU (p<0.002). In contrast, eradication of small intestinal bacterial overgrowth was not associated with any clinical improvement in CSU, despite the fact that these patients had statistically significant more urticaria activity at baseline. Thus there is no evidence to support the eradication of small intestinal bacterial overgrowth in CSU, but eradication of H. pylori infection may result in an improvement of the disease.

Diagnosis of liver cirrhosis by transit-time analysis at contrast-enhanced ultrasonography

PurposeThe aims of this prospective study were to evaluate analysis of sulfur-hexafluoride-filled microbubble contrast agent (Sonovue) transit times as a tool for differentiating liver cirrhosis from the noncirrhotic stage of liver disease and to compare its performance with that of conventional B-mode and Doppler ultrasonography (US).Materials and methodsContrast-enhanced hepatic ultrasonography with the US contrast agent Sonovue was performed on 38 patients with diagnoses of hepatic cirrhosis based on unequivocal clinical signs or liver biopsy findings (Child-Pugh classes A in 19, B in 16 and C in three), 31 patients with noncirrhotic diffuse liver disease (biopsy confirmed) and 14 controls without diffuse liver disease. Time curves of hepatic-vein signal intensity were analysed using objective criteria to determine the time of enhancement onset (hepatic-vein arrival time) and peak enhancement (hepatic-vein peak enhancement). Accuracy in diagnosing cirrhosis was compared with that based on B-mode and Doppler data.ResultsHepatic-vein arrival time in cirrhotic patients was significantly shorter (p<0.01) than in noncirrhotic (chronic liver disease and controls) patients. Peak enhancement times in these three groups were not significantly different. An arrival-time cutoff of 17 s distinguished cirrhotic from noncirrhotic patients with high accuracy (100% sensitivity, 93.3% specificity, positive and negative predictive values 92.6% and 100%, respectively) and excellent reproducibility (kappa coefficients of 1.0 and 0.93 for intraand interobserver agreement). Contrast-enhanced US showed better sensitivity than the B-mode and Doppler data.ConclusionsAnalysis of the time of onset of US contrast enhancement of the hepatic vein appears to be a potentially useful noninvasive supplement to conventional sonography and Doppler in the follow-up of patients with chronic diffuse liver disease.RiassuntoObiettivoLo scopo di questo studio prospettico è stato quello di valutare l’analisi del tempo di transito delle microbolle di mezzo di contrasto costituito da esafluoruro di zolfo (Sonovue) come mezzo per differenziare la cirrosi epatica dagli stadi non cirrotici di malattia e di comparare la sua performance con quella dell’esame B-mode standard e Doppler.Materiali e metodiÈ stato effettuato l’esame ecografico del fegato con mezzo di contrasto Sonovue in 38 pazienti con diagnosi di cirrosi basata su segni clinici inequivocabili o su biopsia epatica (Child-Pugh A in 19 pazienti, B in 16 e C in 3). Sono stati analizzati inoltre 31 pazienti con patologia cronica diffusa non cirrotica (confermata dalla biopsia), e 14 pazienti di controllo senza patologia epatica. Sono stati analizzati i diagrammi tempo/intensità di segnale della vena sovraepatica utilizzando criteri oggettivi per determinare il tempo di inizio dell’enhancement (tempo di arrivo della vena sovraepatica) e il picco di enhancement (tempo di picco della vena sovraepatica). L’accuratezza nella diagnosi di cirrosi è stata confrontata con quella basata sui dati B-mode e Doppler.RisultatiIl tempo di arrivo della vena sovraepatica nei pazienti cirrotici è risultato significativamente pi`u breve (p<<0,01) rispetto ai pazienti non cirrotici (patologia epatica cronica e controlli). Il tempo di picco di enhancement in questi tre gruppi non è risultato significativo. Un tempo cut-off di arrivo di 17 secondi ha distinto pazienti cirrotici da quelli non cirrotici con elevata accuratezza (100% sensitività, 93,3% specificità, valore predittivo positivo e negativo: 92,6% e 100%, rispettivamente) ed eccellente riproducibilità (coefficiente κ di 1,0 e 0,93 per l’accordo intra-ed interosservatore). L’ecografia con mezzo di contrasto ha mostrato una migliore sensibilità rispetto all’esame B-mode e Doppler.ConclusioniL’analisi del tempo di inizio dell’enhancement contrastografico della vena sovraepatica appare un potenziale e non invasivo supplemento all’ecografia convenzionale e all’esame Doppler nel follow-up di pazienti con patologia epatica cronica diffusa.

Ethyl caffeate from Verdicchio wine: chromatographic purification and in vivo evaluation of its antifibrotic activity.

Ethyl caffeate (CfE, caffeic acid ethyl ester) was extracted from dealcoholized Verdicchio, a white wine from Marche (Italy) with ethyl acetate and then purified with semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC) using an ODS2 column (25 cm x 20 mm id) at an isocratic flow of 5 mL/min (the mobile phase A was formic acid 4.5% in water and the mobile phase B was acetonitrile). The CfE extract administered intraperitoneally at 1 mumol/L in rats previously treated with 10 mg/kg dimethylnitrosamine was able to prevent the dimethylnitrosamine-induced loss in body and liver weight, as well as to reduce the degree of liver injury, as determined by alanine aminotransferase values and necroinflammatory score, after a 1-week treatment. This was associated with a reduced hepatic stellate cells activation (from 16.8 to 8.3% of smooth muscle actin positive parenchyma) and proliferation (from 11.3 to 5.5 cells/mm(2)). The collagen synthesis was also reduced: the percentage of Sirius Red positive parenchyma decreased from 21.7 to 7.2%. The CfE levels of Verdicchio wine determined with RP-HPLC-DAD were about 14 times the active levels tested in the in vivo test. CfE can be considered as a promising natural compound for future application in chronic liver disease.

Helicobacter pylori infection in psoriasis: results of a clinical study and review of the literature

Data from the literature concerning the role of Helicobacter pylori (H. pylori) infection in psoriasis are still conflicting. This study was carried out to evaluate prevalence of H. pylori in patients with mild to severe psoriasis, correlation between H. pylori infection and severity of psoriasis, and effect of H. pylori eradication on the clinical course of psoriasis.

Biologic Drugs in Crohn's Disease and Ulcerative Colitis: Safety Profile.

Ulcerative Colitis (UC) and Crohns Disease (CD) are chronic, progressive and disabling disorders characterized by a heterogeneous clinical course. Some years ago the main goal of the therapy was to achieve and maintain clinical remission, whereas at present the main goal of therapy is represented by the deep remission, characterized by sustained clinical remission, complete mucosal healing and normalization of serological markers of inflammation. In the last years new therapeutic approaches have been introduced which have led to a reduction in the mortality rate and have modified the natural history of Inflammatory Bowel Diseases (IBD). In addition, several prognostic factors have been identified which have allowed to better stratify the disease and to choose the most appropriate therapy for the single patient. Moreover, early treatment with immunosuppressive drugs and/or biologics has changed, at least in the short term, the course of the disease by reducing hospitalization rate and the need for surgery. Therefore, the development of biologic therapies has represented an important step in the treatment of IBD, since these drugs induce remission and response rates that are not achieved by other therapies. Since their use can result in significant adverse events that increase morbidity, patients must be aware of the risks associated with treatment and must be strictly monitored. Although treatment with biologic drugs is not successful in all patients and many of them lose clinical response, new therapies are currently under evaluation.

Endoscopic Retrograde Cholangiopancreatography and Endoscopic Sphincterotomy in Acute Pancreatitis: Indications and Technique

Acute pancreatitis is a disease of increasing annual incidence which is associated with significant morbidity and mortality. While many patients need only a general supportive care, about one out of five patients will develop severe acute pancreatitis, and 20% of these patients may die [1, 2].