Selective inhibition of ion transport mechanisms regulating intracellular pH reduces proliferation and induces apoptosis in cholangiocarcinoma cells.

BACKGROUND: Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH through the activity of the Na(+)/H(+) exchanger and the Na(+) dependent Cl(-)/HCO(3)(-) exchanger. The inhibition of these mechanisms could therefore inhibit cancer cell growth. AIM: We evaluated the effect of two selective inhibitors of these transporters (cariporide and S3705) on proliferation and apoptosis of human cholangiocarcinoma cells (HUH-28 and Mz-ChA-1 cells) as a function of external pH (7.4 and 6.8). METHODS/RESULTS: HUH-28 cells incubated for 24h at external pH 7.4 or 6.8 without inhibitors maintained intracellular pH at physiological level, whereas incubation with cariporide and/or S3705 caused the intracellular pH of cells to drop. Incubation of HUH-28 cells with cariporide and/or S3705 was able to reduce proliferation, evaluated by a colorimetric ELISA method, and to induce apoptosis, evaluated by measuring caspase-3 activity and Annexin-V staining, and these effects were more evident at external pH 6.8. S3705 but not cariporide was able to inhibit serum-induced phosphorylation of ERK1/2, AKT and BAD, intracellular molecules involved in cancer cell proliferation and survival. Similar results were obtained in Mz-ChA-1 cells. CONCLUSIONS: (1) Inhibition of intracellular pH regulatory mechanisms by cariporide and S3705 reduces proliferation and induces apoptosis in cholangiocarcinoma cells; and (2) these drugs might have potential therapeutic value against cholangiocarcinoma.

Effect of cyanidin 3-O-beta-glucopyranoside on hepatic stellate cell proliferation and collagen synthesis induced by oxidative stress.

BACKGROUND: Reactive oxygen species play a role in the pathogenesis of hepatic fibrosis, mainly through the activation of hepatic stellate cells. Cyanidin-3-O-beta-glucopyranoside is a natural antioxidant compound distributed in several fruits and vegetables. AIM: To evaluate the effect of cyanidin-3-O-beta-glucopyranoside on hepatic stellate cells proliferation and collagen synthesis induced by a pro-oxidant agent. METHODS/RESULTS: Oxidative stress was induced by incubation of hepatic stellate cells with a ferric nitrilotriacetate complex (100 micromol/L). Incubation with ferric nitrilotriacetate induced an increased intracellular hydroperoxide formation, which was completely inhibited by cyanidin-3-O-beta-glucopyranoside at a concentration of 50mumol/L. Similarly, cyanidin-3-O-beta-glucopyranoside was able to inhibit ferric nitrilotriacetate-induced hepatic stellate cells proliferation, evaluated by an ELISA method, with a maximal effect at 50mumol/L. Incubation of hepatic stellate cells with cyanidin-3-O-beta-glucopyranoside inhibited ferric nitrilotriacetate-induced extracellular signal-regulated kinase 1/2 activation, evaluated by western blot, whereas it did not affect p70S6 kinase and AKT expression. Finally, cyanidin-3-O-beta-glucopyranoside reduced ferric nitrilotriacetate-induced Na(+)/H(+) exchange activation, evaluated by a spectrofluorimetric method, and collagen type I synthesis, evaluated by northern blot. CONCLUSION: Cyanidin-3-O-beta-glucopyranoside is able to modulate hepatic stellate cells proliferation and type I collagen synthesis induced by a pro-oxidant agent, thus suggesting a potential role for this antioxidant compound in the prevention of fibrosis in chronic liver diseases.

Hepatoprotective and antifibrotic effect of a new silybin-phosphatidylcholine-Vitamin E complex in rats.

BACKGROUND: Silybin, the main active component of silymarin, has been reported to reduce hepatic fibrosis by 30% in bile duct ligated rats, whereas Vitamin E alone does not significantly modify liver damage and collagen deposition in chronic liver injury. AIM: The aim of the present study was to evaluate the hepatoprotective and the antifibrotic properties of a new silybin-phosphatidylcholine-Vitamin E complex, characterised by elevated oral bioavailability and lipophilicity, on rat hepatic fibrosis induced by dimethylnitrosamine administration and by bile duct ligation. METHODS/RESULTS: The complex was administered by gastric gavage at a dose of 250 and 75 mg/kg (as silybin and Vitamin E, respectively). Treatment with the complex 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. This was associated with reduced hepatic stellate cells proliferation both after 1 and 5 weeks of treatment. Treatment with the complex reduced also hepatic stellate cells activation and collagen deposition. Treatment with dimethylnitrosamine induced an increase in alpha1(I) procollagen, TGF(beta1), tissue inhibitor of metalloproteinase 1 and metalloproteinase 2 mRNA expression, which were significantly reduced by administration of the complex. In the bile duct ligation model, the administration of the complex was able to reduce hepatic stellate cells proliferation and activation, as well as collagen deposition and alpha1(I) procollagen mRNA expression. CONCLUSIONS: These results suggest that this new silybin-phosphatidylcholine-Vitamin E complex could be an interesting drug to be tested in patients with chronic liver disease.

Influence of pattern of clinical presentation and of gluten-free diet on bone mass and metabolism in adult coeliac disease.

Since no information is available on bone derangements in subclinical coeliac disease (CD), we evaluated bone mineral density (BMD, expressed as z score) at lumbar spine, by X-ray dual-photon absorptiometry, and serum indices of bone metabolism and remodeling in 14 subclinical or silent patients, 10 classical patients, and 15 healthy volunteers all on a gluten-containing diet. In the subclinical group, BMD at lumbar spine was significantly higher than in the classical group (-1.3 +/- 0.8, 73% vs. -2.6 +/- 0.6, 88%, respectively; p < 0.001), but significantly lower than in volunteers (+0.4 +/- 1.1, 104%; p < 0.001). Similar changes were observed in serum calcium, whereas, as regards parathyroid hormone, no significant difference was found between subclinical and classical patients. 25-vitamin D was significantly lower, and 1,25-vitamin D was significantly higher in subclinical and classical patients than in healthy volunteers. Indices of bone remodeling were higher in the subclinical and classical groups than in the volunteers, but lower in the subclinical than in classical patients. Eight subclinical and 8 classical patients were reexamined after a period of gluten-free diet (GFD), and in both groups BMD had significantly improved. Our results show that osteopenia is a frequent feature also in subclinical CD, although the extent of bone and mineral metabolism derangements is lower than in classical CD. GFD is able to normalize BMD in subclinical and to significantly improve it in classical patients.

The anti-fibrotic effect of pirfenidone in rat liver fibrosis is mediated by downregulation of procollagen alpha1(I), TIMP-1 and MMP-2.

BACKGROUND: Pirfenidone (5 methyl-1-phenyl-2(1H)-pyridone) is a novel anti-fibrotic agent, which has been shown to decrease collagen deposition in a variety of animal models in vivo, and recently in hepatic fibrosis also. At cellular level, we have recently demonstrated that pirfenidone is able to inhibit proliferation of hepatic stellate cells induced by platelet-derived growth factor, as well as collagen type I accumulation and alpha1(I) procollagen mRNA expression. AIMS: To evaluate if pirfenidone maintains its anti-fibrotic properties also when administered after the induction of hepatic damage and to further investigate the molecular mechanisms leading to the anti-fibrotic effect of pirfenidone. METHODS AND RESULTS: Rats treated with dimethylnitrosamine (10 mg/kg) for 5 weeks received a liquid diet containing 0.5% pirfenidone starting from the third week. Pirfenidone treatment reduced the degree of liver injury, as determined by alanine aminotransferase values and necro-inflammatory score, which was associated with reduced hepatic stellate cells proliferation and collagen deposition. Treatment with dimethylnitrosamine increased transcripts levels for transforming growth factorbeta1, procollagen alpha1(I), tissue inhibitors of metalloproteinase-1 and matrix metalloproteinase-2 by 7-, 7-, 4- and 15-fold, respectively. Pirfenidone administration downregulated elevated levels of those transcripts by 50-60%, and this was associated with a 70% reduction in collagen deposition. CONCLUSIONS: (1) Pirfenidone is effective also if administered after the induction of the hepatic damage; (2) the anti-fibrotic effect of pirfenidone is mainly due to the reduced expression of profibrogenic procollagen alpha1(I) and TIMP-1, most likely through the downregulation of transforming growth factorbeta1 mRNA, and of matrix metalloproteinase-2, which is mainly implicated in the degradation of the normal extracellular matrix.

Diagnosis of liver fibrosis.

Hepatic fibrosis and cirrhosis represent the main consequence of chronic liver diseases of different origin. Therefore, the clinical assessment of disease severity is a must in the management of patients with chronic liver damage. Although liver biopsy may be associated with sampling error, interobserver variability and potential complications, it still remains the gold standard for establishing the severity of hepatic necroinflammation and fibrosis. In the last years, several non-invasive tests for the assessment of disease activity and stage have been proposed. However, at present all these tests are not totally accurate and reliable and need further evaluation.

The effect of etanercept on hepatic fibrosis risk in patients with non-alcoholic fatty liver disease, metabolic syndrome, and psoriasis.

BACKGROUND: Patients with psoriasis show a greater prevalence of non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome than the general population. Moreover, patients with NAFLD and psoriasis are at higher risk of severe liver fibrosis than their counterparts with NAFLD and without psoriasis. The link between these three pathological conditions is a chronic low-grade inflammatory status. In this study, we aimed to evaluate the effect of etanercept versus psoralen and UVA (PUVA) therapy on the hepatic fibrosis risk in patients with psoriasis, metabolic syndrome, and NAFLD (with NAFLD diagnosed by ultrasonography). METHODS: Eighty-nine patients with chronic moderate-to-severe plaque-type psoriasis, metabolic syndrome, and NAFLD received etanercept or PUVA treatment. The two groups of patients were compared for anthropometric variables (body mass index and waist/hip ratio), lipid profile, glucose homeostasis, inflammatory status, risk of hepatic fibrosis, and ultrasonographic aspect of the liver, both at baseline (time [T] 0) and after 24 weeks of treatment (T24). RESULTS: After 24 weeks of treatment, only in the group receiving etanercept, we detected significant reductions (p < 0.05) in the aspartate transaminase (AST)/alanine transaminase (ALT) ratio, C-reactive protein (CRP) serum levels, fasting insulin levels, and homeostasis model assessment (HOMA) index, and a significant increase in the Quantitative Insulin-Sensitivity Check Index (QUICKI) (p < 0.05). CONCLUSIONS: In patients with psoriasis, metabolic syndrome, and NAFLD, the risk of the development of hepatic fibrosis seems to be directly correlated with insulin resistance. Etanercept could be more efficacious to reduce the risk of developing hepatic fibrosis than PUVA therapy, and this preventive effect could be related to its anti-inflammatory and glucose homeostatic properties. We note that a limitation of the study was that the diagnosis of NAFLD was conducted by ultrasonography.

Methodology and indications of H2-breath testing in gastrointestinal diseases: the Rome Consensus Conference.

BACKGROUND: Breath tests represent a valid and non-invasive diagnostic tool in many gastroenterological conditions. The rationale of hydrogen-breath tests is based on the concept that part of the gas produced by colonic bacterial fermentation diffuses into the blood and is excreted by breath, where it can be quantified easily. There are many differences in the methodology, and the tests are increasingly popular. AIM: The Rome Consensus Conference was convened to offer recommendations for clinical practice about the indications and methods of H2-breath testing in gastrointestinal diseases. METHODS: Experts were selected on the basis of a proven knowledge/expertise in H2-breath testing and divided into Working Groups (methodology; sugar malabsorption; small intestine bacterial overgrowth; oro-coecal transit time and other gas-related syndromes). They performed a systematic review of the literature, and then formulated statements on the basis of the scientific evidence, which were debated and voted by a multidisciplinary Jury. Recommendations were then modified on the basis of the decisions of the Jury by the members of the Expert Group. RESULTS AND CONCLUSIONS: The final statements, graded according to the level of evidence and strength of recommendation, are presented in this document; they identify the indications for the use of H2-breath testing in the clinical practice and methods to be used for performing the tests.

Osteonecrosis of the femoral head in refractory coeliac disease.

Osteonecrosis has been described occurring in many clinical conditions that require steroid administration. Mechanisms by which steroids produce osteonecrosis are not well known and the importance of the underlying disease has been recently emphasized. We report on a 48-year-old woman with coeliac disease who developed osteonecrosis of the femoral head after being treated with steroids for non-response to gluten withdrawal. We stress the possible role of osteoporosis and osteomalacia, frequently found in patients with coeliac disease, in the pathogenesis of this complication, and advise using drugs other than steroids in the treatment of refractory coeliac disease.

Transforming growth factor beta 1 increases the number of apoptotic bodies and decreases intracellular pH in isolated periportal and perivenular rat hepatocytes.

Transforming growth factor beta 1 (TGF beta 1) is involved in promoting cell death by apoptosis in the liver, whereas the activation of Na+/H+ exchanger has been related to cell proliferation. The aim of this study was to gain information on the effects of TGF beta 1 on intracellular pH and Na+/H+ exchange activity in isolated periportal (PP) and perivenular (PV) rat hepatocytes using the pH-sensitive dye BCECF in a perfused subconfluent hepatocyte monolayer. Steady-state intracellular pH (pHi) in a bicarbonate-free solution (HEPES) were 7.17 +/- 0.031 in PP and 7.15 +/- 0.041 in PV cells. Treatment with TGF beta 1 (120 pmol/L) for 7 hours increased the number of apoptotic bodies by 25% and 38%, and decreased steady-state pHi to 7.11 +/- 0.018 (P = .05) and to 7.07 +/- 0.021 (P < .02), respectively, in PP and PV hepatocytes. In HEPES, cells recovered from an acid load, extruding protons at a rate (JH) of 4.85 +/- 1.01 mmol/L/min in PP cells and of 4.91 +/- 0.99 mmol/L/min in PV hepatocytes. This recovery appeared amiloride inhibitable (1 mmol/L). Culture with TGF beta 1 for 7 hours induced (in HEPES) a decrease of pHi recovery rate from an acid load more in PV (by 46%) than in PP hepatocytes (by 35%, P < .05). Acute administration of epidermal growth factor (EGF) (10 to 100/mL) induced an increase in Na+/H+ exchange activity by 32% and 27%, respectively, in PP and PV cells compared with controls. In contrast, in cells cultured for 7 hours with 120 pmol/L TGF beta 1, the acute administration of EGF slightly increased Na+/H+ exchange activity (by 18%, P < .05) only in PP cells. This study demonstrates that pHi and Na+/H+ exchange activity are decreased by TGF beta 1, which increases the number of apoptotic bodies in periportal and perivenular rat hepatocyte primary cultures.

Observer agreement in endoscopic assessment of ulcerative colitis.

BACKGROUND: Colonoscopy is the investigation of choice to evaluate ulcerative colitis, but the reliability of the assessment of endoscopic signs is not clear. AIMS: The aim of this study was to evaluate interobserver agreement for the identification of endoscopic lesions typical of ulcerative colitis, and the influence of training. MATERIAL AND METHODS: Four experienced observers and 11 endoscopists under training assessed 49 still images selected from endoscopic video recordings. RESULTS: The agreement rate between experienced observers was excellent or good (k > 0.39) for recognition of 10 out of 14 signs or patterns (loss of vascular pattern, erythema, oedema, granular mucosa, blood, pseudopolyp, erosion, ulcer, normal pattern, severe activity), and was poor for pus, stricture, mild activity, moderate activity. The rates between endoscopists under training were excellent or good for 6 items (loss of vascular pattern, erythema, oedema, pseudopolyp, normal pattern, severe activity). CONCLUSIONS: Trained observers can reproducibly record most endoscopic signs of ulcerative colitis. A reliable overall scoring of severity should be based on a simple three-grading scale, i.e. normal pattern, moderate activity, severe activity. Acceptable agreement rates can be obtained by endoscopists under training on some well-defined endoscopic appearances.

Bone mass and metabolism in patients with celiac disease.

BACKGROUND & AIMS: Several aspects of the pathogenesis of osteopenia in celiac disease are still unclear. Therefore, bone mass and metabolism were evaluated in adults with celiac disease in a cross-sectional study. METHODS: Bone mineral density (BMD), assessed by total body dual-photon absorptiometry, and serum indices of bone metabolism and remodeling were evaluated in 17 patients with untreated celiac disease, 14 with celiac disease on a gluten-free diet, and 24 healthy volunteers. RESULTS: BMD, expressed as a z score, was significantly lower in patients with untreated celiac disease than in patients with treated celiac disease and volunteers and lower in patients with treated celiac disease than in volunteers. Similar changes were observed in serum calcium level, whereas intact parathyroid hormone level was significantly higher in untreated than in treated patients with celiac disease and volunteers, and no difference was found between the latter two groups. 25-Vitamin D level was significantly lower and 1,25-vitamin D level significantly higher in untreated celiac disease than in treated celiac disease and volunteers. Indices of bone remodeling were significantly higher in untreated than in treated patients and volunteers and significantly and positively correlated with iPTH in untreated patients with celiac disease. CONCLUSIONS: BMD is almost invariably low in patients with untreated celiac disease. Results in treated patients suggest that gluten-free diet improves but does not normalize BMD. Untreated celiac disease is characterized by high levels of 1,25-vitamin D and by increased bone turnover, caused by the increase in intact parathyroid hormone level.

Lactose intolerance and bone mass in postmenopausal Italian women.

Previous studies on the role of lactose malabsorption in the pathogenesis of postmenopausal osteoporosis have yielded conflicting results and further information is needed. To date, all studies have been carried out on populations with a low prevalence of lactose malabsorption and the lactose intestinal absorptive capacity was tested using a non-physiological dose of lactose. In fifty-eight Italian postmenopausal women (mean age 57 (SD 7) years), bone mineral density (BMD) at lumbar spine, H2 breath response after ingestion of 20 g lactose, intensity of symptoms of intolerance after a lactose load and daily Ca intake were evaluated. No differences were found between women with or without a positive H2 breath test with regard to BMD (-1.2 (SD 0.9) v. -0.9 (SD 0.8)) and Ca intake (509 (SD 266) v. 511 (SD 313) mg/d). On the contrary, both BMD and Ca intake were significantly lower in women with lactose malabsorption and symptoms of intolerance (-1.5 (SD 0.7) and 378 (SD 220) mg/d) than in those with malabsorption without symptoms (-0.9 (SD 0.9) and 624 (SD 254) mg/d). Moreover, in lactose malabsorbers Ca intake was correlated inversely with symptom score (rs -0.31, P < 0.05) and positively with BMD (rs 0.42, P < 0.005). Our results show that in Italian postmenopausal women Ca intake and BMD are not influenced directly by lactose malabsorption; the appearance of symptoms of intolerance seems to influence BMD unfavourably through a reduced Ca intake.

Subclinical coeliac disease: an anthropometric assessment.

OBJECTIVES: To evaluate the prevalence of malnutrition in patients with untreated coeliac disease (CD) according to their pattern of presentation, and the effect of gluten-free diet (GFD) upon nutritional status. DESIGN: Cohort prospective study. SETTING: All subjects were seen at the outpatient 'malabsorption' clinic of the Department of Medical Pathology I, University of Bologna (referral centre), Bologna. SUBJECTS: Eighty consecutive patients with CD (48 with classical and 32 with subclinical presentation), 15 patients with dermatitis herpetiformis (DH) and 40 healthy volunteers (members of the hospital staff). MAIN OUTCOME MEASURES: The nutritional status was evaluated by anthropometric measurements (percentage of ideal body weight for height and sex, percentage of standard triceps skinfold thickness and percentage of ideal arm-muscle circumference). RESULTS: The overall prevalence of malnutrition in our series of CD patients was 53%. Prevalence of malnutrition (actual body weight less than 90% of the ideal) was significantly higher in classical coeliacs (67%) than in subclinical ones (31%, P < 0.002), in patients with DH (13%, P < 0.0003) and in healthy volunteers (13%, P < 0.0001). At diagnosis, percentage values of ideal body weight, triceps skinfold thickness and arm-muscle circumference were significantly lower (P < 0.0001, P < 0.0002 and P < 0.0003, respectively) in classical coeliacs (84.5 +/- 10.6, 71.2 +/- 28.1 and 87.1 +/- 10.8, respectively) than in subclinical coeliacs (95.5 +/- 9.1, 105.6 +/- 41.0 and 94.8 +/- 10.6, respectively). After GFD, 33% of classical and only 3% of subclinical coeliacs were still malnourished. CONCLUSIONS: Prevalence of malnutrition in CD is lower than was previously thought. CD patients with classical presentation may require a longer period of GFD to achieve a significant improvement of their nutritional status, with respect to those with subclinical presentation, probably because of a greater extent of intestinal damage. Finally, a careful evaluation of dietary habits is usually sufficient to identify incomplete adherence to GFD as the reason for nonimprovement of the nutritional status in patients with CD.

Intracellular signaling pathways involved in acetaldehyde-induced collagen and fibronectin gene expression in human hepatic stellate cells.

Ethanol induces liver fibrosis by several means that include, among others, the direct fibrogenic action of acetaldehyde on hepatic stellate cells (HSC). However the mechanisms responsible for this effect are not well understood. In this communication we investigated signal transduction pathways triggered by acetaldehyde leading to upregulation of alpha2(I) collagen and fibronectin gene expression in human HSC. Run-on assays showed that acetaldehyde-enhanced transcription of these 2 genes as early as 2 hours, via de novo protein synthesis-independent and -dependent mechanisms. It also stimulated a time-dependent induction in phosphorylation of pp70(S6K) and extracellular-regulated kinase (1/2) (ERK1/2). These effects were completely prevented by calphostin C, a protein kinase C inhibitor. As expected, acetaldehyde-elicited ERK1/2 phosphorylation was inhibited by PD98059, a MEK inhibitor, but not by wortmannin, a PI3K inhibitor. On the other hand, both of these inhibitors partially inhibited phosphorylation of pp70(S6K) induced by acetaldehyde suggesting that its activation is ERK1/2- and PI3K-dependent. Acetaldehyde-elicited fibronectin and alpha2(I) collagen upregulation was inhibited by calphostin C. However, while PD98059, wortmannin and rapamycin (a pp70(S6K) inhibitor) completely abrogated alpha2(I) collagen upregulation, they had no effect on fibronectin expression. Overall, these data suggest that protein kinase C is an upstream component from which acetaldehyde signals are transduced to other pathways such as PI3K and ERK1/2. In addition, differential activation of these pathways is needed for the increase in fibronectin and alpha2(I) collagen gene expression induced by acetaldehyde in human HSC.

Measurements of the jejunal unstirred layer in normal subjects and patients with celiac disease.

Normal intestinal absorption of nutrients requires efficient luminal mixing to deliver solute to the brush border. Lacking such mixing, the buildup of thick unstirred layers over the mucosa markedly retards absorption of rapidly transported compounds. Using a technique based on the kinetics of maltose hydrolysis, we measured the unstirred layer thickness of the jejunum of normal subjects and patients with celiac disease, as well as that of the normal rat. The jejunum of humans and rats was perfused with varying maltose concentrations, and the apparent Michaelis constant (Km) and maximal velocity (Vmax) of maltose hydrolysis were determined from double-reciprocal plots. The true Km of intestinal maltase was determined on mucosal biopsies. Unstirred layer thickness was calculated from the in vivo Vmax and apparent Km and the in vitro Km of maltase. The average unstirred layer thickness of 11 celiac patients (170 micron) was seven times greater than that of 3 controls (25 micron). The unstirred layer of each celiac exceeded that of the controls. A variety of factors could account for the less efficient luminal stirring observed in celiacs. Although speculative, villous contractility could be an important stirring mechanism that would be absent in celiacs with villous atrophy. This speculation was supported by the finding of a relatively thick unstirred layer (mean: 106 micron) in rats, an animal that lacks villous contractility. Because any increase in unstirred layer slows transport of rapidly absorbed compounds, poor stirring appears to represent a previously unrecognized defect that could contribute to malabsorption in celiac disease and, perhaps, in other intestinal disorders.

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.

Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways.

Insulin and insulin-like growth factor (IGF-1) are mitogenic for fibroblasts and smooth muscle cells. IGF-1 increases in inflamed and fibrotic tissues and induces proliferation of rat hepatic stellate cells (HSC). This study evaluates the potential roles of these hormones in the development of liver fibrosis. Insulin and IGF-1 receptor expression was evaluated by immunohistochemistry in both cultured human HSC and human liver tissue. Phosphorylation of both 70-kd S6 kinase and extracellular-regulated kinase (ERK), cell proliferation, type I collagen gene expression, and accumulation in HSC culture media were evaluated by Western blot, immunohistochemistry for bromodeoxyuridine (BrdU), Northern blot, and enzyme-linked immunosorbent assay, respectively. Insulin and IGF-1 receptors were detected in HSC in vitro and in liver sections from patients with chronic active hepatitis. Insulin and IGF-1 induced 70-kd S6 kinase phosphorylation in HSC, whereas IGF-1 only induced ERK phosphorylation. Insulin and IGF-1 stimulated HSC proliferation in a dose-dependent fashion, with IGF-1 being four to five times more potent than insulin. Cell exposure to specific inhibitors showed that both phosphatidylinositol 3-kinase (PI3-K) and ERK are involved in IGF-1-induced mitogenesis, whereas insulin stimulated mitogenesis through a PI3-K-dependent ERK-independent pathway. IGF-1 increased type I collagen gene expression and accumulation in HSC culture media through a PI3-K- and ERK-dependent mechanism. In conclusion, insulin and IGF-1, which stimulate HSC mitogenesis and collagen synthesis, may act in concert to promote liver fibrosis in vivo by a differential activation of PI3-K- and ERK1-dependent pathways.

Scalloped duodenal folds in childhood celiac disease.

Unlike the adult form, childhood celiac disease has not hitherto been associated with a particular endoscopic pattern. The upper gastrointestinal tracts of 46 children with various stages of celiac disease and of 27 children with conditions other than celiac disease were examined by an endoscopist unaware of the clinical details of the patients. The scalloped configuration of duodenal folds was shown to have a sensitivity of 88% and a specificity of 87% in the diagnosis of subtotal villus atrophy. Attention to this finding, described here for the first time in childhood celiac disease, may be helpful for the diagnosis of this condition.