Antitumour efficacy of MEK inhibitors in human lung cancer cells and their derivatives with acquired resistance to different tyrosine kinase inhibitors.

BACKGROUND: To study the molecular mechanisms regulating cancer cell resistance to four different tyrosine kinase inhibitors (TKIs): erlotinib, gefitinib, vandetanib and sorafenib. METHODS: An in vitro model of acquired resistance to these TKIs was developed by continuously treating the human lung adenocarcinoma cell line CALU-3 with escalating doses of each drug. Transcriptional profiling was performed with Agilent whole genome microarrays. Western blot analysis, enzyme-linked immunosorbent (ELISA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation, migration, invasion and anchorage-independent colony growth assays were conducted in vitro and experiments with established xenografts in athymic nude mice were performed in vivo in parental (P) and TKI-resistant (R) CALU-3 cell lines. RESULTS: As compared with P-CALU-3 cells, in TKI-R CALU-3 cell lines a significant increase in the expression of activated, phosphorylated MET, IGF-1R, AKT, MEK, MAPK and of survivin was observed. Downregulation of E-cadherin and amphiregulin mRNAs and upregulation of vimentin, VE-cadherin, HIF-1α and vascular endothelial growth factor receptor-1 mRNAs were observed in all four TKI-R CALU-3 cell lines. All four TKI-R CALU-3 cells showed increased invasion, migration and anchorage-independent growth. Together, these data suggest epithelial to mesenchymal transition (EMT) in TKI-R CALU-3 cells. Treatment with several agents that target AKT, MET or IGF-1R did not affect TKI-R CALU-3 cell proliferation. In contrast, treatment with MSC19363669B and selumetinib, two selective MEK inhibitors, caused inhibition of cell proliferation, invasion, migration, anchorage-independent growth in vitro and of tumour growth in vivo of all four TKI-R CALU-3 cell lines. CONCLUSION: These data suggest that resistance to four different TKIs is characterised by EMT, which is MEK-inhibitor sensitive in human CALU-3 lung adenocarcinoma.

The effect of a new symbiotic formulation on plasma levels and peripheral blood mononuclear cell expression of some pro-inflammatory cytokines in patients with ulcerative colitis: a pilot study.

BACKGROUND: During intestinal inflammation white blood cells are recruited from the blood, and they represent the major contributors to tissue perpetuation of inflammation via their production of chemokines and proinflammatory cytokines. OBJECTIVES: Investigate the effect of a symbiotic formulation containing Lactobacillus Paracasei B 20160 versus placebo, on serum levels of interleukin (IL)-6, tumor necrosis factor (TNF)alpha, IL-8, IL-1beta and IL-10 and on mRNA lymphomonocyte expression of TNFalpha, IL-8 and IL-1beta in patients with ulcerative colitis. MATERIALS AND METHODS: Eighteen patients entered the study with histologically proven not complicated ulcerative colitis, treated with mesalazine. Patients were treated for 8 weeks (9 with symbiotic and 9 with placebo). Serum levels of IL-6, TNFalpha, IL-8, IL-1beta and IL-10 were measured using a commercially available sandwich ELISA kit. RT-PCR analysis was performed on total RNA isolated from peripheral lymphomonocytes. RESULTS: In basal condition, there was an increase of serum levels of TNFalpha, IL-6, and IL-8. The treatment with symbiotic significantly decreased serum levels of the last two cytokines (IL-6 and IL-8). In lymphocytes, the treatment with the symbiotic don't significantly reduced the mRNA expression of TNFalpha and IL-1beta, while that of IL-8 was strongly and significantly decreased. CONCLUSION: Our preliminary results suggest that a symbiotic formulation containing Lactobacillus paracasei significantly improves the plasma and lymphocyte content of some proinflammatory cytokines.

Peptide Hp(2-20) accelerates healing of TNBS-induced colitis in the rat.

BACKGROUND AND AIMS: Hp(2-20), a Helicobacter pylori-derived peptide interacting with N-formyl peptide receptors (FPRs), accelerates the healing of gastric injury in rats. Whether Hp(2-20) affects the recovery of inflamed colonic mucosa is unknown. We evaluated whether Hp(2-20) accelerated the healing of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis and explored the mechanism(s) underlying any such effect. METHODS: Fifteen rats underwent rectal administration of Hp(2-20) 250-500 µg/kg/day, or of its control peptide Hp1 for 10 days, following induction of colitis with TNBS. Macroscopic and histological damage was quantified using predetermined injury scores. FPR1, COX-2, TNF-α, TGF-ß, HB-EGF and tissue transglutaminase (t-TG) messenger RNA (mRNA) expression in colonic tissue was determined by quantitative polymerase chain reaction; FPR1, TNF-α and COX-2 protein levels by Western blotting. RESULTS: (1) Hp(2-20) accelerated healing of TNBS-induced colitis compared to controls consistently with the expression of FPRs in colonic mucosa; (2) TNBS upregulated mRNA mucosal expression of COX-2, TNF-α, TGF-ß, HB-EGF and t-TG and (3) this, with the exception of HB-EGF, was significantly counteracted by Hp(2-20). CONCLUSIONS: Hp(2-20), an FPR agonist, accelerates the healing of TNBS-induced colitis in the rat. This effect is associated with a significant reduction in colonic tissue levels of COX-2, TGF-ß, TNF-α and t-TG. We postulate that FPR-dependent pathways may be involved in the repair of inflamed colonic mucosa.

Role of bisphenol A as environmental factor in the promotion of non-alcoholic fatty liver disease: in vitro and clinical study.

BACKGROUND: Bisphenol A is an endocrine disrupting chemical associated with type 2 diabetes mellitus (T2DM), cardiovascular disease and liver enzyme abnormalities. AIM: To evaluate bisphenol A plasma and urine levels in non-alcoholic fatty liver disease (NAFLD) patients compared to healthy subjects. Furthermore, we evaluated, in human HepG2 cells, the effects of exposure to different concentrations of bisphenol A on both oxidative stress induction and cell proliferation. METHODS: We enrolled 60 patients with histological diagnosis of NAFLD with or without T2DM and sixty healthy subjects. In vitro, the proliferation of bisphenol A-exposed HepG2 cells at two different concentrations (0.025 and 0.05 µM) was evaluated, both at high (H-HepG2) and at low (L-HepG2) glucose concentrations for 48 h. Lipoperoxidation was assessed by thiobarbituric acid reactive substances (TBARS) assay. RESULTS: Bisphenol A levels were significantly higher in 60 NAFLD subjects, both in urine and in plasma (P < 0.0001) when compared to controls and, in this group, it appeared to be higher in 30 non-alcoholic steatohepatitis patients compared to 30 simple steatosis subjects (P < 0.05), independently from the presence of T2DM. After a bisphenol A-free diet for 1 month, NAFLD patients showed a significant reduction in bisphenol A circulating levels (P < 0.05), without a significant reduction in urine levels. H-HepG2 cells treated with bisphenol A (0.05 µM) increased proliferation compared to controls at 48 h (P < 0.0001). Bisphenol A increased TBARS levels at 48 h versus controls. CONCLUSIONS: Our study reveals a possible role of bisphenol A as an environmental factor involved in the promotion of NAFLD, particularly in T2DM patients.

Platelet aggregation is affected by nitrosothiols in patients with chronic hepatitis: in vivo and in vitro studies.

AIM: To investigate the relationship among the number of platelets and plasma levels of S-nitrosothiols (S-NO), nitrite, total non-protein SH (NPSH), glutathione (GSH), cysteine (CYS), malondialdehyde (MDA), 4-hydroxininenal (4HNE), tumor necrosis factor-alpha (TNFalpha) and interleukin (IL)-6 in patients with chronic hepatitis C (CH). METHODS: In vitro the aggregation of platelets derived from controls and CH patients was evaluated before and after the addition of adenosine diphosphate (ADP) and collagen, both in basal conditions and after incubation with nitrosoglutathione (GSNO). RESULTS: In vivo, S-NO plasma levels increased significantly in CH patients and they were significantly directly correlated with platelet numbers. Patients with platelet counts < 150000/microL, had a smaller increase in S-NO, lower levels of GSH, CYS, NPSH, TNFalpha, and IL-6, and higher levels of nitrite, MDA, and 4-HNE relative to those of patients with platelet counts > 150000/microL. In vitro, the ADP and collagen aggregation time was increased in platelets from patients and not from controls; in addition, platelets from CH patients but not from controls also showed a latency time after exposure to collagen. CONCLUSION: The incubation of platelets with GSNO improved the percentage aggregation and abolished the latency time.

IGF-II/IGF-I receptor pathway up-regulates COX-2 mRNA expression and PGE2 synthesis in Caco-2 human colon carcinoma cells.

Nonsteroidal anti-inflammatory drugs reduce the risk of colon cancer and this effect is mediated in part through inhibition of type 2 prostaglandin endoperoxide synthase/ cyclo-oxygenase (COX-2). In the present study, we demonstrate that COX-2 expression and PGE2 synthesis are up-regulated by an IGF-II/IGF-I receptor autocrine pathway in Caco-2 colon carcinoma cells. COX-2 mRNA and PGE2 levels are higher in proliferating cells compared with post-confluent differentiated cells and in cells that constitutively overexpress IGF-II. Up-regulation of COX-2 expression by IGF-II is mediated through activation of IGF-I receptor because: (i) treatment of Caco-2 cells with a blocking antibody to the IGF-I receptor inhibits COX-2 mRNA expression; (ii) transfection of Caco-2 cells with a dominant negative IGF-I receptor reduces COX-2 expression and activity. Also, the blockade of the PI3-kinase, that mediates the proliferative effect of IGF-I receptor in Caco-2 cells, inhibits IGF-II-dependent COX-2 up-regulation and PGE2 synthesis. Moreover, COX-2 expression and activity inversely correlate with the increase of apoptosis in parental, IGF-II and dominant-negative IGF-I receptor transfected cells. This study suggests that induction of proliferation and tumor progression of colon cancer cells by the IGF-II/IGF-I receptor pathway may depend on the activation of COX-2-related events.

The treatment of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is becoming an increasing cause of chronic liver damage. The decision of start a medical treatment is based on the documented risk of progression to cirrhosis and liver cancer, when steatohepatitis (NASH) occurs. The therapy of this syndrome requires, as obviously, some considerations on the natural history of the condition, on the efficacy and safety of various therapeutic options, as well as on the costs. Treatment of patients with NAFLD has typically been focused on the management of associated conditions such as obesity, diabetes mellitus and hyperlipemia. Weight loss improves insulin sensitivity, and NASH may resolve with weight reduction. Insulin resistance seems to be the common denominator in many cases of NAFLD. Two classes of drugs have been shown to correct insulin resistance: biguanides (e.g., metformin) and thiazolidinediones (e.g., rosiglitazone and pioglitazone). The last two decades have witnessed a considerable progress in the understanding of the mechanisms respon-sible for the fibrogenic progression of chronic liver diseases. Several drugs believed to be hepatoprotective or antifibrotic agent as UDCA, betaine, vitamin E, lecithin, beta-carotene and selenium have been used in patients with NASH. Silybin is the main component of silymarin that is absorbed when linked whith a phytosome. This substance reduces in rats the lipid-peroxidation and the activaction of hepatic stellate cells. In humans, some non controlled data show that silybin is able to reduce insulin resistance, liver steatosis and plasma markers of liver fibrosis.

[Effects of a new pharmacological complex (silybin + vitamin-E + phospholipids) on some markers of the metabolic syndrome and of liver fibrosis in patients with hepatic steatosis. Preliminary study]. / Effeto di un nuovo complesso farmacologico (sillibina vitamina E+ fosfolipidi) su alcuni indicatori di sindrome metabolica e di fibrosi epatica in pazienti con epatopatia steatosica. Studio pilota.

AIM: This open preliminary pilot study was aimed to evaluate the effect of a new pharmaceutical complex (silybin+vitamin E+phospholipids - RealSIL-IBI-Lorenzini Pharmaceutical, Italy) on some parameters of metabolic syndrome and of liver fibrosis in patients with non alcoholic fatty liver disease (NAFLD) with or without the contemporaneous presence of hepatitis C virus (HCV)-related chronic hepatitis. METHODS: Eighty five patients were consecutively enrolled in the study and divided in 2 groups; the first group was represented by 59 patients affected by NAFLD, negative for other known causes of chronic liver damage (M/F= 39/20; median age and range: 44 years, 22-76, group A); the second group was represented by 26 patients (M/F=19/7; median age and range 51 years, 20-75, group B) with HCV-related chronic hepatitis associated to NAFLD. Adverse events and drop-outs were absent in all group and compliance at the study was absolute. RESULTS: This open preliminary study shows that the new compound silybin+vitamin E+ phospholipids is active, in vivo, and produces some therapeutic effects in patients with different forms of chronic liver damage. In particular, it improves insulin resistance and plasma levels of markers of liver fibrosis in patients in whom these parameters are particularly altered. CONCLUSIONS: Our data have a role of suggestion to further evaluate, through a controlled trial, a possible therapeutic use of this new compound in the management of patients with NAFLD.

A pilot study on the ability of clinoptilolite to absorb ethanol in vivo in healthy drinkers: effect of gender.

Zeolites are microscopic minerals of volcanic origin, and the zeolite most commonly used in medicine is clinoptilolite. Over the years, clinoptilolite has been tested in several ways: as an antioxidant, as an adjuvant in anticancer therapy due to its ability to capture chemotoxins, as an antidiarrhoeal agent and as a chelating agent for heavy metals. The aim of this study was to evaluate the ability of clinoptilolite to absorb ethanol in vivo in healthy drinkers. We enrolled 12 healthy drinkers in this study. The study was conducted as follows: phase 1: consumption of a hydroalcoholic solution containing 25 g of ethanol; phase 2: use of a 16.25 mL medical device containing clinoptilolite (2.5 g of clinoptilolite within a single-dose sachet) + consumption of a hydroalcoholic solution containing 25 g of ethanol; phase 3: use of a 32.5 mL medical device (5 g of clinoptilolite within a single-dose sachet) + consumption of a hydroalcoholic solution containing 25 g of ethanol. At the time of blood sampling, alcohol ingestion was also measured using an Alcolmeter instrument, and the results showed that the two methods overlapped. Reductions of 43%, 35%, 41% and 34% in blood ethanol at 30, 60, 90 and 120 minutes, respectively, were observed after the consumption of 5 g of clinoptilolite + 25 g of ethanol in both males and females, whereas the consumption of 2.5 g of clinoptilolite did not result in a statistically significant reduction in blood ethanol. In particular, the blood ethanol reduction was more significant in males. Our study highlights and confirms the ability of clinoptilolite to decrease the absorption of ingested ethanol by reducing blood alcohol levels. This effect was statistically significant at a dose of 5 g.

Helicobacter pylori infection but not small intestinal bacterial overgrowth may play a pathogenic role in rosacea.

BACKGROUND AND AIMS: Recent studies suggest a potential relationship between rosacea and Helicobacter pylori (H. pylori) infection or small intestinal bacterial overgrowth (SIBO), but there is no firm evidence of an association between rosacea and H. pylori infection or SIBO. We performed a prospective study to assess the prevalence of H. pylori infection and/or SIBO in patients with rosacea and evaluated the effect of H. pylori or SIBO eradication on rosacea. METHODS: We enrolled 90 patients with rosacea from January 2012 to January 2013 and a control group consisting of 90 patients referred to us because of mapping of nevi during the same period. We used the (13)C Urea Breath Test and H. pylori stool antigen (HpSA) test to assess H. pylori infection and the glucose breath test to assess SIBO. Patients infected by H. pylori were treated with clarithromycin-containing sequential therapy. Patients positive for SIBO were treated with rifaximin. RESULTS: We found that 44/90 (48.9%) patients with rosacea and 24/90 (26.7%) control subjects were infected with H. pylori (p = 0.003). Moreover, 9/90 (10%) patients with rosacea and 7/90 (7.8%) subjects in the control group had SIBO (p = 0.6). Within 10 weeks from the end of antibiotic therapy, the skin lesions of rosacea disappeared or decreased markedly in 35/36 (97.2%) patients after eradication of H. pylori and in 3/8 (37.5%) patients who did not eradicate the infection (p < 0.0001). Rosacea skin lesions decreased markedly in 6/7 (85.7%) after eradication of SIBO whereas of the two patients who did not eradicate SIBO, one (50%) showed an improvement in rosacea (p = 0.284). CONCLUSIONS: Prevalence of H. pylori infection was significantly higher in patients with rosacea than control group, whereas SIBO prevalence was comparable between the two groups. Eradication of H. pylori infection led to a significant improvement of skin symptoms in rosacea patients.

Long-term outcome of interferon-alpha-induced thyroid autoimmunity and prognostic influence of thyroid autoantibody pattern at the end of treatment.

Thyroid autoimmunity and dysfunction have been widely reported as side effects of interferon-alpha (IFN-alpha) treatment, but the literature lacks data regarding the long-term course of these complications, clinical observation being limited to 6-12 months off therapy. Our study is the first that has aimed to evaluate the natural history of IFN-related thyroid autoimmunity during a 6.2-yr follow-up after the IFN-alpha withdrawal as well as to investigate the potential role of the autoantibody pattern at the end of treatment to predict the long-term outcome. Our study group included 114 patients (79 males, 35 females), mean age 48 yr (range 23-67 yr) with no preexisting thyroid disease, undergoing a 12-month treatment with recombinant IFN-alpha for C virus-related chronic active hepatitis. Thyroid autoimmunity (serum TgAb and TPOAb) and function (serum FT(4), FT(3), TSH) were retrospectively evaluated at the end of IFN therapy, 6 months after IFN withdrawal and after a median period of 6.2 yr (range 5.5-8.4 yr). At the end of treatment, 78 patients were negative for thyroid autoantibodies (Abs-) and all but one of them remained so for the following evaluations. The remaining 36 patients had thyroid autoantibodies (Abs+) at the end of treatment, and they subsequently showed a heterogeneous behavior: 16 patients remained Abs+ for the whole length of the study (persistent thyroiditis); 10 patients became Abs- 6 months off therapy but were again Abs+ 6.2 yr later (remitting/relapsing thyroiditis); 10 patients reverted to autoantibody negativity at different observation times (transient thyroiditis). The absence of thyroid autoantibodies at the end of treatment was a protective factor for the successive development of thyroiditis (odds ratio: 0.02, confidence interval (CI) 95%: 0-0.1). On the contrary, the positivity for TgAb and/or TPOAb at high titers at the end of IFN treatment was significantly related to the highest risk of having chronic thyroiditis (odds ratio: 17.3, CI 95%: 3.2-91.7 for TgAb levels > 50 degree percentile; odds ratio: 7.3, CI 95%: 1.5-35.2 for TPOAb levels > 50 degree percentile). None of the patients showed overt thyroid dysfunction throughout the study, whereas a subclinical hypothyroidism was found in 12 patients. In all 12 cases, the functional abnormality was accompanied by the presence of thyroid autoantibodies. Eight of these 12 patients belonged to the group with persistent thyroiditis (P < 0.05). The absence of thyroid autoantibodies at the end of treatment was a protective factor for the successive development of thyroid dysfunction (odds ratio: 0.06, CI 95%: 0.01-0.56). On the contrary, the positivity for both TgAb and TPOAb at the end of IFN therapy was significantly correlated with the highest risk of having subclinical hypothyroidism 6.2 yr. later (odds ratio: 38.7; CI 95%: 6.2-242). Our study demonstrates that in patients undergoing an IFN-alpha therapy for chronic hepatitis C and with no evidence of preexisting thyroid disease: 1) the negativity for thyroid autoantibodies after IFN treatment is a protective factor for the developing thyroid autoimmunity and/or dysfunction in following years; 2) the IFN-alpha-related thyroid autoimmunity is not a complete reversible phenomenon because some patients can develop chronic thyroiditis; 3) high autoantibody levels at the end of IFN therapy are related to the risk of having chronic thyroid autoimmunity; and 4) the coexistence of TgAb and TPOAb at the end of treatment is a predictive factor for the presence of thyroid dysfunction, even if subclinical, many years after IFN withdrawal.

Serum interleukin-10 is an independent prognostic factor in advanced solid tumors.

Interleukin (IL)-10 is a Th2 type pleiotropic cytokine that has been found to be produced at the tumor site and to be increased in sera of patients suffering from different types of cancer. IL-10 has been shown to hinder a number of immune functions, i.e., T lymphocyte proliferation, Th1 type cytokine production, antigen presentation, and lymphokine-activated killer cell cytotoxicity. To assess its prognostic value, we measured serum levels of IL-10 in 118 patients with advanced solid tumors before treatment, after completion of therapy, and during follow-up. Other prognostic variables, to which IL-10 results were compared, were analyzed as well. IL-10 serum levels were found significantly elevated in cancer patients with respect to healthy controls. Of interest, a significant decrease in IL-10 serum levels was observed in the responder group, whereas a significant increase was recorded in the non-responder group. Using univariate and multivariate analyses, a significant relationship was shown between IL-10 serum levels and both overall survival (OS) and time to treatment failure (TTF). Stepwise regression analysis selected IL-10 serum level, performance status (PS), and stage as the best association of variables with significant impact on OS and TTF. In conclusion, this study shows that IL-10 has an independent prognostic significance in patients with advanced solid tumors and may be useful for monitoring disease progression.

Serological and clinical markers of autoimmune disease in HCV-infected subjects with different disease conditions.

OBJECTIVE: To investigate whether the serological markers of autoimmunity and the clinical features of autoimmune disease which occur in hepatitis C virus (HCV)-infected subjects are correlated to each other and/or to the clinical pattern of the disease. METHODS: Seventeen symptom-free, anti-HCV antibody positive subjects, 17 patients with chronic hepatitis C, 21 patients with mixed cryoglobulinemia (MC), and as controls 17 anti-HCV negative patients with dyspepsia were enrolled in a prospective study. A patient history, clinical examination, self-administered questionnaire and laboratory investigations (hepatic enzyme levels, serum HCV-RNA and anti-HCV antibody testing, and serum autoantibody profile) were performed to detect liver and/or autoimmune disease. RESULTS: Serological markers of autoimmunity and clinical findings of autoimmune disease were found to be more frequent in the HCV-infected patients considered as a whole than in controls. However, rheumatoid factor and clinical findings of autoimmune disease were more frequent in MC patients, while anti-smooth muscle antibodies not linked to symptoms or signs of autoimmune disease were detected in all groups of HCV-infected individuals, including healthy carriers and subjects who had recovered from a previous HCV infection. CONCLUSION: Anti-smooth muscle antibodies, a serological marker of autoimmunity, are detectable in HCV-infected subjects whatever their clinical status. Clinical findings of autoimmune disease prevalently occur in patients with mixed cryoglobulinemia.

Chronic hepatitis C long-term responders to human leukocyte interferon-alpha therapy: persistence of a sustained biochemical and virological response during 5 years of surveillance.

OBJECTIVES: To define the biochemical and virological course and IgM response to HCV-core protein in long-term responders (LTRs) during a long surveillance (5 years). DESIGN: From 1989 to 1991, 98 patients (pts) with biopsy-proven chronic hepatitis C were enrolled into this study. These pts underwent human leukocyte interferon-alpha (LE-IFN alpha) therapy at the prolonged schedule (3 MU thrice weekly for 1 year). METHODS: Serum alanine-aminotransferases (ALTs) were assessed monthly during and until 1 year after treatment, then every 3 months during the observation period. Qualitative and quantitative HCV RNA and HCV IgM were measured in all pts on baseline samples and in LTRs also after treatment and every following year. RESULTS: Based on serum ALT course, the pts were defined as: LTRs (14 pts), if their serum ALT levels returned to the normal range during therapy and remained so for at least 1 year afterwards; responders with relapse (RRs, 20 pts), if their serum ALT levels returned to the normal range during therapy but increased after ending treatment; and non-responders (NRs, 64 pts), if their serum ALT levels remained abnormal throughout therapy. No significant differences were seen regarding IgM anti-HCV positivity and serum ALT levels among the three groups. LTRs (12 HCV-RNA negative and two HCV-RNA positive at the end of treatment) maintained their virological status and not one of them experienced an elevation of serum ALT levels throughout the surveillance. CONCLUSION: Patients affected by chronic hepatitis C and treated with interferon, but who did not experience a biochemical or virological relapse within the first year of follow-up would not relapse later on; thus, we are able to conclude that these subjects made a complete recovery.

Non-alcoholic fatty liver disease: a multicentre clinical study by the Italian Association for the Study of the Liver.

AIM: To define the characteristics of the Italian patient presenting non-alcoholic fatty liver disease. PATIENTS AND METHODS: A total of 305 patients with abnormally high plasma aminotransferase and/or gamma-glutamyl-transpeptidase levels for at least 12 months, with no known cause of chronic liver damage, were consecutively enrolled in the study. Clinical, routine biochemical and liver histology investigations were carried out in all patients. Also evaluated were: (a) oral glucose load; (b) insulinaemia and insulin-resistance using the HOMA test model; and (c) plasma endotoxaemia, total antioxidant plasma capability, tumour necrosis factor-alpha, plasma interleukin-6 and -10 levels. Malondialdehyde and 4-hydroxynonenal content were determined on liver samples from 120 patients. RESULTS: The majority of patients were young overweight or obese males, with dyslipidaemia (20-60%), diabetes (10.5%), hyperinsulinaemia (40%), hyperferritinaemia (35%). Endotoxaemia was negative in all patients and cytokines were only sporadically altered. Total antioxidant plasma capability was decreased in 38.4% of the patients. Eighty percent of the cases had histological steatosis with a mild degree of inflammation and fibrosis. Seven patients had cirrhosis. Lipid peroxidation markers were increased in 90% of the cases, inversely correlated with fibrosis. Even if at univariate analysis, age, ferritin and tissue 4-hydroxynonenal were independent factors of steatosis (P < 0.01), and insulin, HOMA and ferritin of inflammation and fibrosis (P < 0.01), at multivariate analysis no single factor was found to be an independent predictor of hepatic lesions. CONCLUSIONS: The typical Italian patient with non-alcoholic fatty liver disease is a young male, obese, not diabetic, with a variable incidence of dyslipidaemia and hyperinsulinaemia. Only liver biopsy may define the type of liver damage.

Subjects positive for hepatitis C virus RNA with normal aminotransferase levels, a "trompe l'oeil" clinical picture?

BACKGROUND: Quite often subjects affected by chronic hepatitis C virus infection have no clinical signs of liver disease and serum aminotransferase values never go beyond the upper limit of normal. Yet these subjects, defined "asymptomatic HCV carriers", often have active viral replication and various degrees of histological damage. AIMS: To verify, in a population of antibody to hepatitis C virus carriers, if normal serum aminotransferase values in hepatitis C virus-RNA positive differed considerably from those in hepatitis C virus-RNA negative subjects. SUBJECTS/METHODS: We followed 24 anti-hepatitis C virus-positive subjects (15 hepatitis C virus-RNA positive and 9 negative) by measuring alanine and aspartate aminotransferase levels at 3-month intervals for a median of 40 months (range 6-77). RESULTS: Determinations resulted repeatedly and rigorously within the normal range in all participants. Alanine aminotransferase values were higher in hepatitis C virus-RNA positives than in negatives (mean +/- SD: 0.609+/-0.172 vs 0.434+/-0.153 times the upper limit of normal; p

Interferon-related thyroid autoimmunity and long-term clinical outcome of chronic hepatitis C.

BACKGROUND: A high incidence of thyroid autoantibodies and/or disorders was observed in subjects with hepatitis C virus-related chronic hepatitis during interferon-alpha therapy. AIM: To evaluate whether thyroid autoimmunity and dysfunction, induced by interferon-alpha therapy, could be viewed as predictors for treatment response and as valid prognostic markers of liver disease progression. PATIENTS: A total of 136 subjects (96 males/40 females; median age 48 years; range 23-64) affected by biopsy-proven chronic hepatitis C (33.1% with compensated liver cirrhosis). METHODS: All subjects were treated with interferon-alpha therapy at 6 MU 3 times weekly for 12 months and then followed up for an average period of 60 months (range 12-108). Routine laboratory tests, virological assessment, liver ultrasound, thyroid function tests (serum free-triiodothyronine, free-thyroxine, serum thyrotropin), and autoimmunity were performed for all subjects. RESULTS: Percentage of thyroid autoimmunity and thyroid dysfunction in long-term responders was not significantly different compared to that in non-responders (47.0% and 11.8% vs 35.3% and 5.9%, respectively; non significant). The multivariate model demonstrated that the absence of cirrhosis was the only factor significantly related to successful response to therapy (odds ratio: 14.9; 95% confidence interval: 1.9-115.0 for chronic hepatitis C vs presence of cirrhosis). Moreover, the occurrence of thyroid autoimmunity during interferon therapy was similar both in patients with or without worsening of liver disease (33.3% and 39.8%, respectively; p = not significant). No subject with on-going liver disease developed thyroid dysfunction during treatment, as opposed to the 10/118 (8.4%) with a better course of liver disease; however, this difference was not statistically significant. The multivariate model showed that age was the only covariate significantly associated with unfavourable outcome of liver disease (odds ratio: 18.6; 95% confidence interval: 2.3-151.9, for those over 48 years vs younger patients). CONCLUSIONS: There is no evidence that the immune mechanism involved in the pathogenesis of thyroid autoimmune phenomena is the same as that regulating the therapeutic clearance of HCV or modulating the unfavourable course of HCV-related chronic hepatitis. However, our study confirmed that liver disease seems to progress more slowly in younger subjects.

Up-regulation of heparin binding epidermal growth factor-like growth factor and amphiregulin expression in Helicobacter pylori-infected human gastric mucosa.

BACKGROUND: Host response plays a major role in pathogenesis of Helicobacter pylori-induced gastroduodenal disease including adenocarcinoma of distal stomach. Epidermal growth factor-related growth factors are important modulators of gastric homeostasis in normal and damaged gastrointestinal mucosa. AIM: To evaluate expression of heparin binding epidermal growth factor and amphiregulin in antral mucosa of Helicobacter pylori-infected and non-infected dyspeptic patients and to correlate levels of heparin binding-epidermal growth factor and amphiregulin mRNA with mitogenic activity of gastric epithelial cells. METHODS: A total of 10 Helicobacter pylori-infected and 15 Helicobacter pylori non-infected (10 with and 5 without gastritis) dyspeptic patients were studied. Diagnosis of Helicobacter pylori infection was based on rapid urease test and histology. Heparin binding-epidermal growth factor and amphiregulin mRNA expression in antral mucosa were assessed by reverse transcriptase-polymerase chain reaction. Protein expression and localization of both peptides were determined by immunohistochemistry. Mitogenic activity of antral gastric mucosa was assessed by determination of proliferating cell nuclear antigen labelling index by immunohistochemistry. RESULTS: Heparin binding-epidermal growth factor and amphiregulin mRNA expression increased in Helicobacter pylori-infected vs Helicobacter pylori non-infected patients. Heparin binding-epidermal growth factor and amphiregulin immunostaining was more intense and deeper in gastric gland compartment in infected mucosa than in non-infected mucosa. Increase in heparin binding-epidermal growth factor and amphiregulin mRNA expression significantly correlated with increase in proliferating cell nuclear antigen labelling index. CONCLUSIONS: Helicobacter pylori gastritis is associated with up-regulation of heparin binding-epidermal growth factor and amphiregulin which correlates with increased mitogenic activity of gastric mucosa. Increased heparin binding-epidermal growth factor and amphiregulin expression is postulated to contribute to reparative response of gastric mucosa to Helicobacter pylori infection.

The significance of alpha-glutathione S-transferase determination in patients with chronic liver diseases.

BACKGROUND: Alpha-glutathione S-transferases (alpha-GST) are the ''basic'' class of a large family of enzymes, ubiquitarly distributed in the cells, particularly in the hepatocytes. Their principal biological function is the detoxification of the cells by conjugating glutathione with various electrophiles and hydroperoxides. METHODS: We evaluated plasma alpha-GST in comparison to aminotransferase values (ALT) in 234 patients: 18 healthy volunteers, 51 chronic alcohol abusers with (22) or without (29) chronic liver damage and 165 with chronic HCV infection (32 ''apparently'' healthy carriers of HCV infection, 88 with a biopsy-proven chronic hepatitis (CH), 48 treated with interferon, 15 with well compensated and 12 with decompensated liver cirrhosis (LC) and 18 with hepatocellular carcinoma (HCC). Alpha-GST were determined on the venous blood samples after an overnight fast by Hepkit Alpha-Biotech (Biotrin International, Dublin, Ireland). RESULTS: Control values: <8 ng/ml (range 2.7-6.3). In alcoholics we found a constant increase in 31% of patients without liver disease, in which ALT levels proved constantly normal, and in 13.6% of cirrhotics, without significant correlation among plasma values of alpha-GST, gamma-GT or alcoholemia. In ''healthy'' HCV-carriers, HCV-RNA determination selected 21 subjects positive for viral replication, while 11 were negative. HCV-RNA negative subjects had constant normality of plasma alpha-GST values, while in those HCV-RNA positive alpha-GST increased in 57% of cases. In patients with HCV-related chronic liver disease, alpha-GST proved higher than normal in 80.6% patients with CH, in 40% well-compensated and in 8.3% decompensated cirrhotics and in 33.3% of patients with HCC. No statistically significant correlation was observed between alpha-GST and other liver tests in all groups studied. In patients treated with interferon (6 MU x 3/weekly of alpha-interferon) alpha-GST plasma values were significantly higher in relapsers with respect to responders or not to interferon treatment and during the treatment only in relapsers, while ALT returned to normal values, alpha-GST did not change and, in some cases, increased further. No significant correlation was observed between response to treatment, ALT, alpha-GST and HCV-RNA plasma levels in all groups of CH patients. CONCLUSIONS: Data indicate that in patients with chronic liver disease with different aetiologies, plasma alpha-GST determination may be useful for discovering a liver involvement also when ALT are normal. In patients with HCV-related CH, plasma alpha-GST values may be utilized as reliable markers in monitoring the response to interferon treatment.

Alcoholic beverages and gastric epithelial cell viability: effect on oxidative stress-induced damage.

UNLABELLED: Alcohol is known to cause damage to the gastric epithelium independently of gastric acid secretion. Different alcoholic beverages exert different damaging effects in the stomach. However, this has not been systematically evaluated. Moreover, it is not known whether the non-alcoholic components of alcoholic beverages also play a role in the pathogenesis of gastric epithelial cell damage. Therefore, this study was designed to evaluate whether different alcoholic beverages, at a similar ethanol concentration, exerted different damaging effect in gastric epithelial cells in vitro. Moreover, we evaluated whether pre-treatment of gastric epithelial cells with alcoholic beverages prevented oxidative stress-induced damage to gastric cells. Cell damage was assessed, in MKN-28 gastric epithelial cells, by MTT assay. Oxidative stress was induced by incubating cells with xanthine and xanthine oxidase. Gastric cell viability was assessed following 30, 60, and 120 minutes incubation with ethanol 17.5-125 mg/ml(-1) or different alcoholic beverages (i.e., beer, white wine, red wine, spirits) at comparable ethanol concentration. Finally, we assessed whether pre-incubation with red wine (with or without ethanol) prevented oxidative stress-induced cell damage. Red wine caused less damage to gastric epithelial cells in vitro compared with other alcoholic beverages at comparable ethanol concentration. Pre-treatment with red wine, but not with dealcoholate red wine, significantly and time-dependently prevented oxidative stress-induced cell damage. CONCLUSIONS: 1) red wine is less harmful to gastric epithelial cells than other alcoholic beverages; 2) this seems related to the non-alcoholic components of red wine, because other alcoholic beverages with comparable ethanol concentration exerted more damage than red wine; 3) red wine prevents oxidative stress-induced cell damage and this seems to be related to its ethanol content.