EFFETTO DELLA GLIADINA SUL BILANCIO OSSIDATIVO E DANNO AL DNA NELLA LINEA CELLULARE CACO-2 E IN PAZIENTI CELIACI

In recent years a significant increase of gluten-related disorders (GRDs) has been observed. Two factors seem at the basis of this increase. The first set would be related to the diffusion of serological tests. The second set of data associated with the increased prevalence of CD and GRDs could be related to an increase in the global consumption of wheat in recent decades; Cereals that contain gluten are widely consumed and current wheat varieties have a higher content in gluten compared to the past due to changes directed by both technology and nutritional reasons. In the past, classification of GRDs was very simple, because CD and Dermatitis Herpetiformis (DH) were the only known diseases with a well-documented role of gluten in their pathogenesis. Increasing complexity in the nomenclature and clinical presentation of GRDs has led to the development of a consensus document by a panel of experts on new classification including: CD, Non-Coeliac Gluten Sensitivity (NCGS), Wheat Allergy (WA) DH, and gluten ataxia (GA) although there can show considerable overlap in the clinical presentation. Each gluten-related disorder exhibits a specific pathophysiological response to gluten ingestion. Gluten is the main structural protein complex of wheat with equivalent toxic proteins found in other cereals, including rye and barley. The toxic protein fractions of gluten include gliadins and glutenins, with gliadins containing monomeric proteins and glutenins containing aggregated proteins. In wheat allergy (WA) and celiac disease (CD) the reaction to gluten is mediated by T-cell activation in the gastrointestinal mucosa. However, in WA it is the cross-linking of immunoglobulin (Ig)E by repeat sequences in gluten peptides (for example, serine-glutamine-glutamine -glutamine-(glutamine-) proline-proline-phenylalanine) that triggers the release of chemical mediators, such as histamine, from basophils and mast cells. Whereas in CD it is characterized as a chronic duodenal inflammation in which the increased secretion of inflammatory cytokines may in turn derange intestinal permeability and produce large amounts of reactive oxygen species (ROS), altering the redox state at the cellular level. Oxidative stress has been defined as the imbalance between the production of ROS and the antioxidant defenses of the cells in favor of the oxidants, leading to potential damage. ROS are produced during the cellular metabolic processes; if the production of ROS overwhelms a cell\u2019s antioxidant (AO) capability, a condition known as oxidative stress occurs. The impairment of redox equilibrium proved to cause severe damage in proteins, lipids and DNA. In the last decade several studies showed that gluten exposure reflects in an intracellular oxidative imbalance, characterized by: increased levels of lipid peroxidation products (4-hydroxy-2(E)-nonenal (4-HNE)), increased oxidized (GSSG)/reduced (GSH) glutathione ratio and decreased number of protein-bound sulfhydryl groups. Finally, ROS can induce the formation of oxidative DNA lesion products (8-oxodG), which is considered as a mutagenicity marker. Oxidative damage can lead to single or double-strand breaks, point and frameshift mutations and chromosome abnormalities. There is considerable circumstantial evidence that oxidative DNA damage may play an important role not only in carcinogenesis, being used as a predictive marker of cancer development, but also in aging. Nowadays, studies concerning oxidative damage on DNA have not been developed with regard to CD. The aim of this study was to investigate a possible gliadin-induced genotoxic damage and its correlation with oxidative stress in vitro and in vivo. The in vitro models consist of Caco-2 cell line of heterogeneous human epithelial colorectal adenocarcinoma cells generally used for this type of study. Whereas the in vivo model has been used serum and duodenal biopsies of patients. For this study the Caco-2 cells have been exposed for maximum 24h to increasing concentrations (250\ub5g/mL\u20121000\ub5g/mL) of digested gliadin (PT gliadin). Starting from a 500\ub5g/mL dose (with a 12-hour contact time), an increase of reactive oxygen species (ROS) (DCFDA probe) was observed. The alkaline Comet assay showed DNA damage at a concentration of 1000 \uf06dg/mL after 24h, characterized by a significant increase of DNA in the tail. Furthermore, the enzyme-modified Comet assay showed oxidative damage mainly with endonuclease-III, calculated as 06 tail moment after 24h treatment at 1000\ub5g/mL. Moreover, immunohistochemistry &3-H2AX detection (focal phosphorylation of histone H2AX at serine 139 to generate &3-H2AX in response to double-strand breaks) demonstrated an increase of the number of foci at 500\ub5g/mL and 1000 \ub5g/mL as genotoxic signature. The transglutaminase type 2 (TG2) activity was evaluated by western blot and ELISA method. The results show an increase of the enzyme expression in the chromatin and cytoskeleton at different doses (250, 500, and 1000\ub5g/mL) compatible with apoptosis, as confirmed by annexin V (cytofluorometric staining method). Has been observed an increase of apoptotic cells starting from a 250 \ub5g/ml dose. The oxidative stress has been evaluated through the analysis of biomarkers into the serum of patients: na\uefve celiac disease (nCD), celiac patient responders, undergoing to gluten free diet at least 12 months (CD-GDF), refractory celiac patient non responders, undergoing to gluten free diet at least 12 months (RCD) and healthy subjects (CTRL). The results demonstrated that Total Antioxidant Capacity (TAC) appeared to be significantly decreased in patients RCD respect to CD-GDF (p<0.05) and to CTRL (p<0.001). Plasmatic concentrations of TBARS, assessed as marker of lipid peroxidation and protein carbonyls (PC), assessed as marker of protein oxidation, were found significantly increased in RCD respect to CD-GDF, while the plasma levels of CD-GDF resulted similar to CTRL in both plasma biomarkers. Whereas the genotoxic damage was confirmed in vivo at the duodenal biopsy of celiac patients by means of H2AX e 8-OHG immunohistochemistry. In conclusion digested gliadin induces an increase of ROS production in Caco-2 cells with an alteration of the cellular redox state. Moreover, the high concentration of ROS induces DNA damage and the stimulation of the apoptotic process. This mechanism seems present also in vivo as demonstrated by the findings from CD patients

Le emissioni in atmosfera dalle combustioni in Lombardia

Il lavoro presenta le stime delle emissioni in atmosfera di polveri fini, precursori di polveri fini e gas serra in Lombardia provenienti dalle attività di combustione di combustibili e carburanti, come stimate dall’inventario regionale INEMAR per l’anno 2003. I dati dei consumi elaborati nell’inventario derivano da informazioni raccolte sia mediante questionari relativi alle principali sorgenti puntuali della regione, sia consultando le diverse fonti statistiche. Le emissioni in atmosfera sono stimate sia attraverso dati derivanti da misure effettuate presso gli impianti che tramite fattori di emissione proposti in letteratura e utilizzati in ambito EMEP-Corinair e IPCC (Intergovernmental Panel on Climate Change). La combustione nel settore del trasporto stradale risulta essere la principale sorgente per numerosi inquinanti, fra cui PM10, NOx, SO2 e CO. L’olio combustibile utilizzato per la produzione di energia è la maggiore fonte di SO2 (56% delle emissioni totali regionali), mentre il gasolio per autotrazione fornisce il principale contributo alle emissioni di NOx (44% del totale). Per le emissioni di polveri (sia PM10 che PM2,5), oltre ai determinanti contributi della combustione del gasolio per autotrazione e della legna in ambito domestico, sono rilevanti anche i processi di usura nel traffico stradale (pneumatici, freni, abrasione dell’asfalto) e le attività relative al settore agro-zootecnico. La benzina è la principale fonte di CO (50%), mentre il metano costituisce una sorgente di grande importanza (45%) per la CO2 e di interesse anche per gli ossidi di azoto (21% del totale regionale). Il lavoro fornisce altresì una valutazione dei fattori di emissione medi dalle attività di combustione in Lombardia a diverse scale di dettaglio

Assessment of Toxicity of Myristicin and 1&#8217;-Hydroxymyristicin in HepG2 Cell Line

Background and Objective: Myristicin belongs to a class of potentially toxic chemicals (alkoxy substituted allylbenzenes) and despite the structural analogy with safrole, data on this compound are very controversial and unclear. In this study assessed the cytotoxic and genotoxic potential of myristicin and 1\u2019-hydroxy-myristicin after 24 h of exposure in HepG2 cells. Methodology: The compounds were tested up to 600 \u3bcM concentration, for 24 h. The genotoxicity was assessed with alkaline and neutral comet assay and micronucleus assay. The data were analysed by one-way ANOVA. Results: It is to be emphasized that only the synthetic Phase 1 metabolite (1\u2019-hydroxymyristicin) showed a genotoxic effect starting from the concentration of 150 \u3bcM both in comet and micronucleus tests. However, it is important to point out that the same concentration cause a statistically significant (p<0.001) apoptotic process. Conclusion: The consumption of a traditional diet determines very low levels of exposure to the parent myristicin. This fact implies as the primary metabolic pathway the O-demethylation (5-allyl-2,3-dihidroxyanisole) and not to Phase I metabolism, which leads to the conclusion that this substance could not present a significant risk to humans

Oxidative stress as a biomarker for monitoring treated celiac disease article

Introduction: High levels of reactive oxygen species (ROS) and impaired antioxidant defense systems lead to oxidative stress (OxS) and tissue injury in different intestinal and extra intestinal conditions, including celiac disease (CD). The aim of the present study was to investigate the role and potential use of ROS and other biomarkers of OxS in the clinical management of CD. Methods: We collected duodenal specimens and blood samples from na\uefve patients (N-CD), patients on a gluten free diet (GFD) including responders (CD-GFD) and non-responders (NRCD). We measured plasmatic ROS production (electron paramagnetic resonance, EPR), lipid peroxidation (thiobarbituric acid-reactive substances, TBARS), protein oxidation (protein carbonyl, PC), total antioxidant capacity (TAC), nitric oxides and glutathione (GSH) in erythrocytes. Results: Fifty-four patients affected by CD were enrolled (17 N-CD, 18 CD-GFD and 19 NRCD; 44 F; age 44 \ub1 13 years). A significant increase of plasmatic OxS biomarkers (ROS, peroxidated lipids, oxidized proteins, and nitrate concentrations) and decrease of antioxidant species (TAC and GSH levels) were found in NRCD and N-CD compared to CD-GFD. Comparably, a significant direct relationship between the severity of duodenal atrophy, ROS production rates and TBARS was found; conversely, TAC and GSH presented an inverse correlation. Discussion: OxS is involved in CD tissue damage and correlates with the degree of duodenal atrophy. These findings suggest the possible role of OxS biomarkers as indicators of CD activity during the clinical follow-up

Rischi e benefici delle vaccinazioni anti-influenzale ed anti-pneumococcica in individui HIV-positivi

Influenza and Streptococcus pneumoniae diseases can cause severe complications in HIV-1 infected individuals leading to increases in hospital admission and even death. Both vaccinations are recommended for such individuals, but some studies reported that such immunizations may stimulate an increase of HIV-1 viral load and decrease of CD4+ cells count. A review of published studies, including our studies carried out in HIV-1 infected former drugs addicts, indicates that influenza and pneumococcal vaccinations are well tolerated in individuals with HIV-1, and do not induce deterioration of the course of HIV-1 infection, even though the immune response to vaccination is lower than that one observed in immunocompetent individuals. Therefore the lack of significant changes of virological and immunological parameters indicates that such immunizations can be safely administrated to HIV-1 infected individuals