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- Nutraceuticals: a new therapeutic approach againstPublication . Santos, António Mário; Oleastro, Mónica; Vale de Gato, Inês; Lopes, Teresa; Seixas, Elsa; Machado, Jorge; Guerreiro, António SousaBackground and Aim: H. pylori induces severe gastric chronic inflammation and is the cause of gastritis, peptic ulcer and a major risk factor for gastric cancer. The aim of the study was to investigate the anti-inflammatory effect of two nutraceuticals in Hp-infected mucosa. Materials and Methods: Eighteen C57BL/6 mice were inoculated with Hp SS1 by gavage three times with 3 · 109 viable cells. Mice were then treated with either PBS, curcumin (10 mg/mouse) or Symbiotic 2000 (50 mg/mouse), three times per week. Half of the infected and three non-infected mice were euthanized at week 6, the remaining at week 18. Gastric samples were removed for immunohistochemistry and PCR array (inflammatory response and immunity pathway) analysis (Sabiosciences, Qiagen). Results: All the 18 mice were Hp positive by immunohistochemistry. The production of the chemokines CCL2, CCL5, CCL20, CCL25, CXCL1 and CXCL11 was significantly up-regulated at both week 6 (range of fold-change 4.3–718) and week 18 (range of fold-change 16–1192). Similarly, the expression of the proinflammatory cytokines IL-1b, IL6, IL9, IL10, IL23, TNFa and INFc was significantly augmented (range of fold-change 1338–8251). The treatment with either curcumin or symbiotic drastically decreased the expression of all these mediators, restoring their levels to those similar to the non-infected mice. Conclusions: The present study confirmed that Hp infection induces a strong inflammatory response. Curcumin and Symbiotic treatments exerted a significant anti-inflammatory effect in Hp-infected mucosa. The supplementation of diet with these nutraceuticals may be a novel clinical approach against gastric inflammation induced by Hp infection.
- Regulation of the human erythropoietin gene expression by an upstream open reading framePublication . Barbosa, Cristina; Romão, Luísa
- Helicobacter pylori infection: the role of intestinalPublication . Machado, Jorge; Gato, Inês; Lopes, Teresa; Pereira, Teresa; Santos, Mário; Oleastro, Mónica; Chaves, Paula; Guerreiro, AntónioIntestinal microbiota may influence inflammation in the host. The aim of the present study was to explore the role of modulation of intestinal microbiota in the outcome in the Helicobacter pylori (Hp) gastric inflammation. Twenty five C57BL/6 male mice were separated in three groups: Control group (CG) n = 5 Infected group (IG) n = 10 and Synbiotic 2000TM (SG) n = 10. CG received PBS by gavage; IG and SG were inoculated intragastrically with H. pylori SS1 cell suspension (109 CFU/mL). Then, mice were treated either with PBS (CG and IG) or Synbiotic 2000TM (SG). Five mice from each group were sacrificed at week 6 and the other at week 18. At each time samples were collected from: gastric tissue to immunohistochemistry and histological evaluation (HE) and faeces to evaluate intestinal microbiota composition by FISH, targeting 14 bacterial groups. IG and SG groups were H. pylori positive by immunohistochemistry. Microbiota analysis: In IG there were significant changes in the microbiota composition, comparing to CG. At week 6 there were changes in 12 of 14 (85.7%) bacterial groups, while at week 18 there was a change in 6/14 (42.9%). In SG, there were changes in 7/14 (50.0%) at week 6, and in 4/14 (28.6%) at week 18, comparing to CG. Histology: IG at weeks 6 and 18 has 40% (2/5) of intramucosal inflammation and SG at the same end points has 0% (0/5). These results suggest that modulation of the intestinal microbiota by Synbiotic 2000TM may influence the outcome of Hp gastric inflammation.
- Ulcerogenic profile of helicobacter pylori pediatric strains: a contribution to get insight into the virulence of the BacteriaPublication . Vitoriano, Inês; Saraiva-Pava, Kathy; Rocha-Gonçalves, Alexandra; Santos, Andrea; Lopes, Ana Isabel; Oleastro, Mónica; Roxo-Rosa, Mónica
- Characterization of new human gastric epithelial cell linesPublication . Saraiva-Pava, Kathy; Navabi, N.; Lindén, Sarah; Oleastro, Mónica; Roxo-Rosa, MónicaThe lack of a cellular model which correctly mimics the natural niche of the pathogen Helicobacter pylori is still limitative for the study of this infection. Aiming to overcome this limitation, we have previously isolated clones of a subpopulation of the widely used heterogenic NCI-N87 (ATCC CRL-5822) gastric cell line1, those presenting typical epithelial markers and a progenitor-like phenotype (simultaneous synthesis of mucus and zymogens). For that, we stably-transduced the NCIN87 cells with human telomerase reverse-transcriptase (hTERT) catalytic subunit (pGRN145 plasmid, ATCC MBA-141), using the FuGENE-HD reagent (Roche). The two most promising NCI-N87-derived clones (C5 and C6) were shown to be composed of cells with homogenous phenotype with ability to grow in adherent monolayers, to produce gastric zymogens (hematoxylin staining) and to produce and secrete neutral mucins (Periodic-Acid-Schiff staining). Preliminary results have also shown that they are able to generate transepithelial electrical resistance and the ability of C5 to produce and secrete acidic mucins (Alcian-Blue staining). We are now clarifying the identity of the mucin species C5 and C6 produce by immunohistochemical analysis and zymogens (Pepsinogen) by western-blot. Moreover, the subcellular localization (immunocytochemistry) of adherens and tight-junctions’ proteins (E-cadherin and ZO-1) and the polarization status of both clones is now under evaluation. Due to their improved properties, compared to the heterogeneous parental line, these NCI-N87-derived clones are promising models of the human gastric epithelium.
- Proteome analysis of clustered helicobacter pylori strainsPublication . Vitoriano, Inês; Vítor, Jorge; Oleastro, Mónica; Roxo-Rosa, Mónica; Vale, FilipaGenomic-methylation typing method, based on strains’ Restriction/Modification systems, confirmed the genetic variability of Helicobacter pylori. According to this, strains isolated from patients of the same family, or from the same geographic region, cluster together. The analysis of proteome’s variability of these clusters has been a missing topic. We applied the Minimum-Common-Restriction-Modification (MCRM) algorithm to genomic-methylation data of 30 H. pylori strains, isolated from Portuguese patients, presenting different gastric diseases. 100% of generated dendrograms presented three incipient clusters (C1, C2 and C3), which is characteristic of strains sharing the geographic origin. The same pattern was observed when the MCRM algorithm was applied to a subset of strains (2 of C1, 2 of C2, 4 of C3 and two outsiders). These were heterogeneous regarding their cagA and vacA genotypes and in terms of patient’s age, gender and gastric disease. Comparative analysis of two-dimensional-gel-electrophoresis (2-DE) maps, obtained for total-protein extracts of each strain, revealed that among 70 matched protein spots (in a universe of 300), 16 were differently abundant (p < .05) among clusters. These proteins’ abundance was then compared having the 2DE-maps regrouped according to the strain’s cagA-genotype or its association with gastric disease. We concluded that abundance variations of at least 12 proteins were dictated by differences in virulence, rather than cluster proximity. Therefore, although the genome-methylation typing method discriminates differences in restriction/modification enzymes, strains of each generated cluster do not share a marked particular proteome, arguing that strains with common geographic origin vary greatly in virulence.
- Nonsense-mediated decay resistance of AUG-proximal nonsense-mutated transcripts relies on the link between translation initiation and premature stop codon definitionPublication . Peixeiro, Isabel; Barbosa, Cristina; Silva, Ana Luísa; Romão, LuísaThe nonsense-mediated decay (NMD) is a surveillance mechanism that recognizes and rapidly degrades transcripts bearing a premature termination codon (PTC). Although in mammals the location of a PTC more than 50 nucleotides upstream the terminal exonexon junction has been pointed as a mark for NMD, it is now known that the physical distance between the PTC and cytoplasmic poly(A)-binding protein 1 (PABPC1) is a crucial determinant in PTC definition. We have previously reported that mRNAs carrying an AUG-proximal PTC evade NMD although apparently fulfilling the defined criteria for PTC definition. This unexpected NMD-evasion reflects an AUG-proximity effect. To test the hypothesis that the AUG-proximity effect on NMD resistance could be the result of a positional effect of PABPC1 relative to the PTC due to the inherent nature of the process of short ORF translation process, the PABPC1/eRF3 interaction was inhibited by RNA interference. Under conditions of overexpression of a PABPC1 mutant lacking the eRF3-interaction domain (PABPC1DelC), levels of beta15 transcripts were quantified by RT-qPCR and showed to be significantly decreased. The reciprocal experiment, i.e. conditions of overexpression of an eRF3 mutant lacking the PABPC1-interaction domain (eRF3DelN) was also performed. Results showed that the NMD-resistance of an AUG-proximal nonsense-mutated mRNA can be converted to NMD-sensitive when PABPC1/eRF3 interaction is impaired. Moreover, inhibition of PABPC1-eIF4G interaction by overexpression of PAIP2 protein, as well as the knockdown of the initiation factor eIF3h and eIF3f subunit, targets these transcripts for NMD. Our findings support a role for PABPC1 and associated initiation factors during translation in NMD-resistance of AUG-proximal nonsense-mutated transcripts, providing evidence for a link between translation initiation and PTC definition.