Ana Magalhães

Helicobacter pylori induces β3GnT5 in human gastric cell lines, modulating expression of the SabA ligand sialyl–Lewis x

Chronic Helicobacter pylori infection is recognized as a cause of gastric cancer. H. pylori adhesion to gastric cells is mediated by bacterial adhesins such as sialic acid-binding adhesin (SabA), which binds the carbohydrate structure sialyl-Lewis x. Sialyl-Lewis x expression in the gastric epithelium is induced during persistent H. pylori infection, suggesting that H. pylori modulates host cell glycosylation patterns for enhanced adhesion. Here, we evaluate changes in the glycosylation-related gene expression profile of a human gastric carcinoma cell line following H. pylori infection. We observed that H. pylori significantly altered expression of 168 of the 1,031 human genes tested by microarray, and the extent of these alterations was associated with the pathogenicity of the H. pylori strain. A highly pathogenic strain altered expression of several genes involved in glycan biosynthesis, in particular that encoding beta3 GlcNAc T5 (beta3GnT5), a GlcNAc transferase essential for the biosynthesis of Lewis antigens. beta3GnT5 induction was specific to infection with highly pathogenic strains of H. pylori carrying a cluster of genes known as the cag pathogenicity island, and was dependent on CagA and CagE. Further, beta3GnT5 overexpression in human gastric carcinoma cell lines led to increased sialyl-Lewis x expression and H. pylori adhesion. This study identifies what we believe to be a novel mechanism by which H. pylori modulates the biosynthesis of the SabA ligand in gastric cells, thereby strengthening the epithelial attachment necessary to achieve successful colonization.

Fut2-null mice display an altered glycosylation profile and impaired BabA-mediated Helicobacter pylori adhesion to gastric mucosa

Glycoconjugates expressed on gastric mucosa play a crucial role in host-pathogen interactions. The FUT2 enzyme catalyzes the addition of terminal alpha(1,2)fucose residues, producing the H type 1 structure expressed on the surface of epithelial cells and in mucosal secretions of secretor individuals. Inactivating mutations in the human FUT2 gene are associated with reduced susceptibility to Helicobacter pylori infection. H. pylori infects over half the worlds population and causes diverse gastric lesions, from gastritis to gastric cancer. H. pylori adhesion constitutes a crucial step in the establishment of a successful infection. The BabA adhesin binds the Le(b) and H type 1 structures expressed on gastric mucins, while SabA binds to sialylated carbohydrates mediating the adherence to inflamed gastric mucosa. In this study, we have used an animal model of nonsecretors, Fut2-null mice, to characterize the glycosylation profile and evaluate the effect of the observed glycan expression modifications in the process of H. pylori adhesion. We have demonstrated expression of terminal difucosylated glycan structures in C57Bl/6 mice gastric mucosa and that Fut2-null mice showed marked alteration in gastric mucosa glycosylation, characterized by diminished expression of alpha(1,2)fucosylated structures as indicated by lectin and antibody staining and further confirmed by mass spectrometry analysis. This altered glycosylation profile was further confirmed by the absence of Fucalpha(1,2)-dependent binding of calicivirus virus-like particles. Finally, using a panel of H. pylori strains, with different adhesin expression profiles, we have demonstated an impairment of BabA-dependent adhesion of H. pylori to Fut2-null mice gastric mucosa, whereas SabA-mediated binding was not affected.

The role of N-acetylglucosaminyltransferase III and V in the post-transcriptional modifications of E-cadherin

It has long been recognized that E-cadherin dysfunction is a major cause of epithelial cell invasion. However, very little is known about the post-transcriptional modifications of E-cadherin and its role in E-cadherin mediated tumor progression. N-acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, and has been pointed as a metastasis suppressor. N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6 GlcNAc branching of N-glycans, and has been associated to increase metastasis. The regulatory mechanism between E-cadherin expression and the remodeling of its oligosaccharides structures by GnT-III and GnT-V were explored in this study. We have demonstrated that wild-type E-cadherin regulates MGAT3 gene transcription resulting in increased GnT-III expression. We also showed that GnT-III and GnT-V competitively modified E-cadherin N-glycans. The GnT-III knockdown cells revealed a membrane de-localization of E-cadherin leading to its cytoplasmic accumulation. Further, the GnT-III knockdown cells also caused modifications of E-cadherin N-glycans catalyzed by GnT-III and GnT-V. Altogether our results have clarified the existence of a bidirectional crosstalk between E-cadherin and GnT-III/GnT-V that was, for the first time, reproduced in an in vivo model. This study opens new insights into the post-transcriptional modifications of E-cadherin in its biological function, in a tumor context.

Can Dogs Prime Autistic Children for Therapy? Evidence from a Single Case Study

BACKGROUND AND OBJECTIVES Canine-assisted therapy has been receiving growing attention as a means of aiding children with autism spectrum disorder (ASD). Yet, only limited studies have been done and a great deal of literature related to this intervention is anecdotal. The present study aims at providing additional quantitative evidence on the potential of dogs to positively modulate the behavior of children with ASD. SETTINGS/LOCATION, SUBJECTS, AND INTERVENTIONS A 12-year-old boy diagnosed with ASD was exposed, at his usual treatment location (the Portuguese Association for Developmental Disorders and Autism at Vila Nova de Gaia, Portugal), to the following treatment conditions: (1) one-to-one structured activities with a therapist assisted by a certified therapy dog, and (2) one-to-one structured activities with the same therapist alone (as a control). To accurately assess differences in the behavior of the participant between these treatment conditions, the therapist followed a strict research protocol. The behavior of the participant was continuously video-recorded during both treatment conditions for further analysis and comparison. Treatment outcomes: In the presence of the dog, the participant exhibited more frequent and longer durations of positive behaviors (such as smiling and positive physical contacting) as well as less frequent and shorter durations of negative behaviors (such as aggressive manifestations). CONCLUSIONS These findings are in accordance with previous experimental work and provide additional support for the assertion that dogs can prime autistic children for therapy. Ultimately, this study may contribute toward a change for full acceptance of canine-assisted therapy programs within the medical milieu. Additional studies using a similar research protocol on more autistic children will certainly help professionals to work on the most effective methods to individually serve this population through canine-assisted interventions.

Gastric cancer: adding glycosylation to the equation

Gastric cancer has a high incidence and mortality, so there is a pressing need to understand the underlying molecular mechanisms in order to discover novel biomarkers. Glycosylation alterations are frequent during gastric carcinogenesis and cancer progression. This review describes the role of glycans from the initial steps of the carcinogenesis process, in which Helicobacter pylori adheres to host mucosa glycans and modulates the glycophenotype, as well as how glycans interfere with epithelial cell adhesion by modulating epithelial cadherin functionality in gastric cancer progression. Other mechanisms regulating gastric cancer malignant behavior are discussed, such as increased sialylation interfering with key signaling pathways and integrin glycosylation leading to an invasive phenotype. Applications of these glycosylation alterations in the clinical management of gastric cancer patients are discussed.

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ Proximity Ligation

Mucin glycoproteins are major secreted or membrane‐bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O‐glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O‐glycan modifications. We therefore aimed to identify the combined mucin and O‐glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer‐associated carbohydrate antigens Tn, Sialyl‐Tn (STn), T, Sialyl‐Lea (SLea) and Sialyl‐Lex (SLex) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLea/SLex‐MUC1 and STn/SLea/SLex‐MUC2 glycoforms in

Expression of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 in gastric mucosa, intestinal metaplasia, and gastric carcinoma.

50% of the cases, with a variable distribution among organs. Some new glycoforms‐T/SLea‐MUC2, STn/T/SLea/SLex‐MUC5AC and STn/T/SLea/SLex‐MUC6‐were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O‐glycan haptens alone.

Helicobacter pylori adhesion to gastric epithelial cells is mediated by glycan receptors

Aberrant mucin O-glycosylation is often observed in cancer and is characterized by the expression of immature simple mucin-type carbohydrate antigens. UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6) is one of the enzymes responsible for the initial step in O-glycosylation. This study evaluated the expression of ppGalNAc-T6 in human gastric mucosa, intestinal metaplasia, and gastric carcinomas. Our results showed that ppGalNAc-T6 is expressed in normal gastric mucosa and in intestinal metaplasia. A heterogeneous expression and staining pattern for this enzyme was observed in gastric carcinomas. ppGalNAc-T6 was expressed in 79% of the cases, and its expression level was associated with the presence of venous invasion. Our results provide evidence that ppGalNAc-T6 is an IHC marker associated with venous invasion in gastric carcinoma and may contribute to the understanding of the molecular mechanisms that underlie aberrant glycosylation in gastric carcinogenesis and in gastric carcinoma.

Helicobacter pylori and the BMP pathway regulate CDX2 and SOX2 expression in gastric cells

Helicobacter pylori adhesion to gastric epithelial cells constitutes a key step in the establishment of a successful infection of the gastric mucosa. The high representation of outer membrane proteins in the bacterial genome suggests the relevance of those proteins in the establishment of profitable interactions with the host gastric cells. Gastric epithelial cells are protected by a mucous layer gel, mainly consisting of the MUC5AC and MUC6 mucins. In addition to this protective role, mucins harbor glycan-rich domains that constitute preferential binding sites of many pathogens. In this article we review the main players in the process of H. pylori adhesion to gastric epithelial cells, which contribute decisively to the high prevalence and chronicity of H. pylori infection. The BabA adhesin recognizes both H-type 1 and Lewis b blood-group antigens expressed on normal gastric mucosa of secretor individuals, contributing to the initial steps of infection. Upon colonization, persistent infection induces an inflammatory response with concomitant expression of sialylated antigens. The SabA adhesin mediates H. pylori binding to inflamed gastric mucosa by recognizing sialyl-Lewis a and sialyl-Lewis x antigens. The expression of the BabA and SabA adhesins is tightly regulated, permitting the bacteria to rapidly adapt to the changes of glycosylation of the host gastric mucosa that occur during infection, as well as to escape from the inflammatory response. The growing knowledge of the interactions between the bacterial adhesins and the host receptors will contribute to the design of alternative strategies for eradication of the infection.

Probing the O-glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery

Helicobacter pylori infection is the main risk factor for intestinal metaplasia (IM) and gastric cancer development. IM is a pre-neoplastic lesion, induced by the transcription factor CDX2, where the gastric mucosa is converted to an intestinal phenotype. We previously demonstrated that key elements of the bone morphogenetic protein (BMP) pathway co-localize with CDX2 in IM and upregulate CDX2 expression in gastric cell lines. These observations, together with the hypothesis that CDX2 could be repressed by SOX2, led us to test whether H. pylori, through BMPs, SOX2 and CDX2 could participate in a molecular network critical for the development of IM. AGS cells with and without SMAD4 knock-down were co-cultured with H. pylori or BMP2 to assess the expression of BMP pathway members as well as CDX2 and SOX2 by qPCR and western blot. Proximity ligation assay (PLA) was also performed to evaluate SMAD proteins interaction. Immunohistochemistry and western blot were performed in gastric samples from mice infected with Helicobacter spp. to measure Smad4, pSmad1/5/8, Cdx2 and Sox2 expression in vivo. Increased expression and activity of the BMP pathway accompanied by CDX2 upregulation and SOX2 downregulation were observed in AGS cells co-cultured with H. pylori or BMP2. These effects were impaired by downregulation of the BMP pathway. Finally, infected mice present BMP pathway upregulation, focal Cdx2 expression and decreased Sox2. These results provide a novel link between H. pylori infection and the BMP pathway in the regulation of intestinal and gastric-specific genes that might be relevant for gastric IM.