Elisabete Silva

Expression of intestine-specific transcription factors, CDX1 and CDX2, in intestinal metaplasia and gastric carcinomas.

Intestinal metaplasia (IM) is part of a stepwise sequence of alterations of the gastric mucosa, leading ultimately to gastric cancer, and is strongly associated with chronic Helicobacter pylori infection. The molecular mechanisms underlying the onset of IM remain elusive. The aim of this study was to assess the putative involvement of two intestine‐specific transcription factors, CDX1 and CDX2, in the pathogenesis of gastric IM and gastric carcinoma. Eighteen foci of IM and 46 cases of gastric carcinoma were evaluated by immunohistochemistry for CDX1 and CDX2 expression. CDX1 was expressed in all foci of IM and in 41% of gastric carcinomas; CDX2 was expressed in 17/18 foci of IM and in 54% of gastric carcinomas. In gastric carcinomas, a strong association was observed between the expression of CDX1 and CDX2, as well as between the intestinal mucin MUC2 and CDX1 and CDX2. No association was observed between the expression of CDX1 and CDX2 and the histological type of gastric carcinoma. In conclusion, these results show that aberrant expression of CDX1 and CDX2 is consistently observed in IM and in a subset of gastric carcinomas. The association of CDX1 and CDX2 with expression of the intestinal mucin MUC2, both in IM and in gastric carcinoma, indirectly implies that CDX1 and CDX2 may be involved in intestinal differentiation along the gastric carcinogenesis pathway. Copyright

Prevalence of lysosomal storage diseases in Portugal

Lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders individually considered as rare, and few data on its prevalence has been reported in the literature. The overall birth prevalence of the 29 different LSDs studied in the Portuguese population was calculated to be 25/100 000 live births, twice the prevalence previously described in Australia and in The Netherlands. The comparison of the prevalence profile of the LSDs presenting a prevalence higher than 0.5/100 000 in the Portuguese, Dutch and Australian populations showed, in the Portuguese, the existence of a higher prevalence of GM2 gangliosidoses (B variant), mucolipidoses (II and III), Niemman-Pick type C and metachromatic leukodystrophy (MLD), and a lower prevalence of Pompe and Fabry. The highest prevalence value for a single LSD is the one of GM2 gangliosidoses (B variant), corresponding to 3/100 000, a value which is significantly higher than the prevalence of the most frequent LSD in Dutch, Pompe disease (2/100 000) and Australians, Gauchers disease (GD) (1.8/100 000). It is worth noting that the highest prevalence of GM2 gangliosidoses found in the Portuguese is mainly due to the existence of a unique subtype, the rare juvenile B1 variant.

Human MUC2 Mucin Gene Is Transcriptionally Regulated by Cdx Homeodomain Proteins in Gastrointestinal Carcinoma Cell Lines

In intestinal metaplasia and 30% of gastric carcinomas, MUC2 intestinal mucin and the intestine-specific transcription factors Cdx-1 and Cdx-2 are aberrantly expressed. The involvement of Cdx-1 and Cdx-2 in the intestinal development and their role in transcription of several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and gastric carcinoma. To clarify the mechanisms of transcriptional regulation of the MUC2 mucin gene in gastric cells, pGL3 deletion constructs covering 2.6 kb of the human MUC2 promoter were used in transient transfection assays, enabling us to identify a relevant region for MUC2 transcription in all gastric cell lines. To evaluate the role of Cdx-1 and Cdx-2 in MUC2 transcription we performed co-transfection experiments with expression vectors encoding Cdx-1 and Cdx-2. In two of the four gastric carcinoma cell lines and in all colon carcinoma cell lines we observed transactivation of the MUC2 promoter by Cdx-2. Using gel shift assays we identified two Cdx-2 binding sites at –177/–171 and –191/–187. Only simultaneous mutation of the two sites resulted in inhibition of Cdx-2-mediated transactivation of MUC2 promoter, implying that both Cdx-2 sites are active. Finally, stable expression of Cdx-2 in a gastric cell line initially not expressing Cdx-2, led to induction of MUC2 expression. In conclusion, this work demonstrates that Cdx-2 activates the expression of MUC2 mucin gene in gastric cells, inducing an intestinal transdifferentiation phenotype that parallels what is observed both in intestinal metaplasia and some gastric carcinomas.

Mucins as key molecules for the classification of intestinal metaplasia of the stomach

Abstract. Mucins and mucin-associated carbohydrates have a distinct expression pattern that can be modified under pathological conditions. Normal gastric mucosa expresses MUC1 and MUC5AC in foveolar epithelium and MUC6 in the glands. Lewis type-1 chain antigens (Lea and Leb) are expressed in foveolar epithelium, whereas Lewis type-2 chain antigens (Lex and Ley) are expressed in the glands. In this study we used monoclonal antibodies to evaluate the pattern of mucins and Lewis type-1 carbohydrates in intestinal metaplasia (IM) and compared it with IM types determined using histochemistry. In type-I or complete IM we found expression of MUC2 intestinal mucin and decreased/absent expression of MUC1, MUC5AC and MUC6. In type-II/III or incomplete IM there was co-expression of MUC2 and the mucins expressed in the stomach. No major differences were detected among the three IM types regarding expression of Lewis antigens. Furthermore we observed that sialylated compounds other than sialyl-Lea are responsible for histochemical detection of sialomucins and that sulpho-Lea/c is expressed in the presence or absence of sulphomucins detected using histochemistry. We conclude that mucin immunohistochemistry may replace classic histochemistry for the classification of IM into complete and incomplete types. The present study challenges the distinction of type-II from type-III IM since we did not observe major differences in the expression profile of mucins and Lewis type-1 carbohydrates. Finally, it seems necessary to evaluate the predictive value of IM according to the presence of specific sulphated carbohydrates (e.g. sulpho-Lea/c) rather than histochemically detected sulphomucins.

OCT-1 is over-expressed in intestinal metaplasia and intestinal gastric carcinomas and binds to, but does not transactivate, CDX2 in gastric cells

Intestinal metaplasia (IM) is a preneoplastic lesion of the stomach in which there is transdifferentiation of the gastric mucosa to an intestinal phenotype. The caudal‐related homeobox gene CDX2 encodes an intestine‐specific transcription factor crucial for the regulation of proliferation and differentiation of intestinal cells. In addition, CDX2 is involved in the induction of IM in the stomach. The aim of this study was to access the putative involvement of OCT‐1 in the induction of CDX2 expression de novo in gastric mucosa leading to the onset of IM. OCT‐1 protein expression was evaluated by immunohistochemistry in 31 biopsies with chronic gastritis, 15 biopsies with foci of IM and adjacent gastric mucosa and 42 gastric carcinomas. Furthermore, we evaluated OCT‐1 binding by electrophoretic mobility shift assay and activation of the CDX2 promoter by co‐transfecting a CDX2 promoter/reporter construct with an OCT‐1 expression vector in two gastric carcinoma cell lines, GP220 and MKN45. Our results show that OCT‐1 is expressed in chronic gastritis, particularly when it is adjacent to IM and is expressed in 87% of IM foci. Furthermore, 74% of the gastric carcinomas were positive for OCT‐1 and a strong association was observed between OCT‐1 expression and intestinal‐type carcinoma. We identified that OCT‐1 binds to the CDX2 promoter, although we could not see a transactivation effect in gastric carcinoma cell lines. In conclusion, we observed increased OCT‐1 expression in IM and in intestinal gastric carcinomas and identified the capacity of OCT‐1 to bind to the CDX2 promoter, although we could not demonstrate a direct effect of OCT‐1 in the transactivation of CDX2. Copyright

Age-related changes in renal expression of oxidant and antioxidant enzymes and oxidative stress markers in male SHR and WKY rats

Oxidative stress has been hypothesized to play a role in aging and age-related disorders, such as hypertension. This study compared levels of oxidative stress and renal expression of oxidant and antioxidant enzymes in male normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) at different ages (3 and 12 months). In the renal cortex of 3-month old SHR increases in hydrogen peroxide (H(2)O(2)) were accompanied by augmented expression of NADPH oxidase subunit Nox4 and decreased expression of antioxidant enzymes SOD1 and SOD3. A further increase in renal H(2)O(2) production and urinary TBARS was observed in 12-month old WKY and SHR as compared with 3-month old rats. Similarly, expressions of NADPH oxidase subunit p22(phox), SOD2 and SOD3 were markedly elevated with age in both strains. When compared with age-matched WKY, catalase expression was increased in 3-month old SHR, but unchanged in 12-month old SHR. Body weight increased with aging in both rat strains, but this increase was more pronounced in WKY. In conclusion, renal oxidative stress in 12-month old SHR is an exaggeration of the process already observed in the 3-month old SHR, whereas the occurrence of obesity in 12-month old normotensive rats may partially be responsible for the age-related increase in oxidative stress.

New insights into the regulation of Na+,K+-ATPase by ouabain.

Cardiac glycosides have been widely used in the treatment of congestive heart failure and cardiac arrhythmias. The mechanism of their positive ionotropic effect is well characterized and is based on the inhibition of Na(+),K(+)-ATPase. However, recently it has been discovered that circulating endogenous cardiotonic steroids bind to Na(+),K(+)-ATPase, activate cell signaling pathways, and regulate diverse cellular functions. Thus, they play an important role in both physiological and pathological processes. Circulating endogenous ouabain has been reported to activate several signaling pathways upon binding to Na(+),K(+)-ATPase, and altered plasma levels of this endogenous cardiotonic steroid have been found in several pathologies. This review focuses on ouabain-mediated activation of cell signaling pathways and secondary messengers that conversely may modulate Na(+),K(+)-ATPase function.

Overexpression of Na(+)/K (+)-ATPase parallels the increase in sodium transport and potassium recycling in an in vitro model of proximal tubule cellular ageing.

Na+/K+-ATPase plays a key role in the transport of Na+ throughout the nephron, but ageing appears to be accompanied by changes in the regulation and localization of the pump. In the present study, we examined the effect of in vitro cell ageing on the transport of Na+ and K+ ions in opossum kidney (OK) cells in culture. Cells were aged by repeated passing, and Na+/K+-ATPase activity and K+ conductance were evaluated using electrophysiological methods. Na+K+-ATPase α1– and β1-subunit expression was quantified by Western blot techniques. Na+/H+ exchanger activity, changes in membrane potential, cell viability, hydrogen peroxide production and cellular proliferation were determined using fluorimetric assays. In vitro cell ageing is accompanied by an increase in transepithelial Na+ transport, which results from an increase in the number of Na+/K+-ATPase α1- and β1-subunits, in the membrane. Increases in Na+/K+-ATPase activity were accompanied by increases in K+ conductance as a result of functional coupling between Na+/K+-ATPase and basolateral K+ channels. Cell depolarization induced by both KCl and ouabain was more pronounced in aged cells. No changes in Na+/H+ exchanger activity were observed. H2O2 production was increased in aged cells, but exposure for 5 days to 1 and 10 μM of H2O2 had no effect on Na+/K+-ATPase expression. Ouabain (100 nM) increased α1-subunit, but not β1-subunit, Na+/K+-ATPase expression in aged cells only. These cells constitute an interesting model for the study of renal epithelial cell ageing.

AGEs, contributors to placental bed vascular changes leading to preeclampsia

Abstract Glycation of proteins or other biomolecules and their further long-term degradation result in the formation of advanced glycation end products, AGEs. AGEs and other ligands interact with their receptors, RAGEs, localized to a variety of tissues, but mainly in endothelium and vascular wall cells. This interaction triggers diverse signaling pathways that converge on the activation of NF-κB and the initiation of a local inflammatory reaction that, when prolonged, results in dysfunctional features. Preeclampsia is a serious vascular disorder centred at the placenta–uterine interface, the placental bed, but the condition extends to the mother´s circulation. RAGEs have notorious expression in the placental bed tissues along pregnancy but, in addition, RAGEs and their ligands are expressed in the fetal membranes and are found in the amniotic fluid and the mother´s serum. Disorders complicating pregnancies and having an important vascular involvement, as preeclampsia and diabetes mellitus, have additional enhanced AGE/RAGE expression variation. This indicates that for their assessment, the assay of RAGEs or their ligands may become useful diagnostic or prognostic procedures.

Lewis enzyme (α1–3/4 fucosyltransferase) polymorphisms do not explain the Lewis phenotype in the gastric mucosa of a Portuguese population

AbstractThe human α-1,3/4 fucosyltransferase III (FucT III) catalyses the synthesis of Lewis antigens including Leb antigen which is a ligand for Helicobacter pylori adhesion. Several polymorphisms have been described in the FUT3 gene affecting both the transmembrane and catalytic domains, some of which affect the enzyme activity. The aim of the present work was to study the Lewis gene polymorphisms in a Caucasian Portuguese population, with a high rate of H. pylori infection, and to evaluate the implications of mutant enzymes in Leb expression in the gastric mucosa. We studied 460 asymptomatic or dyspeptic individuals from northern Portugal. Screening for Lewis gene polymorphisms was performed by SSCP and direct sequencing. Lewis phenotype in gastric mucosa was determined by immunohistochemistry. In 47 individuals with a Lewis negative blood group, we found FUT3 gene polymorphisms that were previously described in other populations: 59T>G, 202T>C, 314C>T, 508G>A and 1067T>A. Among the 47 Lewis negative individuals in blood, only nine were also negative in gastric mucosa, suggesting the existence of another α 1-4 fucosyltransferase that is responsible for Lea and Leb synthesis in gastric mucosa.