Ulderico Ventura

Helicobacter pylori vacuolating toxin accumulates within the endosomal-vacuolar compartment of cultured gastric cells and potentiates the vacuolating activity of ammonia.

This study explored the relationship between vacuolating toxin and ammonia in the genesis of Helicobacter pylori‐induced vacuolation in cultured human gastric cells and investigated the intracellular sites of toxin accumulation. Neutral red dye uptake and electron microscopy were used in the investigation of the respective roles of, and of the reciprocal interaction between, toxin and ammonia in cell vacuolation and ultrastructural immunocytochemistry was used for the identification of the intracellular sites of internalized toxin. Toxin was found to cause an expansion of the endosomal compartment, where it accumulates after cellular internalization. However, toxin does not form large, neutral red‐positive vacuoles unless combined with ammonia, whose moderate vacuolating activity is markedly potentiated by the toxin. It is concluded that the toxin accumulated within the endosomal compartment alters the morphology and function of this organelle and plays a permissive role towards cell vacuolation, possibly by increasing the accumulation of protonated ammonia within endosomes. In turn, ammonia induces excessive dilatation of the endosomes with reciprocal fusion of their membranes, thus causing cytoplasmic vacuolation.

Helicobacter pylori upregulates expression of epidermal growth factor-related peptides, but inhibits their proliferative effect in MKN 28 gastric mucosal cells.

Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.

Expression and immunolocalization of aquaporin-7 in rat gastrointestinal tract.

Background information. In the gastrointestinal tract of mammals, water can either be secreted with digestive juices or absorbed by the small and large intestine. Transcellular water movement can be mediated by the transmembrane protein family of AQPs (aquaporins), as has also been recently identified in the gastrointestinal tract. However, the localization, expression and functioning of AQPs in the gastrointestinal tract have not been completely characterized. For the present study, we investigated: (1) the expression of AQP7 in some portions of rat gastrointestinal tract by semiquantitative reverse transcriptase—PCR and by immunoblotting and (2) the cellular and subcellular localization of AQP7 by immunohistochemistry.

Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells

The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium. Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H. pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell. The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting. We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than approximately 90 kDa. Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity.The vacuolating toxin A (VacA) is one of the most important virulence factors in Helicobacter pylori-induced damage to human gastric epithelium. Using human gastric epithelial cells in culture and broth culture filtrate from a VacA-producing H. pylori strain, we studied 1) the delivery of VacA to cells, 2) the localization and fate of internalized toxin, and 3) the persistence of toxin inside the cell. The investigative techniques used were neutral red dye uptake, ultrastructural immunocytochemistry, quantitative immunofluorescence, and immunoblotting. We found that VacA 1) is delivered to cells in both free and membrane-bound form (i.e., as vesicles formed by the bacterial outer membrane), 2) localizes inside the endosomal-lysosomal compartment, in both free and membrane-bound form, 3) persists within the cell for at least 72 h, without loss of vacuolating power, which, however, becomes evident only when NH4Cl is added, and 4) generally does not degrade into fragments smaller than ∼90 kDa. Our findings suggest that, while accumulating inside the endosomal-lysosomal compartment, a large amount of VacA avoids the main lysosomal degradative processes and retains its apparent molecular integrity.

Cytotoxicity of Helicobacter pylori on human gastric epithelial cells in vitro: role of cytotoxin(s) and ammonia

Objective To evaluate the respective roles of vacuolating cytotoxin(s) and urease-mediated ammonia production in Helicobacter pylori-induced cytotoxicity on cultured human gastric epithelial cells. Methods Monolayers of MKN 28 cell line were incubated with broth culture filtrates of two urease-positive H. pylori strains: CCUG 17874, cytotoxin-producing and C21, non-cytotoxin-producing. After incubation, highly sensitive quantitative assays were used to measure the ammonia levels in the cell medium, cell vacuolation and cell death. In order to evaluate the cytotoxic role of ammonia more accurately, we performed an ‘ammonia simulation’ by measuring cell vacuolation and cytolethality induced by incubation with definite concentrations of ammonium chloride. Cell damage was also studied morphologically. Results Both H. pylori strains produced ammonia and induced cell vacuolation and cytolethality. With the G21 strain, both cell vacuolation and cytolethality appeared virtually identical to ammonia simulation. With the CCUG 17874 strain, which induced a higher degree of cell vacuolation and cell death compared with the G21 strain, cell vacuolation and cytolethality appeared largely ammonia-independent. The vacuoles induced by each H. pylori strain and by ammonia simulation were similar to each other. In less severely affected cells, vacuole formation and ammonia simulation were regularly limited by smooth membranes and morphologically and topographically resembled dilated endosomes or trans Golgi vesicles. In more severely affected cells, large autophagic-like vacuoles containing cell debris were also prominent. Conclusions The vacuolating cytotoxin(s) and ammonia are major H. pylori virulence factors. The cytotoxin(s) seem to play a more prominent role than ammonia in cell vacuolation. Nevertheless, ammonia seems to damage cells by other mechanisms in addition to vacuolation. A common pathway for cytotoxin- and ammonia-induced cell vacuolation seems likely.

Solute transporters and aquaporins are impaired in celiac disease

Background information. Celiac disease is a chronic inflammatory disorder of the small bowel induced in genetically susceptible subjects by gluten ingestion. Diarrhoea, weight loss and malabsorption represent the major clinical presentation of the disease. Here we examined the possible alteration in the expression and localization of water channels [AQPs (aquaporins)] and some solute transporters in duodenal mucosa of celiac disease patients. Duodenal biopsies from untreated celiacs, treated celiacs, healthy controls and disease controls were considered in the present study. The expressions of some AQPs and transporter mRNAs in human duodenal biopsies were determined by semi‐quantitative RT—PCR (reverse transcription PCR) and real‐time RT—PCR. The localization of AQPs 3, 7 and 10 and of SGLT1 (Na+/glucose co‐transporter 1), PEPT1 (H+/oligopeptide transporter 1) and NHE3 (Na+/H+ exchanger 3) was evaluated by immunohistochemistry.

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine

BackgroundSeveral aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel.ResultsRT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding.Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein.The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells.ConclusionThe results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi.

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.

Significance of Ammonia in the Genesis of Gastric Epithelial Lesions Induced by Helicobacter pylori: An in vitro Study with Different Bacterial Strains and Urea Concentrations

Two Helicobacter pylori products cause cell damage both in vivo and in vitro: ammonia, from bacterial urease activity, and a vacuolating toxin named VacA. In this in vitro study, the vacuolating effect of H. pylori broth culture filtrate from a VacA-positive/urease-positive strain is compared with that of a VacA-negative/urease-positive strain and a VacA-negative/urease-negative strain. The effect of VacA and ammonia was evaluated with and without addition of 10 mM urea, a physiological concentration for the human stomach, and with and without addition of 0.5 mg/ml acetohydroxamic and (AHA), an urease inhibitor. Our data show that: (1) both urease-positive H. pylori strains caused cell vacuolation in the absence of urea, the VacA-positive strain being approximatively twice as potent as the VacA-negative strain; (2) addition of urea to the culture medium caused an approximatively 3-fold increase in the vacuolating activity of both urease-positive strains; (3) a VacA-negative/urease-negative strain did not exert any vacuolating effect, either in the presence or in the absence of urea; (4) the ratio between cell vacuolation induced by VacA-positive and VacA-negative strains was enhanced by the presence of AHA: ratio was about 2 in the absence of AHA and about 6 in the presence of AHA, either with or without urea added. The increment of vacuolation is likely due to an interaction between AHA and VacA. In conclusion, a VacA-negative/urease-positive strain becomes highly cytotoxic when physiological levels of urea are present in the incubation medium. This finding suggests that all urease-positive H. pylori strains, both with and without VacA expression, should be considered as potentially cytotoxic for the human gastric mucosa, although VacA enhances the severity of cell damage.

Na+,K+‐ATPase of gastric cells A target of Helicobacter pylori cytotoxic activity

The present study shows a direct impairing action of a cytotoxin‐producing Helicobacter pylori strain on the Na+,K−‐ATPase (evaluated as K+‐dependent phosphatase activity) of human gastric epithelial cells in culture. The toxin itself is likely involved in this action which may also account for the cell edema found in vivo in Helicobacter pylori‐colonized stomach.