Selective inhibition of intestinal guanosine 3',5'-cyclic monophosphate signaling by small-molecule protein kinase inhibitors.

The guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.

Impact of the freeze-drying process on product appearance, residual moisture content, viability, and batch uniformity of freeze-dried bacterial cultures safeguarded at culture collections.

In this study, causes of collapsed bacterial cultures in glass ampoules observed after freeze-drying were investigated as well as the influence of collapse on residual moisture content (RMC) and viability. Also, the effect of heat radiation and post freeze-drying treatments on the RMC was studied. Cake morphologies of 21 bacterial strains obtained after freeze-drying with one standard protocol could be classified visually into four major types: no collapse, porous, partial collapse, and collapse. The more pronounced the collapse, the higher residual moisture content of the freeze-dried product, ranging from 1.53 % for non-collapsed products to 3.62 % for collapsed products. The most important cause of collapse was the mass of the inserted cotton plug in the ampoule. Default cotton plugs with a mass between 21 and 30 mg inside the ampoule did not affect the viability of freeze-dried Aliivibrio fischeri LMG 4414(T) compared to ampoules without cotton plugs. Cotton plugs with a mass higher than 65 mg inside the ampoule induced a full collapsed product with rubbery look (melt-back) and decreasing viability during storage. Heat radiation effects in the freeze-drying chamber and post freeze-drying treatments such as exposure time to air after freeze-drying and manifold drying time prior to heat sealing of ampoules influenced the RMC of freeze-dried products. To produce uniform batches of freeze-dried bacterial strains with intact cake structures and highest viabilities, inserted cotton plugs should not exceed 21 mg per ampoule. Furthermore, heat radiation effects should be calculated in the design of the primary drying phase and manifold drying time before heat sealing should be determined as a function of exposure time to air.

Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. RESULTS: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. CONCLUSIONS: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.

Optimization of a small intestinal segment perfusion model for heat-stable enterotoxin A induced secretion in pigs.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of infectious diarrhea both in human and pigs. After ingestion of contaminated food or water, ETEC bacteria colonize the small intestine where they produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, which induce watery diarrhea. We investigated the possibility of eliciting STa-induced secretion in jejunal segments of anesthetized pigs using a small-intestinal segment perfusion (SISP) model. Five consecutive mid-jejunal segments of anaesthetized piglets were perfused for 6h with different concentrations of STa in a physiologic salt solution. Changes in intestinal net fluid absorption were measured. From the results we could conclude that the STa response was dose-dependent and that continuous perfusion with 50 nM of STa or more was required to reduce net absorption. This concentration was sufficient to reduce net absorption compared to control segments in 12 out of 14 piglets. STa-induced responses however showed relative high variation between different jejunal segments of one pig, similar to the inter-segment variation seen in control animals where segments were perfused with physiologic salt solution. These results indicate that more optimization is required before this model could be used to test compounds that could interfere with the STa-induced fluid secretion.

Protective immunization with homologous and heterologous antigens against Helicobacter suis challenge in a mouse model.

Helicobacter (H.) suis colonizes the stomach of more than 60% of slaughter pigs and is also of zoonotic importance. Recently, this bacterium was isolated in vitro, enabling the use of pure cultures for research purposes. In this study, mice were immunized intranasally or subcutaneously with whole bacterial cell lysate of H. suis or the closely related species H. bizzozeronii and H. cynogastricus, and subsequently challenged with H. suis. Control groups consisted of non-immunized and non-challenged mice (negative control group), as well as of sham-immunized mice that were inoculated with H. suis (positive control group). Urease tests on stomach tissue samples at 7 weeks after challenge infection were negative in all negative control mice, all intranasally immunized mice except one, and in all and 3 out of 5 animals of the H. cynogastricus and H. suis subcutaneously immunized groups, respectively. H. suis DNA was detected by PCR in the stomach of all positive control animals and all subcutaneously immunized/challenged animals. All negative control animals and some intranasally immunized/challenged mice were PCR-negative. In conclusion, immunization using antigens derived from the same or closely related bacterial species suppressed gastric colonization with H. suis, but complete protection was only achieved in a minority of animals following intranasal immunization.

Peptic ulcer disease associated with Helicobacter felis in a dog owner.

The aim of this study was to investigate the identity of the Helicobacter heilmannii-like bacteria found in the stomach of a human patient suffering from stomach ulcers and her asymptomatic pet dog. An elderly woman was referred for gastroscopy because of right hypochondrial pain, nausea, anorexia and vomiting. Gastric ulcers were observed and histology revealed the presence of multiple H. heilmannii-like bacteria. Multiplex polymerase chain reaction (PCR) identified the bacteria as H. felis. Her pet dog was also examined gastroscopically. Only mild gastric lesions were found but PCR showed the presence of H. felis as well as H. bizzozeronii and Candidatus H. heilmannii. This report associates H. felis infection in humans with severe gastric ulceration. Moreover, the suggestion can be made that the patient contracted H. felis from her dog.

Helicobacter felis and Helicobacter bizzozeronii induce gastric parietal cell loss in Mongolian gerbils.

Non-pylori helicobacter infections are associated with gastritis, gastric ulcers and MALT lymphomas in man. Approximately 50% of these are caused by helicobacters commonly found in dogs and cats, including Helicobacter felis, Helicobacter bizzozeronii and H. salomonis. In contrast to Helicobacter pylori, the virulence mechanisms of these species are unknown. In this study the virulence of H. felis, H. bizzozeronii and H. salomonis was investigated in Mongolian gerbils. Female SPF gerbils were inoculated intragastrically with H. felis, H. bizzozeronii or H. salomonis and sacrificed 3 weeks later. Fundus and antrum samples were taken for bacterial detection by PCR. A longitudinal strip covering all stomach regions was taken for histology. Gastric colonization, inflammation, apoptosis, loss of parietal cells and cell proliferation were assessed. Controls and H. salomonis inoculated gerbils were negative in PCR. H. felis and H. bizzozeronii inoculated animals were positive. H. felis inoculated animals showed loss of parietal cells extending from the limiting ridge into the fundus. A high cell proliferation rate was noticed in the mucosal area devoid of parietal cells. A dense band of apoptotic cells and large numbers of Helicobacter bacteria were seen at the transition zone between affected and normal parietal cells. In H. bizzozeronii infected gerbils, this was less pronounced. Focal apoptotic loss of gastric epithelial cells was spatially associated with the presence of bacteria especially in H. felis and to a lesser extent in H. bizzozeronii infected gerbils. This loss of cells may lead to intestinal metaplasia.