NOD1 mediates interleukin-18 processing in epithelial cells responding to Helicobacter pylori infection in mice.

The interleukin-1 family members, IL-1ß and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1ß processing in myeloid cells have been defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here we report that the host defence molecule NOD1 regulates IL-18 processing in mouse epithelial cells in response to the mucosal pathogen, Helicobacter pylori. Specifically, NOD1 in epithelial cells mediates IL-18 processing and maturation via interactions with caspase-1, instead of the canonical inflammasome pathway involving RIPK2, NF-κB, NLRP3 and ASC. NOD1 activation and IL-18 then help maintain epithelial homoeostasis to mediate protection against pre-neoplastic changes induced by gastric H. pylori infection in vivo. Our findings thus demonstrate a function for NOD1 in epithelial cell production of bioactive IL-18 and protection against H. pylori-induced pathology.

Long-Term Evolution of Patients Treated for Paroxysmal Atrial Fibrillation with First and Second Generation Cryoballoon Catheter Ablation with a Prospective Protocol Guided by Complete Bidirectional Left Atrium-Pulmonary Veins Disconnection after Adenosine as Main Target end Point to achieved. Seven Years Follow-up of Patients with a rough estimation profile of Low ALARMEc Score. A Single Center Report.

INTRODUCTION: Cryoballoon ablation (CB) has proven effective for treating patients with paroxysmal atrial fibrillation (PAF). We analyzed our seven year follow-up of patients, treated for PAF with first (CB1) and second generation (CB2), with demonstration of LA-PV disconnection with bidirectional block (BB) after adenosine (AD). METHODS: Since November 2008 to May 2015, 128 patients, 97 male (58±7 years), without heart disease, highly symptomatic, refractory to antiarrhythmic drugs (AAD) were treated, and follow-up (1411 ±727 days). Left atrial size: 37±6 mm. RESULTS: A total of 439 PV were successfully isolated (91.9%). Acute reconduction: 44 PV (9%): 16 after CB; 16 unmasked by AD; 12 extrapulmonary muscular connections (EMC). Main complication was phrenic nerve palsy (PNP): 9 (7 %). On follow-up, 114 patients (89%) remain asymptomatic in sinus rhythm (SR), free of medication. Fourteen patients (11%) had arrhythmia recurrence: 12 male (52±8 years). Early recurrences occurred in 9 male. Late recurrences presented 3 male at 24, 27 and 60 months, and 2 female at 7 and 40 months respectively. All recurrence patients were Redo, and remain in SR without medication during follow-up. CONCLUSIONS: CB alone is very effective and safe for the definitive treatment of patients suffering PAF with 72.6% success rate, increasing up to 89.1% when this protocol is applied in a single procedure. After Redo, all population group (100%), remain in sinus rhythm, freedom of arrhythmia, without AAD, in this very long term follow-up. Checking for BB, AD protocol, and ruling out EMC allowed-us to identified 14.8% of patients with underlying substrate for potential arrhythmia recurrence. CB2 applications entail a highest risk of PNP. Patients with a rough estimated profile of low ALARMEc score (≤ 1) have an excellent long term outcome, being this series the largest follow-up described so far, for patients treated for PAF with CB.

Genotype and phenotype correlations in diabetic patients in Uruguay.

To differentiate among different types of diabetes is becoming an increasingly challenging task. We investigated whether the patient's genetic profile is useful to identify the particular type of diabetes, to determine the corresponding hyperglycemia pathogenesis and treat accordingly. Three hundred and thirty-eight diabetic patients, diagnosed according to American Diabetes Association criteria, were recruited from 2004 to 2008 in diabetes health reference centers. We analyzed the major gene for type 1 diabetes susceptibility (HLA DQ/DR). In order to improve our understanding of the pathogenesis of the resulting hyperglycemia and to implement a more adequate treatment for the patients, we reclassified our sample according to the presence or absence of the genetic markers. We found that a higher percentage of people than expected have immunological disease, independent of their phenotype, with a relative risk of 4.62 (95% confidence interval). This methodology allowed us to establish an association between the genotype and its resulting phenotype. We found significant differences; the phenotypic classification did not reflect immunological disease based on genotype. Moreover, when we examined markers, body mass index and age of onset, we found that many people have an intermediate phenotype between type 1 and type 2. This genetic data can help provide an accurate definition of the disease and would therefore provide the physician a better possibility of providing adequate treatment.

Innate immune recognition of the extracellular mucosal pathogen, Helicobacter pylori.

Toll-like receptor (TLR) molecules play a frontline role in the defence of the host against infection by microbial pathogens. These molecules, together with the recently described Nod family proteins, have been shown to trigger innate immune responses in host cells via the recognition of highly conserved microbial structures. TLR4, which is the best-characterised of these "pathogen-recognition molecules" (PRMs), was the first to be shown to recognise a specific microbial component: the lipopolysaccharide (LPS) from Gram-negative bacteria. The molecular specificities of the remaining PRMs have, in nearly all cases, now also been elucidated. Host cells belonging to the myeloid cell lineage are known to be particularly responsive to these microbial constituents. Conversely, other cell types such as epithelial cells, were generally thought to be hypo-responsive to stimulation by such molecules. New evidence suggests that these cells are in fact likely to play a fundamental role in host defence against pathogenic micro-organisms. Indeed, epithelial cells afford an initial barrier against the host microflora, and appear to be able to differentiate between pathogenic and commensal micro-organisms. This review article will discuss current knowledge regarding innate immune responses in epithelial and myeloid cells to the model non-invasive pathogen, Helicobacter pylori, which is a major cause of upper gastrointestinal tract disease in humans.

Hydrogen bonding interactions between indole and benzenoid-pi-bases.

The NH-pi interactions of indole with benzene, naphthalene, phenanthrene, toluene, m-xylene, and mesitilene, in carbon tetrachloride solutions, have been studied by Fourier transform infrared spectroscopy. The experiments, carried out on the NH stretching band of indole, prove the formation of 1:1 complexes in which the NH bond of indole is engaged. The NH frequency shifts are independent of the number of rings in the base, but they progressively increase as the electron density is enhanced by methylation. The association constants increase with the increase of both, the number of rings and the methyl groups on the base. At higher base concentrations, further shifts on the free NH and associated bands indicate the formation of 1:2 complexes, which suggest hybride NH-pi and van der Waals interactions between one indole ring and two benzene acceptor molecules.

Helicobacter pylori-induced activation of human endothelial cells.

Helicobacter pylori infection causes active chronic inflammation with a continuous recruitment of neutrophils to the inflamed gastric mucosa. To evaluate the role of endothelial cells in this process, we have examined adhesion molecule expression and chemokine and cytokine production from human umbilical vein endothelial cells stimulated with well-characterized H. pylori strains as well as purified proteins. Our results indicate that endothelial cells actively contribute to neutrophil recruitment, since stimulation with H. pylori bacteria induced upregulation of the adhesion molecules VCAM-1, ICAM-1, and E-selectin as well as the chemokines interleukin 8 (IL-8) and growth-related oncogene alpha (GRO-alpha) and the cytokine IL-6. However, there were large variations in the ability of the different H. pylori strains to stimulate endothelial cells. These interstrain variations were seen irrespective of whether the strains had been isolated from patients with duodenal ulcer disease or asymptomatic carriers and were not solely related to the expression of known virulence factors, such as the cytotoxin-associated gene pathogenicity island, vacuolating toxin A, and Lewis blood group antigens. In addition, one or several unidentified proteins which act via NF-kappaB activation seem to induce endothelial cell activation. In conclusion, human endothelial cells produce neutrophil-recruiting factors and show increased adhesion molecule expression after stimulation with certain H. pylori strains. These effects probably contribute to the continuous recruitment of neutrophils to H. pylori-infected gastric mucosa and may also contribute to tissue damage and ulcer formation.

Prolonged exposure to YC-1 induces apoptosis in adrenomedullary endothelial and chromaffin cells through a cGMP-independent mechanism.

YC-1, a benzyl indazole derivative, is an NO-independent direct activator of soluble guanylyl cyclase (sGC), which presents a synergistic action with NO in stimulating cGMP synthesis. These properties have served to suggest YC-1 as an attractive therapeutic agent by permitting the reduction of nitrovasodilator dosage and regulating endogenous cGMP metabolism. Here we studied the effect of prolonged exposure of adrenomedullary endothelial and chromaffin cells to YC-1. We found that YC-1 increased cGMP in the two types of cells and this action was blocked by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cells underwent apoptotic death in association with increased caspase-3-like activity, DNA fragmentation, cytoskeletal disorganization and changes in membrane permeability after prolonged incubation with YC-1. Caspase-3-like protease activity and DNA fragments in the cytoplasm were increased in a dose-dependent manner by 16 h YC-1 treatment. The specific and cell permeable caspase-3-like protease inhibitor DEVD-CHO effectively inhibited YC-1-mediated caspase-3-like activation and DNA fragmentation. Moreover, YC-1 also induced cell shape changes accompanied by actin filament disorganization and alterations in membrane permeability. Cells incubated for 24h with YC-1 showed damaged membranes by binding to nucleic acid of a dye excluded by the intact plasma membrane of live cells. YC-1 also induced a decrease in the intracellular non-specific esterase activity, another indication of cell toxicity. Apoptotic phenomena were not prevented by the presence of ODQ although it effectively inhibited the YC-1-elicited cGMP increases. These findings indicate that YC-1 induces apoptosis by activating caspase-3-like protease through a mechanism independent of sGC activation.

Actividades preventivas y esquizofrenia. ¿Lo hacemos bien? / Preventive activities and esquizofreny. Do we do it well?

Fundamento : los esquizofrénicos son grandes frecuentadores en Atención Primaria, ¿aprovechamos para realizar actividades preventivas o se convierten en visitas burocráticas ? Objetivo : conocer si se practican las mismas actividades preventivas en esquizofrénicos que en población general.Diseño : estudio casos-controles .Material y métodos: casos (n=36): esquizofrénicos >15 años. Controles (n=72): extracción aleatoria de la base de datos, apareados por edad sexo .Variables: registro en HCAP de datos de filiación, frecuentación, actividades preventivas (peso, talla, vacuna antigripal, vacuna antitetánica, presión arterial, colesterolemia, alcohol y tabaco, según recomendaciones PAPPS) durante 1993-1998; recogida datos mayo-junio 1999.Explotación estadística: SPSS, estadística descriptiva de variables, Chi cuadrado o análisis de la varianza para análisis bivariante, odds ratio para medidas de asociación.Resultados : en el registro de actividades preventivas no hay diferencias estadísticamente significativas entre casos y controles, excepto mejor registro del hábito enólico (p=0,04) en los controles . Las actividades preventivas registradas, excepto hábito tabáquico, presentan mejores resultados en mujeres. Mejor registro de peso (p=0,01), talla (p=0,07), hábito alcohólico (p=0,04), mejor vacunadas de la gripe (p=0,006) y tétanos (p=0,03), y mejores resultados de cribado de colesterol (p=0,09) y presión arterial (p=0,005). Las mujeres son más frecuentadoras (6,8; DE:9,8) que los hombres (2,8; DE:3,2) (p=0,02). La mayor frecuentación se relaciona con un mejor re g i s t ro de vacuna antigripal (p=0,01), cribaje de c o l e s t e rol (p=0,05), peso (p=0,02), talla (p=0,04) y hábito enólico (p=0,03).Conclusiones : no se observan diferencias en la práctica de actividades preventivas entre esquizofrénicos y población general, siendo en ambos muy bajo el registro (AU)

Effect of low-dose antigen exposure on development of immunity to Helicobacter pylori infection in mice.

The effect of low-dose antigen exposure on the development of immunity to Helicobacter pylori infection was studied in outbred mice. Animals that were primed with a subinfectious number of H. pylori bacteria exhibited significantly lower bacterial loads after challenge with an infectious dose of pathogen (versus controls, P < 0.05).

Use of bioclimatic indexes to characterize phenological phases of apple varieties in Northern Italy.

The research was designed to characterize the phenological behaviour of different apple varieties and to compare different bioclimatic indexes in order to evaluate their adaptability in describing the phenological phases of fruit species. A field study on the requirement for chilling units (winter chilling requirement) and the accumulation of growing degree hours of 15 native apple cultivars was carried out in a fruit-growing area in North West Italy (Cuneo Province, Piedmont). From 1991 to 1993, climatic data were collected at meteorological stations installed in an experimental orchard (Verzuolo, Cuneo). Four methods were compared to determine the winter chilling requirement: Hutchins, Weinberger-Eggert, Utah and North Carolina. The Utah method was applied to determine the time when the chilling units accumulated become effective in meeting the rest requirements. A comparison of the different methods indicated that the Weinberger-Eggert method is the best: as it showed the lowest statistical variability during the 3 years of observations. The growing degree hour requirement (GDH) was estimated by the North Carolina method with two different base temperatures: 4.4 degrees C and 6.1 degrees C. More difficulties were met when the date of rest completion and the beginning of GDH accumulation was determined. The best base temperature for the estimation of GDH is 4.4 degrees C. Phenological and climatic characterizations are two basic tools for giving farmers and agricultural advisors important information about which varieties to choose and which are the best and the most correct cultivation practices to follow.

Nitric oxide-sensitive guanylyl cyclase activity inhibition through cyclic GMP-dependent dephosphorylation.

The soluble form of guanylyl cyclase (sGC) plays a pivotal role in the transduction of inter- and intracellular signals conveyed by nitric oxide. Here, a feedback inhibitory mechanism triggered by cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) activation is described. Preincubation of chromaffin cells with C-type natriuretic peptide, which increased cGMP levels and activated PKG, or with cGMP-permeant analogue (which also activates PKG), in the presence of a broad-spectrum phosphodiesterase inhibitor, resulted in a decrease in subsequent sodium nitroprusside (SNP)-dependent cGMP elevations. This inhibitory effect was mimicked by activating a protein phosphatase and counteracted by the selective PKG inhibitor KT-5823 and by different protein phosphatase inhibitors. Immunoprecipitation of sGC from cells submitted to different treatments followed by immunodetection with antiphosphoserine antibodies (clone 4A9) showed changes in phosphorylation levels of the beta subunit of sGC, and these changes correlated well with differences in SNP-elicited cGMP accumulations. Pretreatment of cells with several PKG inhibitors or protein phosphatase inhibitors produced an enhancement of SNP-stimulated cGMP rises without changing the SNP concentration required to produce half-maximal or maximal responses. Taken together, these results indicate that the catalytic activity of sGC is closely coupled to the phosphorylation state of its beta subunit and that the tonic activity of PKG or its stimulation regulates sGC activity through dephosphorylation of the beta subunit.

Evaluation of nitrofurantoin combination therapy of metronidazole-sensitive and -resistant Helicobacter pylori infections in mice.

The main objectives of this study were to determine whether the nitroreductase enzyme encoded by the rdxA gene of Helicobacter pylori was responsible for reductive activation of nitrofurantoin and whether a triple-therapy regimen with nitrofurantoin was able to eradicate metronidazole-sensitive and -resistant H. pylori infections from mice. The susceptibilities to nitrofurantoin of parent and isogenic rdxA mutant strains (three pairs), as well as a series of matched metronidazole-sensitive and -resistant strains isolated from mice (30) and patients (20), were assessed by agar dilution determination of the MIC. Groups of mice colonized with the metronidazole-sensitive H. pylori SS1 strain or a metronidazole-resistant rdxA SS1 mutant were treated with either metronidazole or nitrofurantoin as part of a triple-therapy regimen. One month after the completion of treatment the mice were sacrificed and their stomachs were cultured for H. pylori. The nitrofurantoin MICs for all strains tested were between 0.5 and 4.0 microg/ml. There was no significant difference between the susceptibility to nitrofurantoin of the parental strains and those of respective rdxA mutants or between those of matched metronidazole-sensitive and -resistant H. pylori isolates. The regimen with metronidazole eradicated infection from all eight SS1-infected mice and from one of eight mice inoculated with the rdxA mutant (P < or =0.001). The regimen with nitrofurantoin failed to eradicate infection from any of the six SS1-infected mice (P < or =0.001) and cleared infection from one of seven mice inoculated with the rdxA mutant. These results demonstrate that, despite the good in vitro activity of nitrofurantoin against H. pylori and the lack of cross-resistance between metronidazole and nitrofurantoin, eradication regimens involving nitrofurantoin are unable to eradicate either metronidazole-sensitive or -resistant H. pylori infections from mice.

Outbred mice with long-term Helicobacter felis infection develop both gastric lymphoid tissue and glandular hyperplastic lesions.

Experimental infection of mice with Helicobacter felis reproduces many aspects of the gastritis observed in Helicobacter pylori-infected humans. The development of gastric inflammatory lesions in chronically infected inbred mice is host-dependent; in BALB/c mice, gastric B-cell MALT lymphomas were observed, whilst other murine hosts (e.g. C57BL/6) developed severe glandular hyperplasia. The aims of this investigation were to characterize and immunophenotype Helicobacter-induced inflammatory lesions in mice with an outbred genetic background. Swiss mice (n=10 per group) were either inoculated with a suspension of H. felis or left untreated. H. felis-inoculated mice and age-matched control animals were killed 13 months later. The severity of gastric inflammatory lesions in the animals was graded and the number and distribution of B (CD45R(+)) and T (CD3(+)) lymphocytes in lymphoid tissues was determined by immunohistochemistry. Compared with control mice, animals with long-term H. felis infection developed severe hyperplastic gastritis (0.80+/-0.63 vs. 2.7+/-0.68), with epithelial dedifferentiation (0. 40+/-0.52 vs. 2.3+/-0.82) and lengthening of the pits and glands (0. 46+/-0.05 vs. 0.8+/-0.19). Gastric CD45R(+) and CD3(+) lymphocyte scores were significantly elevated (r=0.803) in infected animals, while lymphoepithelial lesions and polymorphonuclear leucocyte infiltrates were absent. Although prominent lymphoid follicles were present in the tissues of all infected animals, and in one control animal, only a proportion (55%) of the mucosal follicles had a dominant B-cell phenotype (defined as > or =75% CD45R(+) labelling), and all were poorly labelled with anti-mouse immunoglobulin antibodies. It was concluded that the lesions in outbred Swiss mice differed from B-cell MALT lymphomas. In contrast to inbred mice, outbred animals developed both glandular and lymphoid tissue lesions to chronic H. felis infection. It is suggested that the default T-helper phenotype of the host influences glandular lesion formation or B-cell lymphomagenesis in this model of infection.

Gastric helicobacters in cats.

The types of helicobacter which are found in the stomachs of carnivorous pets, especially cats, have been traditionally referred to as 'gastric helicobacter-like organisms' (GHLOs). These are microaerophilic, Gram-negative, spiral bacteria with multiple terminal flagellae and are endowed with high-level urease activity which allows them to survive in an acidic environment. Certain species have one or more periplasmic fibrils. The two GHLOs most commonly found in cats are Helicobacter felis and a species related to H heilmannii which was recently cultured from dogs. All phenotypic and genotypic (16S RNA gene sequences) evidence suggests that both of these bacteria belong in the genus Helicobacter. Whether or not helicobacters can be transmitted to humans from carnivorous pets is controversial but the recent discovery of H pylori -infected cats may be evidence of an animal reservoir for this pathogen. Although the role of H pylori in inducing antral gastritis and perpetuating pyloric ulcers in humans is well established, whether or not Helicobacter spp are causally involved in any feline gastric inflammatory conditions is unknown.

Helicobacter pylori rocF is required for arginase activity and acid protection in vitro but is not essential for colonization of mice or for urease activity.

Arginase of the Helicobacter pylori urea cycle hydrolyzes L-arginine to L-ornithine and urea. H. pylori urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved in H. pylori nitrogen metabolism. The roles of arginase in the physiology of H. pylori were investigated in vitro and in vivo, since arginase in H. pylori is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The H. pylori gene hp1399, which is orthologous to the Bacillus subtilis rocF gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three H. pylori strains were made by using two different constructs: 236-2 and rocF::aphA3. In contrast to wild-type (WT) strains, all rocF mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of H. pylori, the rocF::aphA3 mutant was approximately 1, 000-fold more sensitive to acid exposure. The acid sensitivity of the rocF::aphA3 mutant was not reversed by the addition of L-arginine, in contrast to the WT, and yielded a approximately 10, 000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that rocF is not required for urease activity in vitro. Finally, H. pylori mouse-adapted strain SS1 and the 236-2 rocF isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the rocF::aphA3 mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of H. pylori recovered from these mice (in log(10) CFU) were 6.1, 5.5, and 4.1, respectively. Thus, H. pylori rocF is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.

Cloning and allelic exchange mutagenesis of two flagellin genes of Helicobacter felis.

Helicobacter felis has been used extensively in animal model studies of gastric Helicobacter infections. Attempts to manipulate H. felis genetically have, however, been unsuccessful and, consequently, little is known about the pathogenic mechanisms of this bacterium. In common with other Helicobacter spp., H. felis is a highly motile organism. To characterize the flagellar structures responsible for this motility, we cloned and sequenced the two flagellin-encoding genes, flaA and flaB, from H. felis. These genes encode two flagellin proteins that are expressed simultaneously under the control of putative sigma28 and sigma54 promoters respectively. Isogenic mutants of H. felis in flaA and flaB were generated by electroporation-mediated allelic disruption and replacement, showing for the first time that H. felis could be manipulated genetically. Both types of H. felis flagellin mutants exhibited truncated flagella and were poorly motile. H. felis flaA mutants were unable to colonize the gastric mucosa in a mouse infection model.

The role of the rdxA gene in the evolution of metronidazole resistance in Helicobacter pylori.

It was recently demonstrated that inactivation of the rdxA gene, which encodes an oxygen-insensitive NADPH nitroreductase, is associated with the development of resistance to metronidazole by Helicobacter pylori. In order to further evaluate the contribution of rdxA to metronidazole resistance, the sequence of the rdxA gene was determined for a series of metronidazole-sensitive and -resistant isolates derived from a single, metronidazole-sensitive strain using an H. pylori mouse model. These strains were cultured from the stomachs of mice experimentally infected with H. pylori strain SS1 and then treated orally with metronidazole. The sequence of the rdxA gene of all 10 sequenced metronidazole-sensitive and two (7%) of the 27 metronidazole-resistant isolates was identical to that of the parental strain. In contrast, the rdxA gene of the other 25 metronidazole-resistant isolates contained between one and three frameshift or missense mutations. This suggests that while the development of metronidazole resistance in H. pylori is frequently associated with mutational inactivation of the rdxA gene, other mechanisms of resistance are likely to exist in this bacterium.