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.

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.

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.

In situ characterization of Helicobacter pylori arginase.

The properties of Helicobacter pylori arginase activity in metabolically competent cells and lysates were investigated with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium. One-dimensional 1H- and 13C-nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of l-arginine by the bacterium. Arginase activity was associated with the cell-envelope fraction obtained by centrifugation of lysates. A Km of 22+/-3 mM was determined for the enzyme activity, and differences of Vmax were observed between strains. Divalent cations stimulated arginase activity, and the most potent activators were Co2+>Ni2+>Mn2+. The activity was highly specific for l-arginine and did not catabolize analogs recognized by other arginases of prokaryote and eukaryote origin. The Ki of several inhibitors was measured and served also to characterize the enzyme activity. The presence of bicarbonate enhanced the hydrolysis of l-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations. Amino acid sequence analyses revealed important differences between the deduced structures of H. pylori arginase and those of other organisms. This finding was consistent with experimental data which showed that H. pylori arginase has unique properties.

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.

Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production.

1. Sodium nitroprusside, S-nitroso-N-acetyl-D,L-penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38 +/- 0.02 microM. 2. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half-life (t1/2) of 3.9 +/- 0.2 min. The t1/2 for SPER/NO was 37 +/- 3 min. SNAP decomposed more slowly (t1/2 = 37 +/- 4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. 3. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 microM and maximal effective concentration between 2.5 and 5 microM. 4. For SNAP, threshold activation was seen at 1 microM, whereas full activation required a higher concentration (500-750 microM). The dose-response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide-deficient SNAP analogue or other NO donors. 5. Although NO-donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.

Influence of marrow CFU-GM content on engraftment and survival after allogeneic bone marrow transplantation.

The influence of the CFU-GM content of donor marrow on the outcome of allogeneic marrow transplantation (BMT) has been debated. We now report 38 patients (25 acute leukemias, 10 chronic myeloid leukemias, two myeloma; 24 in first CR/CP and 14 in more advanced phases of their disease) undergoing unmanipulated HLA-identical sibling BMT following conditioning with cyclophosphamide and total body irradiation (TBI). The median number of nucleated cells infused was 4.3 x 10(8)/kg (range 1.5-8.4); median CFU-GM numbers were 2.4 x 10(4)/kg (range 0.1-46). End-points of the study were (1) speed of neutrophil and platelet engraftment; (2) quality of engraftment beyond day +50 after BMT; and (3) transplant-related mortality in patients stratified according to whether they had received less than (n = 18) or more than (n = 20) 2.4 x 10(4)/kg CFU-GM. These two groups were comparable for diagnosis, disease status, donor sex, donor age, recipient sex, recipient age, CVHD prophylaxis, number of cells infused and CMV serology. Neutrophil counts were comparable at all time intervals. There was also no difference in platelet counts on days +7 to +50. However, patients who had received higher CFU-GM numbers had significantly higher platelet counts on day +80 (149 vs. 75 x 10(9)/L; P = 0.002), day +100 (153 vs. 77 x 10(9)/L; P = 0.0009) and day +150 (179 vs. 95 x 10(9)/L; P = 0.01). The 2-year actuarial transplant-related mortality was 5% vs. 53% (P = 0.007) in patients receiving high or low numbers of CFU-GM.(ABSTRACT TRUNCATED AT 250 WORDS)

The urease enzymes of Campylobacter pylori and a related bacterium.

The urease enzyme of Campylobacter pylori was studied and compared with that of a related spiral-shaped bacterium, St1, isolated from the rodent ileum. Both bacteria possessed constitutive urease enzymes with activities up to 20-70 times that of Proteus vulgaris. This activity was retained on SDS-polyacrylamide gels. A major catalytic subunit of mol. wt 300,000 was located for all (six) strains of C. pylori subjected to SDS-PAGE whereas St1 had two active forms of mol. wts 140,000 and 150,000. Western-blot analysis indicated the presence of anti-urease antibodies in the sera of patients with C. pylori-associated gastritis. The response to C. pylori urease was not strain-specific but no cross-reactivity was detected between the C. pylori enzyme and that of St1. The very high urease activity of these bacteria is likely to be important in colonisation of the host. Possession of glutamate dehydrogenase activity by both organisms suggests that one role of the urease may be to assimilate the available urea nitrogen. Modification of the local environment to facilitate long-term colonisation is another possible function. Protection from acid is unlikely to be a primary role as the natural habitat of the organism St1 is the non-acid-secreting tissue of the small intestine.

Mobilization of cytogenetically 'normal' blood progenitors cells by intensive conventional chemotherapy for chronic myeloid and acute lymphoblastic leukemia.

Various lines of evidence suggest that substantial numbers of very primitive normal hematopoietic cells persist in the marrow of most patients with CML, despite the presence of an expanded Philadelphia-Chromosome (Ph) positive population, and that normal clones might, in certain circumstances, have a proliferative advantage over leukemic populations. We have recently demonstrated in 5/8 CML patients with blastic phase (BP) that the blood progenitor cells/(BPC) harvested during early recovery from marrow aplasia were Ph-negative on cytogenetic analysis, suggesting that leukapheresis may provide a useful source of 'normal' progenitors for subsequent reinfusions. We report here an update on 40 patients with Ph + CML and 9 patients with ALL in first or subsequent relapses with associated cytogenetic translocations including t(8;14) t(4;8) t(4;11) and t(9;22). All these patients received intensive conventional chemotherapy and during early recovery from marrow aplasia, when the WBC reached 0.5-2.0 x 10(9)/L, BPC were collected by 4-8 leukapheresis and tested for the persistence of the marker translocations and, when possible, for the presence of the hybrid bcr/abl transcripts by polymerase chain reaction (PCR). In seven out of 10 patients with chronic phase CML, BPC were Ph-negative and in 5 PCR negative. In both accelerated phase patients, BPC were Ph-negative but PCR-positive and in eight out of 28 blastic CML patients, BPC were Ph-negative and in two cases also PCR-negative. Six out of 9 ALL patients, lost the cytogenic translocations. After complete recovery, 16 patients were subsequently given high-dose therapy followed by reinfusion of 'normal' BPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of ferritins in human milk secretions: similarities to serum and tissue isoferritins.

The ferritins present in the first day human colostrum and mature milk were compared with serum and tissue ferritins on the basis of their iron content, immunological reactivity and glycosylation. Both had a low iron content. The degree of glycosylation and immunochemical properties of colostrum ferritin showed strong similarities with serum ferritin. The isoelectric points and immunochemical properties of milk ferritin were similar to heart ferritin. The concentration of colostrum ferritin was more significantly correlated to body iron stores than milk ferritin.

Patients' group experiences.

In 11 years of experience with dialysis, we have tried to supply a kind of treatment in our center that takes the patient's physical, psychological, and social needs into account. We have been able to observe how important it is to share the common problems connected with a chronic illness in order to make them appear less dramatic. This led to the idea of organizing a group holiday in which the patients and their families would be able to spend some time together. As a result of this positive experience, we decided to organize group encounters in which all the problems could be freely discussed. The group is open to all the patients on dialysis or who have undergone transplants and their families. The group, which has been meeting once every 3 weeks for the last 2 years, is coordinated by a psychologist. This has proved to be important both for the operators, who have been able to expand their own knowledge, and for the patients and their families for whom these gatherings represent a way of helping themselves face up to and accept the limits imposed by chronic illness.

Popliteal artery entrapment syndrome (report on seven cases).

In connection with 7 cases of the popliteal artery entrapment syndrome observed, the Authors discuss certain anatomical, clinical, diagnostic and therapeutic aspects of the cases.

Indobufen vs acetylsalicylic acid plus dipyridamole in long-term patency after femoropopliteal bypass.

To compare the effects of indobufen (INB) with those of ASA+dipyridamole (DP) on graft patency, 113 patients undergoing femoropopliteal bypass surgery were randomly and blindly assigned to treatment with INB 400 mg daily or with ASA 900 mg daily plus DP 225 mg daily. Treatment started 2 days before surgery and lasted for 12 months. All patients underwent two angiographic examinations: the first early after surgery (mean 6 days) and the second at the end of the study (mean 368 days). The 1 year cumulative patency rate for INB was 60% higher but not statistically different from the ASA-DP group (53.2%). The relative risk (INB/ASA+DP) calculated by the Mantel-Haenszel test was 0.86 (confidence limits 0.54-1.35). Only the site of operation (above-knee or below-knee) has a significant prognostic value on the fate of the graft.

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.

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.