Gender-related differential effect of tachykinin NK2 receptor-mediated visceral hyperalgesia in guinea pig colon.

BACKGROUND AND PURPOSE: The tachykinin NK2 receptor antagonist ibodutant is under Phase III clinical investigation to treat female patients with irritable bowel syndrome. The aim of this study was to investigate the NK2 receptor-related gender specificity in a model of colitis. EXPERIMENTAL APPROACH: Colitis was induced by rectal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 0.5 mL, 30 mg·mL(-1) in 30% ethanol) in female and male guinea pigs. Electromyographic recording of the responses to colorectal distension (CRD) was made 3 days later. Ibodutant (0.33 , 0.65, 1.9 and 6.5 mg·kg(-1) ) was given s.c., 30 min before CRD. Release of neurokinin A and substance P from isolated mucosal and smooth muscle tissues following treatment with KCl (80 mM) or capsaicin (10 µM) was measured by EIA. Plasma pharmacokinetics of ibodutant following a single s.c. administration (0.73 or 2.1 mg·kg(-1) ) were measured over 24 h. KEY RESULTS: Ibodutant did not affect abdominal contractions in control animals. After TNBS-induced colitis, ibodutant prevented the increased visceral hypersensitivity to CRD in females, at lower doses than in males. Ibodutant pharmacokinetics did not differ between females and males. Tachykinins release was greater in smooth muscle than in mucosal samples. Capsaicin-stimulated release of tachykinins from inflamed mucosal samples from females was significantly lower than in males. CONCLUSIONS AND IMPLICATIONS: Ibodutant prevented abdominal nociception in a model of visceral hypersensitivity in guinea pigs with a greater efficacy in females than in males. Our results highlight a gender-related difference in colonic visceral hypersensitivity and mucosal nerve activation.

Cow's milk allergy and neonatal short bowel syndrome: comorbidity or true association?

BACKGROUND/OBJECTIVES: Neonatal short bowel syndrome (SBS) follows early intestinal resections that may expose the children to increased intestinal contact with undigested food proteins and to the risk of food allergy. We report three consecutive cases of cow's milk allergy (CMA) in SBS infants. SUBJECTS/METHODS: We reviewed three cases of CMA developed in 37 children with neonatal SBS followed up in the last 10 years. The setting of the survey was the Gastroenterology-Hepatology and Nutrition Unit of the Pediatric Hospital 'Bambino Gesù' in Rome. The diagnosis of CMA was based on the oral food challenge and was supported by the results of the skin prick tests (SPT) and/or the specific immunoglobulin (Ig) E. RESULTS: Two patients had persistent liquid stools and periodic episodes of vomiting when they were fed with an intact milk protein-based formula, that disappeared with extensively hydrolyzed formula and amino-acid-based formulae, respectively. The third patient developed maculo-papular rash, flushing and angioedema, when he was introduced a regular formula. The challenge-confirmed CMA in all patients. Positive specific IgE for milk proteins was documented in all the three patients. Two out of the three patients had positive familial history for allergy and positive SPT. CONCLUSIONS: Our findings suggest that the SBS patients require a careful clinical monitoring of the tolerance for the cow's milk proteins, because CMA could be more frequent than expected. A prospective regular assessment for the potential cow milk sensitization by SPT and specific IgE may clarify the nature of the association and support the clinical surveillance. Multicenter studies are required to better evaluate this comorbidity.

Phase transitions and interface phenomena in the cryogenic temperature domain of a niobate nanostructured ceramic.

Powder neutron diffraction and dielectric spectroscopy were used to investigate both crystallographic and dielectric permittivity properties of a Sr2KNb5O15 single phase ferroelectric oxide with nanosized grains ranging from 35 nm to 90 nm. Measurements were carried out in the temperature range from 10 K (cryogenic) to 550 K. All neutron diffraction data were indexed on the basis of a tetragonal double unit cell. From 10 K to room temperature the space group of the Sr2KNb5O15 ferroelectric phase was considered to be P4bm. The refinement of the paraelectric phase (at 550 K) was determined in the centrosymmetric space group P4/mbm. Dielectric spectroscopy measurements were performed in a thermal cycle. A set of four phase transitions non-related to symmetry changing was detected from Rietveld analysis of neutron powder diffraction data. During a thermal cycle, in the cryogenic temperature domain, strong thermal hysteresis is developed. Both phase transition and thermal hysteresis were correlated. These phenomena are associated with Nb-cation atomic displacements in the NbO6 octahedra along the c-axis direction and of the domain with different frequencies involving grains as well as an excess of interfaces ascribed to the grain boundary. The bulk/grain boundary interfaces in nanostructured ceramics are correlated with the thermal stability phenomenon.

Synovial fluid levels of bradykinin correlate with biochemical markers for cartilage degradation and inflammation in knee osteoarthritis.

OBJECTIVE: To determine the content of bradykinin (BK) and markers of cartilage degradation and inflammation in the synovial fluid (SF) of patients with knee osteoarthritis (OA), and to evaluate correlations with biomarkers or clinical parameters. METHODS: SFs were obtained from 30 patients with knee OA. Levels of basal and generated BK, cartilage oligomeric matrix protein (COMP), interleukin (IL) 1, IL-6, IL-8 and matrix metalloprotease (MMP) 1, MMP-3, MMP-13 and sulfated glycosaminoglycans (GAGs) were measured by enzyme-linked immunosorbent assay (ELISA) or colorimetric assays. RESULTS: The mean concentration of basal BK (in the presence of peptidase and protease inhibitors to avoid degradation and de novo formation of BK) was 422 pg/ml (95% confidence interval, CI, 281-563) whereas that of in vitro generated BK (in the presence of peptidase inhibitors SFs were incubated 60 min at 37°C to measure the potential capability to generate BK) was 3427 pg/ml (2591-4264). The content of MMP-13, IL-1α, and IL-1ß was under assay sensitivity. Basal BK levels positively correlated (Spearman's rank correlation) with GAGs (40 µg/ml, 26-54, r = 0.4834, P = 0.0308) and IL-6 (553 pg/ml, 171-935, r = 0.3946, P = 0.0377) similarly to the generated BK (GAGs, r = 0.4563, P = 0.0431; IL-6, r = 0.5605, P = 0.0019). Statistical analysis of basal BK and biomarkers was significant (P = 0.0483). When applying a stepwise logistic regression analysis considering biomarkers together with clinical parameters, results indicated that K/L radiographic OA grade and COMP improved the model (P = 0.0032). CONCLUSION: The presence of BK in the knee OA SF and its correlations with cartilage degradation and inflammation markers of OA support its participation in OA pathology.

Resolving sphincter of Oddi incontinence for primary duodenal Crohn's disease with strictureplasty.

INTRODUCTION: Crohn's involvement of duodenum is a rare event and may be associated to proteiform symptoms and uncommon pathological aspects which make diagnosis and treatment complex. PRESENTATION OF CASE: The peculiar aspect of this case was a suspected duodeno-biliary fistula. The patient (female, 22 years old) was affected by duodenal Crohn's disease. Magnetic resonance imaging showed a dilated common bile duct, whose final part linked to a formation containing fluid, and characterized by filling of the contrast medium in the excretory phase. Abdominal ultrasound showed intra-hepatic and intra-gallbladder aerobilia. At surgery, the duodenum was mobilized showing an inflammatory stricture and a slight dilatation of the common bile duct, with no signs of fistulas. The opened duodenum was anastomized side to side to a transmesocolic loop of the jejunum. After surgery, the general condition of the patient improved. DISCUSSION: Only two cases of fistula between a narrow duodenal bulb and the common bile duct have been described in literature and the Authors were not be able to verify the occurrence of a duodenal biliary fistula at surgery. The association between duodenal Crohn's disease and Sphincter of Oddi incontinence is a very rare finding with different etiology: chronic intestinal pseudo-obstruction, common bile duct stones, progressive systemic sclerosis. CONCLUSION: The treatment to resolve Sphincter of Oddi incontinence for primary duodenal Crohn's disease is not clear. Strictureplasty could be the treatment of choice, because, resolving the stricture, the duodenal pressure is likely to decrease and the reflux through the incontinent sphincter can be avoided.

Preparation and structural characterization of vulcanized natural rubber nanocomposites containing nickel-zinc ferrite nanopowders.

Single-phase polycrystalline mixed nickel-zinc ferrites belonging to Ni0.5Zn0.5Fe2O4 were prepared on a nanometric scale (mean crystallite size equal to 14.7 nm) by chemical synthesis named the modified poliol method. Ferrite nanopowder was then incorporated into a natural rubber matrix producing nanocomposites. The samples were investigated by means of infrared spectroscopy, X-ray diffraction, scanning electron microscopy and magnetic measurements. The obtained results suggest that the base concentration of nickel-zinc ferrite nanoparticles inside the polymer matrix volume greatly influences the magnetic properties of nanocomposites. A small quantity of nanoparticles, less than 10 phr, in the nanocomposite is sufficient to produce a small alteration in the semi-crystallinity of nanocomposites observed by X-ray diffraction analysis and it produces a flexible magnetic composite material with a saturation magnetization, a coercivity field and an initial magnetic permeability equal to 3.08 emu/g, 99.22 Oe and 9.42 x 10(-5) respectively.

Fasitibant prevents the bradykinin and interleukin 1ß synergism on prostaglandin E2 release and cyclooxygenase 2 expression in human fibroblast-like synoviocytes.

This study investigates the effect of the selective and potent B(2) receptor antagonist fasitibant (MEN16132) on the proinflammatory effect of bradykinin (BK) and its interaction with interleukin 1ß (IL-1ß) in human synoviocytes. PGE(2) content was detected in the surnatants and COX-2 and COX-1 gene and protein expression determined in the cells. Radioligand binding ([(3) H]BK) and BK-induced inositolphosphate experiments were performed. Incubation of synoviocytes with BK induced a sustained production of PGE(2) and transient COX-2 gene expression that were prevented by pretreatment with fasitibant (1 µM, 30 min preincubation). IL-1ß increased PGE(2) release and COX-2 expression more than BK alone. The combined treatment of cells with BK and IL-1ß induced an even increase of released PGE(2) and COX-2 gene and protein expression indicating a synergistic rather than an additive effect, not related to an increase of B(2) receptors density or its coupling. These potentiating effects of BK on PGE(2) production and increased COX-2 expression produced by IL-1ß were B(2)-receptor-mediated as fasitibant could prevent them. None of the treatments induced changes in the COX-1 expression. The synergistic PGE(2) production was abolished by the specific NF-kappaB inhibitor (BAY-117085), whereas specific inhibitors for the p38 (SB203580), JNK (SP600125), and ERK1/2 (PD98059) mitogen-activated protein kinases could prevent the prostanoid release. BK can potentiate the COX-2 gene expression and consequent prostanoid production induced by IL-1ß. The prevention of this synergism by fasitibant indicates BK B(2) receptor blockade as an alternative symptomatic therapy for osteoarthritis.

Bradykinin and B2 receptor antagonism in rat and human articular chondrocytes.

BACKGROUND AND PURPOSE: In osteoarthritis (OA), bradykinin (BK) is known to contribute to pain and synovitis, but not to cartilage degradation. Here, we investigated effects of BK and its antagonists on chondrocytes, cells involved in cartilage homeostasis. EXPERIMENTAL APPROACH: BK receptor density and affinities of BK, its analogues and antagonists were measured in cultured human and rat chondrocytes by radioligand binding. Effects of BK were assessed by accumulation of inositol phosphates (IP) and release of interleukin (IL)-6 and IL-8. KEY RESULTS: Density of [³H]-BK binding sites was higher (13-30-fold) and BK evoked a greater (48-fold) IP production, in human than in rat chondrocytes. The BK B2 receptor antagonists MEN16132 and icatibant displayed similar binding affinity. MEN16132 was 40-fold more potent than icatibant in the IP assay. In human chondrocytes, BK increased release (over 24 h) of IL-6 and IL-8, effects blocked by MEN16132 but not by the B1 receptor antagonist Lys-[Leu8][desArg9]BK. BK-induced release of IL-6, but not of IL-8, was partially inhibited by indomethacin (10 µM) and nordihydroguaiaretic acid (10 µM). Antagonists for the prostanoid EP receptors (AH6809 10 µM; L-798,196, 200 nM; L-161,982, 1 µM) were ineffective. Dexamethasone (100 nM) partially inhibited release of both IL-6 and IL-8. Inhibitors of intracellular downstream signalling pathways (SB203580 10 µM; PD98059, 30 µM; SP600125, 30 µM; BAY-117085, 5 µM) indicated the involvement of p38 MAPK and the activation of NF-κB. CONCLUSION AND IMPLICATIONS: BK mediated inflammatory changes and cartilage degradation and B2 receptor blockade would, therefore, be a potential treatment for OA.

Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B2 receptor.

BACKGROUND AND PURPOSE: Icatibant is a well-known kinin B2 receptor antagonist currently used for angiooedema attacks. MEN16132 is a non-peptide B2 receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists. EXPERIMENTAL APPROACH: Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B2 receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B2 receptors. KEY RESULTS: Recovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [³H]-BK or the B2 receptor antagonist [³H]-MEN11270 binding site) was compared to that of MEN16132 using a panel of point-mutated receptors with mutations located at the transmembrane regions of the B2 receptor, previously shown to decrease MEN16132 high affinity interaction. No consistent decrease of icatibant affinity was observed. From the different affinity of MEN16132 derivatives at wild type and W86A (transmembrane 2 region) receptors, and by evaluating its antagonist profile at the D266A/D284A double mutant receptor, a model of the MEN16132-B2 receptor complex is proposed. CONCLUSIONS AND IMPLICATIONS: MEN16132 dissociated from the B2 receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.

Novel effects mediated by bradykinin and pharmacological characterization of bradykinin B2 receptor antagonism in human synovial fibroblasts.

BACKGROUND AND PURPOSE: Bradykinin (BK) and B2 receptors have been implicated in the pathophysiology of osteoarthritis (OA), and synovitis is one of its hallmarks. Here, the selective B2 receptor antagonists MEN16132 and icatibant have been pharmacologically characterized in human synovial cells. EXPERIMENTAL APPROACH: Radioligand and functional studies (inositol phosphate (IP) accumulation, interleukin (IL)-6 and IL-8 release) were performed in cultured synoviocytes. KEY RESULTS: [3H]-BK saturation studies indicated receptor density (Bmax) and K(d) values of 121,550 sites per cell and 1.14 nM respectively. In synoviocytes, MEN16132 (pK(I) 8.9) was threefold more potent than icatibant (pK(I) 8.4). Both antagonists showed competitive antagonism in the BK-induced IP assay (control EC50 0.45 nM), with pK(B) values of 9.9 (MEN16132) and 8.1 (icatibant). 24h incubation with BK induced IL-6 (EC50 216 nM) and IL-8 (EC50 53 nM) release. Both MEN16132 (IL-6: pIC50 8.1; IL-8: pIC50 8.4) and icatibant (IL-6: pIC50 6.6; IL-8: pIC50 6.7) completely prevented this BK-induced release. Indomethacin did not affect the basal or the IL-6/IL-8 release induced by BK, whereas nordihydroguaiaretic acid decreased the basal release, although BK still increased IL-6 and IL-8 production. BK-induced IL-8 release was attenuated by inhibitors of phospholipase C (U73122), p38 (SB203580), JNK (SP600125), ERK 1/2 (PD98059) MAPKs, phosphoinositide 3-kinase (LY294002), NF-kappaB (BAY-117085) and by the glucocorticoid dexamethasone. CONCLUSIONS AND IMPLICATIONS: Bradykinin via B2 receptors can participate in inflammatory events in synovitis. MEN16132 is a highly potent B2 receptor antagonist capable of blocking pro-inflammatory responses to BK evoked in human synoviocytes.

Comparative antagonist pharmacology at the native mouse bradykinin B2 receptor: radioligand binding and smooth muscle contractility studies.

BACKGROUND AND PURPOSE: The aim was to characterize the recently discovered non-peptide antagonist MEN16132 at the mouse B2 receptor, relative to other antagonists. EXPERIMENTAL APPROACH: [3H]-BK binding experiments used mouse lung and ileum tissue membranes and antagonist potency was measured in the isolated ileum contractility assay. KEY RESULTS: Two BK binding sites resulted from saturation and homologous competition experiments. A role for the B1 receptor was excluded because of the poor affinity of B1 receptor ligands (pIC50<5). MEN16132, and the other reference antagonists, inhibited only one portion of BK specific binding, and the rank order of potency was (pIC50): Icatibant (lung 10.7; ileum 10.2)=MEN11270 (lung 10.4; ileum 9.9)=MEN16132 (lung 10.5; ileum 9.9).>LF16-0687 (lung 8.9; ileum 8.8)>FR173657 (lung 8.6; ileum 8.2). BK homologous curves performed with lung membranes after treatment with the antagonist MEN16132 or Icatibant (10 nM) displayed only the low affinity site. The functional antagonism by MEN16132 (pA2 9.4) and Icatibant (pA2 9.1), towards BK (control EC50 6.1 nM) induced ileum contractions, was concentration-dependent and surmountable, but the Schild plot slope was less than unity. CONCLUSIONS AND IMPLICATIONS: In mouse tissue, radiolabelled BK recognizes two binding sites and B2 receptor antagonists can compete only for the higher affinity one. The pharmacological profile of the novel non-peptide antagonist MEN16132 indicates that it exhibits subnanomolar affinity and potency for the mouse B2 receptor and is suitable for further characterization in in vivo pathophysiological models.

Characterization of kinin receptors in human cultured detrusor smooth muscle cells.

BACKGROUND AND PURPOSE: Kinins have an important role in inflammatory cystitis and in animal pathophysiological models, by acting on epithelium, fibroblasts, sensory innervation and smooth muscle. The aim of this study was to characterize the receptors responsible for direct motor responses induced by kinins on human detrusor. EXPERIMENTAL APPROACH: Human detrusor cells from biopsies were isolated and maintained in culture. B(1) and B(2) kinin receptors were characterized by means of radioligand and functional experiments (PI accumulation and PGE(2) release). KEY RESULTS: [(3)H]-[desArg(9)]-Lys-BK and [(3)H]-BK saturation studies indicated receptor density (B(max)) and K (d) values of 19 or 113 fmol mg(-1), and 0.16 or 0.11 nM for the B(1) or B(2) receptors, respectively. Inhibition binding studies indicated the selectivity of the B(1) receptor antagonist [desArg(9)Leu(8)]-Lys-BK and of the B(2) receptor antagonists Icatibant and MEN16132. [DesArg(9)]-Lys-BK and BK induced PI accumulation with an EC(50) of 1.6 and 1.4 nM and different maximal responses (E(max) of [desArg(9)]-Lys-BK was 10% of BK). BK also induced prostaglandin E(2) release (EC(50) 2.3 nM), whereas no response was detected with the B(1) receptor agonist. The incubation of detrusor smooth muscle cells with interleukin 1beta (IL-1beta) or tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1)) induced a time-dependent increase in radioligand-specific binding, which was greater for the B(1) than for the B(2) receptor. CONCLUSIONS AND IMPLICATIONS: Human detrusor smooth muscle cells in culture retain kinin receptors, and represent a suitable model to investigate the mechanisms and changes that occur under chronic inflammatory conditions.