Central substance P NK(1) receptors are involved in fever induced by LPS but not by IL-1 beta and CCL3/MIP-1 alpha in rats

Substance P (SP) is a neuropeptide that can modulate inflammatory mediator release through activation of NK(1) receptors (NK(1)R). Some studies have also suggested the involvement of SP in lipopolysaccharide (LPS)-induced fever. However, the precise contribution of this neuropeptide to the pathways activated during fever is unknown. In this study we investigated the effect of a selective NK(1)R antagonist, SR140333B, on the febrile response induced by LPS and cytokines. Our results show that the systemic injection of SR140333B did not modify the fever induced by LPS at a dose that is able to reduce protein extravasation induced by SP in the skin. On the other hand, intracerebroventricular administration of 5R140333B significantly reduced the fever induced by peripheral injection of LPS. These data emphasize an important role for SP in the central nervous system during the febrile response to LPS, and are reinforced by the fact that intracerebroventricular injection of SP also induced fever in a dose-dependent manner in captopril-treated rats. Considering that the febrile response can result from the generation of several endogenous pyrogens, among them interleukin (IL)-1 beta and macrophage inflammatory protein-1 alpha (CCL3/MIP-1 alpha), we also examined the effect of SR140333B on the fever induced by these cytokines which act through prostaglandin-dependent and independent mechanisms, respectively. Surprisingly, SR140333B did not modify the febrile response to IL-1 beta or CCL3/MIP-1 alpha. Altogether these data suggest that the central action of SP is essential for LPS-, but not for IL-1 beta- or CCL3/MIP-1 alpha-induced fever. (C) 2011 Elsevier B.V. All rights reserved.

Il-1 beta breaks tolerance through inhibition of regulatory T cell function

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn’s disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1beta drives proliferation and cytokine production by CD4+CD25+FoxP3– effector/memory T cells, attenuates CD4+CD25+FoxP3+ regulatory T cell function, and allows escape of CD4+CD25– autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

Immunoexpression of adrenergic receptors in detrusor from patients with prune belly syndrome: a digital quantification

Introduction: Prune belly syndrome (PBS) presents with large-capacity bladders, high compliance and post-void residual volumes. Operative and conservative treatments are controversial. When histologically compared to normal bladder, bladder outlet obstruction results in an up- or down-regulation of adrenoceptors. Our goal was to study the immunoexpression of adrenoceptors in detrusor from patients with PBS. Materials and methods: Bladder domes from PBS patients (n = 14) were studied (PBG). For normal controls, bladder specimens were obtained at adult surgery (n = 13) (CG1) and at child autopsy (n = 5) (CG2). Staining was performed using antibodies to alpha 1a, alpha 1b, alpha 1d and beta 3 adrenoceptors. Five to 10 images were captured on an optic microscope with a digital camera and analysed with Photoshop(R). The immunocyhistochemical index with arbitrary units was calculated and compared. Results: Mean age was 1.28, 64 and 1.41 years for PBG, CG1 and CG2, respectively. The immunohistochemical index with arbitrary units of alpha 1a receptors was 0.06 in PBG, 0.16 in CG1 and 0.14 in CG2 (p = 0.008); of alpha 1b 0.06, 0.06 and 0.07 (p = 0.781); and of alpha 1d 0.04, 0.04 and 0.05 (p = 0.618). Regarding beta 3 the respective values were 0.07, 0.14 and 0.10 (p = 0.378). Conclusion: Our results show a decrease in ala-adrenoceptor immunostaining intensity in detrusor from children with PBS. Further in vitro studies are needed to determine whether these observations are physiologically significant. (C) 2009 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

Prognostic Value of Dobutamine Stress Echocardiography With Early Injection of Atropine With Versus Without Chronic Beta-Blocker Therapy in Patients With Known or Suspected Coronary Heart Disease

Although a new protocol of dobutamine stress echocardiography with the early injection of atropine (EA-DSE) has been demonstrated to be useful in reducing adverse effects and increasing the number of effective tests and to have similar accuracy for detecting coronary artery disease (CAD) compared with conventional protocols, no data exist regarding its ability to predict long-term events. The aim of this study was to determine the prognostic value of EA-DSE and the effects of the long-term use of beta blockers on it. A retrospective evaluation of 844 patients who underwent EA-DSE for known or suspected CAD was performed; 309 (37%) were receiving beta blockers. During a median follow-up period of 24 months, 102 events (12%) occurred. On univariate analysis, predictors of events were the ejection fraction (p <0.001), male gender (p <0.001), previous myocardial infarction (p <0.001), angiotensin-converting enzyme inhibitor therapy (p = 0.021), calcium channel blocker therapy (p = 0.034), and abnormal results on EA-DSE (p <0.001). On multivariate analysis, the independent predictors of events were male gender (relative risk [RR] 1.78, 95% confidence interval [CI] 1.13 to 2.81, p = 0.013) and abnormal results on EA-DSE (RR 4.45, 95% CI 2.84 to 7.01, p <0.0001). Normal results on EA-DSE with P blockers were associated with a nonsignificant higher incidence of events than normal results on EA-DSE without beta blockers (RR 1.29, 95% CI 0.58 to 2.87, p = 0.54). Abnormal results on EA-DSE with beta blockers had an RR of 4.97 (95% CI 2.79 to 8.87, p <0.001) compared with normal results, while abnormal results on EA-DSE without beta blockers had an RR of 5.96 (95% CI 3.41 to 10.44, p <0.001) for events, with no difference between groups (p = 0.36). In conclusion, the detection of fixed or inducible wall motion abnormalities during EA-DSE was an independent predictor of long-term events in patients with known or suspected CAD. The prognostic value of EA-DSE was not affected by the long-term use of beta blockers. (C) 2008 Elsevier Inc. All rights reserved. (Am J Cardiol 2008;102:1291-1295)

Copper-mediated amyloid-beta toxicity is associated with an intermolecular histidine bridge

Amyloid-beta peptide (A beta) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic A beta-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/ peptide ratios of > 0.6:1 by EPR spectroscopy. The toxicity of the A beta-Cu2+ complex to cultured primary cortical neurons was attenuated when either the pi- or tau-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl- 1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. P-31 magic angle spinning solid-state NMR showed that A beta and A beta-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the A beta toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.

IL-1 beta breaks tolerance through expansion of CD25(+) effector T cells

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn's disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1 beta drives proliferation and cytokine production by CD4(+)CD25(+)FoxP3(-) effector/memory T cells, attenuates CD4(+)CD25(+)FoxP3(+) regulatory T cell function, and allows escape of CD4(+)CD25(-) autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1 beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

Estradiol`s Salutary Effects on Keratinocytes Following Trauma-Hemorrhage Are Mediated by Estrogen Receptor (ER)-alpha and ER-beta

Although administration of 17 beta-estradiol (estrogen) following trauma-hemorrhage attenuates the elevation of cytokine production and mitogen-activated protein kinase (MAPK) activation in epidermal keratinocytes, whether the salutary effects of estrogen are mediated by estrogen receptor (ER)-alpha. or ER-beta is not known. To determine which estrogen receptor is the mediator, we subjected C3H/HeN male mice to trauma-hemorrhage (2-cm midline laparotomy and bleeding of the animals to a mean blood pressure of 35 mmHg and maintaining that pressure for 90 min) followed by resuscitation with Ringer`s lactate (four times the shed blood volume) At the middle of resuscitation we subcutaneously injected ER-alpha agonist propyl pyrazole trial (PPT; 5 mu g/kg), ER-beta agonist diarylpropionitrile (DPN; 5 mu g/kg), estrogen (50 mu g/kg), or ER antagonist ICI 182,780 (150 mu g/kg). Two hours after resuscitation, we isolated keratinocytes, stimulated them with lipopolysaccharide for 24 In (5 mu g/mL for maximum cytokine production), and measured the production of interleukin (IL)-6, IL-10, IL-12, and INF-alpha and the activation of MAPK. Keratinocyte cytokine production markedly increased and MAPK activation occurred following trauma-hemorrhage but were normalized by administration of estrogen, PPT and DPN. PPT and DPN administration were equally effective in normalizing the inflammatory response of keratinocytes, indicating that both ER-alpha. and ER-beta mediate the salutary effects of estrogen on kerotinocytes after trauma-hemorrhage.

Biologically active conformer of the effector region of human C5a and modulatory effects of N-terminal receptor binding determinants on activity

A conformationally biased decapeptide agonist of human C5a (C5a(65-74)Y65,F67,P69,P71,D-Ala73 or YSFKPMPLaR) was used as a functional probe of the C5a receptor (C5aR) in order to understand the conformational features in the C-terminal effector region of C5a that are important for C5aR binding and signal transduction. YSFKPMPLaR was a potent, full agonist of C5a, but at higher concentrations had a superefficacious effect compared to the natural factor. The maximal efficacy of this analogue was 216 +/- 56% that of C5a in stimulating the release of beta-glucuronidase from human neutrophils. C5aR activation and binding curves both occurred in the same concentration range with YSFKPMPLaR, characteristics not observed with natural C5a or more conformationally flexible C-terminal agonists. YSFKPMPLaR was then used as a C-terminal effector template onto which was synthesized various C5aR binding determinants from the N-terminal core domain of the natural factor. In general, the presence of N-terminal binding determinants had little effect on either potency or binding affinity when the C-terminal effector region was presented to the C5aR in this biologically active conformation. However, one peptide, C5a(12-20)-Ahx-YSFKPMPLaR, expressed a 100-fold increase in affinity for the neutrophil C5aR and a 6-fold increase in potency relative to YSFKPMPLaR. These analyses showed that the peptides used in this study have up to 25% of the potency of C5a in human fetal artery and up to 5% of the activity of C5a in the PMN enzyme release assay.

Characterisation of five missense mutations in the cystathionine beta-synthase gene from three patients with B-6-nonresponsive homocystinuria

Homocystinuria, due to a deficiency of the enzyme cystathionine beta-synthase (CBS), is an inborn error of sulphur-amino acid metabolism, This is an autosomal recessive disease which results in hyperhomocysteinaemia and a wide range of clinical features, including optic lens dislocation, mental retardation, skeletal abnormalities and premature thrombotic events, We report the identification of 5 missense mutations in the protein-coding region of the CBS gene from 3 patients with pyridoxine-nonresponsive homocystinuria. Reverse-transcription PCR was used to amplify CBS cDNA from each patient and the coding region was analysed by direct sequencing, The mutations detected included 3 novel (1058C --> T, 992C --> A and 1316G --> A) and 2 previously identified (430G --> A and 833C --> T) base alterations in the CBS cDNA, Each of these mutations predicts a single amino acid substitution in the CBS polypeptide, Appropriate cassettes of patient CBS cDNA, containing each of the above defined mutations, were used to replace the corresponding cassettes of normal CBS cDNA sequence within the bacterial expression vector pT7-7. These recombinant mutant and normal CBS constructs were expressed in Escherichia coli cells and the catalytic activities of the mutant proteins were compared with normal. All of the mutant proteins exhibited decreased catalytic activity in vitro, which confirmed the association between the individual mutation and CBS dysfunction in each patient.

beta-Blocker Therapy and Mortality of Patients With Chagas Cardiomyopathy A Subanalysis of the REMADHE Prospective Trial

Background-Peculiar aspects of Chagas cardiomyopathy raise concerns about efficacy and safety of sympathetic blockade. We studied the influence of beta-blockers in patients with Chagas cardiomyopathy. Methods and Results-We examined REMADHE trial and grouped patients according to etiology (Chagas versus non-Chagas) and beta-blocker therapy. Primary end point was all-cause mortality or heart transplantation. Altogether 456 patients were studied; 27 (5.9%) were submitted to heart transplantation and 202 (44.3%) died. Chagas etiology was present in 68 (14.9%) patients; they had lower body mass index (24.1+/-4.1 versus 26.3+/-5.1, P=0.001), smaller end-diastolic left ventricle diameter (6.7+/-1.0 mm versus 7.0+/-0.9 mm, P=0.001), smaller proportion of beta-blocker therapy (35.8% versus 68%, P<0.001), and higher proportion of spironolactone therapy (74.6% versus 57.8%, P=0.003). Twenty-four (35.8%) patients with Chagas disease were under beta-blocker therapy and had lower serum sodium (136.6+/-3.1 versus 138.4+/-3.1 mEqs, P=0.05) and lower body mass index (22.5+/-3.3 versus 24.9+/-4.3, P=0.03) compared with those who received beta-blockers. Survival was lower in patients with Chagas heart disease as compared with other etiologies. When only patients under beta-blockers were considered, the survival of patients with Chagas disease was similar to that of other etiologies. The survival of patients with beta-blockers was higher than that of patients without beta-blockers. In Cox regression model, left ventricle end-diastolic diameter (hazard ratio, 1.78; CI, 1.15 to 2.76; P=0.009) and beta-blockers (hazard ratio, 0.37; CI, 0.14 to 0.97; P=0.044) were associated with better survival. Conclusions-Our study suggests that beta-blockers may have beneficial effects on survival of patients with heart failure and Chagas heart disease and warrants further investigation in a prospective, randomized trial.

The Carvedilol`s Beta-Blockade in Heart Failure and Exercise Training`s Sympathetic Blockade in Healthy Athletes during the Rest and Peak Effort

In recent years, beta-blocker therapy has become a primary pharmacologic intervention in patients with heart failure by blocking the sympathetic activity. To compare the exercise training`s sympathetic blockade in healthy subjects (athletes) and the carvedilol`s sympathetic blockade in sedentary heart failure patients by the evaluation of the heart rate dynamic during an exercise test. A total of 26 optimized and 49 nonoptimized heart failure patients in a stable condition (for, at least, 3 months), 15 healthy athletes and 17 sedentary healthy subjects were recruited to perform a cardiopulmonary exercise test. The heart rate dynamic (rest, reserve, peak and the peak heart rate in relation to the maximum predicted for age) was analyzed and compared between the four groups. The heart rate reserve was the same between optimized (48 +/- 15) and nonoptimized (49 +/- 18) heart failure patients (P < 0.0001). The athletes (188 +/- 9) showed a larger heart rate reserve compared to sedentary healthy subjects (92 +/- 10, P < 0.0001). Athletes and healthy sedentary reached the maximum age-predicted heart ratefor their age, but none of the heart failure patients did. The carvedilol`s sympathetic blockade occurred during the rest and during the peak effort in the same proportion, but the exercise training`s sympathetic blockade in healthy subjects occurred mainly in the rest.

Activation of the Wnt/Beta-catenin Signaling Pathway during Oral Carcinogenesis Process Is Not Influenced by the Absence of Galectin-3 in Mice

Background/Aim: Galectin-3 has been associated with activated Wnt pathway, translocating beta-catenin into the nucleus. However, it is still unknown whether this lectin drives the Wnt signaling activation in lesions from galectin-3-deficient (Gal3(-/-)) mice. The purpose was to study beta-catenin expression in tongue lesions from Gal3(-/-) and wildtype (Gal3(+/+)) mice and the status of Wnt signaling. Materials and Methods: Twenty Gal3(-/-) and Gal3(+/+) male mice were challenged with 4-nitroquinolin-1-oxide and killed at week 16 and 32. Tongues were processed and stained with H&E to detect dysplasias and carcinomas. An imunohistochemical assay was performed to evaluate beta-catenin expression. Results: Carcinomas were more evident in Gal3(+/+) than Gal3(-/-) mice (55.5% vs. 28.5%, respectively; p>0.05). Elevated expression of non-membranous beta-catenin was observed in dysplasias and carcinomas from both groups (p>0.05). Conclusion: Absence of galectin-3 does not interfere in the pattern of beta-catenin expression and therefore in the mediation of the Wnt signaling pathway.

Insulin-like growth factor-1 and 17 beta-estradiol down-regulate prostate apoptosis response-4 expression in MCF-7 breast cancer cells

The PKC apoptosis WTI regulator gene, also named prostate apoptosis response-4 (PAR-4), encodes a pro-apoptotic protein that sensitizes cells to numerous apoptotic stimuli. Insulin-like growth factor-1 (IGF-1) and 17 beta-estradiol (E2), two important factors for breast cancer development and progression, have been shown to down-regulate PAR-4 expression and inhibit apoptosis induced by PAR-4 in neuronal cells. In this study, we sought to investigate the mechanisms of regulation of PAR-4 gene expression in MCF-7 cells treated with E2 or IGF-1. E2 (10 nM) and IGF-1 (12.5 nM) each down-regulated PAR-4 expression in MCF-7 cells after 24 h of treatment. The effect of E2 was dependent on ER activation, as demonstrated by an increase in PAR-4 expression when cells were pretreated for 1 h with 1 mu M ICI-182,780 (ICI) before receiving E2 plus ICI. The effect of IGF-1 was abolished by pre-treatment for 1 h with 30 mu M LY294002 (a specific PI3-K inhibitor), and significantly inhibited by 30 mu M SB202190 (a specific p38MAPK inhibitor). We also demonstrated that E2 acts synergistically with IGF-1, resulting in greater down-regulation of PAR-4 mRNA expression compared with E2 or IGF-1 alone. Our results show for the first time that E2 and IGF-1 inhibit PAR-4 gene expression in MCF-7 cells, suggesting that this down-regulation may provide a selective advantage for breast cancer cell survival.

Glycosaminoglycan chains from alpha(5)beta(1) integrin are involved in fibronectin-dependent cell migration

alpha(5)beta(1) integrin from both wild-type CHO cells (CHO-K1) and deficient in proteoglycan biosynthesis (CHO-745) is post-translationally modified by glycosaminoglycan chains. We demonstrated this using [(35)S]sulfate metabolic labeling of the cells, enzymatic degradation, immunoprecipitation reaction with monoclonal antibody, fluorescence microscopy, and flow cytometry. The alpha(5)beta(1) integrin heterodimer is a hybrid proteoglycan containing both chondroitin and heparan sulfate chains. Xyloside inhibition of sulfate incorporation into alpha(5)beta(1) integrin also supports that integrin is a proteoglycan. Also. cells grown with xyloside adhered on fibronectin with no alteration in alpha(5)beta(1) integrin expression. However, haptotactic motility on fibronectin declined in cells grown with xyloside or chlorate as compared with controls. Thus, alpha(5)beta(1) integrin is a proteoglycan and the glycosaminoglycan chains of the integrin influence cell motility on fibronectin. Similar glycosylation of alpha(5)beta(1) integrin was observed in other normal and malignant cells, suggesting that this modification is conserved and important in the function of this integrin. Therefore, these glycosaminoglycan chains of alpha(5)beta(1) integrin are involved in cellular migration on fibronectin.