Endo-PDI is required for TNFα-induced angiogenesis

Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function

INFLAMMATORY RESPONSE AND NEUTROPHIL FUNCTIONS IN PLAYERS AFTER A FUTSAL MATCH

de Moura, NR, Cury-Boaventura, MF, Santos, VC, Levada-Pires, AC, Bortolon, JR, Fiamoncini, J, Pithon-Curi, TC, Curi, R, and Hatanaka, E. Inflammatory response and neutrophil functions in players after a futsal match. J Strength Cond Res 26(9): 2507-2514, 2012-Futsal players suffer injuries resulting from muscle fatigue and contact or collision among players. Muscle lesions can be detected by measuring muscle lesion markers such as creatine kinase (CK) and lactate dehydrogenase (LDH) in plasma. After an initial lesion, there is an increase in the plasma levels of C-reactive protein (CRP) and proinflammatory cytokines. These mediators may activate neutrophils and contribute to tissue damage and increase susceptibility to invasive microorganisms. In this study, we investigated the effect of a futsal match on muscle lesion markers, cytokines, and CRP in elite players. The basal and stimulated neutrophil responsiveness after a match was also evaluated based on measurements of neutrophil necrosis, apoptosis, phagocytic capacity, reactive oxygen species (ROS) production, and cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-8, IL-1 beta, IL-10, and IL-1ra) production. Blood samples were taken from 16 players (26.4 +/- 3.2 years, 70.2 +/- 6.9 kg, 59.7 +/- 5.1 ml.kg(-1).min(-1), sports experience of 4.4 +/- 0.9 years) before and immediately after a match. Exercise increased the serum activities of CK (2.5-fold) and LDH (1.3-fold). Playing futsal also increased the serum concentrations of IL-6 (1.6-fold) and CRP (1.6-fold). The TNF-alpha, IL-1 beta, IL-8, IL-1ra, and IL-10 serum levels were not modified in the conditions studied. The futsal match induced neutrophil apoptosis, as indicated by phosphatidylserine externalization (6.0-fold). The exercise induced priming of neutrophils by increasing ROS (1.3-fold), TNF-alpha (5.8-fold), and IL-1 beta (4.8-fold) released in nonstimulated cells. However, in the stimulated condition, the exercise decreased neutrophil function, diminishing the release of ROS by phorbol myristate acetate-stimulated neutrophils (1.5-fold), and the phagocytic capacity (1.6-fold). We concluded that playing futsal induces inflammation, primes and activates neutrophils, and reduces the efficiency of neutrophil phagocytosis immediately after a match.

GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

Comparison between the C5 or C6-Cz electrode assembly and C3 or C4-Cz assembly for transcranial electric motor activation of muscular response of the contralateral facial nerve

We used an assembly of electrodes C3 and C4-Cz in order to activate the motor cortical area of the corticobulbar tract to elucidate the motor-evoked potential of the contralateral mentalis muscle. We compared this setup to that of an assembly with electrodes C5 or C6-Cz using a train of electrical pulses and a single electrical pulse. This analysis was made in 23 consecutive patients who underwent several varied surgeries and were prospectively operated on at Santa Paula Hospital between January and June 2011. The results showed that the assembly with C5 or C6-Cz produced a multisynaptic motor-evoked potential in the contralateral mentalis muscle in 86.9 % of the patients, whereas 82.6 % of patients stimulated at points C3 or C4-Cz presented the same response. However, both assemblies showed similar behavior with the use of a single electrical pulse for peripheral contralateral nerve stimulation. We concluded that the C5 or C6-Cz assembly was similar to C3 or C4-Cz in obtaining a multisynaptic response in the contralateral mentalis muscle, although it required less intensive stimulation than the C3 or C4- Cz assembly.

Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient Little mice

OBJECTIVE: To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormone-releasing hormone receptors. MATERIALS AND METHODS: The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/litmice, which represent a model of GH deficiency arising frommutated growth hormone-releasing hormone-receptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice. RESULTS: After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3 +/- 1.5 ng/ml was observed compared with 1.04 +/- 1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5 +/- 9.7 ng/ml and a higher growth hormone release of 163 +/- 46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions. CONCLUSIONS: Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.

Production of a xylose-stimulated beta-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of beta-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 +/- A 411.2 U g(-1), while beta-glucosidase production was increased about 2.6-fold, reaching 20.7 +/- A 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis beta-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for beta-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The beta-glucosidase maintained about 95 % of its activity after 26 h in water at 55 A degrees C, with half-lives of 15.7 h at 60 A degrees C and 5.1 h at 65 A degrees C. The presence of xylose during heat treatment at 65 A degrees C protected beta-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 A degrees C. Xylose stimulated beta-glucosidase activity up to 1.7-fold, at 200 mmol L-1. The notable features of both xylanase and beta-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Maternal immune activation in late gestation enhances locomotor response to acute but not chronic amphetamine treatment in male mice offspring: Role of the D1 receptor

Exposure to elevated levels of maternal cytokines can lead to functional abnormalities of the dopaminergic system in the adult offspring, including enhanced amphetamine (AMPH)-induced locomotion. Therefore, it seems reasonable to consider that offspring of challenged mothers would behave differently in models of addictive behavior, such as behavioral sensitization. Thus, we sought to evaluate the effects of prenatal exposure to lipopolysaccharide (LPS) on the locomotor response to acute and chronic AMPH treatment in male mice offspring. For this purpose, LPS (Escherichia coli 0127:B8; 120 mu g/kg) was administered intraperitoneally to pregnant Swiss mice on gestational day 17. At adulthood, male offspring were studied under one of the following conditions: (1) locomotor response to acute AMPH treatment (2.5 or 5.0 mg/kg) in an open field test; (2) behavioral sensitization paradigm, which consists of a daily injection of AMPH (1.0 mg/kg) for 10 days and observation of locomotion in the open field on days 1, 5, 10 (development phase), 15 and 17 (expression phase). The LPS stimulated offspring showed enhancement of the locomotor-stimulant effect after an acute AMPH challenge in comparison to baseline and saline pre-treated mice. They also showed development of behavioral sensitization earlier than the saline pre-treated group, although no changes between saline and LPS pre-treated groups were observed on development or expression of locomotor behavioral sensitization to AMPH. Furthermore, there was up-regulation of D1 receptor protein level within striatum in the LPS-stimulated offspring which was strongly correlated with increased grooming behavior. Taken together, our results indicate that motor and dopaminergic alterations caused by maternal immune activation are restricted to the acute AMPH challenge, mostly due to up-regulation of the D1 receptor within the mesolimbic and nigrostriatal pathways, but no locomotor differences were observed for behavioral sensitization to AMPH. (C) 2012 Elsevier B.V. All rights reserved.

Soil-nutrient availability modifies the response of young pioneer and late successional trees to elevated carbon dioxide in a Brazilian tropical environment

The aim of this work was to determine the impact of three levels of [CO2] and two levels of soil-nutrient availability on the growth and physiological responses of two tropical tree species differing in their ecological group: Croton urucurana Baillon, a pioneer (P), and also Cariniana legalis (Martius) Kuntze, a late succession (LS). We aimed to test the hypothesis that P species have stronger response to elevated [CO2] than LS species as a result of differences in photosynthetic capacity and growth kinetics between both functional groups. Seedlings of both species were grown in open-top-chambers under high (HN) or low (LN) soil-nutrient supply and exposed to ambient (380 mu mol mol(-1)) or elevated (570 and 760 mu mol mol(-1)) [CO2]. Measurements of gas exchange, chlorophyll a fluorescence, seedling biomass and allocation were made after 70 days of treatment. Results suggest that elevated [CO2] significantly enhances the photosynthetic rates (A) and biomass production in the seedlings of both species, but that soil-nutrient supply has the potential to modify the response of young tropical trees to elevated [CO2]. In relation to plants grown in ambient [CO2], the P species grown under 760 mu mol mol(-1) [CO2] showed increases of 28% and 91% in A when grown in LN and HN, respectively. In P species grown under 570 mu mol mol(-1) [CO2], A increased by 16% under HN, but there was no effect in LN. In LS species, the enhancement of A by effect of 760 mu mol mol(-1) [CO2] was 30% and 70% in LN and HN, respectively. The exposure to 570 mu mol mol(-1) [CO2] stimulated A by 31% in HN, but was no effect in LN. Reductions in stomatal conductance (g(s)) and transpiration (E), as a result of elevated [CO2] were observed. Increasing the nutrient supply from low to high increased both the maximum rate of carboxylation (V-cmax) and maximum potential rate of electron transport (J(max)). As the level of [CO2] increased, both the V-cmax and the J(max) were found to decrease, whereas the J(max)/V-cmax ratio increased. In the LS species, the maximum efficiency of PSII (F-v/F-m) was higher in the 760 mu mol mol(-1) [CO2] treatment relative to other [CO2] treatments. The results suggest that when grown under HN and the highest [CO2], the performance of the P species C. urucurana, in terms of photosynthesis and biomass enhancement, is better than the LS species C. legalis. However, a larger biomass is allocated to roots when C. legalis seedlings were exposed to elevated [CO2]. This response would be an important strategy for plant survival and productivity of the LS species under drought stresses conditions on tropical environments in a global-change scenario. (C) 2011 Elsevier B.V. All rights reserved.

Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy

Abstract Background Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. Methods Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. Results Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R. Conclusions We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.

Growth hormone response to growth hormone-releasing peptide-2 in growth hormone-deficient Little mice

OBJECTIVE: To investigate a possible direct, growth hormone-releasing, hormone-independent action of a growth hormone secretagogue, GHRP-2, in pituitary somatotroph cells in the presence of inactive growth hormonereleasing hormone receptors. MATERIALS AND METHODS: The responses of serum growth hormone to acutely injected growth hormone-releasing P-2 in lit/litmice, which represent a model of GH deficiency arising frommutated growth hormone-releasing hormonereceptors, were compared to those observed in the heterozygous (lit/+) littermates and wild-type (+/+) C57BL/6J mice. RESULTS: After the administration of 10 mcg of growth hormone-releasing P-2 to lit/lit mice, a growth hormone release of 9.3±1.5 ng/ml was observed compared with 1.04±1.15 ng/ml in controls (p<0.001). In comparison, an intermediate growth hormone release of 34.5±9.7 ng/ml and a higher growth hormone release of 163±46 ng/ml were induced in the lit/+ mice and wild-type mice, respectively. Thus, GHRP-2 stimulated growth hormone in the lit/lit mice, and the release of growth hormone in vivo may be only partially dependent on growth hormone-releasing hormone. Additionally, the plasma leptin and ghrelin levels were evaluated in the lit/lit mice under basal and stimulated conditions. CONCLUSIONS: Here, we have demonstrated that lit/lit mice, which harbor a germline mutation in the Growth hormone-releasing hormone gene, maintain a limited but statistically significant growth hormone elevation after exogenous stimulation with GHRP-2. The present data probably reflect a direct, growth hormone-independent effect on Growth hormone S (ghrelin) stimulation in the remaining pituitary somatotrophs of little mice that is mediated by growth hormone S-R 1a.