A New Goniothalamin N-acylated Aza-derivative Strongly Downregulates Mediators Of Signaling Transduction Associated With Pancreatic Cancer Aggressiveness.

In this study, a novel concise series of molecules based on the structure of goniothalamin (1) was synthesized and evaluated against a highly metastatic human pancreatic cancer cell line (Panc-1). Among them, derivative 8 displayed a low IC50 value (2.7 μM) and its concentration for decreasing colony formation was 20-fold lower than goniothalamin (1). Both compounds reduced the levels of the receptor tyrosine kinase (AXL) and cyclin D1 which are known to be overexpressed in pancreatic cancer cells. Importantly, despite the fact that goniothalamin (1) and derivative 8 caused pancreatic cancer cell cycle arrest and cell death, only derivative 8 was able to downregulate pro-survival and proliferation pathways mediated by mitogen activated protein kinase ERK1/2. Another interesting finding was that Panc-1 cells treated with derivative 8 displayed a strong decrease in the transcription factor (c-Myc), hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) protein levels. Notably, the molecular effects caused by derivative 8 might not be related to ROS generation, since no significant production of ROS was observed in low concentrations of this compound (from 1.5 up to 3 μM). Therefore, the downregulation of important mediators of pancreatic cancer aggressiveness by derivative 8 reveals its great potential for the development of new chemotherapeutic agents for pancreatic cancer treatment.

Muscle Hyperalgesia Induced By Peripheral P2x3 Receptors Is Modulated By Inflammatory Mediators.

ATP, via activation of P2X3 receptors, has been highlighted as a key target in inflammatory hyperalgesia. Therefore, the aim of this study was to confirm whether the activation of P2X3 receptors in the gastrocnemius muscle of rats induces mechanical muscle hyperalgesia and, if so, to analyze the involvement of the classical inflammatory mediators (bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines and neutrophil migration) in this response. Intramuscular administration of the non-selective P2X3 receptor agonist α,β-meATP in the gastrocnemius muscle of rats induced mechanical muscle hyperalgesia, which, in turn, was prevented by the selective P2X3 and P2X2/3 receptors antagonist A-317491, the selective bradykinin B1-receptor antagonist Des-Arg9-[Leu8]-BK (DALBK), the cyclooxygenase inhibitor indomethacin, the β1- or β2-adrenoceptor antagonist atenolol and ICI 118,551, respectively. Also, the nonspecific selectin inhibitor fucoidan. α,β-meATP induced increases in the local concentration of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 1β (IL-1β), which were reduced by bradykinin antagonist. Finally, α,β-meATP also induced neutrophil migration. Together, these findings suggest that α,β-meATP induced mechanical hyperalgesia in the gastrocnemius muscle of rats via activation of peripheral P2X3 receptors, which involves bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines release and neutrophil migration. It is also indicated that bradykinin is the key modulator of the mechanical muscle hyperalgesia induced by P2X3 receptors. Therefore, we suggest that P2X3 receptors are important targets to control muscle inflammatory pain.

Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues

OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

Lung hyperinflation stimulates the release of inflammatory mediators in spontaneously breathing subjects

Lung hyperinflation up to vital capacity is used to re-expand collapsed lung areas and to improve gas exchange during general anesthesia. However, it may induce inflammation in normal lungs. The objective of this study was to evaluate the effects of a lung hyperinflation maneuver (LHM) on plasma cytokine release in 10 healthy subjects (age: 26.1 ± 1.2 years, BMI: 23.8 ± 3.6 kg/m²). LHM was performed applying continuous positive airway pressure (CPAP) with a face mask, increased by 3-cmH2O steps up to 20 cmH2O every 5 breaths. At CPAP 20 cmH2O, an inspiratory pressure of 20 cmH2O above CPAP was applied, reaching an airway pressure of 40 cmH2O for 10 breaths. CPAP was then decreased stepwise. Blood samples were collected before and 2 and 12 h after LHM. TNF-α, IL-1β, IL-6, IL-8, IL-10, and IL-12 were measured by flow cytometry. Lung hyperinflation significantly increased (P < 0.05) all measured cytokines (TNF-α: 1.2 ± 3.8 vs 6.4 ± 8.6 pg/mL; IL-1β: 4.9 ± 15.6 vs 22.4 ± 28.4 pg/mL; IL-6: 1.4 ± 3.3 vs 6.5 ± 5.6 pg/mL; IL-8: 13.2 ± 8.8 vs 33.4 ± 26.4 pg/mL; IL-10: 3.3 ± 3.3 vs 7.7 ± 6.5 pg/mL, and IL-12: 3.1 ± 7.9 vs 9 ± 11.4 pg/mL), which returned to basal levels 12 h later. A significant correlation was found between changes in pro- (IL-6) and anti-inflammatory (IL-10) cytokines (r = 0.89, P = 0.004). LHM-induced lung stretching was associated with an early inflammatory response in healthy spontaneously breathing subjects.

Female sex hormones mediate the allergic lung reaction by regulating the release of inflammatory mediators and the expression of lung E-selectin in rats

Background: Fluctuations of estradiol and progesterone levels caused by the menstrual cycle worsen asthma symptoms. Conflicting data are reported in literature regarding pro and anti-inflammatory properties of estradiol and progesterone. Methods: Female Wistar rats were ovalbumin (OVA) sensitized 1 day after resection of the ovaries (OVx). Control group consisted of sensitized-rats with intact ovaries (Sham-OVx). Allergic challenge was performed by aerosol (OVA 1%, 15 min) two weeks later. Twenty four hours after challenge, BAL, bone marrow and total blood cells were counted. Lung tissues were used as explants, for expontaneous cytokine secretion in vitro or for immunostaining of E-selectin. Results: We observed an exacerbated cell recruitment into the lungs of OVx rats, reduced blood leukocytes counting and increased the number of bone marrow cells. Estradiol-treated OVx allergic rats reduced, and those treated with progesterone increased, respectively, the number of cells in the BAL and bone marrow. Lungs of OVx allergic rats significantly increased the E-selectin expression, an effect prevented by estradiol but not by progesterone treatment. Systemically, estradiol treatment increased the number of peripheral blood leukocytes in OVx allergic rats when compared to non treated-OVx allergic rats. Cultured-BAL cells of OVx allergic rats released elevated amounts of LTB(4) and nitrites while bone marrow cells increased the release of TNF-alpha and nitrites. Estradiol treatment of OVx allergic rats was associated with a decreased release of TNF-alpha, IL-10, LTB4 and nitrites by bone marrow cells incubates. In contrast, estradiol caused an increase in IL-10 and NO release by cultured-BAL cells. Progesterone significantly increased TNF-alpha by cultured BAL cells and bone marrow cells. Conclusions: Data presented here suggest that upon hormonal oscillations the immune sensitization might trigger an allergic lung inflammation whose phenotype is under control of estradiol. Our data could contribute to the understanding of the protective role of estradiol in some cases of asthma symptoms in fertile ans post-menopausal women clinically observed.

Effect of bench press exercise intensity on muscle soreness and inflammatory mediators

This study compared four different intensities of a bench press exercise for muscle soreness, creatine kinase activity, interleukin (IL)-1, IL-6, tumor necrosis factor- (TNF-), and prostaglandin E2 (PGE2) concentrations in the blood. Thirty-five male Brazilian Army soldiers were randomly assigned to one of five groups: 50% one-repetition maximum (1-RM), 75% 1-RM, 90% 1-RM, 110% 1-RM, and a control group that did not perform the exercise. The total volume (setsrepetitionsload) of the exercise was matched among the exercise groups. Muscle soreness and plasma creatine kinase activity increased markedly (P0.05) after exercise, with no significant differences among the groups. Serum PGE2 concentration also increased markedly (P0.05) after exercise, with a significantly (P0.05) greater increase in the 110% 1-RM group compared with the other groups. A weak but significant (P0.05) correlation was found between peak muscle soreness and peak PGE2 concentration, but no significant correlation was evident between peak muscle soreness and peak creatine kinase activity, or peak creatine kinase activity and peak PGE2 concentration. All groups showed no changes in IL-1, IL-6 or TNF-. Our results suggest that the intensity of bench press exercise does not affect the magnitude of muscle soreness and blood markers of muscle damage and inflammation.

Tityus serrulatus venom and toxins Ts1, Ts2 and Ts6 induce macrophage activation and production of immune mediators

Scorpion envenomation induces a systemic immune response, and neurotoxins of venom act on specific ion channels, modulating neurotransmitter release or activity. However, little is known about the immunomodulatory effects of crude venom from scorpion Tityus serrulatus (TsV) or its toxins (Ts1, Ts2 and Ts6) in combination with lipopolysaccharide (LPS). To investigate the immunomodulatory effects of TsV and its toxins (Ts1, Ts2 and Ts6), J774.1 cells were stimulated with different concentrations (25, 50 and 100 mu g/mL) of venom or toxins pre-stimulated or not with LPS (0.5 mu g/mL). Macrophage cytotoxicity was assessed, and nitric oxide (NO) and cytokine production were analyzed utilizing the culture supernatants. TsV and its toxins did not produce cytotoxic effects. Depending on the concentrations used, TsV, Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in J774.1 cells, which were enhanced under LPS co-stimulation. However, LPS + Ts2 inhibited NO, IL-6 and TNF-alpha production, and Ts2 alone stimulated the production of IL-10, suggesting an anti-inflammatory activity for this toxin. Our findings are important for the basic understanding of the mechanisms involved in macrophage activation following envenomation: additionally, these findings may contribute to the discovery of new therapeutic compounds to treat immune-mediated diseases. (C) 2011 Elsevier Ltd. All rights reserved.

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions, adhesion molecule expression and inflammatory mediators release

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1 beta, tumor necrosis factor-alpha (TNF-alpha) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders. (C) 2009 Elsevier GrnbH. All rights reserved.

Prostaglandin E(2)-loaded microspheres as strategy to inhibit phagocytosis and modulate inflammatory mediators release

PGE(2), an arachidonic acid metabolite produced by various type of cells regulates a broad range of physiological activities in the endocrine, cardiovascular, gastrointestinal, and immune systems, and is involved in maintaining the local homeostasis. In the immune system, PGE(2) is mainly produced by APCs and it can suppress the Th1-mediated immune responses. The aim of this study was to develop PGE(2)-loaded biodegradable MS that prolong and sustain the in vivo release of this mediator. An o/w emulsion solvent extraction-evaporation method was chosen to prepare the MS. We determined their diameters, evaluated the in vitro release of PGE(2), using enzyme immunoassay and MS uptake by peritoneal macrophages. To assess the preservation of biological activities of this mediator, we determined the effect of PGE(2) released from MS on LPS-induced TNF-alpha release by murine peritoneal macrophages. We also analyzed the effect of encapsulated PGE(2) on inflammatory mediators release from HUVECs. Finally, we studied the effect of PGE(2) released from biodegradable MS in sepsis animal model. The use of this formulation can provide an alternative strategy for treating infections, by modulating or inhibiting inflammatory responses, especially when they constitute an exacerbated profile. (C) 2008 Elsevier B.V. All rights reserved.

Cyclooxygenase-derived mediators regulate the immunological control of Strongyloides venezuelensis infection

The aim of this study was to define the immunoregulatory role of prostaglandins in a mouse model of Strongyloides venezuelensis infection. Strongyloides venezuelensis induced an increase of eosinophils and mononuclear cells in the blood, peritoneal cavity fluid, and bronchoalveolar lavage fluid. Treatment with the dual cyclooxygenase (COX-1/-2) inhibitors indomethacin and ibuprofen, and the COX-2-selective inhibitor celecoxib partially blocked these cellular responses and was associated with enhanced numbers of infective larvae in the lung and adult worms in the duodenum. However, the drugs did not interfere with worm fertility. Cyclooxygenase inhibitors also inhibited the production of the T-helper type 2 (Th2) mediators IL-5, IgG1, and IgE, while indomethacin alone also inhibited IL-4, IL-10, and IgG2a. Cyclooxygenase inhibitors tended to enhance the Th1 mediators IL-12 and IFN-gamma. This shift away from Th2 immunity in cyclooxygenase inhibitor-treated mice correlated with reduced prostaglandin E(2) (PGE(2)) production in infected duodenal tissue. As PGE(2) is a well-characterized driver of Th2 immunity, we speculate that reduced production of this lipid might be involved in the shift toward a Th1 phenotype, favoring parasitism by S. venezuelensis. These findings provide new evidence that cyclooxygenase-derived lipids play a role in regulating host defenses against Strongyloides, and support the exploration of eicosanoid signaling for identifying novel preventive and therapeutic modalities against these infections.

Changes in inflammatory mediators following eccentric exercise of the elbow flexors

The aims of this study were to examine the plasma concentrations of inflammatory mediators including cytokines induced by a single bout of eccentric exercise and again 4 weeks later by a second bout of eccentric exercise of the same muscle group. Ten untrained male subjects performed two bouts of the eccentric exercise involving the elbow flexors (6 sets of 5 repetitions) separated by four weeks. Changes in muscle soreness, swelling, and function following exercise were compared between the bouts. Blood was sampled before, immediately after, 1 h, 3 h, 6 h, 24 h (1 d), 48 h (2 d), 72 h (3 d), 96 h (4 d) following exercise bout to measure plasma creatine kinase (CK) activity, plasma concentrations of myoglobin (Mb), interleukin (IL)-1 beta, IL-1 receptor antagonist (IL-1ra), IL-4, IL-6, IL-8, IL-10, IL-12p40, tumor necrosis factor (TNF)-alpha, granulocyte colony-stimulating factor (G-CSF), myeloperoxidase (MPO), prostaglandin E-2 (PGE(2)), heat shock protein (HSP) 60 and 70. After the first bout, muscle soreness increased significantly, and there was also significant increase in upper arm circumference; muscle function decreased and plasma CK activity and Mb concentration increased significantly. These changes were significantly smaller after the second bout compared to the first bout, indicating muscle adaptation to the repeated bouts of the eccentric exercise. Despite the evidence of greater muscle damage after the first bout, the changes in cytokines and other inflammatory mediators were quite minor, and considerably smaller than that following endurance exercise. These results suggest that eccentric exercise-induced muscle damage is not associated with the significant release of cytokines into the systemic circulation. After the first bout, plasma G-CSF concentration showed a small but significant increase, whereas TNF-alpha and IL-8 showed significant decreases compared to the pre-exercise values. After the second bout, there was a significant increase in IL-10, and a significant decrease in IL-8. In conclusion, although there was evidence of severe muscle damage after the eccentric exercise, this muscle damage was not accompanied by any large changes in plasma cytokine concentrations. The minor changes in systemic cytokine concentration found in this study might reflect more rapid clearance from the circulation, or a lack of any significant metabolic or oxidative demands during this particular mode of exercise. In relation to the adaptation to the muscle damage, the anti-inflammatory cytokine IL-10 might work as one of the underlying mechanisms of action.

Assisted ventilation modes reduce the expression of lung inflammatory and fibrogenic mediators in a model of mild acute lung injury

The goal of the study was to compare the effects of different assisted ventilation modes with pressure controlled ventilation (PCV) on lung histology, arterial blood gases, inflammatory and fibrogenic mediators in experimental acute lung injury (ALI). Paraquat-induced ALI rats were studied. At 24 h, animals were anaesthetised and further randomized as follows (n = 6/group): (1) pressure controlled ventilation mode (PCV) with tidal volume (V (T)) = 6 ml/kg and inspiratory to expiratory ratio (I:E) = 1:2; (2) three assisted ventilation modes: (a) assist-pressure controlled ventilation (APCV1:2) with I:E = 1:2, (b) APCV1:1 with I:E = 1:1; and (c) biphasic positive airway pressure and pressure support ventilation (BiVent + PSV), and (3) spontaneous breathing without PEEP in air. PCV, APCV1:1, and APCV1:2 were set with P (insp) = 10 cmH(2)O and PEEP = 5 cmH(2)O. BiVent + PSV was set with two levels of CPAP [inspiratory pressure (P (High) = 10 cmH(2)O) and positive end-expiratory pressure (P (Low) = 5 cmH(2)O)] and inspiratory/expiratory times: T (High) = 0.3 s and T (Low) = 0.3 s. PSV was set as follows: 2 cmH(2)O above P (High) and 7 cmH(2)O above P (Low). All rats were mechanically ventilated in air and PEEP = 5 cmH(2)O for 1 h. Assisted ventilation modes led to better functional improvement and less lung injury compared to PCV. APCV1:1 and BiVent + PSV presented similar oxygenation levels, which were higher than in APCV1:2. Bivent + PSV led to less alveolar epithelium injury and lower expression of tumour necrosis factor-alpha, interleukin-6, and type III procollagen. In this experimental ALI model, assisted ventilation modes presented greater beneficial effects on respiratory function and a reduction in lung injury compared to PCV. Among assisted ventilation modes, Bi-Vent + PSV demonstrated better functional results with less lung damage and expression of inflammatory mediators.

Risk stratification in neonates and infants submitted to cardiac surgery with cardiopulmonary bypass: A multimarker approach combining inflammatory mediators, N-terminal pro-B-type natriuretic peptide and troponin I

Low cardiac output syndrome (LCOS) is a common problem following cardiac surgery with cardiopulmonary bypass (CPB) in neonates and infants, and its early recognition remains a challenging task. We aimed to test whether a multimarker approach combining inflammatory and cardiac markers provides complementary information for prediction of LCOS and death in children submitted to cardiac surgery with CPB. Forty-six children younger than 18 months with congenital heart defects were prospectively enrolled. No intervention was made. Blood samples were collected pre-operatively, during CPB and post-operatively (PO) for measurement of interleukin (IL)-6, IL-8, IL-10, tumor necrosis factor (TNF)-alpha, cardiac troponin I (cTnI) and N-terminal pro-B-type natriuretic peptide (NT-proBNP). Clinical data and outcome variables were recorded. Logistic regression was used to identify predictors of LCOS and death. Multivariate logistic regression identified pre-operative NT-proBNP and IL-8 4 h PO as independent predictors of LCOS, while cTnI 4 h PO and CPB length were independent predictors of death. The use of inflammatory and cardiac markers in combination improved sensitivity, negative predictive value and accuracy of the models. In conclusion, the combined assessment of inflammatory and cardiac biochemical markers can be useful for identifying young children at increased risk for LCOS and death after heart surgery with CPB. (C) 2008 Elsevier Ltd. All rights reserved.

Neutrophil activation induced by ArtinM: Release of inflammatory mediators and enhancement of effector functions

The D-mannose binding lectin ArtinM from Artocarpus integrifolia, previously known as KM+ and artocarpin. is considered a stimulant of Th1-type immunity, which is able to confer resistance to some intracellular pathogens. In addition, ArtinM induces neutrophil migration by haptotaxis through simultaneous interactions of its carbohydrate recognition domains (CRDs) with glycans expressed on the extracellular matrix and the neutrophil surface. In the present study, we have expanded the characterization of ArtinM as a neutrophil activator. Exposure of neutrophils to ArtinM for 15 min resulted in tyrosine phosphorylation of intracellular proteins, a process that was selectively inhibited by D-mannose or mannotriose. Shortly after stimulation, neutrophils secreted high levels of LTB(4) and underwent shedding of L-selectin from their surface. Exposure to ArtinM enhanced neutrophil functions, such as respiratory burst and zymozan and Listeria monocytogenes phagocytosis. In addition, ArtinM-stimulated neutrophils displayed increased CXCL-8 secretion and TLR2 gene transcription. These results demonstrate that ArtinM is able to induce potent neutrophil activation, a feature that should be strongly considered in the assessment of the lectin capacity to confer resistance against infections. (C) 2009 Elsevier B.V. All rights reserved.