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Biomedical subjects

C Barja-Fidalgo

Publications and source records attributed to C Barja-Fidalgo.

At least 19 recordsLinked to original sources
PubMed2006

Heme oxygenase/carbon monoxide-biliverdin pathway down regulates neutrophil rolling, adhesion and migration in acute inflammation.

BACKGROUND AND PURPOSE: Heme oxygenase (HO) activity is known to down-regulate inflammatory events. Here, we address the role of HO and its metabolites, carbon monoxide (CO) and biliverdin (BVD), in leukocyte rolling, adhesion and neutrophil migration during inflammatory processes. EXPERIMENTAL APPROACH: Intravital microscopy was used to evaluate leukocyte rolling and adhesion in the mesenteric microcirculation of mice. TNFalpha and IL-1beta were determined by ELISA and HO-1 protein expression by Western blot. KEY RESULTS: Intraperitoneal challenge with carrageenan enhanced HO-1 protein expression in mesentery and bilirubin concentration in peritoneal exudates. Pretreatment of mice with a non-specific inhibitor of HO (ZnDPBG) or with a HO-1 specific inhibitor (ZnPP IX) enhanced neutrophil migration, rolling and adhesion on endothelium induced by carrageenan. In contrast, HO substrate (hemin), CO donor (DMDC) or BVD reduced these parameters. The reduction of neutrophil recruitment promoted by HO metabolites was independent of the production of chemotactic cytokines. Inhibitory effects of CO, but not of BVD, were counteracted by treatment with a soluble guanylate cyclase (sGC) inhibitor, ODQ. Furthermore, inhibition of HO prevented the inhibitory effect of a nitric oxide (NO) donor (SNAP) upon neutrophil migration, while the blockade of NO synthase (NOS) activity by aminoguanidine did not affect the CO or BVD effects. CONCLUSIONS AND IMPLICATIONS: Metabolites of HO decreased leukocyte rolling, adhesion and neutrophil migration to the inflammatory site by a mechanism partially dependent on sGC. Moreover, inhibition by NO of neutrophil migration was dependent on HO activity.

Animals↗
PubMed2006

Neutrophil function in severe sepsis.

Sepsis and septic shock continue to be a major cause of morbidity and mortality in critically ill patients. During the onset of sepsis, several inflammatory mediators, including cytokines, chemokines and nitric oxide are released systemically and mediate most of the pathophysiological events present in sepsis and septic shock, such as cardiovascular dysfunction and target-organ lesions. Polymorphonuclear leukocytes are critical effector cells during the inflammatory process and their migration to the infection focus is extremely important for the local control of bacterial growth and consequently for the prevention of bacterial dissemination. In experimental models and in human sepsis a profound failure of neutrophil migration to the infection focus is observed. It seems that the failure of neutrophil migration is dependent on toll-like receptor 4 (TLR4) and mediated by cytokines and chemokines, which induce the production of nitric oxide that inhibits neutrophil adhesion to venular endothelium and also the neutrophil chemotactic ability.

Animals↗
PubMed2005

Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions.

Extracellular matrix proteins and cell adhesion receptors (integrins) play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp) motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

Cell Adhesion↗
PubMed2005

Novel lipid mediator aspirin-triggered lipoxin A4 induces heme oxygenase-1 in endothelial cells.

Lipoxins (LX) and aspirin-triggered LX (ATL) are eicosanoids generated during inflammation via transcellular biosynthetic routes that elicit distinct anti-inflammatory and proresolution bioactions, including inhibition of leukocyte-mediated injury, stimulation of macrophage clearance of apoptotic neutrophils, repression of proinflammatory cytokine production, and inhibition of cell proliferation and migration. Recently, it was reported that aspirin induces heme oxygenase-1 (HO-1) expression on endothelial cells (EC) in a COX-independent manner, what confers protection against prooxidant insults. However, the underlying mechanisms remain unclear. In this study, we investigated whether an aspirin-triggered lipoxin A(4) stable analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A(4) (ATL-1) was able to induce endothelial HO-1. Western blot analysis showed that ATL-1 increased HO-1 protein expression associated with increased mRNA levels on EC in a time- and concentration-dependent fashion. This phenomenon appears to be mediated by the activation of the G protein-coupled LXA(4) receptor because pertussis toxin and Boc-2, a receptor antagonist, significantly inhibited ATL-1-induced HO-1 expression. We demonstrate that treatment of EC with ATL-1 inhibited VCAM and E-selectin expression induced by TNF-alpha or IL-1beta. This inhibitory effect of the analog is modulated by HO-1 because it was blocked by SnPPIX, a competitive inhibitor that blocks HO-1 activity. Our results establish that ATL-1 induces HO-1 in human EC, revealing an undescribed mechanism for the anti-inflammatory activity of these lipid mediators.

Acute-Phase Reaction↗
PubMed2005

Jararhagin, a snake venom metalloproteinase, induces a specialized form of apoptosis (anoikis) selective to endothelial cells.

Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40microg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-xL. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.

Actins↗
PubMed2003

Impairment of inflammatory response in adult rats submitted to maternal undernutrition during early lactation: role of insulin and glucocorticoid.

OBJECTIVE: Early nutritional environment may program permanent metabolic alterations, predisposing to later diseases. We have investigated the interference of maternal malnutrition during lactation with the development of acute inflammation in adult rats. MATERIALS AND METHODS: Adult rats, offspring of dams fed with either protein-free diet (UN group) or 22% protein diet (C group) during the first 10 days of lactation, were submitted to pleurisy with carrageenan (500 microg/cavity). Pleural edema, neutrophil migration and ICAM expression, were evaluated 4 h after and correlated with alterations in plasma insulin and corticosterone. Leukocyte-endothelium interactions were evaluated by intravital microscopy 1 h after inflammation. RESULTS: Compared to controls, UN rats showed a decrease in pleural edema formation (50%), neutrophil migration (50%), endothelial ICAM-1 expression on pulmonary tissue, and impairment in leukocyte adhesion (50%) and migration (80%) through endothelium. Circulating insulin was lower (42%) and corticosterone was higher (34%) in UN, compared to controls. Pre-treatment of UN with insulin (5 IU/day) or RU486 (20 mg/kg/day), inhibitor of glucocorticoid receptor, restored leukocyte functions and ICAM-1 expression. CONCLUSION: Postnatal maternal malnutrition, programming for permanent alterations in insulin and glucocorticoid secretion in progeny, that were unable to properly mount an inflammatory response, probably predisposes to chronic diseases in adult life.

Animals↗
PubMed2001

Canatoxin, a toxic protein from jack beans (Canavalia ensiformis), is a variant form of urease (EC 3.5.1.5): biological effects of urease independent of its ureolytic activity.

Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD(50)=2 mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184 kDa) is a non-covalently linked dimer of a 95 kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and K(m) of 2-7 mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn(2+) matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10 mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea.

Amino Acid Sequence↗
PubMed2001

Production of reactive oxygen species by hemocytes from the cattle tick Boophilus microplus.

We have investigated the phagocytic activity and the production of reactive oxygen species (ROS) by hemocytes from the cattle tick Boophilus microplus. Two main types of hemocytes were detected in tick hemolymph: plasmatocytes and granulocytes. The plasmocytes were the most abundant cells, being responsible for the in vivo phagocytosis of yeast. ROS production was evaluated by luminol-amplified luminescence and phenol red oxidation. The luminescence increased when hemocytes were incubated with bacteria, zymosan, or phorbol 12-miristate 13-acetate (PMA). The luminescence was inhibited by superoxide dismutase and catalase, which are antioxidant enzymes that remove superoxide and hydrogen peroxide, respectively. The phenol red oxidation assay also showed an increase in the level of hydrogen peroxide produced by hemocytes stimulated with bacteria and PMA. Taken all together, our data indicate that tick hemocytes are able to produce ROS during the phagocytic process similarly to vertebrate phagocytes.

Animals↗
PubMed2000

Hypoxemia modifies circulating and exudate neutrophil number and functional responses in carrageenin-induced pleurisy in the rat.

To assess the effect of hypoxemia on the responses of polymorphonuclear neutrophils (PMN) during an inflammatory response, rats were maintained in a low F1O2 atmosphere (9% O2) or room air for 12 h before intrathoracic injection of carrageenin or intradermal injections of agonists. After 4 h, hypoxemic rats had 50% more circulating PMN in blood and 25% less PMN in pleural exudate, whereas the number of PMN in skin biopsies did not differ from controls. Following hypoxemia, basal adhesion of blood PMN to serum-coated plastic wells was unchanged, whereas fMLP-stimulated adhesion was 50% greater. In contrast, basal adhesion of exudate PMN was 72% greater. In hypoxemic rats, exudate PMN produced 64% more PMA-stimulated superoxide than blood PMN; furthermore, blood and exudate PMN produced 4.5- and 2-fold more LPS-stimulated nitric oxide than controls, respectively. These results show that a moderate level of hypoxemia may trigger mechanisms that will interfere with PMN emigration yet prime these cells for enhanced responses upon stimulation.

Animals↗
PubMed2000

Virulence of Sporothrix schenckii conidia and yeast cells, and their susceptibility to nitric oxide.

The involvement of nitric oxide (NO) in macrophage (M phi) fungicidal activity against Sporothrix schenckii, and the relationship between NO susceptibility and the differential virulence of conidia and yeast cells, were investigated. Confirming a previously reported correlation between the length of time in culture and virulence of S. schenckii, conidia isolated from 12-day mycelial cultures (Ss-12) were less virulent to mice than conidia from 7-day cultures (Ss-7) or yeast cells. Indicative of NO production, infected animals showed a significant increase in serum levels of nitrite that was lower in mice infected with Ss-12 than in mice infected with Ss-7 or yeast. Stimulation of murine M phi with interferon-gamma (IFN-gamma) induced NO production and inhibition of fungal growth. The cytotoxic activity of M phi against Ss-12 was significantly greater than against Ss-7 or yeast cells, the highly virulent fungal forms. The addition of NO synthase inhibitors abrogated M phi cytotoxic activity against all fungal forms. The phagocytic activity of M phi against Ss-7 was significantly lower than against Ss-12 or yeast cells. Although the ingestion of fungal cells triggered the oxidative burst in M phi, the fungicidal activity was not altered in the presence of superoxide dismutase (SOD) and catalase. In addition, Ss-12 and yeast cells were more susceptible than Ss-7 to the direct fungicidal activity of the NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholinosydnonimine (SIN-1). The results of this study indicate that NO is a key cytotoxic mediator involved in the murine M phi defence against S. schenckii, and that the virulence of Ss-7, Ss-12 and yeast cells may be related to a differential susceptibility to NO.

Animals↗
PubMed1999

Effects of jarastatin, a novel snake venom disintegrin, on neutrophil migration and actin cytoskeleton dynamics.

A new disintegrin, an RGD-containing peptide of 6 kDa called jarastatin, was purified from Bothrops jararaca venom. It is a potent inhibitor of platelet aggregation induced by ADP, collagen, and thrombin. The effect of jarastatin on neutrophil migration in vivo and in vitro and on the actin cytoskeleton dynamics of these cells was investigated. Incubation in vitro with jarastatin significantly inhibited, in a concentration-dependent manner, the chemotaxis of human neutrophils toward fMLP, IL-8, and jarastatin itself. Despite this inhibitory effect, jarastatin induced neutrophil chemotaxis. A significant increase of F-actin content was observed in jarastatin-treated neutrophils. Furthermore, as demonstrated by confocal microscopy after FITC-phalloidin labeling, these cells accumulated F-actin at the plasmalemma, a distribution similar to that observed in fMLP-stimulated cells. Pretreatment of mice with jarastatin inhibited neutrophil migration into peritoneal cavities induced by carrageenan injection. The results suggest that binding of jarastatin to neutrophil integrins promotes cellular activation and triggers a dynamic alteration of the actin filament system and that this is one of the first event in integrin-mediated signaling.

Actins↗
PubMed1999

Induction of NOS in rat blood PMN in vivo and in vitro: modulation by tyrosine kinase and involvement in bactericidal activity.

Intravenous administration of lipopolysaccharide (LPS) to rats increased the production of nitric oxide (NO) metabolites (NOx) by blood polymorphonuclear neutrophils (PMN) in vitro. Both dexamethasone and L-NMMA, added in vitro to neutrophil cultures, inhibited the production of NO. On the other hand, the production of NO was not affected by the treatment, in vivo or in vitro, with different inhibitors of cyclooxygenase or 5-lipoxygenase or with a platelet-activating factor (PAF) antagonist. The incubation of blood PMN from normal rats in vitro with neutrophil activators (PAF, leukotriene B4, and interleukin-8) and different cytokines [interleukin-1, tumor necrosis factor alpha, and interferon-gamma (IFN-gamma)] showed that only IFN-gamma was able to induce the production of high amounts of NO. This induction was directly correlated with the expression of iNOS and an increase in in the enzyme activity in blood PMN. The tyrosine kinase inhibitor genistein inhibited NO production induced by IFN-gamma, suggesting that the signal transduction pathway leading to NOS induction in rat PMN involves phosphorylation by tyrosine kinase. We also showed that NO produced by IFN-gamma activated rat blood PMN involved in the killing of Pseudomonas aeruginosa.

Animals↗
PubMed1999

Lymphocyte activation and cytokine production by Pisum sativum agglutinin (PSA) in vivo and in vitro.

Mice spleen cells were incubated in vitro for 24 h with Pisum sativum agglutinin (PSA). The addition of these supernatants (SN) to macrophage cultures induced the production of nitric oxide (NO) by these cells in a dose-dependent manner. NO release was blocked in the presence of IFN gamma antibodies and partially inhibited by TNF alpha antibodies. The ability of PSA in inducing the production of IFN gamma and TNF alpha by spleen lymphocytes was confirmed assaying these cytokine levels in the SN. Spleen cells stimulated in vitro with PSA were highly activated showing an increased expression of the earlier activation marker, CD69, and a great proliferative response. On the other hand, spleen cells obtained from mice treated with PSA 24 h earlier, did not produce significant levels of IFN gamma or TNF alpha when incubated in vitro and showed a significantly lower proliferation rate when pulsed in vitro with PSA or Concanavalin A (ConA). The lower responsiveness to mitogens was also evident after 48 and 72 h after the treatment in vivo with the lectin. Nevertheless, the flow cytometric analysis of spleen lymphocytes obtained from PSA-treated animals showed a high degree of activation in cells CD3+. There was a decrease in the expression of L-selectin and VLA-4, when compared to controls, in parallel with a significant increase in the expression of CD69 and CD122 (IL-2R) in lymphocytes recovered from PSA-injected animals. The data point to evidence that PSA induces immunomodulatory effects, activating spleen lymphocytes in vivo, which become unresponsive to a second stimulation in vitro.

Animals↗
PubMed1999

Studies on the anti-allergic activity of Mikania glomerata.

A fraction (MG1) obtained from the ethanolic extract of Mikania glomerata Sprengel (Compositae), popularly known as 'guaco' and used as 'an' anti-allergic and anti-inflammatory agent, was evaluated for these properties on ovalbumin-induced allergic pleurisy and in models of local inflammation induced by biogenic amines, carrageenan and PAF. Plasma exudation as well as neutrophil and eosinophil infiltration evoked by the intrapleural injection of the antigen were significantly reduced by the fraction. Likewise, PAF-induced pleural neutrophil migration was inhibited by the treatment with MG1. On the other hand, pre-treatment of the animals with MG1 failed to modify the pleurisy induced by histamine, serotonin or carrageenan. These results suggest that MG1 is effective in inhibiting immunologic inflammation but did not affect acute inflammatory response caused by other agents.

Animals↗
PubMed1999

Anti-inflammatory profile of N-phenylpyrazole arylhydrazone derivatives in rats.

A series of synthetic N-phenylpyrazole arylhydrazone compounds, rationally designed as mixed-hybrid isosteres of two known inhibitors of prostaglandin synthase and 5-lipoxygenase enzymes, BW-755c and CBS-1108, has been investigated for anti-inflammatory activity in the carrageenan-induced pleurisy model in rats. The compounds have different oxygenated substituent groups in the aryl group of the hydrazone framework to ensure a different range of redox properties. A new arylhydrazone derivative, 2,6-di-tert-butyl-4-(4-nitro-3-methyl-N-phenylpyrazol-5-yl-hydr azonomethyl)phenol, was also synthesized and tested for anti-inflammatory activity. Although all the compounds significantly inhibited (by 30-90%) neutrophil accumulation in the pleural cavity, there was great variability in the anti-oedematogenic effect of the compounds (3-96%). 5-(4'-Hydroxy-3'-methoxybenzylidene)hydrazone-3-methyl-4-nitrop henylpyrazole was the most active compound in this series; it had a remarkable antiinflammatory profile, almost blocking both assays. In contrast, the compound with a 2,6-di-tert-butylated hydroxybenzene ring on the hydrazone group inhibited neutrophil migration only. These results will be useful for further structure-activity relationship studies devoted to improving the dual prostaglandin synthase-5-lipoxygenase activity of these derivatives and determining the minimum structural requirements necessary for this activity.

Animals↗
PubMed1997

Inflammatory and anti-inflammatory effects of soybean agglutinin.

Soybean agglutinin (SBA) lectin, a protein present in raw soybean meals, can bind to and be extensively endocytosed by intestinal epithelial cells, being nutritionally toxic for most animals. In the present study we show that SBA (5-200 micrograms/cavity) injected into different cavities of rats induced a typical inflammatory response characterized by dose-dependent exudation and neutrophil migration 4 h after injection. This effect was blocked by pretreatment with glucocorticoid (0.5 mg/kg) or by co-injection of N-acetyl-galactosamine (100 x [M] lectin), but not of other sugars (100 x [M] lectin), suggesting an inflammatory response related to the lectin activity. Neutrophil accumulation was not dependent on a direct effect of SBA on the macrophage population since the effect was not altered when the number of peritoneal cells was increased or decreased in vivo. On the other hand, SBA showed chemotactic activity for human neutrophils in vitro. A slight increase in mononuclear cells was observed 48 h after i.p. injection of SBA. Phenotypic analysis of these cells showed an increase in the CD4+/CD8- lymphocyte population that returned to control levels after 15 days, suggesting the development of an immune response. SBA-stimulated macrophages presented an increase in the expression of CD11/CD18 surface molecules and showed some characteristics of activated cells. After intravenous administration, SBA increased the number of circulating neutrophils and inhibited in a dose-dependent manner the neutrophil migration induced by i.p. injection of carrageenan into peritoneal cavities. The co-injection of N-acetyl-galactosamine or mannose, but not glucose or fucose, inhibited these effects. The data indicate that soybean lectin is able to induce a local inflammatory reaction but has an anti-inflammatory effect when present in circulating blood.

Animals↗
PubMed1996

The involvement of nitric oxide in the anti-Candida albicans activity of rat neutrophils.

Rat peritoneal neutrophils (PMN) spontaneously release nitric oxide (NO) when incubated in vitro. Addition of the NO synthase inhibitor L-monomethylarginine (L-NMMA) to the PMN reduces NO production and impairs the killing of the yeast Candida albicans, both effects being reversed by L-arginine. These data strongly suggest that oxidative metabolism of L-arginine by PMN is involved in the candidacidal activity of these cells. Rat blood PMN, which do not produce significant amounts of NO, exhibit a reduced killing capacity compared with peritoneal cells, except when they are obtained from lipopolysaccharide (LPS)-treated rats. In this case they produce measurable amounts of nitrite and express high fungicidal activity in vitro. Confirming the candidacidal activity of NO, the exposure of the C. albicans cultures to different concentrations of NO donors leads to a reduction in their survival. The candidacidal activity related to the NO pathway in rat PMN is phagocytosis dependent, since the activity can be inhibited by cytochalasin B. However, the oxidative products of oxygen released by rat PMN do not seem to be involved in their candidacidal activity, as incubation of the cells with phorbol myristate acetate (PMA) increases release of superoxide anion but does not affect the pattern of killing. Our results suggest that NO could be an important candidacidal pathway in rat neutrophils.

Animals↗