Post-transcriptional control of Amblyomin-X on secretion of vascular endothelial growth factor and expression of adhesion molecules in endothelial cells.

Angiogenesis is a pivotal process of homeostasis and tissue repair, but it also favours neovascularisation syndromes and cancer nutrition. The chemical mediation of angiogenesis is complex, involving a balance between serine proteases and their inhibitors. We addressed the mechanisms of action of a Kunitz serine protease inhibitor (KPI) on spontaneous angiogenesis, using Amblyomin-X, a KPI designed from the cDNA library of the Amblyomma cajennense tick. Amblyomin-X treatment (10-1000 ng/10 µL; each 48 h; 3 times) reduced the number of vessels in the subcutaneous dorsal tissue of male Swiss mice, as measured by intravital microscopy, haematoxylin-eosin staining, and PECAM-1 immunofluorescence labeling. Incubation of Amblyomin-X with t-End endothelial cells, a murine endothelial microvascular lineage, did not alter cell proliferation, cell-cycle phases, necrosis and apoptosis, and the production of nitric oxide and prostaglandin E2. Nevertheless, Amblyomin-X treatment reduced t-End migration and adhesion to Matrigel(®), and inhibited the VEGF-A secretion and VCAM-1 and ß3 integrin expressions by posttranscriptional pathways. Together, data herein outline novel posttranscriptional mechanisms of KPIs on endothelial cells during angiogenesis and point out the possible application of Amblyomin-X as a local inhibitor to undesired neovascularisation process.

Actions of the Kunitz-type serine protease inhibitor Amblyomin-X on VEGF-A-induced angiogenesis.

Amblyomin-X is a Kunitz-type serine protease inhibitor (Kunitz-type SPI) designed from the cDNA library of the Amblyomma cajennense tick, which displays in vivo anti-tumor activities. Here, the mechanisms of actions of Amblyomin-X in vascular endothelial growth factor A (VEGF-A)-induced angiogenesis were characterized. Topical application of Amblyomin-X (10 or 100 ng/10 µl; each 48 h) inhibited VEGF-A-induced (10 ng/10 µl; each 48 h) angiogenesis in the dorsal subcutaneous tissue in male Swiss mice. Moreover, similar effect was observed in the VEGF-A-induced angiogenesis in the chicken chorioallantoic membrane (CAM). Additional in vitro assays in t-End cells showed that Amblyomin-X treatment delayed the cell cycle, by maintaining them in G0/G1 phase, and inhibited cell proliferation and adhesion, tube formation and membrane expression of the adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1), regardless of mRNA synthesis. Together, results herein reveal the role of Kunitz-type SPI on in vivo VEGF-A-induced angiogenesis, by exerting modulatory actions on endothelial cell proliferation and adhesion, especially on membrane expression of PECAM-1. These data provide further mechanisms of actions of Kunitz-type SPI, corroborating their relevance as scientific tools in the design of therapeutic molecules.

Nitric oxide modulates lipopolysaccharide-induced endothelial platelet endothelial cell adhesion molecule expression via interleukin-10.

We have shown previously that nitric oxide (NO) controls platelet endothelial cell adhesion molecule (PECAM-1) expression on both neutrophils and endothelial cells under physiological conditions. Here, the molecular mechanism by which NO regulates lipopolysaccharide (LPS)-induced endothelial PECAM-1 expression and the role of interleukin (IL)-10 on this control was investigated. For this purpose, N-(G)-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg/day for 14 days dissolved in drinking water) was used to inhibit both constitutive (cNOS) and inducible nitric oxide (iNOS) synthase activities in LPS-stimulated Wistar rats (5 mg/kg, intraperitoneally). This treatment resulted in reduced levels of serum NO. Under this condition, circulating levels of IL-10 was enhanced, secreted mainly by circulating lymphocytes, dependent on transcriptional activation, and endothelial PECAM-1 expression was reduced independently on reduced gene synthesis. The connection between NO, IL-10 and PECAM-1 expression was examined by incubating LPS-stimulated (1 µg/ml) cultured endothelial cells obtained from naive rats with supernatant of LPS-stimulated lymphocytes, which were obtained from blood of control or L-NAME-treated rats. Supernatant of LPS-stimulated lymphocytes obtained from L-NAME-treated rats, which contained higher levels of IL-10, reduced LPS-induced PECAM-1 expression by endothelial cells, and this reduction was reversed by adding the anti-IL-10 monoclonal antibody. Therefore, an association between NO, IL-10 and PECAM-1 was found and may represent a novel mechanism by which NO controls endothelial cell functions.

Effects of chlorogenic acid on neutrophil locomotion functions in response to inflammatory stimulus.

AIM OF THE STUDY: Species of Lychnophora are used in Brazilian folk medicine as analgesic and anti-inflammatory agents. Chlorogenic acid (CGA) and their analogues are important components of polar extracts of these species, as well in several European and Asian medicinal plants. Some of these phenolic compounds display anti-inflammatory effects. In this paper we report the isolation of CGA from Lychnophora salicifolia and its effects on functions involved in neutrophils locomotion. MATERIALS AND METHODS: LC-MS(n) data confirmed the presence of CGA in the plant. Actions of CGA were investigated on neutrophils obtained from peritoneal cavity of Wistar rats (4h after 1% oyster glycogen solution injection; 10 ml), and incubated with vehicle or with 50, 100 or 1000 µM CGA in presence of lipopolysaccharide from Escherichia coli (LPS, 5 µg/ml). Nitric oxide (NO; Griess reaction); prostaglandin E(2) (PGE(2)), interleukin-1ß (IL-1ß) and tumor necrosis factor-α [TNF-α; enzyme-linked immunosorbent assay (EIA)]; protein (flow cytometry) and gene (RT-PCR) expression of L-selectin, ß(2)integrin and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were quantified. In vitro neutrophil adhesion to primary culture of microvascular endothelial cell (PMEC) and neutrophil migration in response to formyl-methionil-leucil-phenilalanine (fMLP, 10(-8)M, Boyden chamber) was determined. RESULTS: CGA treatment did not modify the secretion of inflammatory mediators, but inhibited L-selectin cleavage and reduced ß(2) integrin, independently from its mRNA synthesis, and reduced membrane PECAM-1 expression; inhibited neutrophil adhesion and neutrophil migration induced by fMLP. CONCLUSIONS: Based on these findings, we highlight the direct inhibitory actions of CGA on adhesive and locomotion properties of neutrophils, which may contribute to its anti-inflammatory effects and help to explain the use of Lychnophora salicifolia as an anti-inflammatory agent.

Endogenous glucocorticoids control neutrophil mobilization from bone marrow to blood and tissues in non-inflammatory conditions.

BACKGROUND AND PURPOSE: We have shown that endogenous glucocorticoids control neutrophil mobilization in the absence of inflammation. In this study the role of the glucocorticoid receptor (GR) in the physiological control of neutrophil mobilization was investigated, focusing on the specific mechanisms for mature neutrophils in bone marrow, circulating neutrophils and endothelial cells. EXPERIMENTAL APPROACH: Male Wistar rats were treated with RU 38486 or adrenalectomized. Cell numbers in bone marrow and circulation were morphologically quantified and expressions of L-selectin determined by flow cytometry. Expressions of P-selectin, E-selectin, PECAM-1, VCAM-1 and ICAM-1 were measured by immunohistochemistry on vessels of cremaster muscle and their mRNA levels quantified in primary cultured endothelial cells. NF-kappaB activity in neutrophils and endothelium was quantified by EMSA. KEY RESULTS: RU 38486 treatment altered the maturation phases of neutrophilic lineage and reduced expression of L-selectin in mature neutrophils from bone marrow; increased the number of neutrophils in the circulation and elevated the expression of L-selectin in these cells. P-selectin and E-selectin expression in endothelial cells was unchanged by adrenalectomy or RU 38486 treatment. Membrane expressions, mRNA levels of ICAM-1, VCAM-1 and PECAM-1 and NF-kappaB translocation into the nucleus were higher in the endothelium of adrenalectomized and RU 38486 treated rats. CONCLUSIONS AND IMPLICATIONS: Endogenous glucocorticoids, through activation of GR on neutrophils, physiologically control the rolling behaviour of these cells and, by modulating endothelial functions, affect their adhesiveness. The molecular mechanism induced by activated GR is different in each cell, as NF-kappaB translocation was only altered in endothelial cells.

In vivo hydroquinone exposure impairs allergic lung inflammation in rats.

Hydroquinone (HQ) is naturally found in the diet, drugs, as an environmental contaminant and endogenously generated after benzene exposure. Considering that HQ alters the immune system and its several source of exposures in the environment, we hypothesized that prolonged exposure of HQ could affect the course of an immune-mediated inflammatory response. For this purpose, male Wistar rats were intraperitoneally exposed to vehicle or HQ once a day, for 22 days with a 2-day interval every 5 days. On day 10 after exposure with vehicle or HQ, animals were ovalbumin (OA)-sensitized and OA-aerosolized challenged on day 23. HQ exposure did not alter the number of circulating leukocytes but impaired allergic inflammation, evidenced by lower number of leukocytes in the bronchoalveolar lavage fluid 24h after OA-challenge. Reduced force contraction of ex vivo tracheal segments upon OA-challenge and impaired mesentery mast cell degranulation after in situ OA-challenge were also detected in tissues from HQ exposed animals. The OA-specificity on the decreased responses was corroborated by normal trachea contraction and mast cell degranulation in response to compound 48/80. In fact, lower levels of circulating OA-anaphylactic antibodies were found in HQ exposed rats, as assessed by passive cutaneous anaphylaxis assay. The reduced level of OA-anaphylactic antibody was not dependent on lower number or proliferation of lymphocytes. Nevertheless, lower expression of the co-stimulatory molecules CD6 and CD45R on OA-activated lymphocytes from HQ exposed rats indicate the interference of HQ exposure with signaling of the humoral response during allergic inflammation. Together, these data indicate specific effects of HQ exposure manifested during an immune host defense.

Melatonin inhibits nitric oxide production by microvascular endothelial cells in vivo and in vitro.

BACKGROUND AND PURPOSE: We have previously shown that melatonin inhibits bradykinin-induced NO production by endothelial cells in vitro. The purpose of this investigation was to extend this observation to an in vivo condition and to explore the mechanism of action of melatonin. EXPERIMENTAL APPROACH: RT-PCR assays were performed with rat cultured endothelial cells. The putative effect of melatonin upon arteriolar tone was investigated by intravital microscopy while NO production by endothelial cells in vitro was assayed by fluorimetry, and intracellular Ca(2+) measurements were assayed by confocal microscopy. KEY RESULTS: No expression of the mRNA for the melatonin synthesizing enzymes, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase, or for the melatonin MT(2) receptor was detected in microvascular endothelial cells. Melatonin fully inhibited L-NAME-sensitive bradykinin-induced vasodilation and also inhibited NO production induced by histamine, carbachol and 2-methylthio ATP, but did not inhibit NO production induced by ATP or alpha, beta-methylene ATP. None of its inhibitory effects was prevented by the melatonin receptor antagonist, luzindole. In nominally Ca(2+)-free solution, melatonin reduced intracellular Ca(2+) mobilization induced by bradykinin (40%) and 2-methylthio ATP (62%) but not Ca(2+) mobilization induced by ATP. CONCLUSIONS AND IMPLICATIONS: We have confirmed that melatonin inhibited NO production both in vivo and in vitro. In addition, the melatonin effect was selective for some G protein-coupled receptors and most probably reflects an inhibition of Ca(2+) mobilization from intracellular stores.

Importance of jararhagin disintegrin-like and cysteine-rich domains in the early events of local inflammatory response.

Jararhagin is a multi-domain SVMP from Bothrops jararaca venom comprising catalytic, disintegrin-like and cysteine-rich domains, which cause a local reaction manifested by hemorrhage, edema, cytokine release and inflammatory cell recruitment. In this study, the importance of disintegrin-like/cysteine-rich domains of jararhagin was addressed by analyzing the effects of jararhagin-C, which lacks the catalytic domain, in induction of leukocyte rolling and release of pro-inflammatory cytokines. Jararhagin-C was isolated from B. jararaca venom conserving the same ability of complete jararhagin molecule in inhibiting collagen-induced platelet-aggregation. Treatment of trans-illuminated cremaster muscle in vivo with jararhagin-C increased number of rolling leukocytes (approximately 250%) in post-capillary venules in all periods analyzed, without interfering with microvasculature haemodynamic, like vessel diameter, the erythrocyte speed or the blood flow rate. The release of pro-inflammatory cytokines TNF-alpha, IL-1beta and IL-6 was significantly enhanced in the local of jararhagin-C injection, showing the maximum levels in periods between 2 and 4 h after treatment. Besides the action of jararhagin-C, the presence of the inactivated catalytic domain in o-phenanthrolin-treated jararhagin was related to a higher increase in the number of rolling leukocytes. Moreover, the levels of IL-6 and IL-1beta induced by catalytically active jararhagin were higher than those induced by jararhagin-C. In conclusion, our findings suggest that the disintegrin-like/cysteine-rich domains of jararhagin are sufficient to locally activate the early events of an acute inflammatory response as leukocyte rolling and pro-inflammatory cytokine release and this action may add to the effect of catalysis, which enhances the primary cell activation.

Effect of in vivo phenol or hydroquinone exposure on events related to neutrophil delivery during an inflammatory response.

Phenol (PHE) and hydroquinone (HQ) are metabolites of benzene that affect leukocytes after solvent intoxication. Hence, we investigated the effects of PHE or HQ exposure on neutrophil mobilization during an inflammatory response. Male Wistar rats received intraperitoneal injections of PHE, HQ or vehicle only and assays were performed 24 h after the last dose. Quantifications of bone marrow or circulating leukocytes showed that only HQ exposure induced neutrophilia, probably due to the accelerated mobilization from the bone marrow compartment, since reduced numbers of segmented cells in the last phase of maturation were detected there. Intravital microscopy showed that circulating leukocytes of HQ-exposed rats increased their rolling behavior and adherence to the mesenteric postcapillary venule wall in vivo. The enhanced leukocyte-endothelium interaction was not dependent on microvascular reactivity or perivascular mast cell degranulation. Instead, it was the result of neutrophil activation, demonstrated by a decrease in L-selectin and an increase in beta2 integrin expression on neutrophil membranes. This pattern of neutrophil activation may have contributed to the higher number of neutrophils in the subcutaneous inflammatory response of HQ-exposed rats after oyster glycogen injection. Taken together, our results indicate that HQ exposure alters neutrophil mobilization, which results in an exacerbated response after an injury. Although PHE is endogenously metabolized to HQ, PHE exposure only induced an increment in rolling behavior, which was not sufficient to alter the inflammatory response.

Chronic blockade of nitric oxide biosynthesis in rats: effect on leukocyte endothelial interaction and on leukocyte recruitment.

INTRODUCTION: Previous studies showed that animals chronically treated with NG-nitro-L-arginine methyl ester (L-NAME) have a reduced inflammatory reaction. Now the role of L-NAME treatment (20 mg/Kg/day/14 days) on leukocyte mobilisation was assessed in rats. METHODS: In vivo leukocyte recruitment evoked by Bothrops jararaca venom (BjV) and nitrite/nitrate (NO2-/NO3-; Griess reaction) were evaluated in the air pouch cavity. Haematological parameters were evaluated in the bone marrow and in the peripheral compartment. Microcirculatory blood flow, number of rolling and adhered leukocytes, vascular reactivity and mast cell activity were studied by intravital microscopy. Blood pressure was measured by the tail-cuff method. L-selectin and beta(2) integrin expressions on peripheral and bone marrow leukocytes were quantified by flow cytometry. RESULTS: When compared with control rats (D-NAME) L-NAME treated rats had reduced PMN cell infiltrate (50%) and NO2-/NO3- (27%) in the air pouch cavity. Rolling leukocytes were decreased (70%) in L-NAME-treated animals, which was reversed by topical application of NO donor (SIN-1). BjV stimulation increased the number of rolling and adhered leukocytes only in control rats. Systemic blood pressure, microcirculatory blood flow and microvascular reactivity was not altered by the treatment. Only the vessel response to acetylcholine was delayed in treated rats. Peripheral PMN cells were increased by L-NAME treatment (100%), but the number of bone marrow cells was not altered. The treatment reduced L-selectin expression on circulating leukocytes, by either with (16%) or without (26%) stimulation with BjV; PMN cells were more affected (32-37%). Impairment of L-selectin expression was also verified in bone marrow cells under stimulation with BjV. CONCLUSIONS: Results show that this schedule of L-NAME treatment promotes a decrease on L-selectin expression. This effect may promote the standstill of leukocytes in the blood compartment and may be responsible, at least in part, for the observed deficient leukocyte-endothelium interactions with subsequent impairment of leukocyte migration to the inflammatory site.

Melatonin and N-acetylserotonin inhibit leukocyte rolling and adhesion to rat microcirculation.

The hormone melatonin produced by the pineal gland during the daily dark phase regulates a variety of biological processes in mammals. The aim of this study was to determine the effect of melatonin and its precursor N-acetylserotonin on the microcirculation during acute inflammation. Arteriolar diameter, blood flow rate, leukocyte rolling and adhesion were measured in the rat microcirculation in situ by intravital microscopy. Melatonin alone or together with noradrenaline did not affect the arteriolar diameter or blood flow rate. Melatonin inhibited both leukocyte rolling and leukotriene B(4) induced adhesion while its precursor N-acetylserotonin inhibits only leukocyte adhesion. The rank order of potency of agonists and antagonist receptor selective ligands suggested that the activation of MT(2) and MT(3) melatonin binding sites receptors modulate leukocyte rolling and adhesion, respectively. The effect of melatonin and N-acetylserotonin herein described were observed with concentrations in the range of the nocturnal surge, providing the first evidence for a possible physiological role of these hormones in acute inflammation.

Role of crotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom, on inflammatory and immune reactions.

BACKGROUND: Crotoxin (CTX) is a potent neurotoxin from Crotalus durissus terrificus snake venom (CdtV) composed of two subunits: one without catalytic activity (crotapotin), and a basic phospolipase A2. Recent data have demonstrated that CdtV or CTX inhibit some immune and inflammatory reactions. AIM: The aim of this paper was to investigate the mechanisms involved in these impaired responses. MATERIALS AND METHODS: Male Swiss mice were bled before and at different intervals of time after subcutaneous injection of CTX or bovine serum albumin (BSA) (control animals). The effect of treatments on circulating leukocyte mobilisation and on serum levels of interleukin (IL)-6, IL-10, interferon (IFN)-gamma and corticosterone were investigated. Spleen cells from treated animals were also stimulated in vitro with concanavalin A to evaluate the profile of IL-4, IL-6, IL-10 or IFN-gamma secretion. Cytokine levels were determined by immunoenzymatic assay and corticosterone levels by radioimmunoassay. To investigate the participation of endogenous corticosteroid on the effects evoked by CTX, animals were treated with metyrapone, an inhibitor of glucocorticoid synthesis, previous to CTX treatment. RESULTS: Marked alterations on peripheral leukocyte distribution, characterised by a drop in the number of lymphocytes and monocytes and an increase in the number of neutrophils, were observed after CTX injection. No such alteration was observed in BSA-treated animals. Increased levels of IL-6, IL-10 and corticosterone were also detected in CTX-injected animals. IFN-gamma levels were not modified after treatments. In contrast, spleen cells obtained from CTX-treated animals and stimulated with concanavalin A secreted less IL-10 and IL-4 in comparison with cells obtained from control animals. Metyrapone pretreatment was effective only to reverse the neutrophilia observed after CTX administration. CONCLUSIONS: Our results suggest that CTX may contribute to the deficient inflammatory and immune responses induced by crude CdtV. CTX induces endogenous mechanisms that are responsible, at least in part, for these impaired responses.

Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper. A new member of the VEGF family of proteins.

During the generation of abundant expressed sequence tags from the Viperidae snake Bothrops insularis venom glands, we identified for the first time a cDNA coding for a putative vascular endothelial growth factor-like (VEGF-like) protein. The deduced primary sequence, after complete sequencing of the longest snake venom VEGF (svVEGF) cDNA, displayed similarity with vertebrate VEGFs and with the hypotensive factor from Vipera aspis venom. Its cDNA was subcloned, expressed in Escherichia coli with a His(6) tag as an insoluble monomer, and purified by Ni(2+)-affinity chromatography after 8 m urea extraction. Antiserum against svVEGF was generated and tested in Western blot against proteins from snake venoms and cellular extracts. The mature svVEGF appears to be ubiquitously distributed throughout snake venoms and was also confirmed by Northern blot studies of other related Viperidae species and by cDNA cloning of svVEGF from Bothrops jararaca pit viper. The produced recombinant protein dimerizes after refolding processes and was biologically characterized, showing ability to increase vascular permeability. These results established that svVEGF is a novel and important active toxin during the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins.

Skeletal muscle necrosis and regeneration after injection of Thalassophryne nattereri (niquim) fish venom in mice.

Stings by Thalassophryne nattereri are responsible for envenomation of fishermen in north-eastern Brazil. Its venom induces prominent local tissue damage, characterized by pain, oedema and necrosis. The pathogenesis of acute muscle damage induced by T. nattereri venom was studied in mice. Intramuscular injection induced myonecrosis within the first hours. Some muscle cells presented a hypercontracted morphology, but most necrotic fibres were not hypercontracted, being instead characterized by a disorganization of myofibrils, with Z line loss, mitochondrial swelling and sarcolemmal disruption. In addition, thrombosis was observed histologically in venules and veins, together with vascular congestion and stasis, evidenced by intravital microscopy. Venom induced a rapid increment in serum creatine kinase (CK) levels, concomitant with a reduction in gastrocnemius muscle CK activity, whereas no increments in muscle lactic acid were detected. A rapid cytolytic effect was induced by the venom on C2C12 murine myoblasts in culture. The inflammatory reaction in affected muscle was characterized by oedema and scarce cellular infiltrate of polymorphonuclear leucocytes and macrophages, with a consequent delay in the removal of necrotic material. Skeletal muscle regeneration was partially impaired, as evidenced by the presence of regenerating fibres of variable size and by the increase of fibrotic tissue in endomysium and perimysium. It is suggested that T. nattereri venom affects muscle fibres by a direct cytotoxic effect, and that the vascular alterations described preclude a successful regenerative process.

In vivo characterization of Lopap, a prothrombin activator serine protease from the Lonomia obliqua caterpillar venom.

Increasing occurrence of hemorrhagic syndrome in man, caused by contact with Lonomia obliqua caterpillars, has been reported in Southern Brazil in the past 10 years. The L. obliqua venom causes a severe consumptive coagulopathy, which can lead to hemorrhagic syndrome. L. obliqua prothrombin activator protease (Lopap) is a 69-kDa prothrombin activator serine protease isolated from L. obliqua caterpillar bristle extract, which is able to evoke thrombus formation, unclottable blood, and fibrinogen depletion when injected into the blood stream of rats. The purified protein generated thrombin from prothrombin, able to clot purified human fibrinogen and plasma. A decrease in platelet count and inhibition of collagen-induced platelet aggregation were observed, as well as leukocyte infiltration in the lungs. In addition, we observed congestion and hemorrhage in renal glomeruli and necrosis in renal distal tubules. These data support the hypothesis that Lopap contributes to the clinical syndrome caused by human contact with L. obliqua, most probably through prothrombin activation, resulting in a consumption coagulopathy.

Alterations of the microcirculatory network during the clearance of epimastigote forms of Trypanosoma cruzi: an intravital microscopic study.

The distribution of epimastigote forms of Trypanosoma cruzi in the microcirculatory network and the vessel alterations were observed using an intravital microscopy technique. Immediately after intravenous inoculation of 2 x 10(6) epimastigote suspension into normal mice, parasites were seen as circulating clumps, and their retention at some sites of the endothelium of venules and capillaries was observed. Injection of 2 x 10(7) and 2 x 10(8) parasite suspensions induced, respectively, intermittent or total stasis of venules and capillaries, probably via obstruction by clumping. The mobility of epimastigotes in the clumps indicates that parasites were alive in the lumen of vessels. The retention of clumps in the capillaries, although intense, could only be observed when labeled parasites were inoculated. These results suggest that the rapid clearance of epimastigote forms of T. cruzi from the blood circulation of mice may be due to the retention of parasites to the endothelium of venules and capillaries that, in turn, may facilitate phagocytosis. This may be a mechanism by which mice are able to eliminate epimastigote forms from the circulation. These findings are consistent with our previous observations showing that epimastigotes are not lysed by complement activation but are phagocytosed and destroyed by a distinct population of blood cells.

Role of kinins and nitric oxide on the rabbit arthritis induced by Bothrops jararaca venom.

The effects of the nitric oxide synthase inhibitor N(omega)Nitro-L-arginine-methyl ester (L-NAME) and of the bradykinin B(2) receptor antagonist HOE 140 were evaluated in the inflammatory reaction induced by Bothrops jararaca venom (BjV) in New Zealand White rabbits. Arthritis was induced by injecting 0.5 ml of a sterile solution of BjV (1-64 microg/ml) into the knee intraarticular cavity. The contralateral joint was injected with bovine serum albumin (BSA) diluted in sterile saline. At selected times thereafter (4, 24 and 48 h), the vascular permeability and the leukocyte influx in both the synovial fluid and synovium were evaluated. BjV caused a dose-dependent increase in both leukocyte influx and protein extravasation which reached a maximal response at 16 microg. Bothrops jararaca venom also induced the increase in the leukocyte accumulation in the synovium and in the concentration of both NO(2)/NO(3) in the synovial fluid. Chronic administration of L-NAME (20 mg/kg/day in the drinking water for 2 weeks) markedly reduced the leukocyte accumulation (90%), protein leakage (44%), and NO(2)/NO(3) (50%) levels in the synovial fluid, measured at the 4th h. Hoe 140, given i.v. (0.3 mg/kg, 30 min before) also reduced leukocyte accumulation (75%), protein leakage (48%), and NO(2)/NO(3) (79%) levels in the synovial fluid, measured at the 4th h. Similar results were obtained with acute administration of L-NAME (30 mg/kg, i.v., 30 min before). These results indicate that arthritis induced by BjV is triggered by kinin formation and that the increase in both vascular permeability and leukocyte accumulation is modulated by NO release.

Bothrops asper snake venom and its metalloproteinase BaP-1 activate the complement system. Role in leucocyte recruitment.

The venom of the snake Bothrops asper, the most important poisonous snake in Central America, evokes an inflammatory response, the mechanisms of which are not well characterized. The objectives of this study were to investigate whether B. asper venom and its purified toxins--phospholipases and metalloproteinase--activate the complement system and the contribution of the effect on leucocyte recruitment. In vitro chemotaxis assays were performed using Boyden's chamber model to investigate the ability of serum incubated with venom and its purified toxins to induce neutrophil migration. The complement consumption by the venom was evaluated using an in vitro haemolytic assay. The importance of complement activation by the venom on neutrophil migration was investigated in vivo by injecting the venom into the peritoneal cavity of C5-deficient mice. Data obtained demonstrated that serum incubated with crude venom and its purified metalloproteinase BaP-1 are able to induce rat neutrophil chemotaxis, probably mediated by agent(s) derived from the complement system. This hypothesis was corroborated by the capacity of the venom to activate this system in vitro. The involvement of C5a in neutrophil chemotaxis induced by venom-activated serum was demonstrated by abolishing migration when neutrophils were pre-incubated with antirat C5a receptor antibody. The relevance of the complement system in in vivo leucocyte mobilization was further demonstrated by the drastic decrease of this response in C5-deficient mice. Pre-incubation of serum with the soluble human recombinant complement receptor type 1 (sCR 1) did not prevent the response induced by the venom, but abolished the migration evoked by metalloproteinase-activated serum. These data show the role of the complement system in bothropic envenomation and the participation of metalloproteinase in the effect. Also, they suggest that the venom may contain other component(s) which can cause direct activation of C5a.

Characterization of local tissue damage evoked by Bothrops jararaca venom in the rat connective tissue microcirculation: an intravital microscopic study.

The aim of this study was to investigate the dynamic of the effects after topical application of Bothrops jararaca venom (BjV) on the microcirculation of the internal spermatic fascia of Wistar rats. The administration of 1 microg of BjV induced marked disturbances in capillaries and postcapillary venules, characterized by vasodilatation, fibrin clot formation, hemorrhagic lesions and alterations in the leukocyte-endothelial interactions. The installation and development of these effects occurred simultaneously. The application of higher dose of venom (10 microg) induced more intense effects, observed by the time-course of the beginning, evolution and intensity of the effects. The time-course of events suggests that an interaction of different effects occurs during the development of local symptoms following the exposure of tissues to BjV.