Bothrops moojeni venom and BmooLAAO-I downmodulate CXCL8/IL-8 and CCL2/MCP-1 production and oxidative burst response, and upregulate CD11b expression in human neutrophils.

Bothrops snake venoms contain biologically active components, including L-amino acid oxidases (LAAO) that induce significant leukocyte accumulation at inflammatory sites characterized by early neutrophil infiltration. As it remains unclear how snake venoms modulate neutrophil activation and chemokine production, here we examined whether Bothrops moojeni crude venom (BmV) and its LAAO (BmooLAAO-I) affect expression of the surface activation markers CD11b and CD66b, production of the chemokines CCL2/MCP-1, CCL5/RANTES, CXCL8/IL-8, CXCL9/MIG, and CXCL-10/IP-10, and activation of oxidative burst in human neutrophils. Cell viability, expression of activation markers, and chemokine production were assessed by flow cytometry, while the oxidative burst response was measured by chemiluminescence. BmV at 50 and 75 µg/mL reduced CXCL8/IL-8 (p < 0.001 and p < 0.01, respectively) and CCL2/MCP-1 production (p < 0.05), while BmooLAAO-I at the same concentrations reduced only CCL2/MCP-1 production (p < 0.01). These effects were accompanied by CD11b upregulation (p < 0.05 for 50 and 75 µg/mL BmV; p < 0.01 for 50 and 75 µg/mL BmooLAAO-I) and CD66b downregulation (p < 0.05 for 50 and 75 µg/mL BmV). Both BmV and BmooLAAO-I at concentrations ranging from 0.625 to 5 µg/mL suppressed the oxidative burst of neutrophils stimulated with phorbol 12-myristate 13-acetate, while BmooLAAO-I at 2.5 and 5 µg/mL also suppressed the neutrophil response stimulated with opsonized zymosan. Considering that neutrophils participate in the pathogenesis of autoimmune and inflammatory diseases, the findings reported herein indicate that BmV and BmooLAAO-I are potential immunomodulating agents.

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways.

BACKGROUND: Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked by Bothrops snakes. The present study aimed to assess whether Bothrops jararacussu and Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways. METHODS: Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t(½) values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model. RESULTS: At the highest concentration tested (120 µg/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 µg/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 µg/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum. CONCLUSION: Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system by B. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 μg/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 μg/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 μg/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.(AU)

Bothrops snake venoms and their isolated toxins, an L-amino acid oxidase and a serine protease, modulate human complement system pathways

Background Activation of the complement system plays an important role in the regulation of immune and inflammatory reactions, and contributes to inflammatory responses triggered by envenomation provoked byBothrops snakes. The present study aimed to assess whether Bothrops jararacussuand Bothrops pirajai crude venoms and their isolated toxins, namely serine protease (BjussuSP-I) and L-amino acid oxidase (BpirLAAO-I), modulate human complement system pathways.Methods Lyophilized venom and toxin samples solubilized in phosphate buffered saline were diluted in appropriate buffers to evaluate their hemolytic activity on the alternative and classical pathways of the complement system. Venom- and toxin-treated normal human serum was added to the erythrocyte suspension, and the kinetic of hemolysis was measured spectrophotometrically at 700 nm. The kinetic 96-well microassay format was used for this purpose. We determined the t ½values (time required to lyse 50 % of target erythrocytes), which were employed to calculate the percentage of inhibition of the hemolytic activity promoted by each sample concentration. To confirm complement system activation, complement-dependent human neutrophil migration was examined using the Boyden chamber model.Results At the highest concentration tested (120 g/mL), B. jararacussu and B. pirajai crude venoms inhibited the hemolytic activity of the classical pathway (65.3 % and 72.4 %, respectively) more strongly than they suppressed the hemolytic activity of the alternative pathway (14.2 and 13.6 %, respectively). BjussuSP-I (20 g/mL) did not affect the hemolytic activity of the classical pathway, but slightly decreased the hemolytic activity of the alternative pathway (13.4 %). BpirLAAO-I (50 g/mL) inhibited 24.3 and 12.4 % of the hemolytic activity of the classical and alternative pathways, respectively. Normal human serum treated with B. jararacussu and B. pirajai crude venoms induced human neutrophil migration at a level similar to that induced by zymosan-activated normal human serum.Conclusion Together, the results of the kinetics of hemolysis and the neutrophil chemotaxis assay suggest that pre-activation of the complement system byB. jararacussu and B. pirajai crude venoms consumes complement components and generates the chemotactic factors C3a and C5a. The kinetic microassay described herein is useful to assess the effect of venoms and toxins on the hemolytic activity of the complement system.

Evaluation of immunomodulatory and anti-inflammatory effects and phytochemical screening of Alternanthera tenella Colla (Amaranthaceae) aqueous extracts.

Alternanthera tenella Colla extracts are used in Brazilian traditional folk medicine to treat a variety of infectious diseases as well as inflammation and fever. In this work, the immunomodulatory, anti-inflammatory and potential toxic effects of cold (CAE) and hot (HAE) aqueous extracts of A. tenella were investigated in vivo. In addition, we analyzed the phytochemical properties of both extracts. BALB/c mice were immunized in vivo with sheep red blood cells and concomitantly inoculated intraperitoneally (i.p.) with each extract (50, 100 or 200 mg/kg). Specific antibody-producing cells were enumerated using plaque-forming cell assays (PFC) and anti-SRBC IgG and IgM serum levels were measured via enzyme-linked immunosorbent assay. Body and lymphoid organ weights were determined after treatments in order to evaluate toxic effects. Carrageenan-induced paw edema was employed to investigate anti-inflammatory activity in mice inoculated i.p. with CAE or HAE (200 or 400 mg/kg). Phytochemical screening was performed using spectrometric and chromatographic approaches and revealed that CAE possessed higher tannin and flavonoid levels than HAE. PFC numbers were increased after treatment with CAE (100 mg/kg) four days after immunization, as were the serum antibody titers after four and seven days, suggesting immunostimulatory activity through modulation of B lymphocyte functions. Body and organ weights did not show major changes, suggesting that extracts administered to mice did not induce significant toxicity. Both extracts had significant anti-inflammatory activity in the paw edema assay. These results suggested that aqueous extracts from A. tenella contained several chemical compounds that possess positive and/or negative modulator effects on the immune system, which appeared to correlate with tannin and flavonoid levels in those extracts. In summary, these studies provide important insight into the biological activities of A. tenella.

Evaluation of immunomodulatory and anti-inflammatory effects and phytochemical screening of Alternanthera tenella Colla (Amaranthaceae) aqueous extracts

Alternanthera tenella Colla extracts are used in Brazilian traditional folk medicine to treat a variety of infectious diseases as well as inflammation and fever. In this work, the immunomodulatory, anti-inflammatory and potential toxic effects of cold (CAE) and hot (HAE) aqueous extracts of A. tenella were investigated in vivo. In addition, we analyzed the phytochemical properties of both extracts. BALB/c mice were immunized in vivo with sheep red blood cells and concomitantly inoculated intraperitoneally (i.p.) with each extract (50, 100 or 200 mg/kg). Specific antibody-producing cells were enumerated using plaque-forming cell assays (PFC) and anti-SRBC IgG and IgM serum levels were measured via enzyme-linked immunosorbent assay. Body and lymphoid organ weights were determined after treatments in order to evaluate toxic effects. Carrageenan-induced paw edema was employed to investigate anti-inflammatory activity in mice inoculated i.p. with CAE or HAE (200 or 400 mg/kg). Phytochemical screening was performed using spectrometric and chromatographic approaches and revealed that CAE possessed higher tannin and flavonoid levels than HAE. PFC numbers were increased after treatment with CAE (100 mg/kg) four days after immunization, as were the serum antibody titers after four and seven days, suggesting immunostimulatory activity through modulation of B lymphocyte functions. Body and organ weights did not show major changes, suggesting that extracts administered to mice did not induce significant toxicity. Both extracts had significant anti-inflammatory activity in the paw edema assay. These results suggested that aqueous extracts from A. tenella contained several chemical compounds that possess positive and/or negative modulator effects on the immune system, which appeared to correlate with tannin and flavonoid levels in those extracts. In summary, these studies provide important insight into the biological activities of A. tenella.

Activation of the complement system and leukocyte recruitment by Tityus serrulatus scorpion venom.

The scorpion Tityus serrulatus is considered one of the most dangerous species in Brazil. Its venom evokes an inflammatory response, although the exact mechanism of this effect is still unknown. The aim of the present study was to investigate the effect of Tityus serrulatus venom (TsV) on the complement system (CS) and on leukocyte recruitment. Complement consumption by TsV was evaluated using in vitro hemolytic assays, immunoelectrophoresis and two-dimensional immunoelectrophoresis of complement components (factor B and C3). In order to evaluate neutrophil migration induced in normal human serum (NHS) in the presence of TsV, in vitro chemotaxis assays were performed using the Boyden chamber model. In vitro TsV induced a concentration- and time-dependent reduction in hemolytic activity of the classical/lectin and alternative complement pathways, with samples of 43.0 microg and 43.4 microg, respectively, inhibiting 50% of the lytic activity. Alterations in C3 and factor B electrophoretic mobility after incubation of NHS with TsV, were identical to those obtained with zymosan (positive control). Incubation of NHS with TsV induced neutrophil chemotaxis similar to that observed with zymosan-activated serum. Our results show that TsV activates the CS, leading to factor B and C3 cleavage, to reduction of serum lytic activity and generation of complement chemotactic factors. Therefore, CS may play an important role in the inflammatory response observed upon scorpion envenomation.