Comparing Traditional and Toxin-Oriented Approaches towards Antivenom Production against Bitis arietans Snake Venom.

Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom ("traditional approach") and immunization with selected key toxins isolated from the snake venom ("toxin oriented" approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

Anti-Metalloproteases: Production and Characterization of Polyclonal IgG Anti-F2 Fraction Antibodies Purified from the Venom of the Snake Bitis arietans.

Bitis arietans is a medically important snake found in Sub-Saharan Africa. The envenomation is characterized by local and systemic effects, and the lack of antivenoms aggravates the treatment. This study aimed to identify venom toxins and develop antitoxins. The F2 fraction obtained from Bitis arietans venom (BaV) demonstrated the presence of several proteins in its composition, including metalloproteases. Titration assays carried out together with the immunization of mice demonstrated the development of anti-F2 fraction antibodies by the animals. The determination of the affinity of antibodies against different Bitis venoms was evaluated, revealing that only BaV had peptides recognized by anti-F2 fraction antibodies. In vivo analyses demonstrated the hemorrhagic capacity of the venom and the effectiveness of the antibodies in inhibiting up to 80% of the hemorrhage and 0% of the lethality caused by BaV. Together, the data indicate: (1) the prevalence of proteins that influence hemostasis and envenomation; (2) the effectiveness of antibodies in inhibiting specific activities of BaV; and (3) isolation and characterization of toxins can become crucial steps in the development of new alternative treatments. Thus, the results obtained help in understanding the envenoming mechanism and may be useful for the study of new complementary therapies.

Comparing traditional and toxin-oriented approaches towards antivenom production against bitis arietans snake venom

Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom (“traditional approach”) and immunization with selected key toxins isolated from the snake venom (“toxin oriented” approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

Anti-Metalloproteases: production and characterization of polyclonal IgG anti-F2 fraction antibodies purified from the venom of the snake bitis arietans

Bitis arietans is a medically important snake found in Sub-Saharan Africa. The envenomation is characterized by local and systemic effects, and the lack of antivenoms aggravates the treatment. This study aimed to identify venom toxins and develop antitoxins. The F2 fraction obtained from Bitis arietans venom (BaV) demonstrated the presence of several proteins in its composition, including metalloproteases. Titration assays carried out together with the immunization of mice demonstrated the development of anti-F2 fraction antibodies by the animals. The determination of the affinity of antibodies against different Bitis venoms was evaluated, revealing that only BaV had peptides recognized by anti-F2 fraction antibodies. In vivo analyses demonstrated the hemorrhagic capacity of the venom and the effectiveness of the antibodies in inhibiting up to 80% of the hemorrhage and 0% of the lethality caused by BaV. Together, the data indicate: (1) the prevalence of proteins that influence hemostasis and envenomation; (2) the effectiveness of antibodies in inhibiting specific activities of BaV; and (3) isolation and characterization of toxins can become crucial steps in the development of new alternative treatments. Thus, the results obtained help in understanding the envenoming mechanism and may be useful for the study of new complementary therapies.

Anti-metaloproteases: produção e caracterização de anticorpos policlonais IgG anti-metaloproteases purificadas do veneno da serpente Bitis arietans

Bitis arietans is a medical importance snake found predominantly in sub-Saharan Africa. The envenomation is characterized by local and systemic reactions, which can lead the victims to death or permanent disabilities. However, the lack of antivenoms makes the treatment worse. Therefore, the present work aimed to identify venom toxins, learn about their properties and develop antitoxins. The F2 fraction obtained from affinity chromatography fractionation of B. arietans venom showed the major proteolytic activity of SVMPs from enzymatic tests. Based on mass spectrometry analysis, 57 different peptides were identified in the F2 fraction. Among the most prevalent peptides are SVMPs. ELISA titration assays performed together with the mouse immunization step (BALB/c) demonstrated the development of anti-F2 fraction antibodies by the animals. The determination of the affinity of antibodies against different Bitis venoms was evaluated, revealing that only the B. arietans venom has peptides recognized by anti-F2 fraction antibodies. In vivo analyzes demonstrated the hemorrhagic capacity of the venom, the effectiveness of the antibodies to inhibit up to 80% of hemorrhage and 0% of the lethality caused by the venom. Taken together, the data indicate: 1) the prevalence of peptides that influence hemostasis and the dissemination of local and systemic effects caused by poisoning; 2) the effectiveness of monospecific antibodies in inhibiting specific activities of the venom; and 3) the isolation of toxins, production and characterization of antibodies is a crucial step in the development of new antivenoms. Therefore, the obtained results help in understanding the mechanism involved in the envenomation and can be useful for the study of new complementary therapies to antivenom in the treatment of accidents by Bitis arietans.

Bitis arietans é uma serpente de importância médica encontrada predominantemente na África Subsaariana. O envenenamento é caracterizado por efeitos locais e sistêmicos, que podem levar a morte ou incapacidades permanentes. No entanto, a carência de antivenenos agravam o tratamento. Portanto, este trabalho teve como objetivo identificar toxinas do veneno, conhecer suas propriedades e desenvolver antitoxinas. A fração F2 obtida do fracionamento por cromatografia de afinidade do veneno de B. arietans, demonstrou a atividade proteolítica majoritária de SVMPs a partir de testes enzimáticos. Com base na análise por espectrometria de massas, foram identificados 57 peptídeos diferentes na fração F2. Entre os peptídeos de maior prevalência estão as SVMPs. Ensaios de titulação por ELISA realizados em conjunto à etapa de imunização de camundongos (BALB/c), demonstraram o desenvolvimento de anticorpos anti-fração F2 pelos animais. A determinação da afinidade dos anticorpos contra diferentes venenos de Bitis foi avaliada, revelando que somente o veneno de B. arietans possui peptídeos reconhecidos pelos anticorpos anti-fração F2. Análises in vivo demonstraram a capacidade hemorrágica do veneno, a eficácia dos anticorpos de inibir em até 80% a hemorragia e em 0% da letalidade causadas pelo veneno. Em conjunto, os dados indicam: 1) a prevalência de peptídeos que atuam influenciando na hemostasia e na disseminação de efeitos locais e sistêmicos causados pelo envenenamento; 2) a eficácia de anticorpos monoespecíficos em inibir atividades específicas do veneno; e 3) o isolamento de toxinas, produção e caracterização de anticorpos é uma etapa crucial no desenvolvimento de novos antivenenos. Desta forma, os resultados obtidos auxiliam na compreensão no mecanismo de envenenamento e podem ser úteis para o estudo de novas terapias complementares ao antiveneno no tratamento dos acidentes por Bitis arietans.

Bitis arietans Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims' death or permanent disability. To better characterize the inflammatory process induced by this snake's venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1ß in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Bitis arietans snake venom and Kn-Ba, a snake venom serine protease, induce the production of inflammatory mediators in THP-1 macrophages

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Bitis arietans Snake Venom Induces an Inflammatory Response Which Is Partially Dependent on Lipid Mediators.

Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO-/-) or platelet-activating factor (PAF) receptor (PAFR-/- the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB4, LTC4, TXB2 and PGE2, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators' functions. However, 5-LO-/- mice did not show a reduction of inflammatory response induced by the venom, while PAFR-/- mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology.

Effect of Bitis gabonica and Dendroaspis angusticeps snake venoms on apoptosis-related genes in human thymic epithelial cells.

BACKGROUND: Certain environmental toxins permanently damage the thymic epithelium, accelerate immune senescence and trigger secondary immune pathologies. However, the exact underlying cellular mechanisms and pathways of permanent immune intoxication remain unknown. The aim of the present study was to demonstrate gene expressional changes of apoptosis-related cellular pathways in human thymic epithelial cells following exposure to snake venom from Bitis gabonica and Dendroaspis angusticeps. METHODS: Snake venoms were characterized by analytical methods including reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, then applied on human thymic epithelial cells (1889c) for 24 h at 10 µg/mL (as used in previous TaqMan Array study). Gene expressional changes restricted to apoptosis were assayed by TaqMan Array (Human Apoptosis Plate). RESULTS: The most prominent gene expressional changes were shown by CASP5 (≈ 2.5 million-fold, confirmed by dedicated quantitative polymerase chain reaction) and CARD9 (0.016-fold) for B. gabonica, and BIRC7 (6.46-fold) and CASP1 (0.30-fold) for D. angusticeps. CONCLUSION: The observed apoptotic environment suggests that pyroptosis may be the dominant pathway through which B. gabonica and D. angusticeps snake venoms trigger thymic epithelial apoptosis following envenomation.

Effect of Bitis gabonica and Dendroaspis angusticeps snake venoms on apoptosis-related genes in human thymic epithelial cells

Certain environmental toxins permanently damage the thymic epithelium, accelerate immune senescence and trigger secondary immune pathologies. However, the exact underlying cellular mechanisms and pathways of permanent immune intoxication remain unknown. The aim of the present study was to demonstrate gene expressional changes of apoptosis-related cellular pathways in human thymic epithelial cells following exposure to snake venom from Bitis gabonica and Dendroaspis angusticeps. Methods: Snake venoms were characterized by analytical methods including reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, then applied on human thymic epithelial cells (1889c) for 24 h at 10 μg/mL (as used in previous TaqMan Array study). Gene expressional changes restricted to apoptosis were assayed by TaqMan Array (Human Apoptosis Plate). Results: The most prominent gene expressional changes were shown by CASP5 (≈ 2.5 million-fold, confirmed by dedicated quantitative polymerase chain reaction) and CARD9 (0.016-fold) for B. gabonica, and BIRC7 (6.46-fold) and CASP1 (0.30-fold) for D. angusticeps. Conclusion: The observed apoptotic environment suggests that pyroptosis may be the dominant pathway through which B. gabonica and D. angusticeps snake venoms trigger thymic epithelial apoptosis following envenomation.(AU)

Bitis arietans snake venom induces an inflammatory response which Is partially dependent on lipid mediators

Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO−/−) or platelet-activating factor (PAF) receptor (PAFR−/− the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB4, LTC4, TXB2 and PGE2, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators’ functions. However, 5-LO−/− mice did not show a reduction of inflammatory response induced by the venom, while PAFR−/− mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology

Effect of Bitis gabonica and Dendroaspis angusticeps snake venoms on apoptosis-related genes in human thymic epithelial cells

Certain environmental toxins permanently damage the thymic epithelium, accelerate immune senescence and trigger secondary immune pathologies. However, the exact underlying cellular mechanisms and pathways of permanent immune intoxication remain unknown. The aim of the present study was to demonstrate gene expressional changes of apoptosis-related cellular pathways in human thymic epithelial cells following exposure to snake venom from Bitis gabonica and Dendroaspis angusticeps. Methods: Snake venoms were characterized by analytical methods including reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, then applied on human thymic epithelial cells (1889c) for 24 h at 10 μg/mL (as used in previous TaqMan Array study). Gene expressional changes restricted to apoptosis were assayed by TaqMan Array (Human Apoptosis Plate). Results: The most prominent gene expressional changes were shown by CASP5 (≈ 2.5 million-fold, confirmed by dedicated quantitative polymerase chain reaction) and CARD9 (0.016-fold) for B. gabonica, and BIRC7 (6.46-fold) and CASP1 (0.30-fold) for D. angusticeps. Conclusion: The observed apoptotic environment suggests that pyroptosis may be the dominant pathway through which B. gabonica and D. angusticeps snake venoms trigger thymic epithelial apoptosis following envenomation.(AU)

Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities.

BACKGROUND: Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). METHODS: Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically. RESULTS: A serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity. CONCLUSIONS: The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO's recommendation of immunotherapy in cases of human accidents with venomous snakes.

Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities

Background: Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). Methods: Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically. Results: A serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity. Conclusions: The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO's recommendation of immunotherapy in cases of human accidents with venomous snakes.(AU)

Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities

Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). Methods: Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically. Results: A serine protease of 33 kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity. Conclusions: The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO's recommendation of immunotherapy in cases of human accidents with venomous snakes.(AU)

Kn-Ba: a novel serine protease isolated from Bitis arietans snake venom with fibrinogenolytic and kinin-releasing activities

Background Bitis arietans is a venomous snake found in sub-Saharan Africa and in parts of Morocco and Saudi Arabia. The envenomation is characterized by local and systemic reactions including pain, blistering, edema and tissue damage, besides hemostatic and cardiovascular disturbances, which can cause death or permanent disabilities in its victims. However, the action mechanisms that provoke these effects remain poorly understood, especially the activities of purified venom components. Therefore, in order to elucidate the molecular mechanisms that make the Bitis arietans venom so potent and harmful to human beings, this study reports the isolation and biochemical characterization of a snake venom serine protease (SVSP). Methods Solubilized venom was fractionated by molecular exclusion chromatography and the proteolytic activity was determined using fluorescent substrates. The peaks that showed serine protease activity were determined by blocking the proteolytic activity with site-directed inhibitors. In sequence, the fraction of interest was submitted to another cycle of molecular exclusion chromatography. The purified serine protease was identified by mass spectrometry and characterized biochemically and immunochemically. Results A serine protease of 33kDa with fibrinogen-degrading and kinin-releasing activities was isolated, described, and designated herein as Kn-Ba. The experimental Butantan Institute antivenom produced against Bitis arietans venom inhibited the Kn-Ba activity. Conclusions The in vitro activities of Kn-Ba can be correlated with the capacity of the venom to provoke bleeding and clotting disorders as well as hypotension, which are common symptoms presented by envenomed victims. Obtaining satisfactory Kn-Ba inhibition through the experimental antivenom is important, given the WHO's recommendation of immunotherapy in cases of human accidents with venomous snakes.

Snake venoms from Angola: intra-specific variations and immunogenicity

Snakebite is a public health problem in many countries of world. These accidents are considered a Neglected Tropical Disease and are responsible for a high morbidity and mortality index in the South and Southeast Asia and Sub-Saharan Africa. Angolan snake venoms are poorly investigated and no specific antivenom against them is available in the country. Thus, the aim of this study was to evaluate biochemical and immunogenic properties of male and female venoms from Naja nigricollis, Bitis arietans and Bids gabonica snakes. These animals were collected during an expedition covering 1350 km of Angola, including the Provinces of Cuanza Sul, Benguela, Huila and Malanje. Results showed that Angolan snake venoms present distinctive immunogenic properties and large intra-specific variations, associated to the gender and the geographic origin of the animals. Thus, it is possible to suggest that for the preparation of a therapeutic antivenom, intra-species variability should be taken into account, in order to obtain an efficient serum to neutralize the toxic effects of the Angolan snake venoms

Effect of geographical variation of Echis ocellatus, Naja nigricollis and Bitis arietans venoms on their neutralization by homologous and heterologous antivenoms.

Two antivenoms prepared by using Echis ocellatus, Bitis arietans and Naja nigricollis venoms from different locations in sub-Saharan Africa were compared for their neutralizing ability. Both antivenoms were similarly effective in the neutralization of the venoms of the three species from different locations. However in the case of E. ocellatus venom, antivenom prepared using venom from Nigerian specimens was more effective than antivenom prepared with venom from Cameroon specimens in the neutralization of coagulant activity.

New proline-rich oligopeptides from the venom of African adders: Insights into the hypotensive effect of the venoms.

BACKGROUND: The snakes from the Bitis genus are some of the most medically important venomous snakes in sub Saharan Africa, however little is known about the composition and effects of these snake venom peptides. Considering that the victims with Bitis genus snakes have exacerbate hypotension and cardiovascular disorders, we investigated here the presence of angiotensin-converting enzyme modulators on four different species of venoms. METHODS: The peptide fractions from Bitis gabonica gabonica, Bitis nasicornis, Bitis gabonica rhinoceros and Bitis arietans which showed inhibitory activity on angiotensin-converting enzyme were subjected to mass spectrometry analysis. Eight proline-rich peptides were synthetized and their potencies were evaluated in vitro and in vivo. RESULTS: The MS analysis resulted in over 150 sequences, out of which 32 are new proline-rich oligopeptides, and eight were selected for syntheses. For some peptides, inhibition assays showed inhibitory potentials of cleavage of angiotensin I ten times greater when compared to bradykinin. In vivo tests showed that all peptides decreased mean arterial pressure, followed by tachycardia in 6 out of 8 of the tests. CONCLUSION: We describe here some new and already known proline-rich peptides, also known as bradykinin-potentiating peptides. Four synthetic peptides indicated a preferential inhibition of angiotensin-converting enzyme C-domain. In vivo studies show that the proline-rich oligopeptides are hypotensive molecules. GENERAL SIGNIFICANCE: Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension.

Neutralization of bitis parviocula (Ethiopian mountain adder) venom by the south african institute of medical research (SAIMR) antivenom / Neutralização do veneno de Bitis parviocula (serpente da Montanha da Etiópia) pelo antiveneno do Instituto Africano de Pesquisa Médica (SAIMR)

BACKGROUND: The Ethiopian mountain adder (Bitis parviocula) is a viperid known only from a few locations in southwestern Ethiopia. METHODS: a total of 30 µg of B. arietans and B. parviocula venoms were run on a 10-20 percent Tricine gel. To assay lethality dose fifty (LD50), five groups of eight mice for each venom were used. Hemorrhagic activity for crude venom was tested. Fibrinogenolytic activity of crude venom was measured using (2.5 mg/mL) of fibrinogen solution and (0.03 mg/mL) of crude venom. Gelatinase activity of the venom was tested on a Kodak X-OMAT TM film. Crude venoms of B. parviocula and B. arietans were tested for their abilities to affect clotting time, clotting rate and platelet function on whole human blood. RESULTS: The (SAIMR) antivenom was confirmed in this study to neutralize the lethal activity of venom from Bitis parviocula. The ED50s of SAIMR antivenom on B. parviocula and B. arietans neutralized half of 18.2 and 66.7 mg of venom, respectively. The hemorrhagic activities (MHDs) of B. parviocula and B. arietans were 0.88 and 1.7 µg, respectively. Bitis arietans and B. parviocula venoms degradated α and β chains at different times. The γ chains remained unaffected. Bitis parviocula venom did not exhibit gelatinase activity, while B. arietans had a MGD of 6.9 µg. At 3 mg/mL, the crude venoms of B. parviocula and B. arietans did not significantly affect clotting time or clotting rate. CONCLUSIONS: The SAIMR antivenom is very effective in neutralizing the venom of B. parviocula and should be considered in treating envenomations by these snakes.

BACKGROUND: Serpente das Montanhas da Etiópia (Bitis parviocula) é um viperídeo conhecido somente em poucas localizações do sudoeste da Etiópia. MÉTODOS: Um total de 30 µg de veneno de B. arietans e B. parviocula foram corridos em gel de 10 a 20 por cento de tricina. Para se estabelecer a quinquagésima dose de letalidade (LD50) foram usados cinco grupos de oito camundongos para cada veneno. A atividade hemorrágica para o veneno cru foi testada. A atividade fibrogenolítica do veneno cru foi medida usando 2,5 mg/mL de solução de fibrinogênio e 0,03 mg/mL de veneno cru. A atividade de gelatinase do veneno foi testada em um filme KODAK X-OMAT TM. Venenos crus de B. parviocula e B. arietans foram testados no que diz respeito à sua capacidade de afetar o tempo de coagulação, a velocidade de coagulação e a função plaquetogênica em sangue humano total. RESULTADO: o antiveneno SAIMR foi confirmado neste estudo no que diz respeito à neutralização da atividade letal do veneno de Bitis parviocula. ED50s do antiveneno SAIMR sobre a B. parviocula e B. arietans neutralizou metade de 18,2 e 66,7 mg respectivamente do veneno. As atividades hemorrágicas (MHDs) de B. parviocula e B. arietans foram respectivamente 0,88 e 1,7 µg. Os venenos de B. arietans e B. parviocula degradaram cadeias α e β em tempos diferentes. A cadeia Γ permaneceu não afetada. O veneno da B. parviocula não mostrou atividade de gelatinase, enquanto o de B. arietans teve um MGD de 6,9 µg. A nível de 3 mg/mL os venenos crus de B. parviocula e B. arietans não afetaram significantemente o tempo e a velocidade de coagulação. CONCLUSÕES: O antiveneno SAIMR é bastante efetivo para neutralizar o veneno da B. parviocula e deveria ser considerado para o tratamento de envenenamentos por estas serpentes.