Comparison of adjuvant emulsions for their safety and ability to enhance the antibody response in horses immunized with African snake venoms.

Adjuvant emulsions are widely used to enhance the antibody response in animals used as immunoglobulin source to produce snake antivenoms. We tested the performance of four commercial emulsion adjuvants (Montanide, Freund, Carbigen, and Emulsigen-D) and an experimental adjuvant (QH-769) in the antibody response of horses towards venoms of the African snakes Bitis arietans, Echis ocellatus, Dendroaspis polylepis and Naja nigricollis. Montanide, Freund and Carbigen adjuvants generated the highest immune response but induced moderate/severe local lesions at the site of injection. In contrast, Emulsigen-D and QH-769 adjuvants generated the lowest immune response and low incidence of local lesions. No evidence of systemic alterations was observed in the horses immunized with any of the adjuvants. It is suggested that the use of Montanide or Freund-based emulsions in the first immunization steps, followed by the use of Emulsigen-D, QH-769 or similar adjuvants in the following injections, could result in a satisfactory immune response against snake venoms, while not inducing serious local deleterious effects.

Pros and cons of different therapeutic antibody formats for recombinant antivenom development.

Antibody technologies are being increasingly applied in the field of toxinology. Fuelled by the many advances in immunology, synthetic biology, and antibody research, different approaches and antibody formats are being investigated for the ability to neutralize animal toxins. These different molecular formats each have their own therapeutic characteristics. In this review, we provide an overview of the advances made in the development of toxin-targeting antibodies, and discuss the benefits and drawbacks of different antibody formats in relation to their ability to neutralize toxins, pharmacokinetic features, propensity to cause adverse reactions, formulation, and expression for research and development (R&D) purposes and large-scale manufacturing. A research trend seems to be emerging towards the use of human antibody formats as well as camelid heavy-domain antibody fragments due to their compatibility with the human immune system, beneficial therapeutic properties, and the ability to manufacture these molecules cost-effectively.

Signaling pathways involved in zymosan phagocytosis induced by two secreted phospholipases A(2) isolated from Bothrops asper snake venom in macrophages

Phagocytosis, a process involved in host defense, requires coordination of a variety of signaling reactions. MT-II, a catalytically-inactive Lys49-PLA(2), and MT-III, an active Asp49-PLA(2) isolated from Bothrops asper snake venom, activate phagocytosis in macrophages. In this study the signal pathways mediating zymosan phagocytosis, focusing in lipidic second messengers, were investigated. Macrophages collected from male Swiss mouse peritoneum were obtained 96 h after i.p. injection of thioglycollate. Phagocytosis was evaluated with non-opsonized zymosan in the presence or absence of specific inhibitors. Data showed that both venom PLA(2)s increased phagocytosis. Zileuton, Etoricoxib, PACOCF3 (5-LO, COX-2 and iPLA(2) inhibitors, respectively), as well as WEB2170 (PAF receptor antagonist) significantly reduced phagocytosis induced by both venom PLA(2)s. However, Indomethacin (COX-1/COX-2 inhibitor) and Montelukast (CysL receptor antagonist) did not affect the toxins-induced phagocytosis. Moreover, while PACOCF3 (iPLA(2) inhibitor), reduced the phagocytosis induced by MT-II and MT-III, AACOCF3 (cPLA(2) inhibitor) significantly reduced the MT-II, but not MT-Ill-induced phagocytosis. These data suggest the effect of both sPLA(2)s depends on IPLA(2) and that the effect of MT-II depends on activation of cPLA(2). COX-2 and 5-W-derived metabolites as well as PAF are involved in the signaling events required for phagocytosis induced by both venom sPLA(2)s.

Signaling pathways involved in zymosan phagocytosis induced by two secreted phospholipases A(2) isolated from Bothrops asper snake venom in macrophages

Phagocytosis, a process involved in host defense, requires coordination of a variety of signaling reactions. MT-II, a catalytically-inactive Lys49-PLA(2), and MT-III, an active Asp49-PLA(2) isolated from Bothrops asper snake venom, activate phagocytosis in macrophages. In this study the signal pathways mediating zymosan phagocytosis, focusing in lipidic second messengers, were investigated. Macrophages collected from male Swiss mouse peritoneum were obtained 96 h after i.p. injection of thioglycollate. Phagocytosis was evaluated with non-opsonized zymosan in the presence or absence of specific inhibitors. Data showed that both venom PLA(2)s increased phagocytosis. Zileuton, Etoricoxib, PACOCF3 (5-LO, COX-2 and iPLA(2) inhibitors, respectively), as well as WEB2170 (PAF receptor antagonist) significantly reduced phagocytosis induced by both venom PLA(2)s. However, Indomethacin (COX-1/COX-2 inhibitor) and Montelukast (CysL receptor antagonist) did not affect the toxins-induced phagocytosis. Moreover, while PACOCF3 (iPLA(2) inhibitor), reduced the phagocytosis induced by MT-II and MT-III, AACOCF3 (cPLA(2) inhibitor) significantly reduced the MT-II, but not MT-Ill-induced phagocytosis. These data suggest the effect of both sPLA(2)s depends on IPLA(2) and that the effect of MT-II depends on activation of cPLA(2). COX-2 and 5-W-derived metabolites as well as PAF are involved in the signaling events required for phagocytosis induced by both venom sPLA(2)s.

A catalytically-inactive snake venom Lys49 phospholipase A2 homolog induces expression of cyclooxygenase-2 and production of prostaglandins through selected signaling pathways in macrophages.

The effects of a snake venom Lys-49 phospholipase A2 (PLA2) homolog named MT-II, devoid of enzymatic activity, on the biosynthesis of prostaglandins and protein expression of cyclooxygenase-2 (COX-2) and signaling pathways involved were evaluated in mouse macrophages in culture and in peritoneal cells ex vivo. Stimulation of macrophages with MT-II leads to production of prostaglandin D2 (PGD2) and prostaglandin E2 (PGE2) and protein expression of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1). Inhibition of cytosolic PLA2 (cPLA2), but not Ca(2+) independent PLA2 (iPLA2) reduced release of PGD2 and PGE2 and expression of COX-2 induced by MT-II. Inhibition of nuclear factor κB (NF-κB) significantly reduced MT-II-induced PGE2, but not PGD2 production and COX-2 expression. Inhibitors of either protein kinase C (PKC), protein tyrosine kinase (PTK), or extracellular signal-regulated kinase (ERK) pathways abrogated MT-II-induced NF-κB activation and reduced COX-2 expression and PGE2 release, whereas the p38 mitogen-activated protein kinase (MAPK) inhibitor reduced MT-II-induced COX-2 expression and PGD2 production. Inhibition of phosphatidylinositol-3-kinase (PI3K) pathway abrogated MT-II-induced NF-κB activation, but affected neither prostaglandins production nor COX-2 expression. MT-II-induced production of PGD2 and PGE2 and COX-2 expression were also observed in vivo after intraperitoneal injection into mice. Collectively, our data demonstrate that a catalytically-inactive PLA2 homolog is capable of inducing prostaglandins biosynthesis and COX-2 expression in macrophages in both in vitro and in vivo models, indicating that the enzymatic activity of PLA2 is not necessary to trigger these effects. MT-II-activated NF-κB, cPLA2 and distinct protein kinases are the principal steps involved in these cellular events.

Envenenamientos por mordeduras de serpientes en América Latina y el Caribe: Una visión integral de carácter regional / Snakebite poisoning in Latin America and the Caribbean: An integral view from a regional perspective

Se analiza la situación de los envenenamientos por mordeduras de serpiente en América Latina y el Caribe, los cuales representan un importante problema de Salud Pública en la región. Esta patología afecta, fundamentalmente, a la población rural de nuestros países, y tiene un alto impacto en sectores desatendidos por los programas de salud. Estos envenenamientos son causados, en su gran mayoría, por especies de la familia Viperidae, especialmente del género Bothrops. Existe un conglomerado de laboratorios públicos y privados productores de antivenenos en la región, aunque en algunos casos la producción no satisface las necesidades de algunos países, por lo que los antivenenos deben ser importados de países vecinos. Las investigaciones científicas y tecnológicas efectuadas en América Latina han generado un gran bagaje de conocimiento sobre las serpientes y sus venenos, así como sobre la clínica de los envenenamientos y el perfil de eficacia y seguridad de los antivenenos, a niveles preclínico y clínico. Pese a los indudables logros obtenidos en el manejo de esta enfermedad en la región, se debe redoblar esfuerzos para garantizar:

(a) un mejor conocimiento de los venenos y sus efectos; (b) una visión más realista de la incidencia de estos envenenamientos; (c) un mejoramiento cualitativo y cuantitativo en la producción de antivenenos; (d) un mejor control de calidad de los antivenenos que se importan en algunos países; (e) una más adecuada distribución de los antivenenos en la región, especialmente en zonas rurales de alta incidencia de envenenamientos; (f) una mayor capacitación del personal de salud en el tratamiento de estos envenenamientos, incluyendo el correcto uso de antivenenos; (g) un seguimiento y atención a las personas que han sufrido secuelas como producto de estos accidentes; y (h) programas comunitarios de prevención y atención de esta patología. Estas tareas deben ser enfrentadas con una filosofía de equidad, solidaridad y cooperación en la región, con la participación de múltiples protagonistas a muy diversos niveles.

The public health problem of envenomings induced by snakebites in Latin America and the Caribbean is analyzed in this work. This pathology affects predominantly the rural population and has a high impact on regions where the provision of health services is insufficient. The majority of envenomings are inflicted by species of the genera Bothrops and Crotalus, classified in the family Viperidae. There are several laboratories in the region which manufacture antivenoms for the treatment of these envenomings, although the volume of production in some cases does not fulfill the national demand and, consequently, antivenoms have to be imported. A significant body of knowledge has been gained in the taxonomy of the snakes and the biochemistry, toxicology and immunology of venoms, as well as in the preclinical and clinical performance of antivenoms. Despite significant advances in the control of this neglected tropical disease in Latin America, there are pending tasks in the region, such as:

(a) To improve our knowledge on snakes and their venoms; (b) to assess the actual incidence and mortality of snakebite envenomings; (c) to increase the volume of antivenom produced and, in some cases, to improve the quality of antivenoms; (d) to improve the national quality control laboratories; (e) to develop more effective strategies of distribution of antivenoms, especially to remote rural areas where snakebites are frequent; (f) to foster permanent education programs for the health staff in charge of the treatment of these envenomings; (g) to follow up and provide support to people that suffer physical or psychological sequelae as a consequence of these envenomings; and (h) to strengthen community programs aimed at improving the prevention and adequate management of snakebites. This conglomerate of tasks should be approached with a philosophy of solidarity, integration and cooperation in the region, with the involvement of multiple actors and institutions.