Denise V. Tambourgi

IgY: A promising antibody for use in immunodiagnostic and in immunotherapy

Abstract Immunoglobulin IgY is the major antibody produced by chickens (Gallus domesticus). After their V-C gene is rearranged in B cells, IgY is continually synthesized, excreted into the blood and transferred to the egg yolk, where it is accumulated. IgY is produced by hens to provide their offspring with an effective humoral immunity against the commonest avian pathogens until full maturation of their own immune system. In this review we aim to give an overview about the generation, structure, properties of IgY, as well as the advantages of chicken antibodies use over mammalian antibodies in immunodiagnostics and immunotherapy.

Increments in serum cytokine and nitric oxide levels in mice injected with Bothrops asper and Bothrops jararaca snake venoms

Changes in serum levels of several cytokines and nitric oxide were studied in BALB/c mice injected intraperitoneally with one median lethal dose (LD(50)) of the venoms of Bothrops asper and Bothrops jararaca, two of the medically most important poisonous snakes of Latin America. Despite differences observed in the time-course of cytokine increments and in serum cytokine levels, both venoms induced prominent elevations of TNF-alpha, IL-1, IL-6, IL-10 and IFN-gamma. There was an early increase in TNF-alpha and IL-1, followed by a more pronounced increment by 18 h. IL-6 levels peaked between 4 and 6 h, and this cytokine probably modulates the secretion of TNF-alpha and IL-1 and the synthesis of acute-phase proteins. Both venoms induced an early increment in serum IL-10, whereas IFN-gamma levels reached higher values in mice injected with B. jararaca venom than in those receiving B. asper venom. Serum nitric oxide concentration increased in mice injected with both venoms rapidly after envenomation, remaining elevated for 24 h. It is concluded that a complex pattern of cytokine and nitric oxide synthesis and secretion occurs in severe experimental envenomation by B. asper and B. jararaca venoms. Furthermore, it is suggested that some of these mediators, particularly TNF-alpha, IL-1 and nitric oxide, might play a relevant role in the pathophysiology of systemic alterations induced by these venoms.

Structural basis for metal ion coordination and the catalytic mechanism of sphingomyelinases d

Sphingomyelinases D (SMases D) from Loxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-Å resolution using the “quick cryo-soaking” technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (α/β)8 barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg2+ ion, which is coordinated by Glu32, Asp34, Asp91, and solvent molecules. In the proposed acid base catalytic mechanism, His12 and His47 play key roles and are supported by a network of hydrogen bonds between Asp34, Asp52, Trp230, Asp233, and Asn252.

Interspecific variation in venom composition and toxicity of Brazilian snakes from Bothrops genus

The genus Bothrops spp. is responsible for 90% of envenomation by snakes in Brazil, and the standard treatment for snakebites is the antivenom therapy. The anti-bothropic serum produced by Butantan Institute is prepared by the hyperimmunization of horses with a pool of venoms from Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi. In this study, the biochemical and biological characteristics of the venoms from nineteen snakes of the genus Bothrops, responsible for human accidents in Brazil, were analysed. Venoms, particularly from Crotalidae and Viperidae snakes, are rich sources of serine proteases and metalloproteases and the ability of the Brazilian anti-bothropic serum to neutralize the proteolytic activity of these venoms were also tested. The results obtained here show the existence of a large range of variation in the composition and activities in Bothrops spp. toxins and demonstrate that the anti-bothropic serum is not able to fully neutralize the toxic activities of all analysed venoms. These suggest that for the preparation of a fully effective therapeutic anti-bothropic serum, other venoms should be included in the immunization mixture.

Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)‐dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD‐induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD‐ and P1‐treated human erythrocytes was C dependent, but while PLD‐mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

Immunological parameters related to the adjuvant effect of the ordered mesoporous silica SBA-15

In 2006, the first report of a nanostructured material as adjuvant was described establishing the effectiveness of the ordered mesoporous SBA-15 silica as an immune adjuvant. The present study evaluated the SBA-15 capacity to modulate the immune responsiveness of High and Low responder mice immunized with BSA encapsulated/adsorbed in SBA-15 by the intramuscular or oral route and the adjuvant effect was compared with the responsiveness induced by BSA in aluminum hydroxide salts or emulsified in Incomplete Freund adjuvant. These results demonstrate the ability of the non-toxic SBA-15 nanoparticles to increase the immunogenicity and repair the responsiveness of the constitutively low responder individuals inducing both the IgG2a and the IgG1 isotypes, independently of the immune cell committed and conditioning the low phenotype. This new adjuvant may reveal novel therapeutic targets for immune modulation and vaccine design.

Endotoxemic-like shock induced by Loxosceles spider venoms: pathological changes and putative cytokine mediators

The systemic symptoms, tissue lesions and release of cytokines were analysed in four isogenic mouse strains with distinct haplotypes injected with various doses of Loxosceles intermedia spider venom. The estimated LD50 were 24.5 microg for C57Bl/6, 17.6 microg for BALB/c, 6.3 microg for C3H/HeJ and 4.6 microg for A/Sn mice. Prostration, acute cachexia, hypothermia, neurological disorders and hemoglobinuria were the signals preceding death. Accumulation of eosinophilic material inside the proximal and distal renal tubules and acute tubular necrosis were the most common histopathological findings. Death was prevented by previous treatment of venom with specific antivenom serum. The protein F35 purified from the whole venom retained the ability to induce the symptoms of the whole venom. The cytokines tumor necrosis factor (TNF), interleukins IL-6 and IL-10 and the radical nitric oxide were detected in serum at different levels after venom injection. These findings indicate that the state of shock produced in mice by whole endotoxin-free L. intermedia venom or by its purified fraction, protein F35, mimics the endotoxemic shock, that susceptibility to the systemic effects of the venom varies among mice of different haplotypes and that the pattern of in vivo cytokine release resembles that of endotoxemic shock.

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 Boydens 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.

Diversity of Micrurus snake species related to their venom toxic effects and the prospective of antivenom neutralization.

Background Micrurus snake bites can cause death by muscle paralysis and respiratory arrest, few hours after envenomation. The specific treatment for coral snake envenomation is the intravenous application of heterologous antivenom and, in Brazil, it is produced by horse immunization with a mixture of M. corallinus and M. frontalis venoms, snakes that inhabit the South and Southeastern regions of the country. However, this antivenom might be inefficient, considering the existence of intra- and inter-specific variations in the composition of the venoms. Therefore, the aim of the present study was to investigate the toxic properties of venoms from nine species of Micrurus: eight present in different geographic regions of Brazil (M. frontalis, M. corallinus, M. hemprichii, M. spixii, M. altirostris, M. surinamensis, M. ibiboboca, M. lemniscatus) and one (M. fulvius) with large distribution in Southeastern United States and Mexico. This study also analyzed the antigenic cross-reactivity and the neutralizing potential of the Brazilian coral snake antivenom against these Micrurus venoms. Methodology/Principal Findings Analysis of protein composition and toxicity revealed a large diversity of venoms from the nine Micrurus species. ELISA and Western blot assays showed a varied capability of the therapeutic antivenom to recognize the diverse species venom components. In vivo and in vitro neutralization assays indicated that the antivenom is not able to fully neutralize the toxic activities of all venoms. Conclusion These results indicate the existence of a large range of both qualitative and quantitative variations in Micrurus venoms, probably reflecting the adaptation of the snakes from this genus to vastly dissimilar habitats. The data also show that the antivenom used for human therapy in Brazil is not fully able to neutralize the main toxic activities present in the venoms from all Micrurus species occurring in the country. It suggests that modifications in the immunization scheme, with the inclusion of other venoms in the antigenic mixture, should occur in order to generate effective therapeutic coral snake antivenom.

Loxoscelism: From basic research to the proposal of new therapies

Loxoscelism is caused by envenomation by spiders from Loxosceles genus. Clinical symptoms only appear a few hours after envenomation and can evolve in local reactions, such as dermonecrosis, and systemic reactions, such as intravascular haemolysis, intravascular coagulation and renal failure. Current therapies are not effective, often not based in scientific research and can be even detrimental. A lack of understanding of the mechanism of action of the venom of the Loxosceles spider had thus far prevented development of effective therapies. In this review we aim to give an overview of our contributions to the understanding of the mechanism of action of the Loxosceles venom and propose targets and therapeutics for medical intervention.