RESUMO
Due to its physicochemical properties, nanostructured mesoporous SBA-15 silica shows great potential as a vaccine adjuvant. This study evaluated the capacity of SBA-15 to encapsulate/adsorb the recombinant purified HBsAg from the Hepatitis B virus and the immunoresponsiveness of mice orally immunized with HBsAg inside SBA-15. A simulation of small angle X-ray scattering experimental results, together with the nitrogen adsorption isotherms data, allowed to determine the appropriate mass ratio of HBsAg:SBA-15, indicating antigen encapsulation into SBA-15 macroporosity. This was also evaluated by bicinchoninic acid assay and gel electrophoresis. The recruitment of inflammatory cells, an increase in production of specific antibodies, and the non-influence of silica on TH1 or TH2 polarization were observed after oral immunization. Besides, SBA-15 enhanced the phagocytosis of ovalbumin by dendritic cells, an important key to prove how this adjuvant works. Thus, it seems clear that the nanostructured SBA-15 is an effective and safe adjuvant for oral immunizations.
Assuntos
Vacinas contra Hepatite B/administração & dosagem , Imunização/métodos , Dióxido de Silício , Animais , Antígenos de Superfície da Hepatite B , Camundongos , VacinaçãoRESUMO
BACKGROUND: An alarming number of fatal accidents involving snakes are annually reported in Africa and most of the victims suffer from permanent local tissue damage and chronic disabilities. Envenomation by snakes belonging to the genus Bitis, Viperidae family, are common in Sub-Saharan Africa. The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies. In this study we have biochemically characterized venoms from three different species of Bitis, i.e., Bitis arietans, Bitis gabonica rhinoceros and Bitis nasicornis, involved in the majority of the human accidents in Africa, and analyzed the in vitro neutralizing ability of two experimental antivenoms. METHODOLOGY/PRINCIPAL FINDINGS: The data indicate that all venoms presented phospholipase, hyaluronidase and fibrinogenolytic activities and cleaved efficiently the FRET substrate Abz-RPPGFSPFRQ-EDDnp and angiotensin I, generating angiotensin 1-7. Gelatinolytic activity was only observed in the venoms of B. arietans and B. nasicornis. The treatment of the venoms with protease inhibitors indicated that Bitis venoms possess metallo and serinoproteases enzymes, which may be involved in the different biological activities here evaluated. Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested. CONCLUSION: These results suggest that the venoms of the three Bitis species, involved in accidents with humans in the Sub-Saharan Africa, contain a mixture of various enzymes that may act in the generation and development of some of the clinical manifestations of the envenomations. We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.
Assuntos
Antivenenos/metabolismo , Inibidores de Proteases/metabolismo , Venenos de Víboras/enzimologia , África Subsaariana , Animais , Antivenenos/uso terapêutico , Reações Cruzadas , Cavalos , Humanos , Venenos de Víboras/antagonistas & inibidores , Venenos de Víboras/metabolismo , Viperidae/metabolismoRESUMO
Envenomation by spiders belonging to the Loxosceles genus (brown spider) often results in local dermonecrotic lesions. We have previously shown that Loxosceles sphingomyelinase D (SMase D), the venom component responsible for all the pathological effects, induced the expression of matrix metalloproteinases (MMPs) in rabbits and in human keratinocytic cells. We also showed that the SMase D-induced apoptosis and MMP expression of keratinocytes was inhibited by tetracyclines. We have further investigated the ability of tetracyclines to inhibit or prevent the dermonecrotic lesion induced by Loxosceles venom in vivo and in vitro models. Primary cultures of rabbit fibroblasts incubated with increasing concentrations of venom or SMase D showed a decrease in cell viability, which was prevented by tetracyclines. In vivo experiments showed that topical treatments with tetracycline of rabbits, inoculated with crude Loxosceles intermedia venom or recombinant SMase D, significantly reduced the progression of the dermonecrotic lesion. Furthermore, tetracyclines also reduced the expression of MMP-2 and prevented the induction of MMP-9. Our results suggest that tetracycline may be an effective therapeutic agent for the treatment of cutaneous loxoscelism.
Assuntos
Diester Fosfórico Hidrolases/toxicidade , Dermatopatias/tratamento farmacológico , Dermatopatias/etiologia , Venenos de Aranha/toxicidade , Tetraciclinas/farmacologia , Animais , Mordeduras e Picadas , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Necrose , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Coelhos , Dermatopatias/patologia , Esfingomielina Fosfodiesterase/toxicidade , AranhasRESUMO
Envenomation by the spider Loxosceles (brown spider) can result in dermonecrosis and severe ulceration. We have previously shown that Loxosceles sphingomyelinase D (SMaseD), the enzyme responsible for these pathological effects, induced expression of matrix metalloproteinase-9 (MMP-9), which is possibly one of the main factors involved in the pathogenesis of the cutaneous loxoscelism. The aim of this study was to further investigate the molecular mechanisms triggered by Loxosceles SMaseD involved in the initiation of the dermonecrotic lesion, using HaCaT cultures, a human keratinocyte cell line, as an in vitro model for cutaneous loxoscelism. We show here that SMaseD from Loxosceles spider venom induces apoptosis in human keratinocytes, which is associated with an increased expression of metalloproteinase-2 and -9, and that the use of metalloproteinase inhibitors, such as tetracycline, may prevent cell death and potentially may prevent tissue destruction after envenomation.
Assuntos
Apoptose , Queratinócitos/efeitos dos fármacos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Diester Fosfórico Hidrolases/farmacologia , Venenos de Aranha/enzimologia , Anexina A5/metabolismo , Biomarcadores/análise , Linhagem Celular Transformada , Humanos , Queratinócitos/enzimologia , Propídio/metabolismoRESUMO
Colubrids involved in human envenomation in Brazil are mainly from the genera Helicops, Oxyrhopus, Thamnodynastes and Philodryas. There is a relatively large number of clinical descriptions involving the Xenodontinae snakes, Philodryas olfersii and Philodryas patagoniensis, in human accidents. The most common manifestations of envenomation are local pain, swelling, erythema and ecchymosis and regional lymphadenopathy with normal coagulation. The aims of this study were to characterize the biochemical and biological properties of P. olfersii and P. patagoniensis venoms, and to investigate their immunological cross-reactivities by using both specific antisera and anti-Bothrops sp serum used for human serum therapy in Brazil, in neutralizing the lethal and hemorrhagic effects of these venoms. We show here that P. olfersii e P. patagoniensis venoms present proteolytic and haemorrhagic activities but are devoid of phospholipase A2 activity. Haemorrhage and lethality induced by P. olfersii and P. patagoniensis are associated with metal-dependent proteinases, since EDTA could block these toxic activities. P. olfersii and P. patagoniensis venoms were immunogenic and the antisera produced were able to recognize several bands in P. olfersii, P. patagoniensis venoms in Bothrops jararaca venom.
Assuntos
Antivenenos/farmacologia , Bothrops , Colubridae/metabolismo , Glândulas Exócrinas/metabolismo , Venenos de Serpentes/antagonistas & inibidores , Animais , Antivenenos/imunologia , Western Blotting , Brasil , Reações Cruzadas , Ácido Edético/farmacologia , Hemorragia/induzido quimicamente , Humanos , Metais/química , Testes de Neutralização , Peptídeo Hidrolases/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A2 , Venenos de Serpentes/metabolismo , Fatores de TempoRESUMO
Envenomation by the spider Loxosceles can result in dermonecrosis and severe ulceration. Our aim was to investigate the role of the complement system and of the endogenous metalloproteinases in the initiation of the pathology of dermonecrosis. Histological analysis of skin of rabbits injected with Loxosceles intermedia venom and purified or recombinant sphingomyelinases showed a large influx of neutrophils, concomitant with dissociation of the collagenous fibers in the dermis. Decomplementation, using cobra venom factor, largely prevented the influx of neutrophils, while influx of neutrophils was also reduced in genetically C6-deficient rabbits, suggesting roles for both C5a and the membrane attack complex in the induction of dermonecrosis. However, C-depletion and C6 deficiency did not prevent the haemorrhage and the collagen injury. Zymography analysis of skin extracts showed the induction of expression of the endogenous gelatinase MMP-9 in the skin of envenomated animals. Rabbit neutrophils contained high levels of MMP-9, expression of which was further increased after incubation with venom, suggesting that these cells may be a source of the MMP-9 found in the skin of envenomated animals. Furthermore, skin fibroblasts also secreted MMP-9 and MMP-2 upon incubation with venom, suggesting that locally produced MMPs can also contribute to proteolytic tissue destruction.
Assuntos
Proteínas do Sistema Complemento/imunologia , Metaloproteinase 2 da Matriz/metabolismo , Dermatopatias/induzido quimicamente , Esfingomielina Fosfodiesterase/toxicidade , Picada de Aranha/imunologia , Picada de Aranha/patologia , Venenos de Aranha/toxicidade , Animais , Proteínas do Sistema Complemento/efeitos dos fármacos , Eritrócitos/imunologia , Fibroblastos/enzimologia , Fibroblastos/patologia , Hemólise , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Necrose , Neutrófilos/enzimologia , Neutrófilos/imunologia , Neutrófilos/patologia , Coelhos , Ovinos , Dermatopatias/imunologia , Dermatopatias/patologia , Picada de Aranha/metabolismo , AranhasRESUMO
Loxoscelism is the clinical condition produced by the venom of spiders belonging to the genus Loxosceles, which can be observed as two well-defined clinical variants: cutaneous loxoscelism and systemic or viscerocutaneous loxoscelism. We have recently identified, purified and characterised the toxins (sphingomyelinases) from Loxosceles intermedia venom that are responsible for all the local (dermonecrosis) and systemic effects (complement dependent haemolysis) induced by whole venom. In the present study, we have cloned and expressed the two functional sphingomyelinases isoforms, P1 and P2, and shown that the recombinant proteins display all the functional characteristics of whole L. intermedia venom, e.g., dermonecrotic and complement-dependent hemolytic activities and ability of hydrolyzing sphingomyelin. We have also compared the cross-reactivities of antisera raised against the toxins from different Loxosceles species and show here that the cross-reactivity is high when toxins are from the same species (P1 and P2 from L. intermedia) but low when the toxins are from different species (L. intermedia versus L. laeta). These data suggest that in order to obtain a suitable comprehensive neutralizing antiserum using the recombinant toxin as an immunogen, a mixture of the recombinant toxins from the different species has to be used. The use of anti-recombinant toxin antisera may have clinical benefits to those individuals displaying acute loxoscelic lesions.