Nanostructured SBA-15 silica: An effective protective vehicle to oral hepatitis B vaccine immunization.

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.

African adders: partial characterization of snake venoms from three Bitis species of medical importance and their neutralization by experimental equine antivenoms.

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.

A humanized antibody that regulates the alternative pathway convertase: potential for therapy of renal disease associated with nephritic factors.

Dysregulation of the complement alternative pathway can cause disease in various organs that may be life-threatening. Severe alternative pathway dysregulation can be triggered by autoantibodies to the C3 convertase, termed nephritic factors, which cause pathological stabilization of the convertase enzyme and confer resistance to innate control mechanisms; unregulated complement consumption followed by deposition of C3 fragments in tissues ensues. The mAb, 3E7, and its humanized derivative, H17, have been shown previously to specifically bind activated C3 and prevent binding of both the activating protein, factor B, and the inhibitor, factor H, which are opposite effects that complicate its potential for therapy. Using ligand binding assays, functional assays, and electron microscopy, we show that these Abs bind C3b via a site that overlaps the binding site on C3 for the Ba domain within factor B, thereby blocking an interaction essential for convertase formation. Both Abs also bind the preformed convertase, C3bBb, and provide powerful inhibition of complement activation by preventing cleavage of C3. Critically, the Abs also bound and inhibited C3 cleavage by the nephritic factor-stabilized convertase. We suggest that by preventing enzyme formation and/or cleavage of C3 to its active downstream fragments, H17 may be an effective therapy for conditions caused by severe dysregulation of the C3 convertase and, in particular, those that involve nephritic factors, such as dense deposit disease.

Sensitive and specific assays for C3 nephritic factors clarify mechanisms underlying complement dysregulation.

C3 nephritic factors are autoantibodies that prolong the half-life or prevent regulation of the alternative pathway C3 convertase, resulting in uncontrolled complement activation. They are strongly associated with renal disease but their role in pathogenesis remains controversial. Here we optimized and compared a panel of assays to identify and interrogate nephritic factor activities. Of 101 patients with histologic or clinically evident disease, 48 were positive in some or all assays. In the presence of properdin, binding of autoantibody was detected in 39 samples and convertase stabilization was detected in 36. Forty-two of 48 nephritic factors tested prevented convertase decay by factor H, and most of these by decay accelerating factor (28) and complement receptor 1 (34). Representative properdin-independent nephritic factors had no effect on C5 cleavage and terminal pathway activity, while properdin-dependent nephritic factors enhanced activity. Biacore analysis of four purified IgG samples confirmed resistance to decay and showed that properdin-independent nephritic factors increased convertase half-life over 50-fold, whereas properdin-dependent nephritic factors increased the half-life 10- to 20-fold and also increased activity of the C3 convertase up to 10-fold. Thus, our study provides a rational approach to detect and characterize nephritic factors in patients.

The development of atypical hemolytic uremic syndrome depends on complement C5.

Gene variants in the alternative pathway of the complement system strongly associate with atypical hemolytic uremic syndrome (aHUS), presumably by predisposing to increased complement activation within the kidney. Complement factor H (CFH) is the major regulator of complement activation through the alternative pathway. Factor H-deficient mice transgenically expressing a mutant CFH protein (Cfh(-/-).FHΔ16-20) that functionally mimics the CFH mutations reported in aHUS patients spontaneously develop thrombotic microangiopathy. To investigate the role of complement C5 activation in this aHUS model, we generated C5-deficient Cfh(-/-).FHΔ16-20 mice. Both C5-sufficient and C5-deficient Cfh(-/-).FHΔ16-20 mice had abnormal C3 deposition within the kidney, but spontaneous aHUS did not develop in any of the C5-deficient mice. Furthermore, although Cfh(-/-).FHΔ16-20 animals demonstrated marked hypersensitivity to experimentally triggered renal injury, animals with concomitant C5 deficiency did not. These data demonstrate a critical role for C5 activation in both spontaneous aHUS and experimentally triggered renal injury in animals with defective complement factor H function. This study provides a rationale to investigate therapeutic inhibition of C5 in human aHUS.

Decay-accelerating factor suppresses complement C3 activation and retards atherosclerosis in low-density lipoprotein receptor-deficient mice.

Decay-accelerating factor (DAF; CD55) is a membrane protein that regulates complement pathway activity at the level of C3. To test the hypothesis that DAF plays an essential role in limiting complement activation in the arterial wall and protecting from atherosclerosis, we crossed DAF gene targeted mice (daf-1(-/-)) with low-density lipoprotein-receptor deficient mice (Ldlr(-/-)). Daf-1(-/-)Ldlr(-/-) mice had more extensive en face Sudan IV staining of the thoracoabdominal aorta than Ldlr(-/-) mice, both following a 12-week period of low-fat diet or a high-fat diet. Aortic root lesions in daf-1(-/-)Ldlr(-/-) mice on a low-fat diet showed increased size and complexity. DAF deficiency increased deposition of C3d and C5b-9, indicating the importance of DAF for downstream complement regulation in the arterial wall. The acceleration of lesion development in the absence of DAF provides confirmation of the proinflammatory and proatherosclerotic potential of complement activation in the Ldlr(-/-) mouse model. Because upstream complement activation is potentially protective, this study underlines the importance of DAF in shielding the arterial wall from the atherogenic effects of complement.

Factor H facilitates the clearance of GBM bound iC3b by controlling C3 activation in fluid phase.

Dense deposit disease (DDD) is strongly associated with the uncontrolled activation of the complement alternative pathway. Factor H (CFH)-deficient (Cfh(-/-)) mice spontaneously develop C3 deposition along the glomerular basement membrane (GBM) with subsequent development of glomerulonephritis with features of DDD, a lesion dependent on C3 activation. In order to understand the role of CFH in preventing renal damage associated with the dysregulation of the alternative pathway we administered purified mouse CFH (mCFH) to Cfh(-/-) mice. 24h following the administration of mCFH we observed an increase in plasma C3 levels with presence of intact C3 in circulation showing that mCFH restored control of C3 activation in fluid phase. mCFH resulted in the reduction of iC3b deposition along the GBM. The exogenous mCFH was readily detectable in plasma but critically not in association with C3 along the GBM. Thus, the reduction in GBM C3 was dependent on the ability of mCFH to regulate C3 activation in plasma. Western blot analysis of glomeruli from Cfh(-/-) mice demonstrated the presence of iC3b. Our data show that the C3 along the GBM in Cfh(-/-) mice is the C3 fragment iC3b and that this is derived from plasma C3 activation. The implication is that successful therapy of DDD is likely to be achieved by therapies that inhibit C3 turnover in plasma.

Factor I is required for the development of membranoproliferative glomerulonephritis in factor H-deficient mice.

The inflammatory kidney disease membranoproliferative glomerulonephritis type II (MPGN2) is associated with dysregulation of the alternative pathway of complement activation. MPGN2 is characterized by the presence of complement C3 along the glomerular basement membrane (GBM). Spontaneous activation of C3 through the alternative pathway is regulated by 2 plasma proteins, factor H and factor I. Deficiency of either of these regulators results in uncontrolled C3 activation, although the breakdown of activated C3 is dependent on factor I. Deficiency of factor H, but not factor I, is associated with MPGN2 in humans, pigs, and mice. To explain this discordance, mice with single or combined deficiencies of these factors were studied. MPGN2 did not develop in mice with combined factor H and I deficiency or in mice deficient in factor I alone. However, administration of a source of factor I to mice with combined factor H and factor I deficiency triggered both activated C3 fragments in plasma and GBM C3 deposition. Mouse renal transplant studies demonstrated that C3 deposited along the GBM was derived from plasma. Together, these findings provide what we believe to be the first evidence that factor I-mediated generation of activated C3 fragments in the circulation is a critical determinant for the development of MPGN2 associated with factor H deficiency.

Tetracycline protects against dermonecrosis induced by Loxosceles spider venom.

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.

Role of matrix metalloproteinases in HaCaT keratinocytes apoptosis induced by loxosceles venom sphingomyelinase D.

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.

Duvernoy's gland secretion of Philodryas olfersii and Philodryas patagoniensis (Colubridae): neutralization of local and systemic effects by commercial bothropic antivenom (Bothrops genus).

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.

Loxosceles sphingomyelinase induces complement-dependent dermonecrosis, neutrophil infiltration, and endogenous gelatinase expression.

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.

Molecular cloning, expression, function and immunoreactivities of members of a gene family of sphingomyelinases from Loxosceles venom glands.

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.