Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors.

BACKGROUND: In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. METHODS: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. RESULTS: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. CONCLUSION: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.

Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)

Targeting Loxosceles spider Sphingomyelinase D with small-molecule inhibitors as a potential therapeutic approach for loxoscelism.

Loxosceles spiders' venoms consist of a mixture of proteins, including the sphingomyelinases D (SMases D), which are the main toxic components responsible for local and systemic effects in human envenomation. Herein, based on the structural information of SMase D from Loxosceles laeta spider venom and virtual docking-based screening approach, three benzene sulphonate compounds (named 1, 5 and 6) were identified as potential Loxosceles SMase D inhibitors. All compounds inhibited the hydrolysis of the sphingomyelin substrate by both recombinant and native SMases D. Compounds 5 and 6 acted as SMases D uncompetitive inhibitors with Ki values of 0.49 µM and 0.59 µM, respectively. Compound 1 is a mixed type inhibitor, and presented a Ki value of 0.54 µM. In addition, the three compounds inhibited the binding of SMases D to human erythrocytes and the removal of glycophorin C from the cell surface, which are important events in the complement-dependent haemolysis induced by Loxosceles venom. Moreover, compounds 5 and 6 reduced the binding of SMases to human keratinocytes membrane and the venom induced cell death. Importantly, compounds 5 and 6 also controlled the development of the necrotic lesion in an in vivo model of loxoscelism. Together, our findings indicate that the novel SMase D inhibitors presented here are able to suppress both local and systemic reactions induced by Loxosceles venoms. Since the number of Loxosceles envenomation accidents is currently growing worldwide, our results indicate that both inhibitors are promising scaffolds for the rational design of new drugs targeting SMases D from these spiders.

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.

Tb II-I, a Fraction Isolated from Tityus bahiensis Scorpion Venom, Alters Cytokines': Level and Induces Seizures When Intrahippocampally Injected in Rats.

Scorpion venoms are composed of several substances with different pharmacological activities. Neurotoxins exert their effects by targeting ion channels resulting in toxic effects to mammals, insects and crustaceans. Tb II-I, a fraction isolated from Tityus bahiensis scorpion venom, was investigated for its ability to induce neurological and immune-inflammatory effects. Two putative β-sodium channel toxins were identified in this fraction, Tb2 II and Tb 4, the latter having been completely sequenced by mass spectrometry. Male Wistar rats, stereotaxically implanted with intrahippocampal cannulas and electrodes, were injected with Tb II-I (2 µg/2 µL) via the intrahippocampal route. The behavior, electrographic activity and cellular integrity of the animals were analyzed and the intracerebral level of cytokines determined. Tb II-I injection induced seizures and damage in the hippocampus. These alterations were correlated with the changes in the level of the cytokines tumoral necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Therefore, the binding of Tb II-I to its target in the central nervous system may induce inflammation resulting in neuropathological and behavioral alterations.

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)

Angiotensin-converting enzyme inhibitors of Bothrops jararaca snake venom affect the structure of mice seminiferous epithelium.

BACKGROUND: Considering the similarity between the testis-specific isoform of angiotensin-converting enzyme and the C-terminal catalytic domain of somatic ACE as well as the structural and functional variability of its natural inhibitors, known as bradykinin-potentiating peptides (BPPs), the effects of different synthetic peptides, BPP-10c (

Erratum to: Angiotensin-converting enzyme inhibitors of Bothrops jararaca snake venom affect the structure of mice seminiferous epithelium.

[This corrects the article DOI: 10.1186/s40409-015-0030-y.].

Homology modeling, vasorelaxant and bradykinin-potentiating activities of a novel hypotensin found in the scorpion venom from Tityus stigmurus.

In a recent work by our group involving a transcriptomics approach applied to the venom glands from Tityus stigmurus we identified a new family of peptides called Hypotensins (TSTI0006C) (Almeida et al., 2012). The cluster TSTI0006C was analyzed in the main 25 amino acid residues and named T. stigmurus Hypotensin (TistH), showing a molecular mass of 2.7 kDa, an absence of cysteines and the presence of two C-terminal proline residues, which are a bradykinin-potentiating peptide (BPP) signature. Here, we describe the homology modeling of the three-dimensional structure of TistH. In addition, we evaluated the cardiovascular effects elicited by TistH in normotensive rats. Firstly, TistH showed no cytotoxic effect on horse erythrocyte. Furthermore, in normotensive rats TistH was able to potentiate the hypotensive action of bradykinin (BK) and induced a vasorelaxant effect in mesenteric artery rings by endothelium-dependent release of nitric oxide (NO) and demonstrated independent inhibition of angiotensin converting enzyme (ACE). Our data can contribute to a better understanding of the structural and functional characteristics of TistH and suggest its potential use in cardiovascular diseases.

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.

Angiotensin-converting enzyme inhibitors of Bothrops jararaca snake venom affect the structure of mice seminiferous epithelium

Background Considering the similarity between the testis-specific isoform of angiotensin-converting enzyme and the C-terminal catalytic domain of somatic ACE as well as the structural and functional variability of its natural inhibitors, known as bradykinin-potentiating peptides (BPPs), the effects of different synthetic peptides, BPP-10c ( ENWPHQIPP), BPP-11e ( EARPPHPPIPP), BPP-AP ( EARPPHPPIPPAP) and captopril were evaluated in the seminiferous epithelium of male mice.Methods The adult animals received either one of the synthetic peptides or captopril (120 nmol/dose per testis) via injection into the testicular parenchyma. After seven days, the mice were sacrificed, and the testes were collected for histopathological evaluation.Results BPP-10c and BPP-AP showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and high degree of seminiferous tubule degeneration, especially in BPP-AP-treated animals. In addition, both synthetic peptides led to a significant reduction in the number of spermatocytes and round spermatids in stages I, V and VII/VIII of the seminiferous cycle, thickness of the seminiferous epithelium and diameter of the seminiferous tubule lumen. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril or BPP-11e.Conclusions The major finding of the present study was that the demonstrated effects of BPP-10c and BPP-AP on the seminiferous epithelium are dependent on their primary structure and cannot be extrapolated to other BPPs.

Angiotensin-converting enzyme inhibitors of Bothrops jararaca snake venom affect the structure of mice seminiferous epithelium

Background Considering the similarity between the testis-specific isoform of angiotensin-converting enzyme and the C-terminal catalytic domain of somatic ACE as well as the structural and functional variability of its natural inhibitors, known as bradykinin-potentiating peptides (BPPs), the effects of different synthetic peptides, BPP-10c (<ENWPHQIPP), BPP-11e (<EARPPHPPIPP), BPP-AP (<EARPPHPPIPPAP) and captopril were evaluated in the seminiferous epithelium of male mice.Methods The adult animals received either one of the synthetic peptides or captopril (120 nmol/dose per testis) via injection into the testicular parenchyma. After seven days, the mice were sacrificed, and the testes were collected for histopathological evaluation.Results BPP-10c and BPP-AP showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and high degree of seminiferous tubule degeneration, especially in BPP-AP-treated animals. In addition, both synthetic peptides led to a significant reduction in the number of spermatocytes and round spermatids in stages I, V and VII/VIII of the seminiferous cycle, thickness of the seminiferous epithelium and diameter of the seminiferous tubule lumen. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril or BPP-11e.Conclusions The major finding of the present study was that the demonstrated effects of BPP-10c and BPP-AP on the seminiferous epithelium are dependent on their primary structure and cannot be extrapolated to other BPPs.(AU)

P-I snake venom metalloproteinase is able to activate the complement system by direct cleavage of central components of the cascade.

BACKGROUND: Snake Venom Metalloproteinases (SVMPs) are amongst the key enzymes that contribute to the high toxicity of snake venom. We have recently shown that snake venoms from the Bothrops genus activate the Complement system (C) by promoting direct cleavage of C-components and generating anaphylatoxins, thereby contributing to the pathology and spread of the venom. The aim of the present study was to isolate and characterize the C-activating protease from Bothrops pirajai venom. RESULTS: Using two gel-filtration chromatography steps, a metalloproteinase of 23 kDa that activates Complement was isolated from Bothrops pirajai venom. The mass spectrometric identification of this protein, named here as C-SVMP, revealed peptides that matched sequences from the P-I class of SVMPs. C-SVMP activated the alternative, classical and lectin C-pathways by cleaving the α-chain of C3, C4 and C5, thereby generating anaphylatoxins C3a, C4a and C5a. In vivo, C-SVMP induced consumption of murine complement components, most likely by activation of the pathways and/or by direct cleavage of C3, leading to a reduction of serum lytic activity. CONCLUSION: We show here that a P-I metalloproteinase from Bothrops pirajai snake venom activated the Complement system by direct cleavage of the central C-components, i.e., C3, C4 and C5, thereby generating biologically active fragments, such as anaphylatoxins, and by cleaving the C1-Inhibitor, which may affect Complement activation control. These results suggest that direct complement activation by SVMPs may play a role in the progression of symptoms that follow envenomation.

A bradykinin-potentiating peptide (BPP-10c) from Bothrops jararaca induces changes in seminiferous tubules

Background The testis-specific isoform of angiotensin-converting enzyme (tACE) is exclusively expressed in germ cells during spermatogenesis. Although the exact role of tACE in male fertility is unknown, it clearly plays a critical function in spermatogenesis. The dipeptidase domain of tACE is identical to the C-terminal catalytic domain of somatic ACE (sACE). Bradykinin potentiating peptides (BPPs) from snake venoms are the first natural sACE inhibitors described and their structure-activity relationship studies were the basis for the development of antihypertensive drugs such as captopril. In recent years, it has been showed that a number of BPPs - including BPP-10c - are able to distinguish between the N- and C-active sites of sACE, what is not applicable to captopril. Considering the similarity between tACE and sACE (and since BPPs are able to distinguish between the two active sites of sACE), the effects of the BPP-10c and captopril on the structure and function of the seminiferous epithelium were characterized in the present study. BPP-10c and captopril were administered in male Swiss mice by intraperitoneal injection (4.7 μmol/kg for 15 days) and histological sections of testes were analyzed. Classification of seminiferous tubules and stage analysis were carried out for quantitative evaluation of germ cells of the seminiferous epithelium. The blood-testis barrier (BTB) permeability and distribution of claudin-1 in the seminiferous epithelium were analyzed by hypertonic fixative method and immunohistochemical analyses of testes, respectively. Results The morphology of seminiferous tubules from animals treated with BPP-10c showed an intense disruption of the epithelium, presence of atypical multinucleated cells in the lumen and degenerated germ cells in the adluminal compartment. BPP-10c led to an increase in the number of round spermatids and total support capacity of Sertoli cell in stages I, V, VII/VIII of the seminiferous epithelium cycle, without affecting BTB permeability and the distribution of claudin-1 in the seminiferous epithelium. Interestingly, no morphological or morphometric alterations were observed in animals treated with captopril. Conclusions The major finding of the present study was that BPP-10c, and not captopril, modifies spermatogenesis by causing hyperplasia of round spermatids in stages I, V, and VII/VIII of the spermatogenic cycle.(AU)

Enzymatic properties of venoms from Brazilian scorpions of Tityus genus and the neutralisation potential of therapeutical antivenoms.

Tityus scorpion stings are an important public health problem in Brazil, where the incidence of such stings exceeds the incidence of the health problems caused by other venomous animals, including snakes. In this study, we have analysed specific enzymatic activities of the venom from the Brazilian scorpions of Tityus genus, i.e., Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. The data presented here revealed that Tityus spp. venoms exhibited significant hyaluronidase activity but no phospholipase activity. All the venom samples exhibited the ability to hydrolyse Abz-FLRRV-EDDnp and dynorphin 1-13 substrates. These activities were inhibited by 1,10-phenanthroline but not by PMSF, indicating the presence of metalloproteinases in the Tityus spp. venoms. The venom peptidase activity on Abz-FLRRV-EDDnp and on dynorphin 1-13 was partially inhibited by therapeutic Brazilian anti-scorpion and anti-arachnidic antivenoms. Dynorphin 1-13 (YGGFLRRIRPKLK) contains two scissile bonds between the residues Leu-Arg and Arg-Arg that are susceptible to cleavage by the Tityus venom metallopeptidase(s). Their cleavage releases leu-enkephalin, an important bioactive peptide. The detection of metalloproteinase(s) with specificity for both dynorphin 1-13 degradation and leu-enkephalin releasing can be important for the mechanistic understanding of hypotension and bradycardia induction in cases of scorpion stings, whereas hyaluronidases might contribute to the diffusion of the toxins present in these venoms. Furthermore, the limited inhibition of the toxic enzymatic activities by commercial antivenoms illustrates the necessity of improvements in current antivenom preparation.

Premolis semirufa (Walker, 1856) envenomation, disease affecting rubber tappers of the Amazon: searching for caterpillar-bristles toxic components.

BACKGROUND: The caterpillar of the moth Premolis semirufa (Lepidoptera: Arctiidae), commonly named Pararama, is endemic of the Amazon basin. Accidental contact with these caterpillar bristles causes local symptoms such as intense heat, pain, edema and itching which last for three to seven days; however, after multiples contacts, it may induce joint-space narrowing and bone alteration, as well as degeneration of the articular cartilage and immobilization of the affected joints. Specific treatment for this disease does not exist, but corticosteroids are frequently administered. Despite of the public health hazard of Premolis semirufa caterpillar poisoning, little is known about the nature of the toxic components involved in the induction of the pathology. METHODOLOGY/PRINCIPAL FINDINGS: Here we have investigated the biological and immunochemical characteristics of the caterpillar's bristles components. Analysis of the bristles extract in in vitro assays revealed the presence of proteolytic and hyaluronidase activities but no phospholipase A(2) activity. In vivo, it was observed that the bristles extract is not lethal but can induce an intense inflammatory process, characterized by the presence of neutrophils in the paw tissues of injected mice. Furthermore, the bristles components stimulated an intense and specific antibody response but autoantibodies such as anti-DNA or anti-collagen type II were not detected. CONCLUSION: The results suggest that Premolis semirufa caterpillar bristles secretion contains a mixture of different enzymes that may act together in the generation and development of the clinical manifestations of the Pararama envenomation. Moreover, the high immunogenicity of the caterpillar bristles components, as shown by the generation of high antibody titers, may also contribute to the induction and establishment of the inflammatory disease.

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.

Administration of Mycobacterium leprae rHsp65 aggravates experimental autoimmune uveitis in mice.

The 60 kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4(+)IL-17(+), CD4(+)IFN-gamma(+) and CD4(+)Foxp3(+) cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4(+)IFN-gamma(+) and CD4(+)IL-17(+) T cells, corroborating with higher levels of IFN-gamma. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

SMase II, a new sphingomyelinase D from Loxosceles laeta venom gland: molecular cloning, expression, function and structural analysis.

Sphingomyelinase D (SMase D) present in the venoms of Loxosceles spiders is the principal component responsible for local and systemic effects observed in the loxoscelism. By using "expressed sequencing tag", it was possible to identify, in a L. laeta venom gland library, clones containing inserts coding for proteins with similarity to SMase D. One of these clones was expressed and the recombinant protein compared with the previously characterized SMase I from L. laeta, in terms of their biological, biochemical and structural properties. The new recombinant protein, SMase II, possesses all the biological properties ascribed to the whole venom and SMase I. SMase II shares 40% and 77% sequence similarity with SMase I and Lb3, respectively; the latter, a SMase D isoform from L. boneti, catalytically inactive. Molecular modeling and molecular dynamics simulations were employed to understand the structural basis, especially the presence of an additional disulfide bridge, in an attempt to account for the observed differences in SMases D activity.

Ctenus medius and Phoneutria nigriventer spiders venoms share noxious proinflammatory activities.

Ctenus medius Keyserling, 1891 (Araneae: Ctenidae) co-occurs in various microhabitats of the Brazilian Atlantic Forest and can be easily misidentified as the medically important spider Phoneutria nigriventer Keyserling, 1981 (Ctenidae). Despite being phylogenetically close to Phoneutria, no data are available about the toxic potential of Ctenus medius venom. Here we show that, although presenting different profile of protein composition, C. medius venom displays some of the toxic properties exhibited by P. nigriventer venom, including proteolytic, hyaluronidasic and phospholipasic activities, as well as the ability of causing hyperalgesia and edema. Moreover, C. medius venom interferes in the activation of the complement system in concentrations that P. nigriventer venom is inactive. Thus, these data show that venoms of spiders from Ctenidae family share important proinflammatory properties and suggest that the C. medius bite may have an important noxious effect in human accidents.