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Accidents with venomous animals pose a health issue in Brazil, and those involving brown spiders (Loxosceles sp.) figure between the most frequent ones. The accidental envenomation by brown spiders causes a strong local dermonecrotic effect, which can be followed by systemic manifestations that in some cases lead to death. The production of antivenoms for the treatments of such accidents relies on a variety of animal experiments, from the spider venom extraction to the production of antivenom in horses. In the present work, there is an attempt to reduce and optimize animal experiments with the construction and production of a chimeric protein, named Lil, containing immunodominant epitopes previously mapped from the main proteins of the Loxosceles venom, the Sphingomyelinases D. The Lil protein contains epitopes from Sphinomyelinases D of the three-main species found in Brazil and this chimeric protein was found capable of inducing antibodies with the potential to partially neutralize the toxic effects of Loxosceles intermedia venom in an animal model. Therefore, in order to reduce spider usage and to improve the lifespan of the horses used for immunization we suggest the Lil protein as a potential candidate to replace the venom usage in the antivenom production protocols.

We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29–36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism.

This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (P Ll v), which is responsible for the largest number of accidents involving venomous animals in Peru. In this work, we observed that the venom of this spider is more lethal to mice when compared with L. laeta venom from Brazil (B Ll v). The LD 50 of P Ll v was 1.213 mg/kg when the venom was intradermally injected. The venom displayed sphingomyelinase activity and produced dermonecrotic, hemorrhagic and edema effects in rabbits. 2-D SDS-PAGE separation of the soluble venoms resulted in a protein profile ranging from 20 to 205 kDa. Anti-P Ll v and anti-B Ll v sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with P Ll v and B Ll v and also with other Brazilian Loxosceles venoms. Western blotting analysis showed that bands corresponding to 25–35 kDa are the proteins best recognized in every Loxosceles spp venoms analyzed. The immunized rabbits displayed protective effect after challenge with P Ll v and B Ll v. In vitro assays with horse anti-loxoscelic antivenoms produced in Brazil and Peru demonstrated that these commercial antivenoms were efficient to inhibit the sphingomyelinase activity of P Ll v and B Ll v.

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Motivation: Antibodies are an important class of biological drugs, but with limitations, such as inadequate pharmacokinetics, adverse immunogenicity and high production costs. Synthetic peptides for the desired target represent an important alternative to antibodies. However, no computational tool exists to guide the design of these peptides. Results: To identify the interacting residues in a given antibody–antigen (Ab–Ag) interface we used Interface Interacting Residue (I2R), a selection method based on computed molecular interactions. The aggregation of all the molecular interactions between epitope and paratope residues allowed us to transform the 3D Ab–Ag complex structures into interface graphs. Based on these data and the probability of molecular interaction we developed EPI-Peptide Designer tool that uses predicted paratope residues for an epitope of interest to generate targeted peptide ligand libraries. EPI-Peptide Designer successfully predicted 301 peptides able to bind to LiD1 target protein (65% of the experimentally tested peptides), an enrichment of 22% compared to randomly generated peptides. This tool should enable the development of a new generation of synthetic interacting peptides that could be very useful in the biosensor, diagnostic and therapeutic fields. Availability and implementation: All software developed in this work are available at http://www.biocomp.icb.ufmg.br/biocomp/ Contact: liza@icb.ufmg.br Supplementary information: Supplementary data are available at Bioinformatics online.

Members of the spider genus Loxosceles pose a marked health risk to humans because of the seriousness of the necrotic and systemic effects of their bite, known as loxoscelism. The recent confirmation of Loxosceles similis in residences of Belo Horizonte in Minas Gerais Province, Brazil increases the local potential risk of loxoscelism at higher levels. The first characterization of the venom from this species showed that its main biological effects had a similar intensity as other species (e.g. Loxosceles intermedia , Loxosceles laeta , and Loxosceles gaucho ). Therefore, we wished to further analyse the biological activity of the L. similis venom as well as the capacity of anti- L. similis -venom serum to reduce dermonecrotic effects to rabbit skin. Histological analysis of rabbit skin 2, 4 and 8 h after intradermal injection of L. similis venom demonstrated a dense inflammatory infiltrate, edema, degeneration and necrosis of the skin muscle, dissociation of collagen fibers, and disruption of reticular fibers. Importantly, pre-incubation of the venom with anti- L. similis -venom serum significantly decreased all of these effects. Anti- L. similis antivenom generated antibodies that were strongly reactive to L. similis venom and capable of neutralizing the dermonecrotic effects in rabbits caused by this venom. Moreover, the antivenom significantly reduced the sphingomyelinase activity of L. similis crude venom. Venoms produced by male and female spiders were equally reactive towards anti- L. similis and anti- L. intermedia antivenoms, but female venom induced larger lesions on rabbits. In contrast, female venom acted as an immunization enhancer and protected animals from L. similis envenomation to a greater degree than male venom. In conclusion, the results shown in this study for L. similis antivenom merits a more in depth study of its properties, which may become a valuable tool against loxoscelism.

Loxosceles spider bites cause many human injuries worldwide. Injections in mice of whole Loxosceles (L.) intermedia venom or a recombinant toxin (rLiD1) produce systemic symptoms similar to those detected in envenomed humans. This animal model was used to characterize the effects of Loxosceles intermedia venom in cardiac tissues. L. intermedia antigens were detected by ELISA in kidney, heart, lung and liver of experimentally envenomed mice. In addition, rLiD1 binding to cardiomyocytes was demonstrated by immunofluorescence and confocal microscopy. Furthermore, isolated perfused heart preparations and ventricular cardiomyocytes from envenomed mice showed heart function impairment, and a significant increase of I(Ca,L) density and intracellular Ca(2+) transients, respectively. Thus, L. intermedia spider venom, as shown through the use of the recombinant toxin rLiD1, causes cardiotoxic effects and a protein from the sphingomyelinase D family plays a key role in heart dysfunction. Thus, L. intermedia spider venom and the Loxtox rLiD1 play a key role in heart dysfunction.

Motivation: Synthetic biology studies how to design and construct biological systems with functions that do not exist in nature. Biochemical networks, although easier to control, have been used less frequently than genetic networks as a base to build a synthetic system. To date, no clear engineering principles exist to design such cell-free biochemical networks. Results: We describe a methodology for the construction of synthetic biochemical networks based on three main steps: design, simulation and experimental validation. We developed BioNetCAD to help users to go through these steps. BioNetCAD allows designing abstract networks that can be implemented thanks to CompuBioTicDB, a database of parts for synthetic biology. BioNetCAD enables also simulations with the HSim software and the classical Ordinary Differential Equations (ODE). We demonstrate with a case study that BioNetCAD can rationalize and reduce further experimental validation during the construction of a biochemical network. Availability and implementation: BioNetCAD is freely available at http://www.sysdiag.cnrs.fr/BioNetCAD. It is implemented in Java and supported on MS Windows. CompuBioTicDB is freely accessible at http://compubiotic.sysdiag.cnrs.fr/ Contact: stephanie.rialle@sysdiag.cnrs.fr; franck.molina@sysdiag.cnrs.fr Supplementary information: Supplementary data are available at Bioinformatics online.

Antibodies raised against recombinant Loxosceles intermedia dermonecrotic protein isoform 1 (rLiD1) display neutralizing capacity for the L. intermedia whole venom. We previously found that an immunodominant continuous B-cell epitope, recognized by these antibodies corresponds to a region of the protein known to be involved in the active site. In this study, we extend previous work by preparing a 27-residue synthetic replica of this epitope ((25)NLGANSIETDVSFDDNANPEYTYHGIP(51)) and using it as an immunogen in mice and rabbits. The immunization process induced antibodies that protected mice from a lethal dose of L. intermedia crude venom and rabbits against the dermonecrotic effects of rLiD1. An Ala scan of the epitope indicated that 4 residues, E44, Y45, T46 and Y47, are essential (over 70% decrease in binding upon replacement with alanine) for antibody recognition. The possible mechanisms of neutralization are discussed in light of these findings.

Scorpion stings cause human fatalities in numerous countries. Serotherapy is the only specific means to try to circumvent the noxious effects of venom toxins. TsNTxP is a natural anatoxin from the venom of the scorpion Tityus serrulatus that may be useful to raise therapeutic anti-venom sera. Linear epitopes recognized by anti-TsNTxP antibodies have previously been mapped. Here, we attempted to identify discontinuous epitopes in TsNTxP since neutralizing epitopes are often associated with such complex entities. One hundred and fifty-three octadecapeptides with the general formula (P1)-(Gly-Gly)-(P2) were synthesized by the Spot method on cellulose membranes. P1 and P2 were octapeptides from the TsNTxP N-terminal and C-terminal sections, respectively. Each sequence of eight amino acids was frameshifted in turn by three residues, in order to cover TsNTxP entire sequence. Binding of neutralizing anti-TsNTxP rabbit antibodies to spotted peptides revealed GREGYPADGGGLPDSVKI as the more reactive peptide sequence. This epitope was made from the first eight residues of the protein (GREGYPAD) and from residues 47 to 54 (GLPDSVKI) of the C-terminal part of TsNTxP. BALB/c mice were immunized with synthetic GREGYPADGGGLPDSVKI peptide conjugated to ovalbumin. One week after the last immunization, in vivo protection assays showed that immunized mice could resist a challenge by an amount of T.serrulatus whole venom equivalent to 1.75 LD100, a dose that killed all control non-immune mice. Based on molecular models of TsNTxP and related Tityus toxins, we found that the above peptide matches with a discontinuous epitope, well exposed at the toxin molecular surface which contains residues known to be important for the bioactivity of toxins.

Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.

A chimeric protein (rCpLi) was constructed expressing three epitopes of rLiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. We have analyzed the neutralization potential of sera obtained by immunization of horses with rCpLi and rCpLi combined with initial doses of venoms and compared these with antivenom traditionally produced in horses using crude Loxosceles gaucho, Loxosceles laeta and L. intermedia venoms as antigens. We have demonstrated by ELISA that horses immunized with three initial doses of crude venom containing mixtures of L. intermedia, L. gaucho and L. laeta followed by nine doses of rCpLi generate antibodies with the same reactivity as those produced following immunization with traditional antivenom, towards the venoms of the three Loxosceles sp. species. Results from in vivo and in vitro neutralization assays showed that the new horse sera are able to neutralize the dermonecrotic activity of Loxosceles venoms, which are of medical importance in Brazil and some of these sera are capable of meeting the necessary potency requirements that could allow for their therapeutic use in humans. This immunization strategy combining both antigens used approximately 67% less crude Loxosceles venoms compared to traditional immunization protocol and can mean the development of Loxosceles antivenoms with the consequent reduction of devastation of arachnid fauna.

The Loxosceles genus spiders (the brown spiders) are encountered in all the continents, and the clinical manifestations following spider bites include skin necrosis with gravitational lesion spreading and occasional systemic manifestations, such as intravascular hemolysis, thrombocytopenia and acute renal failure. Brown spider venoms are complex mixtures of toxins especially enriched in three molecular families: the phospholipases D, astacin-like metalloproteases and Inhibitor Cystine Knot (ICK) peptides. Other toxins with low level of expression also present in the venom include the serine proteases, serine protease inhibitors, hyaluronidases, allergen factors and translationally controlled tumor protein (TCTP). The mechanisms by which the Loxosceles venoms act and exert their noxious effects are not fully understood. Except for the brown spider venom phospholipase D, which causes dermonecrosis, hemolysis, thrombocytopenia and renal failure, the pathological activities of the other venom toxins remain unclear. The objective of the present review is to provide insights into the brown spider venoms and loxoscelism based on recent results. These insights include the biology of brown spiders, the clinical features of loxoscelism and the diagnosis and therapy of brown spider bites. Regarding the brown spider venom, this review includes a description of the novel toxins revealed by molecular biology and proteomics techniques, the data regarding three-dimensional toxin structures, and the mechanism of action of these molecules. Finally, the biotechnological applications of the venom components, especially for those toxins reported as recombinant molecules, and the challenges for future study are discussed.

Background Sydenham's chorea (SC) is a neurological manifestation of rheumatic fever. Autoimmune mechanism of SC is supported by clinical improvement with immunomodulatory therapy; presence of circulating serum anti-basal ganglia antibodies; increase in Th2 group of cytokines in serum and CSF of patients. However, a role of the antibodies in the pathogenesis can only be established by their passive transfer. Chorea is a manifestation clearly related to increased dopaminergic (DA) activity. The purpose of this study was to investigate the potential of antibodies from patients with Sydenham's chorea to cause behavior alterations on rats with unilateral post-synaptic dopamine receptor up-regulation. Methods Rats previously submitted to 6-hydroxidopamine (6-OH-DA) unilateral lesion of substantia nigra pars compacta (SNc) and tested with apomorphine to ensure DA receptors up regulation, received intrastriatal infusion of antibodies from SC patients (n = 4) or healthy controls (n = 3) during 48 h. 24 h post infusion initiation (24PI) and 48 h post infusion initiation (48PI), we registered the occurrence of spontaneous contra lateral rotations (CLR). Findings SC group exhibited significantly higher number of CLR than control group at 24PI (p = 0.049) and 48PI (p = 0.048). Conclusion The limited sample of the present study restricts us to affirm that SC is really an immune-mediated condition. However the significant result of this pilot study points to preliminary evidence that SC antibodies may affect DA activity in rats with up-regulated striatal DA receptors.

The venom of Loxosceles intermedia (Li) spiders is responsible for cutaneous lesions and other clinical manifestations. We previously reported that the monoclonal antibody LimAb7 can neutralize the dermonecrotic activity of crude Li venom. In this study, we observed that this antibody recognizes several proteins from the venom dermonecrotic fraction (DNF), including LiD1. Identifying the epitope of such a neutralizing antibody could help designing immunogens for producing therapeutic sera or vaccination approaches. To this aim, two sets of 25- and 15-mer overlapping peptides that cover the complete amino acid sequence of LiD1 were synthesized using the SPOT technique. None of them was recognized by LimAb7, suggesting that the epitope is discontinuous. Then, the screening of four peptide phage-display libraries yielded four possible epitope mimics that, however, did not show any obvious similarity with the LiD1 sequence. These mimotopes, together with a 3D model of LiD1, were used to predict with the MIMOP bioinformatic tool the putative epitope region (residues C197, Y224, W225, T226, D228, K229, R230, T232 and Y248 of LiD1) recognized by LimAb7. This analysis and the results of alanine-scanning experiments highlighted a few residues (such as W225 and D228) that are found in the active site of different SMases D and that may be important for LiD1 enzymatic activity. Finally, the only mimotope NCNKNDHLFACW that interacts with LimAb7 by SPOT and its analog NSNKNDHLFASW were used as immunogens in rabbits. The resulting antibodies could neutralize some of the biological effects induced by crude Li venom, demonstrating a mimotope-induced protection against L. intermedia venom.

Picadas de aranhas do gênero Loxosceles causam numerosos acidentes em humanos no mundo todo. No Brasil, constitui a terceira maior causa de acidentes envolvendo animais venenosos. Injeção, em modelos animais, do veneno de Loxosceles intermedia ou da toxina dermonecrótica recombinante (LiD1r) produzem alterações cutâneas e sintomas sistêmicos similares àqueles encontrados em humanos. O intuito deste trabalho foi caracterizar os efeitos cardíacos, em camundongos, induzidos pelo veneno de L. intermedia e LiD1r. Antígenos do veneno foram detectados, por ELISA, no rim, coração, pulmão e fígado de camundongos experimentalmente envenenados. A atividade da enzima creatina quinase e sua fração MB encontraram-se elevadas no soro de animais tratados, por isso foi investigada uma possível ação cardíaca deste veneno. A ligação de LiD1r à cardiomiócitos foi demonstrada pela técnica de imunofluorescência usando microscopia confocal. Preparações de coração isolado de animais envenenados, usando o sistema de perfusão Langendorff, mostraram um prejuízo in vitro das funções cardíacas nestes animais. Cardiomiócitos ventriculares isolados de camundongos envenenados apresentaram um aumento significativo da densidade de corrente nos canais de cálcio de tipo L e do transiente intracelular de cálcio. Em suma, os resultados deste trabalho, caracterizam os efeitos cardiotóxicos do veneno da aranha L. intermedia. Uma proteína da família das esfingomielinases, presente neste veneno, apresenta um papel chave na disfunção cardíaca dos camundongos, como constatado pela atividade cardíaca exercida pela toxina recombinante LiD1r. Loxosceles spider bites cause numerous accidents in humans worldwide. In Brasil, loxoscelism brown spider envenomation constitutes the third cause of accidents by venomous animals. Injections of the whole Loxosceles intermedia venom or of recombinant toxin derivated of it (rLiD1) produce in animal models skin-tissue changes and systemic symptoms similar to those detected in humans. The aim of our work was to characterize de cardiac effects induced in mice by L. intermedia venom and rLiD1. L. intermedia antigens were detected in the kidney, heart, lung and liver of experimental envenomed mice by ELISA. Since serum creatine kinase and creatine kinase MB isoenzyme activities were elevated in treated mice, a cardiac action of the venom was further investigated. The binding of rLiD1 to cardiomyocytes was demonstrated by means of an immunofluorescence technique using confocal microscopy. Isolated Langendorff-perfused heart preparations from envenomed mice presented an impairment of the in vitro heart functions. Ventricular cardiomyocites isolated from envenomed mice showed a significant increase of L-type calcium current density and intracellular Ca2+ transients. Taken together, our findings establish the cardiotoxic effects of Loxosceles intermedia spider venom. A protein from sphingomyelinase family plays a key role in the heart mice dysfunction, as attested by cardiac activity of the recombinant toxin rLiD1.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A genética é a área da Biologia que estuda a transmissão dos caracteres hereditários entre os seres vivos. Então, é preciso que os alunos compreendam os principais conteúdos relacionados a ela, como, por exemplo, a primeira lei de Mendel. No entanto, muitos estudantes apresentam dificuldade em aprender genética, pois consideram os conteúdos relacionados a ela complexos demais. Assim, este trabalho teve como objetivo construir e aplicar uma sequência didática (SD) sobre os principais assuntos relacionados à primeira lei de Mendel, para discentes do ensino médio, com o intuito de que eles compreendam esses conceitos e consigam aplicá-los na vida cotidiana. A SD foi dividida em três momentos: o primeiro tem o objetivo de introduzir e contextualizar o tema; o segundo é composto por atividades que permitem aos alunos colocar a teoria em prática; o terceiro apresenta uma atividade com viés investigativo, que possibilita ao aluno refletir, questionar, levantar hipóteses e testá-las, a fim de chegarem a uma solução para o problema apresentado. A análise da SD foi realizada baseando-se nos dados coletados a partir de sua aplicação e à luz da literatura pertinente ao tema. O comportamento e o envolvimento dos estudantes foram observados pelo pesquisador durante as atividades. Utilizou-se a abordagem qualitativa para analisar os dados coletados durante o desenvolvimento da SD. A partir da percepção do pesquisador de dados do desempenho em relação às atividades aplicadas e da bibliografia consultada, chegou-se à conclusão de que uma SD elaborada a partir de atividades lúdicas, investigativas e dinâmicas em grupo pode colaborar, de maneira considerável, com o processo de ensino-aprendizagem dos principais conteúdos relacionados às leis de Mendel. Genetics is a branch of Biology focusing on the hereditary. Although the Genetics basis, as Mendel’s first law, for instance, is quite important for a comprehensive education, it is common to have students presenting difficulties on learning it. Thus, this work aimed to create a teaching sequence for High School students on Mendel’s first law and related issues, using inquiring and ludic activities. It was planned to use this teaching sequence on classes, but this was only partially done due the suspension of regular classes during the pandemic time. The teaching sequence has three moments: the first one introduces and contextualizes the subject; the second one is composed by activities on applied situations and related issues; and the third one brings an inquiring activity to teach the occurrence and implications of lethal alleles. The teaching sequence could be partially analyzed regarding the student’s participation during the classes it was used, observed by the teacher/researcher. Several of its aspects were compared with the literature and discussed using the researcher teaching experience. After that, the conclusion was that the developed teaching sequence using ludic and inquiring activities performed in group dynamic can help the teaching-learning process of Mendel’s laws and related issues and increases the student’s participation on learning.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Acidentes com animais peçonhentos são um problema de saúde de pública no Brasil, sendo a aranha-marrom a segunda maior causadora de tais acidentes. O envenenamento acidental causado pela picada da aranha marrom gera efeitos dermonecróticos que podem ser seguidos de manifestações sistêmicas. Ainda que estas manifestações sistêmicas ocorram em uma parcela pequena dos casos, esta pode ser fatal. A produção de antivenenos para o tratamento de tais acidentes é dependente de uma variedade de experimentos animais, desde a extração do veneno das aranhas, à produção do antiveneno em cavalos, e até à avaliação in vivo do potencial neutralizante destes antivenenos, que é feita em coelhos. O presente trabalho apresenta tentativas de minimizar e otimizar experimentos com animais na produção de antivenenos com duas abordagens. A primeira se baseia na construção e produção de uma proteína quimérica, a Lil, contendo epitopos imunodominantes, previamente mapeados, da principal proteína do veneno de Loxosceles, a Esfingomielinase D. A proteína quimérica Lil contém epitopos encontrados nas Esfingomielinases D das três principais espécies brasileiras. Esta proteína se mostrou capaz de produzir anticorpos com o potencial de neutralizar parcialmente os efeitos tóxicos do veneno de Loxosceles intermedia, em um modelo animal. Por esta razão, de modo a reduzir o uso de aranhas e otimizar a vida útil dos cavalos produtores de antiveneno, a proteína Lil se mostrou um candidato promissor para uso em processos de imunização. A segunda abordagem foi a elaboração de um teste in vitro para a discriminação de soros com alto e baixo potencial neutralizante. A Smase I de Loxosceles laeta se mostrou capaz de discriminar a potência neutralizante dos soros de alta e baixa potência avaliados, em um teste de ELISA. Então, é sugerida a utilização deste teste como uma alternativa aos protocolos in vivo que utilizam coelhos. Accidents with venomous animmals represent a health issue in Brazil, and the brown spider’s accidents are pointed out as the second most frequent ones. The accidental envenomation by brown spiders (Loxosceles sp.) cause a strong local dermonecrotic effect which can be followed by systemic manifestations, and even though the systemic loxoscelism occurs in a small portion of the cases, it can be fatal. The production of antivenoms for the treatments of such accidents relies on a variety of animal experiments, from the spider venom extraction to the production of antivenom in horses and its in vivo neutralizing potency evaluation, which is performed in rabbits. In the present work, there is an attempt to reduce and optimize animal experiments with two different approaches. The first approach relies in the construction and production of a chimeric protein, named Lil, containing immunodominant epitopes that were previously mapped from the main protein of the Loxosceles venom, the Sphingomyelinase D. The Lil protein contains epitopes from Sphinomyelinases D of the three main species found in Brazil. The Lil protein was found capable of inducing antibodies with the potential to partialy neutralize the toxic effects of Loxosceles intermedia venom in an animal model. Therefore, in order to reduce spider usage and to provide a less harmfull life to the horses used for immunization we suggest the Lil protein as a potential candidate to replace the venom usage in the antivenom production protocols. The second approach was to elaborate an in vitro test for the distinction of high and low potency sera. The Loxosceles laeta Smase I was able to correctly diagnose the neutralizing potency of the evaluated sera, in an ELISA test. Thus, we suggest the use of such test as means to discriminate the neutralizing potency of the antiloxosceles sera as an alternative to the in vivo protocols relying on rabbits.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As leishmanioses são doenças com ampla diversidade de formas clínicas, devido as diferentes espécies. A leishmaniose tegumentar (LT) está frequentemente associada à infecção por Leishmania (Leishmania) amazonensis e/ou L. (Viannia) braziliensis, enquanto a forma visceral (LV) é causada por L. (L.) infantum. As proteínas por sua vez, estão entre as principais moléculas que podem determinar as mais diversas variações biológicas. Outro aspecto relevante é que um diagnóstico eficiente contribui para a avaliação não só clínica, mas também da epidemiologia e controle da doença. No presente trabalho, por meio de abordagem proteômica de expressão diferencial em gel (DIGE), idenficamos proteínas diferencialmente abundantes entre as espécies de Leishmania mais frequentes no Brasil, L. (L.) amazonensis, L. (Viannia) braziliensis e L. (L.) infantum. Através da utilização de ferramentas de bioinformática, foram mostradas as interações dessas proteínas em redes biológicas, bem como os processos biológicos nos quais essas moléculas atuam. No nosso trabalho destacamos 6 dessas categorias funcionais: metabolismo do nitrogênio, resposta ao stress, organização do citoesqueleto, transporte, desenvolvimento de estruturas e vias associadas à regulação do sistema imune; sendo que, nessa última, o enriquecimento estava presente somente em L. braziliensis e L. infatum. Os dados revelaram que L.infatum, espécie com maior potencial de visceralização, apresentou maior quantidade de proteínas detectadas para quase todos os processos. Selecionamos dentre essas proteínas quais eram também antigênicas de acordo com a forma clínica, LT ou LV, dos soros utilizados no western blot. As proteínas antigênicas foram submetidas a uma análise in silico para a predição de epitopos para células B que não possuíam similaridades com ortólogos em Trypanosoma cruzi. Das 18 proteínas identificadas com essas características (mais abundantes, específicas e antigênicas em uma espécie em relação às outras) oito são das espécies causadoras da forma tegumentar da doença (L. amazonensis e L. braziliensis) e 10 na espécie que causa a forma visceral (L. infantum). Os 68 epitopos preditos simultaneamente pelos softwares ABCPred e BCPreds nessas proteínas, foram sintetizados em membrana de celulose e avaliados em relação a reatividade. Considerando os dados analisados, podemos destacar duas proteínas de L. amazonensis, que apresentam potencial para diagnóstico específico de LT: metalo-peptidase, Clan MA (E)-Familia M3 e uma proteína hipotética. Ambas são mais abundantes nessa espécie, apresentam reatividade exclusiva ao pool de soros de LT e a maioria dos epitopos não apresenta similaridade com os ortólogos em T. cruzi. Além disso, nove epitopos, provenientes das proteínas selecionadas por reatividade exclusiva de soros de pacientes com LT ao extrato de L. amazonensis, foram considerados como potenciais antígenos para o diagnóstico sorológico da LT em humanos. Leishmaniasis are disease with a wide diversity of clinical forms, due to the different species. The cutaneous leishmaniasis (LT) is frequently associated with Leishmania (Leishmania) amazonensis and / or L. (Viannia) braziliensis infection, while the visceral form (LV) is caused by L. (L.) infantum. Proteins are one of the main molecules that determinete the largest number of biological variations. Another relevant aspect is that an efficient diagnosis contributes not only to clinical evaluation, but also to disease epidemiology and control. In the present study, by means of a proteomic approach (DIGE), we identified differentially abundant proteins among the most frequent Leishmania species in our country, L (L.) amazonensis, L. (Viannia) braziliensis and L. (L.) infantum. Through the use of bioinformatics tools, the interactions of these proteins in biological networks, as well as the biological processes in which these molecules act, were shown. In our study we highlight 6 of these functional categories: nitrogen metabolism, stress response, cytoskeletal organization, transport, development of structures and pathways associated with regulation of the immune system; being in the latter the enrichment was present in L. braziliensis and L. infatum, only. Data revealed that L.infatum, species with greater visceral potential, showed higher amount of proteins detected for almost all the processes. In addition, we selected from these proteins which were also antigenic according to the clinical form, TL or VL, of the sera used in the western blot. Antigenic proteins were subjected to in silico analysis for the prediction of B cell epitopes without similarities with orthologs in Trypanosoma cruzi. Among 18 proteins identified with these characteristics (more abundant, specific and antigenic in one species relative the others) eight belongs to species that cause the cutaneous form of the disease (L. amazonensis and L. braziliensis) and 10 belongs to the species that cause the visceral form (L. infantum). The 68 epitopes predicted simultaneously by the ABCPred and BCPreds software in these proteins were synthesized in cellulose membrane and evaluated for reactivity. Considering the analyzed data, we highlight two proteins of L. amazonensis, which have potential for specific diagnosis of TL: metallopeptidase, Clan MA (E) -Familia M3 and a hypothetical protein. Both are more abundant in this species, they have exclusive reactivity to the sera from TL pool and most of the epitopes no show similarity to orthologs in T. cruzi. In addition, nine epitopes from proteins selected by exclusive reactivity of sera from TL patients to L. amazonensis extract were considered as potential antigens for the serological diagnosis of TL in humans.

No Brasil, durante o ano de 2015 (últimos dados disponíveis), foram registrados 106.983 casos de acidentes com animais peçonhentos. Desse total, 19.495 envolveram aranhas (18,22%). O estado de Minas Gerais apresentou o maior número de acidentes com animais peçonhentos notificados com 19.474 casos registrados, sendo que 2.269 acidentes foram com aranhas (11,6%). Dentre os acidentes com Aranha, destaca-se o gênero Loxosceles (aranha-marrom), responsável por 58% dos acidentes com aranhas em que houve identificação da espécie. No mundo, já foram identificadas cerca de 100 espécies diferentes de Loxosceles, dentre elas, 10 são encontradas no Brasil. As espécies de maior importância médica são: L. intermedia, L. gaucho, L. similis e L. laeta. O veneno da aranha-marrom possui uma composição extremamente complexa, contendo diversas toxinas. Dentre as enzimas foram identificadas: hidrolases, hialuronidases, lipases, peptidases, colagenases, alcalino fosfatase, 5-ribonucleotidase, fosfohidrolases e proteases. Embora, provavelmente todas as enzimas presentes no veneno sejam capazes de contribuir de alguma forma para a progressão da lesão decorrente do acidente, somente as esfingomielinases D (SMase D), lipases presentes no veneno, foram capazes de produzir lesões dermonecróticas, hemólise e agregação plaquetária em animais de laboratório. Foi demonstrado em laboratório que as SMase D possuem diversos substratos e embora tenha sido demonstrado que em meio fisiológico a SMase D de L. laeta é capaz de liberar a colina da lisofosfatidilcolina (LPC), uma molécula de imunosinalização derivada de fosfatidilcolina (PC), assim gerando ácido lisofosfatídico (LPA), conhecido por induzir várias respostas biológicas e patológicas, o mecanismo das SMAses D causador dermonecrose ainda não foi totalmente elucidado. Recentemente foi identificado um motivo bem conservado na porção C-Terminal das SMases D (ATXXDNPW) de diferentes espécies e gêneros, logo após a última -hélice do TIM barril (/)8. Desta forma, o objetivo deste trabalho foi avaliar os efeitos da mutação de aminoácidos conservados D277 e W280 na porção C-Terminal da esfingomielinase rLID1 do veneno de Loxosceles intermedia. As proteínas recombinantes rLID1, rLID1_D (D277A), rLID1_W (W280A) e rLID1_DW (D277A e W280A) foram submetidas a modelagem e dinâmica molecular a fim de avaliar os impactos das mutações in silico. Constatou-se que as mutações aumentam a flexibilidade da alça catalítica da proteína. Após a avaliação in silico, as proteínas foram expressas em BL21 (DE3) Arctic Express, porém, somente as proteínas rLID1, rLID1_D e rLID1_W ficaram solúveis. As proteínas solúveis foram purificadas utilizando cromatografia de afinidade e logo em seguida, cromatografia de gel filtração. Após a obtenção das proteínas puras, foi realizado o teste enzimático para avaliar a atividade das proteínas e foi observado que a proteína mutante rLID1_D possui atividade enzimática similar a proteína wildtype rLID1 enquanto a proteína rLID1_W possui atividade enzimática superior. Em seguida, foi realizado o teste biológico em coelho e foi constatado que as proteínas mutantes são capazes de causar dermonecrose, porém, a proteína rLID1_W, na quantidade testada, possui capacidade muito inferior a proteína wildtype. Nossos dados indicam que as mutações na região C-Terminal da rLID1 impactam na atividade enzimática e biológica da proteína. In Brazil, during the year 2015 (last available data), there were 106,983 cases of accidents with venomous animals. Of this total, 19,495 involved spiders (18.22%). The state of Minas Gerais had the highest number of accidents with venomous animals reported with 19,474 recorded cases, of which 2,269 were with spiders (11.6%). Among the accidents with Spider, the genus Loxosceles (brown spider) stands out, responsible for 58% of the spider accidents in which the species was identified. In the world, about 100 different species of Loxosceles have been identified, of which 10 are found in Brazil. The species of major medical importance are: L. intermedia, L. gaucho, L. similis and L. laeta. The venom of the brown spider has an extremely complex composition, containing several toxins. Among the enzymes were identified: hydrolases, hyaluronidases, lipases, peptidases, collagenases, alkaline phosphatase, 5-ribonucleotidase, phosphohydrolases and proteases. Although probably all the enzymes present in the venom are able to contribute in some way to the progression of the injury resulting from the accident, only sphingomyelinases D (SMase D), a lipase present in the venom, were able to produce dermonecrotic lesions, hemolysis and platelet aggregation In laboratory animals. It has been demonstrated in the laboratory that SMase D has several substrates and although it has been demonstrated that in physiological medium the SMase D of L. laeta is capable of releasing choline from lysophosphatidylcholine (LPC), an immunosin- guishing molecule derived from phosphatidylcholine (PC), thus generating lysophosphatidic acid (LPA), known to induce various biological and pathological responses, the mechanism of SMAse D causing dermonecrosis is still not fully elucidated. Recently, a well conserved motif was identified in the C-Terminal portion of SMasesD (ATXXDNPW) of different species and genera, shortly after the last -helix of the TIM barrel ( / )8. Thus, the objective of this work was to evaluate the effects of the conserved D277 and W280 amino acid mutation on the C-terminal portion of the rLID1 sphingomyelinase from the Loxosceles intermedia venom. The recombinant proteins rLID1, rLID1_D (D277A), rLID1_W (W280A) and rLID1_DW (D277A and W280A) were submitted to modeling and molecular dynamics in order to evaluate the impact of mutations in silico. The mutations were found to increase the flexibility of the protein's catalytic loop. After in silico evaluation, the proteins were expressed in BL21 (DE3) Arctic Express, however, only the rLID1, rLID1_D and rLID1_W proteins were soluble. Soluble proteins were purified using affinity chromatography and then, gel filtration chromatography. After obtaining the purified proteins, the enzymatic assay was performed to evaluate the activity of the proteins and it was observed that the mutant rLID1_D has enzymatic activity similar to the wildtype protein rLID1 while the rLID1_W protein has superior enzymatic activity. After that, it was performed a biological assay and mutant proteins were found to be capable of causing dermonecrosis, but the rLID1_W protein, in the concentration used, has much lower capacity than the wildtype protein. Our data indicates that the mutation on C-Terminal of rLID1 impacts both biological and enzymatic activity of the protein.

Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune do tecido conjuntivo com largo espectro de manifestações clínicas. A desregulação imune leva ao excesso de produção de auto-anticorpos e complexos imunes, ativação do complemento e inflamação tecidual persistente. Considerando que o diagnóstico atual depende da interpretação de complexos critérios estabelecidos pelo American College of Reumatology, que o curso da doença é caracterizado por ativações e remissões imprevisíveis e que cada paciente desenvolve diferentes manifestações, o desenvolvimento de biomarcadores específicos torna-se urgentemente necessário. Nesse contexto, o presente trabalho objetivou identificar candidatos a biomarcadores do LES ativo e LES inativo potencialmente capazes de distinguir laboratorialmente LES de outras doenças autoimunes. Empregou-se, de forma inédita, a técnica proteômica 2D-DIGE para avaliar a abundância diferencial de proteínas entre pacientes com LES ativo, LES inativo, portadores de outras doenças autoimunes e indivíduos saudáveis. Foram identificadas 103 proteínas diferencialmente abundantes. Dessas, 40 com abundância aumentada no LES ativo em relação aos demais grupos e 6 com abundância aumentada no LES ativo e no LES inativo em relação aos demais. Outras proteínas apresentaram aumento de abundância no LES em relação ao controle, mas não em relação a outras doenças autoimunes, o que requer maior rigor nas etapas de validação. Foram selecionadas como promissoras as proteínas alpha-1-acid glycoprotein 1 / Orosomucoid 1, Zn-alpha2-glycoprotein, AMBP preproprotein, alpha-1-acid glycoprotein, apolipoprotein A-IV precursor, immunoglobulin kappa light chain, alpha-2-macroglobulin, chain A, Structure Of Prealbumin e immunoglobulin alpha-2 chain - fragment (Ig2), corroborando dados da literatura. Por outro lado, foram propostas, pela primeira vez, como potenciais biomarcadores do LES, as proteínas: retinol-binding protein 4 isoform X1 e SP-40,40 partial. Vale destacar que este trabalho é um estudo preliminar e mais estudos complementares são recomendados para a confirmação e validação dos resultados. Systemic Lupus Erythematous (SLE) is an autoimmune disease of connective tissue with large spectrum of clinical manifestations. The immune deregulation leads to autoantibody and immune complexes overproduction, complement activation and persistent tissue inflammation as well. Considering that the current diagnosis depends on the interpretation of complex criteria established by the American College of Rheumatology, and that the disease course is characterized by unpredictable activations and remissions, and that each patient develops different manifestations, the discovery of specific biomarkers becomes urgently necessary. In this context, this study aimed to identify putative biomarkers for active and inactive SLE potentially capable to distinguishing laboratorial SLE from other autoimmune diseases. The 2D-DIGE proteomics technique was used in an unprecedented way, to evaluate the differential abundance of proteins between patients with active SLE, inactive SLE, patients with other autoimmune disease and healthy individuals. One hundred and three differentially abundant proteins were identified. From this amount, 40 with increased abundance in active SLE compared to the other groups, and 6 with increased abundance in active and inactive SLE in relation to the other ones. Other proteins showed increased abundance in SLE compared to the control group, but not in relation to other autoimmune diseases, which demand greater rigor in the validation steps. The following proteins: 1-acid glycoprotein 1 / Orosomucoid 1, Znalpha2-glycoprotein, AMBP preproprotein, alpha-1-acid glycoprotein, apolipoprotein A-IV precursor, immunoglobulin kappa light chain, alpha-2-macroglobulin, chain A, Structure Of Prealbumin e immunoglobulin alpha-2 chain - fragment (Ig2) were selected as promising biomarkers, corroborating data in the literature. On the other hand, proteins like retinol-binding protein 4 isoform X1 e SP-40,40 partial have been proposed for the first time, as potential biomarkers for SLE. It must be stressed that this work is a preliminary study. It is recommended that additional studies must be done to confirm and validate these data.

O Avastin® (bevacizumabe) é um anticorpo monoclonal humanizado anti-VEGF aprovado para o tratamento de diferentes tipos de cânceres e utilizado de forma off label no tratamento de doenças oculares. Essa nova aplicação clínica do bevacizumabe associada à relação custo-benefício do medicamento gerou uma demanda pelo seu fracionamento em doses unitárias para atender às baixas doses utilizadas na administração intraocular. Diante disso, vários estudos têm sido conduzidos a fim de verificar a qualidade do medicamento após seu fracionamento. Para o estudo da qualidade do Avastin® fracionado pelas farmácias brasileiras, o serviço de fracionamento foi requisitado para 3 farmácias especializadas em manipulação de produtos estéreis, oftalmológicos ou de medicamentos especiais. Com base nas características particulares das proteínas, a qualidade do bevacizumabe fracionado foi investigada pela associação de técnicas cromatográficas, eletroforéticas, medidas turbidimétricas, análises de tamanho de partículas e ensaio imunoenzimático. Por meio das metodologias adotadas, nenhuma instabilidade física ou química foi encontrada e a atividade biológica foi mantida nas frações de Avastin® adquiridas. Diferenças significativas foram encontradas entre os conteúdos das frações, observando-se volumes abaixo do rotulado em duas das três farmácias e volumes acima do rotulado em uma delas. No geral, pode-se sugerir que houve manutenção das propriedades físico-químicas das proteínas e da estabilidade da formulação após o fracionamento. Avastin®(bevacizumabe) is a humanized anti-VEGF monoclonal antibody approved as a first-line therapy for different types of cancer and used by intravitreal administration as off-label drug for the treatment of ocular diseases. This new clinical application of bevacizumab associated to the cost-benefit of the drug has generated a demand for its division into unit doses to meet the low doses used in intraocular administration. Thus, several studies have been conducted in order to verify the quality of repackaged bevacizumab. To study the quality of repackaged bevacizumab obtained from Brazilian suppliers we purchased the repackaged product from three external pharmacies specialized in ophthalmology, sterile or special compounding. Based on the complexity of the antibody structure, we investigated the quality of repackaged bevacizumab associating size-exclusion chromatography, polyacrylamide gel electrophoresis, turbidimetry, diffraction laser spectroscopy, nanoparticle tracking analysis and an enzyme-linked immunosorbent assay. In general, there were no detectable differences in the physicalchemical properties between repackaged and reference bevacizumab. The only difference observed is the variation in the volume of the samples among the suppliers. This study suggests that if precautions are taken the formulation remains stable after compounding.

Aranhas do gênero Loxosceles representam um risco para a saúde humana, devido aos efeitos necróticos e sistêmicos de sua piacada. Os sintomas do envenenamento com as aranhas marrons variam de dermonecrose na maioria dos casos a hemólise sistêmica e coagulopatia, levando à falência renal agúda na minoria dos acidentes. Ao contrário dos efeitos sistêmicos, que já são bem caracterizados, os mecanismos de morte celular induzidas pela peçonha das aranhas marrons, ainda não são tão bem descritos na literatura. Existem evidências de que células tratadas com a peçonha iniciam uma resposta apoptótica, mas nenhuma via de apopotose foi caracterizada. No presente trabalho estudamos as caspases ativadas durante o processo de apoptose iniciado in vitro pela peçonha de Loxosceles similis em fibroblastos humanos. Utilizando o teste de metabolização de MTT, verificamos que dentre todas as linhagens testadas, apenas fibroblastos primários foram susceptíveis à ação da peçonha. Através das técnicas de microscopia de fluorescência e de citometria de fluxo, verificamos que fibroblastos humanos tratados com a peçonha iniciam uma resposta apoptótica. Fazendo uso ainda da citometria de fluxo, verificamos que a inibição das caspases-9, 6 e 4 influenciaram no curso do processo apoptótico, diminuindo a marcação para anexina V-FITC em relação ao grupo tratado apenas com a peçonha. Por fim, utilizando a microscopia de fluorescência mostramos que as caspases efetoras 3 e 7 também são ativadas durante o processo. Spiders from the Loxosceles genus represent a risk to human health due to the systemic and necrotic effect of their bite. The symptoms of envenomation with the brown spiders may vary from dermonecrosis in the majority of cases to coagulopathy and hemolysis leading to acute renal failure. Even though the systemic effects are well characterized, the cell death mechanisms triggered by the venom of the brown spiders are still not well described in literature. There is evidence in literature that many cells treated with the venom enter in an apoptotic process, however the triggering of this response is not well comprehended. In the present study we assessed the caspases activated during the process of apoptosis begun in vitro by the Loxosceles similis venom. Using the test of MTT metabolization, we verified that among all tested cell lines, only primary fibroblasts were affected by the brown spiders venom. We demonstrate using fluorescence microscopy and flow cytometry that human fibroblasts treated with the whole venom started an apoptotic response. Furthermore, using flow cytometry we verified that the inhibition of caspases-4, -6 and -9 decreased the percentage of cells labeled with the apoptosis marker annexin V-FITC. Ultimately, by fluorescence microscopy we could observe that the effectors caspases-3 and -7 are also activated during the process.