Sertraline associated with gold nanoparticles reduce cellular toxicity and induce sex-specific responses in behavior and neuroinflammation biomarkers in a mouse model of anxiety.

This study aimed to evaluate the effects of sertraline associated with gold nanoparticles (AuNPs) in vitro cell viability and in vivo behavior and inflammatory biomarkers in a mouse model of anxiety. Sertraline associated with AuNPs were synthesized and characterized. For the in vitro study, NIH3T3 and HT-22 cells were treated with different doses of sertraline, AuNPs, and sertraline + AuNPs and their viability was evaluated using the MTT assay. For the in vivo study, pregnant Swiss mice were administered a single dose of lipopolysaccharide (LPS) on the ninth day of gestation. The female and male offspring were divided into five treatment groups on PND 60 and administered chronic treatment for 28 days. The animals were subjected to behavioral testing and were subsequently euthanized. Their brains were collected and analyzed for inflammatory biomarkers. Sertraline associated with AuNPs exhibited significant changes in surface characteristics and increased diameters. Different doses of sertraline + AuNPs showed higher cell viability in NIH3T3 and HT-22 cells compared with sertraline alone. The offspring of LPS-treated dams exhibited anxiety-like behavior and neuroinflammatory biomarker changes during adulthood, which were ameliorated via sertraline + AuNPs treatment. The treatment response was sex-dependent and brain region-specific. These results suggest that AuNPs, which demonstrate potential to bind to other molecules, low toxicity, and reduced inflammation, can be synergistically used with sertraline to improve drug efficacy and safety by decreasing neuroinflammation and sertraline toxicity.

Apoptosis Induction in Murine Melanoma (B16F10) Cells by Mannosylerythritol Lipids-B; a Glycolipid Biosurfactant with Antitumoral Activities.

Mannosylerythritol lipids have drawn attention to cosmetic and pharmaceutical industries due to their non-toxicity and excellent biological interactions with human skin, particularly with the deepest epidermal layer. Lamellar liquid crystal structure, formed by MEL-B, is an interesting feature due to its similarity to the stratum corneum molecular arrangement and cell signaling events involved in the deregulation of the cancerous cell membrane. Thus, this work aimed to evaluate the cytotoxicity of commercial mannosylerythritol lipids-B in murine melanoma, fibroblast, and human erythrocytes cells. Cytotoxic effect was more pronounced on the tumor cells from 20 µg/mL, reducing cell viability by 65%, whereas fibroblast and human erythrocytes cells were more resistant to glycolipid treatment. Fluorescence microscopy and flow cytometer proved that mannosylerythritol lipids-B is an apoptosis inducer in tumor cells related to reactive oxygen species generation.

A proteomic road to acquire an accurate serological diagnosis for human tegumentary leishmaniasis.

Diagnostic tools are important for clinical management and epidemiological evaluation of Tegumentary (TL) and Visceral (VL) Leishmaniasis. Serology is not frequently used for the diagnosis of the TL form because low antibody titers and cross-reaction with VL. Therefore, it is crucial to identify specific and immunogenic antigens from species associated with the TL form. Here we employed a proteomic approach coupled to an in silico analysis and identified the most abundant and immunogenic proteins from Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum. Of 16 species specific proteins, nine were from the species causative of the TL form (L. amazonensis and L. braziliensis). In silico analysis revealed 18 B-cell epitopes with 0% similarity to Trypanosoma cruzi orthologs and, therefore, less likely to crossreact with sera of patients with Chagas disease. Two proteins reacted exclusively with serum from TL patients and presented several B-cell epitopes without similarity to T. cruzi orthologs: the hypothetical protein GI 134063939 and the metallo-peptidase Clan MA(E)-Family M3. The immunoassay revealed nine peptides with strong reactivity to sera from TL patients. These proteins and peptides may be good candidates to improve the specificity and sensibility of serological tests aiming to diagnose the TL of this neglected human disease. BIOLOGICAL SIGNIFICANCE: As no gold-standard test for tegumentary leishmaniasis (TL) exists, a combination of different diagnostic techniques is often necessary to obtain precise results. Thus, the identification of species-specific, highly immunogenic and abundant proteins that stimulate the humoral immune response in the host should help in the development of serological tests for human TL. Herein we searched for these potential antigens in Leishmania species related to American Leishmaniasis (L. amazonensis, L. braziliensis and L. infantum). To this end, we employed an immunoproteomic approach using proteins from these Leishmania species and sera from TL and Visceral Leishmaniasis (VL) patients. Our study unveils specific proteins and peptides that may represent antigens that will help the efforts to improve the accuracy of serological tests to diagnose the TL form.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified.

Anti-loxoscelic horse serum produced against a recombinant dermonecrotic protein of Brazilian Loxosceles intermedia spider neutralize lethal effects of Loxosceles laeta venom from Peru.

In this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production.

Identification and characterization of B-cell epitopes of 3FTx and PLA(2) toxins from Micrurus corallinus snake venom.

The main goal of this work was to develop a strategy to identify B-cell epitopes on four different three finger toxins (3FTX) and one phospholipase A2 (PLA2) from Micrurus corallinus snake venom. 3FTx and PLA2 are highly abundant components in Elapidic venoms and are the major responsibles for the toxicity observed in envenomation by coral snakes. Overlapping peptides from the sequence of each toxin were prepared by SPOT method and three different anti-elapidic sera were used to map the epitopes. After immunogenicity analysis of the spot-reactive peptides by EPITOPIA, a computational method, nine sequences from the five toxins were chemically synthesized and antigenically and immunogenically characterized. All the peptides were used together as immunogens in rabbits, delivered with Freund's adjuvant for a first cycle of immunization and Montanide in the second. A good antibody response against individual synthetic peptides and M. corallinus venom was achieved. Anti-peptide IgGs were also cross-reactive against Micrurus frontalis and Micrurus lemniscatus crude venoms. In addition, anti-peptide IgGs inhibits the lethal and phospholipasic activities of M. corallinus crude venom. Our results provide a rational basis to the identification of neutralizing epitopes on coral snake toxins and show that their corresponding synthetic peptides could improve the generation of immuno-therapeutics. The use of synthetic peptide for immunization is a reasonable approach, since it enables poly-specificity, low risk of toxic effects and large scale production.

Biochemical and immunological characteristics of Peruvian Loxosceles laeta spider venom: neutralization of its toxic effects by anti-loxoscelic antivenoms.

This manuscript describes the general biochemical properties and immunological characteristics of Peruvian spider Loxosceles laeta venom (PLlv), 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 (BLlv). The LD50 of PLlv 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-PLlv and anti-BLlv sera produced in rabbits and assayed by ELISA showed that rabbit antibodies cross-reacted with PLlv and BLlv 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 PLlv and BLlv. 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 PLlv and BLlv.

Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom.

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.

General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus.

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.

Mimotopes of mutalysin-II from Lachesis muta snake venom induce hemorrhage inhibitory antibodies upon vaccination of rabbits.

Mutalysin-II (mut-II) from Lachesis muta snake venom is an endopeptidase with hemorrhagic activity. A mAb against mutalysin-II that neutralized the hemorrhagic effect was produced previously. To identify the mAb epitopes, sets of 15-mer overlapping peptides covering the mut-II amino acid sequence were synthesized using the SPOT method and tested but failed to react with the mAb. Using a phage-display approach seventeen clones reactive with mAb were identified. Additional immunoassays with the peptides and mAb identified the QCTMDQGRLRCR, TCATDQGRLRCT, HCFHDQGRVRCA, HCTMDQGRLRCR and SCMLDQGRSRCR sequences as possible epitopes. Immunization of rabbits with these peptides induced antibodies that recognize mut-II and protected against the hemorrhagic effects of Lachesis venom.

Antigenic, microbicidal and antiparasitic properties of an l-amino acid oxidase isolated from Bothrops jararaca snake venom.

Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined.

Antibodies against synthetic epitopes inhibit the enzymatic activity of mutalysin II, a metalloproteinase from bushmaster snake venom.

Mutalysin II (mut-II), a 22.5kDa zinc endopeptidase isolated from bushmaster (Lachesis muta muta) snake venom, is a direct acting fibrin(ogen)olytic proteinase. It induces monoclonal and polyclonal antibodies which efficiently neutralize the hemorrhagic effect of L. muta and several Bothrops whole venoms. To characterize epitopes of protective antibodies we have used the Spot method of multiple peptide synthesis to prepare 64 overlapping dodecapeptides frameshifted by three residues, covering the complete amino acid sequence of mut-II. The rabbit anti-mut-II antibodies binding pattern to peptides revealed several continuous antigenic regions: one in the N-terminal part, two in the central region and the other in the C-terminal of mut-II. By using homology modelling, a three-dimensional model of mut-II was built which showed that epitopes are surface exposed. Anti-peptide antibodies were raised against three peptides (one representative of each epitope region) covalently coupled as a mixture to keyhole limpet hemocyanin. Purified IgG from the resulting anti- peptide antibodies cross-reacted with mut-II and induced a dose-dependent inhibition of the mut-II catalyzed proteolysis of fibrinogen.

Molecular characterization of a neutralizing murine monoclonal antibody against Tityus serrulatus scorpion venom.

Monoclonal antibodies (mAbs) against Tityus serrulatus venom were obtained by the fusion of SP2/0 murine myeloma cells and spleen cells from BALB/c mice immunized with a toxic fraction (TstFG50) of the Tityus venom (this G50 chromatography fraction represents most of the toxicity of the crude venom) conjugated to bovine serum albumin (BSA) with glutaraldehyde. From the initial screening of over 200 hybridoma fusion wells, a panel of 9 anti-TstFG50 secreting hybridomas was established. The capacity of mAbs to neutralize the TstFG50 toxic fraction toxic was determined by in vitro neutralization assays and by inhibition of the binding of 125I-TsVII to its site on rat brain synaptosomes. Only mAbTs1 neutralized 50% of the toxic effects produced by scorpion venom and showed 35% inhibition of the binding of 125I-TsVII at 10(-7) M. To map the epitope recognized by the protective mAbTs1, we prepared a comprehensive series of overlapping 15-mer synthetic peptides covering the amino acid sequences of the four Tityus proteins. MAbTs1 reacted with peptide 26 of TsIV (KKSKDKKADSGYSYW), peptide 30 of TsVII (KKGSSGYSAWPASYS) and peptide 31 of TsNTxP (KKGSSGYSAWPASYS). MAbTs1 was not reactive with any peptide from TsII. The N-terminal lysine residue from the epitope was found to be critical for mAbTs1 binding. The epitope was positioned on the available three-dimensional structure of TsVII together with the recently identified residues from the pharmacophore of beta-scorpion toxins. The neutralizing properties of mAbTs1 might be explained by spatial vicinity of epitope residues with pharmacophore residues.

Molecular characterization of protective antibodies raised in mice by Tityus serrulatus scorpion venom toxins conjugated to bovine serum albumin.

The possibility of raising a humoral immune response capable of inducing in vivo protection against the lethal effects of Tityus serrulatus (Ts) scorpion venom was evaluated in the mouse model. An immunogen was prepared that consists of a toxic fraction (TstFG(50)) of the Tityus venom (this G(50) chromatography fraction represents most of the toxicity of the crude venom) conjugated to bovine serum albumin (BSA) with glutaraldehyde. TstFG(50) coupled to BSA yielded a thoroughly detoxified immunogen. BALB/c and C57BL/10 mice were immunized with this preparation and all developed an antibody response. In vivo protection assays one week after the last immunization showed that vaccinated mice could resist the challenge by twice the LD(50) of the TstFG(50), a dose which killed all control non-immune mice. The protective effect persisted nine weeks after the end of the immunization protocol. To characterize epitopes of protective antibodies we used the Spot method of multiple peptide synthesis to prepare sets of immobilized 15 mer overlapping peptides, covering the complete amino acid sequences of the main Tityus toxins, TsII and TsVII (both beta-type toxins) and TsIV, an alpha-type toxin that is the major lethal component of the venom. Antibody binding to peptides, revealed one major antigenic region in the C-terminal part of the three toxins and another region in the helical part of TsII and TsIV toxins. It is likely that these epitopes correspond to neutralizing epitopes since they correspond to regions of the toxins that are known to be involved in the active site of the toxins.