RESUMO
Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na+ ion channels of type beta-toxin (ß-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom.
Assuntos
Peptídeos , Venenos de Escorpião , Escorpiões , Anticorpos de Cadeia Única , Animais , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Venenos de Escorpião/imunologia , Peptídeos/química , Anticorpos de Cadeia Única/química , Humanos , Camundongos , Sequência de Aminoácidos , Antivenenos/imunologia , Antivenenos/química , Antivenenos/farmacologia , Animais PeçonhentosRESUMO
Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas Recombinantes/imunologia , Picadas de Escorpião/imunologia , Venenos de Escorpião/imunologia , Escorpiões/imunologia , Anticorpos de Cadeia Única/imunologia , Sequência de Aminoácidos , Animais , Técnicas de Visualização da Superfície Celular/métodos , Feminino , México , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização/métodos , Alinhamento de SequênciaRESUMO
Scorpion envenomation results in a wide range of clinical manifestations that are mostly attributed to the activation of the autonomic nervous system by venom toxins. In fact, sympathetic and parasympathetic disturbances play important roles during poisoning. However, scorpion venom also induces a complex hyperinflammatory state that occurs parallel to systemic inflammatory response syndrome and acute sepsis. After a scorpion sting, innate immune cells are exposed to the venom molecules, which bind to pattern recognition receptors and activate pro-inflammatory pathways that contribute toward the promotion of severe symptoms, such as pulmonary edema, and eventually lead to death. In this review, we highlight studies that pointed out inflammation as a major pathological facet of scorpion envenomation, so as to provide novel targets to improve therapeutics for scorpionism.
Assuntos
Picadas de Escorpião/patologia , Venenos de Escorpião/toxicidade , Humanos , Imunidade Inata , Macrófagos/efeitos dos fármacos , Picadas de Escorpião/imunologia , Venenos de Escorpião/imunologiaRESUMO
Antivenom treatment has been largely used against scorpion stings. Despite their efficacy, the use of mammalian antivenoms may cause adverse effects due to the immune system activation. IgYs from hyperimmunized laying hens against venoms could be a promising alternative to equine IgGs due to the various benefits that these antibodies can provide. Here we report the preparation of specific IgYs by immunizing laying hens with Aah (Androctonus australis hector) scorpion venom. IgYs were isolated from egg yolks by water dilution and salt precipitation methods; they were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot and ELISA. The efficiency of these immunoglobulins on the pathophysiological effects induced by Aah venom was assessed by histological and metabolical analysis of the aorta and the heart. The inflammatory response was assessed by evaluating the granulocyte tissue infiltration and oxidative/nitrosative status. Results revealed high IgYs titers against Aah venom by ELISA. Overall, these IgYs seem to protect efficiently mice against envenomation and neutralized the lethal effects of scorpion venom with a high efficacy; the median effective dose (ED50) was 221⯵l/2 LD50; i.e. an amount of 79.23â¯mg of IgY scan neutralize 1â¯mg of Aah venom. IgY antibodies neutralize effectively the Aah venom lethality and could prevent severe pathological effects induced by scorpion venom and could be used as an effective alternative to equine IgGs against scorpion envenoming.
Assuntos
Aorta/patologia , Galinhas/imunologia , Imunoglobulinas/uso terapêutico , Imunoterapia Adotiva/métodos , Miocárdio/patologia , Picadas de Escorpião/terapia , Venenos de Escorpião/imunologia , Animais , Antivenenos/uso terapêutico , Humanos , Imunização , Masculino , Camundongos , Camundongos Endogâmicos , Picadas de Escorpião/imunologia , Escorpiões/fisiologiaRESUMO
Interleukin (IL)-1ß is a potential target for treatment of several inflammatory diseases, including envenomation by the scorpion Tityus serrulatus. In this context, bioactive lipids such as prostaglandin (PG)E2 and leukotriene (LT)B4 modulate the production of IL-1ß by innate immune cells. Pattern recognition receptors (PRRs) that perceive T. serrulatus venom (TsV), and orchestrate LTB4, PGE2, and cyclic adenosine monophosphate (cAMP) production to regulate IL-1ß release are unknown. Furthermore, molecular mechanisms driving human cell responses to TsV remain uncharacterized. Here, we identified that both CD14 and CD36 control the synthesis of bioactive lipids, inflammatory cytokines, and mortality mediated by TsV. CD14 induces PGE2/cAMP/IL-1ß release and inflammation. By contrast, CD36 shunts eicosanoid metabolism toward production of LTB4, which represses the PGE2/cAMP/IL-1ß axis and mortality. Of importance, the molecular mechanisms observed in mice strongly correlate with those of human cell responses to TsV. Overall, this study provides major insights into molecular mechanisms connecting CD14 and CD36 with differential eicosanoid metabolism and inflammation mediated by IL-1ß.
Assuntos
Antígenos CD36/imunologia , Interleucina-1beta/imunologia , Receptores de Lipopolissacarídeos/imunologia , Picadas de Escorpião/imunologia , Venenos de Escorpião/imunologia , Adulto , Animais , Antígenos CD36/metabolismo , Modelos Animais de Doenças , Eicosanoides/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Interleucina-1beta/metabolismo , Leucócitos Mononucleares , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , Picadas de Escorpião/sangue , Picadas de Escorpião/mortalidade , Escorpiões/imunologia , Transdução de Sinais/imunologia , Adulto JovemRESUMO
Tityus serrulatus (Ts) venom is composed of a mixture of toxins presenting diverse biological functions. However, although this venom has been studied over the past three decades, omics analysis revealed that most of its toxins are not identified or their biological activities are unknown. Ts19 Frag-II is included in this group, which function is still uncertain. This study aimed to expand the biological activities of Ts19 Frag-II through in vivo investigation. Our results demonstrates that mice challenged with Ts19 Frag-II presented biochemical alterations, increasing serum levels of urea, ALT and ß-globulin, besides decreasing γ-globulins. Moreover, this toxin was also able to induce immunological alterations, increasing NO, IL-6, TNF-α and IL-17, being considered a proinflammatory toxin. The increase of IL-17 was unprecedented regarding Ts toxins and could be a result of the overall produced-effect of cells of innate immunity cells (neutrophils, monocytes, natural killers and lymphoid tissue inducers - LTis) as well as of adaptive immunity (Th17 cells). This study expanded the biological activities of Ts19 Frag-II, suggesting that this toxin could be contributing to the Ts envenoming through alterations of biochemical parameters as well as triggering the inflammatory response.
Assuntos
Interleucina-17/sangue , Peptídeos/imunologia , Peptídeos/toxicidade , Venenos de Escorpião/imunologia , Venenos de Escorpião/toxicidade , Alanina Transaminase/sangue , Animais , beta-Globulinas/análise , Imunidade Inata , Interleucina-6/sangue , Masculino , Camundongos Endogâmicos BALB C , Óxido Nítrico/sangue , Escorpiões/química , Fator de Necrose Tumoral alfa/sangue , Ureia/sangue , gama-Globulinas/análiseRESUMO
A previously undescribed toxic peptide named Cl13 was purified from the venom of the Mexican scorpion Centruroides limpidus. It contains 66 amino acid residues, including four disulfide bonds. The physiological effects assayed in 7 different subtypes of voltage gated Na+-channels, showed that it belongs to the ß-scorpion toxin type. The most notorious effects were observed in subtypes Nav1.4, Nav1.5 and Nav1.6. Although having important sequence similarities with two other lethal toxins from this scorpion species (Cll1m and Cll2), the recently developed single chain antibody fragments (scFv) of human origin were not capable of protecting against Cl13. At the amino acid sequence level, in 3 stretches of peptide Cl13 (positions 7-9, 30-38 and 62-66) some differences with respect to other similar toxins are observed. Some of these differences coincide with contact points with the human antibody fragments.
Assuntos
Peptídeos/imunologia , Venenos de Escorpião/imunologia , Canais de Sódio Disparados por Voltagem/imunologia , Sequência de Aminoácidos/genética , Animais , Humanos , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Venenos de Escorpião/genética , Venenos de Escorpião/metabolismo , Escorpiões/química , Escorpiões/genética , Escorpiões/imunologia , Alinhamento de Sequência , Anticorpos de Cadeia Única/imunologia , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismoRESUMO
Androctonus australis hector (Aah) venom and its neurotoxins may affect the neuro-endocrine immunological axis due to their binding to ionic channels of axonal membranes. This binding leads to the release of neurotransmitters and immunological mediators accompanied by pathophysiological effects. Although the hyperglycemia induced by scorpion venom is clearly established, the involved mediators in these deregulations are unknown. The strong relationship between inflammation and the wide variety of physiological processes can suggest that the activation of the inflammatory response and the massive release of IL-6 and TNF-α release induced by the venom may induce hyperglycemia and various biological disorders. We therefore investigated in this study the contribution of IL-6 and TNF-α in the modulation of inflammatory response and metabolic disorder induced by Aah venom. Obtained results revealed that Aah venom induced inflammatory response characterized by significant increase of inflammatory cells in sera and tissues homogenates accompanied by hyperglycemia and hyperinsulinemia, suggesting that the venom induced insulin resistance. It also induced severe alterations in hepatic parenchyma associated to metabolic disorders and imbalanced redox status. Cytokine antagonists injected 30 min prior to Aah venom allowed a significant reduction of inflammatory biomarker and plasma glucose levels, they also prevented metabolic disorders, oxidative stress and hepatic tissue damage induced by Aah venom. In conclusion, IL-6 and TNF-α appear to play a crucial role in the inflammatory response, hyperglycemia and associated complications to glucose metabolism disorders (carbohydrate and fat metabolism disorders, oxidative stress and hepatic damage) observed following scorpion envenoming.
Assuntos
Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Doenças Metabólicas/imunologia , Neurotoxinas/imunologia , Picadas de Escorpião/imunologia , Venenos de Escorpião/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Bloqueadores/administração & dosagem , Humanos , Imunidade/efeitos dos fármacos , Imunomodulação , Mediadores da Inflamação/imunologia , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos , Escorpiões/imunologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Anti-Tityus discrepans F(ab')2 ELISA recognition of T. discrepans toxins was measured with regression analysis and its slope called ELISA recognition value (ERv). Fractions containing toxins affecting mammal macrophages or Na(+)-channels have Ervs >19. Toxins affecting potassium channels or insect NaV channels have ERvs <10. Fractions including curarizing or antineoplasic peptides had ERvs <1. Erv increases in proportion to mammalian toxin toxicity rather than to toxin molecular mass.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Fragmentos Fab das Imunoglobulinas/imunologia , Venenos de Escorpião/análise , Animais , Cavalos , Peso Molecular , Venenos de Escorpião/imunologiaRESUMO
Using phage display and directed evolution, our group has progressed in the construction of a second family of human single chain variable fragments (scFv) which bind to scorpion toxins dangerous to mammals. It was observed that scFv C1 only bound initially to toxin Cn2, which constitutes 6.8% of whole venom from the scorpion Centruroides noxius Hoffman. Only a few amino acid changes were necessary to extend its recognition to other similar toxins and without affecting the recognition for its primary antigen (Cn2 toxin). One variant of scFv C1 (scFv 202F) was selected after two cycles of directed evolution against Cll1 toxin, the second major toxic component from the venom of the Mexican scorpion Centruroides limpidus limpidus Karsh (0.5% of the whole venom). scFv 202F is also capable of recognizing Cn2 toxin. Despite not having the highest affinity for toxins Cll1 (KD = 25.1 × 10(-9) M) or Cn2 (KD = 8.1 × 10(-9) M), this antibody fragment neutralized one LD50 of each one of these toxins. Additionally, scFv 202F moderately recognized Cll2 toxin which constitutes 1.5% of the venom from C. limpidus. Based on our previous experience, we consider that these results are promising; consequently, we continue working on generating new optimized variants from scFv C1 that could be part of a recombinant scorpion anti-venom from human origin, that might reach the market in the near future.
Assuntos
Anticorpos Neutralizantes/química , Fragmentos de Imunoglobulinas/química , Proteínas Recombinantes/química , Venenos de Escorpião/imunologia , Sequência de Aminoácidos , Antivenenos/biossíntese , Técnicas de Visualização da Superfície Celular , Evolução Molecular Direcionada , Ensaio de Imunoadsorção Enzimática , Humanos , México , Dados de Sequência Molecular , Venenos de Escorpião/química , Alinhamento de Sequência , Ressonância de Plasmônio de SuperfícieRESUMO
In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNFα, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antivenenos/imunologia , Proteínas de Insetos/imunologia , Venenos de Escorpião/imunologia , Escorpiões/fisiologia , Anticorpos de Cadeia Única/imunologia , Animais , Anticorpos Monoclonais Humanizados/biossíntese , Anticorpos Monoclonais Humanizados/uso terapêutico , Antivenenos/biossíntese , Antivenenos/uso terapêutico , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Proteínas de Insetos/antagonistas & inibidores , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Biblioteca de Peptídeos , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/toxicidade , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim, causing serious medical problems. Nanobodies (Nbs), the recombinant single-domain antigen-binding fragments of camel-specific heavy-chain only antibodies, offer special advantages in therapy over classic antibody fragments due to their robustness and smaller size, matching the size of the scorpion toxins. Recently, a potent AahII scorpion toxin-neutralizing Nb was identified. However, this NbAahII10 contains a single Cys in its first antigen-binding loop, leading to Nb dimerization upon prolonged storage. In this work, we first investigate the efficacy of NbAahII10 variants in which this Cys was substituted by Ala, Ser or Thr. Second, the NbAahII10 Cys/Ser mutant displaying the best functional properties is subsequently humanized. It is demonstrated that the maximally humanized version of NbAahII10 Cys/Ser maintains its high affinity for the antigen without conceding much on expression yield and stability. More importantly, its neutralizing capacity is preserved as all mice survive injections of seven LD(50) and 50% of mice survived nine LD(50) of the scorpion toxin. Thus, this humanized Nb is the best candidate to develop a therapy in human against the most toxic venom compound of one of the most dangerous scorpions.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Neutralizantes/imunologia , Cisteína/química , Venenos de Escorpião/imunologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/metabolismo , Afinidade de Anticorpos , Camelus , Cromatografia em Gel , Cisteína/genética , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Técnicas de Silenciamento de Genes , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Dose Letal Mediana , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Venenos de Escorpião/metabolismoRESUMO
OBJECTIVE: To study the effects of polypeptide extract from scorpion venom (PESV) on immune escape of Lewis lung carcinomas (LLC) and its mechanism. METHOD: Forty C57BL/6J mice were inoculated with LLC cells suspension (1 x 10(7) cells/ mL) in right armpit subcutaneously. The tumor-bearing mice were randomly divided into two groups: the control group and the PESV group. PESV was intragastrically subjected to the mice of the experimental group for 18 days. The tumor volume and tumor inhibitory rate were determined. The expression levels of VEGF,TGF-beta1 and IL-10 in tumor microenvironment were determined by immunohisto-chemistry-staining and ELISA. Surface co-stimulatory molecules CD80 and CD86 of tumor infiltrating dendritic cells (DC) were determined by immunohistochemistry-staining and flow cytometry. RESULT: The growth inhibitory rate of PESV was 56. 60%. The expression levels of VEGF,TGF-beta1 and IL-10 were decreased in tumor and serum, while the expression of co-stimulatory molecules CD80 and CD86 on DC were increased in tumor. Compared with the control group, the differences were all significant (P < 6.05). CONCLUSION: PESV was effective in recovering immuno-surveillance and intervening immune escape of lung cancer through multi-pathway. And its effects might be attained by decreasing the level of VEGF, TGF-beta1 and IL-10 in tumor microenvironment and increasing the expression of co-stimulatory molecules CD80 and CD86 on DC.
Assuntos
Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Peptídeos/administração & dosagem , Venenos de Escorpião/química , Evasão Tumoral/efeitos dos fármacos , Animais , Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Modelos Animais de Doenças , Humanos , Interleucina-10/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Venenos de Escorpião/imunologiaRESUMO
Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patient, causes serious medical problems. Nanobodies, single-domain antigen-binding fragments derived from dromedary heavy-chain antibodies, have a size that closely matches that of scorpion toxins. Therefore these nanobodies might be developed into potent immunotherapeutics to treat scorpion envenoming. Multiple nanobodies of sub-nanomolar affinity to AahII, the most toxic polypeptide within the Androctonus australis hector venom, were isolated from a dromedary immunized with AahII. These nanobodies neutralize the lethal effect of AahII to various extents without clear correlation with the kinetic rate constants kon or koff, or the equilibrium dissociation constant, KD. One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37000 LD50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.
Assuntos
Anticorpos/imunologia , Camelus/imunologia , Neurotoxinas/imunologia , Peptídeos/imunologia , Venenos de Escorpião/imunologia , Escorpiões/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/uso terapêutico , Formação de Anticorpos , Especificidade de Anticorpos , Epitopos/química , Epitopos/imunologia , Feminino , Camundongos , Dados de Sequência Molecular , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/terapia , Neurotoxinas/química , Neurotoxinas/toxicidade , Peptídeos/química , Peptídeos/toxicidade , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Escorpiões/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Activation of the inflammatory response with the release and activation of pro-inflammatory cytokines is among the factors thought to be important in the pathogenesis of many deleterious inflammatory effects seen in case of scorpion envenomation. The released inflammatory mediators interact in the body with a large number of proteins and receptors; this interaction determines the eventual inflammatory effect of the venom. Thus, in the present study an attempt was made to map the time course of scorpion envenomation and correlate the effects observed on the cardiovascular and respiratory systems with the changes that could take place in the levels of selected cytokines and nitric oxide during the course of experimental envenomation. New Zealand white male conscious rabbits were prepared for blood pressure recording. Arterial blood pressure was measured from the left central ear artery while a cannula was inserted into the right central ear artery and blood samples collected at different time interval after venom injection for biochemical and hematological analyses. In general, subcutaneous injection of Leiurus quinquestriatus quinquestriatus venom caused a significant (P+/-0.05) triphasic effect on BP consisting of an initial transient reduction, followed by an increase that peaked 2h after venom injection, and a gradual terminal hypotensive phase. The significantly high serum level of IL8, TNFalpha (P<0.001) and nitric oxide (P<0.0001) observed in the present study supports the evidence for the role of these potent vasodilators in the terminal hypotension that is usually observed in humans and animals after envenomation.
Assuntos
Pressão Sanguínea/efeitos dos fármacos , Citocinas/imunologia , Óxido Nítrico/sangue , Venenos de Escorpião/toxicidade , Escorpiões , Animais , Glicemia , Peso Corporal/efeitos dos fármacos , Estado de Consciência , Interleucina-8/sangue , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Tamanho do Órgão , Coelhos , Venenos de Escorpião/imunologia , Soro/química , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangueRESUMO
KAaH1 and KAaH2 are non-toxic peptides, isolated from the venom of the Androctonus australis hector (Aah) scorpion. In a previous study, we showed these peptides to be the most abundant (approximately 10% each) in the toxic fraction (AahG50) of the Aah venom. KAaH1 and KAaH2 showed high sequence identities (approximately 60%) with birtoxin-like peptides, which likewise are the major peptidic components of Parabuthus transvaalicus scorpion venom. Here, we report the immunological characterization of KAaH1 and KAaH2. These peptides were found to be specifically recognized by polyclonal antibodies raised against AahII, the most toxic peptide of Aah venom, and represents the second antigenic group, including toxins from different scorpion species in the world. Moreover, KAaH1 partially inhibits AahII binding to its specific antibody, suggesting some common epitopes between these two peptides. The identification of possible key antigenic residues in KAaH1 was deduced from comparison of its 3-D model with the experimental structure of AahII. Two clusters of putative antigenically important residues were found at the exposed surface; one could be constituted of V3 and D53, the other of D10, T15 and Y16. Polyclonal antibodies raised against KAaH1 in mice were found to cross-react with both AahII and AahG50, and neutralizing 5LD(50)/ml of the toxic fraction. Mice vaccinated with KAaH1 were protected against a challenge of 2LD(50) of AahG50 fraction. All these data suggest that KAaH1 has clear advantages over the use of the whole or part of the venom. KAaH1 is not toxic and could produce sera-neutralizing scorpion toxins, not only from Aah venom, but also toxins of other venoms from Buthus, Leiurus, or Parabuthus scorpion species presenting antigenically related toxins.
Assuntos
Peptídeos/imunologia , Peptídeos/farmacologia , Venenos de Escorpião/antagonistas & inibidores , Venenos de Escorpião/toxicidade , Escorpiões , Sequência de Aminoácidos , Animais , Epitopos/química , Epitopos/imunologia , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Venenos de Escorpião/química , Venenos de Escorpião/imunologiaRESUMO
The nucleotide sequence of a type of chlorotoxin-like peptide, an inhibitor of small-conductance Cl(-) channels, from the scorpion, Buthus martensii Karsch, was synthesized (named rBmK CTa) according to the sequence optimized for codon usage in E. coli. It was over-expressed using a pExSecI expression system and purified to homogeneity. Polycolonal antibodies to the purified protein were raised in rats. Overlay assay and pull-down assay showed that this toxin specially binds to two proteins in the glioma cells with corresponding molecular weights of about 80 and 35 kDa. They may serve as candidate receptors or alternative cellular component for interaction with rBmK CTa.
Assuntos
Receptores de Droga/metabolismo , Venenos de Escorpião/imunologia , Animais , Formação de Anticorpos , Western Blotting , Linhagem Celular Tumoral , Glioma/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Ratos , Venenos de Escorpião/metabolismo , EscorpiõesRESUMO
In this study, we have used Amm VIII, a natural anatoxin from the scorpion Androctonus mauretanicus mauretanicus, to elicit specific polyclonal antibodies in rabbit. Using liquid-phase radioimmunoassay, we have studied its selectivity and its neutralizing activity both in vitro and in vivo for the most lethal scorpion alpha-toxins described, in particular the alpha-toxin of reference AaH II. We have shown that the anti-Amm VIII serum prevents the association of 125I-AaH II with its receptor and is able to remove 125I-AaH II already bound to its site (the half-life of the complex 125I-AaH II-receptor site was 12 min in the absence of anti-Amm VIII serum but decreased to only 2 min in the presence of anti-Amm VIII serum). In vivo, the serum also has a protective effect in mice: 42 LD50 of AaH II by millilitre are neutralized, measured by subcutaneous injection.
Assuntos
Anticorpos/farmacologia , Venenos de Escorpião/antagonistas & inibidores , Escorpiões/química , Animais , Anticorpos/imunologia , Técnicas Imunoenzimáticas , Testes de Neutralização , Coelhos , Venenos de Escorpião/genética , Venenos de Escorpião/imunologia , Toxoides/imunologia , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Overlapping pentadecapeptides covering the complete amino acid sequence of TsII, TsVII and TsIV toxins from the venom of scorpion Tityus serrulatus (Ts), were prepared by use of the Spot method of multiple peptide synthesis. Horse anti-Ts antisera for therapeutic use were tested for their binding to peptides. All nine antisera tested showed reactivity with several peptides from the three toxins. Three antigenic regions, one in the very N-terminal, the second in the central part and the other in the C-terminal part of the three toxins were frequently, but not constantly recognized, with an intensity that seemed to be related to the neutralizing potency of the tested antivenom. Thus the corresponding peptides (residues 1-15 and 48-62 of TsII; residues 1-15, 16-30 and 48-62 of TsIV and residues 1-15 and 47-61 of TsVII) were synthesized, coupled to KLH and used as antigens to coat the microtitration plates to determine any relationship between their ELISA reactivity with therapeutic horse antivenoms and the neutralizing potential of these antivenoms. The mixture of the N-terminal peptide of TsII, of the N-terminal TsVII peptide and of the C-terminal of TsIV was found to give a linear relationship with the neutralizing titer of horse serum of low neutralizing potency (< or =1 mg/ml). However, high neutralizing antivenoms did not show the expected response in peptide ELISA. This observation is discussed in the context of the occurrence of continuous and discontinuous epitopes on toxins.
Assuntos
Epitopos/genética , Soros Imunes/imunologia , Soros Imunes/metabolismo , Peptídeos/metabolismo , Venenos de Escorpião/genética , Escorpiões/química , Sequência de Aminoácidos , Animais , Ensaio de Imunoadsorção Enzimática , Cavalos/sangue , Soros Imunes/genética , Imunoensaio , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/genética , Venenos de Escorpião/imunologia , Escorpiões/genéticaRESUMO
BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.