RESUMO
Polybia paulista is a neotropical social wasp related to severe accidents and allergic reactions cases, including anaphylaxis, in southeastern Brazil. Antigen 5 (Poly p 5) is a major allergenic protein from its venom with potential use for component-resolved diagnostic. Therefore, the previous characterization of the immune response profile triggered by Poly p 5 should be evaluated. Recombinant Poly p 5 (rPoly p 5) was used to sensitize BALB/c mice with six weekly intradermal doses, and the specific antibody production and the functional profile of CD4+ T cells were assessed. rPoly p 5 induced the production of specific immunoglobulins (sIg) sIgE, sIgG1 and sIgG2a, which could recognize natural Poly p 5 presented in the venom of four different wasp species. rPoly p 5 stimulated in vitro the CD4+ T cells from immunized mice, which showed a significant proliferative response. These antigen-specific CD4+T cells produced IFN-γ and IL-17A cytokines and increased ROR-γT transcription factor expression. No differences between the control group and sensitized mice were found in IL-4 production and GATA-3 and T-bet expression. Interestingly, increased CD25+FoxP3+ regulatory T cells (Tregs) frequency was observed in the splenocyte cell cultures from rPoly p 5 immunized mice after the in vitro stimulation with both P. paulista venom extract and rPoly p 5. Here we showed that rPoly p 5 induces antigen-specific antibodies capable of recognizing Antigen 5 in the venom of four wasp species and modulates antigen-specific CD4+ T cells to IFN-γ production response associated with a Th17 profile in sensitized mice. These findings emphasize the potential use of rPoly p 5 as an essential source of a major wasp allergen with significant immunological properties.
Assuntos
Anafilaxia , Vespas , Animais , Camundongos , Vespas/metabolismo , Venenos de Vespas/metabolismo , Formação de Anticorpos , Alérgenos , Linfócitos T CD4-PositivosRESUMO
As a result of environmental change by anthropic action, animal species that inhabit these areas may suffer the effects of it on their phenotypes as a consequence of adapting to these conditions. In the case of social wasps, cuticular chemical compounds may be influenced, since these vary depending on genetic and environmental factors. However, few studies have investigated the synanthropic effects over the cuticular surface of social wasps. Therefore, the aim of this study was to investigate how cuticular compounds vary according to the different degrees of human activity and test the hypothesis that cuticular compounds of social wasps are affected by the level of anthropic activity in which their nests are found. Data on the cuticular chemical compounds composition of colonies of 3 species of social wasps were used along with the level of anthropization of their nesting sites in four municipalities in the state of Mato Grosso do Sul, Brazil. From the geographical coordinates of the sampling sites, the percentages of urban construction areas, agriculture, water body, vegetation and exposed land were calculated, and the nesting sites of the colonies were classified as more or less anthropized areas. The chemical profile was determined by extraction of cuticular compounds and analyzed by Gas Chromatography coupled to Mass Spectrometer (GC-MS). The results show that the cuticular chemical composition of the individuals of these species is affected by the level of anthropization in their nesting sites, with a qualitative and quantitative variation that must be tied not only to genetic differences, but, above all, to the local environmental conditions to which their colonies are subjected.(AU)
Assuntos
Animais , Fenótipo , Vespas , Cromatografia Gasosa , Compostos Químicos , Atividades HumanasRESUMO
The social wasp Polybia paulista (Hymenoptera, Vespidae) is highly aggressive, being responsible for many medical occurrences. One of the most allergenic components of this venom is Antigen 5 (Poly p 5). The possible modulation of the in vitro immune response induced by antigen 5 from P. paulista venom, expressed recombinantly (rPoly p 5), on BALB/c mice peritoneal macrophages, activated or not with LPS, was assessed. Here, we analyzed cell viability changes, expression of the phosphorylated form of p65 NF-κB subunit, nitric oxide (NO), proinflammatory cytokines production, and co-stimulatory molecules (CD80, CD86). The results suggest that rPoly p 5 does not affect NO production nor the expression of co-stimulatory molecules in mouse peritoneal macrophages. On the other hand, rPoly p 5 induced an increase in IL-1ß production in non-activated macrophages and a reduction in the production of TNF-α and MCP-1 cytokines in activated macrophages. rPoly p 5 decreased the in vitro production of the phosphorylated p65 NF-κB subunit in non-activated macrophages. These findings suggest an essential role of this allergen in the polarization of functional M2 macrophage phenotypes, when analyzed in previously activated macrophages. Further investigations, mainly in in vivo studies, should be conducted to elucidate Polybia paulista Ag5 biological role in the macrophage functional profile modulation.
Assuntos
Antígenos/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Venenos de Vespas/química , Vespas/fisiologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico , Fosforilação , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Venenos de Vespas/toxicidadeRESUMO
Allergic reactions to Hymenoptera venom, which could lead to systemic and even fatal symptoms, is characterized by hypersensitivity reactions mediated by specific IgE (sIgE) driven to venom allergens. Patients multisensitized to sIgE usually recognize more than one allergen in different Hymenoptera species. However, the presence of sIgE directed against Cross-Reactive Carbohydrate Determinant (CCD), which occurs in some allergens from Hymenoptera venom, hampers the identification of the culprit insects. CCD is also present in plants, pollen, fruits, but not in mammals. Bromelain (Brl) extracted from pineapples is a glycoprotein commonly used for reference to sIgE-CCD detection and analysis. In sera of fifty-one Hymenoptera allergic patients with specific IgE ≥ 1.0 KU/L, we assessed by immunoblotting the reactivity of sIgE to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. We also distinguished, using sera adsorption procedures, the cases of CCD cross-reaction using Brl as a marker and inhibitor of CCD epitopes. The presence of reactivity for bromelain (24-28 kDa) was obtained in 43% of the patients, in which 64% presented reactivity for more than one Hymenoptera venom in radioallergosorbent (RAST) tests, and 90% showed reactivity in immunoblot analysis to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. Sera adsorption procedures with Brl lead to a significant reduction in patients' sera reactivity to the Hymenoptera allergens. Immunoblotting assay using pre- and post-Brl adsorption sera from wasp-allergic patients blotted with non-glycosylated recombinant antigens (rPoly p1, rPoly p5) from Polybia paulista wasp venom showed no change in reactivity pattern of sIgE that recognize allergen peptide epitopes. Our results, using Brl as a marker and CCD inhibitor to test sIgE reactivity, suggest that it could complement diagnostic methods and help to differentiate specific reactivity to allergens' peptide epitopes from cross-reactivity caused by CCD, which is extremely useful in clinical practice.
Assuntos
Alérgenos/imunologia , Venenos de Formiga/imunologia , Venenos de Abelha/imunologia , Carboidratos/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Mordeduras e Picadas de Insetos/imunologia , Venenos de Vespas/imunologia , Adolescente , Adulto , Especificidade de Anticorpos , Bromelaínas/imunologia , Criança , Pré-Escolar , Reações Cruzadas , Epitopos , Feminino , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/diagnóstico , Imunoglobulina E/sangue , Testes Imunológicos , Mordeduras e Picadas de Insetos/sangue , Mordeduras e Picadas de Insetos/diagnóstico , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Adulto JovemRESUMO
Insect venom can cause systemic allergic reactions, including anaphylaxis. Improvements in diagnosis and venom immunotherapy (VIT) are based on a better understanding of an immunological response triggered by venom allergens. Previously, we demonstrated that the recombinant phospholipase A1 (rPoly p 1) from Polybia paulista wasp venom induces specific IgE and IgG antibodies in sensitized mice, which recognized the native allergen. Here, we addressed the T cell immune response of rPoly p 1-sensitized BALB/c mice. Cultures of splenocytes were stimulated with Polybia paulista venom extract and the proliferation of CD8+ and CD4+ T cells and the frequency of T regulatory cells (Tregs) populations were assessed by flow cytometry. Cytokines were quantified in cell culture supernatants in ELISA assays. The in vitro stimulation of T cells from sensitized mice induces a significant proliferation of CD4+ T cells, but not of CD8+ T cells. The cytokine pattern showed a high concentration of IFN-γ and IL-6, and no significant differences to IL-4, IL-1ß and TGF-ß1 production. In addition, the rPoly p 1 group showed a pronounced expansion of CD4+CD25+FoxP3+ and CD4+CD25-FoxP3+ Tregs. rPoly p 1 sensitization induces a Th1/Treg profile in CD4+ T cell subset, suggesting its potential use in wasp venom immunotherapy.
Assuntos
Alérgenos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Dessensibilização Imunológica , Proteínas de Insetos/farmacologia , Fosfolipases A1/farmacologia , Venenos de Vespas/farmacologia , Alérgenos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Feminino , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Hipersensibilidade/terapia , Mordeduras e Picadas de Insetos/imunologia , Mordeduras e Picadas de Insetos/metabolismo , Mordeduras e Picadas de Insetos/terapia , Proteínas de Insetos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Fosfolipases A1/imunologia , Venenos de Vespas/imunologiaRESUMO
Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate-phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy.
RESUMO
Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.
Assuntos
Alérgenos , Venenos de Vespas/química , Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Alérgenos/isolamento & purificação , Humanos , Hipersensibilidade/diagnóstico , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Modelos Moleculares , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Venenos de Vespas/genética , Venenos de Vespas/imunologia , Venenos de Vespas/isolamento & purificaçãoRESUMO
Anxiety disorders are major health problems in terms of costs stemming from sick leave, disabilities, healthcare and premature mortality. Despite the availability of classic anxiolytics, some anxiety disorders are still resistant to treatment, with higher rates of adverse effects. In this respect, several toxins isolated from arthropod venoms are useful in identifying new compounds to treat neurological disorders, particularly pathological anxiety. Thus, the aims of this study were to identify and characterize an anxiolytic peptide isolated from the venom of the social wasp Polybia paulista. The peptide was identified as Polisteskinin R, with nominal molecular mass [M+H](+)=1301Da and primary structure consisting of Ala-Arg-Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg-OH. The anxiolytic effect was tested using the elevated plus maze test. Moreover, adverse effects on the spontaneous behavior and motor coordination of animals were assessed using the open field and rotarod tests. Polisteskinin R induced a dose-dependent anxiolytic effect. Animals treated with the peptide and diazepam spent significantly more time into the open arms when compared to the groups treated with the vehicle and pentylenetetrazole. No significant differences in spontaneous behavior or motor coordination were observed between the groups, showing that the peptide was well tolerated. The interaction by agonists in both known BK receptors induces a variability of physiological effects; Polisteskinin R can act on these receptors, inducing modulatory activity and thus, attenuating anxiety behaviors. The results of this study demonstrated that the compound Polisteskinin R exerted potent anxiolytic effects and its analogues are promising candidates for experimental pharmacology.
Assuntos
Ansiolíticos/uso terapêutico , Ansiedade/tratamento farmacológico , Bradicinina/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Venenos de Vespas/uso terapêutico , Animais , Ansiolíticos/efeitos adversos , Ansiolíticos/isolamento & purificação , Ansiedade/psicologia , Bradicinina/efeitos adversos , Bradicinina/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Masculino , Fragmentos de Peptídeos/efeitos adversos , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Ratos Wistar , Venenos de Vespas/efeitos adversos , Venenos de Vespas/isolamento & purificaçãoRESUMO
To date, there are no allergenic extracts or components available in Brazil to diagnosis and treatment of patients with venom allergy from social wasp (Vespidae Family; Polistinae Subfamily) despite of the great number of existing species. We evaluated the immunogenic potential of the Hyal recombinant protein (Pp-Hyal-rec) which was expressed in an insoluble form in comparison with the allergenic native protein (Pp-Hyal-nat) for recognition of immunoglobulin E (IgE) in the serum of allergic patients to venom of the endemic social wasp Polybia paulista from São Paulo State, Brazil. Hyal cDNA from the venom of the social wasp P. paulista (Pp-Hyal) (GI: 302201582) was cloned into the expression vector pET-28a in Escherichia coli DE3 (BL21) cells. Solubilization and purification of Pp-Hyal-rec from inclusion bodies were performed using Ni(2+) affinity chromatography (Ni-NTA-Agarose) under denaturing conditions. Both the native (Pp-Hyal-nat) and the recombinant (Pp-Hyal-rec) purified allergens were used for Western blotting to assess the levels of Pp-Hyal-IgE specific in the serum of 10 patients exclusively reactive to the venom of the social wasp P. paulista. The immune sera specifically recognized the band corresponding to the Pp-Hyal-rec protein (40 kDa) at a higher intensity than the native allergen (39 kDa). The sera recognized other proteins in P. paulista crude venom extract to a lesser extent, likely corresponding to other venom allergens such as phospholipase (34 kDa), Antigen 5 (25 kDa), and proteases. The recognition pattern of the immune sera to the Pp-Hyal-rec allergen strongly suggests that this recombinant antigen could be used for developing a diagnostic allergy test as well as for specific immunotherapy (IT).
Assuntos
Alérgenos/genética , Alérgenos/imunologia , Hialuronoglucosaminidase/imunologia , Imunoglobulina E/imunologia , Venenos de Vespas/enzimologia , Venenos de Vespas/imunologia , Vespas/imunologia , Animais , Especificidade de Anticorpos , Clonagem Molecular , Reações Cruzadas , Humanos , Hipersensibilidade/imunologia , Corpos de Inclusão/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Venenos de Vespas/genéticaRESUMO
The venoms of the social wasps evolved to be used as defensive tools to protect the colonies of these insects against the attacks of predators. Previous studies estimated the presence of a dozen peptide components in the venoms of each species of these insects, which altogether comprise up to 70% of the weight of freeze-dried venoms. In the present study, an optimized experimental protocol is reported that utilizes liquid chromatography coupled to electrospray ionization mass spectrometry for the detection of peptides in the venom of the social wasp Polybia paulista; peptide profiles for both intra- and inter-colonial comparisons were obtained using this protocol. The results of our study revealed a surprisingly high level of intra- and inter-colonial variability for the same wasp species. We detected 78-108 different peptides in the venom of different colonies of P. paulista in the molar mass range from 400 to 3000Da; among those, only 36 and 44 common peptides were observed in the inter- and intra-colony comparisons, respectively.