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Made available in DSpace on 2022-04-29T08:37:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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-1β 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. Laboratório de Biologia Molecular de Artrópodes—LBMA Instituto de Biologia—IB Universidade Estadual Paulista Câmpus Rio Claro—UNESP-RC, Avenida 24-A, n◦1515, Bela Vista Laboratório de Imunologia Translacional—LIT Departamento de Clínica Médica Faculdade de Ciências Médicas—FCM Universidade Estadual de Campinas—UNICAMP Cidade Universitária “Zeferino Vaz”, Rua Tessália Vieira de Camargo, n◦126 Departamento de Biomedicina Faculdade de Americana—FAM, Avenida Joaquim Bôer, n◦733, Jardim Luciane Faculdade de Medicina Universidade Anhembi Morumbi, Av. Rio das Pedras, 1601-Pompéia Laboratório de Biologia Molecular de Artrópodes—LBMA Instituto de Biologia—IB Universidade Estadual Paulista Câmpus Rio Claro—UNESP-RC, Avenida 24-A, n◦1515, Bela Vista FAPESP: 2017/07988-2 FAPESP: 2017/18422-0 FAPESP: 2019/02298-3

Background: Exercise-induced bronchoconstriction (EIB) is a common cause of breathing difficulty in athletes. Fractional exhaled nitric oxide (FeNO) is an indirect marker of airway inflammation, recommended for the assessment and management of asthma; however, the role of FeNO in detecting and monitoring EIB has yet to be fully established. The aim of this study was therefore to evaluate the predictive value of FeNO to confirm or refute EIB in athletes presenting with exertional dyspnoea. Method: Seventy athletes (male: n = 45) (age: 35 ± 11 years) reporting respiratory symptoms (i.e. wheeze, cough and dyspnoea) during exercise attended the laboratory on a single occasion. All athletes performed resting FeNO and spirometry pre-and-post a eucapnic voluntary hyperpnoea challenge (EVH) in accordance with international guidelines. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated for established FeNO thresholds: (intermediate [≥25ppb] and high [>50ppb]) and evaluated against objective evidence of EIB: (EVH diagnostic cut-off [-10% ΔFEV1 at two consecutive time-points] and [-15% ΔFEV1 at one time-point]). The diagnostic accuracy of FeNO was calculated using receiver operating characteristics area under the curve (ROC-AUC). Results: All had normal resting lung function (>80% FEV1 pred). The prevalence of EIB was 33% (-10% ΔFEV1) and 23% (-15% ΔFEV1) (median (IQR) ΔFEV1 = -7% (10.02)). FeNO values ≥25ppb and >50ppb were observed in 49% and 23% of the cohort, respectively. ROC-AUC for FeNO was 75% (-10% ΔFEV1) and 86% (-15% ΔFEV1). Sensitivity, specificity, PPV and NPV are presented in Table 1. Conclusion: Our findings indicate that FeNO >50ppb provides good specificity for a positive EVH test; however, should not replace indirect bronchoprovocation for diagnostic purposes. The high proportion of athletes reporting breathing difficulty in the absence of EIB highlights the requirement to consider alternative causes of exertional dyspnoea during clinical work-up.

Made available in DSpace on 2020-12-12T02:04:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Center for the Study of Social Insects Department of General and Applied Biology University of Sao Paulo State (UNESP/RC) Center for Genetic Engineering and Biotechnology. Biomedical Research Division System Biology Department, Ave. 31, e/ 158 and 190, Cubanacan, Playa, P.O. Box 6162 Laboratory of Translational Immunology School of Medical Sciences University of Campinas (UNICAMP), Campinas Arthropod Molecular Biology Laboratory-LBMA-IB University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela Vista Venoms and Venomous Animal Studies Center-CEVAP University of São Paulo State (UNESP) Lageado Experimental Farm, José Barbosa de Barros Street, 1780 Center for the Study of Social Insects Department of General and Applied Biology University of Sao Paulo State (UNESP/RC) Arthropod Molecular Biology Laboratory-LBMA-IB University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela Vista Venoms and Venomous Animal Studies Center-CEVAP University of São Paulo State (UNESP) Lageado Experimental Farm, José Barbosa de Barros Street, 1780 FAPESP: # 2014/13936-7

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02202-8) contains supplementary material, which is available to authorized users.

Purpose of Review Stings of Hymenoptera of the superfamily Vespoidea such as yellow jackets, paper wasps or stinging ants are common triggers for severe and even fatal allergic reactions. Antigen 5 allergens are potent allergens in the majority of these venoms with major importance for diagnosis and therapy. Reviewed here are the characteristics of antigen 5 allergens, their role in component-resolved diagnostics as well as current limitations of the available diagnostics for proper therapeutic decisions. Recent Findings Antigens 5 are proteins of unknown function in Hymenoptera venoms with high allergenic potency. They represent key elements in component-resolved diagnosis to discriminate between honeybee and vespid venom allergy. However, due to their pronounced cross-reactivity, there are remaining diagnostic and therapeutic challenges that have to be addressed. Summary Antigens 5 are highly relevant venom allergens of the Vespoidea superfamily. Although their use in component-resolved diagnosis facilitates dissection of cross-reactivity and primary allergy in double sensitization to honeybee and vespid venom, new diagnostic concepts are needed to discriminate between allergies to different vespid species.

Made available in DSpace on 2020-12-12T02:10:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 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- and IL-6, and no significant differences to IL-4, IL-1 and TGF- 1 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. Laboratory of Translational Immunology Medicine School FCM University of Campinas Center of the Study of Social Insects Department of General and Applied Biology Institute of Biosciences of Rio Claro Institute of Biosciences of Rio Claro S o Paulo State University Laboratory of Arthropods Molecular Biology IBRC UNESP S o Paulo State University Venoms and Venomous Animal Studies Center CEVAP S o Paulo State University Laboratory of Arthropods Molecular Biology IBRC UNESP S o Paulo State University

Although systemic reactions caused by allergenic proteins present in venoms affect a small part of the world population, Hymenoptera stings are among the main causes of immediate hypersensitivity responses, with risk of anaphylactic shock. In the attempt to obtain therapeutic treatments and prophylaxis to hypersensitivity responses, interest in the molecular characterization of these allergens has grown in the scientific community due to the promising results obtained in immunological and clinical studies. The present review provides an update on the knowledge regarding the immune response and the therapeutic potential of Antigen 5 derived from Hymenoptera venom. The results confirm that the identification and topology of epitopes, associated with molecular regions that interact with antibodies, are crucial to the improvement of hypersensitivity diagnostic methods.

Made available in DSpace on 2018-12-11T17:07:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-15 Polybia paulista (Hymenoptera: Vespidae) is a clinically relevant social wasp that frequently causes stinging accidents in southeast Brazil. To date, diagnosis and specific immunotherapy (SIT) of allergy are based on the use of crude venom extracts. Production of recombinant forms of major allergens from P. paulista venom will improve diagnosis and SIT of allergic patients by reducing the incidence of cross-reactivity and non-specific sensitization. Here, we describe the molecular cloning, heterologous expression, purification and IgE-mediated immunodetection of phospholipase A1 (Poly p 1), a major allergen from P. paulista venom. The cDNA of Poly p 1 was extracted from venom glands and then cloned, and further expression of the recombinant allergen (rPoly p 1) was achieved in Escherichia coli BL21 (DE3) cells. Purification of rPoly p 1 was performed using immobilized Ni2+ metal affinity chromatography. Also, a single-step chromatographic method allowed the purification of native Poly p 1 (nPoly p 1) from the wasp's venom glands. We used western blotting to evaluate IgE-reactivity of the sera from 10 P. paulista venom-allergic patients to rPoly p 1 and nPoly p 1. High levels of insoluble rPoly p 1 were obtained during heterologous expression. After solubilization of inclusion bodies and purification of the recombinant protein, a unique band of ∼34 kDa was detected in SDS-PAGE analysis. Allergen-specific IgE (sIgE) from allergic patients' sera recognized rPoly p 1, nPoly p 1 and crude venom extract to a similar extent. Our results showed that rPoly p 1 could be used for development of component-resolved diagnosis (CRD) and molecular-defined SIT of P. paulista venom allergy. Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, Bela Vista, CEP Laboratório de Mutagênese Ambiental, Avenida 24-A, nº 1515, Bela Vista, CEP Center for Genetic Engineering and Biotechnology Biomedical Research Division System Biology Department, Ave. 31, e/ 158 and 190, P.O. Box 6162, Cubanacan Laboratório de Imunologia Translacional Faculdade de Ciências Médicas FCM Universidade Estadual de Campinas-UNICAMP, Rua Vital Brasil, nº 300, Cidade Universitária “Zeferino Vaz”, CEP Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, Bela Vista, CEP Instituto de Pesquisa em Bioenergia (IPBEN) (Univ Estadual Paulista), Av. 24-A, nº1515, Bela Vista, CEP Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, Bela Vista, CEP Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, Bela Vista, CEP Instituto de Pesquisa em Bioenergia (IPBEN) (Univ Estadual Paulista), Av. 24-A, nº1515, Bela Vista, CEP Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado

Made available in DSpace on 2018-12-11T17:14:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 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. Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela Vista Laboratório de Imunologia Translacional-LIT Departamento de Clínica Médica Faculdade de Ciências Médicas FCM Universidade Estadual de Campinas-UNICAMP, Rua Tessália Vieira de Camargo n◦ 126, Cidade Universitária “Zeferino Vaz” System Biology Department Biomedical Research Division Center for Genetic Engineering and Biotechnology, Ave. 31, e/158 and 190, P.O. Box 6162, Cubanacan Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP Univ Estadual Paulista, Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela Vista Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP Univ Estadual Paulista, Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado FAPESP: 2013/26451-9 FAPESP: 2014/13936-7 CNPq: 455422/2014-1