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1.
Handb Exp Pharmacol ; 268: 249-264, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34196808

RESUMO

B cells are key players in the mechanisms underlying allergic sensitization, allergic reactions, and tolerance to allergens. Allergen-specific immune responses are initiated when peptide:MHCII complexes on dendritic cells are recognized by antigen-specific receptors on T cells followed by interactions between costimulatory molecules on the surfaces of B and T cells. In the presence of IL-4, such T-B cell interactions result in clonal expansion and isotype class-switching to IgE in B cells, which will further differentiate into either memory B cells or PCs. Allergic reactions are then triggered upon cross-linking of IgE-FcɛRI complexes on basophils and mast cells, leading to cell degranulation and the release of pro-inflammatory mediators.Mechanisms underlying effective allergen-specific immunotherapy (AIT) involve the induction of Tregs and the secretion of blocking IgG4 antibodies, which together mediate the onset and maintenance of immune tolerance towards non-hazardous environmental antigens. However, the importance of regulatory B cells (Breg) for tolerance induction during AIT has gained more attention lately. Studies in grass pollen- and house dust mite-allergic patients undergoing SCIT reported increased frequencies of IL-10+ Breg cells and a positive correlation between their number and the improvement of clinical symptoms. Thus, Breg are emerging as biomarkers for monitoring tolerance to allergens under natural exposure conditions and during AIT. Further research on the role of other anti-inflammatory cytokines secreted by Breg will help to understand their role in disease development and tolerance induction.


Assuntos
Dessensibilização Imunológica , Hipersensibilidade , Alérgenos , Linfócitos B , Humanos , Hipersensibilidade/terapia , Tolerância Imunológica
2.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639235

RESUMO

Nanomaterials have found extensive interest in the development of novel vaccines, as adjuvants and/or carriers in vaccination platforms. Conjugation of protein antigens at the particle surface by non-covalent adsorption is the most widely used approach in licensed particulate vaccines. Hence, it is essential to understand proteins' structural integrity at the material interface in order to develop safe-by-design nanovaccines. In this study, we utilized two model proteins, the wild-type allergen Bet v 1 and its hypoallergenic fold variant (BM4), to compare SiO2 nanoparticles with Alhydrogel® as particulate systems. A set of biophysical and functional assays including circular dichroism spectroscopy and proteolytic degradation was used to examine the antigens' structural integrity at the material interface. Conjugation of both biomolecules to the particulate systems decreased their proteolytic stability. However, we observed qualitative and quantitative differences in antigen processing concomitant with differences in their fold stability. These changes further led to an alteration in IgE epitope recognition. Here, we propose a toolbox of biophysical and functional in vitro assays for the suitability assessment of nanomaterials in the early stages of vaccine development. These tools will aid in safe-by-design innovations and allow fine-tuning the properties of nanoparticle candidates to shape a specific immune response.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Epitopos/imunologia , Ativação Linfocitária/imunologia , Nanopartículas/química , Dióxido de Silício/química , Vacinas/imunologia , Alérgenos/química , Humanos , Hidrogéis , Imunoglobulina E/imunologia , Hipersensibilidade Respiratória/imunologia , Linfócitos T/imunologia
3.
J Vis Exp ; (172)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34279512

RESUMO

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (FcεRI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of ß-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.


Assuntos
Alérgenos , Imunoglobulina E , Animais , Basófilos , Gatos , Degranulação Celular , Humanos , Pólen , Ratos
4.
Allergy ; 76(8): 2555-2564, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33724487

RESUMO

BACKGROUND: Evidence has accumulated that birch pollen immunotherapy reduces rhinoconjunctivitis to pollen of birch homologous trees. Therapeutic efficacy has been associated with IgE-blocking IgG antibodies. We have recently shown that sera collected after 16 weeks of sublingual immunotherapy with recombinant Bet v 1 (rBet v 1-SLIT) display strong IgE-blocking bioactivity for Bet v 1. Here, we assessed whether rBet v 1-SLIT-induced IgG antibodies display cross-blocking activity to related allergens in Fagales pollen. METHODS: IgE, IgG1 and IgG4 reactivity to recombinant Bet v 1, Aln g 1, Car b 1, Ost c 1, Cor a 1, Fag s 1, Cas s 1 and Que a 1 were assessed in pre- and post-SLIT samples of 17 individuals by ELISA. A basophil inhibition assay using stripped basophils re-sensitized with a serum pool containing high Bet v 1-specific IgE levels was established and used to assess CD63 expression in response to allergens after incubation with pre-SLIT or post-SLIT samples. IgG1 and IgG4 were depleted from post-SLIT samples to assess its contribution to IgE-cross-blocking. RESULTS: Sublingual immunotherapy with recombinant Bet v 1 boosted cross-reactive IgE antibodies and induced IgG1 and IgG4 antibodies with inter- and intra-individually differing reactivity to the homologs. Highly variable cross-blocking activities of post-SLIT samples to the different allergens were found. IgG1 and IgG4 antibodies displayed cross-blocking activity with individual variance. CONCLUSIONS: Our mechanistic approach suggested that immunotherapy with the reference allergen Bet v 1 induces individual repertoires of cross-reactive IgG1 and IgG4 antibodies. The cross-blocking bioactivity of these antibodies was also highly variable and neither predictable from protein homology nor IgE-cross-reactivity.


Assuntos
Antígenos de Plantas/imunologia , Antígenos de Plantas/uso terapêutico , Imunoterapia Sublingual , Alérgenos , Anticorpos Bloqueadores , Fagales , Humanos , Imunoglobulina E , Proteínas de Plantas , Proteínas Recombinantes
5.
Allergy ; 76(6): 1718-1730, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33037672

RESUMO

BACKGROUND: Common ragweed has been spreading as a neophyte in Europe. Elevated CO2 levels, a hallmark of global climate change, have been shown to increase ragweed pollen production, but their effects on pollen allergenicity remain to be elucidated. METHODS: Ragweed was grown in climate-controlled chambers under normal (380 ppm, control) or elevated (700 ppm, based on RCP4.5 scenario) CO2 levels. Aqueous pollen extracts (RWE) from control- or CO2 -pollen were administered in vivo in a mouse model for allergic disease (daily for 3-11 days, n = 5) and employed in human in vitro systems of nasal epithelial cells (HNECs), monocyte-derived dendritic cells (DCs), and HNEC-DC co-cultures. Additionally, adjuvant factors and metabolites in control- and CO2 -RWE were investigated using ELISA and untargeted metabolomics. RESULTS: In vivo, CO2 -RWE induced stronger allergic lung inflammation compared to control-RWE, as indicated by lung inflammatory cell infiltrate and mediators, mucus hypersecretion, and serum total IgE. In vitro, HNECs stimulated with RWE increased indistinctively the production of pro-inflammatory cytokines (IL-8, IL-1ß, and IL-6). In contrast, supernatants from CO2 -RWE-stimulated HNECs, compared to control-RWE-stimulated HNECS, significantly increased TNF and decreased IL-10 production in DCs. Comparable results were obtained by stimulating DCs directly with RWEs. The metabolome analysis revealed differential expression of secondary plant metabolites in control- vs CO2 -RWE. Mixes of these metabolites elicited similar responses in DCs as compared to respective RWEs. CONCLUSION: Our results indicate that elevated ambient CO2 levels elicit a stronger RWE-induced allergic response in vivo and in vitro and that RWE increased allergenicity depends on the interplay of multiple metabolites.


Assuntos
Ambrosia , Dióxido de Carbono , Alérgenos , Europa (Continente) , Pólen
6.
Int J Mol Sci ; 21(24)2020 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-33371317

RESUMO

To analyze the impact of Ascaris lumbricoides infection on the pathogenesis and diagnosis of allergic diseases, new allergens should be identified. We report the identification of a new Ascaris lumbricoides allergen, Asc l 5. The aim of this study was to evaluate the physicochemical and immunological features of the Asc l 5 allergen. We constructed an A. lumbricoides cDNA library and Asc l 5 was identified by immunoscreening. After purification, rAsc l 5 was physicochemically characterized. Evaluation of its allergenic activity included determination of Immunoglobulin E (IgE) binding frequency (in two populations: 254 children and 298 all-age subjects), CD203c based-basophil activation tests (BAT) and a passive cutaneous anaphylaxis (PCA) mouse model. We found by amino acid sequence analysis that Asc l 5 belongs to the SXP/RAL-2 protein family of nematodes. rAsc l 5 is a monomeric protein with an alpha-helical folding. IgE sensitization to rAsc l 5 was around 52% in general population; positive BAT rate was 60%. rAsc l 5 induced specific IgE production in mice and a positive PCA reaction. These results show that Asc l 5 has structural and immunological characteristics to be considered as a new allergen from A. lumbricoides.


Assuntos
Alérgenos/imunologia , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Ascaris lumbricoides/imunologia , Asma/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Asma/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Imunoglobulina E/sangue , Lactente , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
7.
Front Immunol ; 11: 2118, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013894

RESUMO

Allergic sensitization to the major allergen Bet v 1 represents the dominating factor inducing a vast variety of allergic symptoms in birch pollen allergic patients worldwide, including the pollen food allergy syndrome. In order to overcome the huge socio-economic burden associated with allergic diseases, allergen-specific immunotherapy (AIT) as a curative strategy to manage the disease was introduced. Still, many hurdles related to this treatment exist making AIT not the patients' first choice. To improve the current situation, the development of hypoallergen-based drug products has raised attention in the last decade. Herein, we investigated the efficacy of the novel AIT candidate BM4, a hypoallergenic variant of Bet v 1, to induce treatment-relevant cross-reactive Bet v 1-specific IgG antibodies in two different mammals, Wistar rats and New Zealand White rabbits. We further analyzed the cross-reactivity of BM4-induced Wistar rat antibodies with the birch pollen-associated food allergens Mal d 1 and Cor a 1, and the functional capability of the induced antibodies to act as IgE-blocking IgG antibodies. Enzyme-linked immunosorbent assay (ELISA) was used to determine the titers of rat IgG1, IgG2a, IgG2b, and IgE, as well as rabbit IgG and IgE antibodies. To address the functional relevance of the induced IgG antibodies, the capacity of rat sera to suppress binding of human IgE to Bet v 1 was investigated by using an inhibition ELISA and an IgE-facilitated allergen-binding inhibition assay. We found that the treatment with BM4 induced elevated Bet v 1-specific IgG antibody titers in both mammalian species. In Wistar rats, high BM4-specific IgG1, IgG2a, and IgG2b titers (104 to 106) were induced, which cross-reacted with wild-type Bet v 1, and the homologous allergens Mal d 1 and Cor a 1. Rat allergen-specific IgG antibodies sustained upon treatment discontinuation. Sera of rats immunized with BM4 were able to significantly suppress binding of human IgE to the wild-type allergens and CD23-mediated human IgE-facilitated Bet v 1 binding on B cells. By contrast, treatment-induced IgE antibody levels were low or undetectable. In summary, BM4 induced a robust IgG immune response that efficiently blocked human IgE-binding to wild-type allergens, underscoring its potential therapeutic value in AIT.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Betula/imunologia , Dessensibilização Imunológica , Imunoglobulina G/biossíntese , Coelhos/imunologia , Ratos Wistar/imunologia , Alérgenos/genética , Alérgenos/uso terapêutico , Substituição de Aminoácidos , Animais , Especificidade de Anticorpos , Reações Antígeno-Anticorpo/imunologia , Antígenos de Plantas/genética , Antígenos de Plantas/uso terapêutico , Betula/genética , Ligação Competitiva , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/genética , Epitopos/imunologia , Feminino , Engenharia Genética , Humanos , Imunização/métodos , Imunização Secundária , Imunoglobulina E/biossíntese , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Masculino , Proteínas de Plantas/imunologia , Receptores de IgE/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Especificidade da Espécie
8.
Clin Transl Allergy ; 10: 36, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32884636

RESUMO

The mechanisms involved in the induction of allergic sensitization by pollen are not fully understood. Within the last few decades, findings from epidemiological and experimental studies support the notion that allergic sensitization is not only dependent on the genetics of the host and environmental factors, but also on intrinsic features of the allergenic source itself. In this review, we summarize the current concepts and newest advances in research focusing on the initial mechanisms inducing pollen sensitization. Pollen allergens are embedded in a complex and heterogeneous matrix composed of a myriad of bioactive molecules that are co-delivered during the allergic sensitization. Surprisingly, several purified allergens were shown to lack inherent sensitizing potential. Thus, growing evidence supports an essential role of pollen-derived components co-delivered with the allergens in the initiation of allergic sensitization. The pollen matrix, which is composed by intrinsic molecules (e.g. proteins, metabolites, lipids, carbohydrates) and extrinsic compounds (e.g. viruses, particles from air pollutants, pollen-linked microbiome), provide a specific context for the allergen and has been proposed as a determinant of Th2 polarization. In addition, the involvement of various pattern recognition receptors (PRRs), secreted alarmins, innate immune cells, and the dependency of DCs in driving pollen-induced Th2 inflammatory processes suggest that allergic sensitization to pollen most likely results from particular combinations of pollen-specific signals rather than from a common determinant of allergenicity. The exact identification and characterization of such pollen-derived Th2-polarizing molecules should provide mechanistic insights into Th2 polarization and pave the way for novel preventive and therapeutic strategies against pollen allergies.

9.
Int J Biol Macromol ; 164: 1545-1553, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32735921

RESUMO

Phospholipase A2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA2inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately ß-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA2 from honeybee (Kd = 1.48 µM) and was able to inhibit the PLA2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA2 activity.


Assuntos
Venenos de Crotalídeos/metabolismo , Crotalus/metabolismo , Inibidores de Fosfolipase A2/metabolismo , Fosfolipases A2/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Escherichia coli/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Estrutura Secundária de Proteína , Proteômica/métodos
10.
Curr Allergy Asthma Rep ; 20(7): 25, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32430735

RESUMO

PURPOSE OF REVIEW: Pathogenesis-related class 10 (PR-10) proteins are highly conserved plant proteins, which are induced in response to abiotic and biotic stress factors. To date, no unique biological function could be assigned to them. Rather a more general role of PR-10 in plant development and defense mechanisms has been proposed. In addition, some PR-10 proteins act as allergens by triggering allergic symptoms in sensitized individuals. Regardless of the diversity of reported activities, all PR-10 proteins share a common fold characterized by a solvent-accessible hydrophobic cavity, which serves as a binding site for a myriad of small-molecule ligands, mostly phytohormones and flavonoids. RECENT FINDINGS: Most of available data relate to the ligand binding activity of allergenic PR-10, particularly for those belonging to Bet v 1 family of allergens. Bet v 1 and its homologues were shown to bind flavonoids with high affinity, but the specificity appears to differ between homologues from different species. The flavonoid Q3O-(Glc)-Gal was shown to specifically bind to hazelnut Cor a 1 but not to Bet v 1. Similarly, Q3OS bound only to the major isoform Bet v 1.0101 and not to other closely related isoforms. In contrast, Bet v 1 and hazelnut Cor a 1 showed very similar binding behavior towards other flavonoids such as quercetin, genistein, apigenin, daidzein, and resveratrol. Recent research findings highlighted the importance of more precise knowledge of ligand binding for understanding the functional diversification of PR-10 proteins.


Assuntos
Alérgenos/química , Proteínas de Plantas/química , Humanos , Ligantes
11.
Clin Exp Allergy ; 50(7): 835-847, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32314444

RESUMO

INTRODUCTION: Allergen-specific immunotherapy (AIT) represents a curative approach for treating allergies. In the tropical and subtropical regions of the world, Blomia tropicalis (Blo t 5 and Blo t 21) is the likely dominant source of indoor allergens. AIM: To generate a hypoallergenic Blo t 5/Blo t 21 hybrid molecule that can treat allergies caused by B tropicalis. METHODS: Using in silico design of B tropicalis hybrid proteins, we chose two hybrid proteins for heterologous expression. Wild-type Blo t 5/Blo t 21 hybrid molecule and a hypoallergenic version, termed BTH1 and BTH2, respectively, were purified by ion exchange and size exclusion chromatography and characterized by physicochemical, as well as in vitro and in vivo immunological, experiments. RESULTS: BTH1, BTH2 and the parental allergens were purified to homogeneity and characterized in detail. BTH2 displayed the lowest IgE reactivity that induced basophil degranulation using sera from allergic rhinitis and asthmatic patients. BTH2 essentially presented the same endolysosomal degradation pattern as the shortened rBlo t 5 and showed a higher resistance towards degradation than the full-length Blo t 5. In vivo immunization of mice with BTH2 led to the production of IgG antibodies that competed with human IgE for allergen binding. Stimulation of splenocytes from BTH2-immunized mice produced higher levels of IL-10 and decreased secretion of IL-4 and IL-5. In addition, BTH2 stimulated T-cell proliferation in PBMCs isolated from allergic patients, with secretion of higher levels of IL-10 and lower levels of IL-5 and IL-13, when compared to parental allergens. CONCLUSIONS AND CLINICAL RELEVANCE: BTH2 is a promising hybrid vaccine candidate for immunotherapy of Blomia allergy. However, further pre-clinical studies addressing its efficacy and safety are needed.


Assuntos
Alérgenos , Proteínas de Artrópodes , Hipersensibilidade , Ácaros , Vacinas , Alérgenos/genética , Alérgenos/imunologia , Alérgenos/farmacologia , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Proteínas de Artrópodes/farmacologia , Citocinas , Feminino , Humanos , Hipersensibilidade/imunologia , Hipersensibilidade/terapia , Masculino , Camundongos Endogâmicos BALB C , Ácaros/genética , Ácaros/imunologia , Vacinas/genética , Vacinas/imunologia , Vacinas/farmacologia
12.
J Allergy Clin Immunol ; 146(3): 583-594.e6, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32272131

RESUMO

BACKGROUND: Pollen exposure induces local and systemic allergic immune responses in sensitized individuals, but nonsensitized individuals also are exposed to pollen. The kinetics of symptom expression under natural pollen exposure have never been systematically studied, especially in subjects without allergy. OBJECTIVE: We monitored the humoral immune response under natural pollen exposure to potentially uncover nasal biomarkers for in-season symptom severity and identify protective factors. METHODS: We compared humoral immune response kinetics in a panel study of subjects with seasonal allergic rhinitis (SAR) and subjects without allergy and tested for cross-sectional and interseasonal differences in levels of serum and nasal, total, and Betula verrucosa 1-specific immunoglobulin isotypes; immunoglobulin free light chains; cytokines; and chemokines. Nonsupervised principal component analysis was performed for all nasal immune variables, and single immune variables were correlated with in-season symptom severity by Spearman test. RESULTS: Symptoms followed airborne pollen concentrations in subjects with SAR, with a time lag between 0 and 13 days depending on the pollen type. Of the 7 subjects with nonallergy, 4 also exhibited in-season symptoms whereas 3 did not. Cumulative symptoms in those without allergy were lower than in those with SAR but followed the pollen exposure with similar kinetics. Nasal eotaxin-2, CCL22/MDC, and monocyte chemoattactant protein-1 (MCP-1) levels were higher in subjects with SAR, whereas IL-8 levels were higher in subjects without allergy. Principal component analysis and Spearman correlations identified nasal levels of IL-8, IL-33, and Betula verrucosa 1-specific IgG4 (sIgG4) and Betula verrucosa 1-specific IgE (sIgE) antibodies as predictive for seasonal symptom severity. CONCLUSIONS: Nasal pollen-specific IgA and IgG isotypes are potentially protective within the humoral compartment. Nasal levels of IL-8, IL-33, sIgG4 and sIgE could be predictive biomarkers for pollen-specific symptom expression, irrespective of atopy.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Adulto , Biomarcadores , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Interleucina-33/imunologia , Interleucina-8/imunologia , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/imunologia , Rinite Alérgica Sazonal/sangue , Estações do Ano , Adulto Jovem
14.
Allergy ; 75(4): 882-891, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31750952

RESUMO

BACKGROUND: Transforming growth factor ß1 (TGFß1) is a cytokine that exerts immunosuppressive functions, as reflected by its ability to induce regulatory T (Treg) cell differentiation and inhibit Th1 and Th2 responses. Hence, peptides that mimic the active core domain of TGFß1 may be promising candidates for modulation of the allergic response. This study aimed to investigate a synthetic TGFß1 mimetic peptide (TGFß1-mim) for its ability to modulate the immune response during allergic sensitization to grass pollen allergens. METHODS: The in vitro action of TGFß1-mim was evaluated in human lung epithelial cells, Jurkat cells, and rat basophilic leukemia cells. The in vivo action was evaluated in a murine model of Phl p 5 allergic sensitization. Additionally, the Th2 modulatory response was evaluated in IL-4 reporter mice. RESULTS: In vitro, TGFß1-mim downregulated TNF-α production, IL-8 gene expression, and cytokine secretion, upregulated IL-10 secretion, and inhibited Phl p 5-induced basophil degranulation. During Phl p 5 sensitization in mice, TGFß1-mim downregulated IL-2, IL-4, IL-5, IL-13, and IFN-γ, upregulated IL-10, and induced Treg cell production. Furthermore, mice treated with TGFß1-mim had lower levels of IgE, IgG1, IgG2a and higher levels of IgA antibodies than control mice. In a reporter mouse, the mimetic inhibited Th2 polarization. CONCLUSION: The TGFß1-mim efficiently modulated various important events that exacerbate the allergic microenvironment, including the production of main cytokines that promote Th1 and Th2 differentiation, and the induction of allergen-specific regulatory T cells, highlighting its potential use in therapeutic approaches to modulate the immune response toward environmental allergens.


Assuntos
Alérgenos , Peptídeos , Fator de Crescimento Transformador beta1 , Animais , Biomimética , Imunoglobulina E , Camundongos , Peptídeos/farmacologia , Poaceae , Pólen/imunologia
15.
Int J Biol Macromol, v. 164, p. 1545-1553, dez. 2020
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IBPROD, Sec. Est. Saúde SP | ID: bud-3419

RESUMO

Phospholipase A2 plays an important role in many diseases. Thus, the production of bioactive molecules, which can modulate PLA2 activity, became an important target for the pharmaceutical industry. Previously, we demonstrated the inhibitory and anti-angiogenic effect of γCdcPLI, the natural PLA2inhibitor from Crotalus durissus collilineatus. The aim of the present study was to recombinantly express the γCdcPLI inhibitor and analyze its biochemical and functional characteristics. Based on the amino acid sequence from the natural protein, we designed a synthetic gene for production of a non-tagged recombinant recγCdcPLI using the pHis-Parallel2 vector. To enable disulfide bond formation, protein expression was performed using E. coli Rosetta-gamiB. The protein was purified by anion and affinity chromatography with a yield of 5 mg/L. RecγCdcPLI showed similar secondary structure in CD and FTIR, revealing predominately β-strands. Analogous to the natural protein, recγCdcPLI was able to form oligomers of ~5.5 nm. The inhibitor was efficiently binding to PLA2 from honeybee (Kd = 1.48 μM) and was able to inhibit the PLA2 activity. Furthermore, it decreased the vessel formation in HUVEC cells, suggesting an anti-angiogenic potential. Heterologous production of recγCdcPLI is highly efficient and thus enables enhanced drug design for treatment of diseases triggered by PLA2 activity.

16.
Allergy ; 74(12): 2382-2393, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31230350

RESUMO

BACKGROUND: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen-derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity. METHODS: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs. RESULTS: We identified E1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity. CONCLUSION: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes.


Assuntos
Alérgenos/imunologia , Antígenos de Plantas/imunologia , Endossomos/enzimologia , Peptídeo Hidrolases/metabolismo , Basófilos/imunologia , Basófilos/metabolismo , Betula/imunologia , Degranulação Celular/imunologia , Ativação Enzimática , Humanos , Imunoglobulina E/imunologia , Ligantes , Pólen/imunologia , Ligação Proteica , Proteínas Recombinantes
17.
Front Physiol ; 10: 35, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30745882

RESUMO

Purpose: We set out to investigate the effectiveness of grounded sleeping on the time course of recovery with respect to muscle soreness and athletic performance after intensive eccentric muscle loading. Methods: Twenty-two healthy participants were recruited for this study and randomly assigned to an experimental group (GRD, grounded sleeping, n = 12) or control group (UGD, sham-grounded sleeping, n = 10) to evaluate the effects of 10 days recovery with GRD vs. UGD following a single intensive downhill treadmill intervention in a triple-blinded (participant, tester, and data analyst) manner. To operationalize recovery a test battery was performed at baseline and on days 1, 2, 3, 5, 7, and 10 post-intervention: (1) perception of muscle soreness (VAS), (2) creatine kinase blood levels (CK), (3) maximum voluntary isometric contraction (MVIC) for both legs, (4) counter movement jump (CMJ) and drop jump (DJ) performance. Furthermore, in four participants blood was sampled for detailed analysis of complete blood counts and serum-derived inflammation markers. Results: The downhill treadmill running intervention led to distinct changes in all measured parameters related to fatigue. These changes were detectable already 5-min post intervention and were not fully recovered 10 days post intervention. GRD led to less pronounced decrease in performance (CMJ, MVIC) and less increase with respect to CK compared with UGD (all P < 0.05). Detailed blood samples demonstrated that grounded sleeping modulates the recovery process by (a) keeping a constant hemoconcentration, as represented by the number of erythrocytes, and the hemoglobin/hematocrit values; and (b) by the reduction of muscle damage-associated inflammation markers such as, IP-10, MIP-1α, and sP-Selectin. Conclusion: The downhill running protocol is a feasible methodology to produce long term muscle soreness and muscular fatigue. GRD was shown to result in faster recovery and/or less pronounced markers of muscle damage and inflammation. GRD might be seen as a simple methodology to enhance acute and long-term recovery after intensive eccentric exercises.

18.
Clin Transl Allergy ; 8: 39, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30338052

RESUMO

Background: The clinical benefit of allergen-specific immunotherapy (AIT) involves induction of blocking antibodies. It is not clear if these antibodies function via steric hindrance alone or a combination of levels, avidities, and epitope specificities, and clinical outcome cannot be predicted. We aim to in-depth characterize serum antibody profiles during birch pollen AIT, investigate therapy-induced antibodies for their capacity to block IgE binding to Bet v 1 and correlate data with clinical outcomes. Methods: Immune responses of five birch pollen allergic patients were monitored during the first year of AIT by nasal provocation tests (NPTs), ImmunoCAP, immunoblots, direct and avidity enzyme-linked immunosorbent assays, mediator release assays, facilitated antigen binding (FAB) assays, and inhibition mediator release assays. Results: There was no correlation between NPT results and therapy-induced changes in levels (IgE, IgG, IgA, IgM), avidities, or mediator release potency of Bet v 1-specific antibodies. In FAB assays, blocking antibodies initiated upon AIT were shown to prevent formation of Bet v 1-IgE complexes of an indicator serum pool and significantly correlated with clinical readout. Inhibition mediator release assays using patient-specific IgE for passive sensitization revealed therapy-induced blocking capacities with very good correlation to NPT results. Notably, this assay was the only one to detect a non-responder during treatment in this pilot study. Conclusions: Clinical outcome of AIT depends on induction of blocking antibodies able to prevent the patient's own IgE from allergen binding. Monitoring of clinical efficacy seems to be best achieved using the inhibition mediator release assay, as development of relevant blocking antibodies can be verified in a patient-tailored manner.

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