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Cross-reactivity between allergens and human proteins could have a clinical impact in allergic diseases. Blo t 13 is an allergen from the mite Blomia tropicalis, which belongs to the fatty acid binding protein (FABP) family and has structural homology with human FABPs. This work aimed to map B cell epitopes on Blo t 13 and to identify epitopes involved in cross-reactivity with human heart FABP (FABP3) and adipocyte FABP (FABP4). Sera from 25 patients with house dust mite (HDM) allergy that were sensitized to Blo t 13 were used for testing the reactivity of immunoglobulin E (IgE) and IgG to FABP. The epitope mapping of Blo t 13 was performed using overlapping peptides, and cross-reactivity between Blo t 13 and human FABP was analyzed using human sera and anti-Blo t 13 monoclonal antibodies. IgE antibodies to all FABPs were detected in 14/25 serum samples, and IgG was detected in 25/25 serum samples. The cross-reactivity of Blo t 13 was 42% with FABP3 and 48% with FABP4. Two IgE-binding regions were identified in Blo t 13; one between residues 54 and 72 (the main cross-reacting region) and another between residues 111 to 129. Our results suggest that exposure to the Blo t 13 allergen could induce an auto-reactive response to endogenous FABP in allergic patients sensitized to Blo t 13.
Assuntos
Alérgenos/metabolismo , Epitopos de Linfócito B/imunologia , Proteína 3 Ligante de Ácido Graxo/imunologia , Proteínas de Ligação a Ácido Graxo/imunologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Adipócitos/metabolismo , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Mapeamento de Epitopos , Proteína 3 Ligante de Ácido Graxo/química , Proteína 3 Ligante de Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/patologia , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Masculino , Ácaros/metabolismo , Miocárdio/metabolismo , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Primary drying is the most critical stage of the freeze-drying process. This work aimed to establish a design space for this process by means of mathematical modeling of the primary drying stage, capable of addressing the thermal characteristics of distinct vial suppliers. Modeling of primary drying was implemented on Microsoft Excel using steady-state heat and mass transfer equations at two extreme conditions as assessed by risk analysis, to predict product temperature and primary-drying time. The heat transfer coefficients (Kv) of four different vial suppliers were experimentally determined, both, at the center and edge of the freeze-dryer's shelf. Statistically significant differences (ANOVA p<0.05) were observed between suppliers throughout the assessed pressure range. Overall, the average Kve/Kvc (edge/center) ratio was higher than 1.6 for all suppliers due to the radiation effect. A design space for the drying process was established using mathematical modeling taking into account the Kv of the worst-case supplier, in the shelf edge. A primary drying cycle was carried out at a shelf temperature of -25 °C and a chamber pressure of 45 mTorr for 8 % sucrose and at -10 °C and 75 mTorr for 5 % NaCl. Freeze-dried products with good cosmetic appearance were obtained for the four vial suppliers both, in the shelf center and edge. The results show that it is possible to predict and establish the critical parameters for the primary drying stage, under a design space concept, considering the differences in the Kv of vial suppliers without adverse consequences on the quality of the finished freeze-dried product.
Assuntos
Liofilização , Liofilização/métodos , Modelos Teóricos , Sacarose/química , Temperatura , Pressão , Temperatura AltaRESUMO
Allergen extracts are used for hyposensitivity and immunotherapy treatments, reducing significantly clinical symptoms of allergic diseases. Because of its wide use in immunoallergen therapy, we evaluated the Dermatophagoides siboney allergen extract to establish the potential toxicity following repeated subcutaneous dosing in Cenp:NMRI mice. Animals were randomly distributed into two groups, control (vehicle) and treated (166.6 UB/animal), and they were observed daily for clinical signs of toxicity following treatment. Body weight was weekly measured. At the end of the study, blood samples were collected for hematology and serum chemistry analysis and animals were euthanized for gross necropsy and histological examination of tissues. There were not significant differences in body weight or hematology parameters between control and treated animals. Differences were noted in uric acid, blood urea nitrogen and glucose; however, these alterations were not considered to be of biologic relevance. Pathology evaluations demonstrated hemorrhagic and inflammatory lesions at the administration site in both experimental groups. We conclude that repeated dosing of 166.6 UB did not cause significant toxic effects in the mouse model.
Assuntos
Alérgenos/toxicidade , Antígenos de Dermatophagoides/toxicidade , Misturas Complexas/toxicidade , Animais , Feminino , Masculino , Camundongos , Testes de Toxicidade SubagudaRESUMO
The outer-membrane-derived proteoliposome (PL) of Neisseria meningitidis has been reported as a potent vaccine adjuvant, inducing a Th1-skewed response. This work aimed to assess the immunogenicity of a novel anti-allergic vaccine candidate based on allergens from Dermatophagoides siboney house dust mite and a combination adjuvant containing PL and Alum. In a preventative experimental setting, BALB/c mice were administered with three doses containing 2⠵g of Der s1 and 0.4⠵g Der s2 allergen, PL and Alum, at 7 days intervals, by subcutaneous route. Furthermore, mice were subjected to an allergen aerosol challenge for 6 consecutive days. Serum IgE, IgG1, and IgG2a allergen-specific antibodies were assessed by ELISA. Cytokine levels in supernatants of D. siboney stimulated lymphocyte cultures and in bronchoalveolar lavage (BAL) were measured by ELISA. Lung tissues were subjected to histological examination. The vaccine prevented the development of both, systemic (IgE) and local allergic responses (featuring lower IL-4, and IL-5 levels in BAL) upon allergen exposure by the inhalant route. Histological examination showed also a diminished allergic inflammatory response in the lungs. After the allergen challenge, cytokine levels in stimulated lymphocyte cultures showed lower values of IL-13 and augmented IFN-γ and IL-10. The vaccine induced a mixed IgG2a/IgG1 antibody response; although only IgG2a was PL-dependent. Both, IgG1/IgE and IgG2a/IgE ratios, showed significantly greater values in vaccinated mice. The findings support a preventative anti-allergic effect associated with the induction of a Th1-like IFN-γ/IL-10 response. IgG1/IgE and IgG2a/IgE ratios could be useful biomarkers for translation into clinical trials.
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Airway obstruction with increased airway resistance in asthma, commonly caused by smooth muscle constriction, mucosal edema and fluid secretion into the airway lumen, may partly be due to a poor function of pulmonary surfactant. Surfacen®, a clinical pulmonary surfactant, has anti-inflammatory action, but its effect on asthma has not been studied. This work aimed to evaluate the effect of Surfacen® in a murine allergen-induced acute asthma model, using house dust mite allergens. In a therapeutic experimental setting, mice were first sensitized by being administered with two doses (sc) of Dermatophagoides siboney allergen in aluminum hydroxide followed by one intranasal administration of the allergen. Then, sensitized mice were administered with aerosol of hypertonic 3% NaCl, Salbutamol 0.15 mg/kg, or Surfacen® 16 mg in a whole-body chamber on days 22, 23, and 24. Further, mice were subjected to aerosol allergen challenge on day 25. Surfacen® showed bronchial dilation and inhibition of Th2 inflammation (lower levels of IL-5 and IL-13 in broncoalveolar lavage) which increased IFN-γ and unchanged IL-10 in BAL. Moreover, Sufacen® administration was associated with a marked inhibition of the serum specific IgE burst upon allergen exposure, as well as, IgG2a antibody increase, suggesting potential anti-allergy effects with inclination towards Th1. These results support also the effectiveness of the aerosol administration method to deliver the drug into lungs. Surfacen® induced a favorable pharmacological effect, with a bronchodilator outcome comparable to Salbutamol, consistent with its action as a lung surfactant, and with an advantageous anti-inflammatory and anti-allergic immunomodulatory effect.
Assuntos
Antialérgicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Asma/tratamento farmacológico , Fosfolipídeos/uso terapêutico , Proteínas Associadas a Surfactantes Pulmonares/uso terapêutico , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Asma/sangue , Asma/imunologia , Asma/patologia , Líquido da Lavagem Broncoalveolar/imunologia , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos Endogâmicos BALB CRESUMO
Changing the immune responses to allergens is the cornerstone of allergen immunotherapy. Allergen-specific immunotherapy that consists of repeated administration of increasing doses of allergen extract is potentially curative. The major inconveniences of allergen-specific immunotherapy include failure to modify immune responses, long-term treatment leading to non-compliance and the potential for developing life-threating anaphylaxis. Here we investigated the effect of a novel liposomal formulation carrying low dose of allergen combined with CpG-ODN, a synthetic TLR9 agonist, on established allergic lung inflammation. We found that challenge with allergen (OVA) encapsulated in cationic liposome induced significantly less severe cutaneous anaphylactic reaction. Notably, short-term treatment (three doses) with a liposomal formulation containing co-encapsulated allergen plus CpG-ODN, but not allergen or CpG-ODN alone, reversed an established allergic lung inflammation and provided long-term protection. This liposomal formulation was also effective against allergens derived from Blomia tropicalis mite extract. The attenuation of allergic inflammation was not associated with increased numbers of Foxp3-positive or IL-10-producing regulatory T cells or with increased levels of IFN-gamma in the lungs. Instead, the anti-allergic effect of the liposomal formulation was dependent of the innate immune signal transduction generated in CD11c-positive putative dendritic cells expressing MyD88 molecule. Therefore, we highlight the pivotal role of dendritic cells in mediating the attenuation of established allergic lung inflammation following immunotherapy with a liposomal formulation containing allergen plus CpG-ODN.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Alérgenos/administração & dosagem , Asma/imunologia , Células Dendríticas/imunologia , Dessensibilização Imunológica/métodos , Sistemas de Liberação de Medicamentos/métodos , Fator 88 de Diferenciação Mieloide/metabolismo , Oligodesoxirribonucleotídeos/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Alérgenos/efeitos adversos , Anafilaxia/imunologia , Anafilaxia/prevenção & controle , Animais , Asma/induzido quimicamente , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Resultado do TratamentoRESUMO
Blomia tropicalis mite is highly prevalent in tropical and subtropical regions and it is associated with allergic diseases such as rhinitis and asthma. By using an OVA-model of allergic lung disease, we have previously shown that sensitization in the presence of toll like receptors (TLRs) agonists attenuates subsequent OVA-induced allergic responses. Here, we evaluated the effect of CpG-ODN, a specific synthetic TLR-9 agonist, on the development of experimental asthma induced by Blomia tropicalis extract, a relevant source of aeroallergens. Among different protocols of Blomia tropicalis extract sensitization, the subcutaneous sensitization in the presence of alum adjuvant induced the highest Th2 responses, including high IgE levels. Adsorption of CpG to Blomia tropicalis extract/Alum attenuated the airway hyperreactivity, the infiltration of inflammatory cells including eosinophils, and the IL-5 content in BAL. In addition, lung peribronchial inflammatory infiltrate, mucus production and IL-5-producing CD3+ CD4+ T cells were significantly reduced in the Blomia tropicalis extract/Alum+CpG group. Importantly, CpG inhibited total IgE production as well as active systemic or cutaneous anaphylaxis reactions. Inhibition of pulmonary Th2 responses was associated with increased IL-10 production but not with IFN-γ production. Notably, in IL-10-deficient mice, sensitization with OVA/Alum+CpG resulted in intense lung neutrophilia and IFN-γ production, indicating that IL-10 is necessary to inhibit subsequent Th1 immunity. Our work highlights the mechanisms of allergy attenuation by CpG and it indicates the potential use of Alum-based formulation with CpG to treat allergic processes.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Compostos de Alúmen/química , Asma/prevenção & controle , Asma/parasitologia , Pyroglyphidae/fisiologia , Receptor Toll-Like 9/agonistas , Adjuvantes Imunológicos/farmacologia , Adsorção , Anafilaxia/complicações , Anafilaxia/imunologia , Anafilaxia/parasitologia , Animais , Asma/complicações , Citocinas/biossíntese , Modelos Animais de Doenças , Eosinófilos/patologia , Feminino , Hipersensibilidade/complicações , Hipersensibilidade/imunologia , Hipersensibilidade/parasitologia , Imunidade/efeitos dos fármacos , Imunização , Interleucina-10/metabolismo , Interleucina-4/biossíntese , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Camundongos Endogâmicos C57BL , Neutrófilos/patologia , Oligodesoxirribonucleotídeos/farmacologia , Pyroglyphidae/efeitos dos fármacos , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Receptor Toll-Like 9/metabolismoRESUMO
The proteoliposome (PL) of Neisseria meningitidis serogroup B has been reported as a safe and potent vaccine adjuvant, inducing a TH1-skewed response. The present study describes a pre-clinical safety evaluation of an allergy therapeutic vaccine candidate based on purified allergens from Dermatophagoides siboney house dust mite and PL as adjuvant, both components adsorbed onto aluminum hydroxide gel. Two separate studies of acute toxicity evaluation were performed in mice and rabbits, and two repeat-dose studies were conducted in non-sensitized and allergen-sensitized Balb/c mice, respectively. The study in sensitized mice intends to model a therapeutic setting. Aerosolized allergen challenge was used in both settings to model natural respiratory exposure. In the therapeutic setting, mice were administered with three doses containing 2 µg allergen at weekly intervals [subcutaneous route] and subsequently challenged with aerosolized allergen for 6 consecutive days. Parameters of general toxicity effects were assessed via measures of behavior, body weight, food and water consumption, and macroscopic evaluation of organs. Histological examination of organs and the injection site was performed. Potential immunotoxicity effects at the systemic level were assessed by blood eosinophil counting and serum allergen specific IgE by ELISA The vaccine did not produce general or functional toxic effects of significance, at a dose up to 100 µg allergen per kg body weight. An expected local reaction at the injection site was observed, which could be attributed mostly to the immunological effect of aluminum hydroxide. The models implemented here suggest an acceptable safety profile of this vaccine for testing in clinical trials of allergy immunotherapy.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Antígenos de Dermatophagoides/imunologia , Dessensibilização Imunológica/métodos , Hipersensibilidade/terapia , Neisseria meningitidis/metabolismo , Proteolipídeos/administração & dosagem , Vacinas/imunologia , Adjuvantes Imunológicos/efeitos adversos , Hidróxido de Alumínio/administração & dosagem , Animais , Eosinófilos/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Camundongos , Proteolipídeos/efeitos adversos , Proteolipídeos/metabolismo , Pyroglyphidae , CoelhosRESUMO
Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.
Assuntos
Alérgenos/toxicidade , Asma/imunologia , Lipopolissacarídeos/toxicidade , Neutrófilos/imunologia , Pyroglyphidae/imunologia , Células Th17/imunologia , Células Th2/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Alérgenos/imunologia , Animais , Asma/induzido quimicamente , Asma/genética , Asma/patologia , Modelos Animais de Doenças , Eosinófilos/imunologia , Eosinófilos/patologia , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Eosinofilia Pulmonar/induzido quimicamente , Eosinofilia Pulmonar/genética , Eosinofilia Pulmonar/imunologia , Eosinofilia Pulmonar/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Células Th17/patologia , Células Th2/patologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genéticaRESUMO
The development of effective vaccines against neglected diseases, especially those associated with poverty and social deprivation, is urgently needed. Modern vaccine technologies and a better understanding of the immune response have provided scientists with the tools for rational and safer design of subunit vaccines. Often, however, subunit vaccines do not elicit strong immune responses, highlighting the need to incorporate better adjuvants; this step therefore becomes a key factor for vaccine development. In this review we outline some key features of modern vaccinology that are linked with the development of better adjuvants. In line with the increased desire to obtain novel adjuvants for future vaccines, the Finlay Adjuvant Platform offers a novel approach for the development of new and effective adjuvants. The Finlay Adjuvants (AFs), AFPL (proteoliposome), and AFCo (cochleate), were initially designed for parenteral and mucosal applications, and constitute potent adjuvants for the induction of Th1 responses against several antigens. This review summarizes the status of the Finlay technology in producing promising adjuvants for unsolved-vaccine diseases including mucosal approaches and therapeutic vaccines. Ideas related to adjuvant classification, adjuvant selection, and their possible influence on innate recognition via multiple toll-like receptors are also discussed.
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BACKGROUND: Allergies represent the most prevalent non infective diseases worldwide. Approaching IgE-mediated sensitizations improved much by adopting allergenic molecules instead of extracts, and by using the micro-technology for multiplex testing. OBJECTIVE AND METHODS: To provide a proof-of-concept that a flow cytometric bead array is a feasible mean for the detection of specific IgE reactivity to allergenic molecules in a multiplex-like way. A flow cytometry Allergenic Molecule-based micro-bead Array system (ABA) was set by coupling allergenic molecules with commercially available micro-beads. Allergen specific polyclonal and monoclonal antibodies, as well as samples from 167 allergic patients, characterized by means of the ISAC microarray system, were used as means to show the feasibility of the ABA. Three hundred and thirty-six sera were tested for 1 or more of the 16 selected allergens, for a total number of 1,519 tests on each of the two systems. RESULTS: Successful coupling was initially verified by detecting the binding of rabbit polyclonal IgG, mouse monoclonal, and pooled human IgE toward three allergens, namely nDer s 1, nPen m 1, and nPru p 3. The ABA assay showed to detect IgE to nAct d 1, nAct d 11, rAln g 1, nAmb a 1, nArt v 3, rBet v 1, rCor a 1, nCup a 1, nDer p 1, nDer s 1, rHev b 5, nOle e 1, rPar j 2, nPen m 1, rPhl p 1, and nPru p 3. Results obtained by ABA IgE testing were highly correlated to ISAC testing (r = 0.87, p<0.0001). No unspecific binding was recorded because of high total IgE values. CONCLUSION: The ABA assay represents a useful and flexible method for multiplex IgE detection using allergenic molecules. As also shown by our initial experiments with monoclonals and polyclonals, ABA is suitable for detecting other human and non-human immunoglobulins.
Assuntos
Alérgenos/imunologia , Citometria de Fluxo/métodos , Hipersensibilidade/diagnóstico , Imunoensaio/métodos , Imunoglobulina E/sangue , Análise em Microsséries , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos/imunologia , Criança , Pré-Escolar , Estudos de Viabilidade , Feminino , Humanos , Hipersensibilidade Imediata/diagnóstico , Imunoconjugados/imunologia , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
One current approach in developing anti allergic vaccines is the use of potent adjuvants, capable of inducing Th1 or T regulatory cells. Proteoliposomes (PL) could be a suitable adjuvant. Purified Dermatophagoides siboney (Ds) allergens were mixed with PL and adsorbed into Al(OH)3 and evaluated in mice. The Th1/Th2 responses were measured at classes, subclasses, cytokines, and DTH levels. Anti Ds response was deviated to a Thl pattern, with the production of IgG2a and gamma1FN. A positive DTH response and a dramatic decrease of specific IgE and IL5 were not detected. The low dose was more effective than high dose. These results clearly support the potential use of PL as possible adjuvants for anti-allergic vaccines.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígenos de Dermatophagoides/imunologia , Hipersensibilidade/prevenção & controle , Proteolipídeos/imunologia , Vacinas/imunologia , Hidróxido de Alumínio/administração & dosagem , Animais , Antígenos de Dermatophagoides/administração & dosagem , Camundongos , Neisseria meningitidis Sorogrupo B/imunologia , Proteolipídeos/administração & dosagemRESUMO
Standardized allergen vaccines have been developed and registered as biopharmaceutical products in Cuba. Three different vaccines were obtained from the most relevant allergenic mite species: Dermatophagoides pteronvssinus, Dermatophagoides siboney, and Blomia tropicalis. Immuno-analytical methods based on murine monoclonal antibodies and human IgE antibodies were developed for assessing allergenic potency, composition, and stability. Preclinical and clinical studies showed efficacy and safety in diagnostic prick-tests and subcutaneous immunotherapy in asthmatic patients. New approaches are now undertaken in order to develop new adjuvanted formulations based on liposomes or proteoliposomes from Neisseria meningitidis, and purified allergens; aiming to overcome the drawbacks of conventional immunotherapy.
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Alérgenos/imunologia , Dessensibilização Imunológica , Ácaros/imunologia , Vacinas , Adjuvantes Imunológicos , Alérgenos/administração & dosagem , Alérgenos/efeitos adversos , Alérgenos/isolamento & purificação , Animais , Cuba , Dessensibilização Imunológica/efeitos adversos , Lipossomos/farmacologia , Camundongos , Neisseria meningitidis , Proteolipídeos/farmacologia , Vacinas/imunologia , Vacinas/uso terapêuticoRESUMO
Liposomes are non toxic and biodegradable lipid vesicles, which are safe and effective adjuvants to induce Th1-skewed immune response. Therefore, the encapsulation of allergens into liposomes could be an attractive alternative for specific allergy immunotherapy. Previously, we obtained DPPC iposomes encapsulating purified allergens from Dermatophagoides siboney, with suitable stability and extremely reduced allergenicity. In this study, Balb/c mice were immunized with allergens ncapsulated into liposomes (LP) and the induced immune response was evaluated in comparison with allergens dissolved in PBS (PBSA) or adsorbed in Alum (AL). The use of Alum or Liposomes induced a strong allergen specific IgG response. However, total IgE serum levels in the AL group were very high, while levels found in LP group were not significantly different from the control group receiving only PBS. The IgG2a/IgG1 subclass ratio was raised in the LP group. Allergen specific IgE, as measured by PCA assay, was similar for LP and PBSA groups, and approximately the half of the reaction size found in AL group. After allergen challenge by inhalation route, peripheral blood and airway eosinophil counts increased significantly in AL, but not in LP group. Additionally, histopathological analysis of lung tissue sections obtained from challenged mice indicated a reduced cellular infiltration in mice immunized with liposomes. These results support the potential use of liposomal formulations for allergen vaccines.
Assuntos
Antígenos de Dermatophagoides/imunologia , Hipersensibilidade/imunologia , Lipossomos , Compostos de Alúmen , Animais , Antígenos de Dermatophagoides/administração & dosagem , Lipossomos/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: The recombinant allergen of Blomia tropicalis, rBlo t 13, shows 11% of IgE reactivity to sera from allergic patients. This allergen belongs to the fatty acid-binding protein family and its natural equivalent remains to be isolated. Monoclonal antibodies (MAbs) are important tools for specific determination and isolation of natural allergens as well as for characterization of recombinant proteins. METHODS: Mice were immunized with partially purified preparation of rBlo t 13 allergen expressed in the yeast Pichia pastoris. Spleen cells were fused with myeloma cells using polyethylene glycol. Hybridoma screening was performed using a direct ELISA with recombinant allergen. MAb specificity to rBlo t 13 was tested by immunoblotting. Topography of binding sites and binding of MAb to native allergen was studied by ELISA. Reactivity of MAb against allergenic extract of B. tropicalis and Dermatophagoides siboney was analyzed by ELISA inhibition. In addition, the reactivity of MAbs against rBlot 13 from Escherichia coli and P. pastoris expression was compared. RESULTS: Two MAbs, 5G3 and 3G4 with IgG1 isotype, were generated. These MAbs specifically recognized the 16-kD band, which corresponds to the molecular weight shown by rBlo t 13 on SDS-PAGE. In ELISA, the binding of 5G3 MAb to B. tropicalis and D. siboney extracts was inhibited by rBlo t 13. Both MAbs showed the highest reactivity when the allergen was expressed in P. pastoris. CONCLUSION: Two MAbs specific for Blo t 13 were obtained. These MAbs recognized the same or close epitopes on the rBlo t 13 molecule. The occurrence of homologous allergens to Blo t 13 in D. siboney is suggested by the ELISA inhibition assay.
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Alérgenos/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Proteínas de Transporte/imunologia , Animais , Antígenos de Plantas , Sítios de Ligação de Anticorpos/imunologia , Ligação Competitiva/imunologia , Reações Cruzadas/imunologia , Relação Dose-Resposta Imunológica , Escherichia coli/imunologia , Escherichia coli/metabolismo , Proteínas de Ligação a Ácido Graxo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Antecedentes: en las personas mayores de un año las alergias respiratorias pueden estudiarse desde el punto de vista etiológico utilizando para ello las pruebas cutáneas con alergenos específicos. Material y método: a 76 pacientes con diagnóstico de rinitis alérgica y pólipos nasales que envió el servicio de Otorrinolaringología al de Alergia de Esmeralda Camagüey, se les realizó un estudio para valorar la reactividad cutánea a los ácaros Dermatophagoides pteronyssinus, Dermatophagoides siboney y Blomia tropicalis. A cada paciente se le hizo la prueba por punción cutánea en duplicado. A los 15 minutos se dibujaron los contornos de los habones y se midieron el diámetro mayor y el ortogonal de cada uno y se calculó la media. Igual se hizo con los habones de los duplicados. Se consideraron positivos cuando el diámetro medio del habón fue > 3 mm y negativo < 3 mm. Resultados: en los pacientes con rinitis la mayor frecuencia de positivos correspondió a Dermatophagoides pteronyssinus y la mayor frecuencia de negativos a Dermatophagoides siboney. En los pacientes con pólipos nasales hubo un predominio (p < 0.05) de positivos a Dermatophagoides pteronyssinus y hubo un predominio (p < 0.05) de negativos a Dermatophagoides siboney. Conclusiones: el ácaro Dermatophagoides pteronyssinus provocó más reacciones positivas que Dermatophagoides siboney y Blomia tropicalis en pacientes con rinitis alérgica y en pólipos nasales.
Assuntos
Humanos , Masculino , Feminino , Adulto , Adolescente , Pré-Escolar , Ácaros/imunologia , Alérgenos/administração & dosagem , Alérgenos/efeitos adversos , Rinite Alérgica Perene/diagnóstico , Testes Cutâneos , Pólipos Nasais/diagnósticoRESUMO
Antecedentes: en Cuba no existen antecedentes de estudios realizados para demostrar la eficacia de una prueba diagnóstica de alergia a Blomia tropicalis a pesar de ser uno de los principales ácaros responsables de esta enfermedad humana que sólo en Cuba afecta a más de 2, 200, 000 personas. Objetivos: evaluar el extracto alergénico Blomia tropicalis producido en el Centro Nacional de Biopreparados (BioCen), en la prueba de punción cutánea como diagnóstico de alergia a este alergeno existente en todos los países tropicales y subtropicales. Material y método: se seleccionaron 50 pacientes con historia clínica positiva de alergia al polvo casero y 50 personas sanas. A todos se le realizó la prueba por punción cutánea en duplicado, con dos diluciones del extracto (20 000 UB/mL y 2 000 UB/mL) y controles positivos y negativos. Se midió el diámetro medio del habón y el diámetro ortogonal y se calculó la media. Se calculó la media del tamaño del habón de los duplicados, lo que constituyó el tamaño de la reacción. La validez de la prueba se estimó calculando la sensibilidad y especificidad. Para ello se utilizó el programa EPITABLE. Resultados: la prueba cutánea fue positiva en 78 por ciento de los enfermos y en 6 por ciento de los sanos, para una sensibilidad de 78 por ciento, intervalo de confianza (IC) 63.7 - 88.0 y una especificidad de 94 por ciento, IC 82.5 - 98.4. El valor predictivo positivo fue de 92.9 por ciento, IC 79.4 - 98.1 y el valor predictivo negativo de 81 por ciento, IC 68.2 - 89.7. Conclusiones: el extracto alergénico de Blomia tropicalis producido en BioCen es eficaz como prueba para el diagnóstico.