RESUMO
A number of adjuvant formulations were assayed in mice immunized with 3.75 µg of A/California/7/2009 (H1N1) pdm09 influenza vaccine with vitamins A, D and/or E in emulsions or B2 and/or B9 combined with Bordetella pertussis MPLA and/or alum as adjuvants. Squalene was used as positive control, as well as MPLA with alum. The immune response was evaluated by a panel of tests, including a hemagglutination inhibition (HAI) test, ELISA for IgG, IgG1, and IgG2a and IFN-γ, IL-2, IL-6 and IL-10 quantification in splenocyte culture supernatant after stimulus with influenza antigen. Immunological memory was evaluated using a 1/10 dose booster 60 days after the first immunization followed by assessment of the response by HAI, IgG ELISA, and determination of the antibody affinity index. The highest increases in HAI, IgG1 and IgG2a titers were obtained with the adjuvant combinations containing vitamin E, or the hydrophilic combinations containing MPLA and alum or B2 and alum. The IgG1/IgG2a ratio indicates that the response to the combination of B2 with alum would have more Th2 character than the combination of MPLA with alum. In an assay to investigate the memory response, a significant increase in HAI titer was observed with a booster vaccine dose at 60 days after immunization with vaccines containing MPLA with alum or B2 with alum. Overall, of the 27 adjuvant combinations, MPLA with alum and B2 with alum were the most promising adjuvants to be evaluated in humans.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas contra Influenza/imunologia , Vitaminas/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Antígenos de Bactérias/administração & dosagem , Citocinas/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Testes de Inibição da Hemaglutinação , Memória Imunológica , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Leucócitos Mononucleares/imunologia , Masculino , Camundongos Endogâmicos BALB C , Esqualeno/administração & dosagemRESUMO
Aprotinin, the most studied serine proteinase inhibitor, was isolated from porcine lung for the first time. The purified porcine aprotinin had an Mr value of approximately 7 kDa. It cross-reacted with polyclonal serum anti-commercial aprotinin. About 1 microg porcine aprotinin inhibited 6 microg trypsin whereas 1 microg commercial soybean inhibitor inhibited only 1 microg trypsin. The aprotinin gene was also isolated from porcine lung: the deduced amino acid sequence showed 74% identity to bovine aprotinin.
Assuntos
Aprotinina/genética , Aprotinina/isolamento & purificação , Pulmão/metabolismo , Sequência de Aminoácidos , Animais , Aprotinina/metabolismo , Sequência de Bases , Bovinos , Cromatografia de Afinidade , DNA/genética , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Suínos , Tripsina/metabolismoRESUMO
PURPOSE: To evaluate surfactant protein A levels in an hepatopulmonary syndrome rat model. To date, there have been no studies aimed at evaluating surfactant levels in the setting of cirrhosis or hepatopulmonary syndrome. METHODS: A total of 35 rats were divided into control, sham, and experimental HPS groups. We evaluated surfactant protein A levels in rats and the experimental model designed to induce hepatopulmonary syndrome was common bile duct ligation. Statistical analysis was performed using GraphPad Prism Software(r). Differences were considered statistically significant when p<0.05. RESULTS: Lung homogenate of surfactant protein A levels were lower in the experimental hepatopulmonary syndrome and sham groups in comparison to the control group (p<0.05). Serum SP-A levels were the same in experimental hepatopulmonary syndrome and control groups but decreased in the sham group compared with the experimental groups (p<0.05). Myeloperoxidase activity was higher in the experimental hepatopulmonary syndrome group than the other two groups (p<0.05). CONCLUSION: Surfactant protein A is present in experimental hepatopulmonary syndrome and leads to an imbalance between serum and pulmonary levels due to systemic inflammatory response.
Assuntos
Modelos Animais de Doenças , Síndrome Hepatopulmonar/metabolismo , Pulmão/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Animais , Gasometria , Ducto Colédoco , Síndrome Hepatopulmonar/patologia , Ligadura , Masculino , Peroxidase/metabolismo , Proteína A Associada a Surfactante Pulmonar/análise , Ratos Wistar , Valores de ReferênciaRESUMO
An improved whole cell pertussis vaccine, designated as Plow, which is low in endotoxicity due to a chemical extraction of lipo-oligosaccharide (LOS) from the outer membrane, was evaluated for safety, immunogenicity and potency, comparatively to a traditional whole cell pertussis vaccine. Current whole cell pertussis vaccines are effective but contain large quantities of endotoxin and consequently display local and systemic adverse reactions after administration. Endotoxin is highly inflammatory and contributes considerably to the reactogenicity as well as the potency of these vaccines. In contrast, acellular pertussis vaccines hardly contain endotoxin and are significantly less reactogenic, but their elevated costs limit their global use, especially in developing countries. In this paper, bulk products of Plow and a traditional whole cell vaccine, formulated as plain monocomponents or combined with diphtheria and tetanus toxoids (DTPlow or DTP, respectively) were compared by in vitro and in vivo assays. Chemical extraction of LOS resulted in a significant decrease in endotoxin content (20%) and a striking decline in endotoxin related toxicity (up to 97%), depending on the used in vitro or in vivo test. The LOS extraction did not affect the integrity of the product and, more importantly, did not affect the potency and/or stability of DTPlow. Moreover, hardly any differences in antibody and T-cell responses were observed. The development of Plow is a significant improvement regarding the endotoxicity of whole cell pertussis vaccines and therefore a promising and affordable alternative to currently available whole cell or acellular pertussis vaccines for developing countries.
Assuntos
Endotoxinas/isolamento & purificação , Vacina contra Coqueluche/efeitos adversos , Vacina contra Coqueluche/imunologia , Potência de Vacina , Animais , Estabilidade de Medicamentos , Endotoxinas/análise , Feminino , Camundongos , Vacina contra Coqueluche/administração & dosagem , Vacina contra Coqueluche/química , CoelhosRESUMO
PURPOSE: To evaluate surfactant protein A levels in an hepatopulmonary syndrome rat model. To date, there have been no studies aimed at evaluating surfactant levels in the setting of cirrhosis or hepatopulmonary syndrome. METHODS: A total of 35 rats were divided into control, sham, and experimental HPS groups. We evaluated surfactant protein A levels in rats and the experimental model designed to induce hepatopulmonary syndrome was common bile duct ligation. Statistical analysis was performed using GraphPad Prism Software(r). Differences were considered statistically significant when p<0.05. RESULTS: Lung homogenate of surfactant protein A levels were lower in the experimental hepatopulmonary syndrome and sham groups in comparison to the control group (p<0.05). Serum SP-A levels were the same in experimental hepatopulmonary syndrome and control groups but decreased in the sham group compared with the experimental groups (p<0.05). Myeloperoxidase activity was higher in the experimental hepatopulmonary syndrome group than the other two groups (p<0.05). CONCLUSION: Surfactant protein A is present in experimental hepatopulmonary syndrome and leads to an imbalance between serum and pulmonary levels due to systemic inflammatory response. .
Assuntos
Animais , Masculino , Modelos Animais de Doenças , Síndrome Hepatopulmonar/metabolismo , Pulmão/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Gasometria , Ducto Colédoco , Síndrome Hepatopulmonar/patologia , Ligadura , Peroxidase/metabolismo , Proteína A Associada a Surfactante Pulmonar/análise , Ratos Wistar , Valores de ReferênciaRESUMO
A pulmonary surfactant reduces surface tension at the air/liquid interface of the alveoli and stabilizes alveoli at low lung volumes. Surfactant deficiency and dysfunction were shown to be present in a number of pulmonary diseases, and surfactant replacement therapy is the common clinical conduct. The hydrophilic SP-A (surfactant protein A) is absent when solvent extraction was used during exogenous surfactant production. Addition of SP-A to the surfactant preparation increases the surface activity and completely counteracts inhibition by blood proteins. SP-A recognizes and binds to carbohydrate structures on the surfaces of pathogenic micro-organisms, and acts as opsonins or cross-linking molecules by binding to a variety of cells that participate in the pulmonary immune response. The purification procedure yielded 206 mg of high-purity SP-A/kg of porcine lung, as judged by gel filtration, SDS/PAGE and Western blotting. The electrophoretic profiles obtained showed that pure SP-A consists of proteins of wide molecular mass in the range 26-36 kDa and a dimer in the range 56-60 kDa. The Western-blot results displayed the same band pattern profile after incubating the membrane using a commercially available polyclonal anti-SP-A antibody produced in goat. Gel-filtration experiments confirmed the molecular mass of SP-A in 10 mM NaCl solution. The isolated SP-A showed mannose-binding ability, representative of its functionality.
Assuntos
Centrifugação/métodos , Cromatografia de Afinidade/métodos , Cromatografia por Troca Iônica/métodos , Proteína A Associada a Surfactante Pulmonar/química , Proteína A Associada a Surfactante Pulmonar/isolamento & purificação , Surfactantes Pulmonares/química , Surfactantes Pulmonares/isolamento & purificação , Animais , BovinosRESUMO
We previously showed that surfactant inhibits the synthesis of type IIA secretory phospholipase A2 (sPLA2-IIA) by alveolar macrophages. These cells have been identified as the main source of this enzyme in an animal model of acute lung injury. The aim of the present study was to identify the surfactant components involved in the inhibition of sPLA2-IIA expression in alveolar macrophages and the signaling pathways that mediate this inhibition. Our results show that various surfactant preparations can inhibit sPLA2-IIA expression in endotoxin-stimulated alveolar macrophages. Both the surfactant protein (SP)-A and the surfactant phospholipid fraction inhibit this expression. The surfactant phospholipid dioleylphosphatidylglycerol (DOPG) abolishes sPLA2-IIA expression, whereas dipalmitoylphosphatidylcholine does not. Chromatographic analysis and confocal microscopy revealed that phosphatidylglycerol was rapidly incorporated and metabolized by alveolar macrophages and that its metabolites accumulate in the cytosol. Nuclear factor-kappaB (NF-kappaB) modulates sPLA2-IIA expression in endotoxin-activated alveolar macrophages, and surfactant preparations, surfactant phospholipid fraction, SP-A, and DOPG indeed suppressed NF-kappaB activation. In summary, our results show that SP-A and DOPG play a role in the surfactant-mediated inhibition of sPLA2-IIA expression in alveolar macrophages and that this inhibition occurs via a downregulation of NF-kappaB activation.
Assuntos
NF-kappa B/fisiologia , Fosfatidilgliceróis/farmacologia , Fosfolipases A/biossíntese , Fosfolipases A/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar/farmacologia , Análise de Variância , Animais , Células Cultivadas , Cromatografia em Camada Fina , Regulação para Baixo , Cobaias , Macrófagos Alveolares/fisiologia , Masculino , Microscopia Confocal , Modelos Animais , Fosfolipases A2 , Probabilidade , Surfactantes Pulmonares/metabolismo , RNA Mensageiro/análise , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/análiseRESUMO
A suscetibilidade da linhagem de células Vero ao vírus do sarampo é bem conhecida e sua utilizaçäo no controle da potência da vacina contra o sarampo é amplamente difundida. Com o objetivo de comparar a suscetibilidade de células Vero empregadas em titulaçöes, amostras provenientes de dois laboratórios controladores (Vero IB e Vero INCQS), foram testadas frente a três cepas vacinais: Moraten Schwarz e Biken CAM-70. Foram titulados 72 lotes de vacinas contra o sarampo, sendo 25 produzidos com a cepa Moraten, 24 com a cepa Schwarz e 23 com a cepa Biken CAM-70. A análise estatística dos resultados obtidos nas titulaçöes, feita através dos testes Limites para uma Média e "t" de Student, mostrou que para as cepas Moraten e Biken CAM-70, as diferenças de títulos näo foram estatisticamente significantes, o mesmo näo ocorrendo com a cepa Schwarz, para a qual as células Vero IB se mostraram mais sensíveis