RESUMO
Streptococcus pneumoniae is an important human pathogen. Currently used conjugate vaccines are effective against invasive disease, but protection is restricted to serotypes included in the formulation, leading to serotype replacement. Furthermore, protection against non-invasive disease is reported to be considerably lower. The development of a serotype-independent vaccine is thus important and Pneumococcal surface protein A (PspA) is a promising vaccine candidate. PspA shows some diversity and can be classified in 6 clades and 3 families, with families 1 and 2 being the most frequent in clinical isolates. The ideal vaccine should thus induce protection against the two most common families of PspA. The aim of this work was to develop a liposome-based vaccine containing PspAs from family 1 and 2 and to characterize its immune response. Liposomes (LP) composed of dipalmitoylphosphatidylcholine (DPPC) and 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) with or without α-galactosylceramide (α-GalCer) were produced by microfluidics, encapsulating PspA from clade 1 (PspA1, family 1) and/or clade 4 (PspA4Pro, family 2) followed by spray-drying with trehalose to form nanocomposite microparticles carriers (NCMP). LP/NCMPs showed good stability and preservation of protein activity. LP/NCMPs containing PspA1 and/or PspA4Pro were used for immunization of mice targeting the lungs. High serum IgG antibody titers against both PspA1 and PspA4Pro were detected in animals immunized with LP/NCMPs containing α-GalCer, with a balance of IgG1 and IgG2a titers. IgG in sera from immunized mice bound to pneumococcal strains from different serotypes and expressing different PspA clades, indicating broad recognition. Mucosal IgG and IgA were also detected. Importantly, immunization with LP/NCMPs induced full protection against strains expressing PspAs from family 1 and 2. Furthermore, CD4+ resident memory T cells were detected in the lungs of the immunized animals that survived the challenge.
Assuntos
Galactosilceramidas , Infecções Pneumocócicas , Streptococcus pneumoniae , Humanos , Animais , Camundongos , Lipossomos , Pós , Infecções Pneumocócicas/prevenção & controle , Proteínas de Bactérias , Imunização , Vacinas Pneumocócicas , Imunoglobulina G , Pulmão , Anticorpos Antibacterianos , Camundongos Endogâmicos BALB CRESUMO
Abstract The aims of this research were: evaluate the chemical composition and the cytotoxicity of the Cuminum cyminum (cumin), Anethum graveolens (dill), Pimpinella anisum (anise) and Foeniculum vulgare (fennel) essential oils, as well as their antifungal activity in vitro against ten Candida spp. isolates. The chemical composition of the oils was analyzed by means of gas chromatography coupled with mass spectrometry (GC/MS). The cytotoxicity assays were performed, using the cell proliferation reagent WST-1 in L929 mouse fibroblasts (20x103 well-1). The determinate the Minimum Inhibitory Concentration (MIC), was performed through the Broth Microdilution technique (CLSI). The chemical main components were the cuminaldehyde (32.66%) for cumin, carvone (34.89%) for the dill, trans-anethole (94.01%) for the anise and anethole (79.62%) for the fennel. Anise and fennel did not were cytotoxic in all the tested concentrations, however the cumin oil was cytotoxic in the concentration of 20 mg.mL-1 and the dill in the concentrations of 20 and 8 mg.mL-1. All yeasts were susceptible against the evaluated essential oils. Cumin presented the lowest MIC against yeasts. We concluded that all the essential oils presented inhibitory action against Candida spp., and C . cyminum, P. anisum and F. vulgare were not cytotoxic in the same minimum inhibitory concentrations for the fungi.
Resumo Os objetivos desta pesquisa foram: avaliar a composição química e a citotoxicidade dos óleos essenciais de Cuminum cyminum (cominho), Anethum graveolens (endro), Pimpinella anisum (erva-doce) e Foeniculum vulgare (funcho), bem como sua atividade antifúngica in vitro contra dez isolados de Candida spp.. A composição química dos óleos foi analisada por meio de cromatografia gasosa acoplada à espectrometria de massa (GC / MS). Os ensaios de citotoxicidade foram realizados, utilizando o reagente de proliferação celular WST-1 em fibroblastos de ratinho L929 (20x103 poço-1). A determinação da Concentração Inibitória Mínima (MIC) foi realizada através da técnica de microdiluição em caldo (CLSI). Os principais componentes químicos foram o cuminaldeído (32.66%) para cominho, carvona (34.89%) para o endro, trans-anetol (94.01%) para erva-doce e anetol (79.62%) para a funcho. O endro e a erva-doce não foram citotóxicos em todas as concentrações testadas, no entanto, o óleo de cominho foi citotóxico na concentração de 20 mg.mL-1 e o endro nas concentrações de 20 e 8 mg.mL-1. Todas as leveduras foram suscetíveis aos óleos essenciais avaliados. O cominho apresentou a menor CIM contra as leveduras. Concluímos que todos os óleos essenciais apresentaram ação inibidora contra Candida spp., e C. cyminum, P. anisum e F. vulgare não foram citotóxicos nas mesmas concentrações inibitórias mínimas para os fungos.
Assuntos
Candida/efeitos dos fármacos , Óleos Voláteis/farmacologia , Apiaceae/química , Antifúngicos/farmacologia , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Cromatografia Gasosa-Espectrometria de Massas , Boca/microbiologiaRESUMO
The aims of this research were: evaluate the chemical composition and the cytotoxicity of the Cuminum cyminum (cumin), Anethum graveolens (dill), Pimpinella anisum (anise) and Foeniculum vulgare (fennel) essential oils, as well as their antifungal activity in vitro against ten Candida spp. isolates. The chemical composition of the oils was analyzed by means of gas chromatography coupled with mass spectrometry (GC/MS). The cytotoxicity assays were performed, using the cell proliferation reagent WST-1 in L929 mouse fibroblasts (20x103 well-1). The determinate the Minimum Inhibitory Concentration (MIC), was performed through the Broth Microdilution technique (CLSI). The chemical main components were the cuminaldehyde (32.66%) for cumin, carvone (34.89%) for the dill, trans-anethole (94.01%) for the anise and anethole (79.62%) for the fennel. Anise and fennel did not were cytotoxic in all the tested concentrations, however the cumin oil was cytotoxic in the concentration of 20 mg.mL-1 and the dill in the concentrations of 20 and 8 mg.mL-1. All yeasts were susceptible against the evaluated essential oils. Cumin presented the lowest MIC against yeasts. We concluded that all the essential oils presented inhibitory action against Candida spp., and C . cyminum, P. anisum and F. vulgare were not cytotoxic in the same minimum inhibitory concentrations for the fungi.
Assuntos
Antifúngicos/farmacologia , Apiaceae/química , Candida/efeitos dos fármacos , Óleos Voláteis/farmacologia , Cromatografia Gasosa-Espectrometria de Massas , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Boca/microbiologiaRESUMO
The aims of this research were: evaluate the chemical composition and the cytotoxicity of the Cuminum cyminum (cumin), Anethum graveolens (dill), Pimpinella anisum (anise) and Foeniculum vulgare (fennel) essential oils, as well as their antifungal activity in vitro against ten Candida spp. isolates. The chemical composition of the oils was analyzed by means of gas chromatography coupled with mass spectrometry (GC/MS). The cytotoxicity assays were performed, using the cell proliferation reagent WST-1 in L929 mouse fibroblasts (20x103 well-1). The determinate the Minimum Inhibitory Concentration (MIC), was performed through the Broth Microdilution technique (CLSI). The chemical main components were the cuminaldehyde (32.66%) for cumin, carvone (34.89%) for the dill, trans-anethole (94.01%) for the anise and anethole (79.62%) for the fennel. Anise and fennel did not were cytotoxic in all the tested concentrations, however the cumin oil was cytotoxic in the concentration of 20 mg.mL-1 and the dill in the concentrations of 20 and 8 mg.mL-1. All yeasts were susceptible against the evaluated essential oils. Cumin presented the lowest MIC against yeasts. We concluded that all the essential oils presented inhibitory action against Candida spp., and C . cyminum, P. anisum and F. vulgare were not cytotoxic in the same minimum inhibitory concentrations for the fungi.(AU)
Os objetivos desta pesquisa foram: avaliar a composição química e a citotoxicidade dos óleos essenciais de Cuminum cyminum (cominho), Anethum graveolens (endro), Pimpinella anisum (erva-doce) e Foeniculum vulgare (funcho), bem como sua atividade antifúngica in vitro contra dez isolados de Candida spp.. A composição química dos óleos foi analisada por meio de cromatografia gasosa acoplada à espectrometria de massa (GC / MS). Os ensaios de citotoxicidade foram realizados, utilizando o reagente de proliferação celular WST-1 em fibroblastos de ratinho L929 (20x103 poço-1). A determinação da Concentração Inibitória Mínima (MIC) foi realizada através da técnica de microdiluição em caldo (CLSI). Os principais componentes químicos foram o cuminaldeído (32.66%) para cominho, carvona (34.89%) para o endro, trans-anetol (94.01%) para erva-doce e anetol (79.62%) para a funcho. O endro e a erva-doce não foram citotóxicos em todas as concentrações testadas, no entanto, o óleo de cominho foi citotóxico na concentração de 20 mg.mL-1 e o endro nas concentrações de 20 e 8 mg.mL-1. Todas as leveduras foram suscetíveis aos óleos essenciais avaliados. O cominho apresentou a menor CIM contra as leveduras. Concluímos que todos os óleos essenciais apresentaram ação inibidora contra Candida spp., e C. cyminum, P. anisum e F. vulgare não foram citotóxicos nas mesmas concentrações inibitórias mínimas para os fungos.(AU)
RESUMO
Streptococcus pneumoniae (pneumococcus) is a bacterium responsible for a wide spectrum of illnesses. The surface of the bacterium consists of three distinctive membranes: plasmatic, cellular and the polysaccharide (PS) capsule. PS capsules may mediate several biological processes, particularly invasive infections of human beings. Prevention against pneumococcal related illnesses can be provided by vaccines. There is a sound investment worldwide in the investigation of a proteic antigen as a possible alternative to pneumococcal vaccines based exclusively on PS. A few proteins which are part of the membrane of the pneumococcus seem to have antigen potential to be part of a vaccine, particularly the PspA. A vital aspect in the production of the intended conjugate pneumococcal vaccine is the efficient production (in industrial scale) of both, the chosen PS serotypes as well as the PspA protein. Growing recombinant Escherichia coli (rE. coli) in high-cell density cultures (HCDC) under a fed-batch regime requires a refined continuous control over various process variables where the on-line prediction of the feeding phase is of particular relevance and one of the focuses of this paper. The viability of an on-line monitoring software system, based on constructive neural networks (CoNN), for automatically detecting the time to start the fed-phase of a HCDC of rE. coli that contains a plasmid used for PspA expression is investigated. The paper describes the data and methodology used for training five different types of CoNNs, four of them suitable for classification tasks and one suitable for regression tasks, aiming at comparatively investigate both approaches. Results of software simulations implementing five CoNN algorithms as well as conventional neural networks (FFNN), decision trees (DT) and support vector machines (SVM) are also presented and discussed. A modified CasCor algorithm, implementing a data softening process, has shown to be an efficient candidate to be part of an on-line HCDC monitoring system for detecting the feeding phase of the HCDC process.
Assuntos
Reatores Biológicos/microbiologia , Reatores Biológicos/estatística & dados numéricos , Escherichia coli/genética , Escherichia coli/imunologia , Redes Neurais de Computação , Vacinas Pneumocócicas/genética , Vacinas Pneumocócicas/imunologia , Algoritmos , Carga Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Técnicas Bacteriológicas , Simulação por Computador , Epoetina alfa , Eritropoetina/genética , Eritropoetina/imunologia , Escherichia coli/crescimento & desenvolvimento , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Software , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Vacinas Conjugadas/genética , Vacinas Conjugadas/imunologiaRESUMO
The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.
Assuntos
Cápsulas Bacterianas/metabolismo , Reatores Biológicos/microbiologia , Meios de Cultura/metabolismo , Microbiologia Industrial/métodos , Vacinas Pneumocócicas , Streptococcus pneumoniae/crescimento & desenvolvimento , Cápsulas Bacterianas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Meios de Cultura/química , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Vacinas Pneumocócicas/genética , Vacinas Pneumocócicas/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismoRESUMO
OBJECTIVE: This study compared the postural control of small (SGA) and appropriate (AGA) for gestational age infants born at term, at the ages of 3, 6, 9 and 12 months. METHOD: This was a cohort study of infants born at term, with birth weight less than the 10th percentile for the SGA group and between the 10th and 90th percentiles for the AGA group. Infants with genetic syndromes, malformations, congenital infections and internment in neonatal intensive care unit were excluded. The evaluation instrument was the Alberta Infant Motor Scale. RESULTS: Comparison of the SGA and AGA groups did not show any significant differences (p>0.05) at the assessment times. However, the postural control of the SGA group was influenced by the gestational age (r=-0.83; p= 0.006) and 5th minute Apgar (r= 0.79; p= 0.01) in the 3rd month, and by maternal occupation (r= 0.67; p= 0.01) in the 6th month. CONCLUSION: It was concluded that the postural control was similar in the groups. However, the postural control of the SGA group was influenced by biological and environmental factors.
OBJETIVO: Este estudo teve por objetivo comparar o controle postural de lactentes nascidos a termo, pequenos (PIG) e adequados para a idade gestacional (AIG) nos 3º, 6º, 9º e 12º meses. MÉTODO: Estudo longitudinal de lactentes nascidos a termo, com peso de nascimento menor que o percentil 10 para o grupo PIG e entre o percentil 10 e 90 para o grupo AIG. Síndromes genéticas, más-formações, infecções congênitas e internados em unidade de terapia intensiva neonatal foram excluídos. O instrumento de avaliação foi Alberta Infant Motor Scale. RESULTADOS: A comparação do grupo PIG e AIG não mostrou diferença significativa (p> 0,05) nos meses avaliados. Entretanto, o controle postural do grupo PIG foi influenciado pela idade gestacional (r= -0,83/p= 0,006) e Apgar de 5' (r= 0,79/p= 0,01) no 3º mês; e pela ocupação materna (r= 0,67/p= 0,01) no 6º mês. CONCLUSÃO: Concluiu-se que o controle postural foi similar nos grupos; porém o controle postural do grupo PIG foi influenciado por fatores biológicos e ambientais.
Assuntos
Lactente , Desenvolvimento Infantil , Retardo do Crescimento Fetal , LactenteRESUMO
AIM: An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed-batch cultivation in bioreactor. METHODS AND RESULTS: Streptococcus pneumoniae serotype 23F was cultivated in a 5-l bioreactor with nitrogen-sparging and followed by addition of air in the stationary phase. The amount of CPS released in the supernatant progressively increased under air sparging. The profile of cellular viability and optical density was similar in both cultures. Immunoelectron microscopy showed that the amount of tightly cell-bound CPS was higher in bacteria cultivated under nitrogen than under air. CONCLUSIONS: The stress caused by the addition of air at the stationary phase promoted a large increase of free CPS into the medium, as a consequence of the morphologic change in the capsule. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of air in the stationary phase of the culture would greatly simplify the subsequent downstream process, allowing CPS purification from the supernatant. The direct consequence of this process improvement is the reduction of vaccine production costs.
Assuntos
Ar , Cápsulas Bacterianas/biossíntese , Streptococcus pneumoniae/metabolismo , Cápsulas Bacterianas/ultraestrutura , Biomassa , Reatores Biológicos , Contagem de Colônia Microbiana , Meios de Cultura , Glucose/metabolismo , Ácido Láctico/biossíntese , Microscopia Imunoeletrônica , Estresse Oxidativo , Vacinas Pneumocócicas/biossíntese , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/imunologiaRESUMO
Aiming at the industrial production of serogroup C meningococcal vaccine, different experimental protocols were tested to cultivate Neisseria meningitidis C and to investigate the related organic acid release. Correlations were established between specific rates of acetic acid and lactic acid accumulation and specific growth rate, during cultivations carried out on the Frantz medium in a 13 l bioreactor at 35°C, 0.5 atm, 400 rpm and air flowrate of 2 l min-1. A first set of nine batch runs was carried out: (1) with control of dissolved oxygen (O2) at 10% of its saturation point, (2) with control of pH at 6.5, and (3) without any control, respectively. Additional fed-batch or partial fed-batch cultivations were performed without dissolved O2 control, varying glucose concentration from 1.0 to 3.0 g l -1, nine of which without pH control and other two with pH control at 6.5. No significant organic acid level was detected with dissolved O 2 control, whereas acetic acid formation appeared to depend on biomass growth either in the absence of any pH and dissolved O2 control or when the pH was kept at 6.5. Under these last conditions, lactic acid was released as well, but it did not seem to be associated to biomass growth. A survey of possible metabolic causes of this behavior suggested that N. meningitidis may employ different metabolic pathways for the carbon source uptake depending on the cultivation conditions.