RESUMO
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
The toxicity of biomolecules obtained from sea anemones in vitro does not necessarily justify their function as toxins in the physiology of the anemone. That is why anatomical and physiological considerations must be taken into account in order to define their physiological role in the organism. In this work, antibodies generated to Sticholysin II, a cytolysin produced by the Caribbean Sea anemone Stichodactyla helianthus, are used as specific markers to explore the sites of production and storage of the cytolysin in the sea anemone. The immunoperoxidase staining developed gave specific dark-brown staining in tentacles and mesenteric filaments as well as in basitrichous nematocysts isolated from tentacles of S. helianthus. These results support the role of these proteins as toxins in the physiology of the anemone, especially in functions such as in predation, defense and digestion.
Assuntos
Venenos de Cnidários/metabolismo , Anêmonas-do-Mar/metabolismo , Animais , Venenos de Cnidários/biossíntese , Imuno-HistoquímicaRESUMO
Se estableció como objetivo la obtención de anticuerpos monoclonales (AcM), mediante la tecnología de fusión celular, capaces de reconocer a la fracción glucuronoxilomanano (GXM), serotipo A Cryptococcus neoformans es un hongo levaduriforme rodeado por una cápsula polisacarídica y agente de micosis profundas en el hombre, que afecta particularmente a los pacientes con el síndrome de inmunodeficiencia adquirida. Fueron inmunizados 3 grupos de ratones Balb/c con GXM acoplado a eritrocitos de carnero con una dosis de 10 µg de GXM vía subcutánea, 50 µ g de GXM vía subcutánea y 50 µ g de GXM vía intraperitoneal. Se obtuvieron 2 hibridomas secretores de AcM murinos contra el polisacárido capsular de Cryptococcus neoformans, de la clase IgG 1 , cadena ligera k , los sobrenadantes de cultivo fueron evaluados por ELISA utilizando el polisacárido capsular sin conjugar. Estos anticuerpos serán utilizados en estudios de protección y fagocitosis in vitro
Assuntos
Ratos , Animais , Anticorpos Monoclonais , Cryptococcus neoformans , PolissacarídeosRESUMO
Our objective was to obtain monoclonal antibodies (Mab), by using the cell fusion technology, capable of recognizing the serotype A glucuronoxylomannan (GXM) fraction. Cryptococcus neoformans is a yeastlike fungus surrounded by a polysaccharide capsule and an agent of deep mycosis in men that affects particularly AIDS patients. 3 groups of Balb/c mice were immunized with GXM coupled to lamb erythrocytes at a dose of 10 microg of GXM by subcutaneous route, 50 microg of GXM by subcutaneous route and 50 microg of GXM y intraperitoneal route. There were obtained 2 secreting hybridomas of murine MAb against the capsular polyssacharide of Cryptococcus neoformans, IgG1, light chain k. The overnadants of culture were evaluated by ELISA, using the capsular polyssacharide without conjugating. These antibodies will be used in studies of protection and phagocytosis in vitro.
Assuntos
Anticorpos Monoclonais , Antígenos de Fungos/imunologia , Cryptococcus neoformans/imunologia , Polissacarídeos/imunologia , Síndrome da Imunodeficiência Adquirida/complicações , Animais , Anticorpos Monoclonais/análise , Criptococose/complicações , Ensaio de Imunoadsorção Enzimática , Humanos , Hibridomas , Injeções Intraperitoneais , Injeções Subcutâneas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Polissacarídeos/administração & dosagemRESUMO
The immunogenicity of sticholysin II (St II), a pore-forming polypeptide from the sea anemone Stichodactyla helianthus, was studied in rabbits using two adjuvants, Freund's and aluminium hydroxide. High titres of antibodies were raised against St II with Freund's adjuvant (FA). The structural homology between sticholysins I and II was also revealed by cross-reactivity assays. Since the oil constituent of FA neutralized the St II haemolytic activity, immunizations with St II-Freund's emulsions were carried out with the inactivated cytolysin. Purified anti-St II IgG also neutralized the St II haemolytic activity.
Assuntos
Anticorpos/imunologia , Venenos de Cnidários/imunologia , Citotoxinas/imunologia , Adjuvante de Freund/farmacologia , Hemólise/efeitos dos fármacos , Anêmonas-do-Mar , Sialiltransferases/imunologia , Hidróxido de Alumínio/imunologia , Hidróxido de Alumínio/metabolismo , Animais , Cromatografia de Afinidade , Venenos de Cnidários/metabolismo , Reações Cruzadas/imunologia , Citotoxinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Adjuvante de Freund/imunologia , Adjuvante de Freund/metabolismo , Lipoproteínas/sangue , Lipoproteínas/metabolismo , Coelhos , Sialiltransferases/metabolismo , EspectrofotometriaRESUMO
A novel immunoenzymatic procedure for the quantitative determination of HIV protease activity is provided. An N-terminal biotinylated peptide (DU1) that comprises an HIV-1 protease (HIV-PR) cleavage sequence was bound to streptavidin-coated microtiter plates. The bound peptide can be quantified by an immunoenzymatic procedure (enzyme-linked immunosorbent assay, ELISA) that includes a monoclonal antibody (Mab 332) against the peptide (DU1) C-terminal. The incubation of the bound peptide with HIV-PR in solution resulted in a signal decrement, as the peptide was hydrolyzed and the released C-terminal segment washed away. An equation that relates the amount of added enzyme to the kinetics of the reaction was written in order to describe this heterogeneous enzyme-quasi-saturable system. This equation allows quantitative determination of protease activity, a feature widely underrated in previous similar assays. The assay also allows evaluation of the inhibitory activity of HIV-PR inhibitors. Due to the intrinsic advantages of the ELISA format, this method could be used in high-throughput screening of HIV protease inhibitors. The assay can be extended to other proteolytic enzymes.
Assuntos
Protease de HIV/análise , HIV-1/enzimologia , Técnicas Imunoenzimáticas , Biotinilação , Cromatografia Líquida de Alta Pressão , Anticorpos Anti-HIV/imunologia , Inibidores da Protease de HIV/farmacologia , HumanosRESUMO
Twenty-six human respiratory syncytial virus strains (subgroup A) isolated from three outbreaks in Havana City during the period 1994/95, 1995/96 and 1996/97 were analyzed to determine their antigenic and genetic relationships. Analyses were performed by monoclonal antibodies and restriction mapping (N gene) following amplification of the select region of the virus genome by polymerase chain reaction. All isolated strains were classified as subgroup A by monoclonal antibodies and they showed a restriction pattern NP4 that belonged to subgroup A. Thus the results obtained in this work, showed a close relation (100 percent) between antigenic and genetic characterization of the isolated strains in our laboratory. These methods permit the examination of large numbers of isolates by molecular techniques, simplifying the researchs into the molecular epidemiology of the virus
Assuntos
Embrião de Galinha , Criança , Lactente , Anticorpos Monoclonais/análise , Anticorpos Antivirais/análise , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Cuba/epidemiologia , Surtos de Doenças , Reação em Cadeia da Polimerase , Infecções por Vírus Respiratório Sincicial , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Mapeamento por RestriçãoRESUMO
A pesar de los avances en el diagnóstico y la intervención temprana con antibióticos, es alta la morbilildad y mortalidad asociada con la sepsis por bacterias gramnegativas. Los mediadores responsables de la patogénesis de la sepsis son componentes derivados de las propias bacterias (endotoxinas) y de las células de la respuesta inmune del hospedero (factor de necrosis tumoral y algunas interleuquinas). El tratamiento que tradicionalmente se ha utilizado en la sepsis, está dirigido sobre todo contra el microorganismo, mediante el uso de antibióticos cada vez más potentes. Sin embargo, está claro que los antibióticos no constituyen una solución definitiva, ya que aun si provocan la muerte bacteriana, no tienen efectos sobre la endotoxina y pueden aumentar su liberación cuando ocurre la lisis celular. A partir de la década de los 80 se han probado nuevos y exitosos tratamientos para la sepsis, que incluyen el uso de anticuerpos policlonales y monoclonales de origen murino y humano, dirigido contra el lípido A de la endotoxina, así como anticuerpos monoclonales contra el factor de necrosis tumoral. Si bien en todos los casos no se puede hablar de una total eficacia de estas moléculas para interrumpir la cadena de acontecimientos indeseables provocados por la endotoxina, sí es válido celebrar el advenimiento de inmunoterapias como tratamiento adjunto para una condición que amenaza la vida
Assuntos
Humanos , Antibacterianos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Endotoxinas/farmacologia , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/etiologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The aim of this study was to develop a polymerase chain reation (PCR) for detection of respiratory syncytial virus (RSV) genomes. The primers were designed from published sequences and selected from conserved regions of the genome encoding for the N protein of subgroups A and B of RSV. PCR was applied to 20 specimens from children admitted to the respirary ward of "William Soler" Pediatric Hospital in Havana City with a clinical diagnosis of bronchiolitis. The PCR was compared with viral isolation and with an indirect immunofluorescence technique that employs monoclonal antibodies of subgroups A and B. Of 20 nasopharyngeal exudates, 10 were found positive by the three assayed methods. In only two cases, samples that yielded positive RNA-PCR were found negative by indirect immunofluorescence and cell culture. Considering viral isolation as the "gold standard" technique, RNA-PCR had 100 per cent sensitivity and 80 per cent specificity. RNA-PCR is a specific and sensitive technique for the detection of the RSV genome. Technical advantages are discussed.
Assuntos
Humanos , Criança , Reação em Cadeia da Polimerase , Vírus Sinciciais Respiratórios , Bronquiolite/diagnóstico , Cuba , Mapeamento por RestriçãoRESUMO
Se purificó una inmunoglobulina G de ratón a partir de suero por cromatografía de afinidad en proteína A. Con esta preparación se inmunizaron los conejos cuyos sueros fueron capaces de reconocer al antígeno inyectado mediante inmunodifusión doble. Los anticuerpos fueron precipitados del suero de conejo y purificados mediante cromatografía de intercambio iónico. Esta preparación fue conjugada a isotiocianato de fluorescencia según la tecnología convencional. El conjugado obtenido fue evaluado con las cepas de referencia de virus Parainfluenza 1, 2, 3; Adenovirus; virus sincitial respiratorio y virus influenza A y B por una técnica de inmunofluorescencia indirecta y muestras positivas de VIH mediante citometría de flujo. En ambos casos se utilizaron anticuerpos monoclonales específicos. Se evaluaron muestras clínicas de pacientes con infección respiratoria aguda