RESUMEN
Streptococcal pyrogenic exotoxins A (SPE-A) and B (SPE-B) have been implicated in the pathogenesis of serious group A streptococcal infections including streptococcal toxic shock-syndrome. Current antibiotics used for the treatment of these infections are penicillin and clindamycin. The effects of sub- and suprainhibitory concentrations of penicillin and clindamycin were evaluated in 14 isolates of Streptococcus pyogenes that were fully susceptible to both antibiotics. Clindamycin was superior to penicillin in reducing the production of SPE-A and SPE-B by invasive and non-invasive Dutch group A streptococcal isolates in vitro.
Asunto(s)
Proteínas Bacterianas , Clindamicina/farmacología , Exotoxinas/biosíntesis , Proteínas de la Membrana , Penicilinas/farmacología , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/metabolismo , Exotoxinas/análisis , Humanos , Pruebas de Sensibilidad Microbiana , Países Bajos , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/crecimiento & desarrolloRESUMEN
As part of a nationwide surveillance in The Netherlands during 1994-1997, 53 patients with invasive group A streptococcal (GAS) infections were evaluated for medical history, symptoms, and outcome. Patients' isolates were tested for the production of pyrogenic exotoxins A (SPE-A) and B (SPE-B). Acute-phase sera from all patients and convalescent sera from 12 patients were investigated for the presence of antibodies against SPE-A and SPE-B. Twenty-three patients developed toxic shock-like syndrome and 16 died. Absence of antibodies against SPE-A and/or SPE-B was a risk factor for developing invasive streptococcal disease. Toxic shock and mortality were associated with a lack of anti-SPE-A antibodies (P<.025). Anti-SPE-A antibodies were found in convalescent sera from all patients infected by speA-positive isolates. Virtually all invasive speA-positive streptococci expressed SPE-A protein in vitro. Thus antibodies against SPE-A appeared vital for mediating the outcome of invasive GAS disease in this population.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas , Exotoxinas/inmunología , Proteínas de la Membrana , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/aislamiento & purificación , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antibacterianos/inmunología , Bacteriemia/diagnóstico , Bacteriemia/epidemiología , Bacteriemia/microbiología , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Exotoxinas/biosíntesis , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Vigilancia de la Población , Pronóstico , Choque Séptico/diagnóstico , Choque Séptico/epidemiología , Choque Séptico/microbiología , Infecciones Estreptocócicas/diagnóstico , Infecciones Estreptocócicas/epidemiología , Streptococcus pyogenes/metabolismoRESUMEN
In this pilot study, we investigated the relative avidities for streptococcal pyrogenic exotoxin A (SPE-A) and SPE-B of antibodies in sera from patients with fatal streptococcal toxic shock-like syndrome and from healthy individuals and in intravenous immunoglobulin (IVIG) preparations. We observed a great variation in the relative avidities of patient, control, and IVIG immunoglobulin G (IgG) (values estimated to be between 10(-7) and 10(-11) M), with mean values for patient IgG about 10-fold lower than those of control IgG.
Asunto(s)
Afinidad de Anticuerpos/inmunología , Proteínas Bacterianas , Exotoxinas/inmunología , Inmunoglobulina G/inmunología , Proteínas de la Membrana , Infecciones Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Adulto , Anciano , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/sangre , Inmunoglobulinas Intravenosas , Persona de Mediana Edad , Proyectos Piloto , Infecciones Estreptocócicas/terapiaRESUMEN
Streptococcal pyrogenic exotoxin A (SPE-A) and SPE-B have been implicated in the pathogenesis of severe group A streptococcal (GAS) disease. We studied 31 invasive GAS strains including 18 isolates from patients with toxic shock syndrome and 22 noninvasive strains isolated in The Netherlands between 1994 and 1998. These strains were associated with the different allelic variants of the gene encoding SPE-A. We selected endemic strains with speA-positive M and T serotypes: speA2-associated M1T1 and M22-60T12 strains, speA3-associated M3T3 strains, and speA4-associated M6T6 strains. Since speA1-positive isolates were not frequently encountered, we included speA1 strains of different serotypes. The GAS strains were compared genotypically by pulsed-field gel electrophoresis and phenotypically by the in vitro production of SPE-A and SPE-B. All strains within one M and T type appeared to be of clonal origin. Most strains produced SPE-A and SPE-B, but only a minority of the speA4-positive isolates did so. Among our isolates, speA1- and speA3-positive strains produced significantly more SPE-A than speA2- and speA4-carrying strains, while SPE-B production was most pronounced among speA1- and speA2-containing strains. There was a marked degree of variability in the amounts of exotoxins produced in vitro by strains that shared the same genetic profile. We conclude that the differences in the in vitro production of SPE-A and SPE-B between our selected strains with identical M and T types were not related to either genetic heterogeneity or the clinical course of GAS disease in the patient from whom they were isolated.
Asunto(s)
Proteínas Bacterianas , Exotoxinas/biosíntesis , Exotoxinas/genética , Genes Bacterianos , Proteínas de la Membrana , Pirógenos/biosíntesis , Pirógenos/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidad , Alelos , Técnicas de Tipificación Bacteriana , Secuencia de Bases , Electroforesis en Gel de Campo Pulsado , Variación Genética , Humanos , Países Bajos , Sondas de Oligonucleótidos/genética , Serotipificación , Choque Séptico/microbiología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/clasificaciónRESUMEN
We used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcus Moraxella (Branhamella) catarrhalis. Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) of M. catarrhalis. HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitive M. catarrhalis strains. Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions. This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.
Asunto(s)
Anticuerpos Antivirales/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Bacteriófagos/inmunología , Proteínas del Sistema Complemento/inmunología , Moraxella catarrhalis/inmunología , Moraxella catarrhalis/virología , Anticuerpos Antivirales/genética , Bacteriófagos/genética , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Citometría de Flujo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/inmunología , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Recently, we showed that complement resistance is an important virulence factor of Moraxella (Branhamella) catarrhalis. Our study used a serum bactericidal assay to determine complement resistance in M. catarrhalis. Although the serum bactericidal assay is considered the "gold standard" for determining complement resistance, it is laborious and time-consuming and therefore not well suited for large-scale studies. Using a large number (n = 324) of M. catarrhalis isolates obtained from the sputa of patients with lower respiratory tract infections (n = 200) and young carriers (n = 124), we assessed the value of a simple "culture-and-spot" test as an alternative to the serum bactericidal assay. For both groups of isolates, the degree of concordance between the two tests used was very significant (P < 0.0001). The agreement between the two assays was estimated to be "excellent beyond chance" (as determined by Cohen's kappa test). The culture-and-spot assay is a valuable alternative to the serum bactericidal assay, not only for screening purposes as shown here but also for studying the mechanism of complement resistance in M. catarrhalis at the molecular level.
Asunto(s)
Moraxella catarrhalis/inmunología , Técnicas Bacteriológicas , Citotoxicidad Inmunológica , Humanos , Inmunidad Innata , Métodos , Moraxella catarrhalis/patogenicidad , Esputo/inmunología , Esputo/microbiología , Virulencia/inmunologíaRESUMEN
The mechanism of resistance to human complement-mediated killing in Moraxella catarrhalis was studied by comparing different complement-sensitive and complement-resistant M. catarrhalis strains in a functional bystander hemolysis assay and an enzyme-linked immunosorbent assay (ELISA) for soluble terminal complement complexes. Complement-resistant stains appeared to activate complement to the same extent as, or even slightly better than, complement-sensitive strains. This indicates that complement-resistant strains do not inhibit classical or alternative pathway activation but interfere with complement at the level of membrane attack complex formation. A clear difference in dose-response curves for resistant and sensitive strains was observed both in the bystander hemolysis assay and in the ELISA. Complement-resistant strains showed optimum curves, whereas complement-sensitive strains gave almost linear curves. We conclude that resistant strains bind and/or inactivate one of the terminal complement components or intermediates involved in membrane attack complex formation. Trypsin, known to abolish complement resistance, changed the optimum dose-response curve of a resistant strain to a linear one, which strongly suggests that complement resistance is mediated by an M. catarrhalis-associated protein. This protein acts directly or through the binding of a terminal complement inhibitor present in serum.
Asunto(s)
Activación de Complemento , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Moraxella catarrhalis/inmunología , Proteínas Bacterianas/inmunología , Citotoxicidad Inmunológica , Humanos , Técnicas In Vitro , Cinética , Especificidad de la EspecieRESUMEN
A specific monoclonal antibody against toxin A from Clostridium difficile was generated that did not show thermolabile binding. Nonspecific murine monoclonal antibodies bound toxin A at 4 degrees C, but less effectively at 37 degrees C. Nonspecific human monoclonal antibodies did not bind to toxin A at 4 degrees C. Cytotoxic properties of purified toxin A were not inhibited by Bandeiraea simplicifolia lectin. This points to a carbohydrate moiety on the cell surface and a multivalent nonspecific carbohydrate binding ligand on toxin A.
Asunto(s)
Toxinas Bacterianas/metabolismo , Metabolismo de los Hidratos de Carbono , Clostridioides difficile , Enterotoxinas/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Especificidad de Anticuerpos , Sitios de Unión , Células Cultivadas , Chlorocebus aethiops , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Células VeroRESUMEN
A simple discriminative typing method for Clostridium difficile has been developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and restriction enzyme analysis are relatively simple techniques but are difficult to evaluate, especially the restriction enzyme analysis. Immunoblotting and restriction fragment length polymorphism typing facilitate simple discrimination of patterns.
Asunto(s)
Técnicas de Tipificación Bacteriana , Clostridioides difficile/clasificación , Niño , Clostridioides difficile/genética , Clostridioides difficile/inmunología , Enzimas de Restricción del ADN , Humanos , Immunoblotting , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
A hybridization assay for detection of toxigenic Clostridium difficile in fecal samples was developed and compared with the classical tissue culture cytotoxicity assay. A DNA fragment probe specific for the toxin B gene of Clostridium difficile was synthesized by the polymerase chain reaction and labelled with digoxigenin. Fecal samples were cultured for 24 hours, replica-plated and hybridized with the probe. The hybridization assay had a sensitivity of 100%, specificity of 96.7%, positive predictive value of 86.7% and negative predictive value of 100% compared with the cytotoxicity assay.
Asunto(s)
Clostridioides difficile/aislamiento & purificación , ADN Bacteriano/análisis , Diarrea/microbiología , Heces/microbiología , Hibridación de Ácido Nucleico , Secuencia de Bases , Pruebas Inmunológicas de Citotoxicidad , ADN Bacteriano/genética , Humanos , Recién Nacido , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
To assess the role of enterovirulent Escherichia coli in The Netherlands, faecal samples of 279 patients (108 children, 171 adults) with diarrhoea and 100 healthy controls were investigated in a prospective study. Enterovirulent Escherichia coli were identified by hybridization with five different non-radioactively labelled DNA probes specific for enteropathogenic Escherichia coli (EPEC), verocytotoxin producing Escherichia coli (VTEC) and enterotoxigenic Escherichia coli (ETEC). The rate of isolation of EPEC was 6.5% in patients with diarrhoea and 2.0% in asymptomatic persons. During the study period, no VTEC were isolated from patients with diarrhoea. ETEC were isolated from two persons, both of whom had experienced diarrhoea and had returned from travel in (sub)tropical areas. Our results suggest that diarrhoea is sporadically caused by ETEC among the indigenous population of The Netherlands, and is mainly associated with travel in endemic areas. Furthermore, the presence of EPEC probe-positive strains in the stool need not always be accompanied by symptoms of diarrhoea.
Asunto(s)
Diarrea Infantil/microbiología , Diarrea/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/patogenicidad , Adulto , Preescolar , Sondas de ADN , Diarrea/epidemiología , Diarrea Infantil/epidemiología , Enterotoxinas/biosíntesis , Escherichia coli/clasificación , Escherichia coli/aislamiento & purificación , Infecciones por Escherichia coli/epidemiología , Heces/microbiología , Humanos , Lactante , Países Bajos/epidemiología , Hibridación de Ácido Nucleico , Estudios Prospectivos , Serotipificación , VirulenciaRESUMEN
To assess the role of diarrhoeagenic Escherichia coli in Southern Spain, faecal samples from 135 patients with diarrhoea and 40 healthy subjects from Seville, Andalusia, were investigated. In this prospective study, enterovirulent E. coli were identified by hybridisation with five non-radioactive DNA probes specific for enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC). Probe-positive strains were isolated from four patients (3%) with diarrhoea and from none of the healthy controls. Two patients harboured ETEC and two patients had EPEC probe-positive strains in their faeces. No VTEC were isolated during this study. Salmonella spp. were the most frequently identified enteric pathogens, accounting for 10% of the cases, followed by Campylobacter jejuni (3%) and diarrhoeagenic E. coli (3%). This study indicates that enterovirulent E. coli play a modest role in the aetiology of diarrhoea among the indigenous population of Southern Spain.
Asunto(s)
Sondas de ADN , Diarrea/microbiología , Infecciones por Escherichia coli/microbiología , Escherichia coli/aislamiento & purificación , Adulto , Niño , Diarrea/epidemiología , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/epidemiología , Humanos , Estudios Prospectivos , España/epidemiologíaRESUMEN
We examined the expression, the signal transduction capacity and mouse IgG-isotype specificity of CD16 on human gamma delta T cells. CD16 is expressed by the majority of gamma delta T cells in peripheral blood and by part of the gamma delta T cell clones. The amount of CD16 expressed on gamma delta T cell clones varied considerably with passaging of the cells, but was always significantly less than on freshly isolated gamma delta T cells. Like CD16 on CD3- CD16+ natural killer (NK) cells, CD16 on gamma delta T cells can act as an activation site triggering cytotoxic activity. CD16+ gamma delta T cell clones exerted antibody-dependent cellular cytotoxicity (ADCC) which could be blocked by anti-CD16 mAb. ADCC activity of gamma delta T cell clones was also inhibited by anti-CD3 mAb, suggesting a functional linkage between the CD16 and CD3 activation pathways. MAb directed against CD16 induced lysis of Fc gamma R+ target cells by CD16+ gamma delta T cell clones. The mouse IgG-isotype specificity of CD16 on gamma delta T cells was analyzed using isotype switch variants of a murine anti-glycophorin A mAb in EA rosette assays, and was found to be identical to that of CD16 on CD3- CD16+ NK cells, i.e., highest affinity for mIgG2a, intermediate affinity for mIgG2b, and undetectable binding of mIgG1-sensitized erythrocytes. CD16 was partly modulated from the cell surface of both gamma delta T cells and NK cells after rosette formation with mIgG2a-sensitized erythrocytes, indicating that the rosette formation was indeed mediated via the CD16 molecule.
Asunto(s)
Antígenos de Diferenciación/análisis , Epítopos , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Receptores Fc/análisis , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Inmunológica , Humanos , Ratones , Receptores de Antígenos de Linfocitos T gamma-delta/fisiología , Receptores de IgGRESUMEN
A total of 39 toxigenic and 20 nontoxigenic strains of Clostridium difficile were tested for the presence of either toxin A or toxin B by the polymerase chain reaction (PCR). All toxigenic strains produced cytotoxin as assayed by using highly sensitive fetal lung fibroblasts and were positive for toxin A as well as toxin B in the PCR assay. All nontoxigenic strains failed to produce toxin and were negative in the PCR assay. This study shows that nontoxigenic strains of Clostridium difficile lack the toxin A as well as the toxin B gene.
Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Enterotoxinas/genética , Secuencia de Bases , Clostridioides difficile/aislamiento & purificación , ADN Bacteriano/genética , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
We have shown previously that certain proteases can modulate the affinity of human Fc gamma RII for IgG. To study whether proteolytic events not only increase FcR affinity, but are essential for Fc gamma R functioning, we evaluated the effect of different protease inhibitors on binding mediated by two classes of human monocyte IgG FcR. These R, Fc gamma RI and Fc gamma RII, can be analyzed selectively in rosetting assays by employing E sensitized by either human IgG or mouse IgG1. Rosetting by both classes of R was inhibited profoundly by incubation of monocytes with different types of serine protease inhibitors such as diisopropylfluorophosphate, PMSF, or N alpha-tosyl-L-lysyl-chloromethylketone. The type II Fc gamma R was much more sensitive to inhibition than Fc gamma RI. We, therefore, studied these effects in more detail by using cell line K562, which expresses only Fc gamma RII. PMSF, diisopropylfluorophosphate, and N alpha-tosyl-L-lysyl-chloromethylketone were, again, inhibiting Fc gamma RII-mediated binding dose-dependently, whereas several inhibitors of metal, aspartic, or thiol proteases proved ineffective. Furthermore, Fc gamma RII-mediated rosetting on both cell types was profoundly inhibited by the addition of different small synthetic substrates of serine esterases. In an attempt to discriminate whether the proteolytic event is an intra- or extracellular process, macromolecular antiproteases such as soybean or ovomucoid trypsin inhibitor or alpha 1-antiprotease were tested. Fc gamma RII-mediated binding by K562 cells was not susceptible to macromolecular antiproteases, in contrast to monocytes. In the presence of drugs which interfere both with receptor recycling and intracellular traffic between endosomal compartments (e.g., primaquine or monensin), the effects of inhibitors were largely abrogated. This showed that endocytosis of inhibitors might be essential, indicating the proteolytic event to be intracellular. Our findings suggest that human monocyte Fc gamma RII-mediated functioning is dependent upon the action of one or more serine proteases.
Asunto(s)
Antígenos de Diferenciación/fisiología , Monocitos/fisiología , Inhibidores de Proteasas/farmacología , Receptores Fc/fisiología , Línea Celular , Humanos , Inmunoglobulina G/metabolismo , Técnicas In Vitro , Lisina/análogos & derivados , Lisina/farmacología , Monensina/farmacología , Primaquina/farmacología , Receptores de IgG , Formación de Roseta , Inhibidores de Serina Proteinasa , Tosilarginina Metil Éster/farmacología , Clorometilcetona Tosilisina/farmacología , Tirosina/análogos & derivados , Tirosina/farmacologíaRESUMEN
A Streptococcus pneumoniae type 14-specific ELISA and ELISPOT assay have been developed based on the use of biotinylated type 14 capsular polysaccharide (S14PS-biotin). A major advantage of this application over other methods is the use of 10-100-fold less antigen than that reported in the literature for other similar assays. Moreover, the prepared biotinylated polysaccharides are very stable and it is possible to use the same procedures for other pneumococcal polysaccharide antigens (e.g., S6BPS) with no major changes necessary in the ELISA and ELISPOT protocols. Furthermore, a simple thin layer chromatography method has been developed as a method for quality control of the biotinylated polysaccharide. Immunization with the thymus-independent antigen S14PS resulted in the induction of IgM spot-forming cells (SFC) and antibodies while S14PS-protein conjugates induced a thymus-dependent response. The immune response to the conjugates was enhanced by the addition of the adjuvant Quil A resulting in high levels of both IgG SFC and antibodies at day 14 after immunization. The developed assays are reliable and reproducible tools for studying the humoral immune response against Streptococcus pneumoniae type 14 capsular polysaccharide derived antigens.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Ensayo de Inmunoadsorción Enzimática , Streptococcus pneumoniae/inmunología , Animales , Antígenos Bacterianos/inmunología , Avidina , Biotina , Femenino , Inmunoglobulina G/análisis , Ratones , Ratones Endogámicos BALB CRESUMEN
Nonionic block polymer surfactants (NBPs) were tested for the capacity to stimulate the antibody response against hexasaccharide (HS), derived from Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS), which was conjugated to proteins. The immune response was evaluated in the (CBA/N x BALB/c)F1 progeny, in which female mice are phenotypically normal whereas male mice carry an X-chromosome-linked immunodeficiency. NBPs L101, L121, 1101, and 1501 were able to increase anti-HS immunoglobulin M (IgM) and IgG levels in both normal and X-chromosome-linked immunodeficient mice (with up to 74-fold stimulation of antibody titers). Distribution of S3PS-specific antibodies over the various IgG isotypes was restricted after immunization with either HS-bovine serum albumin or HS-keyhole limpet hemocyanin (HS-KLH). Addition of NBPs (in particular 1501) resulted in a more diverse immune response with either antigen as judged by isotype distribution. Isoelectric focusing of individual sera and subsequent detection of S3PS-binding antibodies in these sera by immunochemical staining revealed a restricted number of different spectrotypes in the course of the immune response. Upon immunization of mice with HS-KLH, spectra of secreted antibodies were slightly more complex and more densely stained than after immunization with HS-bovine serum albumin. Furthermore, NBPs 1101 and 1501 appeared to be able to stimulate the secretion of antibodies, which were secreted only in small amounts without the use of NBPs. Different explanations for increased spectrotype diversity after immunization with KLH as the carrier and after administration of NBPs as the adjuvant are discussed.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Anticuerpos Antibacterianos/biosíntesis , Proteínas Portadoras/inmunología , Polímeros/farmacología , Polisacáridos Bacterianos/inmunología , Streptococcus pneumoniae/inmunología , Tensoactivos/farmacología , Animales , Anticuerpos Antibacterianos/análisis , Antígenos Bacterianos/inmunología , Sitios de Unión de Anticuerpos , Femenino , Hemocianinas/inmunología , Inmunoglobulina G/biosíntesis , Isotipos de Inmunoglobulinas/biosíntesis , Inmunoglobulina M/biosíntesis , Focalización Isoeléctrica , Masculino , Ratones , Ratones Endogámicos CBA , Albúmina Sérica Bovina/inmunologíaRESUMEN
Streptococcus pneumoniae type 14 capsular polysaccharide-bovine serum albumin (S14PS-BSA) conjugates were prepared by water-soluble-carbodiimide-mediated condensation with or without the use of N-hydroxy-sulfosuccinimide. The immunogenicities of the capsular polysaccharide (S14PS) and of the conjugates were studied in (CBA/N x BALB/c)F1 mice and in female BALB/c mice. The response in these mice indicates that S14PS could be classified as a thymus-independent type 2 antigen. Coupling of S14PS to BSA improved the immunogenicity of this polysaccharide, and an immunoglobulin G memory response was evoked. Conjugation with N-hydroxysulfosuccinimide resulted in a product with a higher polysaccharide/protein ratio. This conjugate induced a greater immune response than did the classical conjugate. Quil A enhanced the immune response to S14PS and to most S14PS-BSA conjugates. The enhancement of the immune response to the conjugates seemed to depend on the coupling procedure. Our results indicate that for the construction of immunostimulating complexes based on polysaccharide or oligosaccharide-protein conjugates, attention should be paid to the degree of cross-linking of the antigens involved.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Anticuerpos Antibacterianos/biosíntesis , Cápsulas Bacterianas , Proteínas Bacterianas/inmunología , Polisacáridos Bacterianos/inmunología , Saponinas/farmacología , Streptococcus pneumoniae/inmunología , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/administración & dosificación , Secuencia de Carbohidratos , Reactivos de Enlaces Cruzados , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Polisacáridos Bacterianos/administración & dosificación , Saponinas de Quillaja , Saponinas/administración & dosificaciónRESUMEN
Non-ionic block polymers (NBPs) have proved to be potent adjuvants for the humoral immune response against liposomes haptenated with tripeptide-enlarged dinitrophenyl groups (hapten J). Since both reversed triblocks and normal octablocks displayed adjuvant activity, reversed octablocks, in which structural properties of both groups are combined, were also tested for their adjuvant activity. The latter compounds displayed very strong adjuvant activity for J-haptenated liposomes, not only in normal BALB/c but also in (CBA/N x BALB/c)F1 progeny. To test the applicability of NBPs as adjuvants in semi-synthetic vaccines, the capacity of NBPs to stimulate the immune response against liposomes haptenated with Streptococcus pneumoniae type 3 capsular polysaccharide-derived oligosaccharides was analysed. In these studies, again NBPs proved potent adjuvants, stimulating antibody production to a large extent. In male (CBA/N x BALB/c)F1 mice, which carry a X-chromosome-linked immunodeficiency (Xid), antibody levels were stimulated to the largest extent by a normal octablock. Stimulation of antibody titres, however, did not result in increased protection in these Xid mice.