RESUMO
Rabbits were immunized with immunotype L3,7,9 phosphoethanolamine (PEA) group containing oligosaccharide-tetanus toxoid conjugates both with and without the addition of the adjuvant Quil A. The epitope specificity of the antibodies present in these antisera was analysed in an immunotype L2 and L3,7,9 specific inhibition ELISA using the homologous and heterologous lipopolysaccharide, oligosaccharide and partial dephosphorylated oligosaccharide as inhibitors. Two groups of antisera could be identified. In one group of antisera, at least two antibody populations are present, namely directed against the PEA group containing determinants on immunotype L3,7,9 lipopolysaccharide and against immunotype L2 specific epitopes in which no PEA group is present. In the second group of antisera, one but probably more antibody populations are detected with a similar specificity towards the conserved epitopes of both immunotypes. In general, immunization with the conjugates only resulted in the induction of antibodies against the PEA group containing epitopes on the L3,7,9 lipopolysaccharide (80%). Antibodies directed against the conserved epitopes of both immunotypes are mainly evoked with the conjugates in combination with the adjuvant Quil A (80%). Although these results suggest that the epitope specificity of the antibodies induced depends on the use of Quil A, the influence of genetic factors cannot be excluded. At the moment it is not known whether the differences in epitope specificities are reflected in biological function of these antibodies. However, the induction of antibodies with clearly different epitope specificities after immunization of different rabbits with the same antigen stresses the importance of this kind of analysis when developing a vaccine based on oligosaccharide-protein conjugates.
Assuntos
Especificidade de Anticorpos/efeitos dos fármacos , Carboidratos/imunologia , Epitopos/efeitos dos fármacos , Imunoglobulina G/biossíntese , Neisseria meningitidis/imunologia , Polissacarídeos Bacterianos/imunologia , Saponinas/farmacologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Sequência de Carboidratos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Etanolaminas/imunologia , Feminino , Imunização , Imunofenotipagem , Dados de Sequência Molecular , Saponinas de Quilaia , CoelhosRESUMO
Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycobacterium tuberculosis were identified by the use of lipopeptides and their cytokine profile studied. Selection of candidate CTL epitopes was based on synthetic peptides derived from the amino acid sequence of the 19-kDa lipoprotein showing major histocompatibility complex class I (MHC-I) binding motifs (H-2D(b) and H-2L(d)). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studies were performed with peptides, in which the anchor amino acid of the H-2D(b) MHC-I motif was replaced by alanine. Three out of five peptides with H-2D(b) or H-2L(d) binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D(b) molecules on RMA-S cells. Replacement of the anchor amino acid residues of the H-2D(b) MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed more to binding of peptide to H-2D(b) molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with incomplete Freund's adjuvant (IFA), induced CTL responses in C57BL/6 (H-2(b)) mice. These CTLs could recognize the naturally processed antigen, i.e. the 19-kDa antigen protein produced and processed by the EX-19 cell line. The capacity of the various lipopeptides to induce CTL correlated well with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D(b) molecules. Lipopeptide LP19c primed spleen cells showed a T helper type one profile after in vitro stimulation with P19c and P19d 19 kDa peptides. The approach to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subunit tuberculosis vaccines.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Lipoproteínas/imunologia , Mycobacterium tuberculosis/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Cromo/metabolismo , Citotoxicidade Imunológica/imunologia , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Interferon gama/análise , Interleucina-4/análise , Lipoproteínas/química , Lipoproteínas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Linfócitos T Citotóxicos/metabolismo , Células Tumorais Cultivadas , Regulação para CimaRESUMO
The reliability of the determination of antibody avidity in polyclonal sera by indirect sandwich ELISA was studied. Binding of IgM and IgG (sub)classes in unpurified serum to Streptococcus pneumoniae type 3 capsular polysaccharide, which was coated onto ELISA plates, was inhibited with different inhibitors. The inhibitor concn at which 50% inhibition of antibody binding to the ELISA coat was achieved, was used as a measure for antibody avidity. As this 50% inhibition value is dependent upon the dilution of the serum and thus upon the initial amount of free antibody, it is necessary to define (a narrow range of) final ELISA absorbance values to which the dilutions of non-inhibited sera have to be adjusted. The shapes of the serum dilution curves have a good correlation with the numerical 50% inhibition values of the antibody avidity. The inhibition ELISA is suitable to compare the avidity values of the different antibody isotypes, but two remarks should be made: (1) antibody heterogeneity should be considered to influence the results and prevent the accurate measurement of absolute numerical avidity values. Because in the ELISA system merely antibody "activity" is measured, comparison of the efficacy of vaccines by means of the 50% inhibition (avidity) value of various antibody (sub)classes can still be performed in a reliable way; (2) results of the determination of the 50% inhibition values of the different antibody (sub)classes showed them to be dependent on the molecular ratio between antibody (sub)class levels. More aspects of the determination should be taken into account, like shapes of simple dilution curves, influences of various inhibitor concns in the diluent and whole (extended) inhibition curves.
Assuntos
Anticorpos Antibacterianos/imunologia , Afinidade de Anticorpos , Ensaio de Imunoadsorção Enzimática/métodos , Streptococcus pneumoniae/imunologia , Animais , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Camundongos , Camundongos EndogâmicosRESUMO
OBJECTIVE: To investigate the possible role of Cryptococcus neoformans in HIV-1 pathogenesis. DESIGN: An in vitro system was developed to study HIV-1 replication in freshly HIV-1-infected peripheral blood mononuclear cells (PBMC) incubated with whole azide-killed C. neoformans. METHODS: Human PBMC or peripheral blood lymphocytes were infected with lymphocytotropic HIV-1 and incubated with azide-killed encapsulated or non-encapsulated C. neoformans for 10 days. Viral replication was followed by HIV-1 p24 enzyme-linked immunosorbent assay and median tissue culture infective dose determination. Tumour necrosis factor (TNF) release by PBMC, induced by C. neoformans, was measured. Anti-TNF monoclonal antibodies or pentoxifylline were used to inhibit TNF bioactivity. RESULTS: Both encapsulated and non-encapsulated C. neoformans enhanced HIV-1 replication in PBMC but not in peripheral blood lymphocytes. C. neoformans induced TNF release by PBMC. Inhibition of TNF bioactivity did not block C. neoformans-enhanced HIV-1 replication in PBMC. CONCLUSIONS: C. neoformans can enhance HIV-1 replication in T cells only in the presence of monocytic cells. This enhancement is not dependent on encapsulation nor can it be attributed to TNF release.
Assuntos
Cryptococcus neoformans/fisiologia , HIV-1/fisiologia , Monócitos/microbiologia , Fator de Necrose Tumoral alfa/metabolismo , Anticorpos Monoclonais/imunologia , Células Cultivadas , Humanos , Monócitos/citologia , Monócitos/fisiologia , Pentoxifilina/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Replicação ViralRESUMO
Cytolytic T cells (CTL) play a critical role in providing protection against the liver stage of malaria infection. Previous investigations have shown that induction of CTL against peptide or proteins can be achieved by attachment of lipids. In the present study, we used the Plasmodium berghei circumsporozoite protein CTL epitope (SYIPSAEKI (PL76)). This peptide with cysteine-serine (CS) as spacer amino acids was coupled to palmitic acid (PA). The same CTL epitope containing only an extra serine was linked to S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-cysteine (tripam-C). Inbred mice [(BALB/c x C57BL/6)F1] were immunized intravenously with the lipopeptides. Both types of lipopeptides induced significant CTL responses after one injection. Immunization of the monopalmitic acid-peptide conjugate intraperitoneally emulsified in Freund's complete adjuvant also induced a significant CTL response, but the magnitude was lower as compared to the intravenous route. The major advantages of the use of the simple monopalmitic acid-peptide conjugates are: (i) low costs of the fatty acid; (ii) coupling of lipid to peptide can be performed using the peptide synthesizer during standard peptide synthesis, and (iii) standard peptide methodology can be used for purification. To investigate whether a spacer amino acid sequence between the actual CTL epitope and PA is required for induction of an optimal CTL response, we prepared monopalmitic acid-peptide conjugates with different spacer amino acids. A lipopeptide without a spacer amino acid and another one containing the CS spacer sequence both induced a CTL response, whereas a lipopeptide with a serine as spacer failed to induce CTL. These results indicate that the amino acid spacer sequences influence the immunological properties of the palmitic acid-peptide conjugates.
Assuntos
Imunoconjugados/farmacologia , Técnicas Imunológicas , Ácidos Palmíticos/farmacologia , Plasmodium berghei/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Citotoxicidade Imunológica , Epitopos/genética , Feminino , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Oligopeptídeos/farmacologia , Ácido Palmítico , Plasmodium berghei/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/farmacologiaRESUMO
A sensitive ELISA has been developed to study immune responses in mice against Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS) and hexasaccharide (HS)-protein conjugates derived therefrom. An advantage of the described system is that the same microtiter plates can be used for both ELISA and ELISPOT tests with a standardized washing procedure and diluent composition. S3PS induced predominantly IgM antibodies and minute amounts of IgG as measured by ELISA in serum. This was accompanied by large numbers (greater than 14000) of IgM spot-forming cells in the spleen. A shift towards IgG production was achieved by addition of lipid A. HS-protein conjugates induced predominantly IgG antibodies after booster immunization(s). Furthermore these conjugates induced large numbers (greater than 40000) of IgG spot-forming cells (SFC) in the spleen. ELISA and ELISPOT assays on microtiter plates are both reliable and highly reproducible assays for the evaluation of immune responses to S. pneumoniae antigens.
Assuntos
Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática , Oligossacarídeos/imunologia , Polissacarídeos Bacterianos/imunologia , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/análise , Especificidade de Anticorpos , Células Produtoras de Anticorpos , Antígenos de Bactérias/análise , Antígenos de Bactérias/normas , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Contagem de Leucócitos , Camundongos , Polissacarídeos Bacterianos/normasRESUMO
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.
Assuntos
Anticorpos Antibacterianos/análise , Ensaio de Imunoadsorção Enzimática , Streptococcus pneumoniae/imunologia , Animais , Antígenos de Bactérias/imunologia , Avidina , Biotina , Feminino , Imunoglobulina G/análise , Camundongos , Camundongos Endogâmicos BALB CRESUMO
A noninternal image monoclonal antiidiotypic antibody (ab2 mAb), designated 1.13A321, that had proved its efficacy as vaccine against infection with Semliki Forest virus (SFV) in BALB/c mice, was used as immunogen to generate a panel of SFV-neutralizing monoclonal anti-anti-idiotypic antibodies (ab3 mAbs) to compare them genetically with ab1 mAb 1.13 (IgG2a). There are various studies that compare ab1 and ab3 mAbs but none that compare virus-neutralizing ab1 and ab3 mAbs. Five SFV-neutralizing ab3 MAbs, all IgG1, were obtained. The Vh gene (36-60), the D gene (Sp2), and the J gene (Jh2) encoding the heavy chain variable regions of all six mAbs, were similar and showed a high homology in the nucleotide sequence. The CDR3 amino acid sequences of four of five ab3 mAbs were identical to that of mAb1. One ab3 differed one amino acid in the CDR3 region. The results suggest that a strict selection criterion (virus neutralization) is sufficient to reach complete homology in the CDR3 region of mAb3. Future experiments are focused on selection of synthetic peptides in the CDR3 region as neutralizing mini-antibodies.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Antivirais/química , Vírus da Floresta de Semliki/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Antivirais/genética , Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/genética , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Homologia de SequênciaRESUMO
We studied the cytokine profile of peripheral blood mononuclear cells after stimulation with various cryptococcal strains or its purified cell wall components. After 3 h of stimulation, tumor necrosis factor (TNF) alpha levels were strongly increased, whereas interferon (IFN) gamma and interleukin (IL) 10 levels were increased only slightly, or not at all (respectively). In contrast, after 18 h, TNF-alpha and IFN-gamma levels were (strongly) decreased, whereas the IL-10 levels were increased. The IL-1beta, IL-6 and IL-8 levels were equally high throughout the experiment. In order to establish which of the cryptococcal envelope components contributed most to the observed cytokine profile induced by whole cryptococci, glucuronoxylomannan, galactoxylomannan and mannoproteins were purified and partially characterized biochemically. All cryptococcal components elicited a similar cytokine pattern despite the differences in structure.
Assuntos
Parede Celular/química , Parede Celular/imunologia , Cryptococcus neoformans/imunologia , Citocinas/biossíntese , Leucócitos Mononucleares/imunologia , Doadores de Sangue , Humanos , Técnicas In Vitro , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , MetilaçãoRESUMO
The study of iron metabolism in human phagocytes requires a continuous source of 59Fe-labelled erythrocytes with high specific activity. Previously, rats and mice have been used for this purpose, but only limited amounts of blood can be obtained and the animals are (often) sacrificed during the experiment. This paper describes the development of a rabbit model for use as a continuous source of highly labelled erythrocytes as an alternative to mice and rats. A chinchilla rabbit was serially injected with 59Fe citrate to maintain a level of labelling of 12 x 10(5) cpm/ml of blood. During the 42-month use of the chinchilla rabbit, a total of 190 MBq was injected and a total of 277 ml blood was withdrawn. During the whole period, the health of the animal was not affected and no important changes were found in any of the haematological parameters studied. The protocol was successfully applied with a second rabbit with the same results. Our model provides a simple continuous source of highly labelled erythrocytes, minimizing the number of animals otherwise needed for this type of experiment.
Assuntos
Eritrócitos/metabolismo , Radioisótopos de Ferro/metabolismo , Coelhos/metabolismo , Animais , Masculino , Coelhos/sangueRESUMO
Although pneumococcal conjugate vaccines are close to being licensed, a more profound knowledge of the virulence factors responsible for the morbidity and mortality caused by Streptococcus pneumoniae is necessary. This review deals with the major structures of pneumococci involved in the pathogenesis of pneumococcal disease and their interference with the defense mechanisms of the host. It is well known that protection against S. pneumoniae is the result of phagocytosis of invading pathogens. For this process, complement and anticapsular polysaccharide antibodies are required. Besides, relatively recent experimental data suggest that protection is also mediated by the removal of disintegrating pneumococci and their degradation products (cell wall, pneumolysin). These structures seem to be major contributors to illness and death caused by pneumococci. An effective conjugate vaccine should therefore preferably include the capsular polysaccharide and at least one of these inflammatory factors.
Assuntos
Vacinas Bacterianas , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/patogenicidade , Adulto , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/fisiologia , Vacinas Bacterianas/imunologia , Criança , Pré-Escolar , Humanos , Lactente , Infecções Pneumocócicas/epidemiologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/química , Streptococcus pneumoniae/ultraestrutura , VirulênciaRESUMO
In available literature, different kinetics for tumor necrosis factor alpha (TNF-alpha) release by peripheral blood mononuclear cells are reported upon stimulation with Cryptococcus neoformans. Results in this study showed that shaking cells gives faster kinetics of TNF-alpha release while large working volumes give lower TNF-alpha concentrations. Different experimental conditions thus influence kinetics of TNF-alpha release by phagocytes.
Assuntos
Cryptococcus neoformans/fisiologia , Leucócitos Mononucleares/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Humanos , CinéticaRESUMO
BACKGROUND: Hereditary haemochromatosis (HH) is a disease of the metabolism of iron characterized by increased iron absorption and heavy parenchymal iron deposition, but with the presence of little iron in the mononuclear phagocytic system (MPS). METHODS: To investigate the role of the MPS, the phagocytic ability of HH monocytes (MNs) and in vitro monocyte-derived macrophages (MDMs) was studied. HH patients with different degrees of iron accumulation were chosen. RESULTS: We observed that HH patients' MNs and MDMs have a significantly decreased ability to phagocytose rabbit red blood cells (RRBCs) and that HH MNs possess a significantly decreased capacity to phagocytose Staphylococcus aureus (S. aureus). The decrease in the ability to phagocytose S. aureus, however, was kinetic in nature, explaining the absence of increased prevalence of bacterial infections among HH patients. Both RRBCs and S. aureus were preopsonized with heat-inactivated serum. No alteration in the complement-dependent phagocytosis of Cryptococcus neoformans was demonstrated when normal human serum was used. The phagocytosis defect was observed in 100% of HH patients and was independent of the magnitude of iron overload, age or liver damage, and affected the antibody-mediated uptake of bacteria and (R)RBCs.
Assuntos
Hemocromatose/imunologia , Hemocromatose/patologia , Macrófagos/imunologia , Monócitos/imunologia , Adulto , Idoso , Animais , Cryptococcus neoformans/imunologia , Eritrócitos/imunologia , Feminino , Humanos , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Fagocitose , Coelhos , Staphylococcus aureus/imunologiaRESUMO
Hereditary haemochromatosis (HH) monocytes have a decreased antibody mediated phagocytosis of rabbit erythrocytes and Staphylococcus aureus compared to control monocytes. In order to investigate whether this decrease could be attributed to a different level of expression of Fc gamma receptors (Fc gamma R) or complement receptors (CR), which cooperate even in the absence of complement, the surface expression of these receptors was determined on monocyte-enriched suspensions. In contrast to what was expected, HH monocytes displayed a significantly higher level of Fc gamma RI and Fc gamma RIIa as compared to healthy donor monocytes, but these differences were very small. The expression of the other receptors studied were similar for both groups. The heat-inactivated mouse serum used for opsonizing the erythrocytes mainly contained mouse IgG1. Two genetically different forms of Fc gamma RIIa are known, each with a different affinity for mouse IgG1 antibodies. Therefore, the Fc gamma RIIa polymorphism in monocytes (MN) of both groups was also investigated. A similar distribution was found for patients and healthy donors. In addition, the extent of erythrophagocytosis of both donors and patients was independent of Fc gamma RIIa allotype. Our results indicate that the altered phagocytosis by HH monocytes cannot be attributed to a different level of expression of receptors involved in phagocytosis or to Fc gamma RIIa polymorphism.
Assuntos
Hemocromatose/imunologia , Proteínas de Membrana , Fagocitose , Receptores de Complemento/sangue , Receptores de IgG/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Membrana Celular/imunologia , Membrana Celular/metabolismo , Eritrócitos/imunologia , Feminino , Antígenos HLA/genética , Hemocromatose/sangue , Hemocromatose/genética , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Soros Imunes/sangue , Soros Imunes/química , Alótipos de Imunoglobulina/genética , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/química , Masculino , Camundongos , Pessoa de Meia-Idade , Monócitos/metabolismo , Mutação/imunologia , Fenótipo , Coelhos , Receptores de Complemento/biossíntese , Receptores de IgG/biossíntese , Receptores de IgG/genéticaRESUMO
Lipopolysaccharides (LPS) are surface components of the outer membrane of Neisseria meningitidis. Today, 12 different types of meningococcal LPS (immunotypes) are known, of which 3 are prevalent in the western world. The differences between these immunotypes are in the oligosaccharide part of the LPS molecule and consist of small differences in the oligosaccharide structure, the amount and location of phosphoethanolamine groups, and the degree of O acetylation of individual monosaccharides. Although the differences between the various immunotypes are small, they have a profound influence on the immunochemical and immunological properties of these molecules. Furthermore, each individual strain synthesizes a number of different LPS molecules. The expression of the various components (protective epitopes) is influenced by growth conditions and growth phase. Meningococci can endogenously sialyate their LPS, which constitutes one of the mechanisms by which N. meningitidis can evade the response of the human host. Meningococcal LPS play a key role in the induction of septic shock and can probably enhance the invasiveness of meningococcal strains and shield protective epitopes. Therefore, incorporation of (detoxified) LPS or oligosaccharide components derived therefrom might be very beneficial for the efficacy of a vaccine against group B meningococci. An overview of the development of vaccines against group B meningococci is given, and the status and potential of meningococcal LPS-derived (synthetic) oligosaccharide-protein conjugate vaccines are discussed.
Assuntos
Vacinas Bacterianas , Lipopolissacarídeos/imunologia , Infecções Meningocócicas/microbiologia , Neisseria meningitidis/patogenicidade , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Infecções Meningocócicas/imunologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis/classificação , VirulênciaRESUMO
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.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Antibacterianos/biossíntese , Cápsulas Bacterianas , Proteínas de Bactérias/imunologia , Polissacarídeos Bacterianos/imunologia , Saponinas/farmacologia , Streptococcus pneumoniae/imunologia , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/administração & dosagem , Sequência de Carboidratos , Reagentes de Ligações Cruzadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Polissacarídeos Bacterianos/administração & dosagem , Saponinas de Quilaia , Saponinas/administração & dosagemRESUMO
In this study, we investigated the influence of different amounts of N-(palmitoyloxy) succinimide (PA-NHS): attachment of lipid tails to the protein and Quil A on the immunogenicity of the 38-kDa mycobacterial protein incorporated into immunostimulating complexes (ISCOMS; 38-kDa ISCOMS). The addition of higher amounts of Quil A during the ISCOMS preparation increased the amount of protein incorporated into ISCOMS, whereas the use of higher amounts of PA did not influence this parameter. Low antibody responses were observed after primary immunization with all 38-kDa ISCOMS preparations which, however, strongly increased after booster injections. IgG2a is the major subclass IgG induced by these ISCOMS preparations. There were only slight differences between the various ISCOMS formulations in their capacity to induce cytotoxic T-lymphocytes (CTLs). Spleen cells primed with ISCOMS prepared with the highest amount of Quil A produced high levels of IFN-gamma after stimulation with T helper cell type one (Th1) peptide of the 38-kDa protein (aa 70-84), 38-kDa protein or purified protein derivate (PPD). Spleen cells primed with ISCOMS prepared with the lowest amount of Quil A only substantial IFN-gamma levels were detected after stimulation with 38-kDa protein. IL-4 secretion was very low or not detectable with all ISCOM preparations. These results therefore demonstrated that all 38 kDa-ISCOMS preparations were: (1) immunogenic by inducing antibodies, Th1 and CTL responses; (2) that the way in which the ISCOMS were prepared, e.g. the amount of Quil A used, modulates the epitope specificity of the Th1 response.
Assuntos
Proteínas de Bactérias/administração & dosagem , ISCOMs/administração & dosagem , Mycobacterium tuberculosis/imunologia , Linfócitos T/imunologia , Adjuvantes Imunológicos , Animais , Formação de Anticorpos/efeitos dos fármacos , Proteínas de Bactérias/imunologia , Citocinas/biossíntese , Feminino , Imunidade Celular/efeitos dos fármacos , Imunização , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos C57BL , Peso Molecular , Saponinas de Quilaia , Saponinas/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/fisiologiaRESUMO
This study investigated the release of erythrocyte-derived iron from purified human monocytes obtained from healthy volunteers and hereditary hemochromatosis (HH) patients. After erythrophagocytosis of 59Fe-labeled erythrocytes, a complete transfer of iron from hemoglobin (Hb) to ferritin was observed within 24 hours in both control and HH monocytes. The iron was released from the monocytes in the form of ferritin, Hb, and as nonprotein bound low molecular weight iron (LMW-Fe). During the initial rapid phase (<1.5 hours), iron release mostly consisted of Hb and LMW-Fe, while in the later phase (>1.5 hours), it was composed of ferritin and LMW-Fe. The kinetics of iron release were identical for HH monocytes. A high percentage of the total amount of iron was released as Hb both by viable normal and HH monocytes, suggesting that iron release as Hb is a physiologic process, which may occur whenever the erythrocyte-processing capacity of macrophages is exceeded. Most remarkably, HH monocytes released twice as much iron in a LMW form as control cells. Iron released in the form of LMW-Fe readily binds to plasma transferrin and may contribute to the high transferrin saturation and the occurrence of circulating nontransferrin-bound iron observed in HH patients.
Assuntos
Eritrócitos/química , Hemocromatose/sangue , Ferro/sangue , Monócitos/metabolismo , Fagocitose , Adulto , Idoso , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Ferritinas/análise , Heme Oxigenase (Desciclizante)/sangue , Hemocromatose/genética , Hemoglobinas/análise , Humanos , Absorção Intestinal , Ferro/farmacocinética , Cinética , Masculino , Pessoa de Meia-Idade , Peso Molecular , Transferrina/metabolismoRESUMO
BACKGROUND: Evidence is accumulating that the alveolar collecting surfactant protein A (SP-A) plays an important role in the first line of defence against infiltrating pathogenic micro-organisms and viruses. The ability of SP-A to facilitate the binding and uptake of acapsular Cryptococcus neoformans by monocyte-derived macrophages, human alveolar macrophages, monocytes and polymorphonuclear leucocytes was investigated. MATERIALS AND METHODS: Binding, competition and phagocytosis experiments were performed using a flow cytometry technique. RESULTS: SP-A bound to both the acapsular and the encapsulated form of C. neoformans in a concentration-dependent manner. SP-A showed a threefold better binding to the acapsular yeast: this binding was partly calcium dependent and could be inhibited by mannose (ID50 = 3 mmol L-1) and glucose (ID50 = 2.1 mmol L-1) but not by galactose (ID50 = 391 mmol L-1). SP-A did not function as an opsonin in phagocytosis of acapsular C. neoformans for any of the phagocytes studied. CONCLUSION: Our results indicate that SP-A binds in a concentration-dependent manner to both encapsulated and acapsular C. neoformans. Despite SP-A binding to the acapsular C. neoformans, phagocytosis by various phagocytes was not enhanced.
Assuntos
Cryptococcus neoformans/metabolismo , Proteínas Opsonizantes , Fagocitose , Proteolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo , Animais , Aspergillus fumigatus , Sítios de Ligação , Cálcio/farmacologia , Metabolismo dos Carboidratos , Galactose/farmacologia , Glucose/farmacologia , Glicoproteínas/metabolismo , Humanos , Vírus da Influenza A , Manose/farmacologia , Fagócitos , Ligação Proteica/efeitos dos fármacos , Proteína A Associada a Surfactante Pulmonar , Proteína D Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , RatosRESUMO
Recently, the structure for pneumococcal polysaccharide (PS) 17F has been revised. Based on the former PS structure, immunogenicities of PS 17F derived synthetic di-, tri- and tetrasaccharide conjugates have been reported in mice. Here, we present additional data on the immunogenicities of these conjugates in rabbits and re-evaluate the immunogenicity results in the light of the revised PS 17F structure.