RESUMO
The association of vaccines with immunostimulants such as ß-glucan, promote the production of cytokines, competent immune cells and antibodies. However, differences between ß-glucan types and trials make it difficult to understand ß-glucan's mechanism of action. In this study, three trials were carried out with control and fish fed ß-glucan, the first trial occurred at 15 days; the second trial occurred at 30 days when we associated ß-glucan and vaccine; and the third trial occurred at 15 days post-challenge with Streptococcus agalactiae in tilapia (O. niloticus) in order to investigate immune-related gene expression in the head kidney and spleen using real-time qPCR. We found increases in HSP70, IL-6, IL-1ß, TNF-α, IL-10, Lys and C3 predominantly in the head kidney, except for IgM expression, which prevailed in the spleen, under vaccinated + ß-glucan action. This demonstrates the trade-off presented by the head kidney and spleen after immunostimulation in order to produce acquired immunity, as well as an increase in HSP70 expression in vaccinated + ß-glucan fish. The results suggest that ß-glucan stimulates the immune response through damage-associated molecular patterns (DAMPs) recognition. Therefore, these dynamics of the immune response promote a more robust defense against disease.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Ciclídeos/imunologia , Rim Cefálico/efeitos dos fármacos , Baço/efeitos dos fármacos , Vacinas Estreptocócicas/administração & dosagem , beta-Glucanas/administração & dosagem , Imunidade Adaptativa , Animais , Ciclídeos/genética , Ciclídeos/microbiologia , Citocinas/genética , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/prevenção & controle , Proteínas de Peixes/genética , Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/genética , Rim Cefálico/imunologia , Muramidase/imunologia , Transdução de Sinais , Baço/imunologia , Infecções Estreptocócicas/genética , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/prevenção & controle , Infecções Estreptocócicas/veterinária , Streptococcus agalactiaeRESUMO
T-cell hybridoma assays have been widely used for the in vitro study of antigen processing and presentation because they represent an unlimited source of cells and they bypass the difficulty of maintaining T-cell clones in culture. One of the most widely used methods to assess hybridoma activation is measurement of CTLL-2 cell proliferation, which is dependent on IL-2. However, continuous culture of this cell line results in a loss of sensitivity, and significant interassay variability can occur. Therefore, our goal was to develop a method to assess T-cell hybridoma activation that was fast and sensitive with low variability based on the IL-2 secretion assay. The assay used flow cytometry detection and employed the hen egg lysozyme (HEL)-specific 3A9 hybridoma as a model. The original murine IL-2 secretion assay protocol from Miltenyi Biotec® was tested and modified; the conjugated capture antibody (anti-CD45-anti-IL-2) was added together with the stimulus at the beginning of the antigen presentation assay instead of after antigenic stimulation. With this modification, the percentage of detectable CD4+IL-2+ cells following HEL stimulation rose from 4.5% with the original protocol (0.8% without stimulus) to 94.1% (0.8% without stimulus) with the newly proposed method under the conditions evaluated in this study. This modification allowed us to evaluate the activation of hybridomas directly and more rapidly (~18h) than the reference method that assayed CTLL-2 cell proliferation using the MTT reduction assay (~48h). In conclusion, the proposed method offered a rapid alternative for screening T-cell hybridomas and evaluating their antigen-specific activation.
Assuntos
Biomarcadores/análise , Imunoensaio/métodos , Interleucina-2/metabolismo , Ativação Linfocitária , Linfócitos T/citologia , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/citologia , Linhagem Celular , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Hibridomas , Camundongos , Camundongos Endogâmicos C3H , Muramidase/imunologiaRESUMO
BACKGROUND: Frogs are a diverse group of vertebrates for which limited genomic resources are available. Natural frog populations face a multitude of threats, including habitat degradation, infectious disease, and environmental change. Characterizing the functional genomics of anuran tissues in general - and the immune system in particular - will enhance our knowledge of genetic and epigenetic responses to environmental threats and inform conservation and recovery efforts. RESULTS: To increase the number of species with genomic datasets and characterize gene expression in immune-related tissues, we sequenced the transcriptomes of three tissues from two frogs (Espadarana prosoblepon and Lithobates yavapaiensis) on the Roche 454 GS FLX platform. Our sequencing produced 8881 E. prosoblepon and 5428 L. yavapaiensis annotated gene products after de novo assembly and Gene Ontology classification. Transcripts of the innate and acquired immune system were expressed in all three tissues. Inflammatory response and acquired immunity transcripts were significantly more diverged between E. prosoblepon and L. yavapaiensis compared to innate immunity and immune system development transcripts. Immune-related transcripts did not show an overall elevated rate of functional evolution, with the exception of glycosyl proteases, which include lysozymes, central bacterial and fungal-killing enzymes of the innate immune system. CONCLUSIONS: The three frog transcriptomes provide more than 600 Mbp of new genomic data, and will serve as a valuable framework for future comparative studies of non-model anurans. Additionally, we show that immune gene divergence varies by functional group and that transcriptome studies can be useful in comparing rates of evolutionary change across gene families.
Assuntos
Sistema Imunitário/fisiologia , Proteínas/genética , Ranidae/genética , Ranidae/imunologia , Transcriptoma , Animais , Anuros/genética , Anuros/metabolismo , Anuros/microbiologia , Costa Rica , Evolução Molecular , Feminino , Ontologia Genética , Genoma/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Inflamação/genética , Inflamação/imunologia , Intestinos/imunologia , Masculino , Muramidase/genética , Muramidase/imunologia , Micoses/microbiologia , Micoses/veterinária , Panamá , Polimorfismo de Nucleotídeo Único , Proteínas/imunologia , Ranidae/microbiologia , Pele/imunologia , Baço/imunologia , Baço/fisiologiaRESUMO
OBJECTIVES: To compare the spectrum, concentration, and activity of host defense proteins (HDPs) on the skin surface of newborns and adults, to assess variation in HDP concentrations in different anatomic regions in newborns, and to examine alteration in HDP concentrations with care practices. STUDY DESIGN: Proteins recovered from tape discs applied to stratum corneum of 25 term newborns (forehead and posterior trunk) and 20 adults (forehead) were analyzed by Western analysis for 5 HDPs and for muramidase activity. Protein concentrations were compared in samples obtained after delivery, after the first bath, and at 24 hours of age. RESULTS: Total protein was 2.8-fold higher in adults compared with newborns. Lysozyme and lactoferrin were detected in all samples. In contrast to total protein, lysozyme concentrations and muramidase activity were 5-fold higher in newborns relative to adults and were not altered after bathing. Lysozyme concentrations were significantly higher over the trunk compared with the forehead in newborns. CONCLUSIONS: The newborn skin surface is replete with prototypical HDPs, lysozyme, and lactoferrin. Bathing does not significantly diminish concentrations. These factors are likely to contribute importantly to the newborn infants' defense against invasive bacterial infections.
Assuntos
Imunidade Inata , Lactoferrina/análise , Lactoferrina/imunologia , Muramidase/análise , Muramidase/imunologia , Pele/imunologia , Adulto , Fatores Etários , Feminino , Humanos , Recém-Nascido , Masculino , Pele/químicaRESUMO
The somatic mutations accumulated in variable and framework regions of antibodies produce structural changes that increase the affinity towards the antigen. This implies conformational and non covalent bonding changes at the paratope, as well as possible quaternary structure changes and rearrangements at the V(H)-V(L) interface. The consequences of the affinity maturation on the stability of the Fv domain were studied in a system composed of two closely related antibodies, F10.6.6 and D44.1, which recognize the same hen egg-white lysozyme (HEL) epitope. The mAb F10.6.6 has an affinity constant 700 times higher than D44.1, due to a higher surface complementarity to HEL. The structure of the free form of the Fab F10.6.6 presented here allows a comparative study of the conformational changes produced upon binding to antigen. By means of structural comparison, kinetics and thermodynamics of binding and stability studies on Fab and Fv fragments of both antibodies, we have determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the V(H)-V(L) interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen.
Assuntos
Anticorpos Monoclonais/química , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Muramidase/química , Muramidase/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos , Galinhas , Dicroísmo Circular , Cristalografia por Raios X , Clara de Ovo , Fluorescência , Ligação de Hidrogênio , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.
Assuntos
Muramidase/imunologia , Penaeidae/imunologia , Penaeidae/microbiologia , Proteínas Recombinantes/imunologia , Vibrio/efeitos dos fármacos , Animais , Muramidase/farmacologia , Nefelometria e Turbidimetria , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Vibrio/imunologiaRESUMO
In the immune response against a typical T cell-dependent protein antigen, the affinity maturation process is fast and is associated with the early class switch from IgM to IgG. As such, a comprehension of the molecular basis of affinity maturation could be of great importance in biomedical and biotechnological applications. Affinity maturation of anti-protein antibodies has been reported to be the result of small structural changes, mostly confined to the periphery of the antigen-combining site. However, little is understood about how these small structural changes account for the increase in the affinity toward the antigen. Herein, we present the three-dimensional structure of the Fab fragment from BALB/c mouse mAb F10.6.6 in complex with the antigen lysozyme. This antibody was obtained from a long-term exposure to the antigen. mAb F10.6.6, and the previously described antibody D44.1, are the result of identical or nearly identical somatic recombination events. However, different mutations in the framework and variable regions result in an approximately 10(3) higher affinity for the F10.6.6 antibody. The comparison of the three-dimensional structures of these Fab-lysozyme complexes reveals that the affinity maturation produces a fine tuning of the complementarity of the antigen-combining site toward the epitope, explaining at the molecular level how the immune system is able to increase the affinity of an anti-protein antibody to subnanomolar levels.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Muramidase/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Afinidade de Anticorpos , Antígenos , Galinhas , Regiões Determinantes de Complementaridade , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Técnicas In Vitro , Cinética , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Muramidase/antagonistas & inibidores , Muramidase/química , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
New research in conditioned enhancement of antibody response requires a general paradigm effective with different antigens. In this experiment series we applied a one-trial protocol using keyhole limpet hemocyanin immunization as an unconditioned stimulus. Several different conditions were tested. Two different times between conditioning and test trial, two relevant antigen doses and the use of an antigen booster during test trial were investigated. We did not find a conditioned effect in any of the conditions used. In contrast, we found a reliable albeit modest conditioned effect using hen egg lysozyme as unconditioned stimulus. By comparing these and other findings we conclude that the number of conditioning trials is a possible requirement for a more reliable conditioning of antibody response.
Assuntos
Formação de Anticorpos/imunologia , Antígenos/imunologia , Condicionamento Psicológico/fisiologia , Neuroimunomodulação/fisiologia , Animais , Relação Dose-Resposta a Droga , Hemocianinas/imunologia , Masculino , Muramidase/imunologia , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
The affinities (Ka) and association rate constants (kon) of 23 mouse (BALB/c) anti-lysozyme mAbs obtained after short and prolonged immunizations have been measured by plasmon resonance techniques. The affinities for the 23 Abs, measured using their Fab, range from Ka = 1.1 x 10(7) to 1.4 x 10(10) M-1. There is no significant correlation between time or dose of immunization and affinity or association rates, indicating no time- or dose-dependent maturation of the response within the doses and times that were explored. IgMs are produced early and late in the response, with intrinsic affinities <10(5) M-1. Two independently derived mAbs, D44.1 (short term) and F10.6.6 (from a longer term response), result from identical or nearly identical somatic recombination events of germline gene segments. F10.6.6 has more mutations and a higher affinity constant (Ka = 1.4 x 10(10) M-1) than D44.1 (Ka = 1.1 x 10(7) M-1). Although higher affinities may result from an accumulation of mutations, they do not correlate with the length and dose of immunogenic challenge.
Assuntos
Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Muramidase/imunologia , Animais , Galinhas , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Ressonância de Plasmônio de Superfície , Fatores de TempoRESUMO
BACKGROUND: This investigation was designed to determine and compare the distribution pattern of anti-neutrophil cytoplasmic antibodies (ANCA) in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in the presence or absence of periodontal disease. METHODS: Sera of 30 patients with SLE and 30 with RA were tested for ANCA utilizing an indirect enzyme immunosorbent assay (ELISA) directed to a neutrophil granular extract and 6 neutrophil granule proteins. A control group of 20 healthy individuals showing neither evidence of periodontal disease nor systemic compromise was also included in this study. RESULTS: For RA, the number of ANCA-positive sera was very low but was evenly distributed among patients with and without periodontitis. Conversely, a high number of ANCA-positive sera in SLE was found mostly in individuals presenting periodontal compromise. A statistically significant association between ANCA and periodontitis in SLE patients was found (P <0.005, chi square test). CONCLUSIONS: A marked difference in the number and distribution of ANCA with respect to periodontitis between RA and SLE was found. Hyperresponsiveness of B cells and polyclonal B activation to periodontopathic bacteria in SLE might be accountable for the high numbers of ANCA and the close association observed between those autoantibodies and periodontitis in SLE.