RESUMO
Lewis rats are susceptible to several forms of experimental arthritis-induced using heat-killed Mycobacterium tuberculosis (adjuvant arthritis, or AA), streptococcal cell walls, collagen type II, and the lipoidal amine CP20961. Prior immunization with the mycobacterial 65-kD heat shock protein (hsp65) was reported to protect against AA, and other athritis models not using M. tuberculosis, via a T cell-mediated mechanism. Hsp65 shares 48% amino acid identity with mammalian hsp60, which is expressed at elevated levels in inflamed synovia. Several studies have reported cross-reactive T cell recognition of mycobacterial hsp65 and self hsp60 in arthritic and normal individuals. We previously described nine major histocompatibility complex class II-restricted epitopes in mycobacterial hsp65 recognized by Lewis rat T cells. Of these only one, covering the 256-270 sequence, primed for cross-reactive T cell responses to the corresponding region of rat hsp60. Here we have tested each hsp65 epitope for protective activity by immunizing rats with synthetic peptides. A peptide containing the 256-270 epitope, which induced cross-reactive T cells, was the only one able to confer protection against AA. Similarly, administration of a T cell line specific for this epitope protected against AA. Preimmunization with the 256-270 epitope induced T cells that responded to heat-shocked syngeneic antigen-presenting cells, and also protected against CP20961-induced arthritis, indicating that activation of T cells, recognizing an epitope in self hsp60 can protect against arthritis induced without mycobacteria. Therefore, in contrast to the accepted concept that cross-reactive T cell recognition of foreign and self antigens might induce aggressive autoimmune disease, we propose that cross-reactivity between bacterial and self hsp60 might also be used to maintain a protective self-reactive T cell population. This discovery might have important implications for understanding T cell-mediated regulation of inflammation.
Assuntos
Artrite Experimental/prevenção & controle , Proteínas de Bactérias , Chaperonina 60/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Linhagem Celular , Chaperoninas/imunologia , Reações Cruzadas , Proteínas de Choque Térmico/imunologia , Imunização , Masculino , Fragmentos de Peptídeos/imunologia , Ratos , Ratos Endogâmicos LewRESUMO
Progression of prostate cancer during endocrine therapy is a major clinical problem, the molecular mechanisms of which remain poorly understood. Amplification of the androgen receptor (AR) gene was recently described in recurrent prostate carcinomas from patients who had failed androgen deprivation therapy. To evaluate the hypothesis that amplification of the AR gene is a cause for the failure of androgen deprivation therapy in prostate cancer, we studied whether AR amplification leads to gene overexpression, whether the amplified AR gene is structurally intact, and whether tumors with AR amplification have distinct biological and clinical characteristics. Tumor specimens were collected from 54 prostate cancer patients at the time of a local recurrence following therapy failure. In 26 cases, paired primary tumor specimens from the same patients prior to therapy were also available. Fifteen (28%) of the recurrent therapy-resistant tumors, but none of the untreated primary tumors, contained AR gene amplification as determined by fluorescence in situ hybridization. According to single-stranded conformation polymorphism analysis, the AR gene was wild type in all but one of the 13 AR amplified cases studied. In one tumor, a presumed mutation in the hormone-binding domain at codon 674 leading to a Gly --> Ala substitution was found, but functional studies indicated that this mutation did not change the transactivational properties of the receptor. AR amplification was associated with a substantially increased level of mRNA expression of the gene by in situ hybridization. Clinicopathological correlations indicated that AR amplification was most likely to occur in tumors that had initially responded well to endocrine therapy and whose response duration was more than 12 months. Tumors that recurred earlier or those that showed no initial therapy response did not contain AR amplification. The median survival time after recurrence was two times longer for patients with AR amplification in comparison to those with no amplification (P = 0.03, Willcoxon-Breslow test). In conclusion, failure of conventional androgen deprivation therapy in prostate cancer may be caused by a clonal expansion of tumor cells that are able to continue androgen-dependent growth despite of the low concentrations of serum androgens. Amplification and the increased expression of a wild-type AR gene may play a key role in this process.
Assuntos
Amplificação de Genes/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/terapia , Receptores Androgênicos/genética , Idoso , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Mutação Puntual , RNA Mensageiro/metabolismo , Análise de Sobrevida , Falha de TratamentoRESUMO
In twenty-five ovine body fluids (serum, lung fluid and cerebrospinal fluid), the concentrations of IgG1I, IgG2, IgM and IgA were determined by laser nephelometry and radial immunodiffusion (RID). When nephelometric assays are carried out, antisera free from any turbidity are essential. Methods ensuring that goat and rabbit anti-sera will satisfy this requirement are described in the present paper. When sheep immunoglobulins were measured by laser nephelometry, adequate and reproducible results were obtained, comparable with those obtained by RID. Advantages of the nephelometric method include the speed of assay and its sensitivity, allowing precise determination of the very low concentrations of immunoglobulins in CSF.
Assuntos
Líquidos Corporais/imunologia , Imunoglobulinas/imunologia , Nefelometria e Turbidimetria/veterinária , Ovinos/imunologia , Animais , Líquidos Corporais/química , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Imunodifusão/veterinária , Imunoglobulinas/análise , Nefelometria e Turbidimetria/métodos , Ovinos/sangue , Ovinos/líquido cefalorraquidianoRESUMO
In 21 dogs with clinical features of German Shepherd dog Pyoderma (GSP) parameters of the specific and aspecific immune system have been examined. Chemotaxis and killing capacities of neutrophilic leucocytes were undisturbed, whereas in skin biopsies no specific immunoglobulin or complement deposits were found with immunofluorescence. With double immunodiffusion, antibodies against Gram-positive bacteria were found. In a laser nephelometric assay significantly elevated levels of IgG, IgGab, IgGd, IgM and bacterial components, associated and non-associated with circulating immune complexes, were detected. However, no relation was found with the disease state. It is concluded that dogs with GSP are immunologically normal reactors. A bacterial hypersensitivity reaction is hypothesized as a possible initiating factor in the pathogenesis of GSP.
Assuntos
Doenças do Cão/imunologia , Pioderma/veterinária , Animais , Anticorpos Antibacterianos/análise , Complexo Antígeno-Anticorpo/metabolismo , Antígenos de Bactérias/análise , Proteínas do Sistema Complemento/metabolismo , Doenças do Cão/etiologia , Cães , Feminino , Bactérias Gram-Positivas/isolamento & purificação , Granulócitos/imunologia , Imunoglobulinas/metabolismo , Testes Intradérmicos , Masculino , Pioderma/etiologia , Pioderma/imunologiaRESUMO
A quantitative enzyme-linked immunosorbent assay (ELISA) has been developed to measure canine von Willebrand factor antigen (vWf-Ag) in plasma of the dog. A vWf-Ag antiserum was raised in rabbits and purified by preabsorption with the low molecular weight vWf-Ag-deficient fraction of canine cryoprecipitate, followed by affinity chromatography on protein-A Sepharose. The rabbit anticanine vWf-Ag IgG was used to bind the vWf-Ag of the test plasmas to the solid phase and to prepare the enzyme-antibody conjugate in ELISA. Normal rat serum was used as blocking agent. The standard curve was linear (r2 greater than 0.98) and reproducible after logit-log transformation. The interassay coefficient of variation (CV) in test plasmas with various vWf-Ag concentrations was never greater than 7.7%. Assayed values in dilutions of pooled normal canine plasma added to canine vWf-Ag-deficient plasma were linear between 0 and 100% (r2 = 0.99) and indicated excellent analytical recovery of vWf-Ag. In 18 dogs with various internal diseases, including von Willebrand's disease and haemophilia A, the coefficient of correlation between the results of the ELISA and those of electroimmunodiffusion (EID) was 0.93.
Assuntos
Doenças do Cão/sangue , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças de von Willebrand/veterinária , Fator de von Willebrand/imunologia , Animais , Doenças do Cão/imunologia , Cães , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Imunoeletroforese , Masculino , Coelhos , Padrões de Referência , Valores de Referência , Doenças de von Willebrand/sangue , Doenças de von Willebrand/imunologia , Fator de von Willebrand/normasRESUMO
The distribution of the immunoglobulins IgG, IgM, and IgA in turkey serum, bile, egg white, egg yolk, intestinal secretions, and some intraocular tissues has been studied. Just as in the chicken and the pigeon, IgG is the most abundant immunoglobulin in the serum and IgA in egg white, intestinal secretions and bile. In addition, IgM was detected in serum and egg white, and IgG in egg yolk, iris, vitreous body, and aqueous humor. The discussion deals with the importance of using purified immunoglobulin fractions and heavy-chain-specific antisera in disease studies in the turkey, specially in the case of infectious diseases which lead to the occurrence of "rheumatoid factors".
Assuntos
Imunoglobulinas/análise , Perus/imunologia , Animais , Bile/imunologia , Líquidos Corporais/imunologia , Olho/imunologia , Feminino , Imunoglobulina A/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Óvulo/imunologiaAssuntos
Columbidae/imunologia , Imunoglobulina A , Animais , Transporte Biológico , Feminino , Imunoglobulina A/isolamento & purificação , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Jejuno/ultraestrutura , Masculino , Leite/imunologia , Faringe/citologia , Faringe/imunologia , Coelhos , Saliva/imunologiaAssuntos
Imunoglobulina A/imunologia , Água/metabolismo , Animais , Bovinos , Galinhas , Columbidae , Cães , Cavalos , Humanos , Imunodifusão , Coelhos , Ovinos , Especificidade da Espécie , Suínos , PerusRESUMO
Pigeon IgA has been isolated from crop-milk and an antiserum was produced. Extracts of the dust in pigeon coops contained detectable amounts of pigeon IgA, but not IgG. The sera of patients with pigeon breeder's disease contained precipitating antibody against pigeon IgA.
Assuntos
Alveolite Alérgica Extrínseca/imunologia , Antígenos , Pulmão do Criador de Aves/imunologia , Imunoglobulina A/imunologia , Animais , Columbidae , Poeira , Plumas/imunologia , Humanos , Soros Imunes/farmacologia , Imunoeletroforese , Imunoglobulina A/isolamento & purificação , Imunoglobulina G/imunologiaRESUMO
One-day-old chickens with maternally derived antibodies to infectious bronchitis virus (IBV) were vaccinated against IB by eye-drop. At 2 and 3 weeks of age the synthesis of immunoglobulins of the IgA isotype in the Harderian gland was demonstrated by immunofluorescence. With an anti IgG conjugate diffuse fluorescence of IgG was observed in the gland at 2, 3 and 4 weeks of age. Involvement of the Harderian gland in the synthesis or the secretion of IgM after IB vaccination could not be demonstrated by immunofluorescence. A quantitative estimation of immunoglobulins was carried out by laser nephelometry. At 2 weeks of age the concentration of antibodies against IBV of the IgA isotype (IgA-IBV) was higher in the tears than in the serum. This and the positive fluorescence indicate a local synthesis of IgA-IBV in the Harderian gland. The highest concentration of IgG antibodies against IBV (IgG-IBV) in the serum was measured at 2 weeks of age, when IgG antibodies could not be detected in the tears. In the tears the IgG-IBV concentration increased from 2 weeks up to and including 5 weeks of age, when the concentration in the tears was higher than in the serum. These findings and the diffuse IgG fluorescence in the Harderian gland suggest a mainly systemic production of IgG-IBV and an active and selective transport of IgG-IBV from the serum to the tears by the Harderian gland. Challenge at 6 weeks of age of the vaccinated chicks caused a sharp increase of IgG-IBV in the serum and a decrease of IgA-IBV in the tears 1 week later. Challenge of the unvaccinated control chicks resulted in a distinct rise of IgA-IBV in the tears and in a low IgG-IBV concentration in the serum after 1 week. These findings and the neutralisation indices measured for sera collected at 5 and 7 weeks are discussed.
RESUMO
Dogs immunized with o-DNCP-BSA or eggs of Toxocara canis developed anaphylactic antibodies other than IgE. The antibodies were heat-resistant at 56 degrees C for 2 hours, not reduced by 2-mercaptoethanol and PCA and P-K reactivity was only observed at 3-4 h sensitized sites of recipient skin. High titres of antigen-specific IgGd were determined by means of an ELISA technique.
Assuntos
Anafilaxia/imunologia , Formação de Anticorpos , Animais , Dinitrofenóis/imunologia , Cães , Eletroforese em Gel de Ágar , Eosinófilos , Feminino , Imunoeletroforese , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Testes Intradérmicos , Contagem de Leucócitos , Masculino , Anafilaxia Cutânea Passiva , Toxocara/imunologiaRESUMO
Allergen specific IgGd antibodies were detected by the enzyme linked immunosorbent assay (ELISA) in 89% of the 62 atopic dogs studied. Antibodies were found most frequently against house dust (47%), human dander (50%), grass pollens (58%) and spring tree pollens (43%). These antibodies were also found in 11 of 20 dogs with atopic symptoms but without immediate skin test reactivity to inhalant allergens. Agreement between the presence of skin reactivity and allergen specific IgGd titres ranged from one of 14 for cat dander to 22 of 34 for house dust. Among dogs with atopic symptoms but without skin test reactivity and specific IgGd titres to the respective allergens, the agreement varied between 28 of 54 for human dander and 67 of 68 for cat dander. In view of the value of the dog as an experimental model of atopic disease in man, further studies of the pathophysiological significance of IgGd antibodies are warranted. In addition, reconsideration of the diagnostic criteria for canine atopic dermatitis, as done by Hanifin & Rajka (1980) in man, seems indicated.
Assuntos
Dermatite Atópica/imunologia , Imunoglobulina G/análise , Alérgenos/imunologia , Animais , Especificidade de Anticorpos , Cães , Poeira , Ensaio de Imunoadsorção Enzimática , Feminino , Hipersensibilidade Imediata , Masculino , Pólen/imunologiaRESUMO
Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis (Mt) in mineral oil induces adjuvant arthritis (AA), associated with T cell responses to residues 180-188 of the mycobacterial 65-kDa heat shock protein (hsp65). Preimmunization with hsp65 protects rats against AA and other forms of arthritis. Several explanations for these protective effects have been proposed, including enhanced responsiveness to protective epitopes in hsp65, down-regulation of T cell responses to the 180-188 epitope, and activation of self-hsp60-reactive T cells. To assess the potential of these hypotheses, we analyzed hsp65 T cell epitopes recognized after immunization of Lewis rats with Mt or hsp65. Here we identify nine RT1.B1-restricted T cell epitopes in hsp65. Mt immunization induced T cell responses in which the 180-188 epitope was dominant, whereas hsp65 immunization resulted in a co-dominance of this and two further epitopes, 216-225 and 226-235. Two minor epitopes were recognized after hsp65 but not Mt immunization. These results indicate that hsp65 preimmunization does not down-regulate responses to the AA-associated epitope, but does enhance responses to several hsp65 epitopes that are minor or absent after the AA-inducing immunization protocol. Cross-reactive T cell recognition of hsp65 and rat hsp60 was limited to a single epitope (256-265), recognized after hsp65 immunization, but poorly recognized after Mt immunization. This study provides the necessary basis for elucidating the T cell events involved in the protective effects of hsp65 preimmunization.
Assuntos
Antígenos de Bactérias/imunologia , Artrite Experimental/imunologia , Proteínas de Choque Térmico/imunologia , Mycobacterium tuberculosis/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Linfonodos/citologia , Ativação Linfocitária , Complexo Principal de Histocompatibilidade/imunologia , Masculino , Dados de Sequência Molecular , Mycobacterium bovis/imunologia , Peptídeos/imunologia , Ratos , Ratos Endogâmicos Lew , Tuberculina/imunologiaRESUMO
The immunoglobulins IgA, IgM and IgG of the turkey were quantitated in individual serum samples as well as in pooled sera. The variability of Ig levels in normal, healthy birds was quite high: IgA: mean value 0.633 mg/ml (4.0 -x - 2.5 -x); IgM: mean value 4.37 mg/ml (0.5 -x - 1.4 -x) and IgG: mean value 8.92 mg/ml (0.6 -x - 1.7 -x). Immunoglobulin levels in egg-yolk, egg-white, bile and some intraocular tissues were quantitated as well. An interesting finding was, that the forementioned variability was by far not so high with respect to IgG levels in 20 egg-yolk samples: mean value 5.1 mg/ml (0.86 x- - 1.17 -x). Though IgG and IgM could be detected in pooled turkey bile, IgA predominated in this secretion. In aqueous humor, iris tissue and vitreous body only IgG could be detected.
Assuntos
Imunoglobulina A/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Perus/imunologia , Animais , Humor Aquoso/imunologia , Bile/imunologia , Gema de Ovo/imunologia , Feminino , Iris/imunologia , Óvulo/imunologia , Corpo Vítreo/imunologiaRESUMO
Prediction, identification and analysis of T cell epitopes in protein antigens has become a central theme in fundamental and applied immunology. However, while for the characterization of linear B cell epitopes the so-called Pepscan procedure was found to be extremely effective, no such technique has so far been available for T cell studies. Recently, we described the identification and localization of a T cell epitope in a mycobacterial 65-kDa shock protein in the model of adjuvant arthritis. This was done by molecular cloning and conventional solid-phase synthesis techniques. We now show that the delineation of such a T cell epitope and its further characterization can be accomplished in a much more rapid and efficient manner by a modification of the existing Pepscan technique. We show for the first time that several hundreds of peptides, simultaneously synthesized in an automated way on activated polyethylene rods, can be easily recovered from these rods in adequate quantities, enabling a systematic analysis of T cell epitopes. Synthesis of sequentially overlapping peptides along the 65-kDa protein revealed that the adjuvant arthritis T cell clones are fully stimulated by peptides that comprise a minimal sequence of seven residues, corresponding to positions 180-186 in the sequence of the 65-kDa protein of M. bovis Bacillus Calmette Guerin (BCG). Detailed examination of the epitope by peptides containing a single amino acid substitution showed that, apart from one conservative replacement (Glu----Asp), the requirement for the native residue at all positions in peptide 180-186 was absolute for full T cell stimulation. Their indispensability was confirmed with deletion and insertion peptides. It is concluded that the occurrence of indifferent or spacer residues in a minimal stimulatory sequence, as observed by others, is not a general feature of T cell epitopes.
Assuntos
Antígenos de Diferenciação de Linfócitos T/isolamento & purificação , Epitopos/isolamento & purificação , Mycobacterium bovis/análise , Oligopeptídeos/síntese química , Mapeamento de Peptídeos , Sequência de Aminoácidos , Variação Antigênica , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/isolamento & purificação , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Dados de Sequência Molecular , Oligopeptídeos/imunologia , Oligopeptídeos/isolamento & purificaçãoRESUMO
Reactivity towards TCR peptides plays an important role in the regulation of several experimental autoimmune diseases. In a previous paper, we showed the TCRAV11 usage by an arthritogenic T cell clone isolated from a rat with adjuvant arthritis (AA). Moreover, we identified three immunogenic peptides in AV11: AV11 24-40, 41-55 and 66-80. In the present study, we show that T cells directed towards all three epitopes are part of the immune repertoire. The strongest delayed-type hypersensitivity (DTH) reaction was observed against the peptide derived from the third framework region, peptide AV11 66-80. DTH reactions to this peptide were detectable in naive rats and increased significantly after AA induction. Interestingly, modulation of the AV11 66-80 T cell response by nasal AV11 66-80 administration resulted in reduced DTH responses and in a strong inhibition of AA. These findings suggest that during the natural course of AA, T cells directed towards the third framework region of AV11 do not have a disease regulatory function, but instead play a role in the deterioration of AA.
Assuntos
Artrite Experimental/prevenção & controle , Receptores de Antígenos de Linfócitos T/administração & dosagem , Administração Intranasal , Animais , Artrite Experimental/imunologia , Modelos Animais de Doenças , Progressão da Doença , Epitopos/administração & dosagem , Masculino , Fragmentos de Peptídeos/administração & dosagem , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/fisiologia , VacinaçãoRESUMO
Serum samples and immunoglobulin fractions of eight mammalian species were applied to a Sepharose--protein A column. As with the human immunoglobulin subclasses IgG1, IgG2 and IgG4, all examined IgG classes and subclasses were bound to a greater or lesser extent to protein A. However, the binding of IgG1 of ruminants was very poor. Polyclonal IgM and IgA of the pig, the dog and the cat may be separated in protein A reactive and protein A non-reactive fractions. In addition, monoclonal canine IgM and IgA partially reacted with protein A. In combination with methods such as ammonium sulphate precipitation, ion exchange chromatography and gel-filtration, affinity chromatography with protein A is recommended for the rapid purification of certain Ig (sub)classes of a number of mammalian species.
Assuntos
Imunoglobulinas , Proteína Estafilocócica A/imunologia , Animais , Gatos , Bovinos , Precipitação Química , Cromatografia de Afinidade , Cromatografia em Gel , Cromatografia por Troca Iônica , Cães , Cabras , Cavalos , Soros Imunes , Imunodifusão , Imunoeletroforese , Imunoglobulinas/isolamento & purificação , Ligação Proteica , Coelhos , Ovinos , Especificidade da Espécie , SuínosRESUMO
Adjuvant arthritis (AA) is a chronic disease inducible in rats by immunization with an antigen of Mycobacterium tuberculosis. After the isolation of arthritogenic T-cell lines and clones, it became possible to demonstrate that the critical M. tuberculosis antigen contained an epitope cross-reactive with a self-antigen in joint cartilage. Like AA rats, patients suffering from rheumatoid arthritis demonstrated specific T-lymphocyte reactivity to the M. tuberculosis fraction containing the cross-reactive epitope. To characterize the critical M. tuberculosis epitope we used AA T-cell clones to screen mycobacterial antigens expressed in Escherichia coli and genetically engineered truncated proteins and synthetic peptides. The AA T-cell clones recognized an epitope formed by the amino acids at positions 180-188 in the sequence of a Mycobacterium bovis BCG antigen. Administration of this antigen to rats induced resistance to subsequent attempts to produce AA.