RESUMO
Pathogenic invasion by Escherichia coli and Salmonellae remains a constant threat to the integrity of the intestinal epithelium and can rapidly induce inflammatory responses. At birth, colostrum consumption exerts numerous beneficial effects on the properties of intestinal epithelial cells and protects the gastrointestinal tract of newborns from pathogenic invasion. The present study aimed to investigate the effect of colostrum on the early and late inflammatory responses induced by pathogens. The short-term (2 h) and long-term (24 h) effects of exposure to heat-killed (HK) E. coli and Salmonella enterica Typhimurium on gene expression in the porcine intestinal epithelial cell (IPEC-J2) model were first evaluated by microarray and quantitative PCR analyses. Luciferase assays were performed using a NF-κB-luc reporter construct to investigate the effect of colostrum whey treatment on the activation of NF-κB induced by HK bacteria. Luciferase assays were also performed using NF-κB-luc, IL-8-luc and IL-6-luc reporter constructs in human colon adenocarcinoma Caco-2/15 cells exposed to dose-response stimulations with HK bacteria and colostrum whey. Bovine colostrum whey treatment decreased the expression of early and late inflammatory genes induced by HK bacteria in IPEC-J2, as well as the transcriptional activation of NF-κB-luc induced by HK bacteria. Unlike that with colostrum whey, treatment with other milk fractions failed to decrease the activation of NF-κB-luc induced by HK bacteria. Lastly, the reduction of the HK bacteria-induced activation of NF-κB-luc, IL-8-luc and IL-6-luc by colostrum whey was dose dependent. The results of the present study indicate that bovine colostrum may protect and preserve the integrity of the intestinal mucosal barrier in the host by controlling the expression levels of early and late inflammatory genes following invasion by enteric pathogens.
Assuntos
Colostro/metabolismo , Enterócitos/metabolismo , Escherichia coli/imunologia , Regulação da Expressão Gênica , NF-kappa B/antagonistas & inibidores , Salmonella typhimurium/imunologia , Soro do Leite/metabolismo , Animais , Células CACO-2 , Bovinos , Linhagem Celular , Enterócitos/imunologia , Enterócitos/microbiologia , Genes Reporter , Humanos , Imunidade nas Mucosas , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/antagonistas & inibidores , Interleucina-8/genética , Interleucina-8/metabolismo , Cinética , NF-kappa B/genética , NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sus scrofaRESUMO
An immuno-culture method has been developed by combination of specific monoclonal antibodies and plate culture to allow detection of viable bifidobacteria. Cell wall proteins were selected as surface antigen to produce antibodies against bifidobacteria. The cell wall proteins were extracted and purified from six ATCC strains of bifidobacteria grown in MRS broth using an anaerobic system. To compare the profile of the protein extracts, all the protein solutions obtained were analyzed by SDS-PAGE. Similar bands corresponding to the major proteins of each species of bifidobacteria were observed. The proteins were tested for their immunogenicity in Balb/c mice after immunization and subsequent analysis using ELISA procedures. High immune responses were generated in mice immunized by proteins from Bifidobacterium bifidum and Bifidobacterium longum. Monoclonal antibodies were produced against B. longum and tested for their specificity, sensitivity and cross reactivity with other bifidobacteria species. All the hybridoma cells selected produced anti-B. longum antibodies cross-reacting with native and purified proteins from five other bifidobacteria species. An epitope supported by a cross-reacting protein of 58 kDa shared by bifidobacteria was revealed by western blot. This was confirmed by immune-transmission electron microscopy observations which showed the specific interaction of these antibodies with bifidobacterial cell wall proteins. Also, the antibody obtained was found to be specific for the genus Bifidobacterium and sensitive, allowing the detection of at least 10(5) target cells/ml. An immuno-culture detection approach was then developed using the selected anti-B. longum antibodies. This method was shown to be very efficient for the detection of viable cells of bifidobacteria suggesting the possibility of its use to quantify these bacteria in various food matrices.
Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas de Bactérias/imunologia , Bifidobacterium/imunologia , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Bifidobacterium/isolamento & purificação , Bifidobacterium/ultraestrutura , Parede Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Isotipos de Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão , Sensibilidade e EspecificidadeRESUMO
Bovine colostrum is well known for its beneficial properties on health and development. It contains a wide variety of bioactive ingredients that are known to promote a number of cellular processes. Therefore the use of colostrum whey as a feed additive to promote intestinal health has been proposed, yet little is known about mechanisms implicated in its beneficial properties on intestinal epithelial cells. In the present paper, casein were removed from bovine colostrum and the remaining liquid, rich in bioactive compounds, was evaluated for its capacity to modulate cellular processes in porcine intestinal epithelial cell line IPEC-J2 and human colon adenocarcinoma cell line Caco-2/15. First, we verified the effect of colostrum whey and cheese whey on processes involved in intestinal wound healing, including cell proliferation, attachment, morphology and migration. Our results showed that colostrum whey promoted proliferation and migration, and decreased specifically the attachment of Caco-2/15 cells on the culture dish. On the other hand, cheese whey induced proliferation and morphological changes in IPEC-J2 cells, but failed to induce migration. The gene expression profile of IPEC-J2 cells following colostrum whey treatment was evaluated by microarray analysis. Results revealed that the expression of a significant number of genes involved in cell migration, adhesion and proliferation was indeed affected in colostrum whey-treated cells. In conclusion, colostrum specific bioactive content could be beneficial for intestinal epithelial cell homoeostasis by controlling biological processes implicated in wound healing through a precise gene expression programme.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Hipersensibilidade/terapia , Imunoglobulina E/imunologia , Alérgenos/imunologia , Animais , Antígenos de Plantas , Venenos de Abelha/imunologia , Humanos , Hipersensibilidade/imunologia , Imunoglobulina E/biossíntese , Idiótipos de Imunoglobulinas/imunologia , Proteínas de Plantas/imunologiaAssuntos
Alérgenos/imunologia , Epitopos/imunologia , Lolium/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Alérgenos/biossíntese , Alérgenos/genética , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Plantas , Sítios de Ligação , Clonagem Molecular , Mapeamento de Epitopos , Epitopos/genética , Humanos , Imunoglobulina E/sangue , Camundongos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/imunologia , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Pólen/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
The intestinal microbiota is the largest source of microbial stimulation that exerts both harmful and beneficial effects on human health. The interaction between probiotic and enterocytes is the initiating event in immunomodulation and merits particular attention. The effects of probiotic is strain dependent and for each new probiotic strain, profiles of cytokines secreted by lymphocytes, enterocytes or dendritic cells that come in contact with the strain should be systematically established. To evaluate the effects of probiotics on the immune system, models that mimic the mucosa, and thus the physiological reality, should be preferred whenever it is possible. Then, the in vitro observed effects should be backed up by properly conducted randomized double bind clinical studies. More detailed studies are needed to determine the precise action mode of probiotics on both mucosal and systemic immunity.
Assuntos
Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/imunologia , Fatores Imunológicos/farmacologia , Probióticos/farmacologia , Ensaios Clínicos como Assunto , Humanos , Imunidade/efeitos dos fármacos , Imunidade/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologiaRESUMO
To study the role of anti-idiotypic antibodies in the regulation of the immune response to Lol p I (the major allergenic component of rye grass pollen), we have recently generated a panel of three MoAbs directed against distinct epitopes of Lolp I and an anti-idiotypic MoAb directed against the idiotype borne by one of the anti-Lol p I MoAbs (290A-167). The effects of pretreatment with this anti-idiotypic MoAb in BALB/c mice before immunization with the antigen have been examined. The anti-idiotypic MoAb or unrelated MoAb were given weekly for 8 weeks intraperitoneally. Mice then received the antigen (2 micrograms) adsorbed with alum (2 mg) at weeks 9, 11 and 13. Serum anti-Lol p I antibodies (IgG or IgE) and specific idiotypic responses were measured. Anti-Lol p I IgG antibodies could be detected before immunization with Lol p I only in mice pretreated with anti-idiotypic MoAb. Immunization with Lol p I induced an anti-Lol p I IgG response in both groups, but this response was higher in mice that received anti-idiotypic MoAb. Similar profiles were seen for specific IgE antibodies and idiotypic responses. Surprisingly, idiotypes borne by other anti-Lol p I MoAbs (539A-6 and 348A-6) had also been enhanced after pretreatment with the anti-290A-167 MoAb. These observations suggested that the pretreatment with this anti-idiotypic MoAb modulates not only the expression of the respective idiotype, but also affects other idiotype responses.
Assuntos
Alérgenos/imunologia , Anticorpos Anti-Idiotípicos/farmacologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Antígenos de Plantas , Epitopos/imunologia , Hipersensibilidade/prevenção & controle , Imunização , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Allergic diseases are characterized by an increased production of specific IgE antibodies. Suppression of IgE antibody production may be accomplished through idiotypic manipulation. OBJECTIVE: Using an animal model, we explored the effects of anti-Lol pI monoclonal antibody administration on the subsequent IgE and IgG antibody response against Lol pI. METHODS: Mice were treated with an anti-Lol pI monoclonal antibody (290A-167), which resulted in the production of anti-idiotypic antibodies as evidenced by their ability to bind to the Fab fraction of 290A-167 and to inhibit the binding of rabbit polyclonal anti-idiotypic antibodies to 290A-167. The animals were then immunized with Lol pI adsorbed onto alum, and the immune response to the protein was analyzed. RESULTS: Antigen-specific IgG1 and IgE responses were strongly suppressed as determined by immunoassay. Suppression of anti-Lol pI IgE antibodies was confirmed by a reduction of end-point titers measured by passive cutaneous anaphylaxis. The suppression of antigen-specific antibody was accompanied by a reduction of anti-Lol pI antibody-producing spleen cells. CONCLUSION: These data indicate that pretreatment with 290A-167 can strongly downregulate the IgE response to the main allergen of ryegrass pollen, which is associated with an increase in anti-idiotypic antibodies. This approach could provide rapid, long-term hyposensitization in patients with grass pollen allergy.
Assuntos
Alérgenos/imunologia , Anticorpos Anti-Idiotípicos/administração & dosagem , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Proteínas de Plantas/imunologia , Animais , Anticorpos Anti-Idiotípicos/biossíntese , Antígenos de Plantas , Feminino , Terapia de Imunossupressão , Camundongos , Camundongos Endogâmicos BALB C , Ratos , Ratos Sprague-Dawley , Baço/imunologiaRESUMO
BACKGROUND: The development of an animal model representative of the in vivo situation of human atopic diseases is always of interest for a better understanding of IgE production and regulation. Along these lines, mice with severe combined immunodeficiency (SCID mice) engrafted with lymphocytes from atopic subjects might be a suitable model for such studies. OBJECTIVE: This study aims to analyze the production of Lol p I-specific IgE and IgG antibodies in SCID mice after transplantation of human peripheral blood mononuclear cells from atopic patients sensitive to grass pollens and from nonatopic donors. METHODS: Peripheral blood mononuclear cells were transplanted into SCID mice, which were then challenged with Lol p I, and antibody responses (IgG and IgE) were analyzed over a 6-week period. RESULTS: Total IgG antibody was measured in each mouse serum after transplantation. Also, most mice (regardless of whether donors were atopic) that were challenged with Lol p I produced specific IgG antibody. Total IgE antibody production was observed only in mice grafted with cells from atopic patients. Lol p I-specific IgE antibodies were also produced after immunization with Lol p I. Although IgG antibody/response tended to plateau, the IgE antibody response increased until it peaked and declined thereafter. Interferon-gamma was detected in sera from mice producing IgE antibody, which supports a possible role of interferon-gamma in the decrease of IgE response. CONCLUSION: This study suggests that the SCID mouse model could represent an interesting approach to studying specific, total IgG and IgE antibody production, and ultimately their regulation.
Assuntos
Alérgenos/imunologia , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Leucócitos Mononucleares/imunologia , Lolium , Linfócitos/imunologia , Proteínas de Plantas/imunologia , Animais , Formação de Anticorpos , Antígenos de Plantas , Humanos , Hipersensibilidade Imediata/patologia , Leucócitos Mononucleares/metabolismo , Transfusão de Linfócitos , Camundongos , Camundongos SCIDRESUMO
This study aims to confirm that cat allergen 1 (CAT-1) is a major allergenic determinant in cat-sensitive patients, and to further define the role of other determinants, as well as to identify the determinants responsible for the cross-reactivity between cat and dog extracts. Firstly, the allergenic determinant with an electrophoretic mobility of 18 kD (corresponding to CAT-1) is indeed a major allergenic determinant being recognized by the majority (75%) of cat-sensitive subjects. Secondly, the cross-reactivity between the two species was confirmed by RAST inhibition. Cat and dog soluble allergens could inhibit, to variable degrees, the binding of serum IgE from cat- and dog-sensitive patients to insolubilized allergens. binding of serum IgE from subjects sensitive only to cats was inhibited by cat extracts only. These observations suggest the presence of determinants common to the two sources of extracts, and others specific for each species. These data were confirmed by immunoblot analysis. Indeed, an allergenic determinant of 69 kD was found in both cat and dog extracts. Conversely the allergenic determinants with an electrophoretic mobility of 18 and 32 kD were found only in cat extracts, and those at 22 and 24 kD were dog specific. However, surprisingly, serum IgE antibodies from patients sensitive only to cats reacted on immunoblot differently from those of both cat- and dog-allergic subjects. Indeed, the 18 kD determinant was the only one recognized by serum IgE antibodies from subjects sensitive to cats only, as opposed to the patients allergic to both species: then, the 69 kD determinant was strongly recognized and the 18 kD only slightly recognized.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Alérgenos , Hipersensibilidade/diagnóstico , Animais , Gatos , Reações Cruzadas , Cães , Eletroforese em Gel de Poliacrilamida , Epitopos/isolamento & purificação , Humanos , Imunoglobulina E/imunologia , Técnicas Imunológicas , Testes CutâneosRESUMO
The role of IL-4 on IgE synthesis has been well established. IL-4 has been shown to promote IgE production by B cells from atopic and non-atopic donors. In this study, the effects of natural exposure to pollens on IL-4-induced IgE synthesis by peripheral blood mononuclear cells of atopic and non-atopic donors were examined. The results confirm production of IgE in an IL-4 dose-dependent manner by PBMC cultures of these two groups. When cultures were performed out of the pollen season, following stimulation by IL-4, no significant differences was observed between the levels of IgE produced by PBMC of atopic and non-atopic donors. In contrast, upon natural exposure to pollens, significant higher levels of IgE were measured in the atopic group than in the non-atopic one. These results show that the pollen season influences the IL-4-induced IgE synthesis by PBMC of allergic patients and are in keeping with seasonal rise of specific IgE antibodies.
Assuntos
Hipersensibilidade/sangue , Imunoglobulina E/biossíntese , Interleucina-4/farmacologia , Monócitos/metabolismo , Estações do Ano , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Poaceae , Pólen , Valores de ReferênciaRESUMO
Upon immunization with an anti-Lol p I (major allergenic component of Lolium perenne pollen) monoclonal antibody, we have previously produced anti-idiotypic monoclonal antibody (A7H2) displaying some internal image properties. The present study was designated to evaluate the capacity of this anti-idiotypic monoclonal antibody to mimic functionally the antigen by triggering histamine release from basophils of patients allergic to Lol p I. Anti-idiotypic monoclonal antibody, as the antigen, could induce histamine release in a dose-response fashion in all of the atopic patients (6/6). The inhibition of this histamine release by the addition of the idiotype (290A-167) confirmed the specificity of the reaction. Binding inhibition of human IgE to Lol p I demonstrated that the anti-idiotypic antibody recognized an idiotope expressed in the antigen-combining site of IgE molecules. Altogether, these data confirmed the internal properties of our anti-idiotypic antibody and it can mimic the original antigen in its capacity to trigger histamine release.
Assuntos
Alérgenos/imunologia , Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Lolium/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Adulto , Reações Antígeno-Anticorpo/imunologia , Antígenos de Plantas , Basófilos/imunologia , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Liberação de Histamina/imunologia , Humanos , Imunoglobulina E/imunologia , Pessoa de Meia-Idade , Rinite/imunologiaRESUMO
Cat and dog albumins from commercial extracts were used to produce monoclonal antibodies (MoAb). Anti-cat albumin MoAb recognized both cat and dog albumin equally, as did anti-dog albumin MoAb; this confirms cross-reactivity between cat and dog. The MoAb were separated into two groups according to their epitopic specificity; they recognized two overlapping epitopes of cat albumin. Furthermore, by competitive inhibition of radio-allergosorbent test (RAST), it was shown that one MoAb group inhibited significantly the binding of human IgE antibodies (from a pool of 13 patients allergic to both cats and dogs) to insolubilized cat or dog extracts. These observations suggest that murine anti-cat or anti-dog MoAb and human IgE antibodies recognize identical or closely related determinants on cat and dog albumin.
Assuntos
Albuminas/imunologia , Gatos/imunologia , Epitopos/análise , Animais , Anticorpos Monoclonais/imunologia , Reações Cruzadas , Cães , Epitopos/imunologia , Teste de Radioalergoadsorção , Especificidade da EspécieRESUMO
We have recently shown that altered peptide ligands influence differentiation of CD4+ T cells into Th1 and Th2 subsets. In the present study, we have examined the biochemical signals in naive CD4+ T cells after priming with altered peptide ligand (APL) that correlate with differences in cytokine expression. Although we observed zeta-chain phosphorylation in APL-stimulated cells, other signaling events such as ZAP70 and Lnk phosphorylation are not initiated. This altered pattern observed in the early phosphorylation events correlates with a distinct Ca2+ mobilization pattern that characterizes APL-stimulated cells. By changing the calcium signaling environment during T cell priming, we present data indicating that qualitative differences in calcium mobilization are associated with differentiation of naive CD4+ T cells into Th1- and Th2-like effector subsets.
Assuntos
Linfócitos T CD4-Positivos/citologia , Peptídeos/agonistas , Peptídeos/metabolismo , Células Th1/metabolismo , Células Th2/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos T CD4-Positivos/metabolismo , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Columbidae , Grupo dos Citocromos c/imunologia , Grupo dos Citocromos c/metabolismo , Interfase/efeitos dos fármacos , Interfase/imunologia , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/imunologia , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Células Th1/citologia , Células Th2/citologia , Tirosina/metabolismoRESUMO
To define the role of CD4 in modulating T cell signaling pathways and regulating Th1 and Th2 differentiation, we have examined the activation and differentiation characteristics of naive T cells from CD4 mutant mice. Using peptides with differing affinities for the moth cytochrome c-specific TCR, we test the hypothesis that differences in coreceptor recruitment and signaling explain the qualitatively distinct signaling pathways seen in CD4 T cells following high affinity agonist and low affinity altered peptide ligand (APL) ligation. We find that the absence of CD4 signaling during stimulation with a strong agonist peptide does not qualitatively change the pattern of early TCR-mediated biochemical signaling events into a pattern resembling the response of CD4+ T cells to APLs. In contrast, the response to APL stimulation, by T cells bearing the same TCR, does require a component of CD4 signaling. The proliferative response and calcium signals normally seen following APL stimulation are markedly diminished in the absence of CD4. In addition, we find that naive T cell differentiation into Th2 effector cells is impaired in the absence of CD4. These data suggest that the altered pattern of biochemical signals generated by APLs require CD4 coreceptor function and that some of these signals may be required to initiate Th2 differentiation.
Assuntos
Antígenos CD4/fisiologia , Ativação Linfocitária , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD4/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cruzamentos Genéticos , Grupo dos Citocromos c/imunologia , Grupo dos Citocromos c/farmacologia , Interleucina-4/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Biológicos , Dados de Sequência Molecular , Mariposas/imunologia , Fragmentos de Peptídeos/agonistas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Células Th1/citologia , Células Th2/citologia , Células Th2/efeitos dos fármacosRESUMO
CD45 is the predominant transmembrane tyrosine phosphatase in lymphocytes and is required for the efficient induction of T cell receptor signaling and activation. However, the regulation of CD45 activity and substrate specificity are poorly understood. In the present study, we demonstrate a basal biochemical association of CD45 with the T cell receptor complex that is regulated in part by CD45 isoform expression. Further, maintenance of CD45/TCR association is differentially regulated following TCR ligation with peptide: a partial agonist peptide induces CD45/TCR dissociation while an agonist peptide promotes sustained association in a CD4-dependent manner. These data suggest that T cell receptor signaling pathways may be modulated by altering access of CD45 to TCR-associated substrates involved in T cell activation.
Assuntos
Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/química , Citoplasma , Antígenos Comuns de Leucócito/biossíntese , Antígenos Comuns de Leucócito/química , Ligantes , Camundongos , Camundongos Endogâmicos AKR , Camundongos Endogâmicos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/imunologia , Ligação Proteica/imunologia , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T/química , Células Tumorais CultivadasRESUMO
Upon immunization of mice with a mAb (290A-167) directed against an epitope of Lol p I (the major allergenic determinant of Lolium perenne), both anti-idiotypic (aId) mAb (Ab2) and anti-aId mAb (Ab3) were produced. The Ab2 displayed the following internal image properties of Lol p I: it can be affinity-purified on an immobilized Id column; its binding to the anti-Lol p I mAb (290A-167) is inhibited by Lol p I; it inhibits in a dose-response fashion the binding of the specific Id to Ag. It is recognized by anti-Lol p I antisera from different species such as mouse, human, and goat. The Ab3 which binds to Lol p I was also produced from the same fusion. This binding was inhibited significantly by aId mAb (Ab2), anti-Lol p I mAb (290A-167) and Lol p I. These data indicate that the two mAb with specificity for Lol p I (290A-167 and Ab3) share similar reactivity to the Ag and that aId mAb is the internal image of the epitope recognized by the Id. We showed also that the capacity of rabbit aId Ab directed against the 290A-167 Id to inhibit the binding of Ab1 and Ab3 to Ag was almost abolished by passage over a Ab3-coated Sepharose column. This would suggest that not only are the two mAb with reactivity to Lol p I (Ab1 and Ab3) directed against identical epitopes, but that they in fact shared identical idiotopes as well. The production of identical mAb upon immunization with either the Ag or the aId mAb supports that the conceptual framework proposed by Jerne finds its biologic application in the course of an immune response.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Animais , Especificidade de Anticorpos , Antígenos/imunologia , Fusão Celular , Hibridomas/imunologia , Idiótipos de Imunoglobulinas/imunologia , Camundongos , Pólen/imunologiaRESUMO
BACKGROUND: Passive infusion of beekeepers' plasma was shown to protect patients against systemic reactions occurring during active immunotherapy by mechanisms still to be clarified. It is tempting to speculate that anti-idiotypic antibodies could play a role because they are found in beekeepers' plasma and are involved in the regulation of IgE synthesis. METHODS: In this report we studied the effects of passive infusion of a beekeeper's plasma rich in anti-idiotypic antibodies to a patient who experienced systemic reactions to honeybee venom. RESULTS: We reported, during the days after the infusion, a decrease of clinical sensitivity to the honeybee venom. Indeed, the patient tolerated a cumulative dose of 280 micrograms of venom without adverse reactions. We also observed decreases in skin mast cell and in basophil sensitivity. After the plasma infusion, a modified rush immunotherapy with honeybee venom was initiated in our patient. In the following 76 weeks, increased levels of anti-idiotypic antibodies in the serum of the patient were associated with a diminution of specific antibodies (IgG and IgE) to honeybee venom. CONCLUSION: These results suggest a dual role of anti-id in our combined protocol of passive and active immunotherapy: an immediate action on clinical sensitivity along with a decrease of skin mast cell and basophil sensitivity and an immunoregulatory role on specific antibody production.
Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Venenos de Abelha/imunologia , Imunização Passiva , Mordeduras e Picadas de Insetos/imunologia , Vacinação , Adulto , Anticorpos Anti-Idiotípicos/sangue , Anticorpos Anti-Idiotípicos/uso terapêutico , Ligação Competitiva , Feminino , Liberação de Histamina , Humanos , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Testes CutâneosRESUMO
The different determinants of birch pollen extracts, as shown by SDS-PAGE analysis, range from 10 to 94 kDa. These determinants were then electrotransferred on nitrocellulose strips and allowed to react with human IgE Ab from sensitive patients in order to identify the allergenic determinants. Several minor (43, 35, 28 and 21 kDa) and the major (17 kDa) allergenic determinants were identified. Murine monoclonal antibodies (mAb) were then produced against the major allergenic determinant (Bet v I) and their specificity confirmed by immunoblot. One of them, mAb 3F10, was used to affinity-purify the Bet v I. The purity of this material was confirmed by SDS-PAGE analysis and its reactivity on immunoblot against human IgE ensured its biological activity. These mAb were then gathered on four families based on their pattern of reactivity with Bet v I. Indeed four different epitopes on the molecule were identified. Binding inhibition studies using two of them (mAb 5F9 and 8F12) suggested that the epitopes of Bet v I recognized by these mAb are not overlapping. On another hand, the binding of 8H7 and 3F10 was partially inhibited by 5F9 and the binding of 3F10, by 8F12. These data suggest that those two latter epitopes are somewhat overlapping. Finally, the mAb 5F9 could inhibit the binding of human IgE on the affinity-purified Bet v I up to 40% and then shares a common idiotope with human specific IgE Ab of allergic patients.
Assuntos
Alérgenos/química , Epitopos/química , Proteínas de Plantas/química , Anticorpos Monoclonais , Antígenos de Plantas , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Imunoglobulina E/metabolismo , Pólen/imunologia , RadioimunoensaioRESUMO
BACKGROUND: Current forms of allergy diagnosis and therapies are based on the use of natural allergenic extracts. Despite strong evidence that higher therapeutic efficacy may be achieved with purified allergens, the purification of multiple allergic components from extracts is a fastidious and sometimes an impossible task. However, the use of recombinant allergens may be an alternative to overcome this problem. OBJECTIVE: In this study, we compared the immunological properties of recombinant (r) Lol p 1 with those of the natural protein. METHOD: We cloned directly the gene encoding Lol p 1 from genomic DNA of ryegrass pollen. This gene was subcloned into the expression vector pMAL-c and expressed as fusion protein. Subsequently, rLol p 1 was cleaved from maltose-binding protein using factor Xa. Using binding inhibition and proliferative assays, we assessed the immunological properties of the recombinant allergens. The capacity of rLol p 1 to trigger basophil histamine release and to elicit a skin reaction was also assessed and compared to those of its natural counterpart. RESULTS: We found that the Lol p 1 gene has no introns since we amplified this gene directly from genomic DNA. We demonstrated that the binding sites of anti-Lol p 1 monoclonal antibody, specific human IgG and IgE antibody are well conserved on rLol p 1 as no difference in the binding inhibition profile was observed when using either natural or recombinant protein. At the T-cell level, rLol p 1 elicited a T-cell response in mice comparable to that observed with the natural protein. In addition, we demonstrated that the biological characteristics of rLol p 1 were comparable to those of the natural counterpart, in that rLol p 1 elicited a skin wheal reaction and induced basophil histamine release in grass-allergic patients only. CONCLUSION: The data indicate that natural Lol p 1 and rLol p 1 shared identical immunological and biological properties.