RESUMO
The larva of the cestode Echinococcus granulosus (hydatid cyst) is protected by the acellular laminated layer (LL). The mechanisms that make this thick coat a poor activator of host complement are incompletely understood. The structure binds, through unknown motifs, the host regulator of the alternative complement pathway (ACP), factor H. A second potential mechanism of ACP regulation, the inhibition of factor B activation, was detected in assays employing purified components (Immunopharmacology 42 : 91). The inhibitor was subsequently identified as myo-inositol hexakisphosphate (InsP(6)), which in the form of nano-deposits is a major component of the LL (Biochem J 362 : 297; J Cell Biochem 93 : 1272; FEBS J 273 : 3192). In this report we show that colloidal InsP(6 )solids inhibit factor B activation, through adsorption and associated impairment of C3b binding. However, this interaction is not relevant in the presence of serum proteins. In serum, InsP(6) deposits instead bind C1q, and initiate complement activation. This activation is curtailed through efficient C3b inactivation, previously shown to be entirely factor H-dependent, and now observed to be independent of the InsP(6) deposits. Therefore the complement resistance of the LL must be based on functional factor H binding sites present on the mucin-based meshwork that is its other major constituent.
Assuntos
Via Alternativa do Complemento , Equinococose/imunologia , Echinococcus granulosus/imunologia , Ácido Fítico/imunologia , Animais , Complemento C1q/imunologia , Complemento C1q/metabolismo , Complemento C3b/imunologia , Fator B do Complemento/antagonistas & inibidores , Fator H do Complemento/imunologia , Humanos , Ácido Fítico/metabolismoRESUMO
In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Especificidade de Anticorpos/genética , Antígenos Glicosídicos Associados a Tumores/imunologia , Antígenos Glicosídicos Associados a Tumores/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos Monoclonais/isolamento & purificação , Fusão Celular , Vetores Genéticos , Humanos , Hibridomas/metabolismo , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/isolamento & purificação , Imunoglobulina M/biossíntese , Imunoglobulina M/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/isolamento & purificação , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/imunologia , Transfecção , Células Tumorais CultivadasRESUMO
Here, Ana Mar a Ferreira and colleagues discuss the interplay between the larval stages of Echinococcus granulosus and an important effector arm of immunity: the host complement system. During early infection, the parasite activates complement, and hence complement-dependent inflammatory responses. However, on differentiation into the hydatid cyst, the parasite exposes to the host a structure - the cyst wall - that does not activate complement strongly. Mechanisms inhibiting complement activation on the cyst wall have been elucidated, contributing to the understanding of how this large, persistent, tissue-dwelling pathogen controls the inflammatory response.
Assuntos
Ativação do Complemento , Equinococose/imunologia , Echinococcus/imunologia , Animais , Bovinos , Echinococcus/crescimento & desenvolvimento , Interações Hospedeiro-Parasita/imunologia , CamundongosRESUMO
In the present study we have investigated and compared in vitro the specific complement (C) activating activity of three metacestode preparations of Echinococcus granulosus. Extracts from hydatid cyst fluid (HCF-ext), protoscoleces (PSC-ext) and hydatid cyst membrane (HCM-ext) activated human C producing C3 conversion and generating the C5b6 complex and the terminal C complex (TCC). HCM-ext showed much lower C activating activity than PSC-ext and HCF-ext. Moreover, its ability to generate C5b6 and TCC was lower than its ability to convert C3. On the other hand, PSC-ext and HCF-ext proved to be good C activators when their specific C activating activities were compared with that of inulin. However, PSC-ext produced lower levels of TCC than those produced by HCF-ext, in spite of the fact that both produced practically the same levels of C3d and C5b6. These results may be consistent with the existence of several mechanisms of C modulation involved in the defence of the parasite against host C damage.