RESUMEN
A hallmark of celiac disease is autoantibodies to transglutaminase 2 (TG2). By visualizing TG2-specific antibodies by antigen staining of affected gut tissue, we identified TG2-specific plasma cells in the lamina propria as well as antibodies in the subepithelial layer, inside the epithelium, and at the brush border. The frequency of TG2-specific plasma cells were found not to correlate with serum antibody titers, suggesting that antibody production at other sites may contribute to serum antibody levels. Upon commencement of a gluten-free diet, the frequency of TG2-specific plasma cells in the lesion dropped dramatically within 6 months, yet some cells remained. The frequency of TG2-specific plasma cells in the celiac lesion is thus dynamically regulated in response to gluten exposure. Laser microdissection of plasma cell patches, followed by antibody gene sequencing, demonstrated that clonal cells were seeded in distinct areas of the mucosa. This was confirmed by immunoglobulin heavy chain repertoire analysis of plasma cells isolated from individual biopsies of two untreated patients, both for TG2-specific and non-TG2-specific cells. Our results shed new light on the processes underlying the B-cell response in celiac disease, and the approach of staining for antigen-specific antibodies should be applicable to other antibody-mediated diseases.
Asunto(s)
Autoanticuerpos/genética , Enfermedad Celíaca/inmunología , Proteínas de Unión al GTP/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Células Plasmáticas/inmunología , Transglutaminasas/inmunología , Autoanticuerpos/biosíntesis , Biopsia , Enfermedad Celíaca/inducido químicamente , Enfermedad Celíaca/dietoterapia , Enfermedad Celíaca/genética , Recuento de Células , Dieta Sin Gluten , Duodeno/efectos de los fármacos , Duodeno/inmunología , Duodeno/patología , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica/inmunología , Glútenes/efectos adversos , Humanos , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Captura por Microdisección con Láser , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/patología , Proteína Glutamina Gamma Glutamiltransferasa 2 , Análisis de Secuencia de ADN , Transglutaminasas/genéticaRESUMEN
UNLABELLED: Several serological diagnostics rely on enzyme-linked immunosorbent assay (ELISA) to detect bacterial infections. However, for some pathogens, including Bartonella henselae, diagnosis still depends on manually intensive, time-consuming assays including micro-immunofluorescence, Western blotting or indirect immunofluorescence. For such pathogens, there is obviously still a need to identify antigens to establish a reliable, fast and high-throughput assay (Dupon et al. ). We evaluated two B. henselae proteins to develop a novel serological ELISA: a well-known antigen, the 17-kDa protein, and GroEL, identified during this study by a proteomic approach. When serum IgG were tested, the specificity and sensitivity were 76 and 65·7% for 17-kDa, respectively, and 82 and 42·9% for GroEL, respectively. IgM were found to be more sensitive and specific for both proteins: 17-kDa protein, specificity 86·2% and sensitivity 75%; GroEL, specificity 97·7% and sensitivity 45·3%. IgM antibodies were also measured in lymphoma patients and patients with Mycobacterium tuberculosis infection to assess the usefulness of our ELISA to distinguish them from B. henselae infected patients. The resulting specificities were 89·1 and 93·5% for 17-kDa protein and GroEL, respectively. Combining the results from the two tests, we obtained a sensitivity of 82·8% and a specificity of 83·9%. Our work described and validated a proteomic approach suitable to identify immunogenic proteins useful for developing a serological test of B. henselae infection. SIGNIFICANCE AND IMPACT OF THE STUDY: A reliable serological assay for the diagnosis of Cat Scratch Disease (CSD) - a pathological condition caused by Bartonella henselae infection - has not yet been developed. Such an assay would be extremely useful to discriminate between CSD and other pathologies with similar symptoms but different aetiologies, for example lymphoma or tuberculosis. We investigate the use of two B. henselae proteins - GroEL and 17-kDa - to develop a serological-based ELISA, showing promising results with the potential for further development as an effective tool for the differential diagnosing of B. henselae infection.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Bartonella henselae/inmunología , Enfermedad por Rasguño de Gato/diagnóstico , Adolescente , Adulto , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/inmunología , Estudios de Casos y Controles , Enfermedad por Rasguño de Gato/sangre , Chaperonina 60 , Niño , Preescolar , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina M/sangre , Linfoma/diagnóstico , Masculino , Datos de Secuencia Molecular , Curva ROC , Tuberculosis Pulmonar/sangre , Tuberculosis Pulmonar/diagnóstico , Adulto JovenRESUMEN
M2 is one of the most conserved influenza proteins, and has been widely prospected as a potential universal vaccine target, with protection predominantly mediated by antibodies. In this paper we describe the creation of a humanized single chain Fv from 14C2, a potent monoclonal antibody against M2. We show that the humanized scFv demonstrates similar activity to the parental mAb: it is able to recognize M2 in its native context on cell surfaces and is able to show protective in vitro activity against influenza, and so represents a potential lead antibody candidate for universal prophylactic or therapeutic intervention in influenza.