RESUMEN
OBJECTIVES: This study describes a new method to detect autoantibodies against actin filaments (AAA) as a serological marker of intestinal villous atrophy (IVA) in celiac disease (CD), and reports the results of an Italian double-blind multicenter study. METHODS: IgA-AAA were analyzed by immunofluorescence using a newly developed method based on intestinal epithelial cells cultured in presence of colchicine. IgA-AAA were blindly evaluated prospectively in 223 antiendomysial antibody (AEA) and/or antitransglutaminase antibody (TGA) positive subjects and in 78 AEA and TGA negative subjects. IgA-AAA positive patients underwent an intestinal biopsy to confirm the diagnosis. Moreover, IgA-AAA were retrospectively investigated in 84 biopsy-proven CD patients and in 2,000 new consecutively collected serum samples from AEA and TGA negative nonbiopsied subjects. RESULTS: IgA-AAA were positive in 98.2% of the CD patients with flat mucosa, in 89% with subtotal villous atrophy, and in 30% with partial villous atrophy. IgA-AAA were present in none of the AEA and TGA negative nonbiopsied controls. In AEA and/or TGA positive CD patients IgA-AAA positivity significantly correlated with IVA (p < 0.000 in the prospective study, p = 0.005 in the retrospective study). In the prospective study, the values of sensitivity, specificity, the positive predictive value, the negative predictive value, and the efficiency of the IgA-AAA test to identify patients with IVA were, respectively, 83.9%, 95.1%, 97.8%, 69.2%, and 87.0%. Furthermore, a significant correlation (p < 0.0001) was found between the IgA-AAA serum titre and the degree of IVA (rs 0.56). CONCLUSIONS: The results of this multicenter study show that the new method for IgA-AAA detection could represent a practical diagnostic tool in AEA and/or TGA positive subjects, which would be especially useful when IVA shows a patchy distribution, when the histological picture is difficult to interpret, or when a biopsy could represent a life-threatening risk.
Asunto(s)
Actinas/inmunología , Autoanticuerpos/sangre , Enfermedad Celíaca/diagnóstico , Enterocitos/metabolismo , Actinas/metabolismo , Adolescente , Adulto , Anciano , Biomarcadores/sangre , Biopsia , Enfermedad Celíaca/patología , Células Cultivadas , Niño , Preescolar , Método Doble Ciego , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunoglobulina A/sangre , Lactante , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Estudios Retrospectivos , Sensibilidad y Especificidad , Transglutaminasas/inmunologíaRESUMEN
BACKGROUND AND AIMS: Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability. METHODS: The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers. RESULTS: Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release. CONCLUSIONS: Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.
Asunto(s)
Toxina del Cólera/inmunología , Enterocitos/efectos de los fármacos , Gliadina/farmacología , Transducción de Señal/inmunología , Actinas/inmunología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/inmunología , Células Cultivadas , Toxina del Cólera/análisis , Cicloheximida/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/inmunología , Enterocitos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Gliadina/inmunología , Haptoglobinas , Masculino , Microscopía Fluorescente/métodos , Polímeros , Proteína Quinasa C/metabolismo , Precursores de Proteínas , Conejos , Ratas , Espectrometría de Fluorescencia/métodosRESUMEN
BACKGROUND: The cytoskeleton actin network of intestinal microvilli has been found to be rapidly impaired after gluten challenge in coeliac disease (CD). The aim of this study was to investigate the presence of an immune reaction towards cytoskeleton structures such as actin filaments in CD. METHODS: Eighty three antiendomysial antibody positive CD patients (52 children and 31 adults) were studied at our outpatient clinics from 1996 to 1998 using indirect immunofluorescence, ELISA, and western blotting for antiactin (AAA) and antitissue transglutaminase (TGA) antibodies before and after a gluten free diet (GFD). Sixteen patients with smooth muscle antibody positive autoimmune hepatitis, 21 with inflammatory bowel diseases, seven with small bowel bacterial overgrowth, and 60 healthy subjects were studied as controls. RESULTS: Fifty nine of 83 CD patients (28/31 adults (90.3%); 31/52 children (59.6%)) were positive for IgA and/or IgG AAA. Seventy seven (92.7%) were positive for IgA TGA. IgA AAA were strongly correlated with more severe degrees of intestinal villous atrophy (p<0.0001; relative risk 86.17). After a GFD, AAA became undetectable within five months. CONCLUSIONS: Apart from the immune reaction against the extracellular matrix, we have described an immune reaction against the cytoskeleton in both children and adults with CD. As AAA are strongly associated with more severe degrees of villous atrophy, they may represent a useful serological marker of severe intestinal atrophy in CD.