Modulation of antigen trafficking to MHC class II-positive late endosomes of enterocytes. within five minutes after uptake as well as ovalbumin after incubation for 10 minutes. These microvillus inclusion bodies correspond to early endosomes because they lack lysosome associated membrane proteins. Late endosomes and lysosomes made up of sucrase-isomaltase did not reveal RG3039 microvillus-like structures. Conclusion: Microvillus inclusion body in MID originate from autophagocytosis of the apical membrane of enterocytes with engulfing of microvilli. test. Biosynthetic labelling Biopsy specimens from one RG3039 patient were cultured and labelled as explained previously.15 Three small pieces of the biopsy were labelled in organ culture with 35S methionine for 30 minutes, four hours, and 18 hours. Triton X-100 detergent extracts of the labelled samples were immunoprecipitated with monoclonal antibodies directed against the brush border protein SI. Immunoprecipitates were finally analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with or without treatment with endoglycosidase H. RESULTS Ultrastructural localisation of actin and villin in MID enterocytes Labelling of thin frozen sections with anti-villin and anti-actin antibodies showed gold particles within microvilli of enterocytes varying in number between the microvilli of single enterocytes from control and diseased biopsies (fig 1 ?). Binding sites for actin were also detected within the terminal web and to a lesser extent at the basolateral membrane of enterocytes. According to the position of the enterocytes around the crypt-villus axis, no difference in staining of actin and villin was found. Open in a separate window Physique 1 RG3039 Thin frozen sections of control (A, C) and patient (B, D) small bowel biopsies labelled by antibodies against villin (A, B) and actin (C, D). Labelling densities did not differ between the microvilli of patient and control samples by qualitative means. Bar=0.1 m. Quantification of actin and villin labelling revealed no significant differences between individual and control biopsies. Labelling density for the actin antibody within the microvilli revealed 35.4C49.8 gold particles/m2 in the biopsies of three patients compared with 38.5 gold particles/m2 in three control biopsies (table 2 ?). Villin labelling density within the microvilli showed 33.6 (patient No 2) and 19.8 (patient No 4) platinum particles/m2 in patient biopsies in comparison with 44.7 particles/m2 in four control biopsies (table 3 ?). The Student’s test for patients versus control showed t=0.7 for actin labelling and t=0.8 for villin labelling. Table 2 Quantification of labelling density for actin have already shown that this direct and indirect constitutive pathways of enterocytes are intact in MID.16 These findings were confirmed by our results demonstrating normal intracellular processing and transport of the brush border protein SI. RG3039 Most of the secretory granules in the apical region of a minority of crypt epithelial cells were significantly labelled by the anti-SI antibody. This is in contrast with Philips who found SI at the apical membrane and in the MIBs but not in secretory granules.16 We suggest in our model of MID (fig 6A ?) that this transport granules deliver SI (and other brush border proteins) to the apical membrane but to a different extent in different patients, most likely depending RG3039 on which secretory protein is induced. This is supported by the observation that (induced) enterocytes with SI positive secretory granules do not contain SI around the apical membrane in contrast with (uninduced) enterocytes with SI unfavorable secretory granules and SI positivity of the brush border. While the presence of immunoreactive SI in the apical membrane supports the concept of an intact biosynthetic pathway in MID, the high number of SI positive granules within the apical region of crypt epithelial cells may be indicative of delayed fusion of these granules within the apical membrane. Open in a separate window Physique 6 A model of the exocytic and endocytic pathways in crypt and villus microvillus inclusion disease (MID) epithelial cells. (A) Secretory granules, which are present in crypt epithelial cells, are labelled by the sucrase-isomaltase (SI) antibody with variable intensity. Crypt epithelial cells whose secretory granules do not contain SI show SI labelling around the apical membrane (AM). In contrast, crypt epithelial cells with no SI labelling on microvilli reveal secretory granules with SI staining. (B) Villus enterocytes with detectable microvillus inclusion Rabbit polyclonal to RAB18 body (MIBs) are characterised by a few and shortened microvilli. Microvilli within MIBs are labelled by the SI.