RESUMEN
Mechanical, enzymatic and chelating methods for isolating rat intestinal epithelial cells were compared to determine the best technique for obtaining high yields of viable cells. The mechanical techniques resulted in consistently poor viability compared with the other methods. There was excellent agreement between viability determinations made by trypan blue exclusion and by cytofluorochromasia. Incubation with EDTA, citrate and dithiothreitol, combined with mechanical extrusion of dissociated cells provided the highest yields of viable cells in these studies.
Asunto(s)
Separación Celular/métodos , Colon/citología , Animales , Recuento de Células , Supervivencia Celular , Citratos/farmacología , Ditiotreitol/farmacología , Ácido Edético/farmacología , Células Epiteliales , Estudios de Evaluación como Asunto , Hialuronoglucosaminidasa/farmacología , Intestino Delgado/citología , Masculino , Ratas , Coloración y Etiquetado , Azul de Tripano/farmacología , Tripsina/farmacologíaRESUMEN
To look for possible HLA linkage in familial Crohn's disease, HLA haplotype segregation patterns were determined in 70 members of five kindreds, each having 2 or more patients with Crohn's disease (total number: 13 with Crohn's disease, 1 with ulcerative colitis). Concordant segregation of HLA haplotypes and disease was not observed. Study of HLA A and B locus phenotypes in 64 additional patients with documented Crohn's disease failed to show any significant disease association. We conclude that in these kindreds, HLA linkage does not account for the familial susceptibiltiy to Crohn's disease and that HLA A and B locus antigens are not associated with Crohn's disease.
Asunto(s)
Enfermedad de Crohn/genética , Antígenos HLA/genética , Colitis Ulcerosa/genética , Ligamiento Genético , Genotipo , Antígenos HLA/análisis , Haploidia , Humanos , Linaje , FenotipoRESUMEN
Lymphocyte subpopulations in peripheral blood (PBL) and intestinal mucosa (IML) of 10 patients with inflammatory bowel disease (IBD) were compared with those of 11 non-IBD controls. PBL were separated on Ficoll/hypaque gradients, and IML were isolated by incubation in dithiothreitol, EDTA, and collagenase. These methods yielded cells of good viability and with intact HLA A and B-antigens. T-cells, identified by neuraminidase-treated sheep RBC rosettes and non-specific esterase staining, comprised approximately 91% of the IML from normal mucosa of all groups. B-cells, identified by erythrocyte-antibody-complement rosettes and surface immunoglobulins, were only 7% of these IML populations. Cell yields were two-fold or more greater from abnormal IBD mucosa, with T-cells ranging from 55 to 95% and B-cells from 2 to 36%. The percentage of Fc receptor bearing cells was low in all specimens. By these methods, T-lymphocytes predominated in intestinal mucosa of both IBD and non-IBD patients, but there is marked increase in the percentage of B-cells isolated from abnormal mucosa in IBD.