A comparison of Verotoxin B-subunit (Stx1B) and CD77 antibody to define germinal centre populations.

We have directly compared the use of a CD77 antibody with the binding subunit of Shiga-like toxin 1, Verotoxin 1, and (Stx1B) for delineation on human tonsil cells. We determined that the Stx1B produced a greater intensity of staining than the CD77 antibody, allowing three sub-populations of germinal centre cells to be seen. The populations express high, medium, and low levels of globotriaosylceramide as determined by the binding of the Stx1B reagent. The strong staining patterns of Stx1B suggest that it may be useful in defining germinal center B cell populations.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is associated with a naïve B-cell phenotype in human tonsils.

In B cells, signaling through the B-cell antigen receptor (BCR) is negatively modulated by the co-ligation of immunoglobulin (Ig)-immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing molecules such as FcgammaRIIB1, B-cell transmembrane protein CD72, paired immunoglobulin-like receptor PIR-B, leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Ig-like transcript ILT2, biliary glycoprotein BGP-1 and B-cell co-receptor CD22. The co-expression of multiple Ig-ITIM receptors may provide B cells with different mechanisms of regulating inhibitory pathways at different stages of differentiation. In this study, we have examined the expression of a newly defined Ig-ITIM receptor, PECAM-1 (CD31) on human B-cells. Human tonsillar B cells were purified using negative selection by depleting T cells with a combination of monoclonal antibodies and magnetic bead separation. Following purification, the pattern of PECAM-1 expression was analyzed in B-cell subpopulations using two- and three-colour fluorescence. To complement this work, PECAM-1 localization in the context of distinct areas of human tonsil was defined by immunohistochemical analysis of tonsil sections. Finally to investigate somatic mutation, Ig variable (V) region genes belonging to the nonpolymorphic VH6 family were amplified by polymerase chain reaction (PCR), subcloned and sequenced from sort-purified CD19+ PECAM-1+ and CD19+ PECAM-1- B cells. Our results demonstrate that PECAM-1 is associated with an unstimulated resting B-cell phenotype, localization to the follicular mantle and marginal zones of human tonsil and expression of unmutated Ig V region genes. These studies suggest that PECAM-1 appears on the cell surface at the naive B-cell stage and is lost as B cells differentiate into memory cells, indicating that PECAM-1 is primarily involved in naive or immature B-cell function.

Temporal changes in TFF3 expression and jejunal morphology during methotrexate-induced damage and repair.

Trefoil factor TFF3 has been implicated in intestinal protection and repair. This study investigated the spatiotemporal relationship between TFF3 expression and morphological changes during intestinal damage and repair in a rat model of methotrexate-induced small intestinal mucositis. Intestinal tissues from rats with mucositis were collected daily for 10 days. Mucosal damage was characterized by an initial decrease in cell proliferation resulting in crypt loss, villus atrophy, and depletion of goblet cells, followed by hyperproliferation that lead to crypt and villus regeneration and mucous cell repopulation. TFF3 mRNA levels increased marginally during histological damage, and the cell population expressing TFF3 mRNA expanded from the usual goblet cells to include some nongoblet epithelial cells before goblet cell repopulation. TFF3 peptide, however, was depleted during histological damage and normalized during repair, mirroring the disappearance and repopulation of goblet cells. Although there is no temporal relationship between TFF3 levels and crypt hyperproliferation, confirming the nonmitogenic nature of TFF3, the coincidental normalization of TFF3 peptide with repopulation of goblet cells and mucin production after proliferative overshoot suggests that TFF3 may play a role in the remodeling phase of repair.

Site-specific changes in transforming growth factor-alpha and -beta1 expression in colonic mucosa of adolescents with inflammatory bowel disease.

BACKGROUND: Transforming growth factors (TGF-alpha and -beta1) play important roles in intestinal growth and repair. To further understand their roles in the pathophysiology of inflammatory bowel disease (IBD), this study examined changes in their expression in colonic mucosa of adolescents with IBD. METHODS: TGF-alpha and -beta1 expression was examined by immunohistochemistry and in situ hybridization. RESULTS: TGF-gamma immunostaining and mRNA labelling appeared unchanged in the epithelium of specimens with active IBD. Similarly, expression of epithelial TGF-beta1 remained unaltered in IBD. However, the numbers of TGF-beta1-positive cells, including T cells, neutrophils, and monocytes/macrophages, in the lamina propria increased during disease activity. CONCLUSIONS: Adolescent IBD is characterized by a normal expression of TGF-alpha and -beta1 peptide and mRNA in the colonic epithelium but by an increased density of TGF-beta1-positive immune cells in the lamina propria during disease activity, suggesting a role in inflammatory modulation in IBD.

Specificity of the localization of transforming growth factor-alpha immunoreactivity in colon mucosa.

Transforming growth factor-alpha (TGF-alpha) plays an important role in gastrointestinal pathophysiology. However, the exact location of its expression in the intestine is still controversial. This study systematically compared the localization of TGF-alpha immunoreactivity in frozen or fixed human colon using three different antibodies and examined specificity of antibodies by using tissues from TGF-alpha knockout mice and by Western blotting. Consistent with the mRNA distribution revealed by in situ hybridization, a similar staining pattern was obtained in frozen sections by all three antibodies, localizing on the surface and along the crypt epithelium. In paraffin sections, although the polyclonal antibodies (raised against recombinant human or rat TGF-alpha) gave minimal staining, the monoclonal antibody (against C-terminal peptide of human TGF-alpha) still gave intense staining on the surface and upper crypt epithelium. By using specimens from TGF-alpha knockout mice in immunostaining and Western blotting, the polyclonal antibodies were shown to be specific. In contrast, specificity of the monoclonal antibody was in doubt in rodent tissues because it gave similar detection between wild-type and knockout mice in both analyses, indicating its crossreaction to non-TGF-alpha molecules. In conclusion, frozen sections and antibodies raised from recombinant TGF-alpha should be used for TGF-alpha immunohistochemistry in the colon.

Expression of functional insulin-like growth factor-1 receptor on lymphoid cell subsets of rats.

It has become evident that insulin-like growth factor-1 (IGF-1) acts as a growth factor for immune cells, yet the precise regulatory role of IGF-1 in the immune system is unknown. The aim of this study was to examine the distribution of IGF-1 receptors on rat lymphoid cells. A flow cytometric method was used, with a biotinylated and functionally active IGF-1 analogue, namely des(1-3)IGF-1, which binds well to IGF-1 receptor but poorly to IGF binding proteins, followed by phycoerythrin-conjugated streptavidin (PE-SA) staining. Our results showed that IGF-1 receptors were readily detectable on a wide variety of the immune cells, including T cells, B cells and monocytes, but the binding capacity for IGF-1 was monocytes > B cells > T cells, as determined by titration experiments. Furthermore, the level of expression on resting CD4+ T lymphocytes was greater than on CD8+ cells, and the concentration of biotin-des(1-3)IGF-1 required to demonstrate the binding to IGF-1 receptor on CD8+ cells (68 nmol/l) was 200-fold higher than for CD4+ cells (0.34 nmol/l), indicating that most of the IGF-1 receptor on CD8+ cells represented lower affinity sites. The level of IGF-1 receptor expression was increased several-fold after concanavalin A stimulation on both CD4+ and CD8+ T-cell subsets. Kinetic analysis of the expression of IGF-1 receptor and its association with interleukin-2 receptors (IL-2R) following activation showed a similar pattern, with no significant differences in the ratio of IGF-1 receptor: IL-2R per cell during the 3 days of cell culture. Our studies suggest that biological activities of IGF-1 include direct stimulation of immune cells, and that expression of IGF-1 receptor may have a role in regulation of T-cell function.