Using the Peggy Simple Western system for fine needle aspirate analysis.

Simple Western™ assays are capillary-based electrophoretic immunoassays, similar in scope to SDS-PAGE (molecular weight separation, "size") and IEF (isoelectric focusing, "charge") immunoblotting. The enhanced sensitivity and automation of the Simple Western makes it better suited to cancer diagnostics and research than the traditional Western platform. Because of its smaller sample volume requirements, primary cells, such as those obtained from fine needle aspirates (FNAs), and solid tumor slices may be used to generate quantitative comparable data. The Peggy™ instrument is capable of performing either size or charge assays on up to 96 samples in a single unattended run.

Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice.

The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.

Vascular development of the brain requires beta8 integrin expression in the neuroepithelium.

We showed previously that loss of the integrin beta8 subunit, which forms alphavbeta8 heterodimers, results in abnormal vascular development in the yolk sac, placenta, and brain. Animals lacking the integrin beta8 (itgbeta8) gene die either at midgestation, because of insufficient vascularization of the placenta and yolk sac, or shortly after birth with severe intracerebral hemorrhage. To specifically focus on the role of integrins containing the beta8 subunit in the brain, and to avoid early lethalities, we used a targeted deletion strategy to delete itgbeta8 only from cell types within the brain. Ablating itgbeta8 from vascular endothelial cells or from migrating neurons did not result in cerebral hemorrhage. Targeted deletion of itgbeta8 from the neuroepithelium, however, resulted in bilateral hemorrhage at postnatal day 0, although the phenotype was less severe than in itgbeta8-null animals. Newborn mice lacking itgbeta8 from the neuroepithelium had hemorrhages in the cortex, ganglionic eminence, and thalamus, as well as abnormal vascular morphogenesis, and disorganized glia. Interestingly, adult mice lacking itgbeta8 from cells derived from the neuroepithelium did not show signs of hemorrhage. We propose that defective association between vascular endothelial cells and glia lacking itgbeta8 is responsible for the leaky vasculature seen during development but that an unidentified compensatory mechanism repairs the vasculature after birth.