Peripheral blood is the primary source of lymphoid cells for investigation of the human immune system. Its use is facilitated by Ficoll-Hypaque density gradient centrifugation-a simple and rapid method of purifying peripheral blood mononuclear cells (PBMC) that takes advantage of the density differences between mononuclear cells and other elements found in the blood sample. Thus, cells are distributed in the solution in layers based on the differences in their density/size. Additional purification methods can be employed as the mononuclear cell sample can be purified from monocytes by adherence or by exposure to L-leucine methyl ester; these methods are described for both procedures. Cord blood and peripheral blood from infants contain immature cells, including nucleated red cells, which can result in significant contamination of the mononuclear cell layer, and removal of these cells requires additional steps that are described. The isolation procedures presented here can also be applied to cell populations derived from tissues.

Material Supplementary 2.DC1http://www.jimmunol.org/content/suppl/2010/06/16/jimmunol.100008

No abstract

Human platelet glycoprotein Ib (GP Ib) i s a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that G P Ib is normally coinplexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GPIb complex (GPIb plus GPIX) was purified to homogeneity in = 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. G P Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/ polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170000 under nonreducing conditions and 135000 (cr subunit) and 25000 (p subunit) under reducing conditions. GP IX had an apparent M , of 22000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-Factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. G P Ib, had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib, and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib, and G P 1X were blocked; G P Ib, had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (2 I: SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ib, (22000 I: 2700, n = 3), WM 23, epitope on G P Ib, (21 000 3400, n = 3), FMC 25, epitope on GP IX (20100 f 2700, n = 3), and FMC 25 (Fab')2 (27100 800, n = 2). The combined evidence suggests that GP Ib is normally bound to GP IX and that the stoichioinetry of this complex is 1 : 1.The initial event in hemostasis in response to vascular injury involves the adhesion of platelets to the exposed vascular subendothelium. The adhesion reaction appears to be dependent on three components [I]: a receptor on platelets which on the basis of a variety of evidence has been defined as the platelet membrane protein, glycoprotein Ib (GP Ib); a plasma component, factor VIII/von Willebrand fac...

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal (GI) tract characterized by pain or discomfort from the lower abdominal region, which is associated with altered bowel habit. Despite its prevalence, there is currently a lack of effective treatment options for patients. IBS has long been considered as a neurological condition resulting from alterations in the brain gut axis, but immunological alterations are increasingly reported in IBS patients, consistent with the hypothesis that there is a chronic, but low-grade, immune activation. Mediators released by immune cells act to either dampen or amplify the activity of GI nerves. Release of a number of these mediators correlates with symptoms of IBS, highlighting the importance of interactions between the immune and the nervous systems. Investigation of the role of microbiota in these interactions is in its early stages, but may provide many answers regarding the mechanisms underlying activation of the immune system in IBS. Identifying what the key changes in the GI immune system are in IBS and how these changes modulate viscerosensory nervous function is essential for the development of novel therapies for the underlying disorder.