In the pathogenesis of the hemolytic uremic syndrome (HUS), endothelial damage of glomeruli and arterioles of the kidney appears to play a central role. Previous studies have shown that verocytotoxin-1 (VT-1) cytotoxicity on human vein endothelial cells require additional stimuli, in particular the inflammatory mediator tumor necrosis factor alpha (TNF alpha). In this study the effects of VT on human glomerular microvascular endothelial cells (GMVEC) were examined. A reproducible method was developed for the isolation and purification of large numbers of highly purified GMVEC. The obtained GMVEC were over 99% pure; their endothelial origin was demonstrated by the expression of the endothelial antigens von Willebrand factor, EN-4, PECAM-1 and V,E-cadherin. Upon stimulation with TNF alpha the cells expressed the endothelial-specific adhesion molecule E-selectin. A limited number of fenestral structures was observed by scanning electron microscopy (SEM), suggesting glomerular origin of the endothelial cells. Cytotoxicity of VT-1 to GMVEC was evaluated by determination of the number of viable adherent cells and by assay of overall protein synthesis after exposure to varying concentrations of VT-1. In non-stimulated GMVEC, cytotoxicity of VT-1 was inversely related to the degree and duration of confluence, subconfluent cells being the most sensitive. In highly confluent GMVEC, VT cytotoxicity required pre-exposure of the cells to the inflammatory mediator TNF alpha, which induced an increase in the number of VT receptors on GMVEC. Thin layer chromatography of extracted glycolipids from the GMVEC showed binding of VT-1 to globotriaosylceramide (Gb3), known to be the functional receptor for VT. There were no major differences in protein synthesis inhibition with equal concentrations VT-1 and VT-2. In conclusion, in this study we provide a reproducible method to isolate, purify and culture well characterized human GMVEC on a routine basis. In vitro studies with these GMVEC demonstrate that VT cytotoxicity depends on the degree of confluence and the additional preexposure to the inflammatory mediator TNF alpha. These observations provide further insight into the complex events that may occur in glomeruli in the pathogenesis of HUS.

IntroductionHistones can mediate the binding of DNA and anti-DNA to the glomerular basement membrane (GBM). In ELISA histone/DNA/anti-DNA complexes are able to bind to heparan sulfate (HS), an intrinsic constituent of the GBM. We questioned whether histone containing immune complexes are able to bind to the GBM, and if so, whether the ligand in the GBM is HS. Monoclonal antibodies (mAbs) complexed to nucleosomal antigens and noncomplexed mAbs were isolated from culture supernatants of four IgG anti-nuclear mAbs. All noncomplexed mAbs showed strong anti-nucleosome reactivity in ELISA. One of them showed in addition anti-DNA reactivity in noncomplexed form. The other three mAbs only showed anti-DNA reactivity when they were complexed to nucleosomal antigens. After renal perfusion a fine granular binding of complexed mAbs to the glomerular capillary wall and activation of complement was observed in immunofluorescence, whereas noncomplexed mAbs did not bind. Immuno-electron microscopy showed binding of complexes to the whole width of the GBM. When HS in the GBM was removed by renal heparinase perfusion the binding of complexed mAb decreased, but did not disappear completely.We conclude that anti-nucleosome mAbs, which do not bind DNA, become DNA reactive once complexed to nucleosomal antigens. These complexed mAbs can bind to the GBM. The binding ligand in the GBM is partly, but not solely, HS. Binding to the GBM of immune complexes containing nucleosomal material might be an important event in the pathogenesis of lupus nephritis. (J. Clin. Invest. 1994. 94:568-577.)

Histologic grading systems are used to guide diagnosis, therapy, and audit on an international basis. The reproducibility of grading systems is usually tested within small groups of pathologists who have previously worked or trained together. This may underestimate the international variation of scoring systems. We therefore evaluated the reproducibility of an established system, the Banff classification of renal allograft pathology, throughout Europe. We also sought to improve reproducibility by providing individual feedback after each of 14 small groups of cases. Kappa values for all features studied were lower than any previously published, confirming that international variation is greater than interobserver variation as previously assessed. A prolonged attempt to improve reproducibility, using numeric or graphical feedback, failed to produce any detectable improvement. We then asked participants to grade selected photographs, to eliminate variation induced by pathologists viewing different areas of the slide. This produced improved kappa values only for some features. Improvement was influenced by the nature of the grade definitions. Definitions based on "area affected" by a process were not improved. The results indicate the danger of basing decisions on grading systems that may be applied very differently in different institutions.

IntroductionAntibodies have been studied extensively for their use in immunotherapy of cancer. [1][2][3][4] It is evident that receptors for the Fc portion of immunoglobulins (FcRs) on myeloid cells are critical in triggering antitumor cytotoxicity in vivo. 5,6 Antibody-dependent cellular cytotoxicity (ADCC), considered crucial for antibody-mediated tumor cell degradation, can be mediated by polymorphonuclear leukocytes (PMNs), monocytes/macrophages, eosinophils, and natural killer (NK) cells. 7,8 These effector cells use different cytotoxic mechanisms, depending on their activation state and the nature of the target. 7,9-12 PMNs, representing the most populous type of white blood cell, exhibit fast recruitment activity in vivo. Potent and very rapid (within 30 minutes) PMN cytotoxicity toward various tumor targets has been documented. 10,[13][14][15] Two classes of immunoglobulin (Ig)G receptors (Fc␥RIIa, CD32, and Fc␥RIIIb, CD16) and one class of IgA receptor (Fc␣RI, CD89) have been identified on human PMNs, whereas Fc␥RI (CD64) expression is inducible on PMNs on stimulation with granulocyte colony-stimulating factor. 16 PMNs can trigger ADCC by engagement of Fc␥RI, Fc␥RIIa, and Fc␣RI. Fc␣RI, however, has been observed to be the most effective FcR for PMN-mediated tumor cell killing. 14,17 Mac-1 (CR3, CD11b/CD18) is a member of the ␤ 2 integrin family, which includes Mac-1, LFA-1 (CD11a/CD18), and gp150/95 (CR4, CD11c/CD18). 18,19 These receptors, sharing a common ␤-chain (CD18), can bind multiple ligands and can regulate various leukocyte functions. Mac-1 represents the predominant ␤ 2 integrin on PMNs and is furthermore expressed on monocytes/macrophages and NK cells. Several PMN functions are regulated by Mac-1, including adhesion, migration, chemotaxis, phagocytosis, respiratory burst activity, and degranulation. 18 On activation, Mac-1 is able to initiate signaling by its linkage to the actin cytoskeleton and associated signaling proteins. 20,21 A number of studies described Mac-1 cooperation with different receptors on PMNs, indicating Mac-1 to be a signaling partner for other receptors. 22 These include FMLP receptors, LPS/LBP receptors (CD14), urokinase plasminogen activator receptor (CD87), and Fc receptors. [22][23][24][25][26] Mac-1 was found to trigger Ab-dependent phagocytosis by Fc␥RIIIb in fibroblasts transfected with both Mac-1 and Fc␥RIIIb, whereas cells expressing only Mac-1 or Fc␥RIIIb were unable to ingest Ab-opsonized particles. 27 Mac-1 cooperation with Fc␥RIIIb in the generation of PMN respiratory burst has been described as well. 28 Furthermore, Mac-1 restored IgG-dependent phagocytosis of transfectants with Fc␥RIIa tail-minus mutants. 29 Importantly, PMNs from patients with leukocyte adhesion deficiency, who lack CD18, were shown to be severely impaired in mediating phagocytosis and ADCC. 18,[30][31][32] Although the relative contribution of individual ␤ 2 integrins remains to be determined, various studies point to an important role of Mac-1 in FcR-mediated cytotoxicity. Targets st...