Defective glycosylphosphatidylinositol anchor synthesis in paroxysmal nocturnal hemoglobinuria granulocytes. (73/413)

To investigate the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor in the granulocytes of paroxysmal nocturnal hemoglobinuria (PNH), the glycolipids of granulocytes from PNH patients and normal volunteers were biosynthetically labeled with [3H]mannose in the presence of tunicamycin. Extracted glycolipids were examined by thin-layer chromatography and compared with known biosynthetic intermediates. Normal granulocytes consistently showed [3H]mannose incorporation into the complete GPI core, several GPI biosynthetic intermediates, and dolichol phosphate mannose (DPM). The granulocytes of 10 patients with PNH that had no expression of CD55 and CD59 on greater than 95% of the cells were able to incorporate [3H]mannose into DPM, but were not able to incorporate detectable amounts into the complete GPI core. THus, PNH granulocytes do not synthesize detectable amounts of the complete GPI core and this defect likely accounts for the absence of GPI-linked membrane proteins on hematopoietic cells in this syndrome.  (+info)

Longer in vivo survival of CD59- and decay-accelerating factor-almost normal positive and partly positive erythrocytes in paroxysmal nocturnal hemoglobinuria as compared with negative erythrocytes: a demonstration by differential centrifugation and flow cytometry. (74/413)

Three populations of erythrocytes have been shown by flow cytometric analysis on complement regulatory proteins: CD59 and decay-accelerating factor (DAF) on erythrocytes in paroxysmal nocturnal hemoglobinuria (PNH). CD59 and DAF in PNH may be completely deficient in CD59- and DAF-negative erythrocytes, they may be decreased varyingly in partly positive erythrocytes, and they may be approximately normal in almost normal positive erythrocytes. Control erythrocytes are always CD59- and DAF-normal positive. CD59- and DAF-negative erythrocytes have been shown to be most sensitive to complement lysis in vitro. However, it has not yet been elucidated whether CD59- and DAF-almost normal positive and partly positive erythrocytes in a patient have a longer in vivo survival than negative erythrocytes. Blood from controls and PNH patients was separated in five fractions by differential centrifugation. CD59 and DAF on the fractionated erythrocytes were determined by flow cytometry using specific antibodies. Ratios of CD59- and DAF-almost normal positive and partly positive cells to negative erythrocytes were increased progressively from the top fraction to the bottom. The erythrocytes in the top fraction are younger and reticulocyte-rich, while those in the bottom are older and reticulocyte-poor. Hence, the present results indicate that CD59- and DAF-partly positive erythrocytes as well as almost normal positive erythrocytes in patients may have a longer in vivo survival than negative erythrocytes.  (+info)

New monoclonal antibodies in CD59: use for the analysis of peripheral blood cells from paroxysmal nocturnal haemoglobinuria (PNH) patients and for the quantitation of CD59 on normal and decay accelerating factor (DAF)-deficient erythrocytes. (75/413)

CD59 is a widely expressed cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein which acts as an inhibitor of the assembly of the membrane attack complex of autologous complement. Four new monoclonal antibodies to CD59 (2/24, 1B2, BRIC 229, BRIC 257) are described. Competitive binding experiments using these antibodies, two known CD59 antibodies (MEM-43, YTH 53.1) and a previously described antibody LICR-LON-Fib75.1 demonstrated that all seven antibodies see related epitopes on human erythrocyte CD59. In common with other GPI-linked proteins, CD59 (as defined by antibody 2/24) was sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) on lymphocytes and monocytes but not on erythrocytes. Flow cytometric analysis using antibody 2/24 identified two populations (CD59 positive and CD59 deficient) of lymphocytes, monocytes and erythrocytes in peripheral blood from a patient with paroxysmal nocturnal haemoglobinuria (PNH). The abundance of CD59 on normal erythrocytes was determined as 21,000 copies/cell when radioiodinated BRIC 229 was used. Other CD59 antibodies gave values of 10,000 (IF5) and 15,000 (2/24) against the same target cells. Radioiodinated Fab fragments of BRIC 229 gave a value of 39,000 copies/cell. Erythrocytes from two individuals with a rare inherited deficiency of decay accelerating factor (DAF), known as the Inab phenotype, expressed normal levels of CD59.  (+info)

CD66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells. (76/413)

Neutrophil adherence to cytokine-activated endothelial cell (EC) monolayers depends on the expression of the endothelial leukocyte adhesion molecule-1 (ELAM-1). The ligand for ELAM-1 is the sialylated Lewis-x antigen (SLe(x)) structure. The selectin LAM-1 (or LECAM-1) has been described as one of the SLe(x)-presenting glycoproteins involved in neutrophil binding to ELAM-1. Other presenter molecules have not yet been described. Our data demonstrate that the carcinoembryonic antigen (CEA)-like surface molecules on neutrophils--known as the nonspecific cross-reacting antigens (NCAs)--are involved in neutrophil adherence to monolayers of IL-1-beta-activated EC. The NCAs are recognized by CD66 (NCA-160 and NCA-90) and CD67 (NCA-95). Because NCA-95 and NCA-90 have previously been found to be phosphatidylinositol (PI)-linked, paroxysmal nocturnal hemoglobinuria (PNH) neutrophils (which lack PI-linked surface proteins) were tested as well. PNH neutrophils showed a diminished binding to activated EC. CD66 (on PNH cells still recognizing the transmembrane NCA-160 form) still inhibited the adherence of PNH cells to IL-1-beta-activated EC, but to a limited extent. Soluble CEA(-related) antigens inhibited normal neutrophil adherence as well, whereas neutrophil transmigration was unaffected. Sialidase-treatment as well as CD66 preclearing abolished the inhibitory capacity of the CEA(-related) antigens. The binding of soluble CEA antigens to IL-1-beta-pretreated EC was blocked by anti-ELAM-1. These soluble antigens, as well as the neutrophil NCA-160 and NCA-90, both recognized by CD66 antibodies, presented the SLe(x) determinant. Together, these findings indicate that the CD66 antigens (i.e., NCA-160/NCA-90) function as presenter molecules of the SLe(x) oligosaccharide structures on neutrophils that bind to ELAM-1 on EC.  (+info)

Synthesis of mannosylglucosaminylinositol phospholipids in normal but not paroxysmal nocturnal hemoglobinuria cells. (77/413)

To identify mannosyl (Man)-containing intermediates of the human glycoinositol phospholipid (GPI) anchor pathway and examine their expression in paroxysmal nocturnal hemoglobinuria (PNH), mannolipid products deriving from in vitro guanosine diphosphate [3H]Man labeling of HeLa cell microsomes were characterized. The defined GPI species were correlated with products deriving from in vivo [3H]Man labeling of normal and (GPI-anchor defective) affected leukocytes. In vitro analyses in HeLa cells showed dolichol-phosphoryl (Dol-P)-[3H]Man and a spectrum of [3H]Man lipids exhibiting TLC mobilities approximating those of Trypanosoma brucei (Tryp) GPI precursors. Iatrobead HPLC separations and partial characterizations of the major isolated [3H]Man species (designated H1-H8) showed that all but H1 (Dol-P-Man) were sensitive to HNO2 deamination and serum GPI-specific phospholipase D digestion but were resistant to phosphatidylinositol-specific phospholipase C digestion unless previously deacylated with mild alkali. [3H]Man label in H3, H4, and H6 but not in H5 or H7 was efficiently released into the aqueous phase by jack bean alpha-mannosidase digestion. BioGel P-4 and AX-5 sizing of the dephosphorylated core glycan fragments of H6 and H7 gave values that coincided precisely with the corresponding glycan fragments from the fully assembled Tryp anchor donor A' (P2). Affected leukocytes from four patients with PNH supported formation of GlcNAc- and GlcN-PI but all failed to express H6 and H7 as well as H8 and two showed complete absence of earlier Man-containing intermediates. These findings argue that human intracellular GPI mannolipids are built on acylated inositol phospholipids, that H6 and H7 contain differentially phosphoethanolamine-substituted Man3-GlcN-inositol cores, and that PNH cells are defective in conversion of GlcN-PI into these more mature mannolipid structures.  (+info)

Syphilitic paroxysmal cold haemoglobinuria. Case report and study of the Coombs test. (78/413)

A case of congenital syphilis which presented with paroxysmal cold haemoglobinuria is described. The patient had aortic incompetence, and the significance of this finding is discussed. The Coombs test was studied, and positive results appear to be due to interaction between antiglobulin serum and complement adsorbed on to the test cells, this adsorption being promoted by the cold antibodies.  (+info)

Glycosyl-phosphatidylinositol-anchored membrane proteins. (79/413)

Many proteins of eukaryotic cells are anchored to membranes by covalent linkage to glycosyl-phosphatidylinositol (GPI). These proteins lack a transmembrane domain, have no cytoplasmic tail, and are, therefore, located exclusively on the extracellular side of the plasma membrane. GPI-anchored proteins form a diverse family of molecules that includes membrane-associated enzymes, adhesion molecules, activation antigens, differentiation markers, protozoan coat components, and other miscellaneous glycoproteins. In the kidney, several GPI-anchored proteins have been identified, including uromodulin (Tamm-Horsfall glycoprotein), carbonic anhydrase type IV, alkaline phosphatase, Thy-1, BP-3, aminopeptidase P, and dipeptidylpeptidase. GPI-anchored proteins can be released from membranes with specific phospholipases and can be recovered from the detergent-insoluble pellet after Triton X-114 treatment of membranes. All GPI-anchored proteins are initially synthesized with a transmembrane anchor, but after translocation across the membrane of the endoplasmic reticulum, the ecto-domain of the protein is cleaved and covalently linked to a preformed GPI anchor by a specific transamidase enzyme. Although it remains obscure why so many proteins are endowed with a GPI anchor, the presence of a GPI anchor does confer some functional characteristics to proteins: (1) it is a strong apical targeting signal in polarized epithelial cells; (2) GPI-anchored proteins do not cluster into clathrin-coated pits but instead are concentrated into specialized lipid domains in the membrane, including so-called smooth pinocytotic vesicles, or caveoli; (3) GPI-anchored proteins can act as activation antigens in the immune system; (4) when the GPI anchor is cleaved by PI-phospholipase C or PI-phospholipase D, second messengers for signal transduction may be generated; (5) the GPI anchor can modulate antigen presentation by major histocompatibility complex molecules. Finally, at least one human disease, paroxysmal nocturnal hemoglobinuria, is a result of defective GPI anchor addition to plasma membrane proteins.  (+info)

In vitro and in vivo evidence of PNH cell sensitivity to immune attack after nonmyeloablative allogeneic hematopoietic cell transplantation. (80/413)

It has been proposed that paroxysmal nocturnal hemoglobinuria (PNH) cells may proliferate through their intrinsic resistance to immune attack. To evaluate this hypothesis, we examined the impact of alloimmune pressure on PNH and normal cells in the clinical setting of nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Five patients with severe PNH underwent HCT from an HLA-matched family donor after conditioning with cyclophosphamide and fludarabine. PNH neutrophils (CD15(+)/CD66b(-)/CD16(-)) were detected in all patients at engraftment, but they subsequently declined to undetectable levels in all cases by 4 months after transplantation. To test for differences in susceptibility to immune pressure, minor histocompatibility antigen (mHa)-specific T-cell lines or clones were targeted against glycosylphosphatidylinositol (GPI)-negative and GPI-positive monocyte and B-cell fractions purified by flow cytometry sorting. Equivalent amounts of interferon-gamma (IFN-gamma) were secreted following coculture with GPI-negative and GPI-positive targets. Furthermore, mHa-specific T-cell lines and CD8(+) T-cell clones showed similar cytotoxicity against both GPI-positive and GPI-negative B cells. Presently, all 5 patients survive without evidence of PNH 5 to 39 months after transplantation. These in vitro and in vivo studies show PNH cells can be immunologically eradicated following nonmyeloablative HCT. Relative to normal cells, no evidence for a decreased sensitivity of PNH cells to T-cell-mediated immunity was observed.  (+info)