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Paroxysmal nocturnal haemoglobinuria (PNH) results from acquired mutations in the PIG-A gene of an haematopoietic stem cell, leading to defective biosynthesis of glycosylphosphatidylinositol (GPI) anchors and deficient expression of GPI-anchored proteins on the surface of the cell's progeny. Some laboratory and clinical findings have suggested genomic instability to be intrinsic in PNH; this possibility has been supported by mutation analysis of hypoxanthine-guanine phosphoribosyltransferase ( HPRT) gene abnormalities. However, the HPRT assay examines lymphocytes in peripheral blood (PB), and T cells may be related to the pathophysiology of PNH. We analysed the molecular and functional features of HPRT mutants in PB mononuclear cells from eleven PNH patients. CD8 T cells predominated in these samples; approximately half of the CD8 cells lacked GPI-anchored protein expression, while only a small proportion of CD4 cells appeared to derive from the PNH clone. The HPRT mutant frequency (Mf) in T lymphocytes from PNH patients was significantly higher than in healthy controls. The majority of the mutant T lymphocyte clones were of CD4 phenotype, and they had phenotypically normal GPI-anchored protein expression. In PNH patients, the majority of HPRT mutant clones were contained within the V β2 T cell receptor (TCR) subfamily, which was oligoclonal by complementarity-determining region three (CDR3) size analysis. Our results are more consistent with detection of uniform populations of expanded T cell clones, which presumably acquired HPRT mutations during antigen-driven cell proliferation, and not due to an increased Mf in PNH. HPRT mutant analysis does not support underlying genomic instability in PNH. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) cells express certain tumour-associated antigens (TAAs) that could serve as targets for active-specific immunotherapy. The aim of the present study was to test the MM/dendritic cell (DC) fusion as a vaccination strategy. We fused MM cells with DC to generate fusion cells (FCs) and tested their antigen presenting cell (APC) function in mixed lymphocyte reactions and cytotoxicity assays. First, the HS Sultan and SK0-007 HAT sensitive human MM cell lines and DCs generated from peripheral blood of normal donors were fused in the presence of 50% polyethylene glycol to form FCs. Next, tumour cells freshly isolated from patients were similarly fused with autologous DCs to generate FCs. The FCs demonstrated a biphenotypic profile, confirmed both by flow-cytometry and dual immunofluorescence microscopy. These FCs induced MM-specific cytotoxicity. FCs, but not MM cells or DCs alone, were potent stimulators of autologous patient T cells. More importantly, FC-primed autologous peripheral blood mononuclear cells demonstrated major histocompatibility complex-restricted MM-specific cytolysis. These studies therefore demonstrated that MM/DC FC can trigger an autologous immune response to MM cells and formed the framework for a clinical trial currently underway. [ABSTRACT FROM AUTHOR]

We have investigated whether intracellular signal transduction molecules can be used as immunohistological markers of normal and neoplastic human leucocytes in routine tissue sections. We obtained selective labelling of white cells for eight such molecules (the ‘linker’ molecules SLP-76 and BLNK, the Src family kinases Lyn, Fyn, Syk and Hck, and the phospholipases PLC- γ1 and PLC- γ2). Antibodies to SLP-76 and PLC- γ1 selectively labelled T cells, and antibodies to BLNK, Lyn, Fyn, Syk and PLC- γ2 labelled B cells (although Fyn immunostaining was restricted to mantle zone B cells). Antibodies to the Syk and Hck kinases labelled probable thymocyte precursors at the periphery of the thymic cortex. In addition to lymphoid cells, several other leucocyte types were immunostained (e.g. SLP-76, Lyn, Syk and Hck were found in megakaryocytes, myeloid cells and/or macrophages, and PLC- γ2 was detected in arterial endothelium). SLP-76 and PLC- γ1 were found in most T-cell lymphomas studied, and some B-cell lymphomas were also positive for PLC- γ1 (e.g. diffuse large cell and Burkitt's lymphoma). The five B cell-associated markers were found in most B-cell non-Hodgkin's lymphomas, although some diffuse large B-cell lymphomas were negative (e.g. for Lyn) and anti-Fyn tended not to stain small B-cell neoplasms. The observation that a range of leucocyte signalling molecules can be detected in routine biopsies offers new possibilities for studying normal and neoplastic human white cells in diagnostic tissue samples. [ABSTRACT FROM AUTHOR]

Ikaros is a Kruppel-type zinc finger protein that is essential for normal lymphocyte development and differentiation. Recently, it has been demonstrated that Ikaros is frequently inactivated in both human and mouse leukaemias/lymphomas. Although this inactivation is thought to be involved in leukaemogenesis, little is known about the molecular mechanisms that lead to neoplastic transformation. To identify the genes that may be controlled by Ikaros, we performed differential display analysis of RNAs from mouse 3T3-L1 cells that had been transfected with the Ikaros gene. Two cDNAs, the Trk-fused gene ( Tfg) and death-associated protein 3 gene ( Dap3) were upregulated in Ikaros-transfected cells. Expression of Tfg and Dap3 was consistently downregulated in radiation-induced T-cell lymphomas that exhibited defective Ikaros expression. These results suggest that Tfg and Dap3 function downstream of Ikaros and may be involved in radiation-induced lymphomagenesis. [ABSTRACT FROM AUTHOR]