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The GM gangliosides and cholesterol components of plasma membrane lipid rafts play an important role in the recruitment and signaling of protein receptors in eukaryotic cells. Herein, we have analyzed at the single-cell level the partitioning and intracellular trafficking of GM gangliosides and cholesterol in quiescent (CD4+CD69-) and CD3-activated (CD4+CD69+) thymic and splenic T cells. First, regardless the gender and the quiescent or activated status of T cells, the GM and cholesterol content in cytosol and plasma membrane as well as the expression levels of GM synthase, Sphingomyelin phosphodiestarase 2 and HMG Co-A reductase genes involved in GM and cholesterol synthesis were constantly lower in CD4 thymocytes than in CD4 splenocytes. Second, we detected variations in the balance between GM and cholesterol in plasma membrane depending on aging, and found that deprivation of cellular cholesterol does not necessarily affect the GM content in both quiescent CD4 thymocytes and splenocytes. Third, CD3 stimulation up-regulated the GM and little if any the cholesterol content in both thymic and splenic CD4 T cells, suggesting a cross talk between the CD3 signaling and GM but not cholesterol biosynthesis pathway. Fourth, partitioning and trafficking of GM to the plasma membrane depended on the transport of ceramide precursors from endoplasmic reticulum to Golgi network, as well as on the synthesis, glycosylation and vesicular assembly in trans-Golgi, and less on the cytoskeleton architecture in both quiescent and activated CD4 thymic and splenic T cells. Together, these findings suggest that the differential partitioning and intracellular trafficking of GM and cholesterol in thymic and splenic CD4 T cells may account for the stage of functional maturation.

The plasma membrane of T cells is made of a combination of glycosphingolipids and protein receptors organized in glycolipoprotein microdomains termed lipid rafts. The structural assembly of lipid rafts was investigated by various physical and biochemical assays. Depending on the differentiation status of T cells, the lipid rafts seclude various protein receptors involved in T cell signaling, cytoskeleton reorganization, membrane trafficking, and the entry of infectious organisms into the cells. This review article summarizes the most common methods, and their limits and advantages for analyzing the composition and assembly of lipid rafts with protein receptors into lipid rafts microdomains in plasma membrane of T cells. It also includes new methods such as ELISA/Polysorp and flow cytometry, and a combined sucrose gradient centrifugation-FPLC-Western blot strategy developed in our laboratory to study non-covalent interactions between the GM1 glycosphingolipid and protein receptors in plasma membrane of T cells.

Fibrillin-1 (Fbn-1) is a ubiquitous protein present in the extracellular matrix of various organs and it is a major component of microfibrils embedded in the core of elastic fibers. In humans, mutations or deletions of the Fbn-1 gene are associated with several genetic diseases. In addition, several microsatellite alleles near Fbn-1 gene were found associated with diffuse scleroderma. In TSK/+ mice, which develop a scleroderma-like syndrome, the Fbn-1 gene exhibits an inframe duplication of exons 17-40. In this study, we report that the synthesis and secretion of wild-type Fbn-1 in TSK/+ is higher than that of the mutated Fbn-1 protein excluding the possibility that TSK genetic defect is due to a loss of the wild allele. We also demonstrate for the first time that TGF-beta, which plays a crucial role in skin fibrosis, binds to both wild-type and mutated Fbn-1. The amount of bound TGF-beta was higher in mutated than wild-type Fbn-1 and appears related to the number of TGF-beta binding motifs.

In this report we investigated lymphokine-activated killer (LAK) cytotoxicity and lymphocyte subpopulations of peripheral blood mononuclear cells (PBMCs) of patients with acute leukemia in complete remission after chemotherapy or autologous bone marrow transplantation (ABMT) and of normal donors. A positive linear correlation was found between the percentage of spontaneous LAK activity and that of lymphocyte subpopulations with phenotypes CD56+, CD3-CD8+ CD3-CD56+ and CD3-CD57+ in both groups of patients. A 3-day culture with IL-2 produced an up-regulation in the expression of the CD25, CD69, and HLA-DR markers proportional to the LAK cytotoxicity levels generated in the culture. Determination of percentages of spontaneous and in vitro generated LAK activity as well as of the above mentioned phenotypic markers contribute to the analysis of the process of LAK activation in patients with acute leukemia and may also be useful in those cases in which immunotherapy with IL-2 and/or LAK cells is anticipated.

We have studied lymphocyte subpopulations and the NK cytotoxicity of the PBMC from acute leukemia patients in complete remission after chemotherapy or ABMT and from normal donors. We have found a positive linear correlation between the percentage of subpopulations with CD3-CD16+, CD3-CD56+ and CD3-CD8+ phenotypes and the percentage of NK cytotoxicity. The slopes of the regression lines in the two groups of patients were lower than in normal donors, indicating a decreased ability of these cells to operate. The percentages of these subpopulations represent parameters for estimating the percentage of NK cytotoxicity both in normal donors and in patients with acute leukemia.

Rearrangements of the N-myc oncogene have not been described in the literature. The authors present a case of ANLL with the N-myc gene rearranged out of 16 ANLLs and 11 ALLs. While the proportion of the blast cells in the case reported was 85%, the rearranged gene gave a hybridization band which was less intense than that of the germline band, making it probable that this gene rearrangement is present in a leukemia subclone. The c-myc oncogene as well as Ig and TcR genes were in germline configurations. The presence of the rearranged N-myc in the reported case could imply an alternative mechanism of mutation of this oncogene in the pathogenesis of hematological malignancies.