Cytochemical, cytogenetic, immunophenotypic and tumorigenic characterization of two hairy cell lines

Two cell lines (EH and HK) were derived from two patients with hairy cell leukemia (HCL). Both patients exhibited a clinical picture characteristic of HCL, including splenomegaly, cytopenias, and tartrate- resistant acid phosphatase (TRAP)-positive "hairy" lymphocytes in blood and marrow. EH and HK were demonstrably of B lineage, as judged by cytochemistry and immunophenotype, including expression of B1, B2, and LEU-12 antigens and of monoclonal surface immunoglobulins (slgs). Monoclonality was confirmed by clonal karyotype abnormality demonstrated in cell line HK. HCL parentage suggested by cytochemistry and electron microscopy was confirmed by the immunophenotypic observation that HCL lines expressed antigens alpha S-HCL1, alpha S- HCL3, and cCLLa. While alpha S-HCL1 and alpha S-HCL3 are nonspecific, their co-expression is characteristic of HCL cells. The cCLLa is a novel 69-kd membrane HCL-associated polypeptide antigen not shared by circulating normal T or B lymphocytes nor by malignant cells from unrelated lymphoid or nonlymphoid malignancies. The doubling time of EH and HK was 24 and 36 hours, respectively. While HK included a small subset of Epstein-Barr virus (EBV) nuclear antigen-positive cells, EH cells were homogeneously negative for the presence of this antigen. Both cell lines were consistently implantable in irradiation- preconditioned immunodeficient mice giving rise to primary tumors and widespread metastasis.     

P-selectin glycoprotein ligand-1 (PSGL-1) is a ligand for L-selectin in neutrophil aggregation

In inflammation, activated neutrophils adhere to endothelial cells and aggregate with one another. While beta 2-integrin and L-selectin are essential for aggregation, their ligands remain to be identified. We have previously shown that L-selectin mediates a carbohydrate-dependent interaction in aggregation (Simon et al: J Immunol 149:2765, 1992; Rochon et al: J Immunol 152:1385, 1994). We have suggested that the L- selectin counter-structure is a mucinlike protein and proposed that aggregation occurs through a two-step process involving L-selectin, beta 2-integrin, and their distinct counter-structures (Bennett et al: J Leuk Biol 58:510, 1995). A candidate ligand for L-selectin is P- selectin glycoprotein ligand-1 (PSGL-1), a mucinlike protein on neutrophils that binds P-and E-selectin. Using flow cytometry we show that the number and size of neutrophil aggregates is reduced with Fab fragments of PL1, an anti-PSGL-1 monoclonal antibody that blocks the interaction between P-selectin and PSGL-1 (Moore et al: J Cell Biol 128:661, 1995). In addition, monoclonal antibodies to L-selectin and PSGL-1 were used simultaneously to modulate the availability of these adhesion molecules on individual cell populations. The inhibition of aggregation by these antibodies is consistent with L-selectin and PSGL- 1 being counter-structures. We suggest that L-selectin and PSGL-1 support a collisional cell-cell interaction that represents the first step in neutrophil aggregation.     

Immunochemical characterization of fibrinogen, fibrin I, and fibrin II in human thrombi and atherosclerotic lesions

Arterial thrombi and atherosclerotic lesions were analyzed immunochemically and examined histologically. The extent of in vivo proteolytic cleavage of the amino-terminal end of fibrinogen by thrombin and plasmin was determined and quantitated by specific radioimmunoassays. The samples were treated with cyanogen bromide (CNBr), and the total amount of fibrinogen and fibrin-derived protein was determined as NDSK, the NH2-terminal disulfide knot of fibrinogen. Thrombin-releasable fibrinopeptides A and B were used to quantitate fibrinogen and fibrin I. Previous plasmin cleavage of the B beta chain was inferred from the amount of B beta 1-42 and B beta 15-42 in undigested NDSK. The results obtained in both acute and organized thrombi indicate that approximately 60% of the total protein (as determined by amino acid analysis) was fibrinogen-derived and that 70% to 80% of the fibrinogen-derived material was fibrin II. These findings support the hypothesis that fibrin II as distinct from fibrin I is the predominant component in a thrombus. In samples from normal and atherosclerotic aortas, fibrinogen-derived protein comprised less than 10% of the total protein. Samples from grossly normal aortas contained only fibrinogen and fibrin I. Fibrinogen concentration decreased and fibrin II concentration increased with increasing severity of the lesions, suggesting that increased fibrin II formation is associated with progression of atheromas.     

p53 negatively regulates the osteogenic differentiation of vascular smooth muscle cells in mice with chronic kidney disease

Aim

To investigate the osteogenic differentiation of vascular smooth muscle cells (VSMCs) in mice with chronic kidney disease (CKD) and to evaluate the effects of p53 on the osteogenic differentiation of the VSMCs.

Methods

Experimental models of CKD-associated vascular calcification generated by five-sixth (5/6) nephrectomy (Nx) and a high-phosphate (HP) diet were used in p53+/+ and p53–/– mice. Following 5/6 Nx, aortic calcification, markers of osteogenic differentiation, VSMCs and p53 protein in aortic tissues were studied.

Results

Aortic calcification was observed after eight weeks following 5/6 Nx in mice of both genotypes, and expression of the markers of osteogenic differentiation in the VSMCs was increased. These changes were continuously observed up to 12 weeks after 5/6 Nx, and particularly after 5/6 Nx + HP. Compared with p53+/+ mice, aortic calcification in p53–/– mice was more severe (p < 0.001). Expression of the markers of osteogenic differentiation was noticeably increased (p < 0.001), while expression of the marker of VSMCs had decreased (p < 0.001). Statistical analysis demonstrated that the markers of osteogenic differentiation were negatively correlated with p53, and the marker of VSMCs was positively correlated with p53 (p < 0.001).

Conclusion

p53 has the potential to negatively regulate the osteogenic differentiation of VSMCs in CKD mice.