Nihon Ketsueki Gakkai zasshi : journal of Japan Haematological Society最新文献

We analyzed the rearrangement of TcR delta chain gene in 179 cases of hematological malignancies. In 17 T-cell lines, RPMI 8402, DND41, Peer, and Molt 13 had delta rearranged band (s). Except for RPMI 8402, these cell lines expressed functional delta gene. All of those gamma delta-T-cell lines had short message (1 kb) of TcR beta gene. These findings suggest differences between alpha beta-T-cells and gamma delta-T-cells. All 9 cases of T-ALL/LBL, of which 4 had neither gamma nor beta gene rearrangement, had a new rearranged band of TcR delta locus. This rearrangement was observed in 63% of B-lineage ALL/LBL. In the other T-lymphoproliferative disorders, only 2 cases of AILD and 1 of T-cell lymphoma had the rearranged band (s), showing derived T-cell neoplasm from gamma delta-T-cell as minority. In B-leukemia/lymphoma and myelocytic leukemia, 15% of the cases had the delta rearrangement. Heterogenous findings of TcR delta locus analysis were observed in ATLL without proviral HTLV-I DNA, T-cell lymphoma, AILD and HD. The J delta 1 region was frequently used and the J delta 2 region was rearranged in one AILD. It is suspected that J delta 3 was used in one T-ALL/LBL. There was no correlation between the phenotypic pattern of CD3, CD4, and CD8 in T cell disorders and the rearrangement of the TcR delta gene. These findings suggest that the newly identified TcR delta chain gene rearranges at a very early stage of T cell ontogeny; prior to the other TcR genes and perhaps at almost the same differentiation level as that of CD7 expression. The TcR delta gene is useful in evaluating clonality for the most immature T cell neoplasms not showing rearrangement of the other TcR genes. This gene is not lineage specific, however, when used in conjunction with IgHC gene, it may be a useful tool for the study of ALL/LBL.

The interaction of plasma clotting factors with vascular endothelial cells was investigated. Human umbilical cord vein endothelial cells generated tissue factor activity after treatment with various stimulators including IL-1. Cycloheximide inhibited production of the tissue factor in the cells, but did not affect the expression of the tissue factor activity on the surface of endothelial cells. Endotoxin-treated vascular endothelial cells activated Factor X in the presence of Factor VII and calcium ion. Activation of Factor X by endothelial cells with Factor VII was enhanced by the presence of both Factors VIII and IX. Binding study revealed that endotoxin-treated endothelial cells bound Factor IX. These data suggest that perturbed vascular endothelium expresses tissue factor activity on the cell surface, binds factor IX and in the presence of Factor VII, activates not only factor X but also Factor IX.

Since myeloperoxidase (MPO) is considered to be a critical marker of differentiating acute myelogenous leukemia (AML) from acute lymphocytic leukemia (ALL), the analysis of MPO gene expression may provide further insight into the leukemia classification and the lineage fidelity of leukemia cells. By Northern blot hybridization using full-length MPO cDNA as a probe, approximately 66% of AML cells (3/4 M1 cases, 2/4 M2 cases, 15/15 M3 cases, 11/15 M4 cases, and 2/12 M5 cases) were found to express MPO mRNAs, whereas none of 18 ALL cases did. MPO mRNA was detectable when AML cells contained at least 10% peroxidase-positive cells. APL (M3) cells expressed high levels of mRNA in accordance with heavy staining for peroxidase.

Cells from 203 children with leukemia/lymphoma were analyzed by the FAB (French-American-British) system using a broad panel of markers such as immunological marker studies, Southern blot and Northern blot analyses to establish a lineage specific classification of childhood leukemia. Phenotypically, they were divided into B-lineage (62.6%), T-lineage (9.8%), non-lymphoid (14.3%) and uncertain lineage (13.3%). Two B-lineage ALL cells and two T-lineage ALL cells studied did not show immunoglobulin (Ig) or T-cell receptor (TCR) gene rearrangements, respectively. Therefore, those four cases were excluded from the final classification. The uncertain lineage leukemia, which includes undifferentiated leukemia and mixed lineage leukemia, were further subclassified at the DNA and RNA levels. The definitions of B-lineage and T-lineage cells, incidence of dual genotypes or spillover, heterogeneity of undifferentiated leukemia, and a new classification for mixed lineage leukemia were discussed.

A substantial number of leukemic blast colonies were formed when conditioned medium of human bladder carcinoma cell line 5637 was added as a stimulator. Recombinant colony-stimulating factor (CSF) also stimulated leukemic blast cell proliferation, leading to colony formation. Furthermore, serum CSF levels in some patients with acute myelogenous leukemia (AML), as detected by sensitive enzyme-linked immunosorbent assay (ELISA), were high. These observations prompted us to study further the expressions of hematopoietic growth factor genes. Granulocyte-macrophage CSF (GM-CSF) mRNA was detected in the leukemic blast cells from about 30% of patients with AML by Northern blot analysis using strict hybridization conditions with or without in vitro blast cell enrichment. These findings suggest that the expression of cytokine genes including the GM-CSF gene reflects in vivo phenomena, although no clear relationship between expression of the genes and serum CSF level has been established. Gene encoding tumor necrosis factor alpha (TNF-alpha), lymphotoxin (LT) and transforming growth factor beta (TGF-beta) were sometimes expressed in some malignant hematological cell lines and also some fresh leukemic cells.

Anticoagulantly active heparin-like glycosaminoglycans are apparently present on the vascular surface. We have tried to modulate heparin-like substances on endothelial cells using an experimental cell culture system. Perturbation of the endothelial proteoglycan metabolism by beta-D-xyloside resulted in a reduced biosynthesis of cell surface heparan sulfate, and impaired antithrombin III binding to endothelial cells in parallel with an inhibition of endothelial cell heparin-like activity. In a separate series of experiments, treatments of endothelial cells with interleukin 1 beta and tumor necrosis factor alpha, physiological mediators of immunologic and inflammatory responses, were shown to cause an inhibition of the synthesis of endothelial cell surface heparan sulfate. The endothelial heparin-like activity was partially diminished by these cytokines, suggesting that cytokine-mediated suppression of heparin-like substance on endothelial cells is another cytokine-inducible endothelial effect affecting coagulation. The modulation of endothelial heparin-like activity by these pharmacological and physiological agents may have pathophysiological implications in thrombosis.

Polymerase chain reaction (PCR) was applied to detect the structural change accompanying the activation of oncogenes in hematological malignancies and preleukemic states. Point mutation of N-ras oncogene was examined by oligonucleotide differential hybridization coupled with PCR. Five out of 17 AML patients were shown to have mutated N-ras gene. These mutations could be used as a genetic marker to diagnose the residual malignant cells. Philadelphia chromosome in CML was examined by cDNA synthesis and PCR with successful results. PCR was shown to be a highly versatile and sensitive method which would be invaluable in clinical diagnosis.