Teruhiko Tsubota

Human B cell, T cell and null cell leukaemic cell lines derived from acute lymphoblastic leukaemias

CONTINUOUS culture of human leukaemic cells is still a difficult task, although several leukaemic cell lines have been derived from patients with acute and chronic myelogenous leukaemia1,2 and acute lymphoblastic leukaemia (ALL) (refs 3–6). We describe here B-cell, T-cell and null-cell leukaemic cell lines newly established from three patients with ALL.

Production of a monoclonal antibody-bleomycin conjugate utilizing dextran T-40 and the antigen-targeting cytotoxicity of the conjugate

Bleomycin (BLM), a potent anticancer glycopeptide antibiotic, was linked covalently to murine monoclonal anti-HLA IgG1 antibody (H-1) with the use of dextran T-40. As determined spectrophotometrically, the conjugate was composed of 57.5 moles BLM per mole antibody. Of the substituted BLM, 18.4% (10.6 moles BLM per mole antibody) exhibited antimicrobial activity. The BLM-(H-1) conjugate showed stronger cytotoxicity than BLM alone against HLA-bearing cells in cultivation after a 30-min exposure to the drugs. In the same experiment, the conjugate was less toxic than BLM against cells lacking HLA.

Production of tumor antibody-neocarzinostatin(NCS) conjugate and its biological activities

SummaryRabbit xenoantiserum was produced against a human leukemia cell line (NALL-1) derived from a patient with acute lymphoblastic leukemia, and IgG was purified. Anti-NALL-1 rabbit IgG was reacted with NCS, an unique membrane-reactive anticancer antibiotic, in the presence of water-soluble carbodiimide. The resulting mixture was concentrated and chromatographed on a Sephadex G-200 column. The first and second fractions were shown by immunoelectrophoresis and the Ouchterlony double-diffusion method to contain NCS-IgG but not free NCS. The conjugates inhibited the growth of Sarcina lutea, and the growth and 3H-TdR incorporation of NALL-1 cells. A membrane immunofluorescent test with FITC-labeled rabbit anti-NCS and goat anti-rabbit IgG antibodies demonstrated specific localization of NCS-IgG on NALL-1 cell surfaces. These results indicate that IgG-bound NCS retained both NCS and antibody activities, and thus should be useful for cancer therapy.

Hairy cell leukemia: establishment of a cell line and its characteristics.

A hairy cell leukemia (HCL) line, ZK‐H, was established from peripheral blood of a 69‐year‐old male patient. The ZK‐H cells and the patients original hairy cells shared the same surface properties; both possessed membrane‐bound IgG with kappa light chains and villous surface structures. The ZK‐H line carried Epstein‐Barr virus (EBV)‐determined nuclear antigen, but the patients fresh leukemic cells lacked this antigen. Morphologically, the ZK‐H cells appeared lymphoblastoid and more primitive than the preculture cells. The ZK‐H line had a hyperdiploid chromosome constitution of 47 and trisomy no. 2. The presence of membrane‐bound immunoglobulin and of B‐cell tropic EBV in this cell line provides further evidence for the B‐cell nature of HCL in this patient.

Determination of aspoxicillin in broncho-alveolar lavage fluid by high-performance liquid chromatography with photolysis and electrochemical detection

A determination method for the penicillin antibiotic aspoxicillin in broncho-alveolar lavage fluid has been developed, involving high-performance liquid chromatography and post-column photolysis. The method enabled the determination of aspoxicillin at concentrations below 100 ng/ml, with a linear calibration curve at concentrations of 1-1000 ng/ml for 100 microliters of lavage fluid. The detection limit was 1 ng/ml at a signal-to-noise ratio of 3:1.

Production of a macromomycin (MCR)-monoclonal antibody conjugate and its biological activity

Macromomycin (MCR), an unique membrane-reactive anticancer antibiotic, was incubated with murine monoclonal anti-HLA IgG1 antibody (H-1) in the presence of carbodiimide. The resulting mixture was fractionated with a Sephadex G-200 column. The first and second fractions were shown to contain MCR-(H-1) conjugate by the elution profile, as well as by the Sarcina lutea growth inhibition assay and Ouchterlony double-diffusion method. A membrane immunofluorescence test with anti-MCR and anti-mouse IgG antibodies demonstrated specific localization of MCR-(H-1) on the surface of HLA-bearing NALL -1 cells. MCR-(H-1) inhibited the growth of HLA-lacking NS-1 cells statistically less effectively than MCR alone (p less than 0.01). On the other hand, the conjugate and free MCR equally inhibited the growth and 3H-TdR incorporation of HLA-bearing NALL -1 cells. These results indicate that the antibody-bound MCR retained both MCR and antibody activities, and thus exerted antibody-targeting MCR cytotoxicity in vitro.

Simultaneous determination of antibody to Epstein-Barr virus in prenatal mothers and new-born infants.

Antikörpertiter gegen EBV wurden in 40 gepaarten Seren von pränatalen Müttern und Neugeborenen gleichzeitig mit indirekter Immunofluoreszenz bestimmt. In 95% hatten sowohl Mütter als auch Neugeborene den Antikörper. Die Titer der Mütter waren mehrheitlich gleich oder höher jenen der Neugeborenen, woraus geschlossen wird, dass der Antikörper gegen EBV von der Mutter diaplacentar auf den Fetus übertragen werden kann.

Propagation of Rauscher leukemia virus to human lymphoblastoid cells by in vivo diffusion chamber cultivation.

Summary Diffusion chambers containing EBV-bearing human lymphoblastoid cells were placed temporarily within the peritoneal cavity of BALB/c mice with Rauscher leukemia. Reestablished human leukocyte cultures became dually infected with C type and EBV particles and have continuously produced abundant C type particles. These virus particles were nonleukemogenic when inoculated into BALB/c mice but afforded some protection against challenge infection with spleen-derived leukemogenic RLV. The authors are indebted to Drs. F. J. Rauscher and R. J. Huebner of the National Cancer Institute for the generous supply of antisera used for immunofluorescence.