Filgrastim (rHuG-CSF)-mobilized peripheral blood progenitor cells (PBPC) in healthy Japanese volunteers were characterized in detail using two clonal cell culture systems and double-colour flow cytometry to detect multilineage colony-forming cells and subsets of CD34+ cells. The kinetics of PBPC during the administration of filgrastim was studied, and possible differences in the character of progenitor cells relative to given doses of filgrastim were investigated. Filgrastim was administered subcutaneously to normal volunteers for 7 d at doses of 100, 200 or 400 microgram/m2 (10 per cohort). Treatment with 100 or 200 microgram/m2 filgrastim was well tolerated; however, the 400 microgram/m2 dose level was not completed because of bone pain and myalgia. The treatment strikingly mobilized various types of progenitor cells, including highly proliferative megakaryocytic colony-forming cells. The number of progenitor cells peaked on days 5 and 6. The fold increase of circulating progenitor cells from the baseline value in the volunteers treated with 200 microgram/m2 filgrastim was more pronounced than in those treated with 100 microgram/m2. Treatment with 200 microgram/m2 also released the less mature progenitor cells (i.e. mixed colony-forming cells CD34+/33- cells, and CD34+/HLA-DR-cells) into circulation better than the 100 microgram/m2 dose. These results suggest that daily subcutaneous injection with 200 microgram/m/2 filgrastim for 5 d will effectively mobilize, both qualitatively and quantitatively, PBPC in healthy donors.

We report a case of a 32-year-old male, an asymptomatic carrier of human T-cell leukemia virus type 1 (HTLV-1), who underwent a renal transplantation and developed adult T-cell leukemia (ATL) during the course of posttransplant immunosuppressive treatment. He was treated with combination chemotherapies consisting of cyclophosphamide, vincristine, doxorubicin, prednisolone, cisplatin, cytosine arabinoside, etoposide, and methyl-prednisolone, without any improvement. Bestrabucil (KM2210), a conjugate of chlorambucil and estradiol, was administered as an alternative therapy; this therapy successfully suppressed his leukemic cell growth, and partial remission was achieved. Posttransplant immunosuppressive therapy with prednisolone, mizoribine, and cyclosporin A might have been the predominant cause of the transition from an asymptomatic HTLV-1 infection to overt ATL. A careful approach is required with HTLV-1 asymptomatic carriers who need organ transplantation followed by immunosuppressive treatment.

Immunosuppressants cyclosporin A (CsA), FK506, and KM2210 modulated colony formations of murine hematopoietic progenitor cells. In a 4-h treatment with CsA, 10 micrograms/ml increased the formation of colony-forming units of mixed lineages (CFU-Mix) but decreased the formation of highly proliferative potential colony-forming units (CFU-HPP); 1 microgram/ml of CsA increased the formations of CFU-HPP, CFU-Mix, and colony-forming units of granulocytes/macrophages (CFU-GM); 0.1 microgram of CsA increased the formation of CFU-Mix and burst-forming units of erythroid lineage (BFU-E). Lower doses of CsA appeared to induce an increase in various colony formations. FK506 increased CFU-HPP and CFU-Mix formations at lower doses. Another immunosuppressant, KM2210, increased CFU-HPP and CFU-GM formations but decreased CFU-Mix and BFU-E formations. In a 24-h treatment, 10 micrograms/ml and 1 microgram/ml of CsA inhibited all the colony formations, but 0.1 microgram/ml of CsA increased CFU-Mix, CFU-GM, and BFU-E formations. Similarly, 100 ng/ml and 10 ng/ml of FK506 decreased all the colony formations but 1 ng/ml of FK506 increased CFU-HPP and CFU-GM formations. KM2210 inhibited all the colony formations. These findings showed that lower doses of CsA and FK506 appeared to increase the colony formations, although higher doses of these drugs decreased the colony formations, similar to the findings in a 4-h treatment. On the other hand, KM2210 showed opposing effects on colony formation with 4-h and 24-h treatments.

A fibroblast-mediated gene delivery method was used for the endogenous expression of human granulocyte colony-stimulating factor (G-CSF) as a model for cytokine supplement therapy. Human G-CSF cDNA was inserted into the plasmid expression vector BMGNeo, which contains a partial sequence of bovine papilloma virus and a selectable marker gene. The recombinant plasmid (BMGNeo-GCSF) was transfected into NIH/3T3 fibroblasts by the calcium phosphate coprecipitation method, and the stably transformed cells were isolated by G418 selection. An appropriate clone producing a large amount of G-CSF was selected by enzyme immunoassay of the culture supernatants. Southern blot analysis suggested that the BMGNeo-GCSF plasmid replicated mainly as an episome, and Northern blot analysis demonstrated the high expression of human G- CSF mRNA in the cells. After the implantation of the G-CSF-producing fibroblasts into nude mice, prominent neutrophilia, about 30-fold the level of normal control, was observed within seven days. Moreover, the number of hematopoietic progenitor cells in spleen remarkably increased for all cell lineages in these mice. To regulate the in vivo expression of G-CSF, we designed a subcutaneous diffusion chamber apparatus that contains the G-CSF-producing fibroblasts. The leukocytosis (neutrophilia) induced in C3H mice after embedding the device quickly disappeared after ethanol treatment of the chamber. Furthermore, reinjection of the G-CSF-producing fibroblasts into the chamber caused a second neutrophilia.

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