Mice were infected intravenously with M. intracellulare (5.2 x 10(6) CFU/mouse) and then were given 0.4 mg of KRM-1648 emulsified in 2.5% gum arabic-0.2% Tween 80 by gavage, once daily 1, 3 or 6 times per week, from 24h after infection to the end of experiment (week 8). Evaluation of the therapeutic efficacy of the drug against the infection was done on the basis of incidence and degree of gross lung lesions, organ weight (square root of organ (mg)/body (g) x 10), and bacterial loads in the lungs and spleen. The lung lesions were not observed in all experimental groups at 4 weeks after infection. At 8 weeks after infection, the lung lesions observed in all control and solute (2.5% gum arabic-0.2% Tween 80) control mice, whereas 3 of the 5 mice given KRM-1648, 3 times per week and all of 5 mice given KRM-1648, 6 times per week showed no lung lesions. Although lung lesions were observed in all mice given KRM-1648 only once per week, the degree of the lesions was much more milder in KRM-1648-treated mice than in solute control mice. The spleen weight of solute control mice and KRM-treated mice differed from each other 4 and 8 weeks after infection, especially in mice administered 6 times per week. The CFUs of organisms in the lungs and spleen were lower in mice treated with the agent than in mice given solute at 4 and 8 weeks after infection, in orders of administration of 6, 3 and 1 times per week.

To distinguish hypoplastic myelodysplastic syndrome (MDS) from aplastic anemia (AA), morphological abnormalities of bone marrow hematopoietic cells in 8 patients with MDS and 39 patients with AA were studied. All the patients with MDS and AA showed prolonged plasma iron disappearance time, (PIDT1/2) > 120 min. Five hundred erythroid and myeloid cells, as well as 20 megakaryocytes were counted. Dysplastic changes were defined if morphological changes were present in more than 1.0% cells with only one lineage, or in more than 0.6% cells with more than two lineages. Twenty six of 39 patients with AA showed morphological abnormalities. In MDS cases, morphological abnormalities were prominent in trilineage cells in some cases, in bilineage (erythroid and megakaryocytic or myeloid and megakaryocytic cells), in others, or solely in myeloid cells or in megakaryocytic cells in other cases. Morphological abnormalities seen solely in erythroid cells, especially those with segmented nuclei were considered to be less significant for the diagnosis of MDS. The findings were considered to be useful to distinguish hypoplastic MDS from AA.

Autologous blood stem cell transplantation (ABSCT) has been increasingly used, as the third generation of hemopoietic stem cell transplantation next to allogeneic and autologous bone marrow transplantation (allo-BMT and ABMT), in the treatment of malignant diseases. As to the principle of treatment, ABSCT is identical to ABSCT. However, ABSCT has some advantages over ABMT as follows: 1) a rapid engraftment, 2) no requirement of general anesthesia for harvesting hemopoietic stem cells, and 3) possibility of less contamination of tumor cells. Accordingly, ABSCT will take place of ABMT in the strategy of cancer treatment and improved results will be expected.

Antitumor activity of cisplatin, carboplatin, etoposide, or human hepatocyte growth factor (hHGF), was compared by examining the colony formation ability of four liver cancer cell lines (PLC/PRF/5 and HuH-7, hepatocellular carcinoma; HuH-6 and HepG2, hepatoblastoma). Antitumor activity was evaluated from the drug concentration causing 50% cell death by a colony assay. PLC/PRF/5 cells were most effectively killed by cisplatin and etoposide, HuH-7 cells by cisplatin and carboplatin, HuH-6 cells by etoposide, and Hep G2 cells by cisplatin. These results indicate that among four liver cancer cell lines, the three were the most sensitive to cisplatin, the two were to etoposide and the only one cell line was to carboplatin. There was no significant relationship between each drug and types of liver cancer. Combined treatment with cisplatin (0.01-1.0 microgram/ml) and etoposide (0.1 microgram/ml) showed synergistic cytotoxic effects on the colony formation of PLC/PRF/5 cells, while combination of carboplatin (0.01-0.1 microgram/ml) and etoposide (0.1 microgram/ml) caused subadditive cytotoxic effects. hHGF stimulated the colony formation of HuH-6 cells, while it inhibited that of Hep G2 cells. The treatment of HuH-6 with cisplatin and hHGF showed a higher cell survival percentage compared with the treatment with cisplatin alone. On the other hand, cell survival of Hep G2 cells was remarkably decreased by the combined treatment with cisplatin and hHGF.

Gaucher disease is the most prevalent lysosomal storage disease. It is caused by deficient activity of a lysosomal enzyme known as glucocerebrosidase, also called glucosylceramidase, resulting from mutations in the gene encoding the enzyme. The numerous mutations in glucocerebrosidase gene from patients were reported and the correlation of phenotype and genotype were studied. However, a given genotype cannot be uniquely correlated with a specific phenotype. In the therapeutic point of view, two successful treatment were developed based on the correction of the enzyme deficiency in macrophages. These are bone marrow transplantation and enzyme infusion therapy. Although patients have responded to these two therapies, inherent problems limit their application. Thus, these problems makes Gaucher disease an excellent candidate for therapy based on gene transfer to hematopoietic cells. We succeeded in efficient transduction and sustained high expression of glucocerebrosidase gene in mouse hematopoietic stem cells and macrophages from long term reconstituted mice. The results of our study strengthen the rationale for gene therapy as a treatment for Gaucher disease.

We report two cases of AIDS whom we have recently experienced. One patient was a 54-year-old man who admitted our hospital due to third degree burn. In this case, we did not know whether or not he was suffered from AIDS, when he was delivered by the ambulance. In autopsy, pneumocystis carinii pneumonia and renal tuberculosis were found in addition to marked decrease of T cells in lymph nodes. The other patient was a 40-year-old man with remarkable symptoms of central nervous system. Route of infection of HIV is unknown. He had dementia, left hemiplegia, bulbar palsy, progressed to rigid decorticate posture and died of respiratory arrest due to involvement of the brain stem, despite of treatment including use of 3'-azido-2',3'-dideoxythymidine (AZT). Magnetic resonance (MR) images showed progressive cerebral atrophy and a diffuse high signal intensity area of cerebral white matter on T2-weighted MR images, suggesting the diagnosis of HIV-induced encephalopathy.

We report a 47-year-old woman with SLE, who developed meningeal signs and consciousness disturbance. She noted an onset of fever, and swelling and pain in her face, hands and feet in 1990. She was seen in another hospital and the diagnosis of SLE was made. She was treated with prednisolone with marked improvement in her symptoms. She was well with 5 mg of oral prednisolone daily until January of 1991, when she developed fever, myalgia and weakness in her legs. She was admitted to the medical service of our hospital on August 5. She was receiving 15 mg of prednisolone daily. Gram positive rods were cultured from her blood on August 5. She became incoherent 2 days later, and had a convulsive episode on August 8. After the convulsion, she lost consciousness from which she did not recover. Her CSF contained 304/3 microliters cells, 29 of which were neutrophils, 6 lymphocytes, 90 others, and 179 destructed cells. The CSF protein content was 345 mg/dl, and glucose 23 mg/dl. A neurological consultation was asked on August 9. Physical examination at that time revealed a semicomatous woman. Respiration was 30/min and regular. BP 132/82 mmHg, heart rate 122/min and regular, and BT 39.6 degrees C. General physical examination was unremarkable. Pertinent neurologic findings were positive Kernig sign and spasticity in all four limbs. Brain stem reflexes were retained. Upon painful stimulation, withdrawal response was elicited both lower extremities. She was treated with pipiracillin, latamoxef and phenobarbital, however, she had frequent seizures. She was deeply comatose on December 10. She became flaccid and no more meningeal signs were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) stimulates the growth of myeloid leukemic cells and increases their susceptibility to cell-cycle specific agents. We treated a patient with acute myelogenous leukemia (AML) in a state of second resistant relapse, with high-dose chemoradiotherapy combined with rhGM-CSF (total body irradiation: TBI 3Gy x 4, on days -8 & -7; cytosine arabinoside: Ara-C 3g/m2, iv, q12h, on days -5-2; rhGM-CSF 250 micrograms/m2/day, cont.iv, on days -5-2) followed by autologous peripheral blood stem cell transplantation (PBSCT). In this case, rhGM-CSF enhanced the proliferation of leukemic cells in vitro. The test dose of rhGM-CSF (84 micrograms/m2 over 8 hours) also promoted leukemic cell proliferation in vivo, resulting in an increase in the percentage of leukemic cells in the peripheral blood and reappearance of chromosomal aberrations in the bone marrow. The toxicity of rhGM-CSF-combined conditioning regimen included fever and mild liver damage. The patient achieved a complete remission lasting for 2 months, then relapsed. The rhGM-CSF-combined conditioning regimen was tolerated by this patient, but further studies will be required to confirm not only its safety but also its effectiveness in the treatment of refractory AML.

The hematological recovery of 11 patients who received autologous peripheral blood stem cell transplantation (PBSCT) followed by recombinant granulocyte colony-stimulating factor (rG-CSF) was studied. After high-dose cytarabine therapy or high-dose etoposide therapy followed by rG-CSF, the peripheral blood stem cells were collected using CS-3000 during bone marrow recovery and subsequently cryopreserved in 8 patients with malignant lymphoma, 2 with multiple myeloma and 1 with ALL. The infused dose of CFU-GM following myeloablative chemotherapy ranged from 1.9 to 18.1 x 10(5)/kg BW. Nine patients received more than 5 x 10(5) CFU-GM/kg BW. The median time to reach 1,000 WBC/microliter, 500 neutrophils/microliter, 5 x 10(4) platelets/microliter and 1% reticulocytes was 9 (range; 8-12), 9 (range; 8-12), 16 (range; 8-27) and 15 (range; 9-38) days, respectively. A negative correlation was found between the logarithm of CFU-GM/kg BW numbers infused and the time post-transplant to reach 500 neutrophils/microliter (r = -0.82, P < 0.005), 5 x 10(4) platelets/microliter (r = -0.48) and 1% reticulocytes (r = -0.57, p < 0.05). Transient decrease in the neutrophil (n = 3) or platelet counts (n = 9) was observed 4 to 8 weeks after transplantation, but the recovery of hematopoiesis became stable thereafter. We concluded that with combined use of rG-CSF, the minimal CFU-GM dose required for safe PBSCT is 2 x 10(5)/kg BW.

In normal hematopoiesis, granulocyte macrophage-colony stimulating factor (GM-CSF) is known to stimulate the proliferation and differentiation of myelo-monocytoid lineage, while Steel factor (SLF) has been supposed to support the proliferation of more primitive hematopoietic progenitors including stem cells. We investigated the stimulatory effect of SLF and GM-CSF in twelve cases of acute myeloid leukemia (AML) cells in vitro by MTT assay. In 10 out of 12 cases, SLF or GM-CSF stimulated the proliferation of leukemic cells in short term liquid culture. In 7 out of these 10 cases, the leukemic cells responded to both cytokines. To investigate the possibility of the clinical application of these cytokines by combination with cell cycle-specific anti-tumor reagents, we assessed the effects of SLF and GM-CSF on the modulation of the cell cycle and sensitivity against cytosine arabinoside (ara-C) in a human factor-dependent leukemic cell line, MO7e. Flow cytometric analysis revealed the effect of cell recruitment into the cell cycle in the quiescent MO7e cells by exposure to these cytokines. The drug-sensitivity test using the MTT assay system demonstrated that pre-treatment with each cytokine enhanced sensitivity of ara-C. Furthermore, by combination of SLF and GM-CSF, the ara-C sensitivity was significantly wore graty enhanced than by each cytokine alone. These data suggest that combined pre-treatment with SLF and GM-CSF may provide new benefits for cure-oriented chemotherapy of AML followed by bone marrow transplantation or peripheral blood stem cell transplantation.

A 36-year-old man was diagnosed as having acute non-lymphocytic leukemia (AML M5b) in 1985 and received allogenic bone marrow transplantation from an ABO-mismatched sibling in January 1987. Recovery of erythropoiesis in this patient was delayed, then the hemoglobin level improved in parallel with disappearance of anti-A antibody in the serum on day 260 post transplantation. However, as anemia occurred again despite no relapse of leukemia on day 350, we tried to determine the presence of erythropoietic inhibitory factor in this patients. Erythroid colony formation was decreased when bone marrow cells were co-cultured with peripheral mononuclear cells from the patient. Further, erythroid colony inhibitory activity was found in conditioned medium of PHA-stimulated T cells from the patient. Sephadex gel fractionation showed that the molecular weight of this inhibitory factor was approximately 11,000 and addition of a high concentration of EPO did not eliminate the inhibitory activity. These findings suggest that the novel inhibitor described in this manuscript, produced by T cells, was differed from previously reported inhibitors such as anti-EPO antibody, spermine and uremic toxins.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia caused by complement-mediated hemolysis. Blood cells from patients with PNH contain abnormal cells that lack complement regulatory proteins, DAF and CD59, both of which protect host cells from action of complement. DAF and CD59 are GPI-anchored and on the abnormal blood cells other GPI-anchored proteins are also deficient. A fundamental abnormality of PNH appeared to be deficient biosynthesis of the GPI-anchor at an early step. We cloned a cDNA of a gene termed PIG-A (for Phosphatidyl Inositol Glycan-class A) that encodes a 484 amino acid putative ER membrane protein which functions at that step and hence a responsible gene for PNH. Analysis of PIG-A transcripts in the abnormal cells from patients with PNH demonstrated various types of abnormalities such as decreased level of the transcript, splicing abnormality and mutations in the coding region. Thus, PNH is caused by a clonal expansion of abnormal blood cells derived from a hematopoietic stem cell bearing a somatic mutation occurred in PIG-A gene.

We studied the effects of several cytokines on the development of granulocyte-macrophage (GM) progenitors using the serum-deprived culture. SCF plays an important role in the GM-CSF- or IL-3-dependent production of neutrophils and macrophages. In vitro colony assay also suggests an increase in sensitivity of GM progenitors to cytokines (GM-CSF, IL-3, G-CSF and/or SCF) in a patient with juvenile chronic myelogenous leukemia. A high level of serum IFN-gamma was associated with leukopenia and thrombocytopenia in a patient with hemophagocytic syndrome. Based on the evidence that IFN-gamma significantly inhibited the proliferation of GM progenitors, IFN-gamma-mediated suppression was suggested as one of the mechanisms causing cytopenia. In patients with aplastic anemia and neutropenia, an increase of serum G-CSF levels was observed when neutrophils decreased remarkably in number. However, the serum SCF levels were constant in these patients. A failure of SCF to enhance colony growth in some patients with aplastic anemia implies qualitative abnormalities of hematopoietic progenitors.

A 35-year-old female was referred to our hospital for fever and anemia. Physical examination was unremarkable. Complete blood count revealed microcytic hypochromic anemia and reticulocytopenia. The bone marrow cellularity was normal. Some giant pronormoblasts were seen but other erythroid cells were absent. No stainable iron was seen. Parvovirus B19 (PVB19) DNA was detectable by polymerase chain reaction. Anti-PVB19 IgM-antibody was also positive in the serum on admission. Antibodies against rubella, measles, mumps, EB virus and HBs were negative and HBs antigen was also negative. Thus the diagnosis of iron deficiency anemia complicated with pure red cell aplasia secondary to PVB19 infection was made. The PVB19 DNA was still positive on days 6 and 11, suggesting that PVB19 virus persists as long as 3 weeks after the onset of PVB19 infection. However, the erythroid cells had recovered by day 6 after admission suggesting that the development of IgM antibody successfully protected the erythroid cells from infection by the residual PVB19. Hence, careful observation for PVB19 DNA and the antibody may be necessary if immunodeficient patients developed anemia of unknown etiology.

Recent progress of molecular biology and gene technology has developed a novel approach of clinical treatment. Several recombinant cytokines are already applied to clinical field. In this symposium, I introduced clinical application of some cytokines including GM-CSF, interleukin (IL)-1 and IL-3. The clinical benefits of IL-1 are; 1) IL-1 has an anti-tumor effect especially on cutaneous lymphoma and brain tumors, and 2) IL-1 has a function as hematopoietic growth factor for very immature hematopoietic stem cells. In the clinical Phase I/II study, IL-1 has been shown to have anti-tumor effect on cutaneous T-lymphoma via immune mechanisms. The side effects of IL-1 were variable including fever, fatigue, skin redness and so on, but they were all tolerable. The clinical phase studies of GM-CSF and IL-3 are now on going. The preliminary studies show that GM-CSF has granulo-poietic activity but not thrombo-poietic activity, and that IL-3 has multi-hematopoietic activity. These cytokines may be useful for treatment of disorders of hematopoietic stem cells such as aplastic anemia and myelodysplastic syndrome. The side effects of both cytokines are resemble, but all are tolerable.

To investigate glial carcinogenesis in vitro, fetal rat brain cells were cultured and exposed to ENU (approximately 200 micrograms/ml). The cells were passaged weekly thereafter. Morphological changes were observed under the phase contrast microscope. When mutant colonies where the cells lost contact inhibition and grew in a multilayer fashion appeared, the cells were cloned. To assess the biological characters of cells, expression of GFAP, vimentin, A2B5 and p53 product were determined by immunohistochemistry and flow cytometry. Tumor forming ability of the cells was evaluated by both colony forming efficiency in low serum medium (LSM; 2% FBS, 300 cells/100 mm dish) and transplantability to nude mice. Both primary cultured and ENU-treated cells were positive for GFAP and vimentin, but population of A2B5 positive cells was less than 5%, thus indicating that these cells were astroglial in origin. The mutant colonies appeared 7 weeks after ENU treatment. These cells grew rapidly with cell doubling time ranging between 18 to 26 hours, while non-ENU-treated astroglias had a longer cell doubling time (48 to 55 hours). The cloned mutant glial cell lines formed large colonies in LSM (efficiency 20-40%), but astroglial cells did not. The mutant astroglial cells also developed tumors in nude mice. p53 protein was never detected in normal astroglia, however, some glial cells treated by ENU abruptly became p53 positive after several passages. These p53 positive cells formed stratified colonies thereafter. These results indicate that mutant astroglial cells can be induced by a single dose of ENU in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Peripheral blood stem cells (PBSC) are increased dramatically during a recovery phase after myelosuppressive chemotherapy. PBSC can be used for hematological reconstitution after marrow-ablative therapy. Autologous blood stem cell transplantation (ABSCT) has increasingly been used for the treatment of malignant diseases. Cytokines, especially hematopoietic cytokines are utilized for mobilization of PBSC, enhancement of chemosensitivity of leukemic cells having cytokine receptors and enhancement of hematologic recovery after ABSCT. We studied effects of granulocyte colony-stimulating factor (G-CSF) in these settings. In cytotoxic drug plus G-CSF mobilization of PBSC, granulocyte/macrophage and erythroid progenitor cells were significantly increased in PBSC harvests compared to cytotoxic drug-induced mobilization. When G-CSF was administered prior to the marrow-ablative conditioning before ABSCT, clinical results suggest that chemosensitivity of leukemic cells may be increased. When G-CSF was given after ABSCT, hematologic recovery from marrow aplasia was enhanced substantially. These observations clearly indicate that G-CSF is useful to mobilize PBSC and to facilitate ABSCT.

The effects of granulocyte-colony stimulating factor (G-CSF) on the white blood cell count and the morphology of peripheral white blood cells were studied during the period of leukocytopenia following chemotherapy in patients with non-hematological malignant tumors (solid tumor group) and those with lymphatic hematological diseases (lymphocytic malignancy group). Cell surface markers were also analyzed to evaluate the usefulness of G-CSF in peripheral stem cell transplantation. After G-CSF administration, the white blood cell count showed remarkable changes beyond the physiologic range; the increase and decrease were 5.0 times and 0.3 times previous values, respectively, in the solid tumor group, and 11.0 times and 0.2 times, respectively, in the lymphocytic malignancy group. Concerning the morphology of peripheral white blood cells, immature leukocytes increased markedly to 133.0 times the previous value. The frequency of immature cells with morphological abnormalities was 0.5 percent in the solid tumor group, but 1.6 percent in the lymphocytic malignancy group. These findings suggest that administering G-CSF affects precision control and microscopic findings in routine laboratory blood tests and that information about whether the patient was treated with G-CSF should be obtained from the clinical staff. A cell surface marker, c-kit, was analyzed to clarify whether stem cells useful for peripheral stem cell transplantation appear in peripheral blood after administration of G-CSF. The incidence of c-kit-positive cells was at 1-10% in 3 of 5 patients in the solid tumor group administered G-CSF and in all 6 patients in the lymphocytic malignancy group.(ABSTRACT TRUNCATED AT 250 WORDS)