A 84-year-old man was treated with antibiotics including erythromycin and a diuretic (furosemide) because of acute heart failure and pneumonia. During the treatment, he developed moderate anemia (Hb 8.7g/dl). His anemia improved after the treatment. He again developed marked anemia (Hb 6.3g/dl) during the second treatment with erythromycin and furosemide and received blood transfusions. Bone marrow aspiration study revealed severe erythroid hypoplasia (0.2%). He was referred to our hospital, but he was not treated because his hemoglobin levels and reticulocyte count increased (80%) and his bone marrow showed increased erythroblasts (41.5%). His anemia gradually improved without any treatment. We diagnosed the case as drug-induced pure red cell aplasia (PRCA). We cultured bone marrow cells obtained from the present case and four normal healthy volunteers by a plasma clot method to determine the effects of two drugs on the number of erythroid colony forming unit (CFU-E). Furosemide strongly inhibited the CFU-E colony formation in the patient, but the inhibition effect of erythromycin was moderate. Furthermore, CFU-E was markedly suppressed by a combination of erythromycin and furosemide in both patient and control materials. These results indicate that both furosemide and erythromycin were related to the occurrence of PRCA in this patient.

We reported the treatment outcome of Protocol 8704T, which included repeated L-asparaginase, for childhood T cell malignancies. Fifteen cases of acute lymphoblastic leukemia (T-ALL) and 11 cases of non-Hodgkin's lymphoma (T-NHL), aged 3 to 14 yrs (median 6 yrs), were enrolled. Twelve T-ALL had mediastinal mass. Murphy's stages of T-NHL were 6 with III and 5 with IV. Types of histology consisted of 8 lymphoblastic and 3 large cell. Treatment was performed for 2 years. Observation periods were from 14 months to 78 months (median 42 months). Twenty-three achieved remission and 6 of them were transplanted with bone marrow or peripheral stem cells in the first remission. The protocol was continued in 17 cases. Fourteen of them remain in first remission, but one died of measles and 2 died of relapse. The 5-year event-free survival was 76.1% for ALL and 65.5% for NHL. In terms of histology, it was 87.5% for lymphoblastic NHL and 33.3% for large cell NHL (p = 0.19). In terms of phenotypes in ALL, it was 88.7% for ALL positive to CD2, 5 and 7, while 2 ALL positive to CD7 alone both failed. Therefore, it was shown that this treatment protocol is very effective for T-lymphoblastic leukemia and lymphoma.

A novel human CD7-positive leukemia cell line (HSM911) derived from the peripheral blood of a patient with acute myelogenous leukemia (AML) was studied for its cellular and biological characterization. Proliferation assay using a variety of cytokines demonstrated that the HSM911 cells proliferate in response to recombinant granulocyte-macrophage-colony stimulating factor (rGM-CSF), recombinant Interleukin-3 (rIL-3) and recombinant stem cell factor (rSCF), but do not in response to recombinant granulocyte-colony stimulating factor (rG-CSF), natural macrophage-colony stimulating factor (M-CSF), rIL-1, rIL-2, rIL-4, rIL-5, rIL-6 or recombinant erythropoietin (rEpo). Polyclonal anti-GM-CSF antibody and polyclonal anti-IL-3 antibody blocked the proliferation of HSM911 stimulated with rGM-CSF and rIL-3, respectively. HSM911 maintained in the presence of rGM-CSF expressed the CD7, CD13, CD33, CD34, CD41a, HLA-DR, VLA1-VLA5, CD11a, CD54, CD44 and LAM1. These findings suggest that HSM911 might be of multipotent progenitor cell origin. GM-CSF receptors and rIL-3 receptors expressed on this cell line were simultaneously suppressed by rGM-CSF or rIL-3, whereas only IL-3 receptors were down-modulated by rSCF. Treatment with 12-o-tetradecanoyl-phorbol-13-acetate (TPA) induced the differentiation of HSM911 cells into macrophage-like cells but not erythroblasts, megakaryocytes or lymphocytes. Interferon-gamma and transforming growth factor-beta (TGF-beta) suppressed the proliferation of HSM911 cells in a dose dependent manner. HSM911 was relatively resistant against anti-cancer drugs compared with fresh AML cells and other leukemic cell line. HSM911 is a useful tool for analyzing CD7-positive acute myelogenous leukemia.

The percentage of CD34-positive cells (the CD34-positive rate) in peripheral blood stem cell harvests (PBSCH) was determined using two color flow cytometric methods, i.e., representation in a histogram (the histogram method) and the two-dimensional side scatter-fluorescence representation (the SSC-FL method). For all samples examined, the CD34-positive rate obtained using the histogram method was higher than that obtained using the SSC-FL method. This finding was probably due to the fact that some monocytes non-specifically reacted with anti-CD34 monoclonal antibody, and the histogram method could not distinguish these non-specifically stained cells from CD34-positive precursor cells. On the other hand, the SSC-FL method seemed to yield a more accurate measurement of the percentage of CD34-positive cells in PBSCH samples. Based on this finding, it is recommended that the histogram method, which is currently used at most commercial laboratories, be reviewed in favor of the SSC-FL method when the CD34-positive cell rate of PBSCH is to be determined.

A 51-year-old man had suffered from massive pleural effusion due to invasion of malignant cells. The analysis of bone marrow aspiration showed the proliferation of myeloperoxidase-positive blasts. The surface marker analysis of the blasts revealed the positivities for CD7 and CD19 as well as CD13, CD33 and CD34, while the karyotypes of 20 cells were normal. Therefore, CD7 positive AML was diagnosed. The patient was treated with araC and daunorubicin as a remission induction therapy. Peripheral blood stem cells were harvested by leukapheresis after first and second consolidation therapies. Then, 3 x 10(4) cells/kg of CFU-GM were infused. Complete remission has been maintained for 8 months after autologous blood stem cell transplantation. Pleural involvement as an initial manifestation is rare in AML. Extramedullary growth of AML cells may be related to their immaturity, indicated by the expression of the cell surface antigens.

Interleukin 3 (IL-3) was initially described in the supernates of cultures of viral-infected murine spleen cells, as a cytokine produced by T lymphocytes can promote differentiation of immature T lymphocytes. Later, it was found that IL-3 exhibited a striking effect on hematopoiesis. The recombinant molecule of murine and human IL-3 can promote the sustained proliferation of clones of mast cells and basophils. It also acts as a colony stimulating factor (CSF) for bone marrow cells. Although other CSFs generally stimulate specific lineages of myeloid or erythroid cells, IL-3 stimulates bone marrow to induce proliferation of a variety of clonal cell populations, including colonies of granulocytes, macrophages, megakaryocytes, eosinophils, basophils, mast cells, normoblasts and erythroblasts. Thus, IL-3 is responsible for promoting proliferation of earlier lineage pluripotent stem cells, of hematopoietic cells and lymphoid cells. Recently, it is also suggested, as to its effects on lymphocytes, that IL-3 may possibly be a factor responsible for T lymphocytes to be differentiating extra-thymically.

We describe here characteristics of animal models for intractable hematologic disorders and discuss the concept of "stem cell disorders." Using these animal models, we have found that bone marrow transplantation can be used to treat both systemic and organ-specific autoimmune diseases, and that transplantation of hematopoietic stem cells from autoimmune-prone mice induces autoimmune diseases in normal mice. Based on these findings, we have proposed that autoimmune diseases are stem cell disorders. It has recently been reported that bone marrow transplantation can be used to treat autoimmune diseases such as rheumatoid arthritis and ulcerative colitis, also in humans.

This is a comprehensive review of autologous peripheral blood stem cell transplantation (PBSCT). Collection of peripheral blood stem cells (PBSC) does not require anesthesia and is less invasive compared to harvesting marrow cells. As the hematopoietic recovery speed after autologous PBSCT is fast, the procedure is associated with less complication compared to marrow transplantation. Thus, high-dose therapy can safely be administered, without the use of aseptic measures, in a larger number of hospitals. Preliminary therapeutic results for the treatment of relapsed childhood acute lymphoblastic leukemia appears to be equivalent to that obtained by application of allogeneic bone marrow transplantation. Alternate use of PBSC includes routine application after consolidation therapy as one of growth factors. Use of PBSC in allogeneic setting has been under intense investigation. Collection, processing and storage of PBSC will shortly become a part of routine procedure in major blood centers and banks.

To study the alteration of nuclear DNA content of cancer cells after peplomycin (PEP) treatment, DNA cytofluorometry was performed in combination with 3H-thymidine (3H-TdR) autoradiography using cultured A431 cells. The cells in the logarithmic growth were treated with PEP (1.25 micrograms/ml) for 24 hr, during the first 4 hr of which they were pulse-labeled with 3H-TdR (2.4 x 10(4) Bq/ml). After washing with PBS, the cells were then cultured without both PEP and 3H-TdR, fixed at different times and stained with propidium iodide (PI) for the auto-stage cytofluorometry, which enabled DNA content analysis for labeled and unlabeled cells by repeated scanning of the same cell population. The nuclear DNA content histograms demonstrated that A431 cells were mostly arrested in G2 phase of 4C stem line by treatment with PEP for 24 hr. This G2 block lasted up to 8 hr after removal of the drug, and thereafter, marked polyploidization associated with DNA synthesis occurred, showing almost no mitotic figures, while only a few cells returned to G1 phase via M phase. During the period of 72-120 hr, however, the fractions of advanced polyploid cells (DNA content > or = 8C) gradually decreased and the DNA content distribution pattern became eventually similar to the original one as seen before PEP treatment. From these results we hypothesized as follows: 1) At S-G2 boundary, there is some control mechanism that checks whether the cells, after S phase, can enter the M phase or not. 2) The cells, which are not permitted to enter mitosis by the control mechanism, show marked polyploidization. 3) Only the cells that enter into mitosis can live and proliferate, though the advanced polyploid cells die shortly. 4) This control mechanism might be related to the precision of DNA repair that is checked at the G2-M checkpoint.

We studied the expression of HRF20, one of the GPI-anchor proteins, on the hematopoietic progenitors and their progeny cells grown in cultures with bone marrow cells from patients with paroxysmal nocturnal hemoglobinuria (PNH). PNH bone marrow cells were sorted by fluorescence-activated cell sorter with anti-HRF20 monoclonal antibody and then cultured in methylcellulose. Every CFU-GM colonies and BFU-E bursts formed from the HRF20-negative fraction were HRF20-negative. On the other hand, there were HRF20-positive colonies and bursts as well as HRF20-negative ones in cultures with bone marrow cells from the HRF20-positive fraction. The results suggested that abnormal events can occur during the several steps of differentiation form PNH hematopoietic stem cells.

A 17-year-old girl was admitted to our hospital because of drowsiness, diplopia and gait difficulty. She had been well until ten days before admission when fever, drowsiness, headache and general fatigue developed. On admission, there were drowsiness, ophthalmoplegia, ataxia and hyporeflexia. CSF cells and anti-CMV antibody titers increased. CMV-DNA was detected in the CSF by the polymerase chain reaction (PCR). Serum anti-GQ1b antibody was positive. During recovery, forced laughing temporarily appeared. The neurological symptoms disappeared completely. CSF anti-CMV antibody titers became normalized and CSF CMV-DNA-PCR became negative. This is the first case report of Bickerstaff's brainstem encephalitis associated with CMV infection.

Clinical effects of peripheral blood stem cell autotransplantation (PBSCT) after ultra high-dose chemotherapy were evaluated in patients with chemotherapy-resistant and/or poor prognostic testicular cancer. Four patients with testicular cancer, who had high-risk malignancy, were treated with high-dose etoposide (500 mg/m2 x 4 days) in order to collect peripheral blood stem cells. After the administration of high-dose etoposide, rG-CSF (250 micrograms/body) was administered from nadir state. Blood mononuclear cells were collected using a Fenwall CS-3000 blood cell separator. Fractions enriched for stem cells were obtained by discontinuous Percoll gradient centrifugation and were stored in liquid nitrogen using patient's sera and DMSO. The mean number of peripheral blood granulocyte-macrophage-colony-forming units (CFU-GM) collected by one apheresis was 22.3 x 10(5)/kg body weight. In addition, CFU-GM more than 2.0 x 10(5)/kg body weight could be collected in each apheresis, which was though to be sufficient dosis to perform PBSCT in safe, based upon our previous studies. All the patients were treated by a combination of cisplatin (20 mg/m2 x 5 days), etoposide (100 mg/m2 x 5 days) and bleomycin (15 mg x 3 days). Three patients responded to BEP therapy and obtained a CR, however, remaining 1 patient failed to achieve CR, who was later treated by ultrahigh-dose chemotherapy including carboplatin (200 mg/m2 x 4 days), etoposide (250 mg/m2 x 4 days) and cyclophosphamide (50 mg/kg x 2 days) followed by PBSCT. He responded to this therapy and obtained a CR for 10 months. The results suggested the method was promising for patients with chemotherapy-resistant and/or poor prognostic testicular cancer.

The myelodysplastic syndromes comprise a heterogeneous group of hematopoietic disorders characterized by peripheral cytopenia, and ineffective and dysplastic hematopoiesis. Patients are predominantly aged, and often develop acute non-lymphocytic leukemia. Bone marrow failure, notably infection and bleeding, is the most common cause of death. Our understanding of the biology of the myelodysplastic syndromes is limited and thus reduces the efficacy and specificity of therapeutic intervention. Available evidence, however, suggests that the myelodysplastic syndromes arise from hematopoietic stem cells. The emergence and progression of the myelodysplastic clone have been reviewed in the light of the multistep theory of human carcinogenesis.

We presented three cases of collagen diseases complicated with myelodysplastic syndrome (MDS). To our knowledge, MDS following collagen disease is very rare, and only seven cases have been reported previously. First case (a 40-years old woman) had suffered from rheumatoid arthritis, systemic lupus erythematosus and Sjögren's syndrome for 16 years. Second case (a 65-years old woman) had suffered from rheumatoid arthritis for 15-years. Third case (a 63-years old woman) had suffered from progressive systemic sclerosis for 16 years. These patients developed MDS. After onset of MDS, cytopenia progressed rapidly within half a year. In the types of MDS, case 1 and case 2 had refractory anemia and case 3 had refractory anemia with excess of blasts and a preleukemic state. Case 2 had a clonal abnormality of haemopoietic stem cells. It is unlikely that MDS in our cases are caused by mutant agents or irradiation. These results suggest MDS may be contributed to long-term immunodysfunction found in these collagen diseases.

This is a report of 45-year-old man with advanced nonseminomatous germ cell tumor (stage IIIB2: embryonal carcinoma, yolk sac tumor, seminoma), who had relapse after PVB (cisplatin, vinblastine, bleomycin) chemotherapy. Peripheral blood stem cells (PBSCs) were taken by two consecutive apheresis using a CS-3000 blood separator after high-dose chemotherapy of cytarabine and mitoxantrone. In total, 6.4 x 10(5)/kg of granulocytic cells (CFU-GM) was collected. He was treated with ultra high-dose chemotherapy consisting of carboplatin (800 mg/m2), etoposide (1,000 mg/m2) and cyclophosphamide (100 mg/kg) from day 1, followed by peripheral blood stem cell autotransplantation (PBSCT) on day 9. We transfused 2.4 x 10(5)/kg CFU-GM, which was enough number of stem cells for safe PBSCT. No serious side effects or complications were encountered. The patient achieved partial remission for more than two months. However, he died of respiratory dysfunction caused by metastatic lung cancer 5 months later. It was thought that ultra high-dose chemotherapy with PBSCT might be a new therapy for refractory testicular cancer.

The Pathophysiological analysis of hematopoietic tumors has advanced markedly owing to the progress in the fields of histomorphology, biochemistry, hematopoietic stem cells including cytokines, cell surface markers, cytogenetics, and molecular biology. This progress has led to the establishment of many new disease concepts and has almost completely changed the recognition of hematopoietic tumors. These basic studies have also supported the development of effective therapy of hematopoietic tumors. Of course, this progress has been reflected in the techniques and knowledge in routine laboratory evaluation.

A 47-year-old man presented with fever, cough and chest pain in January, 1989. He was found to have mediastinal tumor and generalized lymphadenopathy. Peripheral blood and bone marrow findings were typical for the chronic phase of chronic myelogenous leukemia (CML). Although the histological findings of a cervical lymph node were indistinguishable from those of malignant lymphoma, cytogenetic studies of the lymph node cells showed positive Ph1 chromosome and rearrangement of the bcr gene as well as bone marrow cells. Double fluorescence analysis of lymph node cells demonstrated co-existence of CD5, CD7 and CD33 positive cells and of cells sharing both CD5 or CD7 and CD33 antigens. These findings suggest that tumor cells originate from the stage at which the differentiation pathways of hematopoietic stem cells branch into precursor T and myeloid cells. Various combination chemotherapies had only partial effects on lymph node swelling. Chronic daily administration of low dose etoposide was very effective to control both lymphadenopathy and leukocytosis and the patient remained well for over 2 years until July, 1991 when hematological myeloid blast crisis developed. He died of pneumonia in October, 1991. This is a rare case of CML with extramedullary mixed crisis which survived for a long time.

Bronchial asthma has now been recognized as an inflammatory disease. The present study was undertaken to evaluate the role of mast cells and basophils in the pathogenesis of bronchial asthma. Stem cell factor, a growth factor of mast cells, activates rat peritoneal mast cells and human lung mast cells to release histamine. We demonstrated the presence of basophils in lung tissues obtained from fatal asthma cases using immunohistochemical techniques. IL-3, GM-CSF, IL-5 and IGF-I enhanced basophil histamine release. These findings indicate that mast cells and basophils have important roles in the pathogenesis of bronchial asthma.

Acute myelogenous leukemia is characterized by the infinite proliferation of malignant leukemia cells and by the impaired hematopoiesis. The proliferation of leukemia cells is supported by a small subpopulation, leukemic blast progenitors. Leukemic blast progenitors make leukemic blast colonies in methylcellulose culture. To determine the mechanism by which leukemia cells proliferate, we studied the role of several cytokines in the proliferation of leukemic blast progenitors in vitro. The findings indicated that there are at least three types in the regulation by cytokines of the leukemic cell growth. One is the stimulation of leukemic blast progenitors by colony-stimulating factors(CSFs) or interleukins(ILs) added in culture. These cytokines include granulocyte-CSF(G-CSF), granulocyte-macrophage CSF (GM-CSF), IL-3 and IL-1. The second is the autocrine growth mechanism. Leukemia cells by themselves produce and secrete G-CSF and GM-CSF, which stimulate the growth of leukemic blast progenitors. The third is a complex mechanism. IL-1, produced by leukemia cells or other cells, enhances the production of GM-CSF by leukemia cells, which stimulates the growth of leukemic blast progenitors. The precise mechanism by which each cytokine acts on leukemic blast progenitors should be determined to explore the mechanism of leukemia cell growth.