The importance of distinguishing accurately between patients with asymptomatic or stable monoclonal gammopathies and those with overt multiple myeloma cannot be overemphasized. Most patients with asymptomatic monoclonal gammopathy will remain stable without treatment, but in some overt multiple myeloma may develop after several to many years of observation. Multiple myeloma is a malignant disease of the bone marrow plasma cells. Most patients die of progressive disease despite chemotherapy. Thus, accurate differentiation between asymptomatic monoclonal gammopathy and multiple myeloma is essential. Given a diagnosis of overt myeloma, how does the physician decide which treatment regimen to use for an individual patient? Based on the risk assessment, age considerations and risk benefit consideration for the patient, the most proper selection of treatment regimen should be done. Until we can identify a group of patients who will survive longer, intensive therapy, including stem cell transfusion, should be done with caution.

Dose intensive chemoradiotherapy with peripheral blood stem cell transplantation (PBSCT) has emerged as a useful treatment for patients with multiple myeloma. High-dose melphalan (140 mg/m2) combined with total body irradiation is usually an effective regimen for refractory myeloma. Based on the greater efficacy of the tandem autoplants with high-dose melphalan (200 mg/m2) for refractory myeloma, a similar approach is currently under investigation in untreated patients. A considerable relapse rate is still observed after high-dose chemoradiotherapy supported with PBSCT. It is unknown whether these relapses are the consequence of inadequate tumor reduction or tumor cells contamination in the grafts or a combination of both.

Mouse embryonal carcinoma (EC) cells are the stem cells of teratocarcinoma. EC cells are malignant but have pluripotentiality like an early embryo. Therefore, mouse EC cell lines have been used to investigate the gene expression and its regulation in development and differentiation. On the other hand, human EC cells are considered to be the stem cells of human germ cell tumors. More than 50 cell lines derived from human germ cell tumors have been established since 1975. If human EC cells have pluripotentiality like mouse EC cells, human EC cell lines would be useful as an experimental model of human embryogenesis. We have investigated the cell biological characteristics of 3 kinds of cell lines derived from human germ cell tumors and have obtained the following results. 1) PA1/NR cell line derived from a patient with ovarian teratocarcinoma can grow in suspension and the spheroids are characterized by differentiation to primitive neuroectodermal rosette. 2) lMa cell line derived from a patient with ovarian dysgerminoma is consistent with choriocarcinoma. 3) NEC14 cell line derived from a patient with testicular EC/yolk sac tumor shows pluripotentiality.

Peripheral blood stem cells (PBSC) were collected from 24 patients who were treated with high dose etoposide. Studied patients included one with acute lymphoblastic leukemia, 4 with acute myeloid leukemia (AML), 1 with myelodysplastic syndrome, 13 with lymphoma, 1 with malignant histiocytosis, 2 with myeloma, and 4 with testicular tumor. Etoposide was infused at a dose of 500 mg/m2 for 4 days, followed by subcutaneous injection of recombinant human granulocyte-colony stimulating factor from the nadir of leukocyte. PBSC were collected by processing 15-20 liters of blood apheresis in the recovery phase of chemotherapy. In all patients, the number of CFU-GM collected per aphereresis ranged from 0.01 to 59.4 x 10(5)/kg, and more than 5 x 10(5)/kg CFU-GM were collected in 19 of the patients (73%). All leukemia patients treated along with our protocols have remained in complete remission, but one patient with AML relapsed within 1 month after the treatment. Ten lymphoma patients were assessable for antitumor effect, and complete response (CR) was observed in 2, partial response (PR) was 7, and no change (NC) in one patient. Two patients with myeloma were classified to be NC. Three of the 4 patients with testicular tumor were PR, and the other one was NC. Eleven patients subsequently underwent PBSCT. The number of days required to achieve an absolute granulocyte count of 0.5 x 10(9)/l was 7 to 11 days, with a mean of 8.6.

A new therapeutic strategy for advanced ovarian cancer is to administer ultra-high doses of anticancerous drugs, followed by peripheral blood stem cell transplantation (PBSCT) to recover normal marrow functions. There are, however, no clear regimens to induce mobilization of peripheral blood stem cells (PBSCs). We therefore used three different chemotherapies and granulocyte colony-stimulating factor (G-CSF) to produce a rebound increase in PBSCs during myelosuppression by apheresis. Eleven patients with advanced ovarian cancer (FIGO Stage; IIc-1, IIIc-5, IV-4, Recurrence-1) received chemotherapy with CEP (cyclophosphamide: 500-750mg/m2, epirubicin: 50-70mg/m2, cisplatin: 70mg/m2), CEE (cyclophosphamide: 2,000mg/m2, epirubicin: 50mg/m2, etoposide: 300mg/m2, and PEC salvage (cisplatin: 75mg/m2, etoposide: 300mg/m2, cyclophosphamide: 1,000mg/m2) followed by lenograstim (G-CSF; 2 micrograms/kg subcutaneous injection daily for 14-18 days) to mobilize PBSCs. A range of 0-38.2 x 10(4) (mean +/- S.D.; 11.1 +/- 5.0 x 10(4) colony forming unit granulocyte-macrophage (CFU-GM)/kg in the CEP regimen (n = 15), 1.6-40.8 x 10(4) (mean +/- S.D.; 12.3 +/- 3.6 x 10(4) CFU-GM/kg in the CEE regimen (n = 11), and 8.6-11.4 x 10(4) (mean +/- S.D.; 10.0 +/- 2.0 x 10(4) CFU-GM/kg in the PEC salvage regimen (n = 2) were recruited by a single apheresis. Although the CEE regimen to mobilize PBSCs was much more efficient than the CEP regimen, a large number of PBSCs showing 38.2 x 10(4) CFU-GM/kg were mobilized by the CEP regimen with a dose-escalation of cyclophosphamide 750mg/m2 and epirubicin 70mg/m2. The number of CFU-GM/kg correlated well with that of CD34+ (r = 0.693).(ABSTRACT TRUNCATED AT 250 WORDS)

The treatment strategy for adult T-cell leukemia has not been established. CHOP derived combination chemotherapy protocols such as VEPA and VEPAM have failed to yield high complete remission rates and long survivals. Second or third generation combination chemotherapy regimens using alternative non-cross resistant drugs also have not achieved satisfactory results. A new multi-institutional trial attempting to increase dose intensity supported by granulocyte colony-stimulating factor is bringing improved remission rates and survival times. Other strategies using autologous bone marrow or peripheral blood progenitor cells should be studied as a next step.

Radiation therapy causes various degrees of damage to hematopoietic cells. Cepharanthine (CE), a plant alkaloid, showed the most prominent restorative effect on hematopoiesis among the antileukopenia drugs examined in a previous study. Its mechanism of action was further analyzed in this study, and the following results were obtained: Intravenous administration of CE every other day for 10 days after irradiation increased CFU-S two-fold. CE-conditioned medium from bone marrow stroma cells increased CFU-C. Higher concentrations of CE in the culture non-specifically suppressed the growth of various types of cells, including T cells. However, this suppression disappeared in the presence of peritoneal exudate macrophages (PEC). When Con A blasts were co-cultivated on PEC, concentrations between 10(-6) to 10(-12) M promoted growth of both PEC and T cells. 10 pg/ml and 8 pg/ml of IL-1 were produced from 10(6)/ml of PEC and bone marrow stroma cells, respectively, in 24 hrs with CE. A lesser amount was also produced from thymus stroma cells. A detectable amount of IL-6 was produced from CE-stimulated bone marrow stroma cells. These findings suggested that CE acted as a cytokine-inducer on various epithelial cells in the body and the cytokines thus produced stimulated multipotential hematopoietic progenitors and their progeny as growth and/or differentiation factors.

The localization of the glucose transporter 3 (GLUT3) was examined immunohistochemically, using a newly developed polyclonal antibody, in human brainstem and cerebellar tissues from neurologically normal, lacunar stroke and Alzheimer disease cases. In the brainstem, GLUT3 immunoreactivity was limited to the melanized neurons of the paranigral nucleus and substantia nigra, and to neurons in dorsal nucleus of the vagus nerve, and in the oculomotor, pontine, ambigius and hypoglossal nuclei. In the cerebellum, only the dentate nucleus had positive immunoreactivity. Glial cells and endothelial cells were not immunopositive. The results suggest a preferential expression of GLUT3 in particular neurons with a differential glucose need.

Although monocyte influx has been suggested as the primary source of pulmonary alveolar macrophages (AM), increasing evidence from recent studies has indicated that AM may be sustained through a self-renewal mechanism. We evaluated the age-related changes of the clonal growth (colony formation) of AM in mice (C57BL/6N mice and senescence accelerated mice). The colony forming unit (CFU) of AM of 24 month old C57BL/6N mice was lower than that of AM of 4-month-old mice (p < 0.05). In SAMP6 (senescence accelerated mice), CFU of AM was decreased with aging (p < 0.05). In SAMR1 (controls for SAMP6), CFU of AM was decreased with aging (p < 0.001). In SAMR1, CFU of bone marrow (BM) adherent cells of 12-month-old mice was similar to that of 4-month-old mice. In SAMP6, CFU of BM adherent cells of 12-month-old mice was larger than that of 4-month-old mice (P < 0.005). It was concluded that the CFU of AM declined with aging, but the CFU of the BM adherent cells did not. The decline of the AM CFU may be partly responsible for the defect of the immune response of the alveolar space in the elderly.

Effects of oxygen inhalation on hemopoiesis were investigated in mice and following results were obtained. 1) The numbers of pluripotent hemopoietic stem cells (CFU-S) and granulocyte-macrophage progenitor cells (GM-CFC) in murine bone marrow and spleen decreased significantly from day 1 of exposure to 70% and 100% oxygen. 2) The numbers of CFU-S and GM-CFC did not recover in mice that were exposed to 100% oxygen for 1 and 2 days followed by air exposure for 7 days. These results suggest that inhalation of more than 70% oxygen for 1 day induces inhibitory effects of the hemopoietic stem cells, and 100% oxygen exposure over 1 day can cause irreversible damage to the hemopoiesis.

The present study was designed to determine the effects of sex steroid hormones (estradiol, progesterone), gonadotropins (FSH, hCG), and gonadotropin releasing hormone agonist (GnRHa: Buserelin) on the proliferation of the ovarian serous adenocarcinoma cell line KOC-2S in vitro. The results showed: 1. The colony formation rate and fraction growth in the colony stem cell assay were suppressed by estradiol (5,000 pg/ml) and progesterone (50 ng/ml). The colony formation rates were 2.96% (control); 2.49%, 0.99% (estradiol: 500 pg/ml, 5,000 pg/ml); 2.58%, 0.53% (progesterone: 5.0 ng/ml, 50.0 ng/ml); 4.80%, 3.34% (hCG: 10 mIU/ml, 100 mIU/ml); and 1.79%, 2.96% (GnRHa: 1.0 ng/ml, 10 ng/ml). 2. The doubling time (DT) in the growth inhibition test was shortened by FSH and the saturation density (SD) became greater. The SD was suppressed by hCG. 3. The cytogenic features of the cells treated with FSH, estradiol and GnRHa did not show obvious morphologic change. The spontaneous floating cells were observed following treatment with progesterone. Cytoplasmic enlargement was observed following treatment with hCG. 4. Neither erb B nor erb B-2 was expressed in KOC-2S cells, and neither was induced by sex steroid hormones, gonadotropins or GnRHa.

I investigated the effects of a tyrosine phosphatase inhibitor, orthovanadate, on the proliferation of normal and CML hematopoietic progenitor cells stimulated by different colony stimulating factors. Orthovanadate decreased CFU-GM colony formation from normal bone marrow cells stimulated by IL-3 and GM-CSF in a dose dependent manner, except for G-CSF. But, BFU-E colony formation was not affected by the treatment with orthovanadate. In CML cells, CFU-GM colony formation was relatively more resistant to orthovanadate than that in normal bone marrow cells and orthovanadate, surprisingly, increased BFU-E colony formation. Western blot analysis showed that preincubation of CML cells with orthovanadate resulted in the enhancement of tyrosine-phosphorylation of p65 mainly, when stimulated with EPO. These results suggest that the second messenger system of IL-3, G-CSF, GM-CSF, and EPO in progenitor cells in CML is different from that in normal progenitor cells and that there is big difference in the second messenger system between myeloid and erythroid progenitor cells in CML cells.

Colony formation of mouse primitive hemopoietic progenitors with interleukin-6 (IL-6) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA), and their signal transduction were studied. Although IL-6 or TPA alone could not form colonies, their combination gave rise to significant number of colonies from Day-2 post 5-FU bone marrow cells. When colony numbers were compared with those supported by IL-3, IL-6+TPA gave rise to 86 + 47% of colonies formed with IL-3. Time course of colony formation with IL-6+TPA run parallel with that of IL-3. These colonies included not only granulocyte/macrophage (GM) colonies, but also granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies and blast cell colonies. Delayed addition of IL-6 or TPA decreased colony numbers, suggesting that both IL-6 and TPA were needed from the start of cultures for maximal colony formation. When cultures were started with TPA, and IL-6 was added on Day 2 of culture or later, few colonies developed. These data suggested that IL-6 might be essential to the survival of the progenitors in culture. Chronic exposure of progenitors to TPA prior to the culture with IL-6+TPA suppressed colony formation. Addition of calphostin C, a specific protein kinase C (PKC) inhibitor or genistein and herbimycin A, specific tyrosine kinase (TK) inhibitors to the culture also decreased colony numbers formed with IL-6 and TPA. To clarify which effects of IL-6 or TPA on colony formation were blocked by the inhibitors, the inhibitors were added to preincubation of progenitors with IL-6. Both the PKC inhibitor and TK inhibitors blocked the increase of colonies resulted from a pre-incubation with IL-6. Although delayed addition of TPA enhanced IL-6-dependent colony formation, delayed addition of TPA with either the PKC inhibitor or TK inhibitors canceled the increase of colonies. These data suggested that both signals of IL-6 and TPA might be transduced via activation of PKC and TK, but further studies are needed to confirm that.

The role of M-CSF in the process of mammalian development has been drawing attention. Unlike mammalian embryonic development, in which M-CSF is supplied by the maternal body, avian embryonic development begins and proceeds under the influence of a certain amount of M-CSF present in each fertilized egg, and its concentration can be experimentally controlled while observing the embryo's developmental state. Apart from this fact, aves provide us with many other advantages in the study of embryonic development. It is, however, not easy to separate multipotent hematopoietic stem-cell fractions from avian bone marrow. In addition, because a macrophage colony forming assay in soft agar, though it has been widely used, requires a large number of cells and a long culture period, the method of assay is not adequate for the sensitive detection of M-CSF activity in small scale samples. We have established a method for the depletion of nucleated erythrocytes from chicken bone marrow cell suspensions using percoll density gradient centrifugation, and have also developed a new method for determining chicken M-CSF-like activity employing a liquid culture. In this method, a 100 microliters aliquot of fractionated hematopoietic stem cells, 1 x 10(5), was placed in a well of 96-well flat-bottom culture plate, 100 microliters of sample was then added to each well, and the uptake of neutral red was measured after 4 days of culture. These procedures represent a simple and sensitive means of detecting M-CSF-like activity in chicken serum of x60 to x32 dilutions.(ABSTRACT TRUNCATED AT 250 WORDS)

We report a 63-year-old man presenting with dementia and cerebellar ataxia associated with multiple iron deposits in the brain on MRI. Numerous small lesions of low-intensity on both T1- and T2-weighted images were found in the parenchyma and surface of the cerebrum, cerebellum and brain stem. The number and size of lesions were increased on MRI with the method of gradient recalled acquisition in the steady state (GRASS), indicating that they were composed of iron. The similar lesions were not found in any organs on the abdominal GRASS-MRI. Any abnormalities were also not found in the cerebral angiography. Meanwhile, the protein and IgG levels and activated CD4-cells were increased in the cerebrospinal fluid, indicating the involvement of chronic inflammation in the iron deposits in this case.

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.