Mouse embryonic stem cells can be cultured in vitro and still retain their pluripotency. When reintroduced into mouse embryos, these cells can contribute to the germ line of the resultant chimeras. Homologous recombination between DNA sequences residing in the chromosome of embryonic stem cells and newly introduced DNA sequences allows the transfer of any modification of the cloned gene into the genome of a living mouse. This article discusses the current status of knock-out mice preparation, with emphasis on unsettled steps, during the knock-out procedure and introduces some of the newly developed gene targeting techniques by which more ideal animals can be prepared.

We report a case of rapidly spreading myeloma of immature cell morphology treated by peripheral blood stem cell transplantation (PBSCT). A 54-year-old man had a right orbital tumor, which subsequently was removed and proved to be plasmacytoma. Three years later a mass lesion appeared in his left lung and bilateral kidneys. The specimen obtained at lung biopsy confirmed the diagnosis of plasmacytoma. Serum M-protein, IgG lambda was increased, but there was no increase in plasma cells in the bone marrow. Since chemotherapy with VAD did not show any improvement, a high dose etoposide (500 mg/day, 4 days) was administered. When bone marrow suppression recovered, PBSCs were harvested (3.3 x 10(6)/kg). After conditioning therapy with cyclophosphamide (2.0 g/day, 2 days), etoposide (200 mg/day, 3 days) and ranimustine (200 mg/day, 2 days), the stored PBSCs were injected. Minor response was obtained and he was discharged. 2 months thereafter, it was found that plasma cells increased in the bone marrow. He died of pulmonary bleeding soon. Autopsy revealed immature plasma cell infiltration in multiple organs including the heart, liver, spleen, kidneys, intestine, bone and bone marrow.

The aim of this study is to clarify the clinicopathological characteristics of the multisystem degeneration seen in two male siblings with familial amyotrophic lateral sclerosis (FALS). A similar neurological disorder affected their elder sister and paternal uncle, but not their parents. The older brother (case 1) developed muscular weakness at 50 years of age and the younger brother (case 2), at 42 years of age. The duration of illness was 19 months in case 1 and 31 months in case 2. The clinical picture was the common (suspended) form in case 1 and the pseudopolyneuritic form in case 2. Pyramidal tract sign was obscure in both cases and cerebellar sign, sensory disturbance, sphincter disturbance and oculomotor palsy were not observed in either case. Neuropathological examination revealed similar findings in the two cases: 1) marked loss of lower motor neurons in the spinal anterior horn and motor nuclei of the lower brain stem in both cases, with neuronal loss of Onuf's nuclei in case 2; 2) very mild involvement in Clarke's nuclei, the dorsal and ventral spinocerebellar tracts and the middle root zone of the posterior column; 3) relatively well preserved Betz cells in the upper motor cortex with the appearance of a few macrophages, and mild changes in the pyramidal tract of the spinal cord; and 4) mild degenerative changes in the pallidoluysian system and the dentatorubral system. The most characteristic pathological findings common to both cases were the extremely mild involvement of the middle root zone of the posterior column, Clarke's nuclei and spinocerebellar tracts. The pattern of lower motor neuron system degeneration paralleled the development of clinical features. Genetic studies demonstrated no mutations in exons 1, 2 and 4 of Cu/Zn-binding superoxide dismutase gene. We emphasized the existence of mild involvement of middle root zone of posterior column, Clarke's nuclei and spinocerebellar tract in FALS with multisystemic degeneration.

To clarify the common characteristics among P-glycoprotein (P-gp)-expressing hematological malignancies and whether chemotherapies could or could not induce P-gp expression, we analyzed P-gp/MDR1 expression in tumor cells from 200 Japanese patients (104 with acute myeloblastic leukemia (AML); 30 with acute lymphoblastic leukemia (ALL); 66 with mature lymphoid malignancies). Functional P-gp expression was examined by Rhodamine-123 efflux test, and estimated with the data by RT-PCR method. In mature lymphoid malignancies, the cells of T or natural killer (NK) cell malignancies frequently expressed P-gp/MDR1. In AML, frequent P-gp/MDR1 expression was associated with the expression of CD7 or c-kit and with 8; 21 chromosomal translocation (p < 0.01), which were thought to be the characteristics of the hematopoietic stem cell. Though the expression of P-gp/MDR1 was more frequent at onset than at relapse phase, the increase is thought to result from the expansion of blastic fraction expressing P-gp/MDR1. In ALL, P-gp/MDR1 expression was not frequent in B-cell precursor lineage (three of eighteen patients), but the incidence was high in CD7(+) surface CD3(-) cases (seven of the cases). These results indicate P-gp/MDR1 expression is more frequently in the tumor of T, NK cell and stem cell, reflecting the characteristics of its normal counterpart.

Autologous blood stem cell transplantation (ABSCT) has been increasingly used in the treatment of malignant diseases. With the use of hematopoietic cytokines, collection of peripheral blood stem cell (PBSC) has become easier. Contamination of PBSC with tumor cells is supposed to be reflecting the amount of residual tumor cells in the host. G-CSF combined conditioning regimen seems to be effective for ANLL in complete remission (CR) probably by in vivo purging of residual leukemic cells. From our preliminary results, ABSCT can be used as the treatment of choice for standard risk ANLL, and aggressive non-Hodgkin's lymphoma. To clarify the curative potential of ABSCT, prospective clinical trial consisting of remission induction, consolidation of CR, PBSC harvests, and marrow-ablative therapy for ABSCT will be required.

When one hundred of c-Kit+Sca-1+Lin- cells obtained from B6. Ly 5.1 mouse were injected into lethally irradiated B6. Ly 5.2, 100% of the mice injected survived. Sequential analyses of recipient's peripheral blood leukocytes revealed presence of Ly 5.1+ myeloid and lymphoid cells even 12 months after the injection. We also examined the fate of these cells in the recipient mouse after transplantation. Time course analyses of the mouse injected with 500 FACS-purified c-Kit+Sca-1+L- cells revealed that spleen is the primary site for expansion of the injected c-Kit+Sca-1+Lin- cells. By day 28 s/p transplantation, more than 50,000 donor type c-Kit+Sca-1+Lin- cells were found in the spleen and over 15,000 cells in the bone marrow. By day 60, number of c-Kit+Sca-1+Lin- cells decreased in the spleen and returned to normal level in the bone marrow. Stem cell activity of the donor derived c-Kit+Sca-1+Lin- cells in the primary recipient was confirmed by in vitro and in vivo colony formation as well as transplantation to the secondary recipient. These results provide evidence that c-Kit+Sca-1+Lin- cells in the bone marrow have capability of self-renew as well as multilineage differentiation potential and that spleen is the primary site of stem cell expansion after bone marrow transplantation in mouse.

In this study, the newly developed marrow-rescue therapy during myelosuppression is utilized. In this therapy, peripheral blood stem cell transfusion (PBSCT) is administered following high-dose chemotherapy. Harvest of peripheral blood stem cells (PBSC) during myelosuppression following marrow-ablative chemotherapy is a safe, reliable procedure in children with leukemia. And administration of these cryopreserved PBSC is useful in reducing myelosuppression following intensive/ultra high-dose chemotherapy. In this study, several courses of intensive chemotherapy (1 course: VP-16 300mg/m2 x 5 days + carboplatin 400-500mg/m2 x 3 days) and one course of ultra-high dose chemotherapy (1 course: VP-16 400mg/m2 x 8 days + carboplatin 800mg/m2 x 5 days + MCNU 250, 200mg/m2 x each day) with PBSC transfusion were applied in four cases of pediatric malignant brain tumors (2 cases of medulloblastoma, one case of pineoblastoma and anaplastic ependymoma) after surgical reduction. With PBSC transfusion, myelosuppression following high-dose chemotherapy could be overcome without serious complication in all cases. Three cases showed complete remission and one showed partial remission after the operation and intensive chemotherapy. However, CSF dissemination appeared in two cases and they died 20 and 28 months after the onset respectively. Intensive/ultra high-dose chemotherapy with PBSC transfusion is a safe procedure in children with malignant brain tumors. This procedure may enable the postponement of radiation for pediatric malignant brain tumor cases under three years of age.

Clonal analyses using X-chromosome inactivation patterns of the phosphoglycerate kinase (PGK) and DXS255 (M27 beta) genes were performed in women with various hematological diseases or normal hematopoiesis. Four (17%) of 24 hematologically normal females had a skewed Lyonization pattern. It, however, appeared that in the majority of cases clonality determination of myeloid cells would be possible in comparison with lymphocytes. Blasts of acute myeloid leukemia (AML) (21) and granulocytes and bone marrow cells of chronic myeloproliferative disorders (6) were all shown to be clonal, suggesting that this method was useful for the concept formation of a clonal disorder. Twelve of 14 patients with the myelodysplastic syndromes had a clonal pattern and 8 of 10 patients with aplastic anemia had a polyclonal pattern. Three of 34 patients with AML during remission were diagnosed as a clonal hematopoiesis and associated with features of myelodysplasia during remission, when clonal patterns are different in patients with the same disease, it seems important to elucidate their clinical implications.

Ideally, in preoperative intra-arterial infusion chemotherapy (regarding advanced breast cancer) is to obtain the most significant effects concerning histological features. Intra-arterial infusion chemotherapy using epirubicin (EPI), used in conjunction with daily doses of 1,200 mg medroxyprogesterone acetate (MPA), have recently been performed. This procedure has shown remarkable histological effects in the metastatic lymph nodes as well as in the primary lesions. These results were especially remarkable in the patients who were administered MPA (daily) two weeks prior to EPI infusion. These patients showed a complete disappearance of tumor cells. The results were interesting in view of the mechanism of action. Intra-arterial infusion chemotherapy combined with MPA may also be valuable in treating metastatic liver tumors and recurrent lesions in the regional lymph nodes such as supraclavicular tumors. A high response rate was obtained in the chemo-endocrine therapy when combined with MPA as a pretreatment. Thus, MPA may be expected to be available as a systemic therapy, too. In the future, a new development in the field of intra-arterial infusion chemotherapy may be achieved by utilizing a combination of angiogenesis inhibitors and peripheral blood stem cell transplantation.

Preoperative autologous blood donation is widely used in cardiac surgery. However, some patients are unable to store adequate amounts of blood before surgery, and some develop anemia after the operation. We attempted to clarify the limitations of blood donation alone and its influence on erythropoiesis in comparison with those associated with adding recombinant human erythropoietin (rEPO). Subjects were twenty-five patients who were scheduled to undergo elective cardiac surgery. A unit of autologous blood (200 ml) was to be donated every 3 or 4 days for 2 weeks. 200mg of ferrous sulfate was given orally every day in 10 patients (the simple donation group), while 200 U/kg of rEPO was given intravenously 3 times a week in combination with oral ferrous sulfate supplementation in 15 patients (the rEPO-treatment group). After donation, reticulocyte counts increased significantly in both groups. In the simple donation group, hematocrit levels decreased significantly (p < 0.02), while serum iron levels did not change significantly. In the rEPO-treatment group, hematocrit levels remained unchanged and serum ferritin levels decreased significantly (p < 0.02) after the donation; in addition, serum iron levels in the rEPO-treatment group decreased significantly (p < 0.05) than those in the simple donation group during donation. The erythroid/nucleated cell ratio remained almost normal in the simple donation group. This ratio was significantly higher in the rEPO-treatment group than in the simple donation group (36.4 +/- 8.3% versus 26.2 +/- 6.8%, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Recent development of chemotherapy enabled us to cure patients with malignancies including leukemia, malignant lymphoma, choriocarcinoma, ovarian cancer, breast cancer and so on. In order to obtain the definite effect, the quantities of chemotherapeutic agents should be increased and the major lethal side effect caused by bone marrow failure should be avoided. For this purpose, hematopoietic stem cells derived from either bone marrow or peripheral blood can be used for the rapid recovery of neutropenia and thrombocytopenia. We prepared stem cells both from bone marrow and peripheral blood by anti-CD34 monoclonal antibody bound on magnetic beads column and administered into severe combined immunodeficiency (SCID) mouse. Four and 20 weeks after transplantation, mononuclear cells bearing human hematopoietic antigens and human immunoglobulin G were appeared in the circulation, suggesting that stem cells from peripheral blood also possess long-standing capability of maturation as well as those from bone marrow. We next conducted clinical study of high dose chemotherapy combined with peripheral blood stem cell transplantation for patients with poor risk choriocarcioma. By this treatment, 4 complete response (CR) and 4 partial response (PR) were obtained, whereas 2 progressive disease (PD), 1 no change (NC) and 5 PR were obtained by the previous conventional chemotherapies. The results obtained were promising and this treatment regimen may become a good modality for the complete treatment of chemotherapy curable tumors.

The inactivation of hematopoiesis and survival of mice have been used in determining the relative biological effectiveness (RBE) of heavy charged particles of carbon 12 generated by Riken ring-cyclotron. Whole bodies of anesthetized mice were exposed to accelerator-generated carbon 12 of 135 MeV/u or X-rays of 200 kVp in graded doses. At about 5 hours after exposure, exogenous spleen colonies (CFU-S), granulocyte-macrophage progenitor cells (GM-CFC) and megakaryocyte progenitor cells (Meg-CFC) were assayed to determine dose relationships, and then D0 doses were determined. The inactivation of blood cells and lethality of mice (LD50/30) were also monitored for 30 days after irradiation. The RBE values of the 135 MeV/u carbon beam in terms of CFU-S were 1.68 for marrow and 1.41 for spleen, for GM-CFC they were 1.56 for marrow and 3.29 for spleen, for Meg-CFC they were 1.25 for marrow and 3.73 for spleen, while they were about 1.0 for erythrocytes, leukocytes and thrombocytes in mice irradiated with 2 Gy. However, the RBE values of the carbon beam were 1.32 for LD50/30.

It has recently become known that sufficient numbers of multi-potent stem cells transiently appear in the peripheral blood at the time of recovery from myelosuppression after chemotherapy for collection for later transfusion (autologous peripheral blood stem cell transplantation; ABSCT). It is also known that cytokines such as G-CSF or GM-CSF enhance the mobilization of stem cells in peripheral blood. It is, therefore, easy to collect a large number of progenitor cells using the apheresis technique. We have also succeeded in developing an easy preservation method using DMSO-HES-ALBUMIN as a cryoprotective agent for simple storage in a -80 degrees C refrigerator. Furthermore, the development of measurement of CD34 positive cells become easy to estimate the contents of progenitor cells in harvested peripheral mononuclear cells. These techniques contribute to the safe and reliable practice of ABSCT. There are high expectations for ABSCT as a new technique in the cure of patients with such diseases as hematological malignancy and chemotherapy sensitive solid tumor, as it makes possible the administration of super-high dose anti-cancer drugs which cause marrow ablation.