Various modes of administration of ACNU (nimustine hydrochloride) were tried to make clear which mode is the best method to obtain intrathecal diffuse distribution of ACNU to match the condition of killing of glioma cells (10 micrograms/ml; greater than 30 min.). Tried modes of administration included 1)bolus injection into ventricular cavity, 2)bolus injection into cisterna magna, 3)bolus injection into lumbar subarachnoid space, 4)ventriculo-lumbar perfusion, 5)chiasmatic cistern-lumbar perfusion. Used dose of ACNU was 5 mg/body for all modes of administration. ACNU level in CSF was measured by HPLC method specially developed by authors. To make clear intrathecal distribution of ACNU, autoradiography using 14C-ethylene-ACNU was studied after administration of 10 muCi/Kg of radioactive ACNU. The images were studied by image analyzer system (BAS-2,000 system developed by Fuji Film Co. Ltd). Among the modes of administration tried, ventriculo-lumbar perfusion method gave the best results in terms of lumbar, ventricular, cisterna magna, and basal cistern distribution of ACNU to match the cell kill condition experimentally ascertained. Although, bolus injection of ACNU into cisterna magna gave sufficient amount of ACNU in lumbar region, the initial level of ACNU was too high in cisterna magna, and administration of ACNU once a week for three times in a canine cisterna magna resulted in considerable deterioration of brain stem and basal structure. In addition to it, the level of ACNU in ventricular cavity was not detectable. Lumbar bolus injection resulted in also too much ACNU accumulation at the injected lumbar area, and at the cisterna magna region, ACNU was not detectable.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone resorption plays an important role in bone modeling and remodeling. Osteoclasts are the cells responsible for the bone resorption. Osteoclasts are located on endosteal bone surfaces and on the periosteal surface beneath the periosteum. They are multinucleated giant cells highly polarized in their morphology and function. Among the proximal surface, the membrane and the area of the cytoplasm directly oppose to the bone surface, which are specialized into two regions. A central region consisting of many irregular cytoplasmic processes and infoldings, the ruffled border, is known to be the active site of bone resorption. Surrounding the ruffled border, a second region, the clear zone provides an area of close attachment to the mineralized bone surface. The osteoclasts secrete a large amount of protons by the action of H(+)-pump on the ruffled border into the sealed resorption cavity, resulting in the acidified microenvironment under which condition the bone matrix is dissolved. Protons are provided by the intracellular action of carbonic anhydrase. Following the secretion of the protons, several ion-transporting systems, i.e., carbonate-chloride exchanger, chloride-channel, Ca(2+)-transport systems, Na+/K(+)-ATPase, and voltage-dependent Ca(2+)-channel, are sequentially operated on both apical and basolateral cytoplasmic membranes. In addition, osteoclasts contain a large amount of lysosomal enzymes (cathepsin C, beta-glycerophosphatase, beta-glucuronidase, etc.), which contribute to degrade the bone organic matrices exposed in the resorption cavity. These enzymes bind to the mannose-6-phosphate receptor on Golgi apparatus, are transported to the ruffled border and are secreted into the extracellular compartment in an exocytotic manner. Osteoclasts also have a high tartrate-resistant acid phosphatase activity which is currently used as a marker enzyme osteoclastic differentiation. Osteoclasts are considered to develop from hematopoietic stem cells. So far, the following four different pathways of the differentiation of osteoclast are proposed: The precursors of osteoclast develop (1) from multilineage hematopoietic cells via a completely separate differentiation line, (2) from granulocyte macrophage-colony forming cells, (3) from committed but proliferative monocyte-macrophage, and (4) from mature and unproliferative monocyte-macrophage. However, the differentiation line of the osteoclasts has still to be elucidated. The formation of osteoclasts as well as that of other hematopoietic cells is strongly regulated by many cytokines [interleukin (IL)-1,IL-3,IL-6, M-colony stimulating factor (CSF), and GM-CSF]. 1,25-Dihydroxyvitamin D3 and parathyroid hormone also stimulate the differentiation of osteoclast precursors. However, the mature osteoclasts do not possess the receptors for these hormones.(ABSTRACT TRUNCATED AT 400 WORDS)

Growth hormone (GH), which has been extracted from the pituitary gland since early times, has become easily available by the advance of genetic engineering in recent year. Its clinical application to treatment in various fields, involving obesity, wounds, fractures, gastric ulcers and so on, is being increasingly discussed. The presence or absence of the effect of GH on leukopoiesis was studied in vivo and in vitro experiments. In the in vivo experiment, GH was administered to rats whose bone marrow production had been suppressed by the injection of mitomycin C, and time-course changes in the peripheral blood leukocyte count in these rats were compared with those in rats given physiological saline solution alone (control group). The in vitro experiment was performed by colony assay of mouse marrow cells. Insulin growth factor-1 (IGF-1) was also studied in the in vitro experiment. The in vivo experiment revealed that GH promoted recovery of leukocytes from the nadir, and in the in vitro experiments GH and IGF-1 were demonstrated to increase the number of colonies in the presence of granulocyte macrophage colony stimulating factor (GM-CSF). GH was considered to exert effects on myeloid progenitor cells and the hemopoietic microenvironment simultaneously, resulting in an increase in leukocytes.

The case survey of drug-induced hematologic disorders in Shikoku District (Ehime Prefecture) disclosed 21 patients. Cases were 12 rheumatoid arthritis patients, 2 brain tumor, one epilepsy, 2 liver cirrhosis, one neuralgia, one arthralgia, one hyperthyroidism, and one IBL-like T-lymphoma. Causative drugs for aplastic anemia were Metalcaptase, Shiosol, Voltaren and Emeside. Drug-induced aplastic anemia was so severe that 4 out of 5 patients had died of bone marrow dysfunction. Neutropenia was caused by drugs as Rimatil, Cefobit, Sepatren, Mercazole, Sulpyrin, Aleviatin, Cefamedin and Metalcaptase. The real causes of these drug-induced hematologic disorders have not been clear. Remarkably high incidence among rheumatoid arthritis patients is suggestive several reasons as unique reactivity associated with HLA, suppression on hematologic stem cells by abnormal metabolites, and immunologic dysfunction commonly seen in collagen diseases. Further studies of more accurate incidence of drug-induced hematologic disorders are needed in investigating real causes of unhappy side-effects.

The maintenance of mature blood cells requires the presence of hemopoietic stem cells, whose characteristics are their ability both to self-renew and to make differentiated progeny. We proposed a stochastic model for the mechanism of self-renewal and commitment of hemopoietic stem cells using in vitro clonal culture techniques. Recent progress in the molecular biology facilitated isolation and characterization of a number of cytokines that regulate the proliferation of hemopoietic stem cells. In this review, I summarize the current understanding of the mechanisms on interaction between several cytokines including IL-1, IL-3, IL-6, IL-11, G-CSF and stem cell factor (SCF).

Acute unclassified/undifferentiated leukemia (AUL) is classified into following 3 subgroups; 1) cases with coexpression of myeloid and lymphoid antigens on a single blast, 2) cases with coexistence of heterogenous subpopulations, 3) cases showing lineage-switch during the clinical course. Most cases in the groups 2 and 3 were not different from those in group 1 because of the presence of one or more common antigen (s) on the blasts. Accordingly, AUL can be considered as a candidate of leukemia arising form multipotential stem cells and the phenotype and genotype are represent a potential for myeloid and lymphoid differentiation. We should recognize that dual genotypes of IgH and TCR genes in B-precursor cell leukemia differ from such multipotentiality of leukemic cells, because the genotype occurs in re-arrangement process for IgH gene diversity after malignant transformation.

We have carried out the molecular and cell-biological analysis on Ph1-positive leukemias in this study. Five out of nine Ph1-positive ALL cases showed molecular rearrangement within the classical bcr sequence (or M-bcr), similar as those in 47 CML cases. We examined 4 cases of Ph1-positive ALL presenting no rearrangement of M-bcr and found that, in 2 of 4 cases, one showed the breakpoint in a 5 kb segment of the bcr gene first intron (bcr-2) and the other in bcr-1, 16 kb upstream of bcr-2. Ph1-positive ALL frequently showed biphenotypical or biclonal phenotypes of myeloid and lymphoid lineages. Furthermore, we demonstrated the ability of two Ph1-positive ALL cell lines to differentiate into monocytic lineage in vitro, thus suggesting the possibility that these Ph1-positive ALL cells might reside on the stage of multipotent stem cell along the hematopoietic cell differentiation. Two out of 31 CML cases showed the mutations of the ras genes by the polymerase chain reaction; one case in the crisis phase and the other in the chronic phase. However, no mutations of the fms genes was detected. Two cases in the crisis phase of 24 CML patients (11 cases in the chronic phase and 13 cases in the crisis phase) contained rearrangements of the p53 gene by Southern analysis. Furthermore, the transcriptional alteration was found in 2 CML-BC and 2 CML-BC derived cell lines' samples, suggesting a important role of the p53 gene in the transformation of CML into the crisis phase.

To identify the biological characteristics of so called stem cell leukemia (SCL), of which leukemic blast cells should be derived from pluripotent stem cells, immunophenotypical and genotypical analysis and response to several hematopoietic cytokines were studied in 272 cases with acute de novo leukemia. In 132 cases with acute myelogenous leukemia (AML), some cases of CD19+ and/or CD7+ AML were considered as SCL. In cases with myeloperoxidase negative acute lymphoblastic leukemia (ALL), cases of CD7 + CD1 - CD3 - CD4 - CD8 - My-Ag (myeloid antigens) +ALL, considered as those of T-precursor ALLs, and cases of HLA-DR + CD19 + CD20 - My-Ag + ALL, considered as those of B-precursor ALLs, were though to be SCL. We did not think the cases of ALL with dual genotype to be SCL, since dual genotype could not be considered as sings of ability to differentiate to multilineage but as products of the process of active V-DJ rearrangements of Ig heavy chain gene.

One of the most important factors for the proliferation and hemoglobin synthesis of erythroid cells is iron atom. This atom is tightly bound to serum transferrin (Tf) and is taken up by erythroblasts and reticulocytes through transferrin receptor (TfR). Both Tf and TfR are reutilizable and have roles for the efficient intracellular accumulation of iron. In addition to the reutilization (recycling), the expression of TfR is also regulated by cytoplasmic iron concentration; the increase of iron downregulate the synthesis of TfR at the translational level and vice versa. This mechanism was recently explained by the binding between "iron responsive element (IRE)" in the 5' end of TfR mRNA and IRE binding protein by a transacting manner. Johnstone et al, and we found that TfR was externalized from sheep reticulocyte and human erythroleukemia cell, K562, respectively. Furthermore, we confirmed that this shed TfR was detected in blood and concluded that the quantitation of TfR in serum is a useful index for evaluating the erythropoiesis. The serum TfR was increased in iron deficiency anemia, hemolytic anemia and polycythemia and was decreased in aplastic anemia. In renal anemia, it was increased after the administration of erythropoietin (Epo). By the in vitro liquid culture of peripheral blood stem cells using interleukin 3 and Epo, it was found that soluble TfR was derived from the erythroblasts during the maturation process.

Inhibition of hemopoiesis by nitrous oxide was studied in mice and the following results were obtained. 1) During exposure to N2O, the numbers of pluripotent hemopoietic stem cells (CFU-S) and granulocyte-macrophage progenitor cells (GM-CFC) decreased significantly in the murine spleen and bone marrow, but the former more than the latter. 2) The recovery of CFU-S and GM-CFC was delayed in mice that were given low-dose irradiation followed by continuous exposure to N2O. The delay of recovery was significantly more in the spleen than in the bone marrow. 3) The levels of serum granulocyte-macrophage colony stimulating factor (CSF) in N2O exposed mice, induced by endotoxin, decreased significantly compared with those of control mice. Prolonged N2O administration to mice appears to impair the hemopoietic inductive microenvironment as well as hemopoietic stem cells and hence results in hemopoietic death and delay of hemopoietic recovery after irradiation.

The central feature of hematopoiesis is life-long, stable cell renewal. This process is supported by hemopoietic stem cells which, in the steady state, appear to be dormant in cell cycling. The recruitment of the dormant stem cells into cell cycle may be promoted by such factors as interleukin (IL)-1, IL-6, granulocyte-colony stimulating factor (G-CSF), and newly discovered IL-11. The effects of IL-1 on stem cells may be indirect. Once the stem cells leave Go and begin proliferation, the subsequent process is characterized by continued proliferation and differentiation. Though several models of stem cell differentiation have been proposed, micromanipulation studies of individual progenitors suggest that the commitment of multipotential progenitors to single lineages is a stochastic process. The proliferation of early hemopoietic progenitors requires the presence of IL-3 and/or IL-4, and the intermediate process appears to be supported by granulocyte/macrophage-CSF (GM-CSF). Once the progenitors are committed to individual lineages, the subsequent maturation process appears to be supported by late-acting, lineage-specific factors such as erythropoietin (erythropoiesis), G-CSF (neutrophil production), and IL-5 (eosinophilopoiesis). Thus, hemopoietic proliferation appears to be regulated by a cascade of factors directed at different developmental stages.

A 33-year-old man was hospitalized because of thrombocytopenia and severe splenomegaly. On admission 78% of peripheral lymphoid cells were abnormally large, with pale cytoplasm. Flow cytometry of the abnormal lymphocytes showed that they expressed CD 2, CD 3, CD 11, CD 16, and CD 56, but not CD 4 nor CD 8, so they were T-cell large granular lymphocytes (T-LGL). Abnormal lymphocytes obtained from a lymph node expressed CD 2, CD 16, CD 38, and CD 56, but not CD 3, CD 4, and CD 8, so they were natural killer(NK) cells. Splenectomy was performed and the operative specimen showed diffuse infiltration of pleomorphic lymphocytes, probably chronic lymphocytic leukemia cells. After splenectomy, the platelet count returned to normal but the lymphocytosis continued. Two years after discharge, chemotherapy was done because of thrombocytopenia and hepatomegaly. The patient died of disseminated intravascular coagulation arising from sepsis. The differences and similarities between peripheral and lymph-node lymphocytes suggest that LGL and NK cells may be differentiated from the same kind of cell, somewhat differentiated from stem cells.

We presented here a case with ALL transplanted with autologous marrow cells cryopreserved after heat treatment. Bone marrow cells from a 17-year-old female patient in first complete remission were cryopreserved after treatment with heat (42 degrees C, 60 min) and interferon-alpha. Three and a half years later, at her 4th remission, she was treated with busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) followed by autologous marrow infusion. The hemopoietic recovery after marrow infusion was prompt except in thrombopoietic lineage. She was doing well without limitation until leukemic relapse after 6 months. From this experience, marrow cells treated with heat and cryopreservation still include multipotent hemopoietic stem cells.

A single high dose of 5-fluorouracil (5-FU) was administered intravenously in mice to induce the aplastic bone marrow and to seize the morphological characteristics of blood stem cells. Time course observations of histological and cytological changes in bone marrow hematopoiesis and stem cell assay revealed that the day 2 bone marrow contained a number of quiescent blood stem cells. For advanced morphological studies of blood stem cells, the day 2 marrow cells were seeded in methylcellulose culture medium for stem cell assay. Blast cell colonies (BCC) which contained CFU-GEMM (colony-forming unit granulocyte, erythroid, monocyte/macrophage, megakaryocyte) were formed within 2 weeks. BCC-constituting cells (BCC-cell) were lifted and processed for morphological analyses. The BCC-cells were variable in size as well as morphology, and were divided into 3 sub-groups according to their size. Large-sized group (greater than 14 microns) consisted of large blastic cells with a tendency of differentiation into early granulocyte or monocyte/macrophage lineage. Medium-sized group (10-14 microns) consisted of more immature blastic cells which lacked a differentiation. Small-sized group (7-10 microns) consisted of two cell-types. One type (named 'transitional cell' or 'TC cell') had distinctive features as a dark nucleus and a narrow basophilic cytoplasm seemed to be the most immature cell type. The other (named 'lymphoid cell') was morphologically indistinguishable from the small lymphocyte. Blast cell colony-replating experiments disclosed that the proportion of small-sized cells in BCC-cells (about 4%) was equivalent to the incidence of GEMM colonies formed in replated culture (about 2-5%). Furthermore, immunohistochemical staining of wheat germ agglutinin (WGA), anti-Thy-1 antibody and anti-B cell antibody revealed that most of TC cells were positive for WGA suggesting of CFU-GEMM, and negative for anti-B cell antibody. Most of lymphoid cells were positive for anti-B cell antibody, and negative for WGA and anti-Thy-1 antibody, and were estimated to be true B-lymphocytes. Based on these results, it was reasonable to consider that a majority of TC cells were multipotent blood stem cells.

An acridine orange cytofluorometric analysis was used to study the DNA-RNA contents of bladder washing exfoliated cells from 109 patients with transitional cell carcinoma of the urinary bladder. DNA and RNA measurements were performed in each of 200-300 cells/sample, and DNA index (DI) and RNA index (RI) were calculated from the distributions of these values. Presence of aneuploid stem cell line and/or increased mean DNA contents were observed in 83 (76.1%) of the 109 patients. Elevated RNA content in a diploid cell population, was of no additional diagnostic significance. Histologically diagnosed high grade and high stage bladder carcinoma apparently showed an aneuploid stem cell line with high DI and high RI. Thirty patients with superficial bladder cancer (Ta and Tl) who had tumor recurrence within 2 years after surgical interventions showed a significantly increased RI in contrast to 10 patients who had no recurrence for more than 2 years. RNA content measurement was thought to be useful for prediction of tumor recurrence after surgical interventions. Six patients who had a progression from superficial to invasive carcinoma were with high histological malignancy, lamina propria invasion, high DI and high RI. However, there was no significant difference in RI between the 6 patients and the other patients with superficial bladder cancer. Eight patients who had distant metastasis within one year after radical cystectomy also showed a significant increase in RI in contrast to 8 patients who had no metastasis for more than 1 year. Increased RI may be said to be an important risk factor in predicting the development of distant metastasis and recurrent tumor after surgical interventions.

For the study of immunological phenomena peculiar to liver transplantation, it would be important to know well hematolymphoid system in the liver. In this paper, we report the restoring mechanism of hemopoiesis in the murine regenerating liver. 1) We found hemopoietic stem cells (HSC) and wheat germ agglutinin positive cells (WAG-PC) in both fractions of peripheral blood lymphocytes (PBL) and intrahepatic lymphocytes (IHL) from untreated C3H/He mice. The numbers of HSC and WGA-PC in each fraction gradually increased after partial hepatectomy. This change was remarkable in the population of IHL, associated more strongly with the liver. Colonies, which were generated from IHL in a fibrin clot culture system, are mainly composed of granulocytes, macrophages and mast cells. 2) We found colony-stimulating activity not only in the culture supernatant of normal nonparenchymal liver cells but in that of normal parenchymal liver cells. Furthermore, we clarified that one of colony-stimulating factors in both culture supernatants is GM-CSF. This result suggests parenchymal liver cells also release cytokines such as GM-CSF. These facts show that hematolymphoid system in the liver is a complicated network, composed of parenchymal liver cells, nonparenchymal liver cells, hemopoietic stem cells, and other cell populations.