No effects of levamisole on cytotoxic drug-induced changes of human granulopoiesis.

The effect of Levamisole on the human granulopoiesis was studied in patients randomized to receive, in addition to adjuvant chemotherapy for primary breast cancer, either no other treatment or additional unspecific immune therapy with Levamisole. The reaction of granulopoiesis to the cytostatic drugs, as characterized by changes of peripheral blood polymorphonuclear neutrophils (PMN), functional bone marrow granulocyte reserve, serial bone marrow cytology, and granulopoietic stem cells (CFU-C) in marrow and blood, was not affected by administration of Levamisole. The data support the concept that Levamisole has no direct effect on human bone marrow granulopoiesis, but that an allergic mechanism is involved in the pathogenesis of Levamisole-induced agranulocytosis. The expectation that Levamisole exerts a beneficial effect by stimulation of the granulopoiesis, as previously suggested for BCG and Corynebacterium parvum, could not be substantiated in our studies.     

Granulopoiesis in long-term culture by marrow from mice with busulfan-induced chronic latent aplasia

Mice given high-dose busulfan therapy develop a chronic latent marrow aplasia characterized by normal peripheral blood neutrophil numbers, hematocrits and marrow cellularity but reduced numbers of pluripotent hemopoietic stem cells (CFU-s) and granulocyte-monocyte progenitor cells (CFU-gm). To study the pathogenesis of this lesion, bone marrow was propagated in long-term marrow cultures (LTMC). Small amounts of normal marrow readily established and sustained long-term granulopoiesis in vitro. In contrast, inocula of marrow from busulfan-treated animals containing three to five times as many stem and progenitor cells failed to establish long-term granulopoiesis in vitro. These results suggest that high-dose busulfan therapy produces a qualitative defect in either the hemopoietic stem cells, the stromal-forming elements, or both, rendering them incapable of establishing long-term granulopoiesis in vitro. Furthermore, mixing experiments employing normal and busulfan-damaged marrow demonstrate that this qualitative defect is not due to the emergence of a suppressor cell population. LTMC can show types of marrow damage not detectable by other techniques currently available and represent a powerful tool for studying latent bone marrow failure.     

MicroRNA Profiling in Human Neutrophils during Bone Marrow Granulopoiesis and In Vivo Exudation

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development.     

Radioprotection of mice with interleukin-1: relationship to the number of erythroid and granulocyte-macrophage colony-forming cells

This report presents the results of an investigation of changes in the number of erythroid and granulocyte-macrophage colony-forming cells (GM-CFC) that had occurred in tissues of normal B6D2F1 mice 20 h after administration of a radioprotective dose (150 ng) of human recombinant interleukin-1 (rIL-1). Neutrophilia in the peripheral blood and changes in the tissue distribution of GM-CFC demonstrated that cells were mobilized from the bone marrow in response to rIL-1 injection. For example, 20 h after rIL-1 injection marrow GM-CFC numbers were 80% of the numbers in bone marrow from saline-injected mice. Associated with this decrease there was a twofold increase in the number of peripheral blood and splenic GM-CFC. Also, as determined by hydroxyurea injection, there was an increase in the number of GM-CFC in S phase of the cell cycle in the spleen, but not in the bone marrow. Data in this report suggest that when compared to the spleen, stimulation of granulopoiesis after rIL-1 injection is delayed in the bone marrow. Also, the earlier recovery of GM-CFC in the bone marrow of irradiated mice is not dependent upon an increase in the number of GM-CFC at the time of irradiation.     

Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.     

Human granulopoiesis in vitro: an advantage in the use of Iscove's modified Dulbecco's medium

With the aim of determining whether Iscove's Dulbecco's medium (IMDM) provides a growth advantage in the support of granulopoiesis from cultures of human bone marrow in agar, samples from 20 normal subjects were examined in triplicate after 7, 10 and 14 days in parallel cultures containing IMDM or Dulbecco's medium. From every sample, more granulocyte-macrophage colonies were obtained at each culture interval with IMDM. In particular, the number of colonies with IMDM at 14 days (96 +/- 13 per 2x10(5) bone marrow cells) was almost double that with Dulbecco's medium (50 +/- 10). This increment consisted almost entirely of pure granulocyte colonies (P less than 0.001). No significant change in the proportion of eosinophil colonies was observed. These data indicate that IMDM does provide a growth advantage over Dulbecco's medium in the generation of granulocyte (neutrophil and eosinophil) colonies from agar cultures of normal human bone marrow.     

Responsiveness to humoral factors of mouse marrow colony forming cells after treatment with cyclophosphamide.

Humoral factors affecting granulopoiesis have been detected by culturing bone marrow cells in intraperitoneal diffusion chambers. This study investigated the possibility that treatment with drugs alters the ability of cells to respond to these factors. Three days after treatment with 200 mg kg-1 cyclophosphamide, donor marrow cells were more responsive than normal cells to the factors produced in cyclophosphamide pretreated hosts. The optimum timing of host pretreatment for maximum colony stimulation also differed for cells from cyclophosphamide treated and untreated donors and these effects were found to depend on the dose of drug given to the animals.     

Response of multipotent (CFU) and granulocyte (diffusion chamber assay) progenitor cells and differentiating cells of murine haematopoietic tissues to a perturbation of the steady state

Regulation of haematopoiesis was investigated by studying the response of haematopoietic tissues of mice to a perturbation of the steady state by vinblastine (VLB). Progenitor cells were quantified ly limiting dilution analysis of diffusion chamber cultures of haematopoietic cells and by the spleen colony technique. The diffusion chamber technique appears to assay granulocyte progenitor cells and those multipotent progenitor cells that become committed to granulopoiesis during chamber culture. The spleen colony technique probably assays multipotent progenitor cells. Decaying oscillatory responses to VLB were observed for progenitor cells as well as for differentiating cells in bone marrow. The period lengths of the diffusion chamber progenitor cell oscillations might indicate that these were induced by humoral feedback signal(s) from nonproliferative granulocytes. The oscillations of the multipotent progenitor cells of bone marrow were less pronounced and were earlier damped than those of the granulocyte progenitor cells. This may support the hypotesis that multipotent progenitor cells are regulated by more efficient mechanisms, which may depend on short range cell-cell interactions rather than long range humoral regulators.     

RESPONSIVENESS TO HUMORAL FACTORS OF MOUSE MARROW COLONY FORMING CELLS AFTER TREATMENT WITH CYCLOPHOSPHAMIDE

Humoral factors affecting granulopoiesis have been detected by culturing bone marrow cells in intraperitoneal diffusion chambers. This study investigated the possibility that treatment with drugs alters the ability of cells to respond to these factors. Three days after treatment with 200 mg kg^-1 cyclophosphamide, donor marrow cells were more responsive than normal cells to the factors produced in cyclophosphamide pretreated hosts. The optimum timing of host pretreatment for maximum colony stimulation also differed for cells from cyclophosphamide treated and untreated donors and these effects were found to depend on the dose of drug given to the animals.     

Identification of highly elevated levels of melatonin in bone marrow: its origin and significance

Bone marrow is an important tissue in generation of immunocompetent and peripheral blood cells. The progenitors of hematopoietic cells in bone marrow exhibit continuous proliferation and differentiation and they are highly vulnerable to acute or chronic oxidative stress. In this investigation, highly elevated levels of the antioxidant melatonin were identified in rat bone marrow using immunocytochemistry, radioimmunoassay, high performance liquid chromatography with electrochemical detection and mass spectrometry. Night-time melatonin concentrations (expressed as pg melatonin/mg protein) in the bone marrow of rats were roughly two orders of magnitude higher than those in peripheral blood. Measurement of the activities of the two enzymes (N-acetyltransferase (NAT) and hydroxyindole-O-methoxyltransferase (HIOMT)) which synthesize melatonin from serotonin showed that bone marrow cells have measurable NAT activity, but they have very low levels of HIOMT activity (at the one time they were measured). From these studies we could not definitively determine whether melatonin was produced in bone marrow cells or elsewhere. To investigate the potential pineal origin of bone marrow melatonin, long-term (8-month) pinealectomized rats were used to ascertain if the pineal gland is the primary source of this antioxidant. The bone marrow of pinealectomized rats, however, still exhibited high levels of melatonin. These results indicate that a major portion of the bone marrow's melatonin is of extrapineal origin. Immunocytochemistry clearly showed a positive melatonin reaction intracellularly in bone marrow cells. A melatonin concentrating mechanism in these cells is suggested by these findings and this may involve a specific melatonin binding protein. Since melatonin is an endogenous free radical scavenger and an immune-enhancing agent, the high levels of melatonin in bone marrow cells may provide on-site protection to reduce oxidative damage to these highly vulnerable hematopoietic cells and may enhance the immune capacity of cells such as lymphocytes.     

Blast cell and granulocyte production in human leukemia: pathophysiological concepts based on computer simulation using discrete modeling techniques

The granulocyte production of two patients suffering from leukemia was studied extensively by means of the tritiated thymidine method of cellular kinetics. The data obtained (1-h labeling index, pattern of cell labeling, labeling intensity, as well as other conventional parameters of bone marrow and blood) were used to develop a computer model (GPSS-language) to fit the observations. From these models, it was concluded that patients with leukemia may have an abnormal granulopoiesis, characterized by a high degree of inefficiency (premature cell death, skipping of divisions with undisturbed maturation). However, the underlying mechanisms may be quite different. While it cannot be excluded that in acute myelocytic leukemia there is a stem and/or progenitor cell pool that is highly ineffective but still capable of feeding some cells into the granulocytic pathway, it is nevertheless possible, as shown in plasma cell leukemia, that the ineffective granulopoiesis may be the result of direct or indirect interaction between the "leukemic" and the "normal" cell clone.     

重组白介素6-假单胞菌外毒素融合蛋白对正常BN大鼠骨髓粒系造血的作用

本文报道了重组白介素6-假单胞菌外毒素融合蛋白(IL-6-PE40)对正常BN大鼠骨髓粒系造血的体外效应。10ng/ml的IL-6-PE40对高表达IL-6受体的U266骨髓瘤细胞的蛋白质合成抑制率为50％,1000ng/ml则为100％。1～1000ng/mlIL-6-PE40对正常骨髓未纯化细胞的CFU-GM集落形成和DNA合成无明显抑制,但IL-6却具有一定的刺激效应。提示正常骨髓粒系祖细胞和骨髓细胞可能不表达IL-6受体,IL-6-PE40对粒系造血仍具有某些细胞毒作用,但被IL-6-PE40中的IL-6极大地削弱了。     

Relevance of clonogenic assays in hematotoxicology

Clonogenic assays have been established in hematology for 30 years. They have been widely used in fundamental studies on hematopoiesis and they are also routinely used in clinical hematology to confirm diagnosis or to predict time to recovery in cases of bone marrow failure. Their use in toxicological studies is more recent. Adverse effects of xenobiotics can induce hematological problems and pathologies such as neutropenia, thrombocytopenia, anemia, and aplastic anemia. Three clonogenic assays are proposed for granulopoiesis, megakaryopoieisis and erythropoieisis. Hematopoietic progenitors from murine or human origin can be cultured in the presence of xenobiotics using validated protocols to complete standard animal toxicological studies. These clonogenic assays can help to predict adverse effects of drugs or toxicants. Clonogenic assays using white blood cell progenitors (CFU-GM culture) have recently been validated by ECVAM and can be used routinely. Megakaryocyte progenitor (CFU-MK) culture is under development and prevalidation in toxicological studies supported by ECVAM. Red blood cells progenitor culture (BFU-E) has been proposed but needs international validation to be recognized. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reptilian bone marrow. An ultrastructural study in the spanish lizard,Lacerta hispanica

The ultrastructure of hemopoietic bone marrow of the Spanish lizard, Lacerta hispanica, has been studied for the first time. The organ consists of a stroma formed by venous sinuses and reticular cells. Erythropoiesis takes place in the lumen of blood vessels, while granulopoiesis is extravascular. Pluripotent stem cells are structurally differentiated into erythrocytes and granulocytes. Two types of granulocytes, heterophils and acidophils, have been found, and a third granular cell type is tentatively identified as granular leukocyte. Remarkably, plasmacytopoiesis occurs in the bone marrow of Lacerta hispanica. The possible functional significance of these results is discussed with emphasis on their importance for the reptilian immune system.     

Factors that control the differentiation of stem cells. I. The change in direction of hematopoietic stem cell differentiation under the effect of differentiating T-lymphocytes]

A mixed transplantation of bone marrow cells, and lymph nodes or thymic cells of mice CBA strain into lethally irradiated hybrid recipients (CBAXC57B1)F1 is accompanied with changes in the differentiation pattern from a mainly erythroid to a mainly granuloid way. Thymectomy of either donor of bone marrow cells or recipients, or both, destroys the stem cell differentiation in the direction of granulopoieseis. Intact syngeneic lymphocytes normalize differentiation of the stem cells, but in the presence of tissue antigens these provide for the stem cell differentiation mainly in the direction of granulopoiesis. The differentiation of stem haemopoietic cells is accomplished under the thymic and lymphocyte control. T-differentiating lymphocytes (Td) are the lymphocytes controlling the stem cell differentiation.     

REGULATION OF BONE MARROW CELL GROWTH IN DIFFUSION CHAMBERS: THE EFFECT OF GRANULOCYTE EXTRACTS

Hypotonic lysis of mature human blood granulocytes yielded an extract which reduced granulopoiesis and enhanced macrophage formation of mouse bone marrow cells cultured for 7 days in diffusion chambers (DC). The low molecular weight fraction (MW < 15,000–25,000 Daltons) obtained by Amicon filtration of the extract, reduced granulopoiesis without affecting macrophage formation. The high molecular weight fraction (MW > 15,000–25,000 Daltons) reduced the number of granulocytes and increased the number macrophages. Erythrocyte extract increased the macrophage formation in DC but did not alter the number of granulocytes. The spleen colony assay showed that the granulocyte extract increased the number of CFU-S in DC. It is suggested that the granulocyte extract contain an inhibitor of stem cell differentiation to myeloid cells thereby reducing the number of proliferative granulocytes in DC 7 days later. The inhibitor of differentiation may lead to an increased self renewal of the stem cell in the DC system.     

Radiation sensitivity of human and murine peripheral blood lymphocytes,stem and progenitor cells

Immunodeficiency is a severe side effect of radiation therapy, notably at high radiation doses. It may also impact healthy individuals exposed to environmental ionizing radiation. Although it is believed to result from cytotoxicity of bone marrow cells and of immunocompetent cells in the peripheral blood, the response of distinct bone marrow and blood cell subpopulations following exposure to ionizing radiation is not yet fully explored. In this review, we aim to compile the knowledge on radiation sensitivity of immunocompetent cells and to summarize data from bone marrow and peripheral blood cells derived from mouse and human origin. In addition, we address the radiation response of blood stem and progenitor cells. The data indicate that stem cells, T helper cells, cytotoxic T cells, monocytes, neutrophils and, at a high degree, B cells display a radiation sensitive phenotype while regulatory T cells, macrophages, dendritic cells and natural killer cells appear to be more radioresistant. No conclusive data are available for basophil and eosinophil granulocytes. Erythrocytes and thrombocytes, but not their precursors, seem to be highly radioresistant. Overall, the data indicate considerable differences in radiosensitivity of bone marrow and blood normal and malignant cell populations, which are discussed in the light of differential radiation responses resulting in hematotoxicity and related clinical implications.