Endogenous Spleen Colonies Research Articles

Modification of tumor and normal tissue radioresponse in mice by N-methylformamide

The effects of the differentiation-inducing agent N-methylformamide (NMF) on the in vivo response of the murine tumor FSA and its pulmonary metastases to ionizing radiation were investigated. In addition, the radioresponse of acutely responding normal tissues was determined in mice receiving systemic NMF. A dosage of 300 mg/kg administered for 8 days had little effect on the FSA tumor growth, yet enhanced the growth inhibitory actions of ionizing radiation with dose enhancement factors ranging from 1.5 to 1.7. Administration of NMF also enhanced the radiation response of FSA micrometastases. The response to irradiation of hematopoietic tissue, jejunum, and testes in mice receiving NMF was also investigated. NMF administered before or before and after radiation enhanced the formation of endogenous spleen colonies, yet did not influence the LD 50 30 for radiation. Jejunal crypt cell survival after radiation was slightly increased in mice receiving NMF, but the survival of spermatogonia after radiation was not affected. These data indicate that NMF administration results in an increase in the radiosensitivity of the FSA tumor and its metastases with no concomitant increase in the radiation response of the normal tissue tested. Thus, at least in this model system, a therapeutic gain is achieved through the combination of NMF and ionizing radiation.

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.

Radioprotective Properties of Detoxified Lipid A from Salmonella minnesota R595

In the past, the toxicity of bacterial lipopolysaccharide (LPS) or its principal bioactive component, lipid A, has detracted from their potential use as radioprotectants. Recently, a relatively nontoxic monophosphoryl Lipid A (LAM) that retains many of the immunobiologic properties of LPS has been isolated from a polysaccharide deficient Re mutant strain of Salmonella minnesota (R595). The ability of the native endotoxic glycolipid (GL) from S. minnesota (R595) as well as diphosphoryl lipid A (LAD) and nontoxic monophosphoryl lipid A (LAM) derived from GL to protect LPS responsive (CD2F1 or C3H/HeN) and nonresponsive (C3H/HeJ) mice from 60Co gamma irradiation has been studied. Administration of GL, LAD, or LAM to CD2F1 or C3H/HeN mice (400 micrograms/kg) 24 h prior to exposure provided significant radioprotection. No protection was afforded to C3H/HeJ mice. Experiments were also conducted to determine the relative abilities of GL, LAD, and LAM to stimulate hematopoiesis as reflected by the endogenous spleen colony (E-CFU) assay. Protection was not correlated with the ability of these substances to increase E-CFUs or to induce colony-stimulating activity (CSA).

Increased Protective Effect of Hypoxia Against Radiation Following Activation of Haemopoiesis with Dextran Sulphate

The effect of pretreatment of mice with a single injection of dextran sulphate (DS) on the protective capacity of moderate hypoxia was analysed. Dextran sulphate activated haemopoietic stem cell populations, and irradiation under hypoxia one day after DS injection increased the number of haemopoietic stem cells surviving in the bone marrow of the femur, their recovery after irradiation, and the number of endogenous spleen colonies. A moderate hypoxia (15% O2 and 12% O2) significantly reduced the lethal effect of gamma rays in mice after DS injection and increased the value of LD 50/30.

Wound-induced alterations in survival of 60Co irradiated mice: importance of wound timing.

Wounding mice shortly before or shortly after lethal 60Co irradiation enhances survival. Survival of wounded-irradiated mice may be due to enhanced hematopoietic recovery as measured by endogenous spleen colony (E-CFU-s) formation.

Increased Protective Effect of Cystamine and Mexamine Against Radiation after Activation of the Haemopoiesis by Dextran Sulphate in Mice

The effect gained by activation of haemopoietic cell populations by a single injection of dextran sulphate (DS) on the protective effect of cystamine and mexamine administered in small doses was monitored in mice. On irradiation 24 hours after the injection of DS the number of endogenous spleen colonies increased following the administration of mexamine and cystamine, the number of haemopoietic stem cells surviving in the femur after irradiation increased, and recovery of the cellularity of irradiated bone marrow was accelerated. Both substances administered 24 hours after the injection of DS significantly reduced the lethal effects of gamma rays. The maximum values of the dose reduction factor, for combined radiation protection, reached 1.48 as regards decreased lethal effects and 1.6 as regards equieffective E-MSC exposure.

In vivo radioprotective activities of diethyldithiocarbamate (DDC)

Studies were performed to determine whether diethyldithiocarbamate (DDC) protects against radiation damage to bone marrow, jejunal crypts, testicular tubules, hair follicles, tissues in the leg responsible for leg contractures, and a fibrosarcoma (FSA) of C 3Hf/Kam mice. In most experiments, DDC at a dose of 400 mg/kg or 1000 mg/kg body weight was given i.p. 30 minutes before single doses of gamma radiation. DDC (1000 mg/kg) given 30 minutes before whole-body irradiation protected hematopoietic stem cells by a factor (PF) of 1.59, as assessed by the LD50/30 assay, and by PFs of 1.32–1.55, as assessed by the endogenous spleen colony assay. A dose of 400 mg/kg DDC was less effective. Protection was also significant against hair loss and leg contractures; PFs produced by 1000 mg/kg DDC were 1.44 and 1.38–1.51, respectively. Jejunum was protected by 400 mg/kg DDC (PF = 1.2), but not by 1000 mg/kg. The opposite was observed with testis: 1000 mg/kg was protective (PF = 1.2), but not 400 mg/kg. DDC also protected the FSa tumor, either as lung micrometastases or as a solitary tumor in the leg. Both 400 mg/kg and 1000 mg/kg DDC protected 4 day-old micrometastases by a PF of approximately 1.1. DDC at a dose of 1000 mg/kg protected 8 mm leg tumors by a PF of 1.24 at the TCD50 level. Therefore, DDC protected both normal tissues and FSA, but the degree of protection varied greatly. A therapeutic gain was achieved in some instances.

血液系細胞の放射線障害とビスコクラウリン型アルカロイドに関する研究

After a whole-body irradiation at a single dose of 500R, mice were injected intraperitoneally with 0.1mg of cepharanthin (CR) every day for 20 days. Endogenous spleen colonies (CFU-S) were counted on Day 10, and the spleen and thymus weight was measured, peripheral leukocytes counted and hematocrit value determined for 30 days after the irradiation. The number of CFU-S increased remarkably in CR injected mice compared with control mice. Decreased spleen weight by irradiation recovered in excess by Day 30. In CR administered mice, the spleen weight increased transitorily along with the proliferation of CFU-S. After irradiation, the recovery of thymus weight was delayed compared with the spleen. Therefore, the effect of CR administration was not clear. Peripheral leukocytes decreased in number successively up to Day 10 and recovered linearly from Day 20 through Day 30, especially in CR injected mice. The hematocrit value was reduced to a minimum on Day 10 and almost recovered by Day 20 in irradiated mice. CR had no effect on the hematocrit value. The effect of cepharanthin was discussed in relation to radiation damage of hemopoietic tissue.

Mast-cell precursors in various haematopoietic colonies of mice produced in vivo and in vitro.

We have examined mast-cell precursors in various in vivo and in vitro haematopoietic colonies of mice. Cells from haematopoietic colonies of (WB x C57BL/6)F1- + +/+ mouse origin were injected into the skin of the congeneic W/Wv mice which are genetically depleted of tissue mast cells. The appearance of mast-cell clusters at the injection site indicated the presence of mast-cell precursors in the injected cell suspension. More than 40% of 12 d exogenous spleen colonies and 14 d in vitro mixed colonies contained mast-cell precursors, but only a small proportion of 7 d exogenous spleen colonies, 9 d in vitro erythroid bursts or 14 d large in vitro neutrophil-macrophage colonies contained mast-cell precursors. Neither transient endogenous erythroid spleen colonies examined at the fifth day nor 7 d in vitro neutrophil-macrophage colonies contained mast-cell precursors. Among 12 d spleen colonies mast-cell precursors were more frequent in the predominantly neutrophil than the predominantly erythroid colonies but 14 d in vitro mixed colonies, in which erythroid cells were predominant, contained mast-cell precursors more frequently than 14 d in vitro neutrophil-macrophage colonies. Therefore, differentiation of mast cells seems to be independent of either the neutrophil-macrophage or erythroid cell lineages.

Survival and endogenous spleen colonies of irradiated mice after skin wounding and hydroxyurea treatment.

Wound trauma-induced survival from radiation may be related to increased mitosis in hematopoietic cells. This is supported by the cell cycle-dependent drug hydroxyurea, which 1. blocked survival of wounded mice injected 2 or 3 days after 900 rad and 2. reduced the number of endogenous CFU-s in wounded mice injected shortly before 700 rad.

Erythropoietic stimulation enhances, and erythropoietic inhibition suppresses, multidirectional differentiation in 4-day transient endogenous spleen colonies.

Transient endogenous spleen colonies were found to be composed of either erythroid, granuloid or megakaryocytic cells, or mixtures of these cell types. Independently of the directions of differentiation of the colonies their formation was uniformly stimulated by bleeding and almost completely prevented by hypertransfusion. It is suggested that cells which form these colonies constitute a separate class of pluripotential hemopoietic progenitors, whose differentiation in either direction passes the stage sensitive to erythropoiesis-modulating factors.

Effects of environmental temperature on haemopoietic stem cells in the tails of mice.

Using the endogenous spleen colony assay method of Till & McCulloch (1963), the numbers of haemopoietic stem cells present in the bone marrow in the tails of mice were estimated under different environmental temperatures. Compared to animals kept at 22--26 degrees C, mice transferred to an kept at 36.5 degrees C showed a doubling of colony-forming units in the tail in 1--4 weeks. Exposing them to 8 degrees C caused a significant depopulation to approximately one-third in 3--4 weeks. By transferring the mice from one temperature extreme to another these changes could be reversed. Tail marrow depleted of viable stem cells by X-irradiation was repopulated within approximately 3 weeks in animals kept at room temperature or above but this process was inhibited in the cold.

Haematopoiesis in mice heterozygous for the W trait: defective formation of transient endogenous spleen colonies.

It has been determined that W/+ and Wv/+ heterozygous mice, as compared with normal +/+ homozygous littermates, form significantly lower numbers of transient 5-day endogenous spleen colonies in response to X-irradiation. This defect was evident for doses of irradiation between 2-6 Gy (200-600 rad) and was associated with a slightly increased radiosensitivity of the assayed precursor cells (TE-CFU) in W heterozygotic mice. Moreover, the defect was transplantable, i.e., intrinsic to the marrow cells and not to the microenvironment, and was not associated with a similar decrease in cells which form erythropoietic bursts in vitro (BFUe). This study provides a cellular basis for increased radiosensitivity of W/+ and Wv/+ mice and suggests that the 'W' mutation is semi-dominant, both with respect to the white spotting and TE-CFU formation.

Cytotoxic and radiosensitizing effects of misonidazole on hematopoiesis in normal and tumor-bearing mice

Misonidazole is a 2-nitroimidazole hypoxic cell radiosensitizing agent currently undergoing clinical trials. Preliminary studies have shown that misonidazole is toxic to human hematopoietic stem cells. A survey of the effects of misonidazole on a variety of murine hematopoietic stem cells was undertaken. No cytotoxicity or radiosensitization could be demonstrated when misonidazole was administered in vivo, in assays of transplantable spleen colony cells (CFU-S), endogenous spleen colony cells and committed granulocyte-macrophage precursors (CFU-C). The ability of misonidazole to radiosensitize hypoxic CFU-S was confirmed. Misonidazole was toxic to bone marrow when incubated in vitro in liquid cultures for long periods at higher concentrations than that attainable in vivo. CFU-C proliferation was also assessed in animals bearing Lewis lung tumor. No toxicity could be demonstrated when there was a marked increase in CFU-C activity following tumor implantation or when the tumors had grown large enough to have a significant hypoxic portion. These studies indicate that misonidazole is not toxic to murine hematopoiesis in vivo, but there are indications that it may be toxic in situations in which relatively high concentrations are maintained for long periods of time.

Response to Endotoxin of Endotoxin-”Resistant” C3H/HeJ Mice: A Model for Study of Hematopoietic Control

Hematopoietic effects of bacterial Iipopolysaccharide (LPS). specifically S. typhosa LPS extracted by the Boivin (B) or Westphal (W) method, were compared in the C3H/HeJ (resistant) strain and in closely related normal strains C3H eB/FeJ and C3H/HeN. Maximal release of neutrophils from marrow to blood was normal after either LPS-B or LPS-W in C3H/HeJ. Induction of an increase in colony-stimulating factor (CSF) in serum was markedly blunted but was present after either LPS-B or LPS-W. and no evidence for this being due to an inhibitor was elicited. The effect of IPS given before sublethal irradiation on subsequent hematopoietic recovery in marrow and spleen was evaluated. LPS-W had no discernable effect on any measure of postirradiation recovery. while LPS-B influenced some but not all measures of recovery. In C3H/HeJ. LPS-B failed to have any appreciable effect on recovery of marrow neutrophils, on 4-day splenic granulocytic colonies, or on changes in numbers of transplantable spleen colony-forming cells (CFU-S). However, its effect on 1 0-day endogenous spleen colonies (E-CFU). spleen weight. and spleen iron uptake appeared to be normal. These data indicate that the C3H/HeJ mouse is a valuable model for study of control of the response of stem cells. For example, results suggest that progenitor cell response is not linked by a recognition system to changes in blood neutrophil level nor to the size of the (postmitotic) marrow pool of neutrophils. Postirradiation granulocyte recovery also appears not to be linked to the number of E-CFU. The results indicate that endotoxin has effects on multiple individual control systems and further that there is a lack of linkage between some of the control systems. T HE C3H/HeJ MOUSE is a C3H substrain that developed a genetic resistance to bacterial lipopolysacchanide (endotoxin, LPS). The present study was undertaken to explore the use of the C3H/HeJ mouse as a model for studies of control factors in hematopoiesis. The control of neutrophil production and distnibution is, at best, poorly understood, and we have even less understanding of control of the plunipotent stem cell system. LPS has been used in studies of control because it produces profound perturbation of the entire hematopoietic system. The possibility that LPS plays an in vivo role in hematopoietic regulation is under study.’ Two difficulties arise in studies of control neutrophil and plunipotent stem cell systems. First, it is difficult to separate the primary from subsequent events after endotoxin injection. Second, any putative control factor thought to be causing one of these hematologic events may contain endotoxin as a contaminant. A national sequence of events relative to the pentur

Defective transient endogenous spleen colony formation in S1/S1d mice

WCB6F1 mice of the genotype S1/S1d did not form transient 5-day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE-CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.

Radiation sensitivity change of hemopoietic cells induced by hydroxyurea.

Hemopoietic tissues of mice contain cells capable of proliferation and differentiation to form endogenous spleen colonies (Endo-CFU-S) in irradiated animals. Hydroxyurea (HU) given before irradiation of C57BL mice for endogenous CFU assay increased the number of surviving CFU-S twofold. Either HU or HSATT given after the initial stimulus abolished this rise. In vivo and in vitro 'suicide' assays of spleen cells indicated that cells were stimulated by the HU or the initial stimulus to enter into DNA synthesis shortly after stimulation. The resting CFU cell population appeared to be positioned close to DNA-S phase and responded to the stimulus by entry into S phase. Whole body irradiation at intervals after HU revealed a changing radiation sensitivity of the endogenous CFU-S consistent with drug-induced progression with decreased radiation sensitivity of the S phase cohort of cells.

Radioprotective activity of interferon inducers

The radioprotective activity of interferon inducers (tilorone, Acranil, poly I:C and LPS) was investigated against acute X-ray irradiation and prolonged 60Co-gamma rays irradiation. The endogenous spleen colony formation test and percentage of surviving mice 30 days after irradiation were used as indicators. All interferon inducers investigated proved to have radioprotective activity.

Activation of precursors of T killers of hematopoietic stem cells by thymosine

Lymph node cells from normal CBA mice, from CBA→CBA syngeneic radiation chimeras, and B mice were incubatedin vitro with fraction 5 of thymosine, and transplanted into sublethally irradiated (CBA x C57BL)F1 recipients, and the number of endogenous colonies in the recipients' spleen was determined. Thymosine was shown to potentiate the killer activity of lymph node cells of normal CBA mice and of CBA→CBA syngeneic radiation chimeras, but not of B mice. It is suggested that the target for the action of thymosine is the subpopulation of T1 lymphocytes.

Mechanisms of hemopoietic stimulation by androgens

Effects of androgens on hematopoiesis in vivo were studied using the growth of endogenous spleen colony forming units (CFU-s) in subleathal (600rad) irradiated mice.Androgens, testosterone and fluoxymesterone, were injected on 1, 3, 5 days after the irradiation and colonies counted on 10 days.Both androgens were found to produce a significant increase in endogenous CFU-s in subleathal irradiated mice, although more stimulation was seen in the mice treated with testosterone. In addition, the type of colonies formed in spleens stained with H. E. were analysed to see effects of androgens on granulopoiesis in vivo. Testosterone stimulated the growth of granuloid colonies as well as erythroid ones in the spleen's, but there was no stimulation seen in the mice treated with fluoxymesterone at the dose used in this study.These results suggest that testosterone, given in vivo, may act on pluripotent stem cells directly to enhance their differentiation into erythroid and granuloid commited stem cell compartments causing an increase in the numbers of erythroid and granuloid cells in spleen.