Spontaneous cell-mediated cytolysis by peripheral blood cells obtained from whole-body chronically irradiated beagle dogs

The level of natural killer (NK) activity of continuously gamma-irradiated (whole body) beagle dogs and their nonirradiated controls was studied. For analytical purposes, irradiated dogs were segregated into groups according to their clinical status: clinically normal, hypocellular, or with acute non-lymphocytic leukemia. Since unirradiated control animals exhibited a wide range of NK responses, the data from each irradiated animal were compared to its own age-matched or litter-matched unirradiated control. Of the eight clinically normal irradiated dogs (median = 146% activity of control) only one animal had a NK activity lower than that of its control. The hypocellular group (n = 5, median = 21.8% of control) and the leukemic group (n = 4, median = 52.5% of control) each contained one responder with higher activity than its control. The difference between the percentage of control of the clinically normal and clinically abnormal dogs was found to be significant (P less than 0.05). There is a negative correlation between the NK results obtained and the total accumulated dose of radiation at the time of sampling (correlation coefficient = -0.739, P less than 0.01), suggesting a radiation effect upon natural killer activity, which is evidence by enhancement at lower doses and depression at higher doses of irradiation.     

Stem cells from peripheral blood and bone marrow: a comparative evaluation of the hemopoietic potential in the dog

The kinetics and pattern of hemopoietic recovery after supralethal total-body irradiation (TBI) were compared after transfusion of cryopreserved autografts derived from peripheral blood and bone marrow. Fractionated TBI was given in three doses of 6 Gy each at intervals of 48 h. Grafts of peripheral blood mononuclear cells (MNC) were collected by means of continuous-flow centrifugation and by using the mobilizing agent, dextran sulphate. Autografts were adjusted to contain equal numbers of committed progenitor cells (CFU-GM). Dogs grafted with blood-derived MNC (group A) and with MNC from bone marrow (group B) all received about 1 X 10(5) CFU-GM per kg body weight. In all dogs consistent hemopoietic engraftment was achieved. Comparing the pattern of regeneration of the granulocytes, group A dogs showed a significant regeneratory advantage over group B dogs, particularly during the first 20 days after transplantation. Lymphoid recovery was more rapid in group A until day 14. In both groups, blood lymphocytes remained below normal values beyond day 100. The regeneration patterns of the platelets and reticulocytes revealed no significant differences. These results are in agreement with the hypothesis that there are differences in the relationship between CFU-GM content and hemopoietic potential of autografts from different sources.     

Experimental bases of the in vitro treatment of leukemic bone marrow by a derivative of cyclophosphamide: ASTA Z 7557

The authors report their experience with the ASTA Z 7557, a derivative of cyclophosphamide, for the in vitro treatment of leukemic bone marrows. They determined the sensitivity of human leukemic progenitors (CFU-L, n = 9) and normal progenitors studied in semi-solid media cultures (CFU-GM, n = 37; BFU-e, n = 11) and in long term marrow culture (pré-CFU-GM n = 41). Data establish: The inhibition of the in vitro proliferation of CFU-L by ASTA Z 7557. The similar sensitivity of CFU-L and normal CFU-GM. The respect, at doses toxic on CFU-L and CFU-GM, of more primitive stem cells, capable of self-renewing and the existence of correlation between the intensiveness of treatment and the regeneration capacity of CFU-GM; therefore, they defined a maximum tolerable dose which spares 5 +/- 5% CFU-GM (DL 95) after treatment. The existence of a wide range susceptibility from patient to patient which requires the determination of the DL 95 for each individual patient.     

Lithium-stimulated recovery of granulopoiesis after sublethal irradiation is not mediated via increased levels of colony stimulating factor (CSF)

We have previously demonstrated that lithium (Li) is an effective agent in accelerating the recovery of granulopoiesis following sublethal (2 Gy) whole body irradiation. In this report, studies are described that further define this Li-mediated recovery by measuring the levels of colony-stimulating factor (CSF) present in serum from mice administered 105 micrograms/mouse (total dose) of ultra-pure Li2CO3 for 3 days immediately following irradiation. On days 1-28 following the last lithium dose, the serum was tested for its CSF activity against both normal non-adherent derived bone marrow target cells and non-adherent marrow cells from mice administered cyclophosphamide (200 mg/kg body weight). Serum was assayed at 0.01, 0.1, 1 and 10 per cent final concentration. No significant difference in the total number of CFU-GM was observed from normal marrow using either serum from irradiated mice or lithium-treated and irradiated mice, although the irradiation did produce a 300 per cent rise in CFU-GM colonies compared to normal serum (days 4 and 10-15). From regenerating marrow, we observed a significant difference (P less than or equal to 0.01) in CFU-GM cultured with serum at 0.1 per cent concentration from irradiated and lithium-treated mice compared to irradiated mice without lithium. The presence of CSF was confirmed by its reduced activity in the presence of anti-(CSF). These results suggest (Li) may increase the sensitivity of CFU-GM to CSF, thereby producing more CFU-GM ultimately providing more circulating granulocytes.     

Sensitivity of CFU-GM from normal human bone marrow and leukaemic clonogenic cells (CFU-L) from blood of patients with myelogenous leukaemia to 3'-deoxy-3'-fluorothymidine in comparison to 3'-azido-3'-deoxythymidine

3'-Deoxy-3'-fluorothymidine (FT), a thymidine analogue highly effective against HIV 1 in vitro was investigated as to its in vitro effect on normal human bone marrow CFU-GM (agar colony assay) and on human peripheral myeloid leukaemic clonogenic cells (CFU-L, colony assay in methylcellulose). For comparison, 3'-azido-3'-deoxythymidine (AZT), structurally related and used in AIDS treatment, was included in the study. Both compounds inhibit the formation of clusters and colonies from bone marrow stem cells with an [IC]50 between 10(-6) and 10(-5)M. In concentrations only 5-10 times lower than the [IC]50, FT begins to stimulate cluster and colony formation. AZT and FT also inhibit the formation of clusters and colonies from CFU-L. Compared to CFU-GM, CFU-L were more sensitive to FT, and a stimulation was not seen. It is concluded that similar side effects on bone marrow could be expected for possible use of FT against AIDS as have been found for AZT and that both compounds are potential candidates for anti-leukaemic drugs.     

Murine and human hematopoietic colony formation: a possible regulatory role for intracellular histamine

Increasing number of data suggests that locally produced histamine is involved in regulation of hematopoiesis. In this study the granulocyte/macrophage (CFU-GM) colony formation by normal murine or human bone marrow cells, leukaemic colony formation (CFU-L) by a murine leukemia cell line (WEHI 3B), and colony formation by bone marrow cells from patients with chronic myeloid leukemia (CML) have been examined. We detected mRNA and protein expression of histidine decarboxylase (HDC), the only enzyme responsible for histamine synthesis both in normal bone marrow progenitor cells and in leukaemic progenitors. The significance of in situ generated histamine was shown on colony formation by inhibitory action of alphaFMH (blocking HDC activity, i.e. de novo histamine formation) and by N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine-HCl (DPPE) disturbing the interference of histamine with intracellular binding sites. These data provide further confirmation of the role of histamine in development and colony formation of bone marrow derived cells.     

Sister chromatid exchanges, during x-irradiation, in the blood lymphocytes of patients with hereditary diseases and radioresistant DNA synthesis

X-ray irradiation induced sister chromatid exchanges (SCE) in blood lymphocytes from patient with Down's syndrome and adult progeria (in both the cases radioresistant DNA synthesis takes place). In these diseases, likely as upon form II of xeroderma pigmentosum (the replicative DNA synthesis is radioresistant), X-ray irradiation lowers the rate of SCE compared with that in the control, then the SCE rate rises with the increase in radiation dose, reaching the rate of SCE in non-irradiated cells. In normal lymphocytes (in which ionizing radiation inhibits the replicative synthesis of DNA) the rate of SCE rises with the rise of radiation dose. Thus, the rate of SCE in X-ray irradiated lymphocytes is in reverse dependence with radioresistance of replicative synthesis of DNA. The data obtained are explained in accordance with the replicative hypothesis of the SCE nature (Painter, 1980a): in cells of patients with Down's syndrome, xeroderma pigmentosum form II and progeria of adults the time of existence of partly replicated clusters of replicons is decreased due to radioresistant replicative synthesis of DNA, but the presence of partly replicated clusters of replicons is necessary for SCE formation. Therefore the rate of SCE in X-irradiated cells of these patients decreases.     

Prenatal and neonatal irradiation in dogs: hematologic and hematopoietic responses

Hematologic and hematopoietic responses were evaluated in beagle dogs following a single prenatal (35 days gestation) or neonatal (10 days postpartum) exposure to 1.5 Gy 60Co gamma radiation. Hematopoiesis was studied by the in vitro culture of bone marrow granulocyte-macrophage progenitors (CFU-GM). Prenatally irradiated dogs exhibited a progressive, significant reduction in CFU-GM which was accompanied by decreases in peripheral blood leukocytes up to 24 weeks of age. Dogs which were neonatally irradiated also demonstrated a significant reduction in CFU-GM which was accompanied by significant alterations in peripheral white and red blood cell parameters. This was transient, however, and these dogs showed partial recovery of CFU-GM and hematologic parameter by 24 weeks of age. The persistent CFU-GM deficit in prenatally irradiated dogs suggests a relatively greater sensitivity of fetal marrow as compared to neonatal bone marrow for long-term damage by ionizing radiation.     

The effect of low-dose irradiation on unstimulated and PHA-stimulated human lymphocyte subsets

A culture system was used to evaluate the radiosensitivity of CD4+ and CD8+ T cells, Leu 19+ cells, and B cells obtained from normal adult males. Unstimulated CD8+ lymphocytes (D0 = 55 cGy) were twice as radiosensitive as CD4+ cells (D0 = 115 cGy). B cells had an intermediate radiosensitivity (D0 = 100 cGy). Leu 19+ cells were much more radioresistant and expressed a D0 of 550 cGy. When lymphoid cells were irradiated 1 or 4 days before phytohemagglutinin (PHA) stimulation, they were more radiosensitive than if they were first stimulated with PHA and then irradiated. When lymphoid cells were irradiated 1 h after PHA stimulation each lymphocyte subset was characterized by an increase in the D0 to 150 cGy for B cells to 290 cGy for CD4+ cells, and to 240 cGy for CD8+ cells. In contrast, Leu 19+ cells exhibited a decrease in their D0 to 290 cGy after they were stimulated by PHA.     

The influence of fibroblast-like cells derived from canine fetal hematopoietic tissues on the regulation of lymphohematopoiesis

Media conditioned by fibroblast-like cells derived from organs active in fetal lymphohematopoiesis were studied for their effects on adult granulocyte/macrophage colony-forming units (CFU-GM). Fibroblasts from fetal liver produced a factor stimulatory for CFU-GM, whereas fibroblasts from fetal marrow produced a factor inhibitory for CFU-GM which was not completely relieved by adding indomethacin to the assay. Our studies indicated that neither fetal marrow nor fetal liver produced factors affecting lymphocyte colony-forming units (CFU-L). Cell-cell interactions between fibroblast-like cells derived from fetal liver or marrow and normal adult CFU-GM were also studied. We observed that fibroblasts derived from both fetal and adult marrow inhibited colony formation, whereas inhibition in the presence of fetal liver fibroblasts was minimal. Loss of inhibitory activity by a liver fibroblast cell line over repeated passages was seen. Differential analysis of colonies formed above an adherent layer of fetal marrow fibroblasts suggested that these fibroblasts suppress myeloid/macrophage differentiation to a far greater degree than did adult marrow fibroblasts. A role in the regulation of fetal lymphohematopoiesis may be played by stromal fibroblasts.     

Cloning stem cells in the bone marrow of irradiated mice]

Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.     

大豆异黄酮对辐射小鼠造血系统损伤防护作用的实验研究

研究大豆异黄酮(SoybeanIsoflavone,SI)对辐射损伤小鼠造血系统功能的影响。雄性昆明小鼠照射前补充SI,经4Gyγ射线照射,观察补充SI对电离辐射损伤小鼠内源性脾结节(CFU-S)数、骨髓细胞粒、巨噬系细胞集落形成单位(CFU-GM)数、骨髓细胞DNA含量及外周血淋巴细胞DNA损伤程度的影响。结果显示SI提高了辐射损伤小鼠CFU-S数,增加了骨髓有核细胞CFU-GM数,降低外周血淋巴细胞DNA损伤程度,对骨髓细胞DNA含量也有一定的增加趋势。表明SI可以提高辐射损伤小鼠造血干祖细胞的增殖能力,减轻辐射对骨髓细胞和外周血淋巴细胞DNA的损害,SI对辐射小鼠的造血系统有一定的防护作用。     

Effects of prodigiozan on post-radiation recovery of hemopoietic precursor cells and colony-stimulating factor level in long-term cultures of mouse bone marrow

Prodigiozan injected to long-term cultures of mouse bone marrow 24 h before irradiation increased CFUs and CFU-GM number and colony-stimulating factor (CSF) level by the time of delivery of ionizing radiation. As early as 60 min following irradiation of bone marrow structures with a dose of 2 Gy the number of CFUs and CFU-GM decreased considerably, and from day 3 on after irradiation the indices under study were gradually restored. By day 14 the cultures preinjected with prodigiozan exhibited higher recovery levels. The decrease in the number of precursor cells 60 min after irradiation was accompanied by a drastic increase in the CSF content of cultures; the CSF release in cultures protected with prodigiozan was more moderate than in the irradiated controls.     

Overexpression of the MnSOD transgene product protects cryopreserved bone marrow hematopoietic progenitor cells from ionizing radiation

We determined whether manganese superoxide dismutase (MnSOD)-plasmid liposome (PL) transfection of C57BL/ 6NHsd mouse bone marrow protected cells irradiated at room temperature (24 degrees C) or in the cryopreserved state. MnSOD-overexpressing hematopoietic progenitor 2C6 cells were radioresistant compared to the parent 32D cl 3 cells when irradiated frozen or at 24 degrees C. Fresh whole marrow from mice injected intravenously with MnSOD-PL prior to explant as well as explanted marrow single cell suspensions transfected in vitro were irradiated at 24 degrees C or -80 degrees C. In vivo or in vitro transfection of marrow with MnSOD-PL produced significant radiation protection of irradiated marrow progenitor cells compared to controls at 24 degrees C or -80 degrees C. (in vivo transfection D(0) 2.19 +/- 0.21 at 24 degrees C, D(0) 2.10 +/- 0.07 at -80 degrees C compared to control D(0) 1.56 +/- 0.06 or 1.66 +/- 0.04, P = 0.047 and 0.017 respectively; in vitro transfection D(0) 2.35 +/- 0.11 at 24 degrees C, D(0) 3.42 +/- 0.13 at -80 degrees C compared to D(0) 1.81 +/- 0.01 or 2.53 +/- 0.05, P = 0.0087 and 0.0026, respectively). Thus the MnSOD transgene product protects frozen marrow cells as well as marrow cells irradiated at 24 degrees C.     

Effects of restoring lethally irradiated mice with anti-Thy 1.2-treated bone marrow: graft-vs-host, host-vs-graft, and mitogen reactivity.

In vitro treatment of A/J mouse bone marrow with anti-Thy 1.2 serum and guinea pig complement (GPC) eliminated its ability to induce graft-vs-host (GVH) mortality in lethally irradiated C57BL/6J x A/F1 (BAF1) mice. The anti-Thy 1.2 and GPC treatment of A/J marrow significantly reduced spleen cell activation by phytohemagglutinin (PHA) but not lipopolysaccharide (LPS) stimulation in A/J mice assayed 6 weeks after lethal irradiation and reconstitution with the treated marrow. However, the anti-Thy 1.2 treatment of A/J bone marrow did not impair the ability of the lethally irradiated, reconstituted, syngeneic mice to reject C57BL/6J skin grafts. We conclude that lymphocytes in bone marrow which are susceptible to inactivation by anti-Thy 1.2 mediate allograft reactions and/or that radioresistant cells which persist in the recipient initiate rejection of allogeneic skin grafts.     

Effects of anesthesia-induced modest hypothermia on cellular radiation sensitivity

To assess the mechanisms of modest hypothermia (MH) and its effects on cellular radiation response, a model of anesthesia-induced modest hypothermia (AIMH) in the adult mice and a model of pure MH in the newborn mice were established. The survival rate of lethally irradiated mice was increased to 72% through AIMH before irradiation. Both apoptosis and necrosis of human fetal bone marrow CD34+ hematopoietic stem cells cultured under MH were significantly decreased as detected by MTT and flow cytometry, with three-color labeled by PE-CD34+/ FITC-AnnexinV /7AAD. The survival and proliferation of mouse bone marrow MNC treated with MH after irradiation were also increased. The MH exerted similar protective effects on the leukemia cell lines A20, HL60, K562 to the normal bone marrow cells, but it enhanced the radiation sensitivity of leukemia cell line FBL3 and mouse melanoma B16F10. No effects have been found on the radiation sensitivity of those cells treated with MH before irradiation. The results also show     

Bone marrow-derived T lymphocytes responsible for allograft rejection

Lethally irradiated mice reconstituted with syngeneic bone marrow cells were grafted with allogeneic skin grafts 6-7 weeks after irradiation and reconstitution. Mice with intact thymuses rejected the grafts whereas the mice thymectomized before irradiation and reconstitution did not. Thymectomized irradiated mice (TIR mice) reconstituted with bone marrow cells from donors immune to the allografts rejected the grafts. Bone marrow cells from immunized donors, pretreated with Thy 1.2 antibody and C', did not confer immunity to TIR recipients. To determine the number of T lymphocytes necessary for the transfer of immunity by bone marrow cells from immunized donors, thymectomized irradiated mice were reconstituted with nonimmune bone marrow cells treated with Thy 1.2 antibody and C' and with various numbers of splenic T lymphocytes from nonimmune and immune donors. Allogeneic skin graft rejection was obtained with 10(6) nonimmune or 10(4) immune T cells. The effect of immune T cells was specific: i.e., immune T cells accelerated only rejection of the relevant skin grafts whereas against a third-party skin grafts acted as normal T lymphocytes.     

Isolation and characterization of highly radioresistant malignant hamster fibroblasts that survive acute gamma irradiation with 20 Gy

To study the acquired radioresistance of tumor cells, a model system of two cell lines, Djungarian hamster fibroblasts (DH-TK-) and their radioresistant progeny, was established. The progeny of irradiated cells were isolated by treating the parental cell monolayer with a single dose of 20 Gy (PIC-20). The genetic and morphological features, clonogenic ability, radiosensitivity, cell growth kinetics, ability to grow in methylcellulose, and tumorigenicity of these cell lines were compared. The plating efficiency of PIC-20 cells exceeded that of DH-TK- cells. The progeny of irradiated cells were more radioresistant than parental cells. The average D0 for PIC-20 cells was 7.4 +/- 0.2 Gy, which is three times higher than that for parental cells (2.5 +/- 0.1 Gy). Progeny cell survival in methylcellulose after irradiation with a dose of 10 Gy was 15 times higher than that of DH-TK- cells. In contrast to parental cells, the progeny of irradiated cells showed fast and effective repopulation after irradiation with doses of 12.5 and 15 Gy. The tumor formation ability of irradiated progeny cells was higher than that of parental cells; after 15 Gy irradiation, PIC-20 cells produced tumors as large as unirradiated progeny of irradiated cells, whereas the tumor development of DH-TK- cells diminished by 70%. High radioresistance of progeny of irradiated cells was reproduced during the long period of cultivation (more than 80 passages). The stability of the radioresistant phenotype of PIC-20 cells allows us to investigate the possible mechanisms of acquired tumor radioresistance.     

Radiosensitivity of pluripotent stem cells detectable by cloning in the spleen of irradiated mice

It was shown that the dose--effect curves describing the radiosensitivity of CFUc of the bone marrow irradiated in vitro (0.04-3.7 Gy) and treated with normal rabbit serum (NRS) and anti-mouse-brain serum (AMBS) has two differently sloping portions indicating that two CFUc populations differing in radiosensitivity are present in the bone marrow. D0 was 0.93 Gy after irradiation with doses of 0.04-0.75 Gy and treatment with NRS, and 0.33 Gy after incubation of the bone marrow with AMBS. The addition of thymus cells "straightened" the dose--effect curve for the bone marrow treated with AMBS: in this case D0 was 1.81 Gy exceeding considerably the values of D0 for intact bone marrow. The CFUc population is suggested to be heterogeneous in radiosensitivity.     

载氢-纳米氧化铈微泡对小鼠造血系统抗辐射作用的分析

为探讨载氢-纳米氧化铈微泡对小鼠辐射损伤的防护作用。本研究检测载氢-纳米氧化铈微泡的表征,并将60只BALB/c小鼠随机分为正常对照组、照射对照组、载氢-纳米氧化铈微泡组。小鼠经6Gy x射线一次性全身照射(剂量率2 Gy/min)。于照射后3 d和8 d处死小鼠,检测其外周血细胞数、脾脏和胸腺指数、骨髓和脾脏组织病理学变化。结果显示,照射后3 d和8 d,与正常对照组相比,载氢-纳米氧化铈微泡组和照射对照组的白细胞均明显下降,相比照射对照组,载氢-纳米氧化铈微泡组有改善(p<0.05或p<0.01);而载氢-纳米氧化铈微泡组和照射对照组的红细胞数和血红蛋白均略有下降,但差异无统计学意义。与正常对照组相比,微泡组的胸腺指数、脾脏指数均有下降,和照射对照组相比,载氢-纳米氧化铈微泡组的胸腺指数明显改善(p<0.05或p<0.01)。照射后3 d,与正常对照组相比,照射对照组的骨髓细胞较少,存在细胞碎片,载氢-纳米氧化铈微泡组骨髓细胞数量略有减少,存在细胞核松散现象。而照射后8 d,与正常对照组相比,照射对照组的骨髓细胞几乎找不到,载氢-纳米氧化铈微泡组骨髓细胞有一定数量,存在细胞凋亡现象。本研究表明,载氢-纳米氧化铈微泡通过保护造血组织、改善造血功能,对机体起到一定的辐射防护作用。