Wilhelm Nothdurft

Dose- and time-related quantitative and qualitative alterations in the granulocyte/macrophage progenitor cell (GM-CFC) compartment of dogs after total-body irradiation

The effects of single-dose total-body X irradiation (TBI) on the granulocyte/macrophage progenitor cell (GM-CFC) population in bone marrow and blood of dogs were studied for dose levels of 0.78 and 1.57 Gy up to 164 days after irradiation. The blood GM-CFC concentration per milliliter was depressed in the first 7 days in a dose-dependent fashion to 5-16% of normal after 0.78 Gy and to between 0.7 and 5% after 1.57 Gy. The bone marrow GM-CFC concentration per 10(5) mononuclear cells, on the other hand, was initially reduced to about 45% of the average pre-irradiation value after 0.78 Gy and to 23% after 1.57 Gy. The regeneration within the first 30 to 40 days after TBI of the blood granulocyte values and the repopulation of the bone marrow GM-CFC compartment was associated with both a dose-dependent increase in the S-phase fraction of the bone marrow GM-CFC and a dose-dependent increase in colony-stimulating activity (CSA) in the serum. The slow repopulation of circulating blood GM-CFC to about only 50% of normal even between days 157 and 164 after TBI could be related to a correspondingly delayed reconstitution of the mobilizable GM-CFC subpopulation in the bone marrow.

In Vitro Studies on the Sensitivity of Canine Granulopoietic Progenitor Cells (GM-CFC) to Ionizing Radiation: Differences between Steady State GM-CFC from Blood and Bone Marrow

The radiosensitivity of the granulopoietic progenitor cells (GM-CFC) from blood and bone marrow of dogs under steady state conditions was studied by in vitro irradiation with 280kV X-rays (approximately 0.56 Gy/min). The dose-effect relationship for colony formation was determined for the dose range from 0 to 3 Gy by means of three different models. A simple exponential function revealed an optimal approximation to the experimental data obtained for the clonogenic cells from the two different sources. The D0 values are 0.261 +/- 0.009 Gy and 0.600 +/- 0.011 Gy for the GM-CFC from blood and bone marrow, respectively. Irradiation of blood-derived GM-CFC in the presence of pre-irradiated bone marrow cells or irradiation of bone marrow cells as a mixture with pre-irradiated blood cells led to small changes only in the survival curves. According to the dose-effect relationship obtained from these studies the GM-CFC of the dog seem to be the most radiosensitive clonogenic haemopoietic cells among the different mammals.

Cell cycle and growth response of CHO cells to X-irradiation: threshold-free repair at low doses.

PURPOSE To test the hypothesis of a threshold for induced repair of DNA damage (IR) and, secondarily, of hyperradiosensitivity (HRS) to low-dose X-irradiation. METHODS AND MATERIALS Exponentially growing Chinese hamster ovary cells (CHO) were X-irradiated with doses from 0.2 to 8 Gy. Survival data were established by conventional colony-forming assay and flow-cytometric population counting. The early cell cycle response to radiation was studied based on DNA-profiles and bromodeoxyuridine pulse-labeling experiments. RESULTS Colony-forming data were consistent with HRS. However, these data were of low statistic significance. Population counting provided highly reproducible survival curves that were in perfect accord with the linear-quadratic (LQ) model. The dominant cell cycle reaction was a dose-dependent delay of G2 M and late S-phase. CONCLUSION There was no evidence for a threshold of IR and for low-dose HRS in X-irradiated CHO cells. It is suggested that DNA damage repair activity is constitutively expressed during S-phase and is additionally induced in a dose-dependent and threshold-free manner in late S-phase and G2. The resulting survival is precisely described by the LQ model.

Haematological Effects of RhGM-CSF in Dogs Exposed to Total-body Irradiation with a Dose of 2·4 Gy

It was the specific aim of this study to test the stimulatory effects of recombinant human GM-CSF (rhGM-CSF) on haemopoietic regeneration in dogs which had received total-body irradiation (TBI) with a dose of 2.4 Gy. In normal dogs rhGM-CSF given subcutaneously at 10 microgram/kg per day or 30 microgram/kg per day for 21 days caused strong but transient increases in the peripheral blood neutrophils. The monocyte counts also showed a transient rise during treatment in a dose-dependent fashion, whereas the lymphocyte counts increased only at the higher dose of rhGM-CSF and the platelet counts were transiently depressed during the course of the treatment. In the irradiated animals treatment with rhGM-CSF decreased the severity and shortened the duration of neutropenia but had no significant influence on monocyte or lymphocyte recovery. The granulocyte values showed a characteristic pattern of fluctuations with the first peak occurring at the same time (day 10 to day 13) when the abortive rise was observed in the untreated dogs. In contrast the GM-CFC in the peripheral blood remained depressed during the whole treatment course, similar to the untreated irradiated controls. These results indicate that treatment with GM-CSF can be an effective biological monotherapy for radiation-induced bone marrow failure, but that for higher radiation doses the number of GM-CSF responsive target cells will become a critical determinant of therapeutic efficacy.

THE DEVELOPMENT OF RADIATION LATE EFFECTS TO THE BONE-MARROW AFTER SINGLE AND CHRONIC EXPOSURE

The marrow is a tissue distributed in numerous skeletal parts and works as an organ which is composed of a haemopoietic cell parenchyma and a supporting stroma. The pathophysiological mechanisms involved in the radiation-induced late effects depend mainly on the damage produced to each of these elements. Parenchymal cell damage ends with a failure of the stem cell pool to supply an adequate number of highly differentiated functional blood cells and is clinically manifested as aplastic anaemia or leukaemia. The effects of radiation on the haemopoietic stem cell can be measured by means of spleen colony forming units (CFU-S) in rodents. The self-maintaining capacity of the CFU-S was found to be lower than normal 16 weeks after a dose of 0.64 Gy. In larger animals it is only possible to measure the activity of some of the progenitor cells, estimating the number of granulocyte-macrophage colonies in culture (CFU-GM) as an indicator of stem cell changes. Their number in the blood is about 50 per cent of normal even 160 days after about 0.78 Gy. The stromal cells are also radiosensitive if measured with respect to their capacity to support long-term cell replication in vitro. Marrow fibrosis develops after single, repeated and chronic radiation exposure, and a dose of 40 Gy impairs the capacity of the marrow to support haemopoiesis.

Acceleration of hemopoietic recovery in dogs after extended-field partial-body irradiation by treatment with colony-stimulating factors: rhG-CSF and rhGM-CSF

PURPOSE The influence of treatment with the two colony-stimulating factors, rhG-CSF and rhGM-CSF, on the hemopoietic recovery in aplastic bone marrow sites after extended-field irradiation was studied in a canine model. METHODS AND MATERIALS The dogs received irradiation of the cranial part of their body with a single dose of 11.7 Gy, comprising approximately 72% of the total bone marrow mass. Anatomically this type of exposure corresponds to upper body irradiation (UBI) as employed under clinical conditions. Treatment with both the CSFs was employed for 7 days by daily injections of 30 microg/kg, starting 24 hr after irradiation. RESULTS Treatment with rhGM-CSF did not completely prevent the initial decrease of the granulocyte counts, but caused an accelerated, though incomplete, recovery in the period from day 5 to day 15. In contrast, treatment with rhG-CSF caused two phases of granulocytosis and an early recovery to normal levels at day 11 after irradiation. Treatment with rhG-CSF, but not with rhGM-CSF, was associated with a strong supra-normal increase of progenitor cells in the blood within the first 8 days and an accelerated hemopoietic recovery in the irradiated sites particularly within the first 7 days after the exposure. CONCLUSIONS These results indicate that under conditions of partial-body irradiation short term treatment with G-CSF is superior to GM-CSF in initiating the hemopoietic recovery on the basis of endogenous stem cell seeding.

Short-Term Effects of Early-Acting and Multilineage HematoPoietic Growth Factors on the Repair and Proliferation of Irradiated Pure Cord Blood (CB) CD34 Hematopoietic Progenitor Cells

PURPOSE Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. METHODS AND MATERIALS We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34+ cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34+ cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose-response curves were fitted to the data. RESULTS The radiobiological parameters (D[0] and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34+ cells compared to optimal two-factor combinations. The D[0] values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56-1.15, 0.41-2.24, and 0.56-1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n=1 for all tested GF(s). DNA repair capacity of CD34+ cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34+ cells was similar. CONCLUSION Our data indicate that increased survival of irradiated CB CD34+ cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.

Acute and long-term alterations in the granulocyte/macrophage progenitor cell (GM-CFC) compartment of dogs after partial-body irradiation: irradiation of the upper body with a single myeloablative dose

The acute and long-term effects of a single dose of partial-body irradiation on the granulocyte/macrophage progenitor cell compartment were studied in dogs. A myeloablative dose of 11.7 Gy (dose rate 6.5 cGy/min) was given to the upper body which contains approximately 70% of the total bone marrow mass. The lower part of the body (pelvis, lower extremities and tail) was shielded by a lead box. In the non-irradiated bone marrow, the concentration of the GM-CFC/10(5) mononuclear cells was slightly decreased within the first 7 days and showed some fluctuations around the normal value for several weeks thereafter. In the irradiated bone marrow, virtually no GM-CFC could be detected on day 1 after exposure. Beginning on day 7, a continuous increase took place up to day 21 when the GM-CFC concentration reached between 25% (sternum) and 43% (humerus) of the initial value. No further increase took place up to day 80. Between day 120 and 380 a secondary increase was observed which reached near-normal bone marrow GM-CFC concentrations. The blood GM-CFC concentration first showed a strong depression followed by a transient increase between day 10 and 30. This coincided with GM-CFC normalization in the protected bone marrow as well as with the initial phase of regeneration in the irradiated sites. A prolonged secondary long-lasting depression between day 33 and 120 amounted to 20 to 50% of normal values. This depression was closely related to the stagnation in the GM-CFC recovery in the irradiated bone marrow sites. The GM-CFC concentration in the blood was found to be supranormal at day 380 when the bone marrow GM-CFC had recovered. The colony stimulating activity in the serum showed an increase within the first 20 days after exposure, that is, within the same interval the bone marrow GM-CFC concentration experienced the strongest alterations, and was inversely related to the changes in the blood granulocyte values.

The role of dextran sulfate in increasing the CFUc-concentration in dog blood.

Summary Intravenous administration of 10 mg or 15 mg dextran sulfate per kg body weight into dogs results in a marked increase of CFUc in the blood within 3 hr and, to a considerably lesser extent, of mononuclear leukocytes (MNC). The increase after the higher dose is more than tenfold, from less than 200 CFUc/ml blood to about 2000 CFUc/ml, within 3-5 hr; the CFUc gradually decrease thereafter to control levels several hours later. On the other hand, the MNC population experiences a two- to threefold increase which is maintained for several hours. The increase in CFUc after 10 mg/kg is less than after 15 mg/kg, indicating dose-dependence. There is also a cumulative increase observed in the CFUc concentration in blood after repeated injections of dextran sulfate; there is no significant alteration in PMN and MNC levels and there is a slight decrease in the hematocrit and in the thrombocyte level. The mechanism responsible for this phenomenon has not yet been clarified. Due to the probable action of polyanions on cell membranes affecting their adherence capabilities, one may consider both an increased rate of release of CFUc from a marginal or extravascular pool into the blood, as well as a prolongation of the CFUc blood transit time as being contributing factors to the observed blood CFUc increase.

Radioprotective effect of N-acetylcysteine on granulocyte/macrophage colony-forming cells of human bone marrow

N-Acetylcysteine, known as a radical scavenger, was examined for its influence on the radiotolerance of progenitor cells of granulocytopoiesis. Added before and after irradiation in a dose of 2 mg/ml to suspension cultures of non-adherent low-density human bone marrow cellsN-acetylcysteine (AcCys) clearly improved the survival. TheD0 value of the survival curve for granulocyte/macrophage colony-forming cells increased by a factor of 1.56 as compared to non-treated control suspensions. The improvement of radiation tolerance is probably not only based on the radical scavenger properties (radioprotective component) of AcCys, but also on the support of repair processes.