Biomedical subjects
Publications and source records attributed to V Svoboda.
The influence of an initial salt concentration, phi(0), on the gradient separation of proteins using hydrophobic interaction chromatography on Separon HEMA 1000 was investigated. The results obtained were compared with the retention times and peak widths calculated according to a mathematical model.
Mitotic index of the bone marrow cells was studied in femoral bone marrow of mice given 313 kBq 239Pu kg-1. The attention was turned to the femoral midshaft and the mitosis concentration, intensified by Colcemid stathmokinetic effect, was evaluated in a sampling field from endosteal surface to the central venous canal, throughout 68 weeks. It has been found that the plutonium effect in the sampling band is rather uniform except the points in subendosteal zone early after plutonium injection, where the mitotic index was reduced in such a way that the mitotic gradient, observed in controls, was affected. The mitotic activity in femoral diaphysis of plutonium injected mice was mobilized approximately till the 30th week of contamination. Later it deteriorated progressively. The results are discussed and should not be regarded as representative for the entire bone marrow hemopoiesis.
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The preparative separation of an enzymatic hydrolysate by a step gradient of a lithium-sodium citrate buffer was studied. Using a two-column arrangement, 10 GBq of 14C-labelled enzymatic hydrolysate or 250 mg of inactive hydrolysate were separated within 36 h. After the chromatographic separation, the fractions of amino acids were isolated from the citrate eluents on weak cation exchangers with an ethylene glycol-poly(methyl methacrylate) matrix. The use of volatile organic acids as an eluent was possible due to the weak interactions with the cation matrix.
The effects of long-term internal contamination with 13.3 kBq kg-1 239Pu injected intravenously were studied in 10-week-old ICR (SPF) female mice. Radiosensitivity of spleen colony-forming units (CFU-S) and 125IUdR incorporating into proliferating cells of vertebral bone marrow and spleens were determined in plutonium-treated and control animals one year after nuclide injection. The CFU-S in 239Pu-treated mice were more sensitive to X-rays (D0 = 0.52 +/- 0.01 Gy) than in controls (D0 = 0.84 +/- 0.02 Gy). 125IUdR incorporation into bone marrow and spleen cells was reduced after plutonium contamination. At one year following plutonium injection, the occurrence of chromosome aberrations was evaluated in metaphase figures of femoral bone marrow cells. The frequency of aberrations increased early after plutonium treatment, at later intervals it tended to decrease but not below the control level. While the relative numbers of vertebral marrow CFU-S decreased significantly, but only to 86 per cent of normal, cellularity of vertebral bone marrow, peripheral blood counts and survival of 239Pu-treated mice did not differ from the control data.
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The self-renewal capacity of murine pluripotent hemopoietic stem cells (CFU-S) of vertebral bone marrow was studied under conditions of short-term and long-term internal contamination with 239Pu or 241Am in female mice. Measurement of the CFU-S self-renewal capacity was carried out using double transplantation assay. To evaluate the production of differentiated progeny of stem cells average erythroblast numbers/visible spleen colony and 59Fe-uptake/colony were computed. The marrow cellularity/vertebra and the number of CFU-S/vertebra were decreased and affected more by 239Pu than by 241Am. The production of erythroblasts per a single CFU-S and the 59Fe-uptake/colony were reduced, similarly the numbers of secondary spleen colonies and of secondary CFU-S in primary colonies. The above changes resulting from impaired functions of surviving CFU-S were more serious with 241Am than with 239Pu. The biological effects of plutonium and americium appeared independent of the phase of contamination.
In the EORTC Radiotherapy Group, the feasibility of multiple fractions per day (MFD) was tested in a pilot study from 1978 to 1980. Three daily fractions of 1.6 Gy were given (4 h interval) during 2 weeks (total dose 48 Gy), with a boost to about 70 Gy after 3-4 weeks. In 53 of the 179 patients, misonidazole was given on every irradiation day (1 g/m2, total 13-14 g/m2). Data on tolerance and early treatment results were published previously; results with a minimum follow-up period of 3 years are now available. Survival (actuarial is 21% and locoregional tumor control was obtained in 34% of patients; no significant differences were seen between the subregions in the head and neck area. Survival is better in patients treated with misonidazole (probably due to selection), but locoregional control was identical as in patients treated without the sensitizer. In February 1984, 38 patients were alive, 35 without evidence of local tumor (5 after rescue surgery). Metastases were seen in 16% and a second tumor in 7% of patients. Seventeen patients (9%) died of causes, possibly related to treatment; necrosis was observed in 8 (4 with local tumor). Late effects in the long survivors were comparable to what is usually seen after high-dose radiotherapy.
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The radiosensitivity of pluripotent hemopoietic stem cells was studied in ICR "Swiss" mice (28 g/mouse) given i.v. 198.6 kBq 239Pu/kg as citrate complex or 208.6 kBq 241 Am/kg as nitrate at the age of 10 weeks. The bone marrow cells were examined at the early and late phases of radionuclide contamination. To obtain data for survival curves and D0 of stem cells the CFU-S assay was used and the donor vertebral marrow cells were exposed to the complementary X-irradiation either early after injection to the heavily irradiated recipients or to the "in vitro" irradiation given before the transplantation. To determine the iron uptake in splenic erythroid progeny the recipients given marrow cells unexposed to the X-rays received 37 kBq 59Fe 6 h before they were killed and the relative activity per colony was calculated. The radiation effect of the used actinides on the bone marrow cells resulted in decreased cellularity and seriously altered both relative and absolute CFU-S numbers. The radiosensitivity of CFU-S increased in all intervals examined (D0 from 0.60 to 0.86 Gy, in controls 0.97 to 1.06 Gy) and was more expressed when the CFU-S were exposed to the X-rays immediately after the bone marrow cell transplantation to the heavily irradiated hosts. The stem cell pool appeared, especially at older age, to be affected also in its ability to produce erythrocytic progeny.
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Expression of Thy-1.2 specificities in cells from 29 primary spontaneous leukemias of random-bred ICR Swiss mice was examined by cell membrane and cytoplasmic immunofluorescence with monoclonal HO-13-4 antibody [1]. The Thy-1.2 epitopes were detected in all thymic lymphomas and were absent in the lymphomas of non-thymic origin. Unexpectedly, the Thy-1.2 epitopes were also detected in 71% (5/7) of myeloid leukemias and 40% (4/10) of reticulum cell sarcomas examined.
Survival, radiosensitivity and capability to produce differentiated progeny were followed in CFU-S from lumbar vertebrae of mice injected with 198.6 kBq 239Pu/kg or 208.6 kBq 241Am/kg. The CFU-S assay and 59Fe uptake into spleen colonies were used. The number of CFU-S from treated mice was significantly lower than in controls. Higher radiosensitivity of CFU-S from 239Pu- or 241Am-treated mice was demonstrated using additional exposure to 0.5 Gy X-rays 1, 24, 48, 72 hrs after cell transplantation and expressed more precisely by survival curves obtained 1 hr after the marrow cell injection. The effect of 239Pu on CFU-S was characterized by Do 0.58 Gy (n = 0.91) and that of 241Am by Do 0.64 Gy (n = 0.91); corresponding control values were Do 0.89 Gy, n = 1.11. Lower iron utilization due not only to the decreased CFU-S numbers, but also to the defective production of erythroid cells per one CFU-S was found. Complexity of radiation effect on hemopoietic stem cells was demonstrated by the present study.
From 1978 to the end of 1980, 179 patients with advanced head and neck tumors were accrued in a multicenter pilot study of the EORTC Radiotherapy Group, investigating the feasibility of high dose multiple daily fractionation (MDF) and its combination with misonidazole. The irradiation scheme consisted of three daily fractions of 1.6 Gy (four hour intervals) to a total dose of 48 Gy in two weeks, followed 3 to 4 weeks later by a boost to a total of about 70 Gy in 6 to 7 weeks. Misonidazole was given in daily doses of 1 g/m2 (total 13 or 14 g/m2) to 53 patients, thus sensitizing every radiation session. All patients had large head and neck tumors, with a poor prognosis. Acute reactions were well tolerated. Skin reactions were very moderate; mucosal reactions started at day 10 to 12. All patients had a confluent mucositis that lasted for one to two weeks. When the whole oral cavity was irradiated, reactions lasted somewhat longer. The boost caused no significant symptoms. The radiosensitizer did not modify the reaction pattern. Tumor regression was very impressive, so that palliation was obtained quickly. Nine patients died from treatment related causes. It is difficult to assess local control at this time, but at the time of analysis (August 1981), the actuarial control rate was 48% at 20 months, with misonidazole 57%. This difference, however, is not statistically significant. Survival of the total group is 31% at 20 months. In these patients with a heavy tumor burden the early results were considered a success by all participants. For patients with sufficient follow-up, late reactions can be evaluated. Some edema and fibrosis is seen, but did not exceed a degree which could be expected with single daily fractionation to the same dose. This study demonstrates the possibility of giving highly concentrated treatments to total doses equal to those used in conventional fractionation. For assessment of a possible benefit in local control and ultimate survival, a randomized study is necessary. Such a study is now underway in the EORTC Radiotherapy Group, comparing single daily radiotherapy with multiple daily fractionation with or without misonidazole.
Some results of long-term experiments performed in mice contaminated with 226Ra and 239Pu, especially the damage to the hemopoietic system and the incidence of osteosarcoma, are reported. Different degree of damage to the bone marrow hemopoiesis, depending on the arrangement of marrow cavities and the capability of stem cells to produce differentiated progeny are evaluated. This ability decreases with age and with duration of the contamination due to radionuclides. From the above aspects of radiation injury the ability of hemopoiesis to compensate the decreased production of differentiated peripheral cell forms is discussed. It is remarkable that myeloid leukemia occurred earlier and more frequently in 239Pu-contaminated animals than in controls. After the injection of plutonium-239, myeloid and lymphatic leukemias were diagnosed in most of the animals dying without osteosarcoma. These diseases represented a danger not lower than that of osteosarcoma which appeared in average later and the expected incidence of which was estimated to rise linearly with age. The findings are discussed from the aspect of their application in evaluating the risk resulting for man from the incorporated bone-seeking alpha emitters.