Proliferative activity of Shvetz tumor following single and repeated irradiation]

Autoradiographic study of an experimentally-induced tumour following local irradiation in a dose of 600 rad showed no retardation of the cell cycle 6 to 12 hours after the irradiation. Marked reduction of the mitotic index (MI) and of the labeled nuclei index (LNI) was noted to the 96th hour after the irradiation. In repeated irradiation in a dose of 1200 rad at an interval of 18 hours there was revealed a marked reduction of the MI and of the LNI as a result of the block of the passage of cells from the G1-period into S. However, restoration of the cell proliferation uas noted by the 24th-48th hours. A high MI revealed at all the periods of investigation after repeated tumour irradiation at an interval of 24 hours was possibly caused by an increase in the time of mitosis proper, this also being confirmed by a significant accumulation of the number of late mitotic phases.     

X-irradiation of equine peripheral blood lymphocytes stimulated with phytohaemagglutinin in vitro.

Small lymphocytes were isolated from the peripheral blood of horses and incubated at 37 degrees C in Eagle's medium supplemented with 20 per cent foetal calf serum. The addition of phytohaemagglutinin (PHA) to the cultures resulted in: increased RNA and protein synthesis; the enlargement of the small lymphocyte into a lymphoblast-like cell; the initiation of DNA synthesis, and cell division. When survival was measured 24 hours after X-irradiation by means of phase-contrast microscopy, the lymphoblast-like cell was much more radio-resistant (D0 = 250 rad) than the small lymphocyte (D0 = 20 rad). This increase in radioresistance, however, was not observed until 12-24 hours after PHA treatment. To investigate which of the changes occurring during the transformation of the small lymphocyte was responsible for the increased resistance to irradiation, the percentage of cells surviving irradiation was compared with the percentage of cells incorporating significant amounts of 3HTdR, 3H-UR, or 3H-leucine at the time of irradiation. For this comparison, a dose of 100 rad was used because 100 rad killed essentially all of the small lymphocytes, but less than 35 percent of the cells which had become radioresistant from the PHA treatment. The results indicated that the increase in radioresistance was not associated with DNA synthesis, but instead correlated with the increase in RNA and protein synthesis which the cells had attained at the time of irradiation.     

Effects of gamma irradiation on histomorphology of some endocrine glands of the rain quail, Coturnix coromandelica (Gmelin)

Effects of single, whole-body 60Co-gamma irradiation in different doses (250 rad to 15 k rad) on histology of thyroid, adrenal and pancreatic islets of the rain quail were studied. A low dose of 250 rad failed to evoke any change in histology of the glands studied. Doses of 500 rad and 1 k rad resulted in hypoactivity of thyroid but could not affect adrenal and pancreatic islets. Exposure to 1.5 k rad and higher doses caused hyperplasia and hypertrophy of thyroid and hypertrophy of adrenal gland. Thyroid was injured by heavy irradiation. Doses up to 3 k rad did not bring about any change in islet cells, however, higher doses resulted in degenerative changes in islet cells. alpha-islets were affected by 7 and 15 k rad but necrotic changes in beta-islets were observed only after exposure to 15 k rad. The results indicate that thyroid is the most sensitive and pancreatic islet, highly resistant to gamma radiation.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0–30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.     

Dose-Dependency of radiation on enzyme production inTrichoderma reesei

Effect of irradiation dose on the production of cellulase and amylase related enzymes inTrichoderma reesei was studied, in which post-irradiation time responce pattern was measured. The damage of the cells irradiated with certain irradiation doses (1.40±0.20x10⁵, 2.20±0.10x10⁵, 3.00±0.50 x 10 and 3.50±0.20 x 10 rad) was rapidly recovered. The increased enzyme production in the culture of the irradiated cells resulted from the recovery of radiation damage after irradiation. The function of cell growth was not affected by irradiation below dose of 5 x 10⁵ rad, though the function of enzyme synthesis was drastically affected.     

Mitotic viability and metabolic competence in UV-irradiated yeast cells

Colony formation is the classic method for measuring survival of yeast cells. This method measures mitotic viability and can underestimate the fraction of cells capable of carrying out other DNA processing events. Here, we report an alternative method, based on cell metabolism, to determine the fraction of surviving cells after ultraviolet (UV) irradiation. The reduction of 2,3,5-triphenyl tetrazolium chloride (or TTC) to formazan in mitochondria was compared with cell colony formation and DNA repair capacity in wt cells and two repair-deficient strains (rad1Delta and rad7Delta). Both TTC reduction and cell colony formation gave a linear response with different ratios of mitotically viable cells and heat-inactivated cells. However, monitoring the formation of formazan in non-dividing yeast cells that are partially (rad7Delta) or totally (wt) proficient at DNA repair is a more accurate measure of cell survival after UV irradiation. Before repair of UV photoproducts (cis-syn cyclobutane pyrimidine dimers or CPDs) is complete, these two assays give very different results, implying that many damaged cells are metabolically competent but cannot replicate. For example, only 25% of the rad7Delta cells are mitotically viable after a UV dose of 12 J/m(2)75% of these cells are metabolically competent and remove over 55% of the CPDs from their genomic DNA. Moreover, repair of CPDs in wt cells dramatically decreases after the first few hours of liquid holding (L.H.; incubation in water) and correlates with a substantial decrease in cell metabolism over the same time period. In contrast, cell colony formation may be the more accurate indicator of cell survival after UV irradiation of rad1Delta cells (i.e., cells with little DNA repair activity). These results indicate that the metabolic competence of UV-irradiated, non-dividing yeast cells is a much better indicator of cell survival than mitotic viability in partially (or totally) repair proficient yeast cultures.     

Qualitative study of chromosomal lesions induced by neutrons and neon ions in human lymphocytes at G0 phase

Chromosomal lesions, mitotic index and cell cycle progression delay induced by neutron (protons 34 MeV on beryllium) and neon (250 MeV/i) particles are studied in human lymphocytes. The cell cycle progression is slightly decreased at a dose of 1 Gy. Mitotic indexes are significantly decreased after irradiation by the different particles, except neon in 52-h cultures. By comparison to chromosome damages caused by gamma-rays, previously published, it is found that the lesions observed here are frequently more complex: the number of breaks is higher per abnormal mitosis and higher per rearrangement on the average. This complexity is higher for neon ions than for neutron beams.     

Cell Proliferation and Ageing In Mouse Colon

The descending colon of 4 month and 2 year old mice was exposed to 1250 rad X-rays. This killed most of the epithelial cells. the surviving cells formed new crypts and surface epithelium in animals of both ages. Not all the crypts were replaced. the irradiated area contained no more than 80% of the control number of crypts per section for at least 6 weeks after irradiation. In the young mice new crypts were much larger and the labelling index (LI) was much higher than in unirradiated animals during the first week after irradiation. In the old mice the overshoot in LI and crypt size began later and continued longer than in young animals. This may be because the control of cell proliferation was much less precise in old than in young mice. The irradiation was repeated, in an attempt to age prematurely the epithelial cells by increasing the number of divisions they underwent. the overshoot in LI and cells per crypt was smaller after a second dose than after the first in both young and old mice. There was almost no overshoot after a third dose was given to young mice. Increasing the number of divisions undergone by the surviving epithelial cells did not change the timing of repopulation in young mice compared to that found in old mice. Little evidence was found for the presence of a limited proliferative lifespan in colon epithelial cells.     

Evaluation of cell regeneration of bone marrow after fractional irradiation of the mouse in toto]

We have studied the recovery for mice bone marrow cells after fractionated irradiation of the whole body. The additional dose (Dr) to obtain a given biological effect if the irradiation is split in two equal subfractions (2 Di) separated by a short interval of time (i) is 40 rad per day when the interval of time between the two irradiations is lengthened of one day.     

Cryopreservation of embryonic stem cell‐derived multicellular neural aggregates labeled with micron‐sized particles of iron oxide for magnetic resonance imaging

Magnetic resonance imaging (MRI) provides an effective approach to track labeled pluripotent stem cell (PSC)‐derived neural progenitor cells (NPCs) for neurological disorder treatments after cell labeling with a contrast agent, such as an iron oxide derivative. Cryopreservation of pre‐labeled neural cells, especially in three‐dimensional (3D) structure, can provide a uniform cell population and preserve the stem cell niche for the subsequent applications. In this study, the effects of cryopreservation on PSC‐derived multicellular NPC aggregates labeled with micron‐sized particles of iron oxide (MPIO) were investigated. These NPC aggregates were labeled prior to cryopreservation because labeling thawed cells can be limited by inefficient intracellular uptake, variations in labeling efficiency, and increased culture time before use, minimizing their translation to clinical settings. The results indicated that intracellular MPIO incorporation was retained after cryopreservation (70–80% labeling efficiency), and MPIO labeling had little adverse effects on cell recovery, proliferation, cytotoxicity and neural lineage commitment post‐cryopreservation. MRI analysis showed comparable detectability for the MPIO‐labeled cells before and after cryopreservation indicated by T₂ and T₂* relaxation rates. Cryopreserving MPIO‐labeled 3D multicellular NPC aggregates can be applied in in vivo cell tracking studies and lead to more rapid translation from preservation to clinical implementation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:510–521, 2015     

Skin response to X-irradiation in the guinea-pig.

Skin reaction to X-irradiation has been studied in the albino guinea-pig; early response in limited-field irradiations of the flank is comparable to that commonly seen in rodents, swine and man, and is dose-dependent with a dynamic range from mild erythema to moist desquamation. The peak early skin reaction is seen between 14 and 21 days after irradiation, and declines before 30 days except at the highest doses used. Fractionation of the X-ray dose at 24 hours results in a 'sparing' of about 340 rad. Permanent partial epilation is detectable at doses in excess of 1400 rad, and complete epilation at 1 year occurs in 50 per cent of irradiated fields at 1740 rad. Twenty-four hour two-dose fractionation results in a 'sparing' of about 500 rad for epilation. Palpable dermal 'fibrosis' is detectable at 3 months after irradiation in fields given more than 2070 rad, and at 1 year after irradiation in fields given more than 1800 rad; 50 per cent of fields showed palpable 'fibrosis' at 1 year at 1930 rad. Unlike domestic swine and man, skin fields in the guinea-pig showed no dimensional contraction after X-ray doses which produced gross early skin damage.     

Repair of potentially lethal damage in yeast induced by fast neutrons

Survival curves of 3 diploid (D7) yeast strains: one wild-type, one deficient in excision of pyrimidine dimers (UV-sensitive) and one blocked in DNA double-strand-break repair (X-ray-sensitive), were compared after irradiation with cyclotron-produced fast neutrons. It was observed that both the UV-sensitive (rad3/rad3) and the X-ray-sensitive (rad52/rad52) mutants were more sensitive to neutrons than the wild-type. The role of DNA double-strand-breaks in neutron-induced cell death was further studied by comparing the relative sensitivity of the rad52/rad52 mutant to gamma-rays and fast neutrons. A comparison of the dose modification factors revealed that the deficiency in DNA double-strand-break repair did not make the yeast cells more sensitive to neutrons than to photons, which suggests that lesions of a different type may also be produced by neutrons. Survival curves obtained upon immediate plating and after delayed plating of neutron-irradiated cells showed that all 3 yeast strains were efficient in liquid holding recovery. The role of different repair pathways in cellular recovery from neutron-induced lethal damage is discussed.     

QUANTITATIVE CHANGES IN THE CELLULARITY OF THE RAT SUBEPENDYMAL PLATE AFTER X-IRRADIATION

The brains of young adult rats were irradiated with a single dose of 8 Gy (800 rad) of 250 kVp X-rays. Within 2 weeks of treatment the cell population of the subependymal plate was reduced by 30%. During this period the cell cycle time remained unchanged but the labelling index was reduced. The cell population subsequently returned to normal after 39-52 weeks. Damage and subsequent recovery of the plate was due mainly to changes in the number of cells with small dark nuclei. Cells with small and large light nuclei were little affected. A model for the production and differentiation of cells in the subependymal region is proposed on the basis of age-related changes in the total number and proportions of the various cell types in the subependymal plate of normal rats. This is discussed both in terms of the radiation response of cells in the plate and the manifestation of delayed white matter necrosis after higher doses.     

The effect of irradiation on the antibody enhancing helper T cells

Helper T cells specific for sheep erythrocytes were generated in lethally irradiated mice. The helper effect exerted by these cells was determined in vitro in spleen cell cultures. Irradiation of the helper cells in vivo or in vitro with 1-9 Gy resulted in a bimodal relation between radiation dose and helper effect when measured either immediately or 6 hours after irradiation. The dose-effect curve became nearly linear when tested 20 hours after irradiation. These results indicated that the helper population consisted of about 80 per cent of cells inactivated shortly after irradiation, possibly due to interphase death, and a second type showing a more retarded cell death. The latter cells were able to exert a helper function during the interval between irradiation and cell death.     

Correlation among the rates of dimer excision, DNA repair replication, and recovery of human cells from potentially lethal damage induced by ultraviolet radiation.

The kinetics of excision repair in confluent cultures of diploid human fibroblasts after ultraviolet irradiation at varying doses was measured by three different methods: (a) removal of thymine-containing dimers, (b) DNA excision repair synthesis, and (c) biological recovery of cells from the potentially lethal effects of the irradiation. Each method gave similar results and indicated that the excision rate was dependent upon the number of thymine-containing dimers induced (substrate concentration). For example, at a dose of 40 J/m2 (0.2% dimerization), the repair rate was 1.6 J/m2 per h as determined by a modified method to measure the number of thymine-containing dimers remaining in DNA and 1.65 J/m2 as measured by excision repair synthesis. At a dose of 7.5 J/m2, the repair rate was 0.5 J/m2 per h as measured by biological recovery, and at a dose of 7 J/m2, the repair rate was 0.46 J/m2 per h as measured by excision repair synthesis.     

Effect of D-valine and cytosine arabinoside on [3H]thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by [3H]thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine medium was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures.     

Analysis of time dependent changes of lymphopoietic activity in organ cultures of embryonic chicken thymus.

The time dependent development of lymphocytes in organ cultures of the thymus obtained from 10-day-old chick embryos was characterized by an initial phase of exponential increase of the number of lymphocytes per thymus followed by a plateau phase with no further increase in cell number. The proportion of cells in DNA synthesis dropped rapidly during the first 10 days of culture. Simultaneously the lymphocytes turned progressively smaller, as evidenced by both cell diameter and dry mass and constituted a homogeneous population of small cells at the end of the culture period. Thymic anlagen partially depleted of lymphoid precursor cells by a short hot pulse with 3H-TdR showed a prolonged exponential phase and reached normal plateau cell numbers 2-4 days later than usual. Furthermore, at least in the first part of the plateau phase, a reduction in the number of lymphoid cells per thymus resulted in a recovery in terms of the cell number which was associateed with increased DNA synthesis. These results are compatible with the regulation of thymic lymphopoiesis in organ culture through a mechanism operating via cell density.     

Changes in the leukocyte phagocytic activity of donor blood after its UV irradiation. I. Its relation to the irradiation dose and initial level of phagocytic activity

Phagocytic activity of human mono- and granulocytes increased markedly after UV blood irradiation in the apparatus "Izolda" used in hospitals of the USSR for medical treatment. With the rise of irradiation dose the ratio of cells ingesting latex particles increased, although the average number of particles ingested per cell decreased. The integrative phagocytic index poorly depended on the irradiation dose. In patients with a low initial level of phagocytic index, after UV blood irradiation it became more pronounced than in those with the initial elevated level. The enhancement of phagocytic activity is the result of a direct UV-stimulation of cells. This stimulation not mediated by irradiated blood plasma is known to inhibit the phagocytic activity of leucocytes. A possible mechanism of phagocytic activity stimulation is discussed.     

Protection by superoxide dismutase of white blood cells in X-irradiated mice

In X-irradiated mice the loss of white blood cells increases exponentially with dose. The dose x_o, which reduces the cell number to 1/e, is 115 ± 15, 205 ± 30, 108 ± 5, and 235 ± 18 rad for leucocytes, granulocytes, lymphocytes and platelets, respectively. The x_o value increases with the amount of endogenous superoxide dismutase per cell. Intravenous bovine superoxide dismutase has no effect on the x_o for granulocytes and platelets but increases it to 145 ± 15 and 143 ± 20 rad for leucocytes and lymphocytes, respectively. The exogenous enzyme also shortens the delay in recovery of the white blood cells, particularly after X-ray doses of 550 and 675 rad. The earlier hematological recovery is attributable to the known radioprotective effect of the enzyme on bone marrow stem cells.