The relationship between 59Fe uptake in marrow and 239Pu deposition in bone.

The marrow in the left femur of each of 17 mice was destroyed by X-irradiation and 59Fe and 239Pu uptake into both femurs was measured 1, 3 and 7 days later. Uptake of 59Fe into marrow was depressed in the left femur 1 and 3 days after irradiation but was enhanced in the right unirradiated femur 3 days after the left femur was irradiated. There was no corresponding depression of 239Pu uptake into the left irradiated femur or enhancement into the right unirradiated femur. These results do not support the view that a functioning erythropoietic marrow is necessary for 239Pu to be deposited in bone.     

Relative number and proliferation kinetics of hemopoietic stem cells in the mouse.

A model calculation of the hemopoiesis of the mouse based on known hematologic data leads to the conclusion that approximately 3% of all nucleated bone marrow cells are stem cells (pluripotent plus committed stem cells). By a new 125IUdR labeling technique on radiation chimeras, a relative number of 2%-7% stem cells was determined. In previous studies with test systems for stem cells using colony formation in vivo or in vitro, a relative number of stem cells of at least one order of magnitude lower has been estimated. In this study the stem cells are found to have a turnover time of about 4.3 days in the donor mice. This turnover time remained unchanged even after transfusion of marrow cells into lethally irradiated recipient mice. Radiosensitivity determinations yielded a D0 of 80 rad for stem cells in S-phase and D0 of 185 rad for stem cells distributed throughout the entire cell cycle. The respective extrapolation numbers were 1.23 and 1.14. Experiments using an 3H-TdR suicide technique revealed different cell cycle parameters for bone marrow stem cells seeding to the spleens and to the femurs of lethally irradiated recipients, primarily a shortening of S-phase in cells seeding to femurs. The method described here provides a new approach to hematologic stem cell research.     

Suppression of antibody responses in allogeneic mice by products of lymphoid tissue. I. Allogeneic suppressive factor (ASF) from spleens repopulated with thymus cells.

A cellfree extract prepared from the spleen cells of C3H mice is capable of suppressing antibody responses to SRBC when extract material is exposed to alloantigens. The observed immunosuppression was attributed to a soluble factor in the extract. This allogeneic suppressive factor (ASF) was detected in extracts prepared from the spleen cells of unirradiated mice as well as those of irradiated mice repopulated with thymocytes, provided that mice were previously immunized with SRBC. Donors of actively suppressive ASF preparations did not need to be previously exposed to alloantigens. Extracts from thymus and marrow cells of unirradiated mice and the spleen cells of irradiated mice repopulated with marrow cells (or no cells) did not contain ASF. C3H thymocytes stimulated with SRBC generated more ASF activity in spleens of C3BF1 hosts than in those of C3H hosts, indicating that alloantigenic stimulation enhances the production or activity of ASF. Once produced, C3H ASF was able to suppress antibody responses in cell transfer experiments only if exposed to C3BF alloantigens of either donor lymphoid cells or irradiated hosts. Once exposed to alloantigens, ASF appears to be capable of suppressing antibody responses of syngeneic C3H or semi-allogeneic C3BF cells. When both donor lymphoid cells and hosts were syngeneic with the donor of the ASF, there was enhancement of antibody formation in cell transfer experiments. C3H ASF did not interfere with education of C3BF thymocytes to SRBC or with the generation of precursors of anti-SRBC antibody-forming cells by C3BF1 marrow cells. ASF may interfere with cellular cooperative events necessary for humoral immune responses or with terminal differentiation of B cells. Production of ASF could partially account for the suppression of antibody responses observed during graft-vs-host reactions.     

Residual radiation effect in the murine spleen. In situ measurement by 125-iodo-deoxyuridine retention

Summary To investigate whether residual radiation damage in hematopoietic tissue is measurable in situ by a change in cell turnover, the retention of the thymidine analogue 5-(125-I)iodo-2-deoxyuridine (125-IUdR) following incorporation into DNA of cells in bone marrow and spleen of mice was measured 35 days after 0–500 rad whole body gamma irradiation.In the bone marrow a rapid and a slow turnover component of 125-IUdR retention were found. Both components were almost identical for unirradiated and irradiated mice. In the spleen the 125-IUdR retention curves exhibited three components with increasingly prolonged half-times. In the second component the half-time was longer in irradiated than in unirradiated mice. This was dose-dependent.The increased half-time of 125-IUdR retention in irradiated spleens may be caused by direct cellular damage of long-lived cells (lymphocytes, early hematopoietic progenitor cells) or/and by diminished stimulation of proliferation by microenvironmental or long-range factors.     

THE ROLE OF BONE MARROW OF X-IRRADIATED MICE IN THYMIC RECOVERY

The influence of the bone marrow on the repopulation of the thymus in X-irradiated mice has been investigated.
It was observed that the thymus and a certain population of bone marrow lymphocytic cells were repopulated in parallel in a cyclic fashion. This occurred either after a single exposure of mice to 400 R or after serial weekly X-ray treatments with 170 R. Lethally irradiated recipients which were grafted with bone marrow cells obtained 12-24 days after four weekly irradiations of donor mice with 170 R also exhibited a cyclic repopulation of both the thymus and the bone marrow lymphocytic population. In contrast, mice which were transplanted with bone marrow cells from unirradiated donors, containing an equal number of stem cells (CFU), exhibited a continuous rather than a cyclic recovery of both cell populations. the bone marrow stem cells of mice recovering from X-irradiation were found to have a decreased proliferative activity, since they produced significantly smaller spleen colonies in lethally irradiated recipients than marrow cells from unirradiated mice.
The results were interpreted as indicating that the bone marrow lymphocytic cells may act as thymic precursor cells and that thymic lymphopoiesis is dependent on the presence of such cells. Evidently, the production of lymphocytic cells will decrease when the stimulus for granulocyte production increases due to the limited proliferative activity of the surviving bone marrow stem cells after irradiation. This may result in a cyclic variation of the production of bone marrow lymphocytic cells and it follows that thymic lymphopoiesis will run parallel.     

Lipoprotn lipase content and triglyceride fatty acid uptake in adipose tissue of rats of differing body weights

Increasing body weight appears to alter lipid metabolism in adipose tissue. We have measured the content of lipoprotein lipase and the uptake of chylomicron triglyceride fatty acids in epididymal fat pads of rats of different weights. In order that the results might be expressed in terms of cell numbers, the relationship between the weights of fat pads and the numbers and volumes of fat cells isolated from them was determined. Highly significant correlations were found between fat pad weight and both the number and the volume of the individual adipocytes. In rats weighing from 140 to 350 g, the increase in the size of fat pads was attributable almost equally to increases in cell size and in cell number. Lipoprotein lipase activity was measured in acetone powders of whole fat pads and of isolated fat cell preparations. With both, lipoprotein lipase activity per cell diminished significantly as the weight of fat tissue increased, i.e., larger fat cells contained less enzyme per cell than smaller cells. The uptake of triglyceride fatty acid radioactivity was measured after incubation of fat pads with radiolabeled rat lymph chylomicrons in flasks containing either buffer alone or with added glucose or glucose plus insulin. The addition of glucose and insulin led to a mean increase of 70% in the uptake of radioactivity, but larger adipocytes were stimulated less than smaller cells. This resulted in a significant negative correlation between the weights of fat pads and the uptake of radioactivity. Enlargement of fat cells also led to a diminution in their capacity to esterify fatty acids.     

Differential proliferation of erythroid and granuloid spleen colonies following sublethal irradiation of the bone marrow donor

Spleen colonies produced by sublethally irradiated mouse bone marrow cells were compared to those produced by unirradiated marrow cells in lethally irradiated mice. Sublethally irradiated marrow cells gave rise to many fewer spleen colonies. At seven days of colony age, the ratio of erythroid colonies to granuloid colonies was lower (< 1) than for colonies formed by unirradiated marrow (2 to 3 or more). Delay of harvest of colonies to day 10 or 12 resulted in 6 to 11 fold increase in the ratio of erythroid to granuloid colonies due largely to the belated appearance of erythroid colonies.     

Genetically determined resistance to foreign bone marrow transplantation in mice: characterization of the effector cells

Mice of most strains show a genetically determined ability to reject a variety of foreign marrow grafts even after lethal irradiation. The phenomenon is both host strain and donor marrow graft-dependent. To characterize the effector cell responsible for graft rejection, attempts were made to 1) determine to what morphologic subclass it belongs; 2) determine its life span; and 3) establish whether genetically different host environments influence the functioning of the effector cell. Mice of the 129/J strain (normally nonresistant), C57BL/6 strain (made non-resistant), and the homozygous mutants of C57BL/6, i.e., C57BL/6 (bg/bg), were recipients of C57BL/6 marrow or spleen cells. After lethal irradiation, hosts were given marrow or spleen cells from normal, strongly resistant C57BL/6 donors pretreated with a) 950 R whole body irradiation or b) twice daily injections for 4 days of the cell cycle toxic drug hydroxyurea followed by 950 R. In other cases, hosts were recipients of the lymphoid cell-rich fraction of marrow from irradiated C57BL/6 donors or adherent cells taken from cultures of marrow cells of unirradiated C57BL/6 donors. Three hours after receiving C57BL/6 marrow or spleen cells, irradiated hosts were given allogeneic DBA/2 marrow (always strongly rejected by C57BL/6 mice and always accepted by 129/J strain mice). Seven days later, host spleens were removed and the numbers of microscopic colonies were counted from subserial sections. The results demonstrate that 1) mice either normally or rendered nonresistant to a marrow allograft can be made to develop resistance by the administration of either whole spleen cells or marrow lymphoid cells from lethally irradiated strongly resistant donors; 2) adherent cells from cultures of marrow from strongly resistant mice are ineffective in conferring resistance; 3) the cell effective in conferring resistance has a life span greater than 4 but less than 7 days; and 4) the effector cell can function in genetically different environments of nonresistant strains.     

Radiation studies of Acholeplasma laidlawii: the role of membrane composition

Acholeplasma laidlawii, a mycoplasma, is unable to synthesize unsaturated fatty acids but it will incorporate them into its plasma membrane if they are supplied exogeneously. Thus the fatty acid composition of the cell membrane can be defined by growing the organism in media containing specific fatty acids. We obtained cells with predominantly one type of unsaturated fatty acid (either oleic, linoleic or linolenic acid) or cells with only saturated fatty acid in the cell membrane. The cells were irradiated with 7 MeV electrons and the effect of membrane fatty acid composition on cell survival was examined. At 200 Gy/min and 0.5 degrees C (melting ice) there was little difference in the radiation sensitivities of the cells grown in unsaturated fatty acids either in aerated or anoxic radiation conditions. However, the cells containing saturated fatty acids irradiated in anoxic conditions were markedly more sensitive than the cells containing unsaturated fatty acids. At 200 Gy/min and 37 degrees C the two types of cells were of similar sensitivity both in aerated and anoxic radiation conditions. At 5 Gy/min at 0.5 degrees C the cells containing linolenic acid (18:3) were less sensitive than those containing solely saturated fatty acids. However, at 5 Gy/min at 37 degrees C there was no difference in sensitivity between these two types of cell. Our results strongly argue against the involvement of lipid peroxidation as a molecular change leading to cell death.     

Effect of alkali on the size dispersity of mammalian DNA measured by filter elution

DNA from unirradiated and irradiated cultured 9L rat brain tumor cells was held for varying times in low ionic strength solutions at pH 11.0, 12.3, or 12.9. The effect of this exposure to alkali on the DNA size distribution was determined by comparing the DNA filter elution profiles obtained experimentally with those theoretically predicted for monodispersed and random distributions. At pH 12.3 or 12.9, DNA from cells irradiated with 300 rad eluted with first-order kinetics corresponding to a random DNA size distribution. The median size of the distribution decreased if the irradiated DNA was exposed to pH 12.3 for 24 h. At pH 12.3 or 12.9, DNA from unirradiated cells eluted initially with complex kinetics that later became linear (18-21 h for pH 12.3 or 13-15 h for pH 12.9), characteristic of a monodispersed DNA size distribution. Holding either unirradiated or irradiated DNA at pH 11.0, below the critical unwinding pH, produced no effect on the elution profiles. Analysis of these filter elution data indicated that after sufficient exposure to pH 12.3 or 12.9, undamaged DNA molecules from mammalian cells elute as a single-stranded monodispersed size distribution of approximately 1 X 10(10) daltons. While the possibility cannot be completely eliminated that this monodispersed size represents an upper limit determined by physical forces, these results, in conjunction with those obtained using other techniques, lend credence to the existence of a nonrandom higher-order structure in mammalian chromosomal DNA.     

Bystander-type effects mediated by long-lived inflammatory signaling in irradiated bone marrow

Radiation-induced bystander and abscopal effects, in which DNA damage is produced in nonirradiated cells as a consequence of communication with irradiated cells, indicate mechanisms of inducing damage and cell death additional to the conventional model of deposition of energy in the cell nucleus at the time of irradiation. In this study we show that signals generated in vivo in the bone marrow of mice irradiated with 4 Gy γ rays 18 h to 15 months previously are able to induce DNA damage and apoptosis in nonirradiated bone marrow cells but that comparable signals are not detected at earlier times postirradiation or at doses below 100 mGy. Bone marrow cells of both CBA/Ca and C57BL/6 genotypes exhibit responses to signals produced by either irradiated CBA/Ca or C57BL/6 mice, and the responses are mediated by the cytokines FasL and TNF-α converging on a COX-2-dependent pathway. The findings are consistent with indirect inflammatory signaling induced as a response to the initial radiation damage rather than to direct signaling between irradiated and nonirradiated cells. The findings also demonstrate the importance of studying tissue responses when considering the mechanisms underlying the consequences of radiation exposures.     

Dissociation of bone resorption and bone formation in adult mice with a non-functional V-ATPase in osteoclasts leads to increased bone strength

Osteopetrosis caused by defective acid secretion by the osteoclast, is characterized by defective bone resorption, increased osteoclast numbers, while bone formation is normal or increased. In contrast the bones are of poor quality, despite this uncoupling of formation from resorption.To shed light on the effect of uncoupling in adult mice with respect to bone strength, we transplanted irradiated three-month old normal mice with hematopoietic stem cells from control or oc/oc mice, which have defective acid secretion, and followed them for 12 to 28 weeks.Engraftment levels were assessed by flow cytometry of peripheral blood. Serum samples were collected every six weeks for measurement of bone turnover markers. At termination bones were collected for μCT and mechanical testing. An engraftment level of 98% was obtained. From week 6 until termination bone resorption was significantly reduced, while the osteoclast number was increased when comparing oc/oc to controls. Bone formation was elevated at week 6, normalized at week 12, and reduced onwards. μCT and mechanical analyses of femurs and vertebrae showed increased bone volume and bone strength of cortical and trabecular bone.In conclusion, these data show that attenuation of acid secretion in adult mice leads to uncoupling and improves bone strength.     

Proliferation inhibition of murine pluripotent haemopoietic stem cells by interferon or poly I:C

The effect of mouse serum interferon (IF) in vitro and an inducer in vivo on the proliferation of a pluripotent stem cell population with high turnover rate was studied. Proliferation rate was characterized by the number of CFUs in the S phase of the cell cycle. Increased proliferation of bone marrow stem cell populations was produced either by irradiating the donor mice with 3.36 Gy (336 rad) 60Co-gamma rays 7 days before the experiment or by incubating normal bone marrow cells with 10(-11) M concentration of isoproterenol. IF considerably reduced the number of CFUs in S phase in both cases without reducing the CFUs content of the samples. Injection of IF inducer (4 mg/kg poly I:C) into regenerating mice also inhibited the proliferation of CFUs without decreasing the femoral CFUs level. Regeneration kinetics of CFUs from irradiated poly I:C-treated mice ran parallel with that of irradiated untreated animals but showed a characteristic delay corresponding to approximately one CFUs doubling. A transient, non-cytotoxic proliferation inhibitory effect of IF or IF inducer is, therefore, proposed.     

Effects of Gamma-irradiation on Lipid Metabolic Changes of Potato Tubers in Response to Mechanical Injury

Linolenic acid contents of glycolipids increased in irradiated potatoes during storage, accompanied by a decrease of linoleic acid. The puncturing of a potato tuber with a needle of a microsyringe caused the similar changes; the elevation of linolenic acid level and decline of linoleic acid were observed within 24 h after puncturing. Irradiation before the puncturing reduced the degree of the increase of linolenic acid in response to the mechanical injury. The rate of [¹³C]acetate incorporation into lipid fractions of irradiated tubers was smaller than that of unirradiated tubers, and polyunsaturated fatty acids (linoleic and linolenic acids) of lipid fractions were weakly labeled in irradiated tubers as compared with unirradiated ones. The results in this study indicate that irradiation retards lipid metabolism in response to mechanical injury.     

Effect of medium on chromatin damage in bystander mammalian cells

In the present study, we examined the potential contribution of irradiated medium to the bystander effect using custom-made double-Mylar stainless steel rings. Exponentially growing human-hamster hybrid (A(L)) cells were plated on either one or both sides of double-Mylar dishes 2-4 days before irradiation. One side (with or without cells) was irradiated with alpha particles using the track segment mode of a 4 MeV Van de Graaff accelerator at the Radiological Research Accelerator Facility of Columbia University. Since alpha particles can traverse only a very limited distance (around 23 microm in water), cells plated on the other side of a medium-filled Mylar dish will not be irradiated by the alpha particles. The results of the cytogenetic assay of unirradiated target cells that were attached to the top Mylar layer indicate that the number of chromatid-type aberrations was higher when there was a bottom layer of cells in the medium-filled chambers than with just medium alone. Furthermore, when the medium was transferred from these cell-irradiated dishes to fresh A(L) cell cultures, chromatid-type aberrations were produced in the unirradiated fresh cells. In contrast, medium irradiated in the absence of cells had no effect on chromatid aberrations. These results suggest that certain unidentified modulating factors secreted from the irradiated cells on the bottom Mylar layer into the medium induce chromatin damage in the unirradiated bystander cells.     

Effect of a single exposure to ultraviolet radiation on Mycobacterium bovis bacillus Calmette-Guerin infection in mice

BALB/c and C3H mice were exposed on the dorsal skin to 45 kJ/m2 of UVB radiation from FS-40 sunlamps 3 days before infection with 1 x 10(6) live units of Mycobacterium bovis bacillus Calmette-Guérin (BCG) (Tice strain) in the footpad. At regular intervals, groups of mice were tested for a delayed type hypersensitivity (DTH) response to the purified protein derivative (PPD) of tubercle bacilli, and the course of infection was monitored by measuring the size of the infected footpad, enlargement of the draining lymph node, and the number of bacteria in the spleen and lymph node. In both strains the DTH response to PPD was significantly delayed in UV-treated mice compared to unirradiated mice, when tested 21 and 42 days after BCG infection. By day 50, no significant difference was detected in the DTH response between irradiated and unirradiated mice. UV treatment reduced the size of the lymph node draining the site of BCG infection in both strains of mice and the size of the infected footpad in C3H mice but not in BALB/c mice. In both strains of mice the total number of bacteria in the spleen and the draining lymph node increased after UV irradiation. When irradiated 3, 5, 18, or 21 days after BCG infection, BALB/c mice also showed a significant decrease in their DTH response to PPD, indicating that the UV-induced suppression of BCG occurs both at the induction and the elicitation stages of the immune response. Thus, mice exposed to a single dose of UV radiation either before or after BCG infection showed an impaired DTH response to mycobacteria, which was accompanied by an increase in the multiplication of bacteria in the tissues, even though the organisms were introduced at an unirradiated site. These studies demonstrate that a systemic effect of UV irradiation can interfere with the development and expression of immunity to pathogenic bacteria in mice.     

Influence of a sublethal irradiation on the immune response of the mouse to sheep erythrocytes]

In the CBA mice, the immunological response of the spleen cells (RFC and PFC direct and indirect) against the sheep erythrocytes is highly depressed by a 400 R dose of X rays. The recovery is not complete at the 30th day after irradiation. The response of the bone marrow cells either irradiated or unirradiated to the antigenic stimulation is very low.     

Hemopoietic colony studies. VI. Increased eosinophil-containing colonies obtained by antigen pre-treatment of irradiated mice reconstituted with bone marrow cells

The cellular response to an intraperitoneal injection of antigen (tetanus toxoid) was studied in reconstituted animals in order to determine the mechanism of control of eosinophil granulocytopoiesis. Antigen treatment of the marrow cell donors did not consistently increase the number of spleen and bone marrow colonies in recipient animals or change the percentage of eosinophil or other hemopoietic colony types. Antigen pre-treatment of the irradiated recipients increased the percentage of eosinophil-containing colonies in the spleen and femoral bone marrow without significantly changing the total number of either spleen or marrow colonies. Antigen treatment of both the bone marrow cell donor and recipient produced a further increase in the percentage of eosinophil-containing colonies in the marrow cavity, but not in the spleen. Antigen treatment of the irradiated recipient increased the number of eosinophilic cells (but not the total number of cells) in both the peritoneal cavity and the bone marrow. Antigen treatment of both the marrow donor and recipient produced a further increase in the number of eosinophilic cells in the peritoneal cavity, but not in a single femur. Since antigen treatment of the marrow recipient, or recipient and donor, but not of the marrow donor alone, results in increased eosinophilic cell and colony numbers, the effect of antigen appears to be mediated through some host factor(s), perhaps the eosinophilic hemopoietic inducing microenvironment (HIM), rather than directly on the hemopoietic stem cells.     

Requirement for Protein Synthesis in rec-Dependent Repair of Deoxyribonucleic Acid in Escherichia coli after Ultraviolet or X Irradiation

Deprivation of amino acids required for growth or treatment with chloramphenicol or puromycin after irradiation reduced the survival of Rec(+) cells of Escherichia coli K-12 which had been exposed to either ultraviolet (UV) or X radiation. In contrast, these treatments caused little or no reduction in the survival of irradiated recA or recB mutants. The effect of chloramphenicol on the survival of X-irradiated cells was correlated with an inhibition of repair of single-strand breaks in irradiated deoxyribonucleic acid (DNA), previously shown to be controlled by recA and recB. In UV-irradiated cells no effect of chloramphenicol was detected on the repair of single-strand discontinuities in DNA replicated from UV-damaged templates, a process controlled by recA but not by recB. From this we concluded that inhibiting protein synthesis in UV or X-irradiated cells may interfere with some biochemical step in repair dependent upon the recB gene. When irradiated Rec(+) cells were cultured for a sufficient period of time in minimal growth medium before chloramphenicol treatment their survival was no longer decreased by the drug. After X irradiation this occurred in less than one generation time of the unirradiated control cells. After UV irradiation it occurred more slowly and was only complete after several generation times of the unirradiated controls. These observations indicated that replication of the entire irradiated genome was probably not required for rec-dependent repair of X-irradiated cells, although it might be required for rec-dependent repair of UV-irradiated cells.     

REGULATION OF HAEMOPOIETIC STEM CELL TURNOVER IN PARTIALLY IRRADIATED MICE

The existence of a possible local control of CFU turnover was studied in mice in which one tibia only was irradiated (LR mice) and in mice in which one tibia was shielded during whole-body irradiation ('TBR'mice). In both the LR and 'TBR'mice, the increased CFU turnover continued in the irradiated tibiae even after the time when in the unirradiated (shielded) tibiae it returned to normal levels. The early temporary decrease in the CFU numbers in the shielded tibiae of 'TBR'mice is attributed to a temporary demand for increased differentiation rather than to migration of CFU. The direct control of CFU turnover appears to be local in nature, in contrast to the stimulus for CFU differentiation.