The effect of recombinant human interleukin-1 beta on the repopulation of bone-marrow colony-forming cells in mice subjected to the action of radiation and burns]

The radioprotective and restorative (therapeutic) effects of human recombinant interleukin-1 beta (IL-1 beta) on the population of bone marrow CFU-S of mice, subjected to either sublethal doses of ionising irradiation itself or the same irradiation in combination with thermal burn, are investigated. Both the effects of the agent are registered under both in vitro and in vivo irradiation in semi-, syn- and allogeneic animals. If the irradiation was combined with thermal burn, the "therapeutic" effect of the agent was demonstrated at irradiation dose equal to 3.06 Gy rather than to 6.12 Gy. If the bone marrow cells were irradiated in vitro in dose 3.06 Gy with the following heat shock at 42 degrees C for 10-20 min, the "therapeutic" effect of IL-1 beta was seen only if it was added to cells before rather than after irradiation. The radioprotective effect of IL-1 beta is maintained under in vitro, as well as in vivo conditions in the allogeneic system of transplantation of the CBA donor bone marrow to the C57BL mice.     

Effect of abrogating hybrid resistance on the survival of radiation chimeras]

A study was made of the effect of the hybrid resistance abrogation by means of the lymphoid cell administration on the survival of the lethally irradiated mice protected by the transplantation of the semiallogeneic bone marrow. Injection to the C57BLxCBA recipients of the C57BL lymphoid cells one day before the irradiation and the transplantation of the bone marrow of the same genotype (C57BL) increased the chimera survival in comparison with the untreated recipients; such pretreatment 7 days before the irradiation decreased the chimera survival. Parental spleen lymphocytes administration produced but an insignificant effect on the radioresistance both of the stem hemopoietic cells (by the endocolonisation test) and of the organism as a whole (by the 30-day survival test) of the F1 hybrid. On this basis a conclusion was drawn that the differences in the splenocyte efficacy, when they were injected at different periods before the irradiation, could not be attributed to the changes in radioresistance.     

MULTIPLICATION OF HAEMOPOIETIC COLONY FORMING UNITS (CFU) IN MICE AFTER X-IRRADIATION AND BONE MARROW TRANSPLANTATION

Kinetics of the multiplication of haemopoietic CFUs was studied in lethally irradiated mice receiving various numbers of syngeneic bone marrow cells. After transplantation of a small number of bone marrow cells, the growth rate of CFU in femoral bone marrow appeared to decrease after about 10 days after transplantation, before the normal level of CFU in the femur was attained. In the spleen it was found that the overshoot which was observed about 10 days after transplantation of a large number of bone marrow cells is smaller or absent when a small number of cells is transplanted. Experiments dealing with transplantation of 50 x 10⁶ bone marrow cells 0, 4 or 10 days after a lethal irradiation indicated that the decline in growth rate of CFUs about 10 days after irradiation could not be attributed to environmental changes in the host.
The results are explained by the hypothesis that a previous excessive proliferation of CFUs diminishes the growth rate thereafter. This hypothesis is supported by experiments in which 50 x 10⁶ bone marrow cells derived from normal mice or from syngeneic chimaeras were transplanted. The slowest growth rate was observed when bone marrow that had been subjected to the most excessive proliferation in the weeks preceding the experiment was transplanted.     

柴胡多糖对照射小鼠骨髓血管机能及造血的影响

本文研究了柴胡多糖对γ线全身照射小鼠骨髓血管机能及GM-CFU-C增殖的影响。结果表明,照前1小时腹腔注射柴胡多糖能减轻照后骨體血管通透性增高的程度,并可加速照射小鼠移植骨髓后股骨内GM-CFU-C的增殖。     

In vivo studies of the relationship between the activation of lipid metabolism, postirradiation bone marrow cell proliferation and radioresistance of mice

The effect of adaptation to intermittent feeding on the in vivo biosynthesis of fatty acids and total lipids in the epididymal adipose tissue, the liver and the bone marrow was studied in adult male mice (CBA/JPh x C57BL@10 ScSnPh)F1. At the same time the effects of the same experimental stimulus on the rate of regeneration (proliferation) of bone marrow cells after sublethal irradiation of animals and on the overall radioresistance of mice expressed as 30 days survival after whole-body gamma irradiation were determined. Intermittent feeding in mice has been shown to have a significant effect on the biosynthesis of fatty acids and total lipids in all the tissues studied, including bone marrow cells, the intensity of the effect being closely dependent on the duration of the experimental stimulus. Maximum stimulation of lipogenesis during realimentation was observed approximately within 1 week of adaptation, with a reduction of the metabolic responses thereafter. The intensity of bone marrow cell proliferation in mice irradiated in the realimentation phase was inversely proportional to the preirradiation degree of biosynthesis of fatty acids and total lipids: in a period of lower lipogenetic capacity of cells in the tissue studied (around the weeks 2-5 of adaptation) an increase in the regeneration potential of bone marrow cells was observed together with increased radioresistance of the mice. During the 1-week of adaptation the opposite proved to be the case. Attention is drawn to the possible participation of prostaglandins and lipid peroxides in the responses observed.     

Effects of pre-irradiation on isogeneic and semi-isogeneic CFU growth: a study on genetic resistance.

The genetic resistance to a parental bone marrow transplant as demonstrated, when transplantation was performed early after irradiation, failed to occur if the interval between irradiation and transplantation was increased to 4 days. A similar radiation induced weakening of genetic resistance to a parental bone marrow graft in spleen and bone marrow could be demonstrated in mice, which had been irradiated with a sublethal dose at 7 days prior to the lethal irradiation and transplantation. The pre-irradiation of the recipient with a sublethal dose induced an enhancement of the growth in spleen and bone marrow of isogeneic transplanted CFU. The pre-irradiation of a single tibia also resulted in a significant weakening of the resistance in the spleen. The experiments with partial body pre-irradiation suggested a local effect of the pre-irradiation, but it could be shown that the enhanced CFU growth is not caused by an enhanced seeding of CFU in pre-irradiated bone marrow. The role of microenvironment in the phenomenon of genetic resistance is discussed.     

Murine cytomegalovirus infection can enhance hybrid resistance through modulation of host natural killer activity

Studies were undertaken to assess the effect of murine cytomegalovirus (MCMV) in two different models involving injection of parental cells into F1 hosts. In both of these systems, MCMV-induced enhancement of hybrid resistance was found. In the first model, parent-into-F1 graft-vs-host reaction, MCMV infection of (C57BL/6 x C3H)F1 (B6C3F1) hosts was found to prevent the GVHR normally induced by injection of B6 parental splenocytes into the F1 hosts. The second model involved injection of parental bone marrow into lethally irradiated B6C3F1 and (C57BL/6 x DBA/2)F1 (B6D2F1) hosts. These irradiated hosts are known to exhibit resistance to engraftment by parental C57BL/6 (B6) bone marrow. This resistance was found to be markedly enhanced by injection of the hosts with MCMV 3 days before irradiation and bone marrow injection. In contrast, engraftment into B6C3F1 hosts of syngeneic marrow, or bone marrow from the C3H parent, was not affected by MCMV infection. Engraftment of DBA/2 marrow into B6D2F1 hosts was reduced at lower doses of injected marrow, suggesting enhanced resistance against the minor Hh Ag Hh-DBA. To test whether the MCMV-induced enhancement of resistance was mediated by NK cells, splenic NK activity (YAC-1 killing) and frequency (NK1.1 staining) were assessed. Both parameters were found to be elevated at 3 days after MCMV infection but to return to normal levels by 9 days. B6 bone marrow engraftment was in fact found to be normal when the marrow was administered to F1 mice 9 days after MCMV infection. Furthermore, anti-asialoGM1 administration prevented MCMV-induced enhancement of resistance to marrow engraftment. Thus, the NK enhancement resulting from MCMV infection appears to play a major role in the enhanced HR observed in the marrow engraftment model. This effect may be of importance in clinical bone marrow transplantation, a situation in which patients are susceptible to viral infection.     

EFFECTS OF PRE-IRRADIATION ON ISOGENEIC AND SEMI-ISOGENEIC CFU GROWTH: A STUDY ON GENETIC RESISTANCE

The genetic resistance to a parental bone marrow transplant as demonstrated, when transplantation was performed early after irradiation, failed to occur if the interval between irradiation and transplantation was increased to 4 days. A similar radiation induced weakening of genetic resistance to a parental bone marrow graft in spleen and bone marrow could be demonstrated in mice, which had been irradiated with a sublethal dose at 7 days prior to the lethal irradiation and transplantation. The pre-irradiation of the recipient with a sublethal dose induced an enhancement of the growth in spleen and bone marrow of isogeneic transplanted CFU. The pre-irradiation of a single tibia also resulted in a significant weakening of the resistance in the spleen. The experiments with partial body pre-irradiation suggested a local effect of the pre-irradiation, but it could be shown that the enhanced CFU growth is not caused by an enhanced seeding of CFU in pre-irradiated bone marrow. The role of microenvironment in the phenomenon of genetic resistance is discussed.     

The stimulating effect of preliminary irradiation of the hematopoietic stroma with small doses on the content of hematopoietic stem cells in the bone marrow in vivo and in a long-term culture system

Preirradiation of mouse recipients with a dose of 1-2 Gy 24 and 48 h before lethal irradiation (8 Gy) made CFUs content of femur increase upon transplantation of bone marrow from exposed and intact donors. The same was with the long-term bone marrow culture: preirradiation of a stromal sublayer increased the number of CFUs in the transplanted bone marrow preirradiated with 6 Gy radiation. Retransplantation of bone marrow to irradiated donors after 5 day cultivation, a sublayer being activated, increased the number of CFUs in the femur in comparison with donors which were injected with the bone marrow from the culture without activation of the sublayer by low-level radiation.     

Allogeneic marrow transplantation after total lymphoid irradiation (TLI): effect of dose/fraction, thymic irradiation, delayed marrow infusion, and presensitization.

BALB/c mice infused with 30 x 10(6) C57BL/Ka bone marrow (BM) cells 1 day after treatment with fractionated total lymphoid irradiation (TLI) (17 fractions of 200 rads each) became stable mixed chimeras without clinical graft-vs-host disease (GVHD). Mice given 18 fractions of 100, 50, or 25 rads each followed 1 day later by C57BL/Ka BM did not become chimeric, indicating that a critical cumulative radiation dose is required for this effect. Animals given TLI with lead shielding placed over the thymus also developed stable chimerism without GVHD. Thus susceptibility to tolerance induction and protection from GVHD after TLI and allogeneic BM transplantation is not due to alteration of the thymic microenvironment by fractionated irradiation. A delay of 7 or 21 days between completion of TLI and BM administration resulted in a high incidence of graft rejection. Sensitization to minor histocompatibility antigens of the BM donor strain by blood transfusion either before or during TLI resulted in marrow graft rejection in a high percentage of animals.     

Correction of a T-immunodeficit in mice by bone marrow transplantation from donors treated with hydrocortisone]

Adult CBA mice were exposed to thymectomy, lethal irradiation, and protection by syngeneic bone marrow transplantation. In some experiments syngeneic bone marrow of donors, treated with hydrocortisone in a dose of 125 mg/kg for 3 days was used. The bone marrow of these donors contained cells with the Q-marker. Thymectomized and lethally irradiated animals subjected to the transplantation of syngeneic bone marrow from hydrocortisone-treated donors rejected the skin allotransplants, and the lymph node cells of these mice suppressed the endogenous colony-formation in the sublethally-irradiated hybrids (CBA X C57Bl/6) F1.     

Formation of hematopoietic colonies of the donor type in the bone marrow of irradiated chickens after transplantation of the cells of the yolk sac and hindlimb of the quail embryo]

The ability of yolk sac and primary bone marrow cells of the quail to form hemopoietic colonies at 6 hours of incubation (i. e. before establishment of circulation) was studied in the bone marrow of 3-week sublethally irradiated chickens. The experiments were based on the possibility of differentiating between quail and chicken cells from the natural cell marker (Pheulgen-positive nucleolus). The number of hemopoietic colonies produced by cells transplanted from the primary bone marrow was three times greater than that consequent on transplantation of yolk sac cells. With the given dose of irradiation the bone marrow shows about 75% exogenous (quail) and 25% endogenous (chicken) hemopoietic colonies.     

Effect of a deficiency in the mononuclear phagocyte system and the administration of yeast polysaccharide on the development of a heterotopic source of hematopoiesis

Mononuclear phagocyte system (MPS) deficiency was induced by repeated peritoneal lavage in (C57Bl x CBA) F1 mice. The animals were then used as donors or recipients in heterotopic bone marrow transplantation. Yeast polysaccharide (YP) produced by Cryptococcus luteolus strain 228 was injected weekly (25 mg/kg) during 30 days after bone marrow transplantation under the kidney capsule. Bone marrow transplantation from MPC-deficient mice to intact mice 30 days later resulted in no variations from the control in cellularity and ossicle weight. YP produced an increase in cellularity, but not in ossicle weight. In the opposite experimental scheme (transplantation from intact mice to MPS-deficient mice) an increase in both cellularity and weight was not noticed. YP injections in this case resulted in the reduction of heterotopic organ size to the control level. Possible mechanisms of this phenomenon are discussed.     

The effect of bone marrow transplantation on the dynamic recovery of the morphofunctional properties of the pancreas in irradiated recipients

Effect of transplantation of syngeneic bone marrow in the dose of 1 X 10(7) cell/ml on the state of pancreatic gland in lethally irradiated recipients has been studied at different stages of posttransplantation period for 3 months using 250 linear male rats G (CBA x C57B). Histological and biochemical investigation, conducted in dynamics, have shown that transplantation of native and cryopreserved bone marrow to lethally irradiated animals facilitates activation of compensatory-restoration processes manifesting themselves in mitotic division of glandular and epithelial cells, as well as optimizes exchange of carbohydrates in the irradiated organism.     

Effect of cyclosporin A on lymphopoiesis. III. Augmentation of the generation of natural killer cells in bone marrow transplanted mice treated with cyclosporin A

The effects of cyclosporin A (CsA) on the generation of NK cells were studied using syngeneic bone marrow transplanted mice subsequently treated with CsA (BMT/CsA mice). In contrast to a severe reduction in T cells that was reported previously, these mice exhibited a marked enhancement of splenic NK activity. The enhanced NK activity was mediated by NK1.1+, Thy-1- cells as assessed by antibody plus complement treatment, and was concomitant with an absolute increase in the numbers of NK1.1+ cells as assessed by flow cytometry. Because the depletion of host-derived, mature NK cells by injection of anti-asialo GM1 antibody before bone marrow reconstitution did not affect the enhancement of NK activity, CsA appeared to augment the generation of NK cells from bone marrow precursors. To investigate a possible relationship between the enhancement of NK activity and the maturational arrest of T cells in the thymus induced by CsA, mice were thymectomized, followed by irradiation, bone marrow reconstitution, and CsA treatment. These mice exhibited as strong enhancement of splenic NK activity as BMT/CsA mice, suggesting that the CsA-induced effect on NK cells is distinct from its effect on T cell development in the thymus. Taken together, these results are the first demonstration of the positive effect of CsA on NK cell generation and may be of importance in clinical bone marrow transplantation.     

Booster irradiation to the spleen following total body irradiation. A new immunosuppressive approach for allogeneic bone marrow transplantation

Graft rejection presents a major obstacle for transplantation of T cell-depleted bone marrow in HLA-mismatched patients. In a primate model, after conditioning exactly as for leukemia patients, it was shown that over 99% of the residual host clonable T cells are concentrated in the spleen on day 5 after completion of cytoreduction. We have now corroborated these findings in a mouse model. After 9-Gy total body irradiation (TBI), the total number of Thy-1.2+ cells in the spleen reaches a peak between days 3 and 4 after TBI. The T cell population is composed of both L3T4 (helper) and Lyt-2 (suppressor) T cells, the former being the major subpopulation. Specific booster irradiation to the spleen (5 Gy twice) on days 2 and 4 after TBI greatly enhances production of donor-type chimera after transplantation of T cell-depleted allogeneic bone marrow. Similar enhancement can be achieved by splenectomy on day 3 or 4 after TBI but not if splenectomy is performed 1 day before TBI or 1 day after TBI, strengthening the hypothesis that, after lethal TBI in mice, the remaining host T cells migrate from the periphery to the spleen. These results suggest that a delayed booster irradiation to the spleen may be beneficial as an additional immunosuppressive agent in the conditioning of leukemia patients, in order to reduce the incidence of bone marrow allograft rejection.     

Mortality of monkeys after exposure to fission neutrons and the effect of autologous bone marrow transplantation.

In order to assess the risk of exposure to ionizing radiation in man, and to evaluate the results of therapeutic measures, the mortality of rhesus monkeys irradiated with X-rays and fission neutrons and the effect of autologous bone marrow transplantation have been investigated. The LD50/30d values for X- and neutron-irradiated monkeys amount to 525 and 260 rad respectively, resulting in an r.b.e. of approximately 2 for the occurrence of the bone marrow syndrome. Protection of the animals by autologous bone marrow transplantation was observed up to doses of 860 rad of X-rays and 440 rad of fission neutrons. After both fission-neutron irradiation and X-irradiation in the lowest range of lethal doses, the bone marrow syndrome was found to occur without the concurrent incidence of the intestinal syndrome. The studies indicate that, for humans accidentally exposed to what would otherwise be lethal doses of fast neutrons, bone marrow transplantation may be beneficial.     

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.     

Irradiation of groups of mice and rats. Studies on the homogeneity of dose distribution in the irradiation field of a Co-60 source

Data are presented on the dose distribution pattern in an irradiation field for the irradiation of groups of small rodents as a preconditioning regimen for experimental bone marrow transplantation. The consequences of this pattern are discussed.     

The facilitation of enduring engraftment of allogeneic bone marrow and avoidance of secondary disease in mice

A method for transplantation of allogeneic bone marrow has been developed and tested in mice. It consists of a treatment preceding supralethal, total-body irradiation (preconditioning) in which a combination of three drugs acting on neuroendocrine regulation are administered, followed by inoculation of a large number of allogeneic bone marrow cells. A second inoculation of allogeneic marrow from the same immunogenetically different donor is given after irradiation. This system provides a high level of protection to mice against radiation damage and facilitates engraftment of the foreign marrow. A large proportion of the engrafted mice become enduring chimeras, manifesting no secondary disease.