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 INFLUENCE OF GENETIC RESISTANCE ON CFU GROWTH KINETICS IN SPLEEN AND FEMUR

An impaired colony formation of C57BL marrow cells transplanted into F_t (C57BL x CBA) mice was observed. In accordance with the literature this phenomenon has been designated as ‘genetic resistance’. Studies to elucidate the mechanism of the genetic resistance demonstrated that the multiplication phase of the CFU growth curve started in the semi-isogeneic combination about 48 hr later than in the isogeneic combination. In the spleen this resulted in a lower ‘dip’. For the spleen as well as for the femur similar CFU doubling times were found during the multiplication phase when both transplantation combinations were compared. Furthermore the percentage of CFU in S-phase (assessed with the ³H-TdR suicide technique) during the first days after transplantation were similar in both combinations. When the spleen was removed 5–6 months before irradiation and bone marrow transplantation was performed the growth curve of parental CFU in the femur was identical with the growth curve of isogeneic CFU (no delay was observed). These results are discussed and a few theories explaining the observations are proposed.     

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.     

In vivo synergistic effect of the immunomodulator AS101 and the PKC inducer bryostatin.

The immunomodulator AS101 has recently been found to have radioprotective properties when injected prior to sublethal and lethal doses of irradiation. In addition, this compound was found to protect mice from hemopoietic damage caused by sublethal doses of cyclophosphamide (CYP) and to increase the rate of survival of mice treated with lethal doses of CYP. AS101 was previously shown to exert a synergistic effect with the PKC-inducer bryostatin in cytokine secretion in vitro. The present studies were designed to evaluate the effects of in vivo combined treatment with AS101 and bryostatin on bone marrow and spleen cellularity and on the number of committed progenitors in the bone marrow at various points of time after their treatment with a sublethal dose of CYP or irradiation. In addition, the combined effect was tested on the survival of mice irradiated with a lethal dose of irradiation. Our data show the presence of synergism which greatly enhances the number of bone marrow and spleen cells 48 hr and 9 days after CYP treatment or irradiation. The combined effect was also demonstrated when bone marrow colony-forming units granulocyte-macrophage (CFU-GM) progenitor cells were evaluated. Moreover, AS101 and bryostatin synergized in their protective effects against lethal damages of irradiation. These results strongly suggest that bryostatin, which lacks tumor-promoting activity, is a particularly good candidate in combination with AS101 for treatment in vivo in counteracting chemotherapy- or radiation-induced hematopoietic suppression or in generally improving the restoration of immune response under conditions involving immune or hemopoietic damage.     

Kinetics of haemopoietic recovery in endotoxin-treated mice.

Kinetics of mouse spleen colony forming units were studied after intra-peritoneal injection of 1 mug/blody weight bacterial endotoxin S. typhosa. When these mice were used as unirradiated and sublethally irradiated donors, it was possible to study the effect of the endotoxin injection upon the cells.Use of the treated mice as irradiated recipients of normal cells gave information about the host effect. In treated unirradiated mice, the total nucleated cell and the CFU counts were disturbed, and 2 days later a large fraction of the CFU were found in the DNA synthesis (S) phase. This meant that injection of endotoxin generated factors affecting the kinetics of the CFU and triggering the resting CFU into the proliferative cycle. If then the mice were given supralethal irradiation and used as recipients of normal bone marrow cells, more CFU seeded to the spleen as compared to normal recipients; but the dip and the growth rate of the CFU were not changed. Hence the endotoxin-generated factors had been eliminated in 2 days. A total body sublethal irradiation by 400 rad X-ray 2 days after endotoxin injection reduced the post-irradiation dip in the recovery curve of the CFU, indicating that though the factors affecting the cell kinetics had been eliminated, the cycling CFU behaved like a growing population. During the first week, the growth rate of the CFU remained the same as in control irradiated mice. The growth rate of the spleen CFU of the endotoxin-treated mice slowed down during the second week, and their self-replicating ability was low. Fluctuations in the DNA synthesizing fraction of the spleen CFU suggested a variability in the ratio of the length of the S phase and the cell generation time.     

Effect of fractionation and rate of radiation dose on human leukemic cells, HL-60

The capacity of HL-60 cells, human acute promyelocytic leukemic cells established in culture, to repair sublethal radiation damage was estimated from the response of the cells to fractionated irradiation or to a single irradiation at different dose rates. The HL-60 cells grown as a suspension culture in RPMI 1640 medium supplemented with 10% calf serum and antibiotics showed a cloning efficiency of about 0.46 in an agar culture bed. After exposure of cells to a single dose of X rays at a dose rate of 78 rad/min, the survival curve was characterized by n = 2.5, Dq = 80 rad, and D0 = 83.2 rad. Split-dose studies demonstrated that the cells were able to repair a substantial portion of sublethal radiation damage in 2 hr. The response of the cells to irradiation at different dose rates decreased with a decrease in the dose rates, which could be attributed to repair of sublethal radiation damage. The radiation response of leukemic cells is only one of the many factors which affect the clinical outcome of total-body irradiation (TBI) followed by bone marrow transplantation. Nevertheless, the possibility that some of the malignant hemopoietic cells, if not all, may possess a substantial capacity to repair sublethal radiation damage should not be underestimated in planning total-body irradiation followed by bone marrow transplantation.     

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.     

KINETICS OF HAEMOPOIETIC RECOVERY IN ENDOTOXIN-TREATED MICE

Kinetics of mouse spleen colony forming units were studied after intra-peritoneal injection of 1 μ/g body weight bacterial endotoxin S. typhosa. When these mice were used as unirradiated and sublethally irradiated donors, it was possible to study the effect of the endotoxin injection upon the cells. Use of the treated mice as irradiated recipients of normal cells gave information about the host effect. In treated unirradiated mice, the total nucleated cell and the CFU counts were disturbed, and 2 days later a large fraction of the CFU were found in the DNA synthesis (S) phase. This meant that injection of endotoxin generated factors affecting the kinetics of the CFU and triggering the resting CFU into the proliferative cycle. If then the mice were given supralethal irradiation and used as recipients of normal bone marrow cells, more CFU seeded to the spleen as compared to normal recipients; but the dip and the growth rate of the CFU were not changed. Hence the endotoxin-generated factors had been eliminated in 2 days. A total body sublethal irradiation by 400 rad X-ray 2 days after endotoxin injection reduced the post-irradiation dip in the recovery curve of the CFU, indicating that though the factors affecting the cell kinetics had been eliminated, the cycling CFU behaved like a growing population. During the first week, the growth rate of the CFU remained the same as in control irradiated mice. The growth rate of the spleen CFU of the endotoxin-treated mice slowed down during the second week, and their self-replicating ability was low. Fluctuations in the DNA synthesizing fraction of the spleen CFU suggested a variability in the ratio of the length of the S phase and the cell generation time.     

In vivo radioprotection by 5-aminosalicylic acid

The radioprotective effect of 5-aminosalicylic acid (5ASA) was investigated in mouse bone marrow. The present study was aimed at investigating the radioprotective effect of pre-irradiation treatment with 5ASA against a range of whole-body lethal (8-11 Gy) and sublethal (1-4 Gy) doses of gamma-radiation (RT) in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 5ASA at a dose of 25mg/kg body weight (b. wt.) 30 min before lethal RT increased survival, giving a dose modification factor (DMF) of 1.08. Injection of 5ASA (25 mg/kg b. wt.) 60 or 30 min before or within 15 min after 3 Gy whole body RT resulted in a significant decrease in the radiation-induced aberrant metaphases, at 24 h post-irradiation. Maximum effect was seen when the drug was administered 30 min before irradiation. 5ASA (25 mg/kg b. wt.) significantly reduced the number of aberrant metaphases and the different types of aberrations at all the radiation doses (1-4 Gy) tested, giving a DMFs of 1.43 for number of aberrant metaphases. 5ASA pretreatment also significantly enhanced the endogenous spleen colonies in mouse exposed to 11 Gy RT. Pretreatment with 5ASA, protected plasmid DNA (pGEM-7Zf) against breakage induced by RT and Fenton reactants. Using nanosecond pulse radiolysis technique, the bimolecular rate constant of the reaction of 5ASA with hydroxyl radical was found to be 6.7x10(9)M(-1)s(-1). The p53 and p21 protein levels of bone marrow and spleen were evaluated to identify the specific molecular mechanisms. Both p53 and p21 increased 24h after 6 Gy irradiation, while treatment with 5ASA inhibited this RT-induced increase. Therefore, the present data suggest that 5ASA pretreatment decreases death caused by RT-induced gastrointestinal and hemopoeitic syndromes. The proposed mechanism of radioprotection by 5ASA is through the inhibition of damage to DNA, lipids, and proteins; and prevention of RT-induced increased expression of p53 and p21.     

Cloning stem cells in the bone marrow of irradiated mice]

Different amount of intact or irradiated bone marrow from syngenous donors was administered to mice irradiated with a lethal dose. There was revealed a linear dependence of the number of the 8-9-day colonies grown in the bone marrow of the femur on the amount of the administered cells, and an exponential dependence on the irradiation dose. Regularity of the stem cell cloning in the bone marrow was analogous to such in the spleen. Radiosensitivity of the colony-forming units (CFU) differed depending on the site (the spleen, the bone marrow) of their colony formation. The CFU settling in the marrow proved to be more radioresistant (D(0) equalled 160-200 P) in comparison with the CFU settling in the spleen (D(0) constituted 80-100 P). It is supposed that a different radiosensitivity of the CFU was caused by the presence of heterogenic population of the stem cells and also by specific peculiarities of the organ (the spleen, the bone marrow) in which the colonies formed.     

THE PROLIFERATIVE CAPACITY OF HAEMOPOIETIC COLONY FORMING UNITS IN THE RAT

After transplantation into rats lethally treated with cytotoxic chemicals both bone marrow and spleen CFU in the spleen and spleen derived CFU in the bone marrow expand with doubling times ( T _d) of approximately 18 hr. However, bone marrow derived CFU in the bone marrow have a T _d of 36 hr. Evidence obtained using tritiated thymidine in vitro and methotrexate in vivo show that the proliferation rate of bone marrow derived CFU is similar in both the bone marrow and spleen and calculations suggest that the different T _d between these two sites is due to the higher loss of CFU through differentiation in the bone marrow compared to the spleen. These findings further support the hypothesis of an environment in the spleen which favours CFU self-maintenance over differentiation with the opposite situation occurring in the bone marrow.     

A Study of the Regenerative Potential of Bone Marrow Cells of Donor Mice that Carry the <Emphasis Type="Italic">egfp</Emphasis> Gene in Irradiated Mice

A study of the regenerative potential of bone marrow cells of donor mice that express the enhanced green fluorescent protein was conducted in mice irradiated at a dose of 7 Gy. Expression of this protein allowed us to carry out monitoring of the presence of donor cells in recipient blood over the entire lifespan of the recipient. The lifespan of young recipients increased by 93% after transplantation; for old recipients it increased by 15%. Total acceptance of the bone marrow, spleen, thymus, and blood of the recipient with donor bone marrow cells was demonstrated over the entire life of the recipient. Only the donor colonies were detected with the studied irradiation dose and number of transplanted cells (11.7 ± 0.4) · 10⁶ on the spleen surface. The percentage of bone marrow and spleen cells that expressed the CD117 and CD34 stem cell markers in the recipient mice was above the control level for a long period of time after the irradiation. More than half of the cells with CD117, CD34, CD90.2, and CD45R/B220 phenotypes in the studied organs were donor cells. Further detailed study of the peculiarities of the engraftment of bone marrow cells, both without preliminary treatment of recipients and after the effects of extreme factors, will allow improvement of the methods of cell therapy.     

Chemical radioprotection of hemopoietic colony-forming cells: comparative effect of AET, anoxia, and urethan

The bone marrow colony-forming unit (CFU) technique of Till and McCulloch was employed to test the radioprotective effect of AET, anoxia, urethan on marrow cells irradiated in vivo. For AET and anoxia, a dose-reduction factor of 1.9 to 2.1 was found. Since the marrow cells were assayed for CFU content immediately after irradiation of the donor, the observed effect can be interpreted as a "true" radiation dose reduction. By contrast, urethan injection did not increase the survival of marrow CFU assayed immediately after whole-body x-irradiation. However, both urethan and AET afforded radioprotection of endogenous CFU content of spleen and bone marrow, but not of endogenous spleen colony count. It is concluded that the mechanism of radioprotection by urethan is fundamentally different from that of AET or anoxia.     

Effect of erythrocyte breakdown products on mast cells and erythropoietin formation]

Experiments were conducted on CBA mice and albino rats. A study was made of the effect of erythrocyte destruction products (EDP) on the content of hemopoietic colony-forming units (CFU), differentiation of stem cells and the erythropoietin production. It was shown that 3 or 4 EDP injections to normal mice or to lethally irradiated (1000 rad) mice after the transplantation of bone marrow cells caused no changes in the CFU level of stem cells differentiation. In case of a daily (for 3 days) administration of EDP to mice before the irradiation (1000 rad) and bone marrow transplantation there was observed an increase of the colonies count in the recipients' spleen on account of the erythroid colonies. EDP injection caused no changes in the erythropoietic activity of the blood serum. A possible role of erythrocyte destruction products in the mechanism of erythropoiesis autoregulation is discussed.     

Effect of age of mouse recipients on the functional activity of transplanted hematopoietic and lymphoid cells]

When transplanting the bone marrow cells from adult C57BL mice to the lethally irradiated (CBA X C57BL) F1 hybrids of different age, the decrease of the colony forming activity of the stem haemopoietic cells was observed in the spleen of the older recipients, as compared with the 3 months old ones. The joint transplantation of the bone marrow and thymus cells resulted in both the cases in the stimulation of the growth of colonies. The number of endogenous colonies of haemopoietic cells arising in the spleen of animals following the sublethal irradiation was greater in younger hybrids. After the induction of the "transplant versus host" reaction by the lymph node or spleen cells from the CBA mice, the relative weight of spleen and regional lymph node, respectively, in the older recipients exceeded those in the younger ones.     

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.     

Increase in endogenous spleen colonies without recovery of blood cell counts in radioadaptive survival response in C57BL/6 mice

The radioadaptive survival response induced by a conditioning exposure to 0.45 Gy and measured as an increase in 30-day survival after mid-lethal X irradiation was studied in C57BL/6N mice. The acquired radioresistance appeared on day 9 after the conditioning exposure, reached a maximum on days 12-14, and disappeared on day 21. The conditioning exposure 14 days prior to the challenge exposure increased the number of endogenous spleen colonies (CFU-S) on days 12-13 after the exposure to 5 Gy. On day 12 after irradiation, the conditioning exposure also increased the number of endogenous CFU-S to about five times that seen in animals exposed to 4.25-6.75 Gy without preirradiation. The effect of the interval between the preirradiation and the challenge irradiation on the increase in endogenous CFU-S was also examined. A significant increase in endogenous CFU-S was observed when the interval was 14 days, but not 9 days. This result corresponded to the increase in survival observed on day 14 after the challenge irradiation. Radiation-inducted resistance to radiation-induced lethality in mice appears to be closely related to the marked recovery of endogenous CFU-S in the surviving hematopoietic stem cells that acquired radioresistance by preirradiation. Preirradiation enhanced the recovery of the numbers of erythrocytes, leukocytes and thrombocytes very slightly in mice exposed to a sublethal dose of 5 Gy, a dose that does not cause bone marrow death. There appears to be no correlation between the marked increase in endogenous CFU-S and the slight increase or no increase in peripheral blood cells induced by the radioadaptive response. The possible contribution by some factor, such as Il4 or Il11, that has been reported to protect irradiated animals without stimulating hematopoiesis is discussed.     

Radioprotection of mice with interleukin-1: relationship to the number of spleen colony-forming units

Compared to saline-injected mice 9 days after 6.5 Gy irradiation, there were twofold more Day 8 spleen colony-forming units (CFU-S) per femur and per spleen from B6D2F1 mice administered a radioprotective dose of human recombinant interleukin-1-alpha (rIL-1) 20 h prior to their irradiation. Studies in the present report compared the numbers of CFU-S in nonirradiated mice 20 h after saline or rIL-1 injection. Prior to irradiation, the number of Day 8 CFU-S was not significantly different in the bone marrow or spleens from saline-injected mice and rIL-1-injected mice. Also, in the bone marrow, the number of Day 12 CFU-S was similar for both groups of mice. Similar seeding efficiencies for CFU-S and percentage of CFU-S in S phase of the cell cycle provided further evidence that rIL-1 injection did not increase the number of CFU-S prior to irradiation. In a marrow repopulation assay, cellularity as well as the number of erythroid colony-forming units, erythroid burst-forming units, and granulocyte-macrophage colony-forming cells per femur of lethally irradiated mice were not increased in recipient mice of donor cells from rIL-1-injected mice. These results demonstrated that a twofold increase in the number of CFU-S at the time of irradiation was not necessary for the earlier recovery of CFU-S observed in mice irradiated with sublethal doses of radiation 20 h after rIL-1 injection.     

PROPERTIES OF HAEMOPOIETIC STEM CELLS IN PHENYLHYDRAZINE TREATED MICE

Recovery of erythropoiesis was fast in Balb/c mice irradiated 700 R 5 days after initiation of phenylhydrazine treatment and took place predominantly in the spleen, which showed numerous large frequently confluent endogenous colonies. Post irradiation phenylhydrazine induced anaemia did not accelerate recovery of erythropoiesis; it did, however, produce a slight but significant rise in endogenous colony formation.
Radiosensitivity of spleen CFU-S from phenylhydrazine treated mice was similar to that of CFU-S in normal mouse spleen.
Spleen CFU-S in mice 5 days after initiation of phenylhydrazine treatment were sensitive to the lethal action of Hydroxyurea, while bone marrow CFU-S were not.
The self-renewal capacity of CFU-S in the endogenously repopulated spleen of phenylhydrazine pretreated 700 R X-irradiated mice was low when compared to that of spleen exogenously repopulated by cells from normal mouse bone marrow, normal and phenylhydrazine treated mouse spleen. CFU circulating in blood of phenylhydrazine treated mice had a low self-renewal capacity.
The marked strain differences in self-renewal capacity of spleen CFU-S, and of the capacity of spleen CFU-S to increase by proliferation are discussed.     

组胺H2受体拮抗剂对骨髓造血重建的影响

用组胺H_2受体拮抗剂(甲氰咪胍或呋喃硝胺)处理正常和亚致死量γ-射线照射小鼠,探讨正常体内造血和再生骨髓中造血重建与组胺受体的关系。发现非毒性剂量的甲氰咪胍对正常小鼠骨髓多能造血干细胞(CFU-s)无抑制作用,但可抑制小鼠体内粒单系祖细胞(CFU-GM)的生长和亚致死量照射后CFU-s产率的恢复。组胺可能与骨髓的再生有关,组胺H_2受体拮抗剂可抑制骨髓的造血重建。