The effect of aluminium upon the activity of selected bone marrow enzymes in rats.

This study was carried out on male Wistar rats. The bone marrow activities of acetylcholinesterase (AchE-EC.3.1.1.7.), glutathione reductase (GR-EC.1.6.4.2.) and glucose-6-phosphate dehydrogenase (G6PD-EC.1.1.1.49.) were determined as affected by aluminium. The present experiments have revealed that within the first days there will be an increase in enzyme activity in the bone marrow. Simultaneously a decline in the activity of bone marrow could be observed which occurred later in the first course of the experiment.     

Effect of mercury, lead and acetylphenylhydrazine (APHZ) on erythrocyte and bone marrow glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). an electrophoretic study

Male and female rats of Wistar strain have been injected with 0.2% HgCl2, 203HgCl2, PbNO3 and 2% APHZ, in the volume of 0.5 ml saline for 4 days. Subbands of G-6-PD enzyme from erythrocytes and bone marrow cells, were separated by disc polyacrylamide electrophoresis. Four subbands of the enzyme resulted electrophoretic separation. In erythrocytes increase of activity of fraction I. and decrease of fraction II were observed, while in bone marrow opposite reaction was visible. In both tissue following administration of Hg and Pb, fractions III and IV disappeared. In the place of disappeared bands increased radioactivity of 203Hg was detected.     

Oxygen metabolism in rabbit bone marrow and liver.

Oxygen metabolism has been quantified in rabbit bone marrow and liver. NADPH-Cytochrome $\text{c}$ reductase activity in bone marrow microsomal and cytosol fractions was about 40% of that found in liver. Superoxide anion and peroxide generation were found to be present in both liver and bone marrow. Catalase and superoxide dismutase activity were measured in liver and in marrow preparations free of erythrocytes; while liver catalase activity was approximately twice that of bone marrow, very low superoxide dismutase activity was observed in erythrocyte free bone marrow homogenates.     

Immunocompetent cells in the chicken. II. Synergism between thymus cells and either bursa or bone marrow cells in the humoral immune response to sheep erythrocytes

Newly hatched F₁ hybrid chicks isogenic for the strong B histocompatibility locus were rendered immunologically incompetent by cyclophosphamide treatment and x-irradiation. They were then injected intravenously with thymus, bone marrow, or bursa cells together with sheep erythrocytes (SE) and received another iv injection of SE 3 days later. Splenic plaque-forming cells (PFC) and serum hemagglutinins were assayed 7 days after transfer. At donor ages of 14–26 days, cells from thymus (T) and bone marrow (BM) showed synergism when injected together, as indicated by a significantly higher geometric mean of PFC per recipient spleen in the BM + T group than in the BM group. The response of the T group was extremely low. With thymus and bursa cells from 6- to 28-day-old donors, significant synergism was demonstrated in 3 of 9 individual experiments. However, almost all the other 6 experiments showed marked differences in the same direction, and the combined probability for all experiments was < 0.001. The most striking demonstration of thymus + bursa synergism was made in 2 experiments using 1-week-old donors. Bone marrow cells from 1-week-old donors failed to cooperate with thymus, as did BM cells from older bursectomized agammaglobulinemic donors. This suggests that B cells from bone marrow originate in the bursa. Thymus-bursa cooperation was somewhat difficult to demonstrate in individual experiments using donors over 1 week of age, owing to the occurrence of some responses with bursal cells alone and to variability of response within bursa or bursa + thymus recipient groups. Synergism between thymus and bursa cells was more consistently demonstrable when irradiated normal spleen or low doses of bone marrow cells were added. These additions led to an increased response and a lowered coefficient of variation in the thymus + bursa recipient groups. The ‘third’ cell type needed for optimal response by the thymus and bursa cells together was tentatively identified as a macrophage.     

Vitamins C and E prevent AZT-induced leukopenia and loss of cellularity in bone marrow. Studies in mice

A major limitation in the use of AZT for AIDS treatment is the occurrence of side effects, such as leukopenia. The effects of antioxidant vitamins C and E on AZT-induced leukopenia were investigated in mice. Mice were divided into four groups: (1) controls; (2) AZT-treated; (3) treated with AZT plus vitamins C and E; and (4) pre-treated with vitamins and then treated with AZT plus vitamins. Our results demonstrate that AZT causes leukopenia in mice, which was abrogated by administration of vitamins C and E in the pre-treated group. These vitamins prevented the decrease in cellular content induced by AZT in bone marrow and diminished peroxide levels in myeloid precursors in bone marrow. AZT also caused an increase in plasma malondialdehyde and blood oxidized glutathione levels, which was prevented by the administration of antioxidant vitamins. In conclusion, oxidative stress is involved in AZT-induced leukopenia which may be prevented by antioxidant treatment.     

Vitamins C and E prevent AZT-induced leukopenia and loss of cellularity in bone marrow. Studies in mice

A major limitation in the use of AZT for AIDS treatment is the occurrence of side effects, such as leukopenia. The effects of antioxidant vitamins C and E on AZT-induced leukopenia were investigated in mice. Mice were divided into four groups: (1) controls; (2) AZT-treated; (3) treated with AZT plus vitamins C and E; and (4) pre-treated with vitamins and then treated with AZT plus vitamins. Our results demonstrate that AZT causes leukopenia in mice, which was abrogated by administration of vitamins C and E in the pre-treated group. These vitamins prevented the decrease in cellular content induced by AZT in bone marrow and diminished peroxide levels in myeloid precursors in bone marrow. AZT also caused an increase in plasma malondialdehyde and blood oxidized glutathione levels, which was prevented by the administration of antioxidant vitamins. In conclusion, oxidative stress is involved in AZT-induced leukopenia which may be prevented by antioxidant treatment.     

Analysis of the tetrahydrobiopterin synthesizing system during maturation of murine reticulocytes

The enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marrow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh-1c/Ldh-1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP-cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6-pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic pathway.     

Diminished genotoxicity of mitomycin C and farmorubicin included in polybutylcyanoacrylate nanoparticles.

The genotoxic effects of mitomycin C (MMC) and farmorubicin (FR) in a free form and included in polybutylcyanoacrylate nanoparticles (PBCN) were studied employing the Salmonella/microsome mutagenicity assay and the micronucleus test in mouse bone marrow as well as in mouse fetal liver. The data obtained clearly indicated that MMC (0.25-2.00 micrograms/plate) was a strong mutagen in S. typhimurium TA102, while the same concentrations of this compound in PBCN were ineffective in inducing his+ revertant mutations in bacterial cells. A similar total suppression of mutagenic activity of FR (1.0-20.0 micrograms/plate) was registered in S. typhimurium TA98 when the drug was included in PBCN. Furthermore, the incorporation of MMC (2.0 or 4.0 mg/kg, i.p.) into PBCN strongly diminished or even abolished its clastogenic activity in the bone marrow of virgin and pregnant mice as well as in mouse fetal liver, respectively. In addition, a lack of genotoxic effect of PBCN only was also established. The toxic activity of MMC in mouse bone marrow was significantly reduced or completely abolished after its inclusion in PBCN. A conclusion might be drawn that the genotoxic activity of some antitumor drugs might be markedly diminished or even abolished after their incorporation in PBCN.     

Stimulation of suppressor cells in the bone marrow and spleens of high dose cyclophosphamide-treated C57Bl/6 mice

Systemic administration of a single dose (300 mg/kg) of cyclophosphamide (Cy) induced the appearance of a population of suppressor cells in the bone marrow and spleens of mice. Suppressor cells were assayed by their capacity to inhibit the concanavalin A (Con A) blastogenesis or the mixed-lymphocyte response of normal C57Bl/6 spleen cells. Cy-induced bone marrow (Cy-BM) suppressor cells were present as early as 4 days following Cy therapy and their activity gradually decreased over the next 2 weeks. Cy-induced splenic (Cy-Sp) suppressor cells were maximally present on Days 6 through 10 following Cy therapy. Studies were performed to characterize the suppressor cells of bone marrow obtained 4 days after Cy treatment and of normal bone marrow (N-BM). Some suppressor activity was present in normal bone marrow. N-BM suppressor cells resembled cells of the monocyte/macrophage lineage in that they were slightly adherent to Sephadex G-10, sensitive to L-leucine methyl ester (LME), and insensitive to treatment either with anti-T-cell antibody and complement or with anti-immunoglobulin antibody and complement. Their suppressive activity was abrogated by incubation with either indomethacin or catalase. Cy-BM suppressor cells were also resistant to treatment with anti-T-cell and anti-immunoglobulin antibody and complement but were not adherent to Sephadex G-10 and not sensitive to LME. Their suppressive activity was partially eliminated by indomethacin alone or in combination with catalase. We conclude that Cy chemotherapy induces the appearance of a population of immune suppressive cells and that these cells appear first in the bone marrow and subsequently in the spleen.     

The dose dependence of the development of compensatory-restorative body reactions to irradiation. The EPR method]

The study deals with the mechanism of organism's adaptive responses to the effect of radiation in widely ranging dose. Post-irradiation metabolic changes were evaluated in canine blood as well as in murine blood, spleen, bone marrow and liver using the EPR spectroscopy. It was shown that the dynamics of changes in transferrin and ceruloplasmin pools and ribonucleotide reductase activity were phase-dependent with the maxima at the 2nd, 6th and 10-12th days after irradiation. Such dynamics was observed at various irradiation doses applied. The data allow us to suggest that the nonspecific compensatory--adaptive reactions of organisms develop as the response to irradiation. The dose-response function of the reaction intensity was found to be linear. The shape of the dose-response curve indicates that the minimum response of organism depends on the dose linearly up to 3.2 Gy (for dogs) as well as the maximum one. However, in the case of low-dose irradiation (0.25 or 0.5 Gy) there were deviations of maximum responses from the linearity, i.e. the amplification of the amplitude of compensatory adaptive reactions. These effect were shown to be dependent upon initial individual characteristics of animal blood and to be related to the "depressed" or "activated" state of organism prior to irradiation. The ribonucleotide reductase activity was measured in bone marrow and spleen of animals by the EPR method. The nature of non-repairable DNA damage is discussed in view of the inactivation of ribonucleotide reductase.     

Bone marrow-derived macrophage expression of endogenous and transfected class II MHC genes during differentiation in vitro

C57BL/6 (H-2b) mice fail to express I-E molecules on the surface of their cells and thus are unable to respond to I-E-restricted antigens such as GL phi and cytochrome c. Previous experiments in our laboratory have involved developing a system for studying differentiation of bone marrow cells into mature macrophage to gain a better understanding of class II MHC gene expression and function. In this study, we have used this system to transfect the E alpha d gene (cosmid 17.2) into C57BL/6 bone marrow cells and subsequently observed I-E expression on bone marrow-derived macrophages (BMDM) after differentiation in vitro. By using a modified calcium phosphate protocol, we found that the optimal period for transfection of the bone marrow cells was after 2 days of culture in vitro. By using the anti-I-E monoclonal antibody (Ia.7) derived from hybridoma 14-4-4, we detected the I-E molecule on the surface of transfected macrophages by a radiobinding assay and immunoprecipitation. BMDM expressed the I-E product maximally at 5 days of differentiation, and expression then declined. Furthermore, we have found that the expression of the I-E molecule on transfected macrophage was dependent upon exposure to interferon-gamma. Expression of I-E molecules was also detected by the generation of an allogeneic response. Transfected BMDM were compared with (CB6)F1 BMDM for their ability to stimulate C57BL/6 T cells and they were found to be equally effective. By using these initial findings, we hope to further optimize the conditions for insertion and expression of class II MHC genes in bone marrow cells.     

大鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的建立较稳定的异基因骨髓移植急性移植物抗宿主病动物模型,为异基因骨髓移植后的急性移植物抗宿主病（aGVHD）的相关研究提供实验参照。方法以雄性SD大鼠为供鼠,雌性Wistar大鼠为受鼠,受体大鼠随机分成A、B、C、D、E 5组,移植当天所有受鼠均接受8.5 GY的全身照射（TBI）,于照射后4～6 h内,A组回输等量培养液,B组经尾静脉输注供鼠骨髓细胞（2×10^8个/kg）,C、D、E组分别回输供鼠骨髓细胞（2×10^8个/kg）＋不同比例的脾细胞。观察各组大鼠生存期、外周白细胞计数、及有无aGVHD的临床及病理表现。结果A组大鼠于15d内全部死亡,外周血白细胞计数明显减低,骨髓病理示造血组织减少,提示死于造血衰竭。B、C、D、E组大鼠外周血白细胞计数均有明显恢复,B组大鼠8只存活超过50 d,C、D、E组大鼠均于50 d观察期内死亡,并有aGVHD的临床表现及病理表现,但C组大鼠aGVHD的程度较轻且时间不集中,其中D、E组大鼠可于相对集中的时间内观察到典型aGVHD临床及病理。结论TBI预处理的方式是可行的,单纯输入异基因骨髓细胞不能引起明显的aGVHD,骨髓细胞与脾细胞1∶1及1∶1.5混合组均可作为异基因骨髓移植后理想的aGVHD动物模型。     

Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes

The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.     

Reconstitution of the complement function in C1q-deficient (C1qa-/-) mice with wild-type bone marrow cells

Besides Ab-independent and Ab-dependent activation of the complement classical pathway in host defense, C1q plays a key role in the processing of immune complexes and in the clearance of apoptotic cells. In humans, C1q deficiency leads to systemic lupus erythematosus-like symptoms in over 90% of the cases, thus making this defect a strong disease susceptibility factor. Similarly, C1q-deficient mice (C1qa-/-) develop systemic lupus erythematosus-like symptoms, such as autoantibodies and glomerulonephritis. We have previously provided evidence that C1q is produced by cells of the monocyte-macrophage lineage. In this study, we have tested whether transplantation of bone marrow cells would be sufficient to reconstitute C1q levels in C1qa-/- mice. C1qa-/- mice received a single graft of 10(7) bone marrow cells from wild-type (wt) donors after irradiation doses of 6, 7, 8, or 9 Gy. Engraftment was monitored by a Y chromosome-specific PCR and a PCR that differentiated wt from C1qa-/- genotype. Serum levels of C1q Ag and C1 function increased rapidly in the recipient mice, and titers reached normal levels within 6 wk after bone marrow transplantation. In wt mice that received C1qa-/- bone marrow, serum levels of C1q decreased constantly over time and became C1q deficient within 55 wk. These data clearly demonstrate that bone marrow-derived cells are the source of serum C1q and are competent to reconstitute normal C1q serum levels in C1q-deficient mice. Therefore, stem cell transplantation could be a therapy for patients with hereditary C1q deficiency.     

Beta-carotene as an inhibitor of benzo(a)pyrene and mitomycin C induced chromosomal breaks in the bone marrow of mice

Female mice of hybrid strain B6C3F1, 8-10 weeks old, were fed on powdered food with or without beta-carotene (100 mg/kg food). After 1 week of these diets, some of each group of mice were injected i.p. with either benzo(a)pyrene (150 mg/kg) in dimethyl sulfoxide, or mitomycin C (1 mg/kg) in distilled water. In the course of separate experiments, bone marrow samples were collected at various intervals after injection for analysis in the in vivo bone marrow micronucleus assay. At the time at which the maximum induction was observed, which coincided between experiments, the frequency of micronuclei induced by benzo(a)pyrene was reduced by 41-61% and that induced by mitomycin C was reduced by 44-71% in the presence of beta-carotene. Beta-carotene is widely distributed in plant material such as carrots and green leafy vegetables and, as such, is a component of the human diet. Our results suggest that beta-carotene provides significant protection against the genotoxicity of benzo(a)pyrene and mitomycin C.     

Preservation of rat bone marrow with dimethylsulphoxide at -150 degrees C.

The authors tested preserving properties of three concentrations of dimethylsulphoxide (15%, 10% and 7.5%) in preservation of rat bone marrow cells at -150 degrees C. Cells of rat bone marrow were frozen at 1 degree C/min to -20 degrees C, 5 degrees C/min to -80 degrees C and then placed directly at -150 degrees C and held at such temperature for 6 months. Vitality of cells was checked monthly for a period of 6 months by means of several vitality tests with dyes (eosin and trypane blue), autoradiography and erythrophagocytosis. It was found that cells capable of cleavage could be equally preserved at such low temperature with all the three DMSO concentrations while mature cells (granulocytes, reticular cells) revealed considerably higher erythrophagocytic activity when preserved at 15% DMSO and lower activity at 10% and 7.5% DMSO.     

Prostaglandin E2 (PGE2) increases the number of rat bone marrow osteogenic stromal cells (BMSC) via binding the EP4 receptor, activating sphingosine kinase and inhibiting caspase activity

Prostaglandin E(2) (PGE(2)) is bone-anabolic, i.e. stimulates bone formation and increases bone mass. In this study, we explored possible intracellular mechanisms of its increase of osteogenic cells in rat bone marrow. Adherent rat bone marrow cells were counted after 12-48 h or cultured for 21 days and mineralized nodules were counted. Also, apoptosis of marrow cells was measured after in vivo PGE(2) injection. PGE(2) (100 nM) increased 2-3 fold the number of adherent BMSC, an effect which was mediated via binding the EP(4) receptor since it was mimicked by forskolin and 11-deoxy-prostaglandin E(1) (PGE(1)) and was blocked by DDA and L-161982 (EP(4) antagonist). PGE(2) stimulated sphingosine kinase (SPK) activity since its effects were blocked by DMS (SPK inhibitor) and mimicked by SPP (SPK product). PGE(2) reduced the activity of caspase-3 and -8 in BMSC and their inhibitors increased BMSC number and nodule formation. In vivo, PGE(2) prevented the increase in the apoptosis of bone marrow cells caused by indomethacin. We propose that PGE(2) exerts an anti-apoptotic effect on BMSC, thereby increasing their number and subsequent osteoblastic differentiation. Such an effect could explain how PGE(2) stimulates bone formation in vivo.     

Hybrid resistance to parental mast cell precursors in the skin of (WB X C57BL/6)F1-W/Wv mice

When bone marrow cells of (WB X C57BL/6)F1-+/+ (WBB6F1-+/+) and WB-+/+ (WB) mice were directly injected into the skin of genetically mast cell-deficient WBB6F1-W/Wv mice, mast cell clusters appeared at the injection sites. However, the number of WB bone marrow cells necessary for appearance of mast cell clusters was significantly larger than when bone marrow cells of WBB6F1-+/+ mice were used. When WB bone marrow cells were mixed either with WB thymus cells or with silica particles, the proportion of injection sites at which mast cell clusters appeared increased to the level that was observed after the injection of the same number of WBB6F1-+/+ bone marrow cells. When suckling WBB6F1-W/Wv mice of less than or equal to 18 days of age were used as recipients, bone marrow cells of WBB6F1-+/+ and WB mice produced mast cell clusters with a comparable efficiency. Both syngeneic thymus cells and silica particles are known to abrogate the hybrid resistance that is observed in the spleen against parental hematopoietic stem cells. The hybrid resistance in the spleen is not detectable in suckling mice, either. Thus, the poor growth of mast cell precursors in the skin and the poor growth of hematopoietic stem cells in the spleen seem to be regulated by the same mechanism.     

Cytokine production by different population of macrophages following radiation or combined radiation injury

Male mice (CBA x C57BL6)F1 were used for the experiments throughout this study. The levels of spontaneous and LPS-stimulated cytokines production (IL-1 beta, IL-6 and TNF-alpha) by peritoneal, splenic, and bone marrow macrophages were evaluated by means of enzyme-linked immunosorbent assay at 3, 6, 24, 48 and 72 hours after irradiation alone or combined injury (irradiation + thermal burn). The results suggest that macrophages, harvested from the main mice hematopoietic organs (bone marrow, spleen), did not increase cytokines production within the first three days following the 7 Gy gamma-irradiation or combined injury. Peritoneal macrophages revealed a capacity to enhance IL-6 and IL-1 production versus normal healthy mice. There were no significant differences of cytokine-producing activity if macrophages were harvested from irradiated or combined injured mice.     

Induction of nuclear lamins A/C in macrophages in in vitro cultures of rat bone marrow precursor cells and human blood monocytes, and in macrophages elicited in vivo by thioglycollate stimulation

Hemopoietic cells from blood and bone marrow of mammals usually do not express lamins A/C but only lamin B, and this feature distinguishes these cells from the vast majority of somatic cells of the adult animal, which reveal lamins A/C as well as lamin B. Here we have cultivated rat bone marrow precursor cells and human monocytes isolated from peripheral blood in tissue culture supplemented with certain growth factors. These conditions allow bone marrow precursor cells and monocytes to differentiate almost quantitatively into accessory cells and/or mature macrophages. The different cell types in the cultures can be identified both morphologically and by other assays. Antibodies specific for mouse A/C lamins, human A/C lamins, or B lamins have been used to define the lamin complement as a function of time in culture and of cell type. A dramatic increase in lamin A/C-positive cells was observed in the first 3 days of culture with both accessory cells and macrophages expressing lamins A/C as soon as such cell types could be identified. Parallel in vivo experiments showed that treatment with thioglycollate caused the percentage of lamin A/C-positive peritoneal macrophages to increase from 5 to 80% between Days 0 and 6.