Inducible protective processes in animal systems: IV. Adaptation of mouse bone marrow cells to a low dose of ethyl methanesulfonate

Ethyl methanesulfonate (EMS), a monofunctiona) alkylating agent,was used in the present investigations to investigate the inductionof adaptive response (inducible protective processes) in mitoticcells of Swiss albino mouse. When a low (conditioning) doseof 80 mg/kg body wt was challenged with a subsequent high (challenging)dose of 240 mg/kg body wt, after different time lags, the yieldof chromosomal aberrations in bone marrow cells was found tobe significantly reduced compared with that of the challengedose. It appears, therefore, that a low dose of EMS offeredresistance to the mitotic cells against further dastogenic effectof any challenge dose of EMS employed. It is clear from theresults that the phenomenon of adaptive response can also beencountered in mammalian in vivo systems. ¹To whom correspondence should be addressed     

Inducible protective processes in animal systems VI. Cross-adaptation and the influence of caffeine on the adaptive response in bone marrow cells of mouse

The effect of caffeine (CAF) (a replicative DNA synthesis inhibitor) given as pre-, inter- and post-treatments on the ethyl methanesulfonate (EMS)-induced adaptive response in in vivo mouse bone marrow cells was studied in order to understand the influence of CAF on the adaptive response. The pre-treatment was given 4 h before a combined treatment with EMS (conditioning + challenge) and in another set CAF was given as a conditioning dose and 4 h later the cells were challenged with a high dose of EMS. In the inter-treatment, CAF (40 mg/kg body wt) was administered 2 or 4 h after the conditioning dose of EMS and 6 or 4 h later the cells were challenged with a high dose of EMS. Similarly, in the post-treatment experiments, CAF was injected 6, 12 or 18 h after a combined treatment with EMS. The results revealed that the pre-, inter- and post-treatments with CAF significantly reduced the frequency of chromosomal aberrations compared with the challenge and combined treatments with EMS. It is interesting to note that CAF pre-treatment resulted in a much greater reduction in chromosomal aberrations compared with the inter- and post-treatments. Thus, this is an example of cross-adaptation induced by CAF in EMS-treated in vivo mouse bone marrow cells and the results also demonstrate an influence of CAF on the adaptive response.     

Inducible protective processes in animal systems: VIII. Enhancement of adaptive response by nicotinamide

The molecular mechanism of the adaptive response or inducible DNA repair process has not been clearly demonstrated in eukaryotic systems. The involvement of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme has been reported in the adaptive response (Shadley and Wolff, 1987; Wiencke, 1987). Hence, the present studies were undertaken to understand the role of PARP in ethyl methanesulfonate (EMS)-induced adaptive response in mouse bone marrow cells by employing the inhibitor of this enzyme, nicotinamide. Inter-, pre- and post-treatments of nicotinamide with EMS were made. The results have revealed that there is a reduction in the frequencies of chromosomal aberrations compared with combined or challenge treatment at the different recovery times tested. These results are discussed with reference to the enhancement of the adaptive response by nicotinamide in mouse bone marrow cells.     

Apoptosis of hemopoietic cells in irradiated mouse bone marrow.

Apoptosis of hemopoietic cells in bone marrow following radiation has been rarely reported, let alone studied quantitatively and pathologically. LACA mice were irradiated with 2.5, 4.0, 5.5, and 7.0 Gy gamma-rays, and the bone marrow was examined at 6 hours, 1 day, and 3 days after radiation. Semi-thin and thin sections were examined by light and electron microscopy. The number and area density of the apoptotic cells were assessed by means of a Cambridge Quantimet 970 Image Analyzer. We found that apoptosis occurred in only a few hemopoietic cells in the control mouse bone marrow, whereas 6 hours after radiation there were many apoptotic hemopoietic cells in each sample of irradiated bone marrow. Compared with controls, both the number and area density of the apoptotic cells markedly increased in the bone marrow of animals in every radiation dose group, and the difference was statistically significant (p < 0.01). Furthermore, the number and area density increased as the radiation dose increased. Our findings suggest that apoptosis is the main mode of radiation-induced hemopoietic cell death.     

Investigation of immunoglobulin-positive cells in mouse bone marrow

The number of immunoglobulin- (Ig-) positive lymphocytes and of their precursors in mouse bone marrow was investigated 6 and 36 h after treatment with hydroxyurea. The number of Ig-positive B-cells in bone marrow so treated was increased a little, whereas dividing and nondividing precursors of B-lymphocytes were virtually absent, with the exception of stem cells.Laboratory of Cytochemistry and Molecular Biology of Immunogenesis, Institute of Human Morphology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. P. Avtsyn.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 10, pp. 460–462, October, 1978.     

Post-irradiation regeneration of early B-lymphocyte precursor cells in mouse bone marrow.

To examine the sequential development of early B-cell precursors in mouse bone marrow, B-lineage cells have been examined during a wave of post-irradiation regeneration. Cell phenotypes have been defined using double-immunofluorescence labelling techniques for (i) terminal deoxynucleotidyl transferase (TdT); (ii) B220 glycoprotein, detected by the binding of mAb 14.8; (iii); mu heavy chains in the cytoplasm (c mu) and at the cell surface (s mu). Three populations of mu- cells (TdT+14.8-; TdT+14.8+; TdT-14.8+) have been proposed to be early B-cell precursors which would give rise to c mu+s mu- pre-B cells and thus to s mu+ B lymphocytes. From 3 to 7 days after a sublethal dose (150 rads) of whole body gamma-irradiation, the B-lineage cells recovered rapidly to exceed normal numbers. The early B-cell precursors increased to peaks of 1.8-2.5 times normal numbers, preceding by 1 day a comparable increase and overshoot of c mu+s mu- pre-B cells, followed by recovery of s mu+ B lymphocytes. The TdT+14.8- cells peaked first at 5 days, subsiding again at 7-10 days with a shift from large- to medium-sized cells. The TdT+14.8+ cells showed a later peak (6 days), a more sustained wave in cell numbers and a delayed shift in cell size. The substantial population of 14.8+ mu- cells reached maximal observed values at 7 days and still maintained a predominantly large cell size profile at 10 days. The timing, cell-size shifts and progressive amplification of the waves of regeneration accord with a dynamic model in which the TdT+14.8-,TdT+14.8+ and TdT-14.8+ cells form three successive stages in B-cell differentiation before the expression of mu chains, presumptively including the stage of mu chain gene rearrangement. In addition, the results provide an experimental system for the enrichment of early B-cell precursors in mouse bone marrow.     

Distribution of bone marrow cells in the mouse skeleton

Details are given of the distribution of nucleated bone marrow cells in 17 parts of the skeleton of laboratory hybrid mice (CBA x C57BL) weighing 18–21 g. The content of bone marrow in the bones of the spine, head, lower limb, pelvis, upper limb, sternum, and ribs was 33.7, 19.6, 11.9, 8.2, and 9.0% respectively of the total.(Presented by Academician of the Academy of Medical Sciences of the USSR P. D. Gorizontov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 5, pp. 483–485, May, 1979.     

反转录病毒载体转染小鼠骨髓细胞

背景：为方便、准确检测外源细胞在体内的存活情况,需在外源细胞转移标记基因。 目的：观察多种细胞因子联合刺激下反转录病毒载体对BALB/C×C57BL F1代小鼠骨髓细胞基因转移效率的影响。 方法：以PA317-GCGPXSN细胞制备病毒上清,NIH3T3细胞测定病毒滴度后,取经过干细胞因子、白细胞介素3、白细胞介素6预培养刺激后的BALB/C×C57BL F1代小鼠骨髓细胞,实验组实施基因转移,流式细胞仪及PCR方法测定基因转移效率。阴性对照组不做基因转移。阳性对照组为PA317-GCGPXSN细胞株。 结果与结论：①病毒滴度为1.9×10⁸ CFU/L。②对照组BALB/C×C57BL F1代小鼠骨髓细胞测定的荧光强度数值为0.63%,实验组为76.04%,两组相比差异有显著性意义(P < 0.01)。PCR方法扩增到了NeoR基因的特异性片断,结果证实细胞因子预刺激后实施基因转移,可有效地将外源基因转移进入BALB/C×C57BL F1代小鼠骨髓细胞基因组中。     

Hidden Thy-1 antigen in a subpopulation of mouse bone marrow cells.

Immunofluorescence and cytolysis studies using antisera against the xeno- and alloantigenic components of the Thy-1 molecule have shown that in adult mouse bone marrow more cells bear the xeno than the alloantigen. Allo+ cells form a subset of a larger, xeno+ population. Xeno+ allo- 'prothymocytes' can be induced to express the alloantigen in vitro; this could result from the unmasking of cryptic antigen, the presence of which is demonstrated, rather than de novo antigen synthesis. Some xeno+ allo- cells are involved in splenic haemopoiesis, either as a subpopulation of precursors, or as accessory helper cells.     

NK细胞在小鼠异基因骨髓移植中的作用

目的:研究自然杀伤(NK)细胞在异基因骨髓移植中对移植物抗宿主病(GVHD)、移植排斥、骨髓植入及造血重建的影响。方法:以近交系小鼠C57/6j(H-2^b)为供鼠、BALB/c(H-2^d)为受鼠,在移植物中增加供者的外周T细胞和/或NK细胞进行异基因骨髓移植,用流式细胞仪检测受鼠的CD₃₄^＋细胞计数和H-2K^b＋细胞表达水平,血细胞自动分析仪检测外周血白细胞计数,并结合临床表现和病理检查,比较不同移植组的存活率、GVHD、植入水平及造血重建等。结果:增加NK细胞组的小鼠存活率显著大于不增加NK细胞组,小鼠出现GVHD的数量少、程度轻,外周血白细胞及骨髓CD₃₄^＋细胞恢复快、H-2K^b＋细胞表达水平高。结论:NK细胞抑制小鼠异基因骨髓移植中的GVHD和移植排斥,促进骨髓植入及造血重建。     

Developmental stages of myeloid dendritic cells in mouse bone marrow

The lineage relationship of dendritic cells (DC) with other hematopoietic cell types has been studied extensively, resulting in the identification of different bone marrow (BM) progenitors that give rise to distinct DC types. However, the identity of the different maturation stages of DC precursors in the BM remains unclear. In this study we define the in vivo developmental steps of the myeloid DC lineage in mouse BM. To this end, BM cells were separated according to their expression of CD31 (ER-MP12), Ly-6C (ER-MP20) and ER-MP58 antigens, and stimulated to develop into myeloid DC, using granulocyte macrophage colony stimulating factor as a specific growth factor. DC developed from three BM subpopulations: ER-MP12(hi)/20(-) (early blast cells), ER-MP12(+)/20(+) (myeloid blasts) and ER-MP12(-)/20(hi) (monocytes). The kinetic and phenotypic features of DC developing in vitro indicate that the three populations represent successive maturation stages of myeloid DC precursors. Within the earliest ER-MP12(hi)/20(-) population, DC precursors exclusively occurred in the myeloid-restricted ER-MP58(hi) subset. By using switch cultures, we show that these BM precursor subpopulations, when stimulated to develop into macrophages using macrophage colony stimulating factor, retain the ability to develop into myeloid DC until advanced stages of maturation. Together, these findings support a common ER-MP12/20-defined differentiation pathway for both macrophages and myeloid DC throughout their BM development.     

Malathion and fenvalerate induce micronuclei in mouse bone marrow cells

Health effects of pesticides are a major public health concern. In this study, the genotoxic effects of two commonly-used pesticides, malathion, and fenvalerate, were investigated in mice in vivo. Induction of micronuclei in bone marrow cells was used as the test parameter following exposure to 2.5, 5 or 10 mg/kg malathion by intraperitoneal (i.p.) or per oral (p.o.) exposure. Exposure by both routes was found to cause a significant increase in micronucleated polychromatic erythrocytes (PCEs) in a dose-dependent manner (r = 0.9769; P < 0.05). The highest dose (10 mg/kg) induced significant (P < 0.05) cytotoxicity. In contrast, fenvalerate caused an increase in micronucleated PCEs only at higher doses (10 and 20 mg/kg) via i.p. injection, and was not associated with cytotoxicity. A significant dose-response correlation was not observed in the dose ranges tested for fenvalerate (r = 0.8704; P > 0.05). The results suggest that technical grade malathion is a genotoxic agent. In contrast, technical grade fenvalerate appears to be a potent genotoxic agent, but this observation should be confirmed with further investigation(s).     

Beta-carotene as a modulator of chromosomal aberrations induced in mouse bone marrow cells.

The inhibitory effects of beta-carotene on cyclophosphamide (CPA)-induced chromosomal aberrations in mouse bone marrow cells were investigated. Male Balb C mice, 8-10 weeks old, were treated with beta-carotene (0.5, 1.0, 2.0, 5.0, 10, 25, 50, 100, and 200 mg/kg) or with corn oil (0.05 ml/10 g b.w.) by gavage for 5 consecutive days. Four hours after the last treatment with or without beta-carotene, the animals were intraperitoneally injected with CPA and killed 24 hr later for cytological preparations and analysis. The results obtained show that beta-carotene provides significant protection against the clastogenicity of CPA. The maximum reduction in the frequency of aberrant metaphases (26.9%) and in total number of chromosomal aberrations were observed when beta-carotene was used at 50 mg/kg. Nevertheless, no direct dose-response relationship was detected, suggesting that beta-carotene might act through different mechanisms at different doses. The results obtained in animals studies have served as part of the basis for the human intervention studies now underway to determine if beta-carotene does indeed function as a chemopreventive agent in human nutrition.     

Participation of adult mouse bone marrow cells in reconstitution of skin

Results of recent studies have indicated that bone marrow cells can differentiate into various cells of ectodermal, mesodermal, and endodermal origins when transplanted into the body. However, the problems associated with those experiments such as the long latent period, rareness of the event, and difficulty in controlling the processes have hampered detailed mechanistic studies. In the present study, we examined the potency of mouse bone marrow cells to differentiate into cells comprising skin tissues using a skin reconstitution assay. Bone marrow cells from adult green fluorescent protein (GFP)-transgenic mice were transplanted in a mixture of embryonic mouse skin cells (17.5 days post-coitus) onto skin defects made on the backs of nude mice. Within 3 weeks, fully differentiated skin with hair was reconstituted. GFP-positive cells were found in the epidermis, hair follicles, sebaceous glands, and dermis. The localization and morphology of the cells, results of immunohistochemistry, and results of specific staining confirmed that the bone marrow cells had differentiated into epidermal keratinocytes, sebaceous gland cells, follicular epithelial cells, dendritic cells, and endothelial cells under the present conditions. These results indicate that this system is suitable for molecular and cellular mechanistic studies on differentiation of stem cells to various epidermal and dermal cells.     

Age-related variations of paracrystalline inclusions in central reticular cells of mouse bone marrow.

The variations in the quantity of paracrystalline cytoplasmic inclusions (PCI) in bone marrow central reticular cells were observed to correlate closely with the maturation and aging of mice. PCI-bearing central reticular cells of mice increased progressively until 12 months of age, after which a sharp reduction of these cells occurred. The crystalloids appeared to be derived from ingested and degenerated granulocytic components within phagosomes. They were remodelled from amorphous, degenerated products into laminar crystalloids exhibiting periodicity. In view of a recent observation (Shaklai and Tavassoli, 1978) that bone marrow central reticular cells bearing PCI may be closely affiliated with the mouse's hemopoietic microenvironment, the present data suggest that aging may affect the production of this essential factor.     

全骨髓培养法扩增小鼠内皮祖细胞

背景：对于小型实验动物,通过分离骨髓单个核细胞进行内皮祖细胞培养、扩增的方法较为繁琐。 目的：探讨采用全骨髓培养方式扩增小型动物内皮祖细胞的可行性。 方法：采用全骨髓培养分离C57BL/6小鼠内皮祖细胞,培养第7天行DiL-acLDL和FITC-UEA-1双荧光染色检测,并利用流式细胞仪检测其CD34、FLK-1表达情况。同时检测其体外血管生成能力,黏附、增殖和迁移能力。设立骨髓单个核细胞培养法为对照。 结果与结论：全骨髓培养至第2天便可见早期内皮祖细胞集落形成,第7天时可见大量短梭状内皮祖细胞,其具有吞噬DiL-acLDL及结合FITC-UEA-1的能力,内皮祖细胞在基质胶上同样能够形成血管样结构,培养至第2周晚期内皮祖细胞集落出现,迅速生长形成典型的铺路石样,并能够在体外传代培养,细胞数量、细胞表面CD34、FLK-1表达、体外黏附、增殖和迁移能力及晚期集落出现时间与对照组差异无显著性意义(P > 0.05)。说明采用全骨髓培养的方式能够实现小型动物内皮祖细胞的筛选扩增,且操作简便。