Use of peritoneal macrophages labeled with Cr51 and cultivated in vitro as target cells for quantitative assessment of the cytotoxic activity of immune T-lymphocytes]

Peritoneal macrophages previously labeled in a suspension with 51Cr and grown in the form of a discontinuous monolayer on glass, pretreated with poly-l-lysin were used as target cells. This permitted to estimate the cytotoxic activity of immune lymphocytes by 51Cr release for the period of 4 and 20 hours of incubation with the target. 51Cr release from such a target incubated with normal lymphocytes for 20 hours did not exceed 10-20% of the maximum release. Application of a 2% solution of sodium dodecylsulfate ensured a 100 per cent solubilization of labeled macrophages growing on the glass surface, and permitted the cytotoxic effect to be determined by measuring the label remaining in the intact cells.     

Production of presumptive humoral haematopoietic regulators in canine cyclic haematopoiesis

Abstract. Conditioned media (CM) were prepared according to previously published techniques from the bone marrow of dogs with cyclic haematopoiesis (CH). CM prepared from day 9 marrows inhibited mouse bone marrow CFU-s proliferation rate while CM from day 10 marrows were stimulatory and also contained an erythroid stimulating factor which appeared to be erythropoietin. In addition a highly significant trend from CM containing CFU-s inhibitory materials to media with CFU-s stimulatory activity was observed through cycles day 1 to 8. These studies further support the concept that CH is due to a defect in factors controlling stem cell proliferation and suggest that a major event occurs in CH dog marrow on days 9 and/or 10 of the cycle. Bone marrow transplantation studies (Dale & Graw, 1974; Weiden et al., 1974; Jones et al., 1975b) have indicated that canine cyclic haematopoiesis (CH) is probably due to a disorder in the multipotential stem cells. Morphological evidence (Scott et al., 1973) and the almost synchronous cycling of CFU-e, CFU-c and diffusion chamber progenitor cells (DCPC) (DUM et al., 1977, 1978a, b) lend support to such a theory. However, efforts to identify the mechanisms controlliig multipotential stem cell proliferation in dogs have been handicapped by the lack of suitable techniques to study these cells in the canine. Recently, Wright and co-workers (Wright & Lord, 1978, 1979; Wright et al., 1979; Lord et al., 1979), on the basis of previous observations (Frindel et al., 1976; Frindel & Guigon, 1977), described the preparation of species non-specific, bone marrow conditioned media (CM) which are capable of influencing the proliferation rate of murine colony forming units-spleen (CFU-s). The studies now reported were designed to determine if CM prepared from canine CH marrow would influence the proliferation rate of murine bone marrow CFU-s. The results indicate that a major event, possibly related to the in vivo control of stem cell proliferation in dogs with CH, occurs on days 9–10 of the cycle; day 1 being the first day when the peripheral blood neutrophil count falls below-1600 mm³.     

IFN-gamma enhances sensitivity of human macrophages to extracellular ATP-mediated lysis

In an effort to determine the mechanism by which autologous monocytes are killed by lymphokine-activated killer cells, soluble mediators were examined for their direct effect on target cells. Extracellular ATP (ATPo), but not ADP, was found to lyse human culture-derived macrophages in a 6-h 51Cr-release assay. Treatment of monocytes with human rIFN-gamma rendered those cells significantly more sensitive to ATPo compared to untreated or granulocyte-macrophage CSF-(GM-CSF) treated cells. In addition, IFN-gamma-treated macrophages released approximately 80% of 51Cr label within 15 min after the addition of ATPo, whereas GM-CSF-treated cells did not release significant levels of radiolabel until 4 to 6 h after initial stimulation with ATPo. Time course studies also demonstrated that 3 days of incubation of macrophages with IFN-gamma induced optimal sensitivity to ATPo, although some effect was noted after 4 h of incubation. Thus, IFN-gamma treatment of macrophages elicited increased sensitivity to ATPo-mediated lysis, a phenomenon characterized by rapid release of 51Cr from labeled cells and which is possibly due to induction or activation of surface ATP-binding receptors different from those present on GM-CSF-treated or untreated macrophages.     

Clastogenic effects of acrylamide in mouse bone marrow cells

Acrylamide, known to induce dominant-lethal mutations (Shelby et al., 1986; Smith et al., 1986) and heritable translocations (Shelby et al., 1987) in rodent germ cells, was hitherto a questionable clastogen in rodent bone marrow (Shiraishi, 1978). Therefore, it was tested for chromosomal aberrations in mouse bone marrow cells, spermatogonia and by the micronucleus test. The intraperitoneally injected doses ranged from 50 to 150 mg/kg. In the chromosomal bone marrow test and the micronucleus assay positive results were obtained with acrylamide, and in the latter test the effect increased linearly with dose. Chromosomal aberrations were not induced in differentiating spermatogonia by the acute acrylamide treatment. Cisplatin was used as a positive control and gave the expected positive response in all 3 tests. The present results demonstrate that acrylamide is no exception among clastogens. It breaks chromosomes not only in mammalian germ cells but also in somatic cells.     

The regulation of hematopoietic stem cell proliferation and differentiation (CFU-S) during antigenic exposure

Hemopoietic stem cell (CFUs) proliferation is controlled by regulatory activities (stimulator and inhibitor) produced by bone marrow macrophages. Previously it has been shown that antigen administration stimulates CFUs proliferation. The data obtained in this study show the possible mechanism of antigen-induced stimulation of CFUs proliferation. 3-4 days after antigen injection bone marrow cells of BDF1 mice cease to produce inhibitory activity in contrast to similar cells of control animals. Therefore, increased CFUs proliferation in immunized mice can be due to decreased production of inhibitory activity and resulting abundance of stimulating factors. In BAlB/c mice CFUs proliferation is not changed after antigen injection and their bone marrow cells continue to synthesize inhibitory substances. Differentiation of CFUs into committed blood precursor cells may depend on the proliferation level in CFUs population since activation of CFUs proliferation in immunized BDF1 mice is accompanied by a decreased number of CFU-GM and CFU-M but an increased number of BFU-E. It should be noted that intact BAlB/c mice show a high level of CFUs proliferation similar to that of immunized BDF1 mice.     

Mechanism of induction of micronuclei and chromosome aberrations in mouse bone marrow by multiple treatments of methotrexate.

Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR), slightly induced micronuclei and this induction of micronuclei was enhanced by multiple treatments with the drug (Yamamoto et al., 1981; Hayashi et al., 1984; CSGMT/JEM.MMS, 1990). More micronuclei and chromosomal aberrations in mouse bone marrow cells were induced by multiple than by single treatment. The MTX level in mouse plasma and bone marrow showed little (or no) differences between single and quadruple treatments several hours after the injection(s). On the other hand, the DHFR activity in bone marrow cells 3 h after one and four injections was decreased to approximately 38 and 0%, respectively, of that in non-treated mice. Furthermore, the intracellular MTX level in the bone marrow cells (but not in total bone marrow) after four injections was about 10-fold higher than that after one injection. The amount of MTX bound to protein 3 h after four injections, as assayed by gel filtration (Sephadex G-25), was approximately 8-fold greater than after one injection. Therefore, the multiple-dose effects of MTX on the induction of micronuclei and chromosomal aberrations may be explained by the intracellular accumulation of MTX resulting in an enhancement of enzyme inhibition.     

猫骨髓间充质干细胞的体外分离培养、纯化、鉴定

目的:分离、培养、纯化家猫的骨髓间充质干细胞,并对获得细胞的表面标志物进行鉴定,为进一步利用骨髓间充质干细胞的细胞移植实验奠定基础。方法:采用全骨髓贴壁法体外分离、培养、纯化家猫骨髓间充质干细胞,通过多次更换培养液获得较纯化的骨髓间充质干细胞,倒置相差显微镜下对细胞形态进行观察;根据第1、3、5、7、9代细胞的镜下增殖情况绘制出生长曲线;通过流式细胞仪检测细胞表面标志抗原CD34、CD44和CD90的表达率。结果:在倒置相差显微镜下观察,分离培养的骨髓间充质干细胞贴壁呈梭形或纺锤形;原代细胞生长丛集成片,5~7 d达到融合,进行传代;培养到第三代以后,细胞出现相对均匀的梭形扁平外观,迅速增殖的细胞呈涡流样排列;第3、5代骨髓间充质干细胞增殖能力强于第7、9代;采用流式细胞仪分析结果显示细胞的CD34、CD44和CD90阳性率分别为17.5%、97.9%和91%,这与骨髓间充质干细胞表面抗原的表达一致。结论:分离培养的细胞具有骨髓间充质干细胞特性,成分相对单一,第3、5代细胞纯度高,增殖能力强,适用于进一步的实验研究。     

Transforming growth factor-beta enhances the M-CSF and GM-CSF-stimulated proliferation of macrophages.

Transforming growth factor-beta (TGF-beta) has been shown to regulate the proliferation and function of several different cell types in the immune system. We have examined the effect of TGF-beta on the proliferation of murine macrophages in liquid culture. TGF-beta by itself did not induce proliferation of differentiated (7 days in culture) bone marrow-derived macrophages (BMM). In the presence of M-CSF, TGF-beta enhanced the proliferation of differentiated BMM and elicited peritoneal macrophages but had an inhibitory effect on the proliferation of nonadherent BMM (3 days in culture). The effect of TGF-beta was not restricted to M-CSF-dependent proliferation but was also observed for GM-CSF-dependent proliferation. The autocrine production of TGF-beta appeared to contribute to the proliferation of BMM. The addition of antibody against TGF-beta inhibited M-CSF- and GM-CSF-dependent proliferation 32% and 28%, respectively. In bone marrow, TGF-beta may be an important negative regulator of macrophage proliferation; whereas, in the tissues, TGF-beta may enhance macrophage proliferation.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.     

Antigen-specific Lyt-2+ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.     

Immune cytokines and dexamethasone influence sensory regeneration in the avian vestibular periphery

Prior studies have shown that macrophages are recruited to sites of hair cell lesions in the avian inner ear in vitro (Warchol, 1997) and in vivo (Bhave et al., 1998). Although the avian ear has a high capacity for sensory regeneration (Oberholtzer & Corwin, 1997; Stone et al., 1998), the role of macrophages in the regenerative process is uncertain. The present study examined the possible influence of macrophages and immune cytokines on regenerative proliferation in the avian utricle, one of the sensory endorgans of the vestibular system. Utricles from post-hatch chicks were placed in organ culture and hair cell lesions were created by incubation in neomycin. The cultures were then maintained for an additional 24–48 hours in vitro, and some cultures were treated with dexamethasone, which inhibits macrophage activation and cytokine production. Following fixation, resident macrophages were identified by immunoreactivity to CD68. Labeled macrophages were present in all specimens and increased numbers of macrophages were observed following neomycin treatment. Regenerative proliferation in dexamethasone-treated specimens was reduced by about 50%, relative to untreated controls. Additional experiments showed that two macrophage secretory products—TGF-α and TNF-α—enhanced the proliferation of utricular supporting cells. The results are consistent with a role for macrophages in hair cell regeneration.     

A monoclonal antibody raised against Alzheimer cortex that specifically recognizes a subpopulation of microglial cells

A monoclonal antibody (MAb), termed AMC30, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer's brain. The antigen to which AMC30 is directed was expressed by microglial cells in senile plaques of Alzheimer's disease (AD). Microglia in the parenchyma surrounding brain tumors or infarctions, multinuclear giant cells, perivascular and parenchymal macrophages throughout the brain of AIDS patients were also labeled. Different non-nervous system lesions in which macrophages participate were also stained. Microglial cells in normal areas of the cortex or white matter were not labeled with MAb AMC30. The antigen to which AMC30 is directed was not detected in normal bone marrow, lymph nodes, lung, or spleen monocytes or macrophages. The epitope recognized by MAb AMC30 was present after formalin fixation and paraffin embedding. Our findings suggest that this MAb is directed against an antigen that is specifically expressed in a subpopulation of microglial cells and macrophages reactive to various pathological conditions.     

Different antigen-presenting cells differ in their capacity to induce lymphokine production and proliferation of an apo-cytochrome c-specific T cell clone

The activation of an apo-cytochrome c-specific T cell clone was found to differ, depending on the antigen-presenting cell population. Whereas total syngeneic spleen cells and bone marrow macrophages could be shown to trigger proliferation, IL 2, and MAF production by the T cell clone, a B cell lymphoma only induced MAF secretion. Further studies demonstrated that this effect was not due to a different antigen processing by the B lymphoma or to limiting amounts of Ia and antigen molecules on the B lymphoma cell surface. The dissociation of induction of MAF production from IL-2 production/proliferation found with the different antigen-presenting cells indicates strongly that molecules other than Ia and antigen may be required for the complete functional activation of antigen-specific T cell clones.     

Role of Nanog in the maintenance of marrow stromal stem cells during post natal bone regeneration

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ～50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ～80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ～3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ～50% decrease was seen in the expression of terminal osteogenic gene expression and a ～50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ～2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.     

Regulatory role of bone marrow in immunogenesis. II. Analysis of various mechanisms of antibody genesis suppression by the bone marrow B cells in vitro]

The suppressive effect of the cells on the bone marrow of the B-lymphocytic series on the production of antibody-forming cells to sheep red blood cells, observed on their addition into the culture of the spleen cells after Mishell and Dutton, was mediated only by the live cells capable of proliferation, and was independent of the histocompatible differences between the bone marrow and the spleen cells and of the preliminary immunization of the bone marrow donors.     

Micronucleus test in bone marrow of mice treated with 1-nitropropane, 2-nitropropane and cisplatin

Micronucleus tests were carried out in bone marrow of mice treated with 1-nitropropane, 2-nitropropane and cisplatin. For 1-nitropropane and 2-nitropropane the results were negative. With cisplatin a dose-dependent increase in the number of polychromatic erythrocytes with micronuclei was observed. The lowest positive dose was 0.1 mg/kg (P less than 0.001, Mann-Whitney-Wilcoxon test). The hepatocarcinogen 2-nitropropane showed clastogenic activity in human lymphocytes in vitro in the presence of S9 (Bauchinger et al., 1987). The negative results in bone marrow suggest that short-lived genotoxic metabolites may be formed in the liver but do not reach the bone marrow.     

The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing: a finite element study based on sheep experiments

During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory (Isakson et al., 2006). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.     

Changes in the homing properties of labeled lymphoid cells caused by solid tumor growth

The effect of a progressively growing fibrosarcoma upon the distribution of ⁵¹Cr-labeled cells from the lymph nodes, spleen, thymus, bone marrow and Peyer's Patches was measured in tumor-bearing recipient mice. Tumor presence caused a uniform depression of migration of labeled cells to the bone marrow. In most cases increased homing of cells to the spleen was also observed. Labeled cells prepared from lymph nodes and Peyer's Patches were generally unaffected by the presence of a growing tumor. Migration of labeled cells from tumor bearing donors into normal syngeneic recipients suggests depletion or incapacitation of parts of the T-cell population of the spleen. These results emphasize the important relationship between splenic function and tumor progression.     

Monoclonal antibody 53.6 recognizes a novel proliferation-associated antigen encoded on human chromosome 11

This paper describes the characterization of a novel cell surface antigen associated with proliferation. Previous work demonstrated that monoclonal antibody 53.6 reacted with every human cell line tested, as well as with subpopulations of normal bone marrow and peripheral blood lymphocytes. Mitogen stimulation of peripheral blood lymphocytes with phytohemagglutinin resulted in increased expression of the antigen recognized by 53.6. Immunoprecipitation of biosynthetically labeled KG-1A cell extracts with 53.6 revealed that the antigen is a nonglycosylated acidic protein of Mr 34,000. Analysis of mouse-human hybrid cell lines indicated that the structural gene for the antigen is encoded on chromosome 11. The antigen recognized by 53.6 is distinct from previously described cell surface antigens based on its distribution on activated cells and biochemical characteristics. These studies indicate that the 53.6 antigen is a novel proliferation-associated antigen, and may be useful in analyzing lymphocyte activation.     

Flow cytometric assay for the measurement of human bone marrow phenotype, function and cell cycle

A flow cytometric assay for the measurement of human bone marrow and blood leukocyte antigen expression, phagocytosis, and proliferation is described. Subpopulations of leukocytes were identified by their light scatter characteristics, and the expression of a myeloid differentiation antigen (designated CDw65) determined following incubation with CDw65 specific fluorescein-isothiocyanate (FITC) conjugated monoclonal antibodies (VIM2). Incubation of leukocytes with ethidium monoazide (EMA) labeled Candida albicans followed by staining with FITC conjugated VIM2 allowed the combined determination of cellular CDw65 expression and phagocytic capacity. In addition, immunostained leukocytes were fixed, and their DNA labeled with propidium iodide (PI), before CDw65 expression was measured for cells in different phases of the cell cycle. The method allows evaluation of phenotypic and functional heterogeneity, as well as cell cycle parameters, within subpopulations of cells during hematopoietic differentiation.