Formation of Mononuclear Phagocyte (Macrophage) Colonies by Mouse Spleen Cells in Liquid Culture

When spleen cells of the adult mouse were tested for the formation of mononuclear phagocyte (macrophage) colonies by the liquid culture technique with an incubation period of 7–8 days, about 100 macrophage colonies were produced from 1 × 10⁶ cells. The number of macrophage colonies appearing after 2 days of incubation was small, but thereafter increased progressively up to at least 8 days. In the later stages of incubation (after day 6) large colonies consisting of more than 100 cells appeared. Macrophage colonies in the early stages consisted almost solely of macrophages. On day 6 significant numbers of small round mononuclear cells with no detectable phagocytic activity were seen in the center of large colonies, and by day 8 marked crowding of these cells had occurred. The peripheral region of the large colonies consisted mainly of macrophages and the intermediate region of middle-sized round or slightly stretched cells with weak phagocytic activity. Approximately two-thirds of the colony-forming cells still remained after glass-adherent cells were removed from the spleen cells by passing over a glass-bead column. In cultures of glass-nonadherent cells macrophage colonies were not generated in the early stage. The number of colony-forming cells did not change significantly even after actively phagocytic cells were rigorously removed from the spleen cells. In addition, no macrophage colonies were generated in cultures of spleen cells treated with mitomycin C.     

单核巨噬细胞铁代谢相关蛋白的表达调控

人类机体的铁代谢表现为受限制的对外界铁的吸收和有效的机体内的铁的再循环利用,单核巨噬细胞系统通过吞噬衰老的红细胞,储存和释放铁,在机体铁的循环再利用方面起到了重要的作用。因此,单核巨噬细胞系统对整个机体铁稳态的维持非常重要。近年来,随着转铁蛋白受体1(transferrin receptor1,TfR1)、铁蛋白(ferritin,Fn)、二价金属离子转运蛋白1(divalent metal transporter1,DMT1)、膜铁转运蛋白1(ferroportin1,FPN1),以及铁调素(hepcidin)等在单核巨噬细胞系统中功能和调控机制研究的不断深入,日益加深了人们对单核巨噬细胞系统的铁代谢过程和调控机制的了解。该文综述了铁水平、NO以及炎症等因素对单核巨噬细胞系统TfR1、Fn、DMT1、FPN1、hepcidin等蛋白表达的调控及其机制研究的最新进展。     

Agar Plaque Formation by Mouse Spleen Cells in Response to Vaccination with Vi Antigen and Typhoid Vaccines

Vi-containing Salmonella typhosa organisms were resistant to killing by immune spleen cells in the presence of complement even when the cells were producing Vi antibody.     

Nonspecific Elicitation of Antibody-Forming Cells in the Mouse Spleen by Bacterial Lipopolysaccharide

Mechanisms of nonspecific elicitation of anti-sheep erythrocyte (SRBC) hemolytic antibody plaque-forming cells (PFC) in mouse spleens with an injection of bacterial endotoxin (lipopolysaccharide (LPS)) were studied in comparison with the genesis of naturally occurring ‘background’ PFC in normal mouse spleens and of rapidly arising PFC in mouse spleens after immunization with SRBC. The cytokinetic pattern of anti-SRBC PFC response after an injection of LPS was quite different from that of the response elicited after immunization with SRBC. In addition, even though LPS nonspecifically elicited anti-SRBC PFC response in mice, LPS could not confer any immunological memory on mouse immunocytes for a ‘secondary-type’ anti-SRBC PFC response to restimulation with LPS or SRBC. The administration of rabbit anti-mouse thymocyte immunoglobulin or anti-SRBC antiserum in mice markedly suppressed the PFC response after immunization with SRBC, but did not do so after stimulation with LPS. Neonatally thymectomized mice could still respond to stimulation with LPS, producing anti-SRBC PFC in their spleens. Injections of actinomycin D or cyclophosphamide into mice resulted in obvious reductions of the PFC responses elicited by either LPS or SRBC. However, injections of these immunosuppressive antisera or drugs did not affect the number of anti-SRBC PFC in normal mouse spleens. These results suggest that the geneses of anti-SRBC PFC developed under different conditions, i.e., background PFC, LPS-stimulated PFC, and antigen-stimulated PFC, are quite different from each other, and that the nonspecific elicitation of anti-SRBC PFC by LPS does not require the helper function of T lymphocytes. No obvious difference, however, was observed in the time of ontogenic maturation among these three different anti-SRBC PFC in the mouse spleens judging from when they were first manifested after birth.     

Growth of Mouse L Cells in Shaken Culture and Mengovirus Plaque Formation on L Cells Suspended in Agar

Procedures are described that require a minimum of equipment and maintenance for growing mouse L cells suspended in liquid medium and for plaquing mengovirus on L cells suspended in agar. Viability of L cells during storage for 1 to 2 hr at relatively high concentrations was better in media at 30 C than at 0 C, as measured by viable counts after growth for 24 hr at 35 C. The number and size of plaques increased with increasing concentration of NaHCO(3) in the agar layers, but the relative difference in plaque size was maintained. Large- and small-plaque-size variants had similar virulence as determined by the rates of viability loss of L cells in liquid suspension cultures.     

Platelet-Activating Factor (PAF) Modulates Peritoneal Mouse Macrophage Infection by Leishmania amazonensis

The effects of platelet-activating factor (PAF) on the infection of peritoneal mouse macrophages by Leishmania amazonensis were investigated. Prior to the infection, the parasites and/or the macrophages were treated with PAF and/or one of the following modulators: WEB 2086 (PAF antagonist), and the modulators of protein kinase C, phorbol-12-myristate-13-acetate (PMA), and sphingosine. The infection was inhibited when the macrophages or both the parasites and the macrophages were treated with PAF, but stimulated by PAF-treated parasites. WEB 2086 abrogated PAF effects in both systems. The infection was stimulated when the macrophages were treated with sphingosine plus PAF, but inhibited when the macrophages were treated with sphingosine and the parasites with sphingosine plus PAF. The infection was inhibited by sphingosine-treated parasites, either in the presence or in the absence of PAF. Leishmania amazonensis–macrophage infection was inhibited by PMA in all systems tested. Received: 27 September 2000 / Accepted: 15 December 2000     

小鼠胚胎干细胞的培养

目的:建立小鼠胚胎干细胞(embryonic stem cells,ES)的培养方法。方法:制备G418抗性的原代小鼠胚胎成纤维细胞,经丝裂霉素C处理后成滋养层细胞,将小鼠胚胎干细胞复苏后,应用含白血病抑制因子的ES细胞培养液,培养小鼠ES细胞,观察集落的生长情况,并在光镜下观察细胞形态。结果:小鼠胚胎成纤维细胞生长良好,ES细胞呈克隆状生长,且保持未分化状态。结论:建立了小鼠胚胎干细胞培养的有效方法,为下一步基因打靶奠定基础。     

Initiation of the Primary Immune Response to Sheep Red Blood Cells in the Dissociated Mouse Spleen Cell Culture

From a suspension of mouse spleen cells were separated two functionally different cell types on the basis of their ability or inability to adhere to plastic dishes during a short period of incubation. Morphological observations of cells of these two fractions were made with the aid of histochemical methods. The majority of cells in the adherent fraction possessed β-glucuronidase, which is one of the lysosomal enzymes rich in macrophages or phagocytic cells. In contrast, almost all cells in the non-adherent fraction were devoid of this enzyme activity and identified morphologically as small lymphocytes. The adherent and non-adherent cells were found to associate in cell clusters during the cultivation. In the cultures stimulated with sheep red blood cells, some of the non-adherent cells which were located peripherically in the cell clusters began to show alkaline phosphatase activity in their cytoplasm. This may perhaps indicate that antibody synthesis is going on in these alkaline phosphatase-positive cells, since in an in vivo study such cells were found to arise in the lymph nodes of immunized animals concomitantly with the appearance of specific serum antibody.     

Rosette Colonies of Choanoflagellates (Salpingoeca rosetta) Show Increased Food Vacuole Formation Compared with Single Swimming Cells

Choanoflagellates exist as both single‐celled and colonial forms and filter‐feed by generating water currents using a single apical flagellum. Hydrodynamic modeling studies have differed in predictions of whether single cells or colonies produce greater fluid flow to enhance feeding, and a recent study reported no increase in feeding efficiency of stalked colonies of choanoflagellates compared with single cells. We report that rosette colonies of Salpingoeca rosetta demonstrate higher rates of food vacuole formation compared with unicellular, slow swimmers.     

Morphological Differentiation induced by Prostaglandin in Mouse Neuroblastoma Cells in Culture

PROSTAGLANDIN is known to affect concentrations of cyclic AMP in some cells¹. Dibutyryl cyclic AMP induces irreversible differentiation of mouse neuroblastoma cells in vitro², which raises the question of whether prostaglandin would mimic this effect. I report here that prostaglandins (PG)E₁ and PGE₂ induce irreversible morphological differentiation of mouse neuroblastoma cells in culture as shown by axon formation, whereas PGF₂α does not.     

Inhibition of Nucleic Acid and Protein Synthesis in Mouse Spleen Cells In Vitro by Azathioprine

The effect of azathioprine on macromolecular biosynthesis was studied in mouse spleen cells cultured in vitro. The rate of incorporation of (3)H-thymidine, (3)H-uridine, and (14)C-leucine into acid-insoluble material was used to measure deoxyribonucleic acid, ribonucleic acid, and protein synthesis. Results indicate that azathioprine inhibited nucleic acid and protein synthesis at levels which did not decrease cell viability.     

Marginal Zone Macrophage Receptor MARCO Is Trapped in Conduits Formed by Follicular Dendritic Cells in the Spleen

The marginal zone (MZ) region of the spleen plays an important role in leukocyte traffic and the removal of blood-borne pathogens by resident macrophages. Macrophage receptor with a collagenous structure (MARCO), expressed by MZ macrophages, recognizes several microbial ligands and is also involved in the retention of MZ B cells. Here, we report that MARCO is also associated with follicular dendritic cells (FDCs) in the spleen. In its FDC-associated form MARCO is arranged in 0.3–0.5-μm diameter granular-fibrillar structures with an appearance similar to the white pulp conduit system formed by fibroblastic reticular cells (FRCs), but with different compartment preference. The follicular display of MARCO resists irradiation and requires the presence of both MZ macrophages and differentiated FDCs. The follicular delivery of MARCO is independent from the shuffling of marginal zone B cells, and it persists after clodronate liposome-mediated depletion of MZ macrophages. Our findings thus indicate that MARCO is distributed to both MZ and follicles within the spleen into conduit-like structures, where FDC-bound MARCO may mediate communication between the stromal microenvironments of MZ and follicles.     

无血清无饲养层条件下培养小鼠胚胎干细胞

目的研究在无血清无饲养层条件下小鼠胚胎干细胞的培养方法,为最终建立无血清无饲养层培养系统打下基础。方法比较小鼠胚胎干细胞ES-S8株在无血清培养体系和有血清培养体系中的生长情况,分析ES-S8细胞克隆形成效率,测定其生长速度;然后在撤去血清和饲养层的条件下培养ES-S8细胞,进行AKP染色和表面标记物SSEA-1免疫荧光检测。结果ES-S8细胞在无血清培养条件下细胞生长速度减缓,克隆形成率降低,但AKP染色、SSEA-1免疫荧光均显阳性;在无血清无饲养层条件下ES-S8细胞培养仍能形成克隆,且AKP染色、SSEA-1免疫荧光均显阳性。结论研究表明ES-S8细胞能够在无血清无饲养层的培养条件下生长,保持其良好的未分化特性。     

Killing of Cells in Bacterial Colonies

Bacterial colonies grown on membrane filters and transferred to plates of inhibitor-containing medium decrease in number of total viable colony-forming cells. The decrease in viable cell count occurs in direct proportion to the concentration of inhibitor present and the length of time the colonies are exposed to the inhibitor, suggesting that the kinetics of cell kill approximate a first-order chemical reaction.     

Haematopoiesis In Mice Heterozygous For the W Trait: Defective Formation of Transient Endogenous Spleen Colonies

It has been determined that W/+ and W^v/+ heterozygous mice, as compared with normal +/+ homozygous littermates, form significantly lower numbers of transient 5-day endogenous spleen colonies in response to X-irradiation. This defect was evident for doses of irradiation between 2–6 Gy (200–600 rad) and was associated with a slightly increased radiosensitivity of the assayed precursor cells (TE-CFU) in W heterozygotic mice. Moreover, the defect was transplantable, i.e., intrinsic to the marrow cells and not to the microenvironment, and was not associated with a similar decrease in cells which form erythropoietic bursts in vitro (BFUe). This study provides a cellular basis for increased radiosensitivity of W/+ and W^v/+ mice and suggests that the ‘W’ mutation is semi-dominant, both with respect to the white spotting and TE-CFU formation.     

Co-Infection of Mouse Spleen Cells with Murine Sarcoma Virus and Guaroa Virus

Enhancement of tumor induction by oncornaviruses through a dual viral infection has been described by several investigators. The mechanism(s) of this enhancement has not as yet been determined. By using a murine sarcoma virus and a Bunyawera group arbovirus (Guaroa virus) in an in vitro system, evidence was obtained for enhancement of the oncogenic potential of the viruses by genetic interaction with the nononcogenic virus as well as the production of increased amounts of the oncogenic virus. These results confirm and extend similar responses obtained in in vivo systems.     

Expression of Myelin Components in Mouse Schwann Cells in Culture

Mouse Schwann cells cultured in vitro are capable of expressing basal levels of the major myelin components P1, P2, P0, and galactocerebroside. Numerical counts of immunostained cultures indicated that between 22 and 40% of the cells are positive up to 21 days for all of the components indicated. Electrophoretic analysis of Schwann cells labeled with a 14C-amino acid mixture revealed the presence of proteins with relative mobilities identical to those of P0 and P1. Positive identification of the two proteins was indicated by immunoprecipitation of P1 and immunoblotting of P0. These data show that in the absence of neurites, Schwann cells in culture can express low levels of myelin characteristic components even in the absence of myelin assembly.     

Synchronization of Mouse Fibroblast LS Cells grown in Suspension Culture

THERE are two classes of method for synchronizing a population of cells in culture¹ and various methods for inducing the synchrony of mitosis² or DNA replication^3–7. We have examined the available means of achieving a synchronous population by selecting a small fraction of the total population^8–13.