Phenotypic characterization of three long-term-cultured murine resident macrophage lines.

The antigenic phenotypes of three long-term cultured murine resident macrophage lines selected in vitro from cell suspensions of different tissues--namely MAY 1 (from the peritoneal cavity), MASP (from the spleen) and MALU (from lung tissue)--were determined using a panel of monoclonal antibodies. The results indicate that all three cell lines belong to the mononuclear phagocyte system and express characteristics indicating a rather high differentiation state. However, there was a significant difference in antigen expression between the two macrophage lines obtained from solid tissues (MASP from spleen and MALU from lung), which were very similar in their antigenic pattern, and the MAY 1 line obtained from the peritoneal cavity, which seemed to be less well differentiated. The antigenic profile of the "mesothelial" cell population associated with the MASP line indicates that this cell population is difficult to characterize and to include in a particular lineage.     

Establishment of replicating long-term lines of rabbit macrophages and lymphocytes.

Macrophages (monocyte derived) and lymphocytes have been successfully cultured from rabbit peripheral blood so that replicating long-term cultures are maintained both in media and in the viable, frozen state. The macrophages are considered as such on the basis of phagocytic capabilities, gamma-globulin receptor sites as evidenced by rosette formation with sensitized red cells and disparate enzyme content from the lymphocytes.     

Mean cell volume of human blood leucocytes and resident and activated murine macrophages

The mean cell volume (MCV) of human blood leucocytes and resident and activated murine macrophages was measured with a Coulter counter connected to a 256 channelyzer. The values found for human blood monocytes, granulocytes, and lymphocytes were 421 +/- 24 femtolitre (fl), 334 +/- 32 fl, and 204 +/- 19 fl, respectively. Resident murine peritoneal macrophages were significantly smaller than rIFN-gamma-activated and BCG/PPD-activated peritoneal macrophages and resident alveolar macrophages.     

Establishment and characterization of factor-dependent macrophage cell lines

Three macrophage cell lines from bone marrow cells of C3H/HeN mice were isolated by successive transfer of the cells in culture with L-cell-conditioned medium (LCM) or WEHI-3 cell-conditioned medium (WEHI-3CM). These cell lines, which express Fc receptors, are involved in Fc-mediated phagocytosis and possess nonspecific esterase activity. Two (BDM-1 and BDM-2) of three cell lines show dependency for growth on either macrophage colony-stimulating factor (M-CSF) (CSF-1) or granulocyte-macrophage colony-stimulating factor (GM-CSF) and do not respond to interleukin 3 (IL-3). The third clone (BDM-3) proliferates in response to IL-3 as well as to GM-CSF and weakly responds to M-CSF and to interleukin 4 (IL-4). GM-CSF, in combination with the suboptimal concentration of M-CSF, acted synergistically on the proliferation of BDM-1 cells. The tumor-promoting phorbol diester, 12-o-tetradecanoyl-phorbol-13-acetate (TPA) also acted synergistically with the three CSFs (IL-3, GM-CSF, and M-CSF) to stimulate the proliferation of BDM-1 cells. The synergistic effect was observed when cells were pretreated with TPA and subsequently stimulated with IL-3. The calcium ionophore A23187 enhanced the proliferation of BDM-1 cells costimulated with TPA and IL-3. These factor-dependent macrophage cell lines should be useful for studying signal transduction mechanisms in the regulation of cell growth.     

Establishment and characterization of two nasopharyngeal carcinoma cell lines

Two human nasopharyngeal carcinoma (NPC) cell lines have been established. One derived from a 64-year-old male, and the other from a 36-year-old female Chinese patient living in Taiwan. Both were keratinizing squamous cell carcinoma in nature and designated as NPC-TW039 and NPC-TW076. Both have been grown in culture system for more than 100 passages. Single cells from both cell lines could form colonies in 0.3% soft agar. In the nude mouse transplantation experiment, both cell lines could produce tumor mass with metastasis. The karyotypic analysis showed multiple chromosomal abnormality. The number of chromosomes ranged between 76 to 109 and 80 to 105 with an average of 98 and 95, respectively. The doubling time was 10.5 hours and 10.8 hours, respectively. The NPC-TW039 cell line has been subcloned and three subclones have been obtained. Ultrastructural studies from those two cell line, three subcloned cell lines and two transplanted tumor masses, all showed two types of morphology: the dark and light cells. This morphologic difference is probably derived from the different metabolic state, but not due to an artifact. Three oncogene probes have been used to check the oncogene expression; none of those five cell lines is positive. Similarly, six Epstein-Barr virus fragments have been labeled to hybridize with NPC cellular DNA preparations, results from the Southern blotting showed no detectable Epstein-Barr virus DNA sequence.     

Establishment and characterization of mouse thymic epithelial cell lines

Primary stromal cell cultures from fetal day-16 thymuses of Swiss mice were developed using a combination of D-valine-containing DMEM and Ham's F-12 medium supplemented with epidermal growth factor, insulin and cortisone. Using cloning cylinders and subsequent limiting dilution techniques, we obtained two clones, MTE-1 and MTE-2. The presence of cytokeratin filaments established their epithelial origin. These cells expressed class I and class II MHC antigens after induction by gamma-interferon, and lacked conventional lymphoid cell-surface markers. Their ability to form rosettes with thymocytes should allow us to identify cell-surface antigens involved in thymocyte-epithelial cell interaction. Moreover, these lines will be used to set up in vitro thymocyte maturation assays.     

Quantitative immunocytochemical characterization of four murine macrophage-like cell lines

The aim of the present study was to obtain quantitative data on the expression of seven cell-surface antigens by the murine macrophage-like cell lines WEHI-3, P388-D1, J774.1, and PU5-1.8, and to compare these data with those obtained for various mononuclear phagocytes. Binding of monoclonal antibodies F4/80, M1/70, 2.4.G.2., 30.G.12, M3/38, M3/84, and 59AD2.2 to cells from these four cell lines was detected by the biotin-avidin immunoperoxidase staining method and quantified by cytophotometry. The results are expressed as the percentage cells expressing a given antigen and the mean specific integrated absorbance per 0.25 micron2 cell-surface area. The results revealed that the phenotypes of the four macrophage-like cell lines differ markedly. Expression of the cell antigens by the cells of the various cell lines did not follow a normal distribution. Although the cell lines divide continuously and have certain characteristics in common with mature mononuclear phagocytes, none of the macrophage-like cell lines accurately resembles any of the mononuclear phagocyte populations. The strongest correlations for membrane were found between on the one hand WEHI-3 and P388-D1 cells and monoblasts and promonocytes (P greater than 0.011) on the other. P388-D1, J774.1 and PU5-1.8 cells expressed four of the six antigens to the same extent as skin macrophages (P greater than 0.012). With respect to expression of antigens recognized by antibodies 2.4.G.2., 30.G.12, M3/38, and M3/84 PU5-1.8 cells resembled activated peritoneal macrophages (P greater than 0.031). It is concluded that WEHI-3 is the most immature cell line, followed by the P388-D1 cell line, while J774.1, and PU5-1.8 are the most mature cell lines.     

Establishment of cell lines latently infected with non-oncogenic murine gammaherpesvirus 76

Murine gammaherpesviruses 68 (MHV-68) and 78 (MHV-78), both inducing tumors in mice and a latent infection in cells in vitro, serve as models for study of human oncogenic gammaherpesviruses, namely Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this work, we succeeded in establishing a latent infection of HeLa and CGL1 cell lines with non-oncogenic murine gammaherpesvirus 76 (MHV-76), which differs from MHV-68 and MHV-78 besides by oncogenicity also by deletion of M1-M4 genes and eight tRNA-like sequences. Viral latency in these cell lines, λHeLa and λCGL1, was demonstrated by the presence of viral DNA, suppression of viral latency-associated ORF73 gene and appearance of low amounts of infectious virus following treatment with phorbol 12-myristate 13-acetate (PMA). Both latently infected cell lines showed irregular presence of viral antigen originating apparently from spontaneous reactivation. The growth of latently infected cells in culture was similar to that of non-infected ones. The latently-infected λHeLa cells did not induce tumors in mice following subcutaneous inoculation. These results (i) confirm that MHV-76 is the only non-oncogenic murine gammaherpesvirus of all the so far tested ones, (ii) suggest that some of the genes deleted in MHV-76 might be responsible for the oncogenicity of murine gammaherpesviruses, (iii) confirm that viral ORF73 is one of major latency-associated genes that is suppressed during virus reactivation, and (iv) present MHV-76 as another murine gammaherpesvirus useful as a model for study of gammaherpesvirus pathogenesis, oncogenicity, latency and reactivation.     

Characterization of surface markers of continuously growing murine resident macrophages

Conditions have been described which allow an in vitro indefinite multiplication of differentiated murine macrophages (Lombard et al: Biol Cell 53, 219, 1985). R. and MAY-1 cell lines, which were obtained, respectively, from mouse (Balb/c) spleen and resident peritoneal macrophages, have been further characterized. They present at their surface, besides the Mac-1 antigen and Fc-receptor, a mannose receptor which was characterized for its binding properties. This receptor is responsive for a specific phagocytosis of mannosylated particles, i.e., mannosylated latex beads or oil droplets containing mannosylated bovine serum albumin. Moreover, R and MAY-1 cells present an ectoenzyme profile (NAD+ glycohydrolase and nucleotide pyrophosphatase) similar to those of the corresponding resident macrophages.     

Establishment and characterization of cell lines from human adenovirus type 12-induced murine tumors producing endogenous virus particles.

Two cell lines designated IC-KMS and D-KMS were established from human adenovirus type 12-induced tumors of C3Hf/OK mouse. The cell lines retained the characteristics of the original tumor i.e., production of numerous C-type and intracisternal A-type particles, integration of Ad12 E1 region DNA and amplification of the myc gene family. Chromosomal analysis revealed chromosome aberrations in both IC-KMS and D-KMS cells. The modal chromosome number of IC-KMS cells was 54 and that of D-KMS cells was 48. Metacentric chromosomes and minichromosomes were found. Trisomy of chromosome 3, 7 and 12 was seen frequently in D-KMS cells. Although DNA aneuploidy was revealed by flow cytometry, the DNA indices of these cells showed no relation to the copy number of integrated Ad12 DNA. These cells have been propagated by serial culture during the past 17 months. Production of endogenous virus particles is a unique characteristic of IC-KMS and D-KMS cells. These cell lines would be useful materials for examining the contribution of Ad12 carcinogenesis to activation of endogenous virus particles, and also the correlation between Ad12 carcinogenesis and cancer-related genes.     

Arginine uptake and metabolism in cultured murine macrophages

Transport ofl-arginine and nitrite production were examined in the murine macrophage cell line J774. Lipopolysaccharide (LPS) induced a dose- and time-dependent stimulation of nitrite production which was further increased in the presence of interferon-γ (INF-γ). Nitrite synthesis was dependent on extracellular arginine and inhibited in the presence ofl-lysine. Treatment of cells with LPS and IFN-γ caused a reduction in intracellularl-arginine concentration which was accompanied by increases in the levels ofl-citrulline in both the cells and culture medium.

These findings indicate that activation of J774 cells with LPS produces an increase in bothl-arginine transport and nitrite synthesis. The elevated rate ofl-arginine transport in activated J774 cells may provide a mechanism for sustained substrate supply during enhanced utilization ofl-arginine for the generation of nitric oxide.

     

Establishment and conventional cytogenetic characterization of three gastric cancer cell lines

Gastric cancer is the fourth most frequent type of cancer and the second most frequent cause of cancer mortality worldwide. Only a modest number of gastric carcinoma cell lines have been isolated thus far. Here we describe the establishment and cytogenetic characterization of three new gastric cancer cell lines obtained from primary gastric adenocarcinoma (ACP02 and ACP03) and cancerous ascitic fluid (AGP01) of individuals from northern Brazil. ACP02, ACP03, and AGP01 cell lines are presently in the 60th passage. The cell lines grew in a disorganized single layer with some agglomerations and heterogeneous divisions (bipolar and multipolar). All cell lines exhibited a composite karyotype with several clonal chromosome alterations. Trisomy 8 was the most frequent alteration. Chromosome 8 aneusomy was confirmed by fluorescence in situ hybridization. All cell lines also exhibited trisomy 7 and deletion of chromosome arm 17p. These results suggest that, although frequent chromosome alterations are commonly observed due to culture process, the ACP02, ACP03, and AGP01 cell lines and primary gastric cancer from individuals of northern Brazil share genetic alterations, supporting use of these cell lines as a model of gastric carcinogenesis in this population.     

抗人成骨肉瘤杂交瘤细胞系的建立及其特性鉴定

目的 建立分泌抗人成骨肉瘤单克隆抗体（mAb)的杂交瘤细胞系，并对其分泌的mAb进行鉴定。方法 用成骨肉瘤细胞系（OSPS-9607）免疫Balb/c小鼠，取脾细胞按常规方法融合，筛选，克隆化及腹水mAb制备，取骨肉瘤手术标本（60例）及正常组织（6种），用mAb进行免疫组化ABC染色。结果 筛选出2株能持续，稳定分泌特异性抗骨肉瘤mAb的杂交瘤细胞株（6E5和3F7），2株杂交瘤细胞经过100d连续培养，分泌mAb的特性稳定，mAb特异性强，效价高。免疫组化染色检测表明，mAb6E5和3F7针对的抗原，在成骨肉瘤组织及成骨肉瘤细胞系均呈高表达（70%）。针对mAb6E5的抗原主要分布于细胞核上，针对mAb3F7的抗原主要分布于细胞浆内，6种正常组织中除胃粘膜可见弱阳性反应外，其余未见明确的阳性反应。结论 此两株杂交瘤细胞分泌的mAb对成骨肉瘤细胞具有特异亲和性，为成骨肉瘤的早期诊断。免疫治疗及发现成骨肉瘤新的标志物奠定了基础。     

Transient expression of virus-specific promoters in murine resident peritoneal macrophages

Monocyte-macrophages (MO), being non-permissive for most viruses, play an important role in resistance to virus infection. In order to establish the mechanism of abortive infection of murine resident peritoneal MO (ResPMO) by herpes simplex virus type 1 (HSV-1), it is desirable to transfect these cells with viral promoters linked to an assayable gene, for example, the bacterial chloramphenicol acetyl transferase (CAT) gene. This will facilitate studies designed to measure levels of promoter activation or repression in these specialized cells. Transient expression of CAT in ResPMO was achieved with DEAE-dextran, but not using either calcium phosphate precipitate or lipofectin. CAT expression driven by various virus-specific promoters was less efficient in ResPMO compared with Vero cells and approximately 50% of input plasmid DNA remained in Vero cells at 48 h post transfection, but only 9% was detectable in ResPMO. However, approximately 6% of ResPMO and 9% of Vero cells contained CAT-specific DNA at 24 h post transfection. In addition, 2% of cells of either cell type contained CAT-specific polypeptide at 48 h. This is therefore the first report that the non-replicating murine ResPMO can be transfected in vitro and more importantly, that these cells express the transfected gene products.     

Enhancement of lung-colonizing potential of murine tumor cell lines co-cultivated with activated macrophages

In order to explore the influence of activated macrophages on tumor cells, we cultured a series of weakly metastatic clones isolated from the murine T3 fibrosarcoma line (T3 clones) and the B16-F10 melanoma cells on feeder layers of C. parvum- or thioglycollate-elicited macrophages, or ÔresidentÕ (unstimulated) macrophages. Co-cultivation with C. parvum-elicited macrophages, but not with resident macrophages, induced an increase of the lung-colonizing potential of T3 clones and B16-F10 cells. An enhancement of lung-colonizing potential was also found in tumor cells grown in media conditioned by C. parvum-elicited macrophages. Thioglycollate-elicited macrophages also generated a pro-clonogenic activity which was however effective only on T3 clones but not on B16-F10 cells. The increase in the lung-colonizing potential of tumor cells stimulated by activated macrophages was retained to some degree after subcultivation in tissue culture medium or transplantation into syngeneic animals. In conclusion, our data support the notion that macrophages at different states of activation may enhance lung colonization of tumor cells by inducing a partially stable change of phenotype.     

Responses of antigen-specific long-term murine T cell lines to wheat gliadin fractions

Recent evidence suggests that the four electrophoretically defined gliadin subfractions (alpha, beta, gamma and omega) of wheat can induce the typical pathological finding of coeliac disease. We have prepared long-term murine T cell lines to gliadin and its four major subfractions. The cell lines were tested in proliferative assays with each homologous gliadin subfraction, and to the other gliadin subfractions. There was some cross-reactivity, with unfractionated gliadin and its alpha-subfraction being the most antigenic, while omega-gliadin was the least. These data demonstrate that gliadin components are effective stimuli for specific T cell responses, and further suggest that the alpha-gliadin subfraction generates the highest specific responses. This accords with observations in man that all four gliadin subfractions exacerbate coeliac mucosa, but that the alpha-subfraction is the most active.     

新生CD-1小鼠雌性生殖干细胞的建系及生物学特征

背景：传统观点认为雌性哺乳动物出生后不再产生新的卵母细胞,近年来多项研究表明出生后哺乳动物卵巢中存在能再生卵子的雌性生殖干细胞。 目的：从新生CD-1小鼠卵巢中分离、纯化雌性生殖干细胞并建系及进行生物学特征研究。 方法：通过两步酶消化法和免疫磁珠分选从新生CD-1小鼠卵巢中分离与纯化雌性生殖干细胞并体外长期培养,然后通过RT-PCR、细胞免疫荧光化学和核型分析鉴定其生物学特征。 结果与结论：从3-5 d龄CD-1小鼠卵巢中分离出雌性生殖干细胞并建立细胞系,目前已在体外长期培养15个月,共传代70多次。该细胞系表达Mvh,Dazl,Oct-4,Stella,Blimp1,Fragilis等生殖细胞标志物,同时通过Fragilis和BrdU的双免疫荧光实验证实了其增殖活性和生殖细胞特性;核型分析发现该细胞系也具有正常的二倍体核型。因此CD-1小鼠卵巢中存在具有有丝分裂活性和正常的核型的雌性生殖干细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：