Astrovirus and Breda virus infections of dome cell epithelium of bovine ileum.

A bovine enteric virus antigenically related to the United Kingdom isolate of bovine astrovirus was isolated from diarrheic feces, also containing rotavirus, of a calf in Florida. The astrovirus infected cell cultures and the epithelial cells of domes in the ileum, and there was cross-immunofluorescence with antiserum to the United Kingdom astrovirus. Calves infected with astrovirus alone did not develop clinical disease, but when astrovirus was mixed with rotavirus or Breda virus 2, the calves developed severe diarrhea and more extensive astrovirus infection of the dome epithelium. The dome epithelial cells showed degeneration associated with astrovirus infection, and a few cells showed degeneration with Breda virus 2 infection. Virions with a 30-nm diameter were seen in astrovirus-infected dome cells, and Breda virus 2 virions were also observed either in separate cells or, on occasion, with both viruses in one cell.     

Cell culture propagation of bovine coronavirus

Summary Although most field strains of bovine coronavirus (BCV) grow poorly in cell culture and fail to produce cytopathic effects (CPE) until after blind passage, primary calf kidney (PCK) and Vero cells have permitted primary isolation of virus. Cell culture-adapted strains of BCV replicate in PCK, bovine embryonic lung, bovine fetal thyroid, bovine fetal brain, bovine skin cells, ovine fetal kidney cells, and the cell lines pig kidney K3 and 15, Vero, human embryonic lung fibroblasts, HRT-18, MDBK and BEK-1, with trypsin useful for enhancing replication. Organ culture as well as suckling mouse, rat, and hamster brains also support the growth of cell culture-adapted BCV strains. Viral growth is most commonly detected by CPE, immunofluorescence, hemagglutination, and hemadsorption assays or electron microscopy of supernatants from infected cells. In this report, the optimal conditions for the growth and plaque assay of the NCDV strain of BCV in MDBK cells are described.     

Prostaglandin F2 alpha can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro.

The effects of PGF2 alpha on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF2 alpha at concentrations equal to or lower than 10(-6) M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10(-5) M PGF2 alpha induced a decrease in cell growth under all culturing conditions. PGF2 alpha did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF2 alpha induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF2 alpha and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE2.     

Two types of morphological transformation of bovine kidney cells infected in vitro with SV40

Summary Two types of morphological transformation of bovine kidney cells were obtained after inoculation with SV₄₀. Primary cultures inoculated were transformed into cultures with epithelioid cells growing mainly in monolayer. When the kidney cells were subcultivated and infected at the 6th passage, another type of transformation, characterized by epithelioid and fibroblastic cells growing in a disorganized multilayer, was seen.Cell lines were obtained from both types of transformed cultures. The epithelioid cell cultures were found to be free of infectious SV₄₀ whereas the cultures composed of epithelioid as well as fibroblastic cells yielded virus even at high passage levels. Both types of transformed cultures contained the complement-fixing tumor antigen. A comparison between the cultures as regards their susceptibility to various viruses showed that the epithelioid cell cultures were more susceptible to parainfluenza virus type 3 than the cultures with both epithelioid and fibroblastic cells. There were no differences in susceptibility to foot-and- mouth disease virus, bovine enterovirus, infectious bovine rhinotracheitis virus, pseudorabies virus, Newcastle disease virus or bovine viral diarrhoea virus.This investigation was supported by grants from the Swedish Cancer Society.     

自体血清与胎牛血清培养兔关节软骨细胞的生物学特性

背景：目前培养软骨细胞多使用胎牛血清。但近年异种血清培养组织向临床应用的安全性受到质疑,因此自体血清培养越来越受到重视。 目的：比较体积分数10%兔自体血清与体积分数10%胎牛血清培养兔关节软骨细胞生物学特性的差异。 方法：兔自体血清培养液制备后,分离培养兔关节软骨细胞,分别在体积分数10%自体血清和体积分数10%胎牛血清中进行单层传代培养至1,3,5代,采用光镜观察细胞形态变化,绘制生长曲线评估细胞增殖速度,观察甲苯胺蓝染色、Ⅰ,Ⅱ型胶原免疫组织化学染色结果,以流式细胞仪分析细胞Ⅰ,Ⅱ型胶原以及CD26,CD44的表达变化。 结果与结论：①在细胞形态上自体血清和胎牛血清培养的软骨细胞差异不大。②自体血清培养的软骨细胞较胎牛血清培养的软骨细胞生长速度更快。③甲苯胺蓝染色结果示,无论是自体血清还是胎牛血清所培养的细胞,染色随代龄的增加逐渐变浅,细胞传至第5代时两组几乎均无异染。对于1代和3代细胞而言,自体血清培养的软骨细胞较胎牛血清所培养的软骨细胞较为深染。④Ⅰ型胶原的表达随代数的增加而增加,而Ⅱ型胶原的表达则随代数的增加而减少。在3代时自体血清培养的软骨细胞Ⅰ型胶原的表达水平低于胎牛血清培养的软骨细胞(P < 0.05);在1代和3代时自体血清培养的软骨细胞Ⅱ型胶原的表达水平高于胎牛血清培养的软骨细胞(P < 0.05)。⑤CD26表达呈现先升高后降低的趋势,而CD44的表达不随传代数的增加而改变。提示同异体体积分数10%的胎牛血清相比,体积分数10%的自体血清培养的兔软骨细胞生长速度快,且在大部分指标上可以较好地保持细胞表型。     

A technique to obviate the risk of inadvertent infection of cell cultures with bovine viral diarrhoea virus

When bovine embryonic kidneys collected at the Gorgie Abattoir, Edinburgh were examined for evidence of infection with bovine viral diarrhoea virus (BVDV), 11 out of 26 kidneys were found to be positive. A technique that detected the presence of inadvertent BVDV in cell cultures consisted of processing and digesting the kidneys to produce cell suspensions, adding dimethyl sulphoxide and dispersing the suspensions in small aliquots that were stored frozen at - 114 degrees C. One aliquot was cultured and screened for BVDV by indirect immuno-fluorescence and interference tests. Bovine embryonic kidney cells so processed retained their viability and virus susceptibility for 15 to 18 months. Selected stocks of "clean" cells only are then used for vaccine production or diagnostic purposes. The cytopathic NADL strain of BVDV multiplied in naturally infected cell cultures but the titres attained were significantly lower than in "clean" cell cultures.     

Rinderpest virus infection in primary bovine skin fibroblasts

Summary.  Rinderpest virus (RPV) replicated to a high titre in primary bovine skin fibroblats. The course of infection was similar to that seen in established cell lines. Virulent field virus grew at a faster rate than the fully attenuated vaccine strain of the virus. Virus antigen expression, as measured by FACScan analysis, correlated with the time course of infection for the two strains in cell cultures. Wild type virus, obtained directly from cattle, infected cells at a slower rate than virus passaged even once in primary bovine skin fibroblasts. This is the first report of a productive infection of primary bovine skin fibroblasts by wild type RPV. Received August 13, 1999 Accepted January 13, 2000     

Kinetics of parvovirus replication,cytopathic effects,and mitotic arrest in synchronized bovine fetal spleen cells

Initiation of autonomous parvovirus replication depends on the S phase of host cells actively traversing the cell cycle. The parvoviruses Lu III and H-1 inhibit synchronized cells from entering mitosis, implying that parvoviruses rapidly shut down cell cycle traverse during G2 phase. Bovine parvovirus (BPV) did not inhibit the entry into mitosis of hydroxyurea synchronized bovine fetal spleen cells. Mitotic indexes of infected cultures were as much as 60-fold higher than those of mock-infected controls. Mitosis in control and infected cultures peaked at 10 hr after infection corresponding to the end of the BPV eclipse period. Cytopathic changes, including morphological alteration of mitotic chromosomes, were detected in mitotic cells from infected cultures by light and electron microscopy. Arrest of BPV-infected cells in mitosis may explain these results. Not all infected cells were killed in mitosis, since some developed intranuclear inclusions and became pycnotic as nucleated, interphase cells. Inclusion formation was coincident with viral morphogenesis in interphase nuclei at 16 to 18 hr postinfection, late in the viral replication cycle. The cell cycle stage at which parvovirus-infected cells are arrested and cytopathic events ensue may be determined by the cellular progression rate from S phase through G2 and M.     

Fetal bovine oviduct epithelial cell monolayers: Method of culture and identification

Summary Bovine epithelial cell monolayers were obtained for culture from fetal oviduct after in situ trypsinization. Isolated ciliated and secretory cells obtained in high yield with good viability were suspended in B2-MENEZO'S medium supplemented with 7.5% fetal bovine serum FBS. The plated primary cultures reached confluency 2 days after initial seeding, producing a monolayer of cohesive polygonal cells with viability of 85 to 95%. Associated with this large epithelial cell population, ciliated cells as well as a few elongated spindle cells were observed. After the first subculture the ciliated cells disappeared and the epithelial cells in the monolayer grew more rapidly to confluence than adult-derived cultures. In addition, frozen-thawed oviduct epithelial cells also maintained a level of 75 to 85% viability during postthaw subculture. The epithelial cells maintained their secretory activity in culture as indicated by electron microscopy and immunocytochemistry. The cell culture monolayers contained keratin, a specific cytoskeletal component of epithelial cells. This culture system may offer benefits for in vitro culture of mammalian embryos. This research was supported by the Mérieux Foundation.     

Enhanced cytopathic effect of human cytomegalovirus on a retinal pigment epithelium cell line, K-1034, by serum-free medium

Summary. Although human cytomegalovirus (HCMV) predominantly infects epithelial cells in vivo, the majority of studies of HCMV gene expression and replication have been conducted using non-epithelial cell lines in part because of the absence of a good experimental system using epithelial cells. To address the nature of epithelial cell infection, we investigated the susceptibility of an epithelial cell line (K-1034) established from the retinal pigment epithelium to HCMV infection. This cell line exhibited high susceptibility to HCMV, as evidenced by detection of one of the immediate early antigens, IE2, in the nuclei of more than 80% of K-1034 cells at 24 h following inoculation at a multiplicity of infection of 3 plaque forming units per cell. However, the yield after one-step growth of HCMV in K-1034 cells was about twenty-fold less than that in human embryonic lung fibroblast cells. Cytopathic effect (CPE) on K-1034 cells was not prominent in medium supplemented with 10% fetal bovine serum and viral late antigens were detected in less than 5% of K-1034 cells. Interestingly, infected cells expressing late antigens and exhibiting CPE were markedly increased in serum-free medium, even though the yield of infectious HCMV and viral genome copy numbers were almost the same in the different serum concentrations, due to viral instability in the absence of serum. Thus, the progression of late antigens expression and the induction of CPE in infected epithelial cells is influenced by physiological conditions, and are negatively regulated by some serum factor. Reeived December 2, 1996 Accepted March 4, 1997     

Viral protein production in homogeneous and mixed infections of cytopathic and noncytopathic BVD virus

Summary Experiments were conducted to examine dual infection of cultured cells with cytopathic and noncytopathic bovine viral diarrhea virus (BVDV). Cell monolayers infected with a noncytopathic BVDV isolate and subsequently superinfected with a cytopathic BVDV isolate were refractive to the cytopathic effects of the cytopathic BVDV isolate, as reported in the literature. Immunofluorescence staining of superinfected cultures with monoclonal antibodies specific for the cytopathic or the noncytopathic viral isolate, demonstrated that cells in superinfected cultures contained both viral biotypes. Immunoprecipitation was used to compare the temporal detection of viral induced polypeptides in superinfected cultures to that of cultures infected with a single viral biotype. In single cytopathic viral infections, viral induced polypeptides of 80 kDa and 53–56 kDa are detected simultaneously, but in superinfections a 4 h gap occurred between detection of the 53–56 kDa polypeptide and detection of the 80 kDa polypeptide.     

犬阴道上皮细胞和平滑肌细胞可在体外长期稳定培养

背景：体外分离培养获得足够活性良好的种子细胞是构建阴道组织工程的关键。文献报道阴道上皮细胞体外纯化培养和传代较为困难,尤其是体外长期培养犬等大动物的阴道种子细胞尚未见报道。 目的：建立体外稳定培养犬阴道上皮细胞和平滑肌细胞方法。 方法：获取犬小块阴道组织,机械分离阴道黏膜上皮,Dispase酶和胰蛋白酶分步消化收集上皮细胞,接种于无血清角化细胞培养液中培养和传代;机械分离阴道平滑肌组织后采用Ⅱ型胶原酶消化获得平滑肌细胞,在含体积分数10%胎牛血清的DMEM培养液中连续培养传代。动态观察上皮细胞和平滑肌细胞生长增殖情况,分别采用特异性抗体行细胞免疫化学染色鉴定。 结果与结论：原代培养的上皮细胞24-36 h后开始贴壁铺展,四五天后呈对数生长,七八天可达70%融合,为单一的上皮细胞,呈典型铺路石样,未见成纤维细胞混杂。每四五天可传代1次,连续传代六七次,细胞免疫化学染色角蛋白AEl/AE3抗体阳性。平滑肌细胞原代培养24 h后贴壁呈梭形,此后呈对数生长,4 d后融合呈典型的“峰和谷”样,每三四天可传代1次,连续传代七八次,细胞免疫化学染色示α-肌动蛋白染色阳性。结果证实,犬阴道上皮细胞和平滑肌细胞可在体外长期稳定培养,可为体外构建组织工程化阴道提供足够的种子细胞。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：     

Fetal and adult bovine interferon production during bovine viral diarrhea virus infection.

Levels of interferon in adult bovine serum and in fetal bovine serum and tissues were examined during the course of transplacental bovine viral diarrhea virus infection. The cows produced circulating interferon between 2 and 9 days after viral inoculation, with mean peak levels in the serum on day 4. Interferon could be routinely detected in fetal tissues (e.g., thymus, spleen, and kidney) between days 4 and 21 after viral inoculation of the cows at 149 to 150 days of gestation (mid-second trimester) and in fetal serum from day 13 through day 21. Interferon was also detectable in the serum and tissues of fetuses from dams infected at day 95 of gestation (the beginning of the second trimester). In general, no differences were found between the ability of the adult and fetus to produce interferon. Fetal lamb kidney cells were more sensitive to the antiviral effects of bovine interferon than were fetal bovine kidney cells. The antiviral substance from the fetal and adult animals was characterized as interferon by standard criteria.     

Defective interfering particles of Sindbis virus. III. Intracellular viral RNA species in chick embryo cell cultures.

Serial undiluted passage of wild-type Sindbis virus (SV-W) in primary chick embryo cell cultures resulted in a cyclic variation in the yield of infectious virus. Cells infected with virus stocks (SV-CP) derived from such passage series directed significantly less viral RNA synthesis than cells infected with SV-W at the same multiplicity of infection (MOI) and produced particles (? = 1.20 g/cc) capable of homologous interference. Cells infected with SV-CP stocks of increasing passage number contained, in addition to 22S dsRNA (found in SV-W infected cells), species of virus-specific dsRNA of decreasing molecular size. The rate of evolution to the smaller dsRNA species was considerably faster during serial undiluted passage of SV-W in BHK-21 cells than in chick cells. Cells infected with SV-CP stocks also contained a new species of ssRNA for each additional species of dsRNA produced suggesting that the two are functionally related.     

Use of human fibroblasts in the development of a xenobiotic-free culture and delivery system for human keratinocytes

Previous work has shown that keratinocytes can be cultured serum-free on an acid-functionalized, plasma-polymerized surface (for subsequent delivery to patients' wound beds) by inclusion of a fibroblast feeder layer. This study seeks to extend this work by substituting human for murine feeder cells in serum-free culture and examining the performance of keratinocytes expanded in this way to transfer to an in vitro human dermal wound bed model. We compared murine and human fibroblasts (both short-term dermal fibroblasts and a fetal lung fibroblast cell line MRC-5, which has a long history in human vaccine production), alternative methods for growth-arresting fibroblasts, establishing culture of cells serum-free, and the impact of culture with fibroblasts on the differentiation of the keratinocytes. Irradiated human and murine fibroblasts were equally effective in supporting initial keratinocyte expansion, both in the presence and absence of serum. Keratinocytes were significantly less differentiated, as assessed by measuring involucrin expression relative to DNA when grown serum-free with fibroblasts than when grown with serum. Initial cultures of fibroblasts and keratinocytes could be initiated serum-free but were much slower to establish than if serum were used. Transfer of keratinocytes from keratinocyte/fibroblast co-cultures cultured on a plasma polymer surface to a human dermal wound bed model was as successful as from monocultures in both serum and serum-free cultures. In summary, we have revisited a well-accepted methodology for expanding human keratinocytes for clinical use and avoided the use of bovine serum and a mouse fibroblast feeder layer by introducing an irradiated human fibroblast feeder layer.     

Chromosome aberration induction in human diploid fibroblast and epithelial cells

The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells; the mean frequency between 1 and 72 h post-irradiation was 25 and 29% aberrant cells, respectively. After 1.5 Gy the corresponding values in skin fibroblasts and epithelial cells were 16.5 and 11.2%, respectively, and after 2.5 Gy, 31.3 and 30%. These observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers.