Interaction between mesenchymal cells and the posterior iris epithelium in chicken embryos

Summary The iris anlage of 3–10 day old chicken embryos was studied by both light and electron microscopy. Serial semithin sections showed that some of the mesenchymal cells overlying the eye cup moved into the primitive eye cavity by the 3rd day of incubation. On the 4th day some of these cells came into close contact with the basement membrane of the dorsal iris epithelium. The bases of the epithelial cells were flat at this stage. Towards the 10th day they formed cytoplasmic processes which did not penetrate the basement membrane. Fine mesenchymal cytoplasmic processes and a large number of extracellular fibrils developed in the epithelial-mesenchymal interface. The fine mesenchymal processes came into close contact with the basement membrane of the posterior iris epithelium but did not penetrate it. Collagen-like material was observed within the cisternae of the rough ER of the mesenchymal cells at certain stages of development. Both, the mesenchymal cells and the collagen fibrils adjacent to the posterior iris layer disappeared by the 10th day when the entire iris epithelium was completely pigmented. The possible origin of the collagen fibrils and the differentiation of the posterior iris epithelium are discussed.     

Feline calicivirus replication induces apoptosis in cultured cells

Infection of Crandell-Rees feline kidney (CRFK) cells by feline calicivirus (FCV) causes rapid cytopathic effects followed by cell death. In this study, we observed that FCV replication in cells results in the induction of changes characteristic of apoptosis, including translocation of phosphatidyl serine to the cell outer membrane, chromatin condensation, and oligonucleosomal DNA fragmentation. FCV infection was associated with increases in the activities of caspase-3, -8, and -9, with the level of activation of caspase-3 higher than those of caspases-8 and -9. Caspase activation in CRFK cells was not observed when cells were inoculated with UV-inactivated FCV or when cycloheximide was present during virus infection, indicating that FCV replication and de novo synthesis of virus proteins are critical for induction of apoptosis.     

鸡胚生殖嵴中原始生殖细胞的分离培养

目的　在鸡胚孵化的 19期以Ficoll密度梯度离心和酶解离两种方法分离生殖嵴中的原始生殖细胞 (PGCs)。探索在生殖嵴中PGCs分离、培养的适宜方式 ,以获得较多数量与较高活力的PGCs作介导生产转基因鸡。方法　在倒置显微镜下进行形态观察 ,台盼蓝染色比较存活时间 ,PAS特异染色法识别鉴定PGCs。结果　两种分离方法均能分离到一定数量的PGCs细胞。与Ficoll密度梯度离心法相比 ,酶解离法分离到PGCs的相对数量较多 ,存活时间较长 ,是一种较可行的分离方法。在鸡胚孵化的第 19期 ,PGCs大量聚集在肢体后端的生殖嵴原基处 ,此时的生殖嵴大小已达一定程度 ,分离其中的PGCs操作简便 ,有较强的可操作性。结论　提取的PGCs为转基因鸡的生产提供了介导材料。     

Avian reovirus sigmaC protein induces apoptosis in cultured cells

The avian reovirus (ARV) infection is associated with various disease conditions in poultry. However, the pathogenesis mechanisms are poorly characterized. In the present study, we clearly demonstrated that the sigmaC of ARV S1133 strain induced apoptosis in both BHK-21 and Vero cells. Five kinds of assays for apoptosis were used in analyzing ARV-infected BHK-21 and Vero cells: (1) assay for DNA ladders, (2) ELISA detection of cytoplasmic histone-associated DNA fragments, (3) nuclear staining with acridine orange, (4) Western blot, Northern blot, and immunofluorescent assay (IFA), and (5) flow cytometric analysis. The sigmaC protein of ARV could elicit apoptosis occurring in a dose- and time-dependent manner. The current results further our understanding of the function of sigmaC in cultured cells and suggest that sigmaC is a viral-encoded apoptin and possesses apoptosis-inducing ability. Furthermore, deletion analysis of the ARV sigmaC protein suggests that the carboxyl-terminus of sigmaC is important in mediating sigmaC-induced apoptosis because its deletion abolished the induction of apoptosis.     

自噬通过抑制凋亡促进体外饥饿状态下脂肪细胞存活

 目的: 观察体外饥饿状态下脂肪细胞的自噬变化,研究自噬在维持体外饥饿状态下脂肪细胞存活中的作用。方法: 将小鼠3T3-L1细胞诱导成为脂肪细胞后,雷帕霉素(rapamycin,RAP)预处理脂肪细胞,在缺糖缺氧(oxygen-glucose deprivation,OGD)模拟的饥饿环境中孵育细胞。应用Western blotting及透射电镜法检测细胞自噬变化,用TUNEL染色及Western blotting检测脂肪细胞凋亡。结果: OGD条件下脂肪细胞的自噬水平明显升高,RAP预处理进一步上调了OGD条件下的细胞自噬。与对照组相比,OGD组细胞cleaved caspase-3的水平及细胞凋亡率明显升高(P<0.01)。RAP预处理降低了OGD 条件下cleaved caspase-3的水平,并明显降低了细胞凋亡率(P<0.05)。结论: 自噬在饥饿状态下脂肪细胞存活中发挥保护性作用,提高自噬水平有助于降低饥饿状态下脂肪细胞的凋亡水平。     

抗Fas抗体诱导人肾小球系膜细胞凋亡

研究抗Ｆａｓ抗体对人肾小球系膜细胞的致凋亡作用，探讨Ｆａｓ－ＦａｓＬ在肾脏损务中的作用。方法：常规方法分离，培养人肾小球系膜细胞，并传代鉴定，用不同浓度的抗Ｆａｓ抗体刺激４－６代Ｍｃｓ１６ｈ后，荧光染色观察Ｍｃｓ凋亡变化，二苯胺法和ＤＮＡ凝胶电泳方法定量和定性分析ＭｃｓＤＮＡ片段化变化。     

African horse sickness virus induces apoptosis in cultured mammalian cells

Infection of mammalian cell cultures with African horse sickness virus (AHSV) is known to result in dramatic cytopathic effects (CPE), but no CPE is observed in infected insect cell cultures despite productive virus replication. The basis for this phenomenon has not yet been investigated, but is suggestive of apoptosis being induced following virus infection of the mammalian cells. To investigate whether AHSV can induce apoptosis in infected mammalian cells, Culicoides variipennis (KC) insect cells and BHK-21 mammalian cells were infected with AHSV-9 and analyzed for morphological and biochemical hallmarks of apoptosis. In contrast to KC cells, infection of BHK-21 cells with AHSV-9 resulted in ultrastructural changes and nuclear DNA fragmentation, both of which are associated with the induction of apoptosis. Results also indicated that AHSV-9 infection of BHK-21 cells resulted in activation of caspase-3, a key agent in apoptosis, and in mitochondrial membrane depolarization. Cumulatively, the data indicate that the intrinsic pathway is activated in AHSV-induced apoptosis.     

Migration and differentiation of human umbilical cord stem cells after heart injury in chicken embryos

Here we have analyzed the behavior and fate of stem cells from human umbilical cord blood (scHUCBs) when grafted into the myocardial wall of normal and damaged hearts of chicken embryos. We started by characterizing the scHUCBs before grafting and we found that they express precardiogenic genes including Nkx2.5, GATA4, MEF-2, and SERCA2a together with undifferentiation markers as CD34 or c-kit. In grafting experiments using scHUCBs labeled with DiI we observed that these cells were not rejected by the host and survived when implanted in chicken hearts, being able to migrate through the myocardial wall. By 3 days after grafting we found labeled cells with morphological characters of myocardiocytes in concordance with the identification of the expression of human genes for myosin light chain 2a (Mlc2a) and myosin heavy chain-beta (Mhc beta) in the chicken heart. When a small injury was applied to the heart wall, grafted scHUCBs were vigorously attracted by the damaged myocardium. This directed migration was only sustained for 12 h after injury, time period required for healing of the damaged heart wall. The rate of myocardial differentiation of scHUCBs in damaged hearts was not significantly increased with respect to that found when implanted in healthy hearts. However, we found stimulation of endothelial differentiation in injured hearts deduced by the increased expression of human genes for platelet endothelial cell-adhesion molecule 1 or vascular endothelial growth factor receptor 2 and the presence of DiI-labeled endothelial cells. Together all these findings support the embryonic chicken heart as a feasible model for experimentation in stem cell therapy and emphasize the relevance of the physiological conditions of the myocardial host tissue for engraftment and differentiation of exogenously applied scHUCBs.     

Evaluation of anticoccidial drugs in chicken embryos

Infections ofEimeria tenella in chicken embryos were used to compare the anticoccidial activity of ten drugs. The minimal inhibitory concentration (MIC) and minimal toxic concentration (MTC) were affected by the time of inoculation into the embryos and by the chemical nature of the compounds. Some compounds (nicarbazin, amprolium) had no effect on the development of coccidia when they were injected into embryos after the day of infection. Drugs that act early in the life cycle of coccidia (robenidine, clopidol, decoquinate, diclazuril, halofuginone, monensin, salinomycin, and lasalocid) were active at 5–125 g/embryo when they were injected on the day of infection. The ionophores and halofuginone were highly toxic to embryos; most synthetic compounds were nontoxic. The incubation of merozoites in drug suspensions prior to the infection of embryos did not result in embryo toxicity, but the resultant MICs were much higher than those obtained when drugs were injected directly into the embryos. Several products were essentially inactive. Neither nicarbazin nor amprolium prevented oocyst formation. The widely divergent endpoints for the MIC and MTC of anticoccidials in embryos seriously limits the application of this technique as a screen for anticoccidial drugs.     

B-cell precursors in early chicken embryos.

The ontogeny of B-cell precursors in chicken embryos from day 3 of incubation onwards has been studied. Purified antibodies to chicken Ig L, gamma, mu, alpha chains were used in a sensitive indirect immunofluorescence assayed on fixed cell smears and wax-embedded tissue sections; the location and morphology of immunoglobulin positive (Ig+) cells were determined either in phase contrast or after histological staining. Lymphoid cells containing small amounts of cytoplasmic immunoglobulin were found in 3 day and older embryonic yolk sac, 11 and 12 day blood, 11, 12 and 13 day bursal mesenchyme. cIg+ large basophilic cells were first seen in 14 day bursal follicles. It is concluded that cells enter the embryonic bursa at different developmental stages: some appear to be uncommitted stem cells, whilst others have already commenced B-cell maturation in an extra-bursal site.     

Primary target cells of virulent strains of type A influenza virus in chicken embryos

Virulent or avirulent strains of type A influenza virus were inoculated into the allantoic cavities of chicken embryos. The antigens of virulent strains appeared initially in the surface epithelium of the allantoic membrane, then in vascular endothelial cells of the chorioallantoic membrane and visceral organs of the embryos, and then spread to parenchymal cells of many organs. In contrast, the antigens of the avirulent strain were confined to the allantoic membrane. These observations indicate that the primary target of virulent influenza viruses in chicken embryos is vascular endothelial cells, and that the embryos died after systemic viral infection.     

TGF-beta antisense oligonucleotides reduce mRNA expression of matrix metalloproteinases in cultured wound-healing-related cells

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.     

Histone deacetylase inhibitors promote apoptosis and senescence in human mesenchymal stem cells

Histone deacetylase inhibitors (HDACi) have received a great amount of attention for their antitumoral properties. Suberoyl anilide hydroxamic acid (SAHA) and MS-275 are among the more promising HDACi for cancer treatments. Although these HDACi compounds exert low toxicity on normal cells, the therapies based on these molecules can cause side effects that can greatly impair the functions of the bone marrow microenvironment. This is a complex system that contains several types of stem cells, such as mesenchymal stem cells (MSCs). We conducted comparative studies on the effects of SAHA and MS-275 on human MSCs in order to ascertain if these compounds can impair the physiology of MSCs. Both SAHA and MS-275 induced an arrest in the cell cycle along with the induction of apoptotic pathways as evidenced by flow cytometry, annexin assay, detection of activated caspase 9, and molecular analysis of Bax/Bcl-2 expression. The MS-275 treatment induced an increase of senescent cells, whereas in cells treated with SAHA, we detected a reduction of senescent cells compared to the control. We hypothesize that SAHA preferentially transactivates apoptotic genes, thereby inducing a great majority of the damaged cells to die by programmed cell death rather than senescence. Following the HDACi treatment, we observed a decrease in the expression of some genes that are involved in the regulation of stem cell properties. This suggests that SAHA and MS-275 could also be involved in the impairment of the stemness characteristics of MSCs. The phenomena that were induced by HDACi treatment were associated with an upregulation of several cyclin kinase inhibitors. By contrast, the p53-p21 pathway is apparently not involved in these processes.     

Bursa lymphocytes and IgM-containing cells in chicken embryos bursectomized at 52-64 hours of incubation

A technique of surgical removal of the bursal primordium ("bursectomy") of chicken embryos at stage 17, approximately 52-64 hours and 29-32 somites, is described. The survival rate of bursectomized (Bx) embryos approached a level of 50% on the 21st day. About 20% of correctly Bx embryos exhibited malformations of the anal sphincter and the large intestine. Using a rabbit anti-bursacyte serum, which did not react with thymocytes, the specific bursa-derived cell (Bu) marker was detected on the surface of bursa, spleen, bone marrow and thymus lymphocytes. Early embryonic bursectomy caused a moderate depletion of Bu marker-bearing and IgM-containing cells. It has been postulated that embryonic Bu cells can be recruited from sites other than the bursa and in the absence of the bursa.     

Cardiac glycoside tolerance in cultured chicken heart muscle cells — A dose-dependent phenomenon

Summary In cultured heart muscle cells from 10–13 day-old chicken embryos, the effects of acute (4 h) and chronic (3 days) exposure of the cells to varying concentrations of ouabain have been studied. In these cells, the cardiac glycoside ouabain binds to a specific cardiac glycoside receptor (K_D=4 × 10^–7 M; 750,000 receptors/cell). Binding to this receptor results in inhibition of active Na⁺/K⁺-transport [EC₅₀ for active (⁸⁶Rb⁺ + K⁺)-influx=4 × 10^–6 M], and in an increase in beating velocity (positive inotropic effect;; EC₅₀=4 × 10^–7 M); toxic signs (arrhythmias) appear at concentrations 6 × 10^–7 M. During exposure of the cells to 3 × 10^–6 M ouabain for 3 days, tolerance develops with respect to both the positive inotropic and the toxic effect. The mechanism underlying this tolerance is identified as an increase in the number of active sodium pump molecules per cell, while the binding properties of the cardiac glycoside receptor remain unchanged. The development of cardiac glycoside tolerance is only observed in the presence of severe impairment of Na⁺/K⁺-homeostasis, due to cardiac glycoside-induced inhibition of active Na⁺/K⁺-transport. This, however, only occurs in the presence of toxic (receptor occupation 60%), but not in the presence of positive inotropic, non-toxic (receptor occupation 20–60%), ouabain concentrations. We conclude that the development of cardiac glycoside tolerance during long-term treatment in patients with heart failure should not occur with submaximal dose regimens, when toxic signs (arrhythmias) are absent.Abbreviations CGP-12177 (4-3-tert-butylamino-2-hydroxy-propoxy)-benzimidazol-2-one hydrochloride) - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid Some of the results were presented at the IXth European Congress of Cardiology, July 1984, Düsseldorf, Germany [49]     

Virulence of Francisella spp. in chicken embryos

We examined the utility of infecting chicken embryos as a means of evaluating the virulence of different Francisella sp. strains and mutants. Infection of 7-day-old chicken embryos with a low dose of F. novicida or F. tularensis subsp. holarctica live vaccine strain (LVS) resulted in sustained growth for 6 days. Different doses of these two organisms were used to inoculate chicken embryos to determine the time to death. These experiments showed that wild-type F. novicida was at least 10,000-fold more virulent than the LVS strain. We also examined the virulence of several attenuated mutants of F. novicida, and they were found to have a wide range of virulence in chicken embryos. Fluorescent microscopic examination of infected chicken embryo organs revealed that F. tularensis grew in scattered foci of infections, and in all cases the F. tularensis appeared to be growing intracellularly. These results demonstrate that infection of 7-day-old chicken embryos can be used to evaluate the virulence of attenuated F. tularensis strains.