Suppression of sheep and goat lymphocyte proliferation by sheep, goat, and sheep x goat hybrid trophoblast tissue cultures.

Immunosuppressive activity of conditioned medium from cultured ovine, caprine, and hybrid trophoblast tissue was examined. Conceptuses were obtained from naturally mated donor ewes and does at d 20 of gestation and trophoblast tissue was cultured for 24 h in medium supplemented with 15% calf serum and 1% antibiotic/antimycotic. Conditioned medium was added to pokeweed mitogen-stimulated sheep and goat lymphocyte cultures. Quantification of [3H]thymidine uptake by cells was used to measure lymphocyte proliferation. Ovine, caprine, and hybrid conditioned medium effectively suppressed sheep and goat lymphocyte proliferation (P less than .01). There were no differences (P greater than .05) between the immunosuppressive activity of the three tissue types on either sheep or goat lymphocytes. For all treatment groups, sheep lymphocytes were suppressed more than goat lymphocytes (P less than .05). These results indicate that, at d 20 of gestation, sheep x goat hybrid trophoblast tissue is capable of suppressing pokeweed mitogen-stimulated lymphocyte proliferation.     

Reduced expression of cyclin-dependent kinase inhibitor p27Kip1 in feline lymphoma.

Expression of p27Kip1 was identified in feline lymphoid tissues by immunohistochemistry. In normal lymphoid tissues, p27Kip1 was detected as a distinct nuclear stain in lymphocytes of the follicular mantle zone and interfollicular small lymphocytes, whereas activated lymphoblasts in the germinal center were negative. Lymphoid hyperplasia was similarly immunolabeled but with an expanded mantle zone and marginal zone of p27Kip1-reactive lymphocytes. Both T- and B-cell lymphomas lacked p27Kip1 immunolabel and were determined to be proliferative based on immunohistochemical detection of the Ki-67 antigen. Scattered p27Kip1-immunolabeled lymphocytes were detected throughout the lamina propria of most specimens characterized as lymphoplasmacytic enteritis. The results of this study suggest that the antiproliferative effect of the cell cycle regulator p27Kip1 is abrogated in feline lymphoma, presumably allowing cells to bypass the G1-S checkpoint of the cell cycle.     

玉米赤霉烯酮对小鼠胸腺上皮细胞的毒性作用

采用台盼蓝计数、流式细胞仪分析等方法,离体研究玉米赤霉烯酮对小鼠胸腺上皮细胞增殖与细胞周期的影响,结果发现:不同质量浓度(1～25 mg/L)玉米赤霉烯酮对小鼠胸腺上皮细胞的增殖均具有显著抑制作用(P<0.05),并表现出与剂量和处理时间依赖性关系.高剂量(10～25 mg/L)ZEA使小鼠胸腺上皮细胞细胞周期显著阻滞于G2/M期(P<0.05),并存在剂量依赖性关系.这些结果表明,玉米赤霉烯酮对小鼠胸腺上皮细胞有直接毒害作用.     

Inhibition of lymphocyte proliferation by bovine trophoblast protein-1 (type I trophoblast interferon) and bovine interferon-alpha I1.

Bovine trophoblast protein-1 (bTP-1) is a Type I interferon secreted by the bovine trophoblast from about Day 15 of pregnancy. It is not known whether bTP-1 has functional properties in common with other interferons. The aim of the present study was to determine whether bTP-1 inhibits proliferation of lymphocytes induced by mitogens, mixed lymphocyte cultures (MLC) and interleukin-2 (IL-2) and, if so, whether this activity is similar to that of a related interferon, bovine interferon-alpha I1 (bIFN-alpha I1). Stimulation of lymphocyte proliferation caused by phytohemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) was inhibited by bTP-1 and bIFN-alpha I1 without any reduction in cell viability. Maximum or near-maximum inhibition (less than 50%) was achieved at concentrations of 0.5-5.0 nM of bTP-1 and bIFN-alpha I1. Cells stimulated with PWM were less inhibited than cells stimulated with PHA and Con A. Both bTP-1 and bIFN-alpha I1 inhibited MLC to a greater degree than lectin-stimulated cells (maximum inhibition was 78% or greater). Also, bTP-1 and bIFN-alpha I1 slightly inhibited incorporation of [3H]thymidine ([3H]TdR) induced by the combination of phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), and calcium ionophore A23187. Finally, bTP-1 and bIFN-alpha I1 had bimodal effects on incorporation of [3H]TdR by IL-2-induced lymphocytes. Incorporation of [3H]TdR was increased at 0.005 nM and 0.05 nM concentrations while higher concentrations caused a slight decrease in [3H]TdR incorporation. Results confirm that bTP-1 inhibits lymphocyte proliferation in a manner similar to that caused by the leukocyte-derived interferon, bIFN-alpha I1. Incomplete inhibition of mitogen-induced proliferation and differences in degree of inhibition between various stimulators suggest that bTP-1 and bIFN-alpha I1 preferentially inhibit certain lymphocyte subpopulations. Local inhibition of lymphocyte proliferation caused by bTP-1 may help protect the allogeneic conceptus from immune responses to fetal antigens or regulate the release of cytokines from endometrial lymphocytes.     

Expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on B-1a cell from persistent lymphocytosis (PL) cows and lymphoma cell induced by bovine leukemia virus

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on B lymphocytes from persistent lymphocytosis (PL) cattle and lymphoma cells induced by bovine leukemia virus (BLV) was studied in vitro. Flow cytometric analysis showed that high levels of receptors to GM-CSF were expressed on these cell types. Proliferation of these B cells was induced in response to bovine GM-CSF. In tumor cell lines, the rate of cell proliferation was correlated with expression of GM-CSF receptors. A monoclonal antibody to GM-CSF inhibited lymphocyte proliferation and blocked the GM-CSF binding of lymphocytes. Cells expressing GM-CSF receptor were Ig positive and both CD5 and CD11 positive (B-1a cell). These results suggest that an abnormal expression of GM-CSF receptors on B lymphocytes from PL and lymphoma cells induced by BLV plays important roles in the PL and proliferation of lymphoma.     

5-羟色胺对肉鸡肺动脉平滑肌细胞增殖和凋亡的影响

采用胶原酶消化法培养原代肉鸡肺动脉平滑肌细胞（PASMC）,检测不同浓度的5-羟色胺（5-HT）对肉鸡PASMC增殖的影响,并用流式细胞仪检测PASMC周期和凋亡情况。结果发现,10-4～10-7 mol/L的5-HT能剂量依赖性地促进肉鸡PASMC的增殖,可能是5-HT加速细胞从G1期向S期过渡,同时5-HT也可促进PASMC凋亡,因此在总体上5-HT促进PASMC增殖的作用更明显。     

鸡p15因子抑制MDCC-MSB1细胞的增殖及端粒酶活性

为探讨鸡p15基因的生物学功能,试验构建了鸡p15基因的真核表达载体pcDNA3.1( )-p15,并转染到鸡MDV转化的淋巴细胞系MDCC-MSB1,应用G418筛选掉未转染的细胞,对存活细胞p15蛋白的表达、细胞增殖力、群体倍增时间、细胞周期和端粒酶的活性进行了检测。结果表明,与转染空质粒pcDNA3.1( )细胞相比,转染了p15基因的细胞稳定表达了p15蛋白;细胞的增殖受到了抑制,抑制率达45%～74%;群体倍增时间从27h延长至416h;流式细胞仪分析细胞周期发现,p15蛋白引起了细胞多停滞于G0/G1期,S和G2/M期细胞比例下降;端粒酶活性受到抑制。     

胸腺素α_1调节鸡脾脏淋巴细胞免疫功能的信号转导途径

在无菌条件下,从健康墟岗黄鸡体内取出脾脏,分离淋巴细胞并进行体外培养。在细胞培养液中分别加入终浓度为0.01 mol/L咖啡碱、0.05 mol/L EDTA、0.01 mol/L氯化锂和5μg/mL甲派氟丙嗪,然后用10μg/mL胸腺素α1(Tα1)处理,再加入20μg/mL ConA,研究Ca2+-肌醇磷脂-蛋白激酶系统和cAMP-肌醇磷脂-蛋白激酶系统在Tα1调节淋巴细胞免疫功能中的作用,揭示Tα1调节免疫功能的信号转导机制。结果表明,咖啡碱处理能显著增强Tα1促进脾脏淋巴细胞增殖转化、IL-2的基因表达,提高培养液中IL-2浓度(P<0.05);而EDTA处理能显著抑制Tα1促进脾脏淋巴细胞增殖转化、IL-2的基因表达与分泌的作用(P<0.05);氯化锂和甲派氟丙嗪对Tα1的免疫调节作用的影响不明显(P>0.05)。结果提示,Tα1调节鸡脾脏淋巴细胞的增殖和IL-2的基因表达与分泌的作用,主要通过cAMP、Ca2+和PKC等第二信使的介导。     

不同刺激剂PHA和ConA对绒山羊淋巴细胞有丝分裂中期化的影响

试验旨在探索使较高比例的淋巴细胞富集在有丝分裂G2/M期的最佳条件。运用植物血凝素(PHA)和刀豆蛋白A(ConA)对淋巴细胞进行刺激使其增殖,培养一定时间后加秋水仙素对淋巴细胞进行同步化处理,用流式细胞仪检测G2/M期的细胞数量进行比对,观察试剂的最佳作用浓度和加秋水仙素的最佳时间。结果表明,采用PHA和ConA刺激淋巴细胞增殖的最佳作用浓度是60和5 μg/mL,且淋巴细胞经ConA刺激培养45 h再加秋水仙素处理5 h G2期的比例最高为58.38%。结果提示,就绒山羊的淋巴细胞来说,ConA刺激绒山羊淋巴细胞增殖的效果比PHA好,且摸索出细胞G2/M期的时间点至关重要。     

Immunity to equine herpesvirus type 1 (rhinopneumonitis): in vitro lymphocyte response.

Twenty-two ponies were examined for serum-neutralizing (SN) antibody to equine herpesvirus type 1 and for in vitro lymphocyte transformation in the presence of viral antigen. Six ponies had undetectable levels of neutralizing antibody (titer less than 1:2) and had lymphocytes which did not respond in culture with viral antigen (stimulation index less than 2.0). Four ponies which had SN antibody to equine herpesvirus type 1 did not manifest lymphocyte transformation in vitro. The 12 remaining seropositive ponies had lymphocyte transformation with viral antigen in vitro (stimulation indexes from 2.0 to 23.5). Lymphocyte transformation was specifically suppressed when cells were grown in medium containing autologous serum. The temporal development of in vitro lymphocyte responsiveness and SN antibody in 3 specific-pathogenfree ponies after experimentally induced infection with equine herpesvirus type 1 is described.     

Inhibition of equine mononuclear cell proliferation and leukotriene B4 synthesis by a specific 5-lipoxygenase inhibitor, A-63162.

The lipoxygenase metabolites of arachidonic acid have an important role in lymphocyte activation. We used a specific 5-lipoxygenase inhibitor, A-63162, to examine the role of 5-lipoxygenase (5-LO) in equine blood mononuclear cell (BMC) proliferation and leukotriene B4 (LTB4) synthesis after stimulation with mitogen (phytohemagglutinin, PHA) or calcium ionophore (A23187). The A-63162 inhibited PHA-induced equine BMC proliferation and, at the same concentration, also inhibited A23187-induced LTB4 synthesis. The presence of exogenous interleukin 2 (IL-2) or the cyclooxygenase inhibitor indomethacin, failed to reverse the immunosuppression caused by A-63162. Further, we found that A-63162, at the concentration that inhibited BMC proliferation and LTB4 synthesis, had no effect on BMC viability. The addition of the specific protein kinase C inhibitor, H-7, did not inhibit A23187-induced LTB4 synthesis. Results indicate that 5-lipoxygenase metabolites may have an important role in equine lymphocyte activation and that protein kinase C has no role in regulating LTB4 production after A23187 stimulation.     

Developmental changes in cell proliferation and apoptosis in the normal duck thymus

Cell proliferation and apoptosis in the normal duck thymus during embryonic and post-embryonic development were studied. The flow cytometry assay shows that the level of G(0)/G(1) thymic cell population and the proportion of apoptotic cells increased with age, while the levels of S phase, G(2) + M phase and the proliferating index decreased with age. Proliferation cell nuclear antigen (PCNA) was mainly detected in the nuclei of lymphocytes. The number of PCNA-positive cells in the cortex and medulla significantly decreased with age. Transferase-mediated dUTP nick-end labelling (TUNEL) reaction stained apoptotic bodies in the cytoplasm of macrophages and free apoptotic bodies or nuclei with condensed chromatin in lymphocytes. The number of TUNEL-positive cells in the cortex and medulla markedly increased with age. The amount of proliferation and apoptotic cells in the thymic cortex was higher than that in the medulla. The balance between proliferation and apoptosis in the duck thymus may account for the process of thymic development and involution.     

Immunomodulation during and after castration under inhalation anaesthetic without genotoxic effects on equine lymphocytes

Genotoxic DNA damage due to inhalation anaesthesia has been demonstrated in human lymphocytes. In order to evaluate anaesthesia-associated changes in cell-mediated immunity on the basis of a potential DNA damage as a health risk in horses, single cell gel electrophoresis and lymphocyte proliferation assay were performed on equine lymphocytes which were obtained before, during and after regular castration under inhalation anaesthetic. No significant lymphocytic DNA damage due to isoflurane anaesthesia was observed, whereas lymphocyte proliferative reactivity and lymphocyte counts decreased significantly (p≤0.05) during and after anaesthesia. The present study thus indicates that the combined anaesthesia does not result in significant DNA damage, which hence cannot be held responsible for the observed changes in the immune response of equine lymphocytes. However, the recognized compromises of immune function ought to be considered especially in immunologically challenged animals.     

Technical aspects of low 3H-thymidine incorporation by mitogen stimulated canine peripheral blood lymphocytes in vitro

The value of [3H]-thymidine incorporation as a measurement for mitogen induced proliferation of dog peripheral blood lymphocytes (PBL) has been examined. The cells were cultured in RPMI 1640, enriched with 10% autologous plasma for 48 hours at 37 degrees C, 5% CO2 and 95% relative humidity. Under these conditions a great variability in [3H]-thymidine incorporation was observed. By analysis of CPM and number of activated cells (G1), it was found that comparable number of G1 cells were generated in human and dog PBL. Also, the membrane transport of thymidine was very similar for lymphocytes of the two species. Nevertheless, a low [3H]-thymidine incorporation by dog PBL was frequently seen, and this phenomenon could be related to a release of soluble substance(s) within the cultures. When the cultured cells were washed and resuspended in fresh medium immediately before pulsing, the expected CPM per G1 cell could be obtained. Since it has been described in the literature that macrophages can produce cold thymidine in macrophage enriched lymphocyte cultures, the in vitro response of non-adherent dog PBL was analyzed. Mitogen stimulation of such non-adherent cells resulted in CPM per G1 cells very similar to those obtained with washed cells. Based on these data, it is suggested that the production of cold thymidine might be one of the technical problems related to cultures of lectin stimulated dog PBL in vitro and it should be taken into consideration, if [3H]-thymidine incorporation is used as the only measure of lymphocyte proliferation.     

Expression of Kit, the receptor for stem cell factor, in bovine peripheral blood

The expression of Kit, the receptor for stem cell factor (SCF), on bovine peripheral blood cells (PBCs) was examined by using monoclonal antibodies against the bovine Kit protein. Flow cytometric analysis showed that approximately 1.5% of PBCs expressed Kit. In cytospin preparations, the morphology of most Kit+ PBCs was similar to that of large lymphocytes. Subsets of Kit+ PBCs coexpressed CD3, IgM, and/or CD11b but not CD14 or G1. SCF did not induce the proliferation of Kit+ PBCs in vitro. These results indicate that Kit is expressed on subsets of lymphocytes in bovine peripheral blood, but the ligand of Kit, SCF, does not directly induce the proliferation of this cell population.     

Leptin inhibits mitogen-induced proliferation of peripheral T lymphocytes from Holstein cows

This study examined the mitogenic response of bovine peripheral T lymphocytes to leptin, a pleiotropic hormone regulating food intake and energy expenditure. Leptin alone slightly suppressed proliferation of T lymphocytes in the presence of concanavalin A (ConA). Leptin also inhibited proliferation of T lymphocytes induced by anti-CD3 antibody. ConA treatment activated some protein kinases, including p44/p42(MAPK) and Akt/PKB, while anti-CD3 antibody treatment increased mRNA expression of suppressor of cytokine signalling (SOCS) 3, interferon (IFN)gamma, interleukin (IL) 2 and IL4 in T lymphocytes. Leptin alone increased only SOCS3 mRNA expression. Simultaneous treatment with mitogens and leptin enhanced IFNgamma mRNA expression but decreased IL2 mRNA expression, without any synergistic effect on phosphorylation of protein kinases or mRNA expression of SOCS3 and IL4. These results suggest that leptin modulates bovine T lymphocyte functions.     

Whey acidic protein (WAP) depresses the proliferation of mouse (MMT) and human (MCF-7) mammary tumor cells

We previously reported that the enforced expression of exogenous whey acidic protein (WAP) significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 and EpH4/H6 cells). This paper presents the first evidence that WAP also depresses the proliferation of mammary tumor cells from mouse (MMT cells) and human (MCF-7 cells). We established WAP-clonal MMT and MCF-7 cell lines, and confirmed the secretion of WAP from the WAP-clonal cells into culture medium. The enforced expression of WAP significantly inhibited the proliferation of MMT and MCF-7 cells in in vitro culture. FACScan analyses revealed that G0/G1 phase cell-cycle progression was disordered and elongated in the WAP-clonal MMT and MCF-7 cells compared to that of the control cells. The expression of cyclin D1 was significantly decreased in the WAP-clonal MMT and MCF-7 cells, suggesting that progression from the G1 to the S phase was delayed in the WAP-clonal cells. The present results indicate that WAP plays a negative regulatory role in the cell-cycle progression of mammary tumor cells via a paracrine mechanism.     

Cyclical endometrial steroid hormone receptor expression and proliferation intensity in the mare

The aims of this study were to investigate the steroid hormone receptor expression and the proliferation intensity during the equine endometrial cycle by immunohistological methods, established for routine examination of formalin-fixed, paraplast-embedded specimens. Endometrial biopsy specimens were obtained during one cycle from 7 mares. In comparison with the blood steroid hormone levels the quantity and distribution of oestrogen (ER) and progesterone receptors (PR) and the proliferation marker Ki-67 antigen expression were investigated. Rising 17beta-oestradiol concentrations in preoestrus induce a synchronous expression of ER, PR and Ki-67 antigen in stromal cells. In the early dioestrus 17beta-oestradiol levels decrease and progesterone levels reach their maxima. This correlates with an intense proliferation activity and the highest hormone receptor expression in epithelial cells. In accordance to the morphological features of asynchronous glandular differentiation in fibrotic areas (endometrosis) their epithelial hormone receptor expression is out of phase.     

Cell proliferation potency is independent of FGF4 signaling in trophoblast stem cells derived from androgenetic embryos

We previously established trophoblast stem cells from mouse androgenetic embryos (AGTS cells). In this study, to further characterize AGTS cells, we compared cell proliferation activity between trophoblast stem (TS) cells and AGTS cells under fibroblast growth factor 4 (FGF4) signaling. TS cells continued to proliferate and maintained mitotic cell division in the presence of FGF4. After FGF4 deprivation, the cell proliferation stopped, the rate of M-phase cells decreased, and trophoblast giant cells formed. In contrast, some of AGTS cells continued to proliferate, and the rate of M-phase cells did not decrease after FGF4 deprivation, although the other cells differentiated into giant cells. RO3306, an ATP competitor that selectively inhibits CDK1, inhibited the cell proliferation of both TS and AGTS cells. Under RO3306 treatment, cell death was induced in AGTS cells but not in TS cells. These results indicate that RO3306 caused TS cells to shift mitotic cell division to endoreduplication but that some of AGTS cells did not shift to endoreduplication and induced cell death. In conclusion, the paternal genome facilitated the proliferation of trophoblast cells without FGF4 signaling.     

RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响

本研究旨在探讨敲低MTPN基因对体外培养水牛颗粒细胞增殖、凋亡及雌激素、孕酮分泌的影响。应用RNAi技术敲低颗粒细胞中MTPN基因的表达水平,通过实时荧光定量PCR方法检测体外培养水牛颗粒细胞中MTPN基因及增殖和周期相关基因的表达情况,CCK-8法检测细胞增殖,借助流式细胞仪检测细胞周期的分布,采用ELISA试剂盒检测细胞培养液中雌激素与孕酮含量。结果显示,经siRNA(si-MTPN)转染颗粒细胞后,MTPN基因相对表达量下降60%(P<0.01);细胞增殖受到显著抑制(P<0.05),G1期细胞数量下降,S期细胞数量上升,G2期细胞数量极显著上升(P<0.01),细胞被阻滞在G2期;增殖与周期相关基因Cyclin D2、Cytochrom C表达量显著上升(P<0.05),Caspase9、Fas基因表达量极显著上升(P<0.01);ELISA检测雌激素和孕酮分泌水平均显著下降(P<0.05)。综上表明,敲低MTPN基因能通过调控相关基因的表达抑制体外培养水牛颗粒细胞的增殖及雌激素、孕酮的分泌水平,为阐明MTPN基因参与家畜卵泡发生的分子机制提供参考。