Interferon-gamma-dependent cytotoxic activation of human astrocytes and astrocytoma cells

Astrocytes and microglia become activated in a broad spectrum of inflammatory neurodegenerative diseases. Activated microglia are widely believed to be the principal source of inflammation-induced neuronal degeneration in these disorders. To investigate the neurotoxic potential of human astrocytes, we exposed them and human astrocytic U-373 MG cells to a variety of inflammatory stimulants. We then assessed the effects of their supernatants on human SH-SY5 cells. When astrocytes and U-373 MG cells were stimulated with interferon (IFN)-γ (150 U/ml), their supernatants significantly reduced SH-SY5Y cell viability. Other powerful inflammatory stimulants such as lipopolysaccharide (0.5 μg/ml), tumor necrosis factor-α (10 ng/ml) and interleukin-1β (10 ng/ml), alone or in combination, were without effect. These combinations were also unable to enhance the IFN-γ effect. The induced cytotoxicities were reversed by JAK inhibitor I, a potent and specific inhibitor of JAKs. This result indicates that the neurotoxic effect was proceeding through the IFN-γ receptor (IFNGR)-JAK-STAT intracellular pathway. To establish that the IFNGR is expressed on both cultured astrocytes and U-373 MG cells, we performed RT-PCR on total RNA extracts to identify a specific IFNGR product. We showed the protein product on these cultured cells by immunocytochemistry using an antibody to IFNGR. Finally, using human postmortem material, we showed sharp upregulation of the IFNGR on activated astrocytes in affected areas in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and multiple sclerosis. These findings suggest that activated astrocytes may become neurotoxic when stimulated by IFN-γ and may therefore exacerbate the pathology in a spectrum of neurodegenerative diseases.     

Purines stimulate chick neuroblasts proliferation in culture

Neuroblasts from cerebral hemispheres of 6-day-old chick embryo are able to divide to a certain extent under suitable culture conditions. It was found that addition of purine bases to the culture medium induced an increase of tritiated thymidine incorporation into the cells, resulting from a stimulation of neuroblast proliferation. Most purines elicited a stimulation, but guanine compounds were the most active. Inosinic acid (IMP), the first purine synthesized, was also active. Folic acid was inactive. These results suggest that neuroblasts in culture are defective in the biosynthesis of purines and that this deficiency is not due to a lack of folic acid. Some other cell types were also tested including glial cells, meningeal cells, whole embryo fibroblasts and heart fibroblasts. Only the latter did not respond to purine bases. These observations show that different cell types in primary culture need exogenous purines for maximal growth.     

脑星形细胞瘤中bFGF及其受体对血管新生 和肿瘤细胞增殖的影响

目的：为探讨碱性纤维母细胞生长因子（ｂＦＧＦ）及其受体（ＦＧＦＲ－２）对人星形细胞瘤的血管新生及细胞增殖的作用。方法：彩和免疫组织化学方法,结果：发现ｂＦＧＦ及其受体,增殖细胞核抗原（ＰＣＮＡ）在瘤细胞及血管内皮细胞中均有表达,高级别星形细胞瘤的表达阳性率高于低级别者,ｂＦＧＦ在瘤细胞中的表达阳性率分别为与ＦＧＦＲ－２及ＰＣＮＡ的表达阳性率呈正相关关系（Ｐ〈０．０１,Ｐ〈０．０５）,ｂＦＧＦ及其受     

Homologous activated platelets stimulate differentiation and proliferation of primary human bone cells

In bone tissue engineering approaches the expansion of bone cells is an essential part. In recent years the search for an appropriate alternative to fetal bovine serum (FBS) in the ex vivo expansion process has increased. This study demonstrates that platelet-rich clot releasate (PRCR) could be an appropriate alternative. The effects of PRCR on bone cell cultures derived from 5 different human donors were analyzed with respect to morphology, proliferation, apoptosis and gene expression. Five different PRCR concentrations were used: 1, 5, 10, 20 and 40%. The population doubling (PD) values were calculated for each concentration. Light microscopy analysis was done after 3 and 9 days. Flow cytometry was used to analyze cell cycle effects. The gene expression of alkaline phosphatase, collagen type 1, osteocalcin, bone sialoprotein and osteopontin was analyzed with RT-PCR. 10% FBS cultures were used as controls. With 10% PRCR the cell morphology resembled the control cultures; however, the PD values were significantly higher (p < 0.01). Concentrations of 20 and 40% had a clear cytotoxic effect, observed with light microscopy analysis and flow cytometry. PRCR had a potent effect on the expression of osteogenic markers and resulted in a concentration-dependent upregulation. We demonstrate that human bone cells derived from the maxillary alveolar ridge can be cultured in medium containing PRCR instead of FBS. The addition of PRCR results in higher proliferative capacity and upregulation of osteogenic markers. These results indicate that FBS could be avoided in future tissue engineering approaches using bone cells from this anatomic site.     

Overexpression of miR-221 inhibits proliferation and promotes apoptosis of human astrocytoma cells

microRNAs (miRNAs) play tumor-promoting roles in a variety of tumors. This study investigated the expression of miRNA-211 (miR-221) in human astrocytoma, and its effect on proliferation and apoptosis of human astrocytoma cells in vitro. miR-221 expression was detected in 10 astrocytoma tissues and 4 adjacent tissues by real-time quantitative PCR (qRT-PCR). miR-221 expression in situ was significantly higher in astrocytoma tissues than in adjacent tissues (P<0.05). To determine whether the upregulation of miR-221 could be associated with tumor development or progression, a synthetic miR-221 mimic was transiently transfected into U251 astrocytoma cells in vitro. qRT-PCR confirmed that the mimic significantly increased the expression of miR-221 in these cells. An MTT colorimetric assay indicated that proliferation was significantly higher in U251 cells transfected with miR-221 mimic than in scramble-transfected control cells (P<0.05). Further analysis of miR-221 transfected cells by flow cytometry revealed an altered cell cycle progression, with more cells in S and G1 phase, as well as an inhibition of apoptosis (P<0.05). These findings indicate that the upregulation of miR-221 in astrocytoma tissues may be associated with development or progression of these tumors. Thus, miR-221 should be explored as a potential molecular marker for the diagnosis and treatment of astrocytoma.     

星形细胞瘤与胚脑星形细胞的电镜对比观察

用光镜及电镜观察了６０例脑星形细胞瘤及８例早期人胚脑及神经管组织。结果显示：瘤细胞超微结构可分五型，即未分化细胞、前星形细胞、星形细胞、胖细胞及瘤巨细胞。与胚脑发生中的星形细胞相比较，瘤细胞有类似发生中星形细胞相应各个阶段的分化。不同之点是瘤细胞常呈不典型性，形态畸变、染色质增多、核浆可呈不同步分化等。基于上述观察，我们对传统的星形细胞瘤命名及分类作了评价，并对分类提出看法。     

肝素结合生长因子的纯化及其刺激人血管内皮细胞增生作用的研究

用40—80%饱和度硫酸铵沉淀、CM-葡聚糖C-50离子交换层析及肝素琼脂糖亲和层析从牛脑中纯化了肝素结合生长因子,SDS-PAGE显示单一带型,分子量约为16kD;以人脐带静脉内皮细胞为靶细胞,对肝素结合生长因子刺激内皮细胞增生作用进行了研究,结果表明,细胞培养时加入肝素结合生长因子,可使内皮细胞长期传代,冷冻后可成功复苏,培养所需血清浓度可降至10—15%。     

脑星形细胞瘤中bFGF及其受体对血管新生和肿瘤细胞增殖的影响

目的：为探讨碱性纤维母细胞生长因子（ｂＦＧＦ）及其受体（ＦＧＦＲ－２）对人星形细胞瘤的血管新生及细胞增殖的作用。方法：彩和免疫组织化学方法,结果：发现ｂＦＧＦ及其受体,增殖细胞核抗原（ＰＣＮＡ）在瘤细胞及血管内皮细胞中均有表达,高级别星形细胞瘤的表达阳性率高于低级别者,ｂＦＧＦ在瘤细胞中的表达阳性率分别为与ＦＧＦＲ－２及ＰＣＮＡ的表达阳性率呈正相关关系（Ｐ〈０．０１,Ｐ〈０．０５）,ｂＦＧＦ及其受     

Adenosine receptor-induced cAMP changes in D384 astrocytoma cells and the effect of bradykinin thereon.

In human D384 astrocytoma cells, cyclic AMP accumulation can be conveniently studied after labelling of the adenosine triphosphate pool (15 fmol cell-1) with [3H]adenine. In this study, adenosine had a biphasic effect on cyclic AMP accumulation, which was scarcely altered by blocking adenosine uptake and metabolism. Low concentrations of adenosine led to an inhibition of cyclic AMP accumulation, and higher concentrations led to stimulation. No effect of adenosine on cyclic AMP was observed unless phosphodiesterase was inhibited by rolipram. The A1 receptor antagonist DPCPX attenuated the inhibitory phase of adenosine response, and enhanced the cyclic AMP accumulation induced by adenosine analogues. The cyclic AMP accumulation was stimulated by NECA greater than ADO greater than CGS 21680 greater than CV 1808 greater than CPA greater than or equal to CHA, indicating mediation by A2 receptors. The stimulatory effect of NECA was much more effectively blocked by the combined A1 and A2 receptor antagonist CGS 15943 (KB 4 nmol l-1) than by the A1 antagonist DPCPX (KB 110 nmol l-1). Treatment of the cells with pertussis toxin (0.2 microgram ml-1 for 2.5 h) potentiated the cyclic AMP response to adenosine analogues significantly. The cyclic AMP response to NECA was enhanced by the protein kinase C activator phorbol dibutyrate even after pertussis toxin treatment. By contrast, nanomolar concentrations of bradykinin, which increases Ca(2+)-levels and protein kinase C activity in D384 cells, reduced NECA-induced cyclic AMP accumulation in control and pertussis toxin-treated cells. Thus, D384 cells possess both A1 and A2 adenosine receptors influencing cyclic AMP in opposite directions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Red blood cells inhibit proliferation and stimulate apoptosis in human lung fibroblasts in vitro

Cell proliferation and apoptosis are both important mechanisms for the regulation of tissue homeostasis. For instance, proliferation is crucial in wound repair, whereas apoptosis is important for removal of damaged cells and resolution of inflammation. Imbalance between cell proliferation and apoptosis can therefore lead to pathological conditions and disease. In inflammatory and fibrotic lung disorders, red blood cells (RBCs) can interact with fibroblasts and connective tissue. In the present study, we therefore hypothesized that the presence of RBCs can affect fibroblast proliferation and apoptosis. Human foetal lung fibroblasts (HFL-1) were cultured in the presence or absence of purified whole RBCs and RBC-conditioned media. RBC significantly decreased fibroblast proliferation as determined both by DNA content analysis (Hoechst 33258 staining, P < 0.01; WST-1, P < 0.001) and BrdU incorporation. After treatment with staurosporine (STS) for 48 h, apoptosis was determined by TUNEL and propidium iodide staining followed by flow cytometry analysis. RBCs augmented STS-induced apoptosis (median: 46.4%; range 12.0-90.4) compared to control cells (median 26.2%; range 7.1-45.5). Thus, our data indicate that the presence of RBCs affects both fibroblast proliferation and susceptibility to undergo apoptosis. Our findings therefore suggest a role for RBCs in regulating fibroblast homeostasis after tissue injury.     

Klebsiella pneumoniae stimulate highly purified human blood B cells to mature into plaque forming cells without prior proliferation.

The plaque forming cell (PFC) and proliferative responses of human peripheral blood lymphocytes and highly purified blood B cells induced by pokeweed mitogen (PWM) and group A streptoccocal cell membranes (A-ScM) were compared with the responses triggered by various cell preparations of Klebsiella pneumoniae K 43 (Klebs). The number of PFC was determined by a protein A plaque assay, and lymphoproliferation was measured by 3H-thymidine incorporation. In cell cultures stimulated with PWM and A-ScM, lymphocyte proliferation appeared to be associated with the generation of PFC. Klebs caused development of PFC without measurable prior proliferation. Whereas the response to PWM and A-ScM was absolutely T cell-dependent, highly purified B cells generated PFC when incubated with Klebs. Moreover, restitution of T cells to the B cell fraction did not augment (or diminish) the number of plaques. These studies establish that Klebs cell envelope structures contain a T cell-independent polyclonal B cell activator for human B lymphocytes in a high stage of differentiation. Use of this probe should provide further insight into the cellular interactions involved in the differentiation of antibody forming cells in humans.     

Adenosine receptor-induced cAMP changes in D384 astrocytoma cells and the effect of bradykinin thereon

In human D384 astrocytoma cells, cyclic AMP accumulation can be conveniently studied after labelling of the adenosine triphosphate pool (15 fmol cell^-1) with [³H]adenine. In this study, adenosine had a biphasic effect on cyclic AMP accumulation, which was scarcely altered by blocking adenosine uptake and metabolism. Low concentrations of adenosine led to an inhibition of cyclic AMP accumulation, and higher concentrations led to stimulation. No effect of adenosine on cyclic AMP was observed unless phosphodiesterase was inhibited by rolipram. The A₁ receptor antagonist DPCPX attenuated the inhibitory phase of adenosine response, and enhanced the cyclic AMP accumulation induced by adenosine analogues. The cyclic AMP accumulation was stimulated by NECA > ADO > CGS 21680 > CV 1808 > CPA ≥ CHA, indicating mediation by A, receptors. The stimulatory effect of NECA was much more effectively blocked by the combined A₁ and A₂ receptor antagonist CGS 15943 (K_B 4 nmol I^-1) than by the A₁ antagonist DPCPX (K_B 110 nmol 1^-l). Treatment of the cells with pertussis toxin (0.2 μg ml^-1 for 2.5 h) potentiated the cyclic AMP response to adenosine analogues significantly. The cyclic AMP response to NECA was enhanced by the protein kinase C activator phorbol dibutyrate even after pertussis toxin treatment. By contrast, nanomolar concentrations of bradykinin, which increases Ca²⁺-levels and protein kinase C activity in D384 cells, reduced NECA-induced cyclic AMP accumulation in control and pertussis toxin-treated cells. Thus, D384 cells possess both A₁ and A₂ adenosine receptors influencing cyclic AMP in opposite directions. A₂ receptor-mediated cyclic AMP accumulation can be stimulated by activating protein kinase C and inhibited by raising Ca²⁺. Neither the effects of protein kinase C activation nor those of bradykinin required pertussis toxin-sensitive G-proteins.     

Adenosine receptors on human inflammatory cells

Adenosine is a natural nucleoside that plays a physiological role in the modulation of human inflammatory cells. We have investigated the presence of adenosine A2/Ra and A1/Ri receptors and of the P-site on human inflammatory cells. Human B and T(OKT4+ and OKT8+) lymphocytes, polymorphonuclear leucocytes, monocytes, basophils and platelets possess a membrane adenosine A2/Ra receptor. The activation of adenosine A2/Ra receptor increases the intracellular level of cyclic AMP in these cells. Human lymphocytes and neutrophils possess also an inhibitory adenosine A1/Ri receptor and a P-site whose activation inhibits the effect of many adenylate cyclase agonists including isoproterenol, PGE1, histamine, adenosine, cholera toxin and forskolin.     

Interactions of monocytic cells with human endothelial cells stimulate monocytic metalloproteinase production.

Monocyte-endothelial cell interactions play an important role in the early stages of atherosclerosis, and it is hypothesized that regulation of metalloproteinase production by these interactions contributes to this pathological process. The effects of monocytic cell-endothelial cell interactions on monocytic metalloproteinase production were investigated using an in vitro system, focusing on the role of endothelial cell secretions and physical contact as effectors in the regulation of monocytic metalloproteinase expression. Human umbilical vein endothelial cells (HUVECs) and the human monocytic cell line THP-1 were used, and changes in the levels of THP-1 metalloproteinase secretion and mRNA were measured. When THP-1 cells were incubated for 18 hours with HUVEC conditioned medium (CM), a four- to eightfold induction of the metalloproteinase MMP-9 was observed at both the mRNA and protein levels; however, levels of another metalloproteinase, MMP-2, were unaffected. The induction of MMP-9 by HUVEC CM was confirmed using freshly isolated human monocytes. A sevenfold increase in MMP-9 levels was observed with apically collected HUVEC CM but not with basally collected CM. THP-1 cells incubated with paraformaldehyde-fixed HUVECs and isolated HUVEC plasma membranes showed an eightfold increase in MMP-9 levels, and measurements of MMP-9 activity found in THP-1 conditioned medium due to either HUVEC contact or HUVEC CM showed a threefold increase. The molecular weight of the endothelial secreted effector molecule(s) was determined to be 30 +/- 6 kd. The data show that endothelial cells through the release of soluble factors and through direct contact with monocytic cells regulate monocytic metalloproteinase production, which has implications for the atherogenic process.     

Adenosine and its derivatives control human monocyte differentiation into highly accessory cells versus macrophages

Human peripheral blood monocytes have been found to undergo a transitory state of high accessory activity before they fully become macrophages. Time kinetics were done to follow this accessory potential. Studying the regulation of accessory activity, we have found that monocyte-derived accessory cells (m-AC) pass through two phases of development, which both are adversely controlled by cyclic nucleotides. Phase I is positively correlated by intracellular cAMP increase and can be arrested by adenosine 3';5' cyclic monophosphate (cAMP) and synergystic agents. In addition to cAMP, non-cyclic adenine nucleotides and adenosine also mimic all cAMP effects. This behavior is explained by the known presence of surface 5' nucleotidase and adenosine receptor, which in turn leads to activation of adenylate cyclase. At phase II serum is required to convert m-AC into macrophages. In the absence of serum, cells were arrested in the m-AC state. Adenine nucleotides effectively counteract the serum induction leading to the development of m-AC even in the presence of serum. Monocyte/macrophage markers such as Fc receptors and non-specific esterase strictly correlate negatively with the expression of accessory activity. Morphologically, the appearance of veils positively correlates with all experimental situations of high accessory activity. Therefore, it is evident that serum contains regulatory factors that strongly modify the accessory potency of the m-AC via the cyclic nucleotide system, thus presenting a potent immunoregulatory principle at the beginning of the immune cascade.     

PTEN在人脑星形细胞瘤中的表达及意义

目的:探讨磷酸酶-张力蛋白基因(phosphatase and tensin homology deleted on chromosome ten,PTEN)在正常脑组织及星形细胞瘤中的表达及其意义.方法:采用免疫组织化学法及RT-PCR方法检测正常脑组织及不同分级星形细胞瘤组织中PTEN蛋白及mRNA的表达水平,探讨并分析其与星形细胞瘤组织学分级的关系,Western印迹法检测正常脑组织及星形细胞瘤脑脊液中PTEN蛋白的表达.结果:免疫组织化学方法显示PTEN在正常脑组织中阳性率较高,为90%(9/10),星形细胞瘤中阳性率较低,为47.5%(19/40),且PTEN在不同的组织学分级中,阳性率不同.RT-PCR结果显示PTEN mRNA在正常脑组织中表达较高,其相对表达量为0.861±0.072,显著高于星形细胞瘤组织中的表达(0.127±0.008),差异有统计学意义(P<0.05),并随着组织学级别增加,其表达呈降低趋势.Western印迹法结果显示PTEN蛋白在非星形细胞瘤组脑脊液中的相对表达量为1.549±0.194,高于星形细胞瘤组脑脊液中的相对表达量(0.602±0.058),差异具有统计学意义(P<0.05).结论:检测PTEN基因蛋白表达有望作为评估人星形细胞瘤生物学行为和预后的参考指标.     

Mesenchymal stem cells stimulate antibody secretion in human B cells

Mesenchymal stem cells (MSC) have immunomodulatory effects and inhibit T-cell responses to alloantigens and mitogens in vitro and in vivo. We wanted to examine the effect of MSC on human B cells. MSC stimulated IgG production, measured in an enzyme-linked immunospot (ELIspot) assay in blood and spleen lymphocytes. MSC only induced a low proliferation. When a semipermeable membrane separated MSC and mononuclear cells, the IgG production was stimulated in unfractionated lymphocytes. In contrast, enriched B cells required cell contact with MSC to produce IgG. Co-cultures of MSC and lymphocytes increased IFN-gamma production. MSC produce IL-6, and addition of MSC to spleen cells dramatically increased IL-6 levels. After lymphocyte stimulation with lipopolysaccharide (LPS), cytomegalovirus or varicella zoster virus, MSC either stimulated or inhibited IgG response, depending on the level of stimulation by LPS or the viral antigens. Similar results were obtained for enriched B cells. To conclude, MSC stimulate B-cell antibody secretion. The IgG secretion by activated B cells may be stimulated or inhibited by the addition of MSC, depending on the level of stimulation.     

Progestogens stimulate prostacyclin production by human endothelial cells

BACKGROUND: The effects of progestogens on endothelial physiology are poorly studied. Prostacyclin is a potent vasodilator synthesized by two isoforms of cyclooxygenase (COX) in endothelium. We examined the effects of two clinically used progestogens, progesterone and medroxyprogesterone acetate (MPA), on prostacyclin production by cultured human umbilical vein endothelial cells (HUVEC) and the possible role of progesterone receptors and both COX enzymes. METHODS: Cells were exposed to 1-100 nmol/l of either progesterone or MPA and prostacyclin production was measured in culture medium. RESULTS: Both progestogens significantly increased prostacyclin release in a time- and dose-dependent manner, being higher than control after 24 h. Progesterone and MPA, both at 10 nmol/l, increased mRNA expression and protein content of both COX. All these effects were mediated through progesterone receptor activation, since they were abolished by treatment of cells with the progesterone receptor antagonist RU-486. Selective inhibitors of COX-1 and -2 (SC-560 and NS-398 respectively) reduced basal prostacyclin release, and eliminated increased production in response to progestogens. In combination with estradiol, progestogens had an additive effect without eliminating estradiol-induced prostacyclin production. CONCLUSIONS: Our results support the hypothesis that progesterone and MPA increased HUVEC prostacyclin production in a progesterone receptor-dependent manner, by enhancing COX-1 and COX-2 expression and activities.     

Lysis-sensitive targets stimulate an elevation of cAMP in human natural killer cells.

Natural killer (NK) cells are lymphocytes that are capable of destroying tumour cells and virally infected cells (cytolysis) without prior sensitization. When cAMP is artificially elevated in NK cells, it is a potent inhibitor of their cytolytic function. We investigated whether NK-cell cAMP levels are modulated in response to tumour target cells to determine the potential of cAMP as a physiological regulator of NK cytotoxic function. When NK cells are exposed to a range of lysis-sensitive (LS) tumour-target cells there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. The peak increase in cAMP (200-400% above control) occurs at 30 min for all LS targets tested. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumour-target cells. The cAMP elevation may be dependent on both LS-target-stimulated adenylyl cyclase (AC) activation and LS-target-stimulated phosphodiesterase (PDE) inhibition. When the NK cells are pretreated with the protein tyrosine kinase (PTK) inhibitor, genistein (30 micrograms/ml), the AC-activation component of the cAMP elevation is abolished. Thus, the AC-activation component appears to require PTK activation. When NK cells are pretreated with the protein kinase C (PKC) inhibitor, chelerythrine chloride (10 microM) the cAMP elevation in response to LS targets was not diminished. This indicates that neither the AC-activation component nor any PDE-inhibition component require PKC activation.