Toxoplasma gondii: Microassay to differentiate toxoplasma inhibiting factor and interleukin 2

Using a sensitive, economical, and reproducible microassay, the relationship of toxoplasma inhibiting factor to interleukin 2 has been examined. The assay developed took advantage of the observation that (1) Toxoplasma gondii tachyzoites replicated efficiently in the murine monocytic cell line, RAW 264; (2) treatment of RAW 264 cells with toxoplasma inhibiting factor prevented intracellular replication of the parasite to an extent similar to that observed with identical treatment of freshly isolated murine peritoneal exudate cells; and (3) [³H]uracil incorporation was an efficacious means to quantify replication (or inhibition of replication) of tachyzoites within the cell line. Although toxoplasma inhibiting factor and interleukin 2 were both present in the same lectin -and antigen-stimulated splenocyte supernatant fluids, results from microassays strongly suggested that the molecules were two distinct entities.     

Identification of TLR2/4-mediated phagocytosis and immune response activation pathways by vacuoles isolated from Saccharomyces cerevisiae

The vacuoles of the yeast Saccharomyces cerevisiae are closely related to mammalian lysosomes and play a role in macromolecular degradation due to the hydrolytic enzymes present inside. The vacuoles also regulate osmotic pressure and control cellular homeostasis. In previous results, vacuoles were shown to activate the immune response of macrophages by promoting the production of immune-mediated transporters nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory cytokines. In this study, the effects of vacuoles on the phagocytosis activity of RAW264.7 cells and their potential as immune enhancers were evaluated, and receptors capable of recognizing vacuoles were examined. An investigation using the phagocytes assay showed that phagocytosis activity increased by the vacuole. Besides, after treatment with TLR2/4 inhibitor, the expression of pro-inflammatory cytokines by vacuoles was significantly reduced and the inducible nitric oxide synthase (iNOS) protein was also significantly reduced. However, treatment with a TLR2 inhibitor did not reduce the production of interleukin-6 (IL)-6, a pro-inflammatory cytokine. As a result of confirming the activation of TLR2/4 using Western blot and immunofluorescence (IF), the TLR2/4 protein expression and fluorescence intensity increased depending on the concentration of vacuoles. Yeast vacuoles significantly upregulate protein expression of p-p65/p-p38 MAPKs. In summary, the vacuoles isolated from S. cerevisiae in macrophages have increased phagocytic ability at a concentration of 20 (µg/ml) and can function as immune-enhancing agent suggesting that TLR2/4 mediated the p38 MAPK/nuclear factor kappa B signaling pathway.     

稳定表达鼠源PLEKHA1细胞系的建立及其对细胞迁移的影响

目的：构建稳定表达鼠源PLEKHA1的RAW264.7小鼠巨噬细胞系,探讨PLEKHA1过表达对巨噬细胞迁移的影响。方法：根据小鼠PLEKHA1基因序列设计引物,克隆其编码区序列,酶切后插入pCDH载体,在293FT细胞中进行病毒的包装,用获得的高滴度慢病毒感染RAW264.7细胞,建立能稳定高效表达PLEKHA1的RAW264.7细胞系;在此基础上,观察PLEKHA1对巨噬细胞迁移的影响。结果：经基因克隆、酶切、连接后,构建了鼠源PLEKHA1重组慢病毒表达载体,包装病毒感染RAW264.7细胞,经嘌呤霉素筛选后免疫印迹检测,RAW264.7细胞中PLEKHA1的蛋白表达提高近30倍;同时,发现PLEKHA1的过表达影响RAW264.7细胞的迁移。结论：构建了稳定表达小鼠PLEKHA1的RAW264.7细胞系,PLEKHA1过表达降低RAW264.7细胞的迁移能力。     

A Transport System for C4-Dicarboxylic Acids in Isolated Membrane Preparations from Escherichia coli

To investigate the mechanism of succinate transport system in Escherichia coli, the isolated membranes were prepared from E. coli W2252 and T5, a mutant defective in succinate uptake derived from W2252. Uptakes of ¹⁴C-substrates by W2252 and T5 membranes and the dilution of accumulated radioactivity by unlabeled C₄-dicarboxylic acids, indicated that C₄-dicarboxylic acids in the tricarboxylic acid cycle are transported by the same system in E. coli which requires a suitable energy source such as NADH, D-lactate or reduced phenazine methosulfate. The uptakes of succinate by W2252 membranes were inhibited by an anaerobic incubation or some of the inhibitors of electron transport chain. Difference spectra of reduced versus oxidized membranes from W2252 and T5 indicated the reduction of flavoproteins and cytochromes by dithionite, NADH or D-lactate. From these results it was concluded that the uptake of the C₄-dicarboxylic acids in isolated membranes is coupled to an electron transport chain involving a specific dehydrogenase system.     

小鼠IL-27真核表达载体的构建及其在RAW264.7 细胞中的表达

目的:构建小鼠白介素27(Interleukin 27,IL-27)单链融合基因的真核表达载体并检验其在RAW264.7细胞中的表达情况。方法:提取小鼠脾细胞总RNA,通过RT-PCR扩增出小鼠EBI3和p28 c DNA。采用重叠延伸PCR(splicing by overlap extension PCR,SOE PCR)通过编码疏水性多肽接头(Gly4Ser)3的DNA序列连接小鼠EBI3和p28基因片段,构建小鼠IL-27单链融合基因(mouse single chain IL-27,msc IL-27),并将其克隆至pc DNA3.1(+)载体。通过酶切和测序鉴定阳性重组载体,将重组质粒pc DNA3.1-IL-27通过脂质体转染法转染小鼠巨噬细胞株RAW264.7,通过RT-PCR方法检测目的基因的表达。结果:测序分析表明,小鼠IL-27单链融合基因中EBI3、linker和p28的连接顺序、方向及碱基序列与预期相符。在转染后的RAW264.7细胞中检测到了小鼠IL-27 m RNA的表达。结论:成功构建了小鼠IL-27单链融合基因及其真核表达载体,并在RAW264.7细胞中实现表达,为进一步探讨IL-27的生物学功能奠定了基础。     

Aminothiazoles inhibit RANKL‐ and LPS‐mediated osteoclastogenesis and PGE2 production in RAW 264.7 cells

Periodontitis is characterized by chronic inflammation and osteoclast‐mediated bone loss regulated by the receptor activator of nuclear factor‐κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). The aim of this study was to investigate the effect of aminothiazoles targeting prostaglandin E synthase‐1 (mPGES‐1) on RANKL‐ and lipopolysaccharide (LPS)‐mediated osteoclastogenesis and prostaglandin E₂ (PGE₂) production in vitro using the osteoclast precursor RAW 264.7 cells. RAW 264.7 cells were treated with RANKL or LPS alone or in combination with the aminothiazoles 4‐([4‐(2‐naphthyl)‐1,3‐thiazol‐2‐yl]amino)phenol (TH‐848) or 4‐(3‐fluoro‐4‐methoxyphenyl)‐N‐(4‐phenoxyphenyl)‐1,3‐thiazol‐2‐amine (TH‐644). Aminothiazoles significantly decreased the number of multinucleated tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclast‐like cells in cultures of RANKL‐ and LPS‐stimulated RAW 264.7 cells, as well as reduced the production of PGE₂ in culture supernatants. LPS‐treatment induced mPGES‐1 mRNA expression at 16 hrs and the subsequent PGE₂ production at 72 hrs. Conversely, RANKL did not affect PGE₂ secretion but markedly reduced mPGES‐1 at mRNA level. Furthermore, mRNA expression of TRAP and cathepsin K (CTSK) was reduced by aminothiazoles in RAW 264.7 cells activated by LPS, whereas RANK, OPG or tumour necrosis factor α mRNA expression was not significantly affected. In RANKL‐activated RAW 264.7 cells, TH‐848 and TH‐644 down‐regulated CTSK but not TRAP mRNA expression. Moreover, the inhibitory effect of aminothiazoles on PGE₂ production was also confirmed in LPS‐stimulated human peripheral blood mononuclear cell cultures. In conclusion, the aminothiazoles reduced both LPS‐ and RANKL‐mediated osteoclastogenesis and PGE₂ production in RAW 264.7 cells, suggesting these compounds as potential inhibitors for treatment of chronic inflammatory bone resorption, such as periodontitis.     

Aminothiazoles inhibit osteoclastogenesis and PGE2 production in LPS‐stimulated co‐cultures of periodontal ligament and RAW 264.7 cells,and RANKL‐mediated osteoclastogenesis and bone resorption in PBMCs

Inflammatory mediator prostaglandin E₂ (PGE₂) contributes to bone resorption in several inflammatory conditions including periodontitis. The terminal enzyme, microsomal prostaglandin E synthase‐1 (mPGES‐1) regulating PGE₂ synthesis is a promising therapeutic target to reduce inflammatory bone loss. The aim of this study was to investigate effects of mPGES‐1 inhibitors, aminothiazoles TH‐848 and TH‐644, on PGE₂ production and osteoclastogenesis in co‐cultures of periodontal ligament (PDL) and osteoclast progenitor cells RAW 264.7, stimulated by lipopolysaccharide (LPS), and bone resorption in RANKL‐mediated peripheral blood mononuclear cells (PBMCs). PDL and RAW 264.7 cells were cultured separately or co‐cultured and treated with LPS alone or in combination with aminothiazoles. Multinucleated cells stained positively for tartrate‐resistant acid phosphatase (TRAP) were scored as osteoclast‐like cells. Levels of PGE₂, osteoprotegerin (OPG) and interleukin‐6, as well as mRNA expression of mPGES‐1, OPG and RANKL were analysed in PDL cells. PBMCs were treated with RANKL alone or in combination with aminothiazoles. TRAP‐positive multinucleated cells were analysed and bone resorption was measured by the CTX‐I assay. Aminothiazoles reduced LPS‐stimulated osteoclast‐like cell formation both in co‐cultures and in RAW 264.7 cells. Additionally, aminothiazoles inhibited PGE₂ production in LPS‐stimulated cultures, but did not affect LPS‐induced mPGES‐1, OPG or RANKL mRNA expression in PDL cells. In PBMCs, inhibitors decreased both osteoclast differentiation and bone resorption. In conclusion, aminothiazoles reduced the formation of osteoclast‐like cells and decreased the production of PGE₂ in co‐cultures as well as single‐cell cultures. Furthermore, these compounds inhibited RANKL‐induced bone resorption and differentiation of PBMCs, suggesting these inhibitors for future treatment of inflammatory bone loss such as periodontitis.     

迷迭香酸抗穿透支原体脂蛋白诱导巨噬细胞凋亡的作用研究

探讨迷迭香酸对穿透支原体脂蛋白诱导巨噬细胞凋亡的保护作用。方法:以穿透支原体脂蛋白损伤RAW264．7细胞为模型,用MTT法检测细胞存活状况,DNA片断化分析观察穿透支原体脂蛋白对RAW264．7细胞DNA降解的影响,碘化丙啶染色流式细胞术检测细胞凋亡率。结果:7．5mg/L穿透支原体脂蛋白可引起RAW264．7细胞存活率降低,出现细胞凋亡特征性“DNA梯带”,流式细胞术检测细胞出现凋亡亚G1期峰,凋亡率为31．9%;100μmol/L迷迭香酸预处理1小时后可以升高RAW264．7细胞的存活率,凋亡细胞特征性的梯状梯带消失,细胞凋亡亚G1期峰消失,并使RAW264．7细胞的凋亡率下降为7．9%。结论:迷迭香酸有抗穿透支原体脂蛋白诱导RAW264．7细胞凋亡的作用。     

Immunomodulatory activity of Ganoderma lucidum immunomodulatory protein via PI3K/Akt and MAPK signaling pathways in RAW264.7 cells

Ganoderma lucidum immunomodulatory protein (FIP-glu) is an active ingredient with potential immunoregulatory functions. The study was conducted to explore the immunomodulatory activities of recombinant FIP-glu (rFIP-glu) and its possible mechanism in macrophage RAW264.7 cells. In vitro assays of biological activity indicated that rFIP-glu significantly activated RAW264.7 cells and possessed proinflammatory and anti-inflammatory abilities. RNA sequencing analysis and Western blot analysis showed that macrophage activation involved PI3K/Akt and MAPK pathways. Furthermore, real-time quantitative polymerase chain reaction indicated that the PI3K inhibitor LY294002 blocked the messenger RNA (mRNA) levels of MCP-1 (CCL-2), the MEK1/2 inhibitor U0126 reduced the mRNA levels of TNF-α and MCP-1 (CCL-2), and the JNK1/2/3 inhibitor SP600125 prevented the upregulation of inducible nitric oxide synthase mRNA in rFIP-glu-induced cells. rFIP-glu did not mediate these inflammatory effects through a general pathway but rather through a different pathway for a different inflammatory mediator. These data imply that rFIP-glu possessed immunomodulatory activity in macrophages, which was mediated through PI3K/Akt and MAPK pathways.     

Nicotine promotes atherosclerosis development in apolipoprotein E-deficient mice through α1-nAChR

α1 Nicotinic acetylcholine receptor (α1nAChR) is an important nicotine receptor that is widely distributed in vascular smooth muscle cells, macrophages, and endothelial cells. However, the role of α1nAChR in nicotine-mediated atherosclerosis remains unclear. The administration of nicotine for 12 weeks increased the area of the atherosclerotic lesion, the number of macrophages infiltrating the plaques, and the circulating levels of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α, in apolipoprotein E-deficient (ApoE^−/−) mice fed a high-fat diet. Nicotine also increased α1nAChR, calpain-1, matrix metalloproteinase-2 (MMP-2), and MMP-9 expression in the aortic tissue. Silencing of α1nAChR with an adenoassociated virus decreased the atherosclerotic size, lesion macrophage content, and circulating levels of inflammatory cytokines and suppressed α1nAChR, calpain-1, MMP-2, and MMP-9 expression in the nicotine group. In vitro, nicotine-induced α1nAChR, calpain-1, MMP-2, and MMP-9 expression in mouse vascular smooth muscle cells (MOVAS) and macrophages (RAW264.7), and enhanced the migration and proliferation of these cells. The silencing of α1nAChR inhibited these effects of nicotine MOVAS and RAW264.7 cells. Thus, we concluded that nicotine promoted the development of atherosclerosis partially by inducing the migration and proliferation of vascular smooth muscle cells and macrophages and inducing an inflammatory reaction. The effect of nicotine on atherogenesis may be mediated by α1nAChR-induced activation of the calpain-1/MMP-2/MMP-9 signaling pathway.