微进化对阿萨希毛孢子菌感染宿主的影响CSCD

目的研究微进化对阿萨希毛孢子菌(Trichosporon asahii,T.asahii)感染宿主情况及感染后机体免疫反应等方面的影响。方法用微进化株(TEVO)与阿萨希毛孢子菌原代株(TO)感染Balb/c小鼠,比较两株菌感染小鼠脏器组织中真菌的相对载量、髓过氧化物酶(myeloperoxidase,MPO)、血清β-葡聚糖水平以及脾脏T细胞分化差异。结果TEVO菌株以孢子状态为主,TO菌株以菌丝生长状态为主;TEVO组小鼠各组织相对载量、血浆(1,3)-β-D-葡聚糖水平及MPO浓度均明显低于TO组;TEVO组小鼠脾脏中CD4^(+)T细胞和CD8^(+)T细胞的比例均高于TO组。结论微进化后的TEVO菌株对宿主的感染能力以及感染前期宿主对TEVO菌株的防御和杀伤能力均低于TO菌株,这可能与TEVO菌株表型变化密切相关。     

RAW264.7小鼠巨噬细胞与阿萨希毛孢子菌相互作用后TNF-α、IL-1β和CCL3的表达变化

目的研究RAW264.7小鼠巨噬细胞与T.asahii相互作用后TNF-α,IL-1β和CCL3的表达水平变化。方法RAW264.7细胞与T.asahii菌液共孵育3 h、6 h、9 h、12 h,对照组不加菌液。在各孵育时间点取上清液,采用双抗体夹心酶联免疫吸附试验(ELISA)试剂盒检测上清液中TNF-α、IL-1β和CCL3的含量。台盼蓝拒染法计各孵育时间点RAW264.7细胞的活细胞数。结果 RAW264.7细胞与T.asahii共孵育3 h、6 h、9 h、12 h后,TNF-α、IL-1β、CCL3的表达水平均明显高于对照组,有显著性差异(P0.05)。3种细胞因子的表达水平随共孵育时间延长而增高,共孵育9 h的表达水平最高。RAW264.7细胞的活细胞数随共孵育时间延长而逐渐下降,共孵育12 h后的活细胞数量与其他各时间点有统计学差异(P0.05)。结论 RAW264.7小鼠巨噬细胞面临T.asahii感染时可分泌TNF-α、IL-1β、CCL3等细胞因子,这些炎性细胞因子可能参与了抗T.asahii感染的免疫反应。     

阻断Kv1.3通道可增强RAW264.7巨噬细胞的吞噬功能

本研究旨在阐明电压门控性钾通道1.3(Kv1.3)在巨噬细胞吞噬功能中的作用.利用RAW264.7巨噬细胞吞噬鸡红细胞的半定量检测系统及吞噬异硫氰酸荧光素标记的大肠杆菌(E.coli)k-12的流式细胞术定量检测系统测定巨噬细胞的吞噬功能.研究发现,用海葵神经毒素(Sh K)(100 pmol/L)选择性阻断Kv1.3通道能显著增强处于静息状态的和被脂多糖(LPS)激活的RAW264.7巨噬细胞吞噬鸡红细胞的能力;Sh K也可增强静息RAW264.7细胞吞噬大肠杆菌的能力,但由于LPS刺激吞噬的效应近乎饱和,Sh K并不能进一步增加被LPS激活的RAW264.7细胞吞噬大肠杆菌的数量.Sh K促进LPS激活的RAW264.7细胞释放一氧化氮(NO),但并不增加静息RAW264.7细胞的NO释放.Sh K(100 pmol/L)自身并不影响静息RAW264.7细胞释放细胞因子,但能抑制LPS激活的RAW264.7细胞释放白细胞介素-1?.Sh K(100 pmol/L)对RAW264.7细胞的活力无明显影响.RAW264.7细胞表达Kv1.3通道蛋白;LPS使RAW264.7细胞的Kv1.3蛋白表达下调,菲律宾菌素Ⅲ(小凹蛋白依赖性内吞途径抑制剂)使Kv1.3蛋白表达上调,细胞松弛素D对Kv1.3蛋白表达无明显影响.研究表明,RAW264.7细胞表达Kv1.3蛋白;阻断Kv1.3通道可增强RAW264.7细胞的吞噬能力和NO生成.结果提示,Kv1.3通道可能是RAW264.7细胞吞噬活动的负调节因子,有可能成为治疗巨噬细胞吞噬功能异常相关疾病的一个靶点.     

肠球菌溶血及非溶血菌株对小鼠巨噬细胞表达TNF-a的影响

【目的】探讨肠球菌溶血菌株及非溶血菌株对小鼠巨噬细胞RAW264.7表达TNF-а的影响。【方法】用多粘菌素B抑制排除内毒素污染对实验的影响。肠球菌溶血菌株、非溶血菌株各11株,以菌/细胞比 30∶1 感染RAW264.7细胞1 h,加入200 mg/mL氨苄青霉素继续培养24 h,分别于感染后3、6、9、24 h,用ELISA方法检测不同观测点细胞培养液中肿瘤坏死因子TNF-a的含量,并用逆转录-聚合酶链反应方法（RT-PCR）比较肠球菌溶血、非溶血菌株感染6 h后TNF-a mRNA表达的差异。【结果】未感染的RAW264.7细胞培养液中检测不到TNF-a。肠球菌溶血株感染组细胞培养上清液中各观测点的TNF-a的平均含量 (pg/mL) 均比非溶血株性组高。经t检验,P<0.01,差别有显著性。RT-PCR法检测其mRNA的表达也有相同结果：TNF-a mRNA在肠球菌溶血株感染细胞中的相对表达量比非溶血株感染的细胞高,经t检验,P<0.05,差别有统计学意义。【结论】肠球菌溶血株比非溶血株更能促进小鼠巨噬细胞RAW264.7产生TNF-а炎症因子。     

恰麻古多糖对巨噬细胞RAW264.7体外免疫调节作用初探

初步探讨恰麻古粗多糖BRP、中性多糖BRNP-1、BRNP-2及酸性多糖BRAP-1、BRAP-2对巨噬细胞RAW264.7的免疫调节作用。实验方法选用CCK-8法检测不同质量浓度各恰麻古多糖组对巨噬细胞RAW264.7细胞增殖率的影响;以中性红法观察各组恰麻古多糖对巨噬细胞RAW264.7吞噬活性的影响;Griess法测定恰麻古多糖致巨噬细胞RAW264.7对NO的释放水平;采用酶联免疫吸附法(ELISA)试剂盒检测细胞因子TNF-α(肿瘤坏死因子)与IL-6(白介素-6)分泌水平。实验结果显示不同质量浓度各恰麻古多糖组能够显著提高巨噬细胞RAW264.7增殖率与对中性红的吞噬活性,并能够刺激巨噬细胞释放NO,且促进其TNF-α及IL-6分泌水平。通过实验,初步验证了各恰麻古多糖具有良好的生物活性,并对巨噬细胞RAW264.7具有免疫调节作用。     

TassC结合过氧化物酶体对早期受染巨噬细胞内的鼠伤寒沙门菌的影响

探讨鼠伤寒沙门菌在感染鼠巨噬细胞早期与细胞器的相互作用。用pTassC-GFP质粒转染鼠巨噬细胞RAW264.7,结合多抗的溶酶体标志物溶酶体相关膜蛋白-1用键合了Alexa594的羊抗鼠二抗显色,以观察标记了绿色荧光蛋白的TassC与溶酶体的关系;用pTassC-GFP和pDsRed2-Perxi质粒共转染RAW264.7细胞,以观察TassC-GFP与过氧化物酶体的关系;用SYTO42标记鼠伤寒沙门菌,感染用pTassC-GFP和pDsRed2-Perxi质粒共转染的RAW264.7细胞,以观察细菌与TassC和过氧化物酶体的关系。免疫荧光显示TassC-GFP不与鼠巨噬细胞RAW264.7中的溶酶体结合,但与标记了红色荧光的过氧化物酶体共定位;感染1 h的RAW264.7胞内SYTO42标记的鼠伤寒沙门菌吞噬泡可招募TassC-GFP和过氧化物酶体。这些发现提示在鼠伤寒沙门菌感染早期过氧化物酶体携带杀菌成分通过TassC介导可参与发挥一定的杀菌作用。     

巨噬细胞冠蛋白-1 siRNA对小鼠巨噬细胞吞噬功能的影响

巨噬细胞冠蛋白-1(Coronin-1)与结核分枝杆菌(Mycobacterium tuberculosis,Mtb)逃逸免疫杀伤有关。该研究探讨了p S-EGFP-SP-Coronin-1si RNA重组质粒靶向抑制巨噬细胞Coronin-1表达后对小鼠巨噬细胞吞噬功能的影响。用该质粒转染小鼠巨噬细胞系RAW264.7,采用RT-PCR法和Western blot检测质粒转染前后细胞Coronin-1的表达水平。以耻垢分枝杆菌(Mycobacterium smegmatis)分别感染转染质粒组细胞和对照组细胞,通过细胞内细菌菌落计数和细胞爬片抗酸染色法评估巨噬细胞转染质粒前后对细菌的吞噬能力;利用流式细胞术检测转染质粒组细胞及对照组细胞吞噬耻垢分枝杆菌后不同时段的细胞凋亡水平。结果显示,该质粒能显著抑制RAW264.7细胞Coronin-1的m RNA水平及蛋白表达水平;感染耻垢分枝杆菌6 h后,转染质粒组细胞内细菌数显著高于对照组细胞(P0.05);感染耻垢分枝杆菌48 h后,转染质粒组细胞凋亡水平显著高于对照组细胞(P0.05)。以上结果表明,p S-EGFP-SP-Coronin-1si RNA重组质粒能显著抑制巨噬细胞Coronin-1的表达,并可显著促进巨噬细胞吞噬细菌和凋亡杀菌,为开发靶向巨噬细胞Coronin-1的抗结核基因治疗措施奠定基础。     

Fucoidan对巨噬细胞RAW264.7的免疫调节作用

目的观察褐藻多糖硫酸酯(Fucoidan)对巨噬细胞RAW264.7体外吞噬活性、细胞因子TNF-α和IL-6分泌,以及Toll样受体4(TLR4)mRNA表达的影响。方法实验分对照组,Fucoidan高、中、低剂量组(浓度分别是200、400和800μg/mL)。药物处理6~48h后,MTT法检测RAW264.7细胞活力;中性红比色法检测细胞吞噬活性;ELISA法检测培养上清中TNF-α和IL-6的分泌水平;实时定量PCR检测Toll样受体4(TLR4)mRNA表达量。结果与对照组相比,Fucoidan显著增强RAW264.7细胞代谢活力和吞噬能力(P0.01),增加TNF-α和IL-6的分泌,上调TLR4的表达,呈剂量依赖关系。结论 Fucoidan可上调TLR4表达,增强巨噬细胞代谢和吞噬活性,增加TNF-α和IL-6的分泌,具有潜在的调节免疫作用。     

蜂蛹多肽对巨噬细胞RAW264.7免疫活性的影响

蜂蛹多肽因具有丰富的营养价值,以及增强免疫、抗肿瘤及抗氧化等生物学活性,而受到了广泛关注,但目前关于蜂蛹多肽纯化组分的体外免疫调节活性的研究尚未见报道。为了探究蜂蛹多肽对巨噬细胞RAW264.7免疫活性的影响,以蜂蛹多肽纯化组分BPP-21为研究对象,研究其在不同浓度（12.5、25、50、100和200 μg·mL-1）下对RAW264.7巨噬细胞的细胞活力、吞噬能力、细胞因子分泌能力、NO分泌能力和氧化应激指标的影响。结果显示,在浓度12.5~200 μg·mL-1范围内,BPP-21对RAW264.7巨噬细胞无明显的细胞毒性作用,可显著提高干扰素-γ（interferon-gamma,IFN-γ）与NO的分泌水平（P<0.05）;在浓度25~200 μg·mL-1范围内,显著增加细胞吞噬能力以及白细胞介素-2（interleukin-2,IL-2）、肿瘤坏死因子α（tumor necrosis factor-alpha,TNF-α）的分泌量（P<0.05）;在浓度50~200 μg·mL-1范围内,显著提高细胞内超氧化物歧化酶（superoxide dismutase,SOD）活力（P<0.05）。研究表明,蜂蛹多肽纯化组分BPP-21可增强RAW264.7巨噬细胞的免疫活性,为蜂蛹多肽免疫调节剂的研究与开发提供了理论依据。     

斗米虫蛋白体外抗肿瘤活性及其对小鼠巨噬细胞免疫调节作用

该文主要为了研究斗米虫蛋白的体外抗肿瘤活性及其免疫调节作用。提取斗米虫总蛋白,逐级盐析分为3个部分,透析除盐后得到不同蛋白部位,并采用SDS-PAGE检测斗米虫不同蛋白部位的分子量;利用MTT法、流式细胞术等方法研究斗米虫蛋白对人胃癌细胞MFC和小鼠乳腺癌细胞4T1增殖、迁移和凋亡的作用。MTT法研究斗米虫蛋白对小鼠单核巨噬细胞RAW264.7和人脐静脉内皮细胞HUEVC增殖的影响;荧光微球吞噬实验检测斗米虫蛋白对RAW264.7细胞吞噬能力的影响;Griess法检测对RAW264.7细胞释放NO能力的影响;ELISA法检测对RAW264.7细胞的IL-6、TNF-α和IL-1β分泌量;RT-PCR法检测不同浓度的斗米虫蛋白作用后RAW264.7细胞中TNF-α、IL-1、TLR4、MIR-7、IFN-γ、TRL-4、IL-6以及4T1细胞中MMP2、MMP9、STAT3、c-Myc和Sdf1 mRNA水平变化。结果显示,斗米虫蛋白主要分子量集中于63 kDa,斗米虫蛋白对人胃癌细胞MFC及小鼠乳腺癌细胞4T1的增殖表现出较好抑制作用,并呈现出一定剂量依赖关系(P<0.05),对HUEVC细胞没有细胞毒性,对RAW264.7细胞表现出较好的促进增殖的作用(P<0.05)。斗米虫蛋白实验组与正常组细胞相比可以抑制4T1细胞的迁移(P<0.01),可诱导MFC和4T1细胞凋亡(P<0.05);斗米虫蛋白能够提高RAW264.7细胞的吞噬活性、NO释放量、TNF-α、1L-1β和IL-6分泌量及TNF-α、IL-1、TLR4、MIR-7、IFN-γ和IL-6细胞因子的mRNA水平以及能显著下调4T1细胞中MMP2、MMP9、STAT3、c-Myc和Sdf1 mRNA水平(P<0.05,P<0.01)。由此推论,斗米虫蛋白具有较好的体外抗肿瘤活性并且具有潜在的免疫调节作用。     

CEL-I, an invertebrate N-acetylgalactosamine-specific C-type lectin, induces TNF-alpha and G-CSF production by mouse macrophage cell line RAW264.7 cells

Our previous studies demonstrated that CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin purified from the marine invertebrate Cucumaria echinata (Holothuroidea) showed potent cytotoxicity to several cell lines such as HeLa, MDCK and XC cells. In this study, we found that CEL-I induced increased secretion of tumour necrosis factor-alpha (TNF-alpha) and granulocyte colony stimulation factor (G-CSF) by mouse macrophage cell line RAW264.7 cells in a dose-dependent manner, whereas this cell line was highly resistant to CEL-I cytotoxicity. The cytokine-inducing activity of CEL-I was stronger than that of phytohaemagglutinin (PHA-L). A binding study using FITC-labelled CEL-I (F-CEL-I) indicated that the amount of bound F-CEL-I on RAW264.7 cells was greater than that of F-PHA-L, suggesting that the greater activity of CEL-I to induce cytokine secretion by RAW264.7 cells is partly due to the higher binding ability. Since the cell binding and cytokine-inducing activity of CEL-I were partly but significantly inhibited by the specific sugar (GalNAc), it is considered that the binding of CEL-I to cell-surface-specific saccharide moieties, which may be recognized by CEL-I with higher affinity than GalNAc, is essential for the induction of cytokine secretion. The secretion of TNF-alpha and G-CSF from CEL-I-treated RAW264.7 cells were almost completely prevented by brefeldin A (BFA), whereas increase in mRNA levels of these cytokines were not affected by BFA. Bio-Plex beads assay suggested that temporal increase in phosphorylation of extracellular-regulated kinase (ERK), c-jun NH(2)-terminal kinase (JNK) and p38 MAP kinase occurred at relatively early time following CEL-I treatment. Furthermore, the secretion of TNF-alpha and G-CSF were inhibited by specific inhibitors for these MAP kinases. These results suggest that the intracellular signal transduction through the activation of MAP kinase system is involved in CEL-I-induced cytokine secretion.     

The two-component system ScnRK of Streptococcus mutans affects hydrogen peroxide resistance and murine macrophage killing

To survive macrophage killing is critical in the pathogenesis of viridians streptococci-induced infective endocarditis (IE). Streptococcus mutans, an opportunistic IE pathogen, generally does not survive well phagocytic killing in murine macrophage RAW 264.7 cells. A putative two-component system (TCS), ScnR/ScnK from S. mutans, was investigated to elucidate the mechanisms underlying bacteria-cellular interaction in this study. Both the wild-type and mutant strains were phagocytosed by RAW 264.7 cells at a comparable rate and an increased intracellular susceptibility during a 5 h incubation period was observed with the scnRK-null mutants. The amount of reactive oxygen species (ROS) in activated macrophages was reduced significantly after ingesting wild-type, but not scnRK-null mutant strains, suggesting that increased macrophage killing of these mutants is due to the impaired ability of S. mutans to counteract ROS. Additionally, both scnR- or scnRK-null mutants were more susceptible to hydrogen peroxide. Interestingly, scnRK expression was unaffected by hydrogen peroxide. These experimental results indicate that scnRK is important in counteracting oxidative stress in S. mutans, and decreased susceptibility to phagocytic killing is at least partly attributable to inhibition of intracellular ROS formation.     

Oxidized lipoprotein induces the macrophage ascorbate transporter (SVCT2): Protection by intracellular ascorbate against oxidant stress and apoptosis

To assess whether ascorbic acid decreases the cytotoxicity of oxidized human low density lipoprotein (oxLDL) in cells involved in atherosclerosis, its interaction with oxLDL was studied in murine RAW264.7 macrophages. Macrophages took up ascorbate to millimolar intracellular concentrations and retained it with little loss over 18 h in culture. Culture of the macrophages with oxLDL enhanced ascorbate uptake. This was associated with increased expression of the ascorbate transporter (SVCT2), which was prevented by ascorbate and by inhibiting the NF-κB pathway. Culture of RAW264.7 macrophages with oxLDL increased intracellular dihydrofluorescein oxidation and lipid peroxidation, both of which were decreased by intracellular ascorbate. Ascorbate also protected the cells against oxLDL-induced cytotoxicity and apoptosis, but it did not affect macrophage accumulation of lipid from oxLDL or oxLDL-induced increases in macrophage cytokine secretion. These results suggest that ascorbate protects macrophages against oxLDL-induced oxidant stress and subsequent apoptotic death without impairing their function.     

2-methoxyestradiol inhibits differentiation and is cytotoxic to osteoclasts

2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17beta-estradiol, is highly cytotoxic to a wide range of tumor cells but is harmless to most normal cells. However, 2-ME prevented bone loss in ovariectomized rats, suggesting it inhibits bone resorption. These studies were performed to determine the direct effects of 2-ME on cultured osteoclasts. 2-ME (2 microM) reduced osteoclast number by more than 95% and induced apoptosis in three cultured osteoclast model systems (RAW 264.7 cells cultured with RANKL, marrow cells co-cultured with stromal support cells, and spleen cells cultured without support cells in media supplemented with RANKL and macrophage colony stimulating factor (M-CSF)). The 2-ME-mediated effect was ligand specific; 2-hydroxyestradiol (2-OHE), the immediate precursor to 2-ME, exhibited less cytotoxicity; and 2-methoxyestrone (2-MEOE1) the estrone analog of 2-ME, was not cytotoxic. Co-treatment with ICI 182,780 did not antagonize 2-ME, suggesting that the cytotoxicity was not estrogen receptor-dependent. 2-ME-induced cell death in RAW 264.7 cells coincided with an increase in gene expression of cytokines implicated in inhibition of differentiation and induction of apoptosis. In addition, the 2-ME-mediated decrease in cell survival was partially inhibited by anti-lymphotoxin(LT)beta antibodies, suggesting that 2-ME-dependent effects involve LTbeta. These results suggest that 2-ME could be useful for treating skeletal diseases in which bone resorption is increased, such as postmenopausal osteoporosis and cancer metastasis to bone.     

二氢青蒿素对超高分子量聚乙烯颗粒诱导的巨噬细胞源性炎性因子释放的影响

目的:研究二氢青蒿素(Dihydroartemisinin,DHA)对超高分子量聚乙烯(Ultra highmolecularweightpolyethylene,UHMWPE)颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放的影响。方法:建立UHMWPE颗粒诱导的小鼠巨噬细胞系RAW264.7细胞源性炎性因子释放模型;施加不同浓度的二氢青蒿素观察药物对细胞的影响,酶联免疫分析法(Enzyme-linked immuno sorbent assay,ELISA)检测细胞培养液上清中TNF-α,IL-1β,IL-6和IL-10含量,MTT法检测细胞毒性反应。结果:酶联免疫分析方法结果表明,二氢青蒿素可以显著抑制由UHMWPE颗粒诱导的小鼠RAW264.7细胞促炎细胞因子TNF-α,IL-1和IL-6的表达,并显著促进抗炎因子IL-10的释放,其效应具有剂量依赖性。结论:二氢青蒿素具有显著的抗炎作用,可以抑制UHMWPE颗粒诱导的巨噬细胞炎症反应,其在预防人工关节置换术后假体无菌性松动的药物治疗方面具有潜在的作用。     

Curcumin enhances non-inflammatory phagocytic activity of RAW264.7 cells

Present study was performed to assess the effect of curcumin treatment on macrophage functions using RAW264.7 cells, a murine macrophage cell line. Phagocytic activity of RAW264.7 cells was enhanced by the treatment with curcumin for 48 h while the nitric oxide synthesis from RAW264.7 cells following lipopolysaccharide exposure was blocked. The incubation of RAW264.7 cells with curcumin dose-dependently inhibited the stimulatory responses of macrophage triggered by lipopolysaccharide; the enhanced secretion of inflammatory cytokines such as TNF-α and IL-1β and the up-regulated expression of surface antigens like CD14 and CD40. Curcumin alone, however, was able to increase the basal level of TNF-α secretion and elevated markedly the expression of CD14 and slightly CD40. The marked enhancement of both phagocytic activity and CD14 was detectable as early as 75 min after curcumin treatment which is the minimum time period required for the phagocytosis and CD14 measurement, suggesting a signaling pathway distinct from that triggered by apoptotic cells. In conclusion, this study elucidates that curcumin treatment enhances the phagocytic activity with blocking nitric oxide synthesis, a scavenger function of macrophages in non-inflammatory condition. In addition, this enhancement of phagocytic activity is triggered directly by the signals from curcumin itself not by apoptotic cells.     

Identification and characterization of monoclonal antibodies specific for macrophages at intermediate stages in the tumoricidal activation pathway

Macrophage activation for tumor cell killing is a multistep pathway in which responsive macrophages interact sequentially with priming and triggering stimuli in the acquisition of full tumoricidal activity. A number of mediators have been identified which have activating capability, including in particular IFN-gamma and bacterial LPS. Although the synergistic functional response of normal macrophages to sequential incubation with these activation signals has been well-established, characterization of the intermediate stages in the activation pathway has been difficult. We have developed a model system for examination of various aspects of macrophage activation, through the use of the murine macrophage tumor cell line, RAW 264.7. These cells, like normal macrophages, exhibit a strict requirement for interaction with both IFN-gamma and LPS in the development of tumor cytolytic activity. In addition, these cells can be stably primed by the administration of gamma-radiation. In the studies reported here, we have used RAW 264.7 cells treated with IFN-gamma alone or with IFN-gamma plus LPS to stimulate the production of rat mAb probes recognizing cell surface changes occurring during the activation process. In this way we have identified three Ag associated with intermediate stages of the activation process. One Ag, TM-1, is expressed on RAW 264.7 cells primed by IFN-gamma or gamma-radiation. This surface Ag thus identifies cells at the primed cell intermediate stage of the tumoricidal activation pathway regardless of the mechanism of activation. A second Ag, TM-2, is expressed on IFN-treated RAW 264.7 cells but not on RAW 264.7 cells primed with gamma-radiation alone. Expression of this Ag can be induced by treatment of irradiated cells with IFN-gamma, but is not induced by IFN-gamma treatment of a noncytolytic cell line, WEHI-3. This Ag thus appears to be an IFN-inducible cell surface protein associated specifically with macrophage activation for tumoricidal activity. Finally, Ag TM-3 is detectable on RAW 264.7 cells primed by either IFN-gamma or gamma-radiation, after subsequent triggering of the primed cells with LPS. The addition of the mAb recognizing this antigen to the function assay of tumor cell killing can inhibit they lytic activity of both triggered cells. Thus, this Ag may play a role in the antitumor effector functions of activated macrophages. Overall, the results suggest that these mAb can serve as useful tools for identification of molecules associated with the process of macrophage activation for tumor cell killing.     

GM1 Induced the inflammatory response related to the Raf-1/MEK1/2/ERK1/2 pathway in co-culture of pig mesenchymal stem cells with RAW264.7

Pig-human xenotransplantation can trigger cell-mediated immune responses. We explored the role of gangliosides in inflammation related to immune rejection in xenotransplantation. Co-culture of xenogeneic cells (pig-MSCs and RAW264.7) was used to emulate xenotransplantation conditions. MTT assay results indicated that cell viability was significantly decreased in pADMSCs co-cultured with RAW264.7 cells. GM1 and GM3 were highly expressed in pADMSCs co-cultured with RAW264.7 cells. pADMSCs co-cultured with RAW264.7 cells strongly expressed pro-inflammatory proteins such as COX-2, iNOS, p50, p65, pIκBα, and TNF-α. GM1-knockdown pADMSCs co-cultured with RAW 264.7 cells did not show significantly altered cell viability, but pro-inflammatory proteins were markedly inhibited. Co-culture of pADMSCs with RAW264.7 cells induced significant phosphorylation (p) of JNK1/2 and pERK1/2. However, pERK1/2 and pJNK1/2 were decreased and MEK1/2 and Raf1 were suppressed in GM1-knockdown pADMSCs co-cultured with RAW264.7 cells. Thus, the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways were significantly upregulated in response to increases of GM1 in co-cultured xenogeneic cells. However, the inflammatory response was suppressed in co-culture of GM1-knockdown pADMSCs with RAW264.7 cells via down-regulation of the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways. Therefore, the ganglioside GM1 appears to play a major role in the inflammatory response in xenotransplantation via the Raf-1/MEK1/2/ERK1/2 and JNK1/2 pathways.     

Induction of cytokines and nitric oxide in murine macrophages stimulated with enzymatically digested lactobacillus strains

Based on observations that lactic acid bacteria have the ability to activate macrophages, we assessed the potential effects of eight different Lactobacillus strains treated with gastrointestinal enzymes on the production of nitric oxide and various cytokines in macrophages. RAW 264.7 murine macrophage cells were cultured with either precipitates or supernatants of Lactobacillus strains digested with pepsin followed by pancreatin. The increased production of nitric oxide and interleukin (IL)-1beta, IL-6, IL-12 and tumour necrosis factor (TNF)-alpha were observed when cultured with precipitates, and this effect was largely strain-dependent. In contrast, the exposure of RAW 264.7 cells to supernatants produced weaker or nearly undetectable effects in comparison to the effects of exposure to precipitates. The induction of nitric oxide appeared to be unaffected. These results demonstrate that nitric oxide and cytokines were effectively induced when the bacterial precipitate was treated with macrophages. The results of the present study also indicate that Lactobacillus strains treated with digestive enzymes are capable of stimulating the production of nitric oxide and cytokines in macrophages, which may modulate the gastrointestinal immune function of the host when it is given as a feed additive.