Immunomodulatory effect of Hypoderma lineatum antigens: in vitro effect on bovine lymphocyte proliferation and cytokine production

This study examines the immunomodulatory effect of a crude larval extract (CLE), obtained from first stage larvae (L1) of H. lineatum , and the purified fractions hypodermin A (HyA), HyB and HyC. Proliferative responses of peripheral blood mononuclear cells (PBMC) from uninfested and previously infested cattle and the production of the cytokines IL-10, IL-4 and IFN-γ, in response to concanavalin A (Con A), were determined. The stimulation index of peripheral blood mononuclear cells from uninfested cattle was significantly lower than that from infested animals with the different antigens assayed. The HyA was the antigen that most inhibited the proliferative response, followed by the HyB, the HyC and the CLE. This hypodermin provoked an increase of IFN-γ and a suppression of IL-10 production that would support a Th1-like cytokine response. The HyB reduced the production of IL-10 stimulated by the Con A in cultures from infested animals. The HyC did not modulate the production of cytokines. Finally, the CLE induced a marked suppression in the production of the different cytokines in cultures from naïve and previously sensitized animals. Our results indicate that Hypoderma larval secretions are comprised of different components (hypodermins) that individually induce distinct but partially overlapping modulatory responses.     

Differential in vitro effects of IL-4, IL-10, and IL-13 on proinflammatory cytokine production and fibroblast proliferation in rheumatoid synovium

The purpose of this study was to compare the potential of interleukin-4 (IL-4), IL-10, and IL-13 to interrupt two major inflammatory pathways in rheumatoid arthritis (RA), i.e., overexpression of proinflammatory cytokines and cytokine-mediated fibroblast growth. IL-4, IL-10, and IL-13 were all able to significantly inhibit the production of IL-1β, tumor necrosis factor-α (TNF-α), IL-6, and IL-8 by freshly isolated RA synovial tissue cells; IL-10 was most effective in terms of IL-1β and TNF-α reduction. The IL-1 receptor antagonist was enhanced by IL-4 and IL-13, but only slightly enhanced by IL-10. Spontaneous interferon-γ secretion was diminished by IL-4 and IL-10 but not by IL-13. Addition of anti-IL-10 neutralizing antibody to RA synovial tissue cells resulted in a substantial increase in IL-1β and TNF-α levels, whereas neither anti-IL-4 nor anti-IL-13 antibody had a significant effect. IL-1β-stimulated proliferation of RA synovial fibroblast cell lines was inhibited by IL-4 and IL-13, but not by IL-10; IL-4 was over tenfold more effective than IL-13. These results suggest that IL-4, IL-10, and IL-13 all have the therapeutic potential to regulate the disease activity mediated by proinflammatory cytokines in RA, but each cytokine may have different potencies. Received: 29 June 2000 / Accepted: 20 July 2000     

Anti-interleukin-4 treatment diminishes secretion of Th2 cytokines and inhibits hepatic fibrosis in murine schistosomiasis japonica

Anti-interleukin-4 (IL-4) treatment o/"Schistosoma japo-nicum-infected mice markedly inhibited in vitro secretion of the Th2 cytokines IL-4 and IL-5 from antigen-stimulated spleen cells, but enhanced the secretion of the Th1 cytokine IFN-γ. IL-2 secretion was unaffected. Hepatic fibrosis was markedly diminished in anti-IL-4-treated-mice at ten weeks of infection while granulomas around S. japonicum eggs in the livers were slightly-to-moderately increased in size. The number of eggs per worm pair in the tissues and feces did not differ significantly in treated and untreated mice. These findings suggest that Th2 cytokine responses are important in the genesis of schistosomal hepatic fibrosis.     

SOX10对胶质瘤细胞增殖及去分化的影响

目的探讨SOX10过表达对胶质瘤细胞增殖和去分化的影响。方法胶质瘤细胞株U87转染SOX10腺病毒48 h后收集细胞进行MTT、流式细胞术、Western blot和Q-RT-PCR。结果 U87细胞感染SOX10过表达腺病毒后细胞的增殖受抑制,DNA合成减少,S期细胞数减少,干细胞标志CD133上调。结论 SOX10可能在胶质瘤细胞的去分化中起重要作用。     

Lymphatic filariasis: parallels between the immunology of infection in humans and mice

Mouse models of Brugia infection have provided much useful quantitative and qualitative information on the immune response elicited by different life cycle stages of filarial worms. Many parallels exist between the immune response in the mouse and the infected human and in this review we highlight areas of topical interest, including the induction of specific cytokine responses and their role in immunomodulation and protective immunity. These studies have reinforced the concept that different life cycle stages of filarial parasites each have their own mechanism of modulating responses so that potentially inflammatory IFN-gamma responses are downregulated. While the precise mechanisms of protective immunity remain to be defined, studies in the mouse have suggested novel pathways, including a possible role for granulocytes.     

Immunoregulation in experimental murine Trypanosoma congolense infection: anti-IL-10 antibodies reverse trypanosome-mediated suppression of lymphocyte proliferation in vitro and moderately prolong the lifespan of genetically susceptible BALB/c mice

We infected highly susceptible BALB/c and relatively resistant C57BL/6 mice with cloned Trypanosoma congolense and followed the effects of these infections on the circulating parasite numbers, mouse mortality and cytokine expression. C57BL/6 mice controlled their parasitaemia and survived for up to 163 ± 12 days, while BALB/c mice could not control their parasitaemia and succumbed to the infection within 8.4 ± 0.5 days. Susceptible BALB/c mice had dramatically higher plasma levels of IL-10 than the resistant C57BL/6 mice from day 7 forward. This was preceded by an earlier and higher level induction of splenic IL-10 messenger RNA (mRNA) expression in the infected BALB/c mice. There was a strong negative correlation between the splenocyte proliferative responses to Concanavalin-A (Con-A) and their production of IL-10 in these infected BALB/c mice. Co-treatment of the Con-A-stimulated spleen cell cultures with monoclonal anti-IL-10 antibodies, but not isotype-matched control antibodies, could completely reverse this suppression of the splenocyte proliferative response. Finally, in three experiments, anti-IL-10 antibody treatment in vivo reduced the peak circulating parasitaemia of infected BALB/c mice by 43% and increased their median survival periods by 38% relative to isotype-matched control antibody-treated mice .     

Interferon-γ signal transduction during parasite infection: modulation of MAP kinases in the infection of human monocyte cells (THP1) by Toxoplasma gondii

We assayed mitogen-activated protein (MAP) kinase phosphorylation in a human monocyte cell line (THP1) during their infection by Toxoplasma gondii . In addition, we tested the effect of specific MAP kinase inhibitors (PD098059 and SB203580) on parasite invasion. MAP kinase phosphorylation was increased in the cytosol and membrane fractions of THP1 infected with T. gondii . The MAP kinase phosphorylation of uninfected THP1 cells was not significantly modified by incubation for 20 h with 1000 U/ml of IFN-γ. However, IFN-γ treatment of infected cells significantly reduces the increase in phosphorylation caused by parasite infection. There was also MAP kinase activity in the cytosol and membrane fractions of extracellular T. gondii tachyzoites. IFN-γ altered the distribution of activity in subcellular fractions of extracellular T. gondii tachyzoites. This indicates that IFN-γ directly affects parasite MAP kinase activity. The results provide evidence that MAP kinase pathways participate in the infection by T. gondii and that the decrease in MAP kinase activity in infected cells caused by IFN-γ may be involved in mediating their protective signals .     

Cytokine profile of protective anti-Trichinella spiralis CD4+ OX22− and non-protective CD4+ OX22+ thoracic duct cells in rats: secretion of IL-4 alone does not determine protective capacity

We analysed the cytokine profile of a T cell subset (CD4⁺ CD45 RC⁻) that confers protection against Trichinella spiralis infection in rats. These CD4⁺ cells are generated in the gut and appear in the thoracic duct lymph within 72 h after infection. Cytokine mRNA levels for IL-2, IL-3, IL-4, IL-5, IL-10 and IFN-γ and functional cytokine secretion for IL-4, IL-5, IFN-γ, TNF-α and mast cell differentiation activity were tested in vitro following stimulation with T. spiralis antigens. Compared to a non-protective T cell population (CD4⁺ CD45 RC⁺ or CD8⁺), also isolated from the same thoracic lymph, no significant differences were observed in the levels of mRNA for IL-2, IL-3, IL-4, IL-5, IL-10 or IFN-γ in the protective CD4⁺ CD45 RC⁻ cells. However, analysis of the cytokine activities in culture supernatant of these T cell subsets following 24 h stimulation in vitro with T. spiralis antigens showed that significant IL-4 and IL-5 activity but little IFN-γ or TNF-α was secreted by the protective CD4⁺ CD45 RC⁻ cells. Whereas the non-protective CD4⁺ CD45 RC⁺ cells secreted significant levels of IL-4, IFN-γ, mast cell differentiating activity and TNF-α but little IL-5 activity. Nonprotective CD8⁺ cells were found to secrete IL-4 but not IL-5. Production of IL-4 was essentially equal for both protective and non-protective T cell subsets. These findings suggest that the presence or absence of IFN-γ secretion, rather than IL-4 alone, determines whether a T cell subset has protective activity against T. spiralis infection in rats.     

生脉注射液对人骨髓造血细胞增殖作用的研究

目的探讨生脉注射液对人骨髓造血细胞的增殖作用。方法2004-10~2005-05在中南大学医学技术与情报学院对采用密度分离法分离人骨髓细胞获得的高活率造血细胞,运用二甲基噻唑-二苯基四唑溴盐[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide,MTT]比色法和体外集落培养法检测生脉注射液对造血细胞的刺激增殖作用。结果(1)当红参质量浓度为5·0、10·0、20·0mg/L,麦冬质量浓度为15·6、31·2、62·4mg/L,五味子质量浓度为7·8、15·6、31·2mg/L时,MTT比色吸光度OD值分别为0·3148、0·4307、0·5298,与对照组0·2227相比显著提高(P<0·01)。(2)无集落刺激因子时,生脉注射液对集落生成无作用。结论体外实验中,生脉注射液通过与集落刺激因子的协同作用在一定浓度范围内对人骨髓造血细胞具有显著的刺激增殖作用,可为临床疾病的有效治疗提供新的思路和方法。     

羟基喜树碱对类风湿关节炎外周血淋巴细胞增殖及凋亡的影响

目的在体外实验中，通过比较羟基喜树碱（HCPT）与甲氨蝶呤（MTX）对类风湿关节炎（RA）患者外周血淋巴细胞（PBL）增殖及调亡的影响，探讨HCPT对RA患者PBL的作用机制。方法①分离纯化淋巴细胞，进行体外培养。②用四甲基偶氮唑蓝（MTF）法检测HCPT与MTX不同浓度（0、0．1、1、5、10、50、100、200μg／ml）、不同时间（24、48、72h）的吸光度值（A），计算细胞生长抑制率（IR％）。③用AnnexinVFITC及碘化丙啶（PI）溶液染色流式细胞术（FCM）检测HCPT与MTX在48h时不同浓度（0、0．1、10、100μg／m1）的细胞凋亡率。结果①HCPT与MTX对RA患者PBL增殖均具有抑制作用，呈时间、剂量依赖关系，时间、剂量的主效应具有统计学意义（P〈0．01）。②HCPT与MTX对PBL细胞增殖的抑制率比较，HCPT优于MTX，差异具有统计学意义（P〈0．01）。③HCPT、MTX均可诱导体外培养PBL早期凋亡，并且有剂量依赖关系，剂量效应具有统计学意义（P〈0．05）。④HCPT与MTX作用于PBL的凋亡率比较，差异无统计学意义（P〉0．05）。结论①HCPT抑制PBL增殖的作用优于MTX。②HCPT诱导PBL凋亡的作用与MTX相当。HCPT有可能在RA治疗方面具有潜在价值。     

不同年龄人骨髓间充质干细胞体外培养增殖的研究

目的本研究分析不同年龄段人骨髓间充质干细胞的生长和增生情况,探讨年龄对于骨髓间充质干细胞的影响。方法采集61例不同年龄人的骨髓,分为少年组、中青年组和老年组,分离间充质干细胞,进行体外培养,观察其形态变化,对比分析其首次传代和每次传代时间、细胞克隆形成数量、细胞增长曲线的变化情况。结果体外培养的干细胞在形态上无年龄差异,均成梭形纤维状,早期呈克隆状生长。细胞的首次传代时间从少年到老年呈逐渐增加的趋势,老年组明显长于另外两组;3组的体外培养倍增时间无明显差异,但老年组的干细胞克隆数量减少（差异尚未达到统计学意义）,细胞的增殖显著低于另外两组（P<0.05）。结论人骨髓间充质干细胞的生长增殖能力随着增龄而逐渐减退,尤其在老年组呈现出更明显的变化。     

地塞米松对体外人骨髓基质细胞增殖及成骨分化的影响

目的观察地塞米松对骨髓基质细胞(MSCs)增殖及成骨分化的影响。方法以离心法分离培养MSCs,分别以10~(-9)、10~(-8)和10~(-7) mol/L地塞米松对细胞进行干预,采用四甲基偶氮唑蓝比色法(MTT法)测细胞增殖率；测细胞质碱性磷酸酶(ALP)含量；利用反转录聚合酶链反应(RT-PCR)技术在转录水平检测成骨细胞标记物骨桥蛋白(OPN)mRNA的表达。结果地塞米松干预8 d后,各干预组的吸光度值均低于对照组,10~(-8)和10~(-7) mol/L地塞米松组与对照组相比差异有统计学意义(P＜0．05)；地塞米松干预12 d后,各干预组细胞质ALP含量呈浓度依赖性增加,与对照组相比差异均有统计学意义(P＜0．05)；地塞米松干预12 d后,各干预组均有OPN mRNA表达,且随浓度增加表达增强。结论地塞米松对体外MSCs增殖有抑制作用,但对MSCs成骨分化有促进作用。     

Comparative studies on immune responses to infection in susceptible B10.BR mice infected with different strains of the murine nematode parasite Trichuris muris

A comparison of immune responses to infection between groups of B10.BR mice infected with different strains of T. muris , S strain (isolated in Sobreda, Portugal), E strain (isolated in Edinburgh), and E-J strain (originally E strain, which has been maintained in our laboratory, Japan), was performed. In mice infected with E and E-J strains, most of the worms were expelled by day 32 after infection, though the expulsion was faster in E-J strain-infected mice. In contrast, no expulsion was observed in S strain-infected mice by day 32 and egg production occurred on day 32. IL-4 production occurred in concanavalin A (Con-A)-stimulated mesenteric lymph node cells (MLNC) from B10.BR mice infected with E and E-J strains, whereas no IL-4 production was observed in S strain-infected mice. IL-4 production did not occur in normal mice. In comparison with normal mice, high levels of IFN-γ production by Con A-stimulated MLNC were detected in mice infected with every strain of  T. muris . IFN-γ production in S strain-infected mice was greater, occurred earlier and was more persistent than in mice infected with E and E-J strains. IgG1 and IgG2a antibodies to T. muris excretory/secretory antigens were observed in B10.BR mice infected with every strain of T. muris . Antibody production showed similar kinetics. These differences in the expulsion kinetics and IL-4 production in B10.BR mice infected with S, E, and E-J strains suggest the involvement of IL-4 in protection against T. muris infection, and confirm the previous conclusion by Else et al . (1994).     

人遗传印记基因PEG10对L02细胞线粒体膜电位的影响

人遗传印记基因PEG10定位于人7号染色体长臂2区1带,是一个反转座子衍生而来的父系表达的遗传印记基因.PEG10基因高表达于肝癌细胞系HepG2和大多数肝细胞癌组织中,在良性肝病组织及非肝脏来源的肿瘤细胞系中没有检测出该基因的表达.PEG10基因的表达水平与肝细胞癌的转移潜能正相关,并且可以促进正常人肝细胞L02增殖,提示PEGl0基因可能在人肝细胞癌的发生过程中起重要作用.本研究通过检测L02细胞转染前后线粒体膜电位及增殖细胞核抗原(PCNA)表达的改变,初步探讨PEG10基因促人肝细胞增殖的机制.     

生脉注射液对人骨髓造血细胞增殖作用的研究

目的 探讨生脉注射液对人骨髓造血细胞的增殖作用。方法 2004—10—2005—05在中南大学医学技术与情报学院对采用密度分离法分离人骨髓细胞获得的高活率造血细胞，运用二甲基噻唑-二苯基四唑溴盐[3-（4，5-dimethyhhiazol-2-y1）-2，5-diphenyl tetrazolium bromide，MTT]比色法和体外集落培养法检测生脉注射液对造血细胞的刺激增殖作用。结果 （1）当红参质量浓度为5．0、10．0、20．0mg／L，麦冬质量浓度为15．6、31．2、62．4mg／L，五味子质量浓度为7．8、15．6、31．2mg／L时，MTT比色吸光度OD值分别为0．3148、0．4307、0．5298，与对照组0．2227相比显著提高（P〈0．01）。（2）无集落刺激因子时，生脉注射液对集落生成无作用。结论 体外实验中，生脉注射液通过与集落刺激因子的协同作用在一定浓度范围内对人骨髓造血细胞具有显著的刺激增殖作用，可为临床疾病的有效治疗提供新的思路和方法。     

In vitro and in vivo effects of zinc on cytokine signalling in human T cells

Aging is associated with changes in the immune response which are collectively called immunosenescence. The changes mainly affect the adaptive immune response and especially the T cell-mediated cellular immune response. There are a few data indicating that the cytokine signalling in T cells is altered with aging. Zinc has been specifically shown to have potent immunomodulatory effects. The aim of the present work was to study the IL-2 and IL-6 cytokine signalling and activation induced cell death (AICD) in T cells of elderly subjects of various ages and from various European countries. These experiments were performed in the frame of European Community financed project called ZINCAGE “Nutritional zinc, oxidative stress and immunosenescence: biochemical, genetic and lifestyle implications for healthy ageing”, assembling 17 laboratories from 8 countries through Europe. The study was carried out in a total of 312 French and a group of 201 (26 from Italy, 63 from France, 57 from Greece, 24 from Poland and 30 from Germany) healthy non-institutionalized men and women older than 60 years of age, with available dietary data. Human peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood and were stimulated in vitro by IL-2 or IL-6 for various periods and the phosphorylation of STAT3 and STAT5 was measured by FACScan. The activation induced cell death (AICD) was measured after anti-CD3 and CD28 restimulation for 48 h by using the Annexin:FITC Apoptosis Kit. We found that there is an IL-2 signalling defect with aging up to 90 years of age which cannot be modulated by zinc. In contrast at 90 years and over the zinc could reverse the negative signalling effect of IL-2. There is also a signalling defect for STAT3 and STAT5 activation in T cells under IL-6 stimulation with aging and the zinc supplementation could potentiate only the STAT5 activation in the age-group 90 years and over. Studying signalling in PBL from different countries we detected less activation in T cells of subjects from France and the most changes occurred in T cells of subjects from Poland, suggesting no correlation with the plasma zinc status observed in these countries. In vivo zinc supplementation had no effect on IL-2 and IL-6-modulated STAT3 and STAT5 activation. Zinc added in vitro to these T cells even inhibited the stimulation either by IL-2 or by IL-6. Zinc supplementation improved the susceptibility of T cells to AICD in both age-groups, with more efficiency in later ages. Our results suggest that zinc can have a potent immunomodulatory effect via the modulation of cytokine signalling and AICD, however this effect depends on the function and the activation status of the T cells.