SPF雏鸡感染网状内皮组织增殖病病毒后白细胞介素2和干扰素诱生 …

对１日龄ＳＰＦ雏鸡人工感染网状内皮组织增殖病病毒（ＲＥＶ）后,胸腺Ｔ淋巴细胞白细胞介素２（ＩＬ－２）诱生活性于１４￣４９ｄ极显著低于对照组（Ｐ〈０．０１）;脾Ｔ淋巴细胞ＩＬ－２诱生活性于１４ｄ明显低于对照组（Ｐ〈０．０５）,感染后２１￣４９ｄ极显著低于对照组（Ｐ〈０．０１）;脾Ｔ淋巴细胞ＩＬ－２诱生活性于１４ｄ极显著降低（Ｐ〈０．０１）;脾淋巴细胞干扰素（ＩＦＮ）的诱生活性于感染后７￣４９ｄ极显著     

人工感梁vMDV雏鸡IL—2活性和淋巴细胞增殖反应的变化

对１日龄ＡＡ肉用雏鸡人工感染马立克氏病强毒后,脾Ｔ淋巴细胞ＩＬ－２诱生活性和Ｔ淋巴细胞增殖反应减弱或显著减弱,;ＭＤ平均发率为３３．７５％,平均死亡率为１１．２５％。     

香菇多糖和黄芪多糖对马立克氏病强毒感染雏鸡白细胞介素2活性和？…

１日龄ＡＡ肉用雏鸡以马立克氏病强毒人工感染后,马立克氏病发病率３３．７５％,死亡率１１．２５％,同健康雏鸡相比,ＩＬ－２诱生活性和Ｔ淋巴细胞增殖反应降低和显著降低。ｖＭＤＶ感染雏鸡注射香菇多糖和黄芪多糖,ＭＤ发病率分别为２０％、１７．５％,死亡率分别为７．５％、５％。同感染组相比,感染／香菇组和感染／黄芪组的ＩＬ－２诱生活性和Ｔ淋巴细胞增殖反应升高和显著升高。     

鸡T淋巴细胞IL－2的体外诱生及活性检测

经Ｌ１６（４５）正交试验选择，并经实验验证，鸡脾脏Ｔ淋巴细胞白细胞介素２（ＩＬ－２）体外诱生和活性检测的最优水平组合及适宜条件为２．５μｇ／ｍＬ伴刀豆蛋白Ａ（ＣｏｎＡ）、ＩＬ－２体外诱生时间２０ｈ、１×１０７／ｍＬ淋巴细胞、１０％小牛血清、靶细胞培养时间４８ｈ、４×１０６／ｍＬ靶细胞、靶细胞接触时间３６ｈ、５ｍｇ／ｍＬＭＴＴ、ＭＴＴ加入时间３ｈ和甲溶解时间２ｈ；胸腺Ｔ淋巴细胞ＩＬ－２体外诱生和活性检测的最优水平组合及适宜条件为５μｇ／ｍＬＣｏｎＡ、ＩＬ－２体外诱生时间４８ｈ，余同脾脏Ｔ淋巴细胞ＩＬ－２体外诱生及活性检测。比较试验表明，ＭＴＴ比色分析法和３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入法有显著的直线回归关系。     

疑核在下丘脑外侧区微量注射IL-6调节机体细胞免疫过程中的作用

为探索外源性ＩＬ－６可否通过反馈作用调节机体的免疫反应,选用３～４月龄、体重１．５～２．５ｋｇ的健康青紫蓝家兔,麻醉后固定于脑立体定位仪上。试验组Ⅰ动物（ｎ＝８）在下丘脑外侧区注射２．５ｐｍｏｌ／ＬＩＬ－６２μＬ;试验组Ⅱ动物（ｎ＝５）在疑核处注入２％盐酸利多卡因０．５μＬ后,再在下丘脑外侧区注射２．５ｐｍｏｌ／ＬＩＬ－６２μＬ;对照组动物（ｎ＝４）在下丘脑外侧区注射人工脑脊液２μＬ。分别在注射前及注射后不同时间无菌采血作细胞免疫指标测定。结果：试验组Ⅰ动物的Ｔ细胞百分率和ＰＨＡ诱导的淋巴细胞转化率,在注射ＩＬ－６后６０ｍｉｎ显著升高;试验组Ⅱ动物的Ｔ细胞百分率和ＰＨＡ诱导的淋巴细胞转化率,在注射ＩＬ－６后１２０ｍｉｎ出现显著升高;对照组动物在注射前后无明显变化;由此证明,下丘脑外侧区一定量的ＩＬ－６具有增强机体细胞免疫功能的作用,而这种细胞免疫的调节作用至少有一部分是通过疑核到达免疫系统的。疑核是下丘脑外侧区ＩＬ－６调节机体细胞免疫的通路之一。     

MD疫苗免疫雏鸡攻击vMDV后IL—2及IL—2R的变化

接种马立克氏病三价疫苗的雏鸡，用马立克氏病强毒攻击，免疫保护指数为７３．３％，脾脏和胸腺Ｔ淋巴细胞ＩＬ－２诱生活性比ｖＭＤＶ感洒雏鸡显著升高，ＩＬ－２Ｒ诱导表达显著增加；接种火鸡疱疹病毒疫苗的雏鸡，用ＶＭＤＶ攻击，免疫保护指数为５６．７％，脾脏和胸腺Ｔ淋巴细胞ＩＬ－２诱生活性比ＶＭＤＶ感染雏鸡显著升高，ＩＬ－２Ｒ诱导表达显著增加。     

雏鸡人工感染马立克氏病强毒后白细胞介素2及其受体的变化

１日龄雏鸡人工感染马立克氏病强毒后，脾脏Ｔ淋巴细胞白细胞介素２诱生活性比健康对照雏鸡显著降低，胸腺Ｔ淋巴细胞ＩＬ－２诱生活性虽见降低但无统计学显著性差异。脾脏和胸腺Ｔ淋巴细胞白细胞介素２受体诱导表达显著减少。     

鸡T淋巴细胞IL—2的体外诱生及活性检测

经Ｌ１６正交试验选择，并经实验验证，鸡脾脏Ｔ淋巴细胞白细胞介素２（ＩＬ－２）体外诱生和活性检测的最优水平组合及适宜条件的２．５μｇ／ｍＬ伴刀豆蛋白Ａ，ＩＬ－２体外诱生时间２０ｈ，１×１０＾７／ｍＬ淋巴细胞、１０％小牛血清、靶细胞培养时间４８ｈ，４×１０＾６／ｍＬ靶细胞，靶细胞接触时间３６ｈ，５ｍｇ／ｍＬＭＴＴ，ＭＴＴ加入时间３ｈ和甲替溶解时间２ｈ；胸腺Ｔ淋巴细胞ＩＬ－２体外诱生和活性检测的最优水平     

香菇多糖和黄芪多糖对马立克氏病强毒感染雏鸡白细胞介素2活性和淋巴细胞增殖反应的影响

１日龄ＡＡ肉用雏鸡以马立克氏病强毒（ｖＭＤＶ）人工感染后,马立克氏病（ＭＤ）发病率３３．７５％,死亡率１１．２５％。同健康雏鸡相比,ＩＬ－２诱生活性和Ｔ淋巴细胞增殖反应降低（Ｐ＞０．０５）和显著降低（Ｐ＜０．０５）。ｖＭＤＶ感染雏鸡注射香菇多糖和黄芪多糖,ＭＤ发病率分别为２０％、１７．５％,死亡率分别为７．５％、５％。同感染组相比,感染／香菇组和感染／黄芪组的ＩＬ－２诱生活性和Ｔ淋巴细胞增殖反应均升高（Ｐ＞０．０５）和显著升高（Ｐ＜０．０５）。     

后海穴对小鼠脾淋巴细胞产生IL—2活性的影响

本文采用Ｃ５７ＢＬ／６Ｊ小鼠胸腺＾３Ｈ－ＴｄＲ掺入法，观察了后海穴对小鼠脾淋巴细胞产季了ＩＬ－２活性的影响。结果表明，针刺后海穴，后海穴注射８３０１多糖，腹腔注射８３０１多糖均能增高脾淋巴细胞产生ＩＬ－２的能力。后海穴注糖量，注糖次数，均比腹腔少，而对ＩＬ－２活性的影响却二者无显著差异，从而间接地证实了后海穴有正向增强小鼠脾淋巴细胞产生ＩＬ－２的能力。     

PRRS病毒感染对猪细胞因子IL-2、IL-4和 IL-10mRNA转录的影响

通过构建猪细胞因子IL-2、IL-4和IL-10的缺失cDNA竞争分子，利用定量竞争PCR技术对猪繁殖与呼吸综合征(PRRS)病毒感染过程中细胞因子IL—2、IL—4和IL—10的mRNA进行转录水平变化的定量检测，研究PRRS病毒感染对猪细胞因子IL—2、IL-4和IL—10mRNA转录的影响。结果 表明猪Th1型细胞因子IL—2的mRNA转录水平分别在PRRS病毒感染后1d、28d和56d出现3个mRNA转录高峰；而Th2型细胞因子IL—4的mRNA转录水平在病毒感染后56d内，一直处于低水平，而后才逐渐升高；IL—10的mRNA转录水平在病毒感染后6d内，一直处于低水平状态，第6d后才逐渐上升，至42d到达高峰，而后下降，恢复正常。结果显示PRRS病毒感染可导致Th2型细胞因子IL-4和IL-10基因转录的抑制。     

广西沼泽型水牛IL-4和IL-5基因的克隆及其序列分析

从刀豆素A(Concanavalin A,ConA)刺激的广西沼泽型成年水牛外周血单个核细胞(Peripheralblood mononuclear cells,PBMCs)中提取总RNA,用逆转录聚合酶链式反应(RT-PCR)方法扩增出白细胞介素-4(Interleukin-4,IL-4)和白细胞介素-5(IL-5)基因,分别克隆到pMD18-T载体上,进行序列分析。结果表明,从培养9 h、17 h的单个核细胞中扩增得到的IL-4基因在核苷酸水平上与GenBank上登录的印度水牛IL-4 cDNA序列同源性为98.8%,与牛、绵羊、猪和马的同源性分别为99.3%、94.1%、84.8%和80.6%;氨基酸水平的同源性分别为96.3%、98.5%、90.4%、78.4%和59.6%。从培养12 h的单个核细胞中扩增得到的IL-5基因在核苷酸水平上与GenBank上登录的牛、绵羊、猪和马IL-5 cDNA序列同源性分别是99.0%、96.5%、89.6%和89.1%;氨基酸水平的同源性分别为97.8%、96.2%、85.9%和86.7%。本研究为进一步了解水牛IL-4和IL-5的结构和水牛免疫学特性奠定了基础。     

子宫内膜炎母牛子宫匀浆IL-2、IL-6、IL-8、TNF-α水平

选用正常子宫标本19例和患子宫内膜炎子宫标本66例,并对其进行分类,测定子宫匀浆中细胞因子IL-2、IL-6、IL-8、TNF-的含量,探讨IL-2、IL-6、IL-8、TNF-变化与子宫内膜炎的关系。结果表明,试验组IL-2、IL-6、IL-8、TNF-的含量均高于对照组(<0.05或<0.01),表明子宫内IL-2、IL-6、IL-8、TNF-含量变化与母牛子宫有无感染,以及免疫状态密切相关。     

枸杞多糖对免疫抑制小鼠血清中IL-6、IL-10和IL-12/IL23 p40分泌水平的影响

通过研究枸杞多糖(LBP)对免疫抑制小鼠血清中细胞因子IL-6、IL-12/IL-23p40和IL-10分泌的影响,探讨LBP的免疫调节机制,为LBP的开发利用提供理论依据。60只ICR雌性小鼠,随机分为5组,对各组小鼠进行连续4d每日腹腔注射环磷酰胺(40mg/kg),间隔3d后,再连续4d每日腹腔注射环磷酰胺(40mg/kg);同时,用高(40mg/1kg)、中(20mg/1kg)、低剂量(10mg/1kg)的LBP给小鼠连续灌胃2周,阳性对照和阴性对照分别用LPS(1mg/kg)和PBS,连续2周。小鼠眼眶取血,分离血清,ELISA检测血清中IL-6、IL-12/IL-23p40和IL-10的分泌水平。结果显示,与阳性对照和阴性对照组相比,LBP能提高免疫抑制小鼠血清中IL-6、IL-12/IL-23p40和IL-10的分泌水平,提示LBP能增强小鼠细胞免疫与体液免疫应答,为枸杞多糖作为免疫增强剂的开发应用奠定了一定的理论基础。     

Cloning and sequence analysis of llama (lama glama) Th2 (IL-4, IL-10 and IL-13) cytokines

This paper describes the cloning and sequence analysis of the cDNAs encoding the T helper (Th) 2 cytokines of llama including interleukin-4 (IL-4), IL-10 and IL-13. The cDNAs encoding for IL-4, IL-10 and IL-13 were amplified using specific primers designed from reported sequences of bovine cytokine genes. The cDNAs for llama IL-4, IL-10 and IL-13 were found to be 402, 537 and 411 bp in length, with open reading frames encoding 133, 178 or 136 amino acids, respectively. Homology analyses of nucleotide and deduced amino acid sequences of llama IL-4, IL-10 and IL-13 and phylogenetic analysis based on their nucleotide sequences indicated the close relationship in these cytokine genes between llama and eutherian mammalian order Artiodactyla (pig, cattle) and Perissodactyla (horse).     

Cellular localization of IL-18 and IL-18 receptor in pig anterior pituitary gland

Pro-inflammatory cytokine interleukin 18 (IL-18) has been proposed to have a role in modulating immuno-endocrine functions. Our previous study showed that IL-18 and IL-18 receptor (IL-18R) colocalized in somatotrophs of the bovine anterior pituitary gland, and the possibility that IL-18 acts on somatotrophs as an autocrine factor. In the present study, we investigated the localization of IL-18 and IL-18R in the pig anterior pituitary gland. RT-PCR analysis showed the expression of IL-18 and IL-18R mRNAin the pig anterior pituitary gland. Immunohistochemistry of IL-18 and specific hormones revealed the presence of IL-18 in somatotrophs, mammotrophs, thyrotrophs and gonadotrophs. IL-18R was localized in somatotrophs and thyrotrophs. Furthermore, the somatotrophs immunoreactive for IL-18 did not contain IL-18R. Thus, IL-18R and IL-18 were not colocalized in an identical somatotroph. These findings suggest that the localization of IL-18 in pig somatotrophs is different from that in bovine somatotrophs, although IL-18 closely associates with somatotrophs in the anterior pituitary glands in both species.     

猪IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系的建立

为了解猪体内细胞免疫应答机制,本研究建立猪细胞因子IL-2、IL-4、IL-10和IFN-γ的TaqMan实时定量RT-PCR检测体系.以五指山小型猪cDNA为模板,扩增IL-2、IL-4、IL-10和IFN-γ的基因保守区,构建相应的重组质粒,并将其作为标准品构建标准曲线.结果显示,各标准曲线的相关系数r值均大于0.990,扩增效率为90%～105%;该检测方法的敏感性较高,IL-4检测下限为1 copies/μL,IL-2、IL-10及IFN-γ检测下限达为102copies/μL;重复性较好,批内重复试验与批间重复试验变异系数均小于5%.本研究为病毒感染后猪体内免疫应答等研究提供方法.     

IL-21及其受体在IBD大鼠结肠、大脑组织中的表达规律

从脑-肠互作的角度出发，研究IL-21及其受体在TNBS化学诱导的IBD模型大鼠的结肠、大脑组织中mR—NA表达水平以及血清蛋白水平变化，以此探讨IL-21在炎症性肠病中的调节作用。建立了大鼠IBD模型，用荧光定量PCR方法检测IL-21及其受体mRNA在结肠、大脑组织中的表达水平变化，并采用ELISA方法测定血清中IL-21在IBD大鼠不同发病时期的蛋白水平。结果显示：IL-21及其受体在IBD大鼠结肠和大脑组织中的mRNA水平的呈现一致变化，即随着炎症的加剧而逐渐增加，在痛变最为严重的时候达到最大值，而随着炎症的消退逐渐恢复至正常水平。在血清中，IL-21蛋白浓度变化也表现出一致的变化趋势。结果表明，IL-21参与了肠道炎症进程，并且IL-21可能通过脑一肠互作来调节IBD的炎症进程。     

Synergistic effects of IL-2, IL-12 and IL-18 on cytolytic activity, perforin expression and IFN-gamma production of porcine natural killer cells

Natural killer (NK) cells are one of the main cellular components of the innate immune system. They play an important role in the immune response against infections as well as tumour cells and therefore have two major properties: production of immune regulatory cytokines and chemokines as well as cytolytic destruction of particular target cells. The existence of NK cells in swine is well known as well as the phenotype of resting NK cells, but their response following activation by cytokines is still poorly understood. Therefore, we tested the influence of the immune regulatory cytokines IL-2, IL-12 and IL-18 on cytolytic activity, phenotype, IFN-gamma production and the accumulation of perforin in cytoplasm of peripheral blood mononuclear cells (PBMC) as well as purified NK cells. NK cells were enriched from PBMC using a magnetic cell separation (MACS) strategy with monoclonal antibodies against CD3, CD21 and SWC3, thereby removing T-, B- and myeloid cells. Respective fractions were used in flow cytometry (FCM) based cytolytic assays with the human tumour cell line K562 as target. After stimulation with the cytokines described above, the NK cell enriched CD3(-)CD21(-)SWC3(-) fraction showed an evident increase in the cytolytic activity compared to PBMC. This enhanced cytolytic activity was accompanied by a strong enrichment of IFN-gamma producing cells when a combination of all three cytokines (IL-2/IL-12/IL-18) was used; as determined in ELISPOT assays and intracellular staining of IFN-gamma in FCM. Also, the combination of these three cytokines led to an accumulation of perforin in the cytoplasm and an up-regulation of CD25 compared to control cultures incubated in medium without cytokines. The experiments performed clearly indicate a stimulatory role and strong synergistic effects of the investigated cytokines in the activation of porcine NK cells in vitro, inducing IFN-gamma, perforin production and cytotoxicity against target cells.     

Blood concentrations of the cytokines IL-1beta,IL-6, IL-10, TNF-alpha and IFN-gamma during experimentally induced swine dysentery

Background

Knowledge of the cytokine response at infection with Brachyspira hyodysenteriae can help understanding disease mechanisme involved during swine dysentery. Since this knowledge is still limited the aim of the present study was to induce dysentery experimentally in pigs and to monitor the development of important immunoregulatory cytokines in blood collected at various stages of the disease.

Methods

Ten conventional pigs (~23 kg) were orally inoculated with Brachyspira hyodysenteriae B204^T. Eight animals developed muco-haemorrhagic diarrhoea with impaired general body condition. Blood was sampled before inoculation and repeatedly during acute dysentery and recovery periods and cytokine levels of IL-1β, IL-6, Il-10, TNF-α and IFN-γ were measured by ELISA.

Results

IL-1β was increased at the beginning of the dysentery period and coincided with the appearance of Serum amyloid A and clinical signs of disease. TNF-α increased in all animals after inoculation, with a peak during dysentery, and IL-6 was found in 3 animals during dysentery and in the 2 animals that did not develop clinical signs of disease. IL-10 was found in all sick animals during the recovery period. IFN-γ was not detected on any occasion.

Conclusion

B. hyodysenteriae inoculation induced production of systemic levels of IL-1β during the dysentery period and increased levels of IL-10 coincided with recovery from dysentery.