Toll样受体9介导的免疫激活研究进展

在细菌和DNA病毒的基因组中广泛存在着以非甲基化的胞嘧啶—鸟嘌呤核苷酸为核心的CpG基序。作为一种病原相关的分子模式（pathogen-associated molecular pattern, PAMP）,含有CpG基序的寡聚脱氧核苷酸（CpG oligodeoxynucleotides,CpG ODN）能激活包括B淋巴细胞和类浆细胞在内的多种免疫细胞,并诱导产生以Th1型细胞免疫反应为主的免疫应答。CpG ODN在哺乳动物细胞中的受体是Toll样受体（Toll-like receptors, TLRs）家族中的Toll样受体 9（TLR9）。TLR9所介导的免疫激活作用在某些传染病的预防、新型疫苗佐剂的开发及过敏性疾病和癌症的治疗中有着巨大的应用前景。     

CpG-DNA免疫刺激活性研究进展

CpG—DNA是一些具有免疫激活功能的以未甲基化的CpG基序(CpG motif)为核心的DNA序列，它包括含CpG基序的人工合成的寡聚脱氧核苷酸(oligodeoxvnucleotides，ODN)和自然界中某些低等生物的基因组DNA。近几年研究证实，CpG基序能在体内诱导强烈的免疫反应，主要包括激活B细胞增殖、分化、分泌白细胞介素-12(IL-12)及其他细胞因子，激活自然杀伤细胞(NK)的裂解活性，分泌     

CpG寡聚核苷酸的免疫激活机理及生物学活性

脊椎动物能够识别入侵的微生物是基于病原体的特定分子结构，这些被认为是病原体相关分子模式（pathogen—associated molecular pattern，PAMP）的结构能够被宿主体内模式识别受体（pattern recognition receptor，PRR）所识别，从而产生免疫应答。细菌和病毒基因组中所携带的非甲基化CpG（胞嘧啶鸟嘌呤二核苷酸）至少是脊椎动物中的20倍，并且脊椎动物的DNA甲基化程度比较高。细菌和脊椎动物这种在DNA上的显著差别使得细菌基因组在进化过程中成为PAMP从而被脊椎动物识别为“危险的入侵信号”，并做出相应的免疫应答。     

羊口疮病毒B2L基因DNA疫苗的构建及其诱导的免疫应答

为研究所构建羊口疮病毒(OrfV)B2L基因DNA疫苗诱导小鼠的免疫应答效果,本研究对pMD18T-B2L质粒进行PCR扩增,克隆B2L片段至pVAX1载体中构建pVAX1-B2L重组质粒,进行酶切和测序鉴定;采用脂质体法将pVAX1-B2L真核表达质粒转染MDBK细胞,RT-PCR和IFA法检测B2L基因在MDBK细胞中的转录和表达;将构建的DNA疫苗免疫KM系小鼠,采用间接ELISA、MTT和FACS法对其诱导的免疫应答进行研究。结果显示,成功构建pVAX1-B2L真核表达质粒,并在MDBK细胞中表达;免疫小鼠后,DNA疫苗能诱导小鼠产生OrfV特异性抗体;脾淋巴细胞增殖、CD4~+、CD8~+T淋巴细胞亚群百分比和IL-2、IFN-γ、IL-4细胞因子均高于pVAX1组和PBS组。结果表明,本研究制备的DNA疫苗能够诱导小鼠产生较高水平的体液免疫和细胞免疫应答。     

苜草素对不同生长阶段蛋鸡免疫系统的增强作用

正白细胞介素2(IL-2),也叫做T细胞生长因子,在机体免疫调节中发挥重要的作用,可以促进T细胞亚群发育,还能够加快B细胞繁殖的速率,因此,它是调节免疫应答的重要因子,它在淋巴细胞的生长和分化中起着重要的作用,如抗体应答、造血和肿瘤监测等。IL-2是所有细胞因子佐剂中研究得最深入最广泛的细胞因子之一,能够直接激活单核细胞,能够加快T细胞分化过程以及繁殖过程,加快B细胞分     

外源siRNA诱导家蚕先天免疫应答

LPS是众所周知的病原体相关分子模式(PAMP),能激活昆虫的先天免疫应答。在LPS和siRNA诱导后相关基因的相似应答说明siRNA在家蚕先天免疫应答中也能充当PAMP。注射siRNA广泛应用于昆虫内源性基因的沉默和研究基因的功能。在这个研究里,我们报道了家蚕在LPS和siRNA诱导后黑化作用相关基因的转录水平。     

外源siRNA诱导家蚕先天免疫应答

LPS是众所周知的病原体相关分子模式(PAMP),能激活昆虫的先天免疫应答。在LPS和siRNA诱导后相关基因的相似应答说明siRNA在家蚕先天免疫应答中也能充当PAMP。注射siRNA广泛应用于昆虫内源性基因的沉默和研究基因的功能。在这个研究里,我们报道了家蚕在LPS和siRNA诱导后黑化作用相关基因的转录水平。     

白细胞介素-2免疫佐剂效应的研究进展

<正>1976年,D.A.Morgan等[1]最先报道,用丝裂原刺激鼠脾淋巴细胞可产生白细胞介素-2(IL-2)。IL-2具有激活杀伤细胞和自然杀伤细胞,刺激T淋巴细胞和自然杀伤细胞产生细胞因子的能力,在机体的免疫应答中起重要作用。IL-2作为新型免疫佐剂,不同于以前的化学佐剂,它不仅能避免常规佐剂的不良反应,而且能增强病毒、细菌和寄生虫疫苗的免疫效果,在改变免疫应答强度的同时改变免疫应答     

IL-18的免疫调节功能及其应用

IL-18是一种具有多向和多层次免疫调节功能的细胞因子,其细胞来源较广。它可通过NF-κB、p56LCK-MAPK、Perforin等多个信号传导途径参与细胞凋亡以及IFN-γ分泌诱导及功能性免疫细胞活性的激活,从而直接或间接激活T细胞、NK细胞、PBMC、DC等免疫细胞的增殖、分化、抗原呈递、CTL反应等,并诱导这些细胞分泌效应性细胞因子或延长免疫保护,进而促进免疫系统增强抗感染、抗肿瘤等免疫效应。IL-18除了主要介导细胞免疫外,在一定条件下,亦能促进抗原特异性中和抗体以及Th2型细胞因子的应答。在某些疾病的防治和诊断中,具有重要临床意义。     

CpG ODN对仔猪淋巴细胞增殖和细胞因子诱生的影响:在体和离体试验

将CpGODN、non CpG ODN分别接种初生仔猪，同时分离仔猪的外周血单核细胞，以CpGODN、non CpG ODN对外周血单核细胞体外培养，分别检测仔猪Th1型细胞因子分泌情况。结果表明，与non CpG ODN和对照组相比，CpG ODN能在体内显著提高仔猪的淋巴细胞白介素-2诱生活性、淋巴细胞增殖、血清中IFN-γ和IL-12水平（P〈0．05），亦能在体外刺激仔猪的外周血单核细胞分泌IFN-γ和IL-12（P〈0．01）。这显示CpG—ODN能显著增强动物的免疫应答能力。     

Th17细胞和IL-17在小鼠寄生虫免疫中的作用

Th17细胞（T helper 17 cells,Th17）是CD4~＋T细胞分化的亚群细胞之一,能够特异性地分泌细胞因子IL-17（interleukin-17,IL-17）,通过IL-17、IL-21、IL-23等细胞因子介导炎症反应和针对细菌的抗感染免疫,同时在寄生虫免疫中发挥重要作用。     

原头蚴外泌体对树突状细胞活化和细胞因子分泌的影响

为分析囊型包虫原头蚴(PSC)分泌的外泌体(Exosome)对树突状细胞(DCs)活化和分泌细胞因子的影响,本研究采用高速离心法结合外泌体提取试剂盒分离原头蚴分泌的外泌体(PE),收集的PE沉淀中加入去离子水低渗裂解离心,提取超滤后的蛋白浓缩液即为原头蚴外泌体超滤裂解物(PEL)。利用免疫磁珠从健康ICR小鼠脾脏中分选DCs,分别加入PE、LPS、PEL、PBS进行刺激培养,24 h后收集培养细胞及培养上清,利用流式细胞术检测DCs表面活化分子的表达水平及细胞因子分泌情况。结果显示:4组DCs表面表达CD40、CD80、CD86、MHCⅡ的比例相比,PEL组结果明显高于PE组,这两组较PBS组均显著升高(p<0.05),但3组均低于LPS组。PE、PEL组DCs培养上清中IL-6、IL-10、IL-12、TNF-α均显著高于PBS组,且PEL组IL-12、TNF-α高于PE组,IL-6、IL-10水平低于PE组。以上结果初步证实PE能刺激DCs活化和释放细胞因子,低渗裂解超滤提纯外泌体蛋白并去除miRNA成分的PEL能够更有效激活DCs活化和促进细胞因子释放,表明外泌体蛋白可诱导抗感染免疫应答,exosomal miRNA成分可能参与虫体免疫逃逸。本研究为后续进一步探索包虫感染及抗感染免疫应答机理提供实验依据。     

白细胞介素-12对犬细小病毒VP2 DNA疫苗的免疫增强作用

犬细小病毒编码的VP2蛋白是该病毒重要的结构蛋白和抗原蛋白。利用VP2基因制备的DNA疫苗能够刺激机体产生免疫应答反应。为进一步提高VP2DNA疫苗的免疫应答水平,本研究在小鼠体内尝试了利用白细胞介素12（IL-12）基因表达载体提高VP2DNA疫苗的免疫应答水平。首先采用RT-PCR方法从小鼠脾淋巴细胞中分别扩增IL-12大亚基（P40）和小亚基（P35）cDNA基因;然后在真核表达载体pcDNA3.1A上通过引入内部核糖体进入位点（IRES）序列,分别将P40基因和P35基因插入到IRES序列的上下游,构建成IL-12（P40和P35双亚基）基因表达载体,pcDNA-P40-IRES-P35。将上述表达载体与本室构建的VP2表达载体通过磷酸钙方法转染HEK 293T细胞进行瞬时表达,以确定构建的表达载体能否介导相应基因在真核细胞中进行分泌表达。然后用VP2载体单免疫和VP2载体和IL-12载体共免疫方法对小鼠进行免疫（用pcDNA3.1A作为对照）。免疫后在特定时间通过ELISA方法检测小鼠血清抗VP2蛋白的抗体水平,并通过淋巴细胞增殖实验检测免疫后35d小鼠脾脏淋巴细胞增殖反应。结果表明,扩增的小鼠IL-12P40和P35亚基基因与GenBank的参考序列基本一致。Western-blot检测结果表明,重组IL-12和VP2均能够在HEK293T细胞中进行分泌性表达。ELISA检测结果表明利用IL-2载体与VP2载体共免疫小鼠,其血清中抗VP2的抗体水平明显高于VP2载体单免疫组（P〈0.01）,抗体水平在第35天高达1：5120。淋巴细胞增殖试验结果表明,免疫小鼠的淋巴细胞刺激指数均明显高于对照组（P〈0.01）,VP2载体与IL2载体共免疫组的刺激指数明显高于VP2载体单免疫组（P〈0.05）。由此可见,在小鼠体内,IL-12基因表达载体可明显提高CPV VP2基因疫苗的免疫应答水平。     

Bovine toll-like receptor 9: a comparative analysis of molecular structure, function and expression

Non-methylated CpG motifs, present in viral and bacterial DNA, are one of many pathogen-associated molecular patterns (PAMP) recognized by the mammalian innate immune system. Recognition of this PAMP occurs through a specific interaction with toll-like receptor 9 (TLR9) and this interaction can induce cytokine responses that influence both innate and adaptive immune responses. Previous investigations determined that both the flanking sequences in synthetic CpG oligodeoxynucleotides (CpG ODN) and the cellular pattern of TLR9 expression can influence species-specific responses to CpG ODN. Therefore, the structure, function and cellular distribution of bovine TLR9 were compared with what is known for mice and human. Analysis of the bovine TLR9 gene revealed greater sequence homology between cattle and humans than cattle and mice Similar CpG motifs induced optimal activation of both human and bovine leukocytes and these motifs were distinct from those which activated mouse leukocytes. Functional analyses with CpG ODN stimulated bovine blood leukocytes revealed that class A CpG ODN were more potent inducers of interferon-alpha (IFN-alpha) than class B CpG ODN. Furthermore, magnetic activated cell sorting of bovine blood leukocyte subpopulations implicated dendritic cells but not monocytes in the regulation of CpG ODN-induced IFN secretion. Thus, the cellular pattern of CpG ODN-induced responses in cattle shared many similarities with human leukocytes. Collectively, these analyses revealed substantial conservation of TLR9 structure and TLR9 function in blood leukocytes of humans, cattle and other domestic species.     

Innate immune response to intramammary infection with Serratia marcescens and Streptococcus uberis

Streptococcus uberis and Serratia marcescens are Gram-positive and Gram-negative bacteria, respectively, that induce clinical mastitis. Once initial host barrier systems have been breached by these pathogens, the innate immune system provides the next level of defense against these infectious agents. The innate immune response is characterized by the induction of pro-inflammatory cytokines, as well as increases in other accessory proteins that facilitate host recognition and elimination of the pathogens. The objective of the current study was to characterize the innate immune response during clinical mastitis elicited by these two important, yet less well-studied, Gram-positive and Gram-negative organisms. The pro-inflammatory cytokine response and changes in the levels of the innate immune accessory recognition proteins, soluble CD14 (sCD14) and lipopolysaccharide (LPS)-binding protein (LBP), were studied. Decreased milk output, induction of a febrile response, and increased acute phase synthesis of LBP were all characteristic of the systemic response to intramammary infection with either organism. Infection with either bacteria similarly resulted in increased milk levels of IL-1 beta, IL-8, IL-10, IL-12, IFN-gamma, TNF-alpha, sCD14, LBP, and the complement component, C5a. However, the duration of and/or maximal changes in the increased levels of these inflammatory markers were significantly different for several of the inflammatory parameters assayed. In particular, S. uberis infection was characterized by the sustained elevation of higher milk levels of IL-1 beta, IL-10, IL-12, IFN-gamma, and C5a, relative to S. marcescens infection. Together, these data demonstrate the variability of the innate immune response to two distinct mastitis pathogens.     

Modulation of innate immunity in chickens induced by in vivo administration of baculovirus

Baculoviruses stimulate cytokine production in mammalian cells. They induce a strong innate immune response in animals and have adjuvant properties. The purpose of this work was to study the in vivo effect of baculovirus on chicken innate immune response. SPF chickens were inoculated intravenously with Autographa californica nuclear polyhedrosis virus (BV). Three hours later, chickens were bled, euthanized and their spleen, duodenum and cecal tonsils were excised in order to take samples for RNA extraction and real time PCR, and to isolate lymphocytes, which were stained and analyzed by flow cytometry. The results obtained showed that baculovirus inoculation up-regulates the expression of IFN-γ, IL-6 and LITAF in spleen cells. This result (IFN-γ) correlated with that obtained by ELISA which showed a very strong increase of IFN-γ in chicken plasma. Flow cytometry analysis revealed that BV inoculation induced in spleen an increase in the percentage of monocyte/macrophage population together with an increase in CD3(+)CD4(+) T lymphocytes. On the other hand, BV inoculation decreased the percentage of CD3(+)CD4(+) T lymphocytes and increased the percentage of NK cells in cecal tonsils. However, intraepithelial lymphocytes of the gut did not show differences between BV and control treated animals. Even though further studies in order to understand the mechanisms by which BVs affect the avian immune response are needed, results obtained in the present work demonstrate the ability of BVs to stimulate the innate immunity in chickens, modifying the expression pattern of related genes and the profile of the immune cells involved.     

Toll样受体信号传导通路的研究进展

Toll样受体（toll like receptors,TLRs）能识别病原微生物相关分子模式,募集含有Toll/白介素-1受体（TIR）结构域的接头蛋白分子,通过髓样分化蛋白88（MyD88）依赖性信号传导通路或由β-干扰素TIR结构域衔接蛋白（TRIF）依赖性信号传导通路启动信号传导,继而引发特异性的免疫应答。作者就TLRs的结构特征、分布、配体识别、参与信号传导的接头蛋白分子及介导的信号传导通路的最新研究进展进行综述。     

Cytokines as markers for infections and their effect on growth performance and well-being in the pig

Exposure to micro-organisms commonly elicit the production of cytokines. These soluble factors enhance several innate immune functions that aim to limit the spread of infection. Further, many of the pro-inflammatory cytokines regulate the ensuing specific immune response. In addition to their effects on cells of the immune system, cytokines also are important regulators in the so called immune-neuroendocrine network. The microbial structures that are necessary for induction of cytokine production are not conclusively determined but in general, bacteria preferentially induce the production of IL-1, TNF-α, IL-6, and IL-8, whereas virus induce the production of Type 1 interferons (IFN-α/β). The onset of production of these cytokines is rapid, and several of them may reach systemic levels during a short period after infection. Thus, cytokines can serve as markers for ongoing infections and be used for discrimination between infections of bacterial or viral origin. Results from experimental and field studies show that serum IFN-α and IL-6 seem to be useful markers for ongoing (subclinical) viral and bacterial infections, respectively, in the pig. Consequently, demonstration of these cytokines can be valuable tools in heard health monitoring programs.     

Salmonella enterica serovar Choleraesuis infection of the porcine jejunal Peyer's patch rapidly induces IL-1beta and IL-8 expression

Salmonella enterica serovar Choleraesuis is an enteric pathogen of swine, producing septicemia, enterocolitis, pneumonia, and hepatitis. The initial molecular events at the site of Salmonella infection are hypothesized to be critical in the initiation of innate and adaptive immune responses; however, the acute immune response elicited by porcine intestinal tissues is not well understood. To address this need, we employed explants of jejunal Peyer's patch (JPP) mucosa from pigs to examine Salmonella-induced immune responses under controlled conditions as well as to overcome limitations of whole animal approaches. JPP explants mounted in Ussing chambers maintained normal histological structure for 2 h and stable short-circuit current and electrical conductance for 2.5 h. After ex vivo luminal exposure to Salmonella serovar Choleraesuis, JPP responded with an increase in mRNA expression of IL-1beta and IL-8, but not TNFalpha. Increased IL-1beta and IL-8 expression were dependent on efficient Salmonella adhesion and internalization, whereas mutant Salmonella did not induce inflammatory cytokine expression. Commensal enteric bacteria, present in some experiments, also did not induce inflammatory cytokine expression. These findings indicate that Salmonella uptake by Peyer's patch is important in the induction of an innate response involving expression of IL-1beta and IL-8, and that ex vivo intestinal immune tissue explants provide an intact tissue model that will facilitate investigation of mucosal immunity in swine.     

不同CpG-DNA序列对鸡ND HI影响的研究

将16种人工合成的CpG-DNA与新城疫Ⅳ弱毒苗(La Sota株)合用,免疫14日龄雏鸡,于免疫前及免疫后7、14、21天测鸡HI抗体效价,筛选出4种CpG-DNA短序并进行SPF鸡重复试验,结果表明:筛选的4种CpG短序(1.AACGTr AACGTr;2.AACGTC AACGTC;3.AGCGTC AGCGTC;4.AGCGCT AGCGCT)可显著提高鸡体的体液免疫水平.