Lentivirus-induced immune dysregulation

FIV/HIV infections are associated with an early robust humoral and cellular anti-viral immune response followed by a progressive immune suppression that eventually results in AIDS. Several mechanisms responsible for this immune dysfunction have been proposed including cytokine dysregulation, immunologic anergy and apoptosis, and inappropriate activation of immune regulatory cells. Studies on FIV infection provide evidence for all three. Cytokine alterations include decreases in IL2 and IL12 production and increases in IFNγ and IL10 in FIV⁺ cats compared to normal cats. The elevated IL10:IL12 ratio is associated with the inability of FIV⁺ cats to mount a successful immune response to secondary pathogens. Additionally, chronic antigenic (FIV) stimulation results in an increase in the percent of activated T cells expressing B7 and CTLA4 co-stimulatory molecules in infected cats. The expression of these molecules is associated with T cells that are undergoing apoptosis in the lymph nodes. As ligation of CTLA4 by B7 transduces a signal for induction of anergy, one can speculate that the activated T cells are capable of T cell–T cell interactions resulting in anergy and apoptosis. The inability of CD4⁺ cells from FIV⁺ cats to produce IL2 in response to recall antigens and the gradual loss of CD4⁺ cell numbers could be due to B7–CTLA4 interactions. The chronic antigenemia may also lead to activation of CD4⁺CD25⁺ T regulatory cells. Treg cells from FIV⁺ cats are chronically activated and inhibit the mitogen-induced proliferative response of CD4⁺CD25⁻ by down-regulating IL2 production. Although Treg cell activation can be antigen-specific, the suppressor function is not, and thus activated Treg cells would suppress responses to secondary pathogens as well as to FIV. Concomitant with the well-known virus-induced immune suppression is a progressive immune hyper-activation. Evidence for immune hyper-activation includes polyclonal B cell responses, gradual replacement of naïve CD4⁺ and CD8⁺ T cell phenotypes with activation phenotypes (CD62L⁻, B7⁺, CTLA4⁺), and the chronic activation of CD4⁺CD25⁺ Treg cells. Thus lentivirus infections lead to severe immune dysregulation manifested as both chronic immune suppression and chronic immune activation. FIV infection of cats provides a number of advantages over other lentivirus infections as a model to study this immune dysregulation. It is a natural infection that has existed in balance with the cat's immune system for thousands of years. As such, the natural history and pathogenesis provides an excellent model to study the long-term relationships between AIDS lentivirus and host immune system function/dysregulation.     

The Influence for Chickens' T Lymphocyte Subsets in Peripheral Blood after Inoculated with the Recombinant Chicken Interferon-α/Chicken Interleukin-2 Complex Protein

To explore the influence of recombinant chicken interferon-α/chicken interleukin-2 fusion protein (rChIFN-α-Linker-ChIL-2 protein, recombinant fusion protein) on the percentage content of peripheral T lymphocyte subsets (PBLC) of SPF chicken,the flow cytometry was used to detected the percentage contents of CD3⁺CD4⁺, CD3⁺CD8⁺ and the CD4⁺/CD8⁺ ratio in PBLC at different days post injection in the 14-day-old SPF chicken injected with rChIFN-α-Linker-ChIL-2 protein (group 2) and recombinant chicken interferon α (rChIFN-α) protein (group 3). The results indicated that the recombinant fusion protein and rChIFN-α protein could increase the percentage content of CD3⁺CD4⁺ T lymphocyte, decrease the percentage content of CD3⁺CD8⁺ T lymphocyte, improve CD4⁺ /CD8⁺ T lymphocyte ratio during 3 to 14 d post injection. The percentage contents of CD3⁺CD4⁺, CD3⁺CD8⁺ and CD4⁺/CD8⁺ ratio in chickens of group 2 were significantly different from the PBS control group (group 1) (P<0.01), and were notably different from group 3 during 3 to 7 d post injection (P<0.05). These results showed that both the rChIFN-α-Linker-ChIL-2 protein and rChIFN-α protein could affect the percentage contents of lymphocyte subsets, improve CD4⁺/CD8⁺ ratio, enhance the cellular immune function in chicken. The influences of rChIFN-α-Linker-ChIL-2 protein on the percentage contents of CD3⁺CD4⁺ and CD3⁺CD8⁺ T lymphocyte, and CD4⁺/CD8⁺ value were notable higher than rChIFN-α, which suggested that recombinant fusion protein play a synergistic role of interferon alpha and interleukin-2 on the cellular immune function by this pathway in chicken.     

伯氏疟原虫ANKA株感染小鼠的T细胞、NK细胞及细胞因子变化

旨在系统分析伯氏疟原虫感染引起宿主T细胞、NK细胞、Tim-3表达及细胞因子的变化。选取64只雌性C57BL/6小鼠随机分为8组,每组8只,分别于感染后0、4、7、9、11、13、16和19 d获取小鼠脾及外周血免疫细胞,利用流式细胞术检测小鼠主要免疫细胞亚群及免疫检查点分子Tim-3表达水平的变化;同时检测血清中细胞因子的变化。结果表明,感染疟原虫后,小鼠脾CD3⁺CD4⁺ T细胞、CD3⁺CD8⁺ T细胞及NK细胞的比例均逐渐降低（P<0.01）,且伴随着3种细胞Tim-3表达量的升高（P<0.01）。小鼠外周血CD3⁺CD4⁺ T细胞的比例呈先降低后升高趋势,CD3⁺CD8⁺ T细胞的比例呈先升高后降低趋势（P<0.05）;小鼠外周血CD3^-NK1.1⁺细胞的比例呈先降低后升高趋势,但感染末期,其比例仍低于未感染组（P<0.05）。Tim-3分子在外周血CD3⁺CD4⁺ T细胞、CD3⁺CD8⁺ T细胞及CD3^-NK1.1⁺细胞的表达均显著升高（P<0.05）。感染疟原虫后,小鼠血清中促炎细胞因子IL-2的分泌量均显著高于未感染组（P<0.05）;促炎细胞因子IFN-γ、TNF-α和IL-6在血清中分泌均呈先升高后降低趋势（P<0.05）;具有免疫抑制作用的细胞因子IL-10呈逐渐升高趋势,且感染后期急剧升高（P<0.001）。以上结果表明,感染疟原虫后,小鼠的特异性免疫反应发挥了一定的杀伤作用,但由于Tim-3免疫检查点分子及一些发挥免疫抑制作用的细胞因子（IL-10）的过度表达,有利于疟原虫逃避宿主的免疫捕杀作用。该研究提示了从宿主免疫抑制角度研究疟原虫感染的重要性。     

新疆栽培与野生一枝蒿粗多糖对口蹄疫疫苗免疫小鼠的佐剂活性比较

基于总物质量和多糖含量比较栽培/野生一枝蒿粗多糖（cultivated/wild Artemisia rupestris L.crude polysaccharides,CARCP/WARCP）作为口蹄疫灭活疫苗（foot-and-mouth disease inactivated vaccine,FMDV）佐剂对小鼠抗体水平及T细胞亚群的影响,探究CARCP/WARCP的佐剂活性和安全性。CARCP/WARCP配伍FMDV肌肉途径免疫ICR小鼠,检测免疫后小鼠血清中FMDV特异性抗体及分型,脾中T细胞亚群比例,血清中IgE水平,观察临床症状和注射部位反应以及小鼠体重。结果显示,总物质量一致时,CARCP1/WARCP1均能极显著提高FMDV特异性IgG、IgG_2a反应（P<0.01）,极显著促进脾T细胞CD3⁺CD4⁺、CD4⁺CD44⁺、CD8⁺CD44⁺CD62L⁺百分比（P<0.05）,显著提高IgG₁、IgG_2a/IgG₁比值,显著促进CD3⁺CD8⁺、CD8⁺CD44⁺、CD8⁺CD44⁺CD62L^-比例（P<0.05）,且除28 d IgG和IgG₁指标外,CARCP1的佐剂活性显著高于WARCP1（P<0.05）。多糖含量一致时,与FMDV相比,CARCP2/WARCP2均极显著增强了28 d IgG水平、IgG_2a/IgG₁比值（P<0.01）,显著提高了21 d IgG、28 d IgG_2a及CD4⁺CD44⁺（P<0.05）,且CARCP2/WARCP2之间差异不显著（P>0.05）。CARCP/WARCP没有引起小鼠脱毛等临床症状,也没有产生肉芽肿、肿胀等注射部位不良反应;CARCP/WARCP免疫后各组小鼠体重之间差异不显著（P>0.05）;各组小鼠血清均没有检测到IgE抗体（P>0.05）;这些结果表明CARCP/WARCP有一定的安全性。综上,当总物质量一致时,CARCP/WARCP均能增强FMDV免疫小鼠体液和细胞免疫反应,且CARCP的佐剂活性优于WARCP;多糖含量一致时,CARCP/WARCP作为FMDV佐剂的免疫增强效果相当,是安全佐剂候选物。     

重组鸡白细胞介素-6/2融合蛋白对新城疫(LaSota株)活疫苗免疫增强作用研究

为探究重组鸡白细胞介素-6/2融合蛋白(rChIL-6-Linker-ChIL-2,重组融合蛋白)对新城疫病毒(NDV)活疫苗(LaSota株)的免疫增强作用,本研究将90只SPF鸡随机分为6组,分别为PBS对照组、NDV弱毒苗对照组、rChIL-6-Linker-ChIL-2免疫增强组、rChIL-6蛋白免疫增强组、rChIL-2蛋白免疫增强组及rChIL-6+rChIL-2混合蛋白免疫增强组,将鸡白细胞介素重组融合蛋白水剂与LaSota株联合接种于SPF鸡,分别采用MTS法、流式细胞术、ELISA和HA/HI法检测接种前后不同时间各组鸡外周血及脾淋巴细胞增殖活性、鸡外周血中CD3+CD4+/CD3+CD8+T淋巴细胞的百分含量、血清中Th1/Th2型细胞因子表达水平及免疫抗体水平等指标。结果显示,接种后7～21d时,与NDV弱毒苗对照组相比,同时接种重组融合蛋白组鸡淋巴细胞增殖活性、外周血CD3+CD4+/CD3+CD8+T淋巴细胞的百分含量比值及血清中重组鸡IL-4蛋白(rChIL-4)、ChIL-6、ChIL-2、重组鸡IFN-α蛋白(ChIFN-α)、ChIFN-γTh1/Th2型细胞因子表达水平明显提升;HI免疫抗体较NDV弱毒苗对照组提高了1.0～1.9个滴度,较单一rChIL-6、rChIL-2对照组分别提高了0.2～0.7、0.2～1.1个抗体滴度。综上所述,rChIL-6-Lin-ker-ChIL-2融合蛋白对NDV(LaSota株)活疫苗在鸡体内具有明显的免疫增强效果。     

鸡传染性支气管炎病毒S1蛋白抗原表位多肽的预测、鉴定及免疫效果的初步研究

旨在对鸡传染性支气管炎病毒（IBV）H120株S1蛋白的B细胞及T细胞可能的表位进行预测并合成相应的多肽,然后免疫小鼠,并分析其免疫效果,以验证候选表位多肽的免疫原性。利用表位预测软件筛选并化学合成针对IBV H120株S1蛋白的5条表位多肽,然后免疫小鼠,通过间接ELISA、中和试验和流式细胞技术检测各表位多肽诱导的特异性抗体、中和抗体和外周血T细胞亚群。ELISA检测结果显示,5条表位多肽均具有良好的反应原性,免疫小鼠的血清效价高低顺序依次为Pep76-106 > Pep240-257 > Pep511-537 > Pep403-421 > Pep135-172;中和试验结果表明,5条多肽免疫小鼠的血清中和滴度均高于空白对照组,其高低顺序依次为Pep240-257=Pep403-421=Pep511-537>Pep76-106=Pep135-172;流式检测结果表明,5条多肽免疫小鼠在CD3⁺、CD4⁺CD8^-、CD8⁺CD4^- T淋巴细胞水平上均极显著高于空白对照组（P<0.01）,CD3⁺T及CD4⁺CD8^-T淋巴细胞数大小顺序依次为Pep403-421 > Pep240-257 > Pep76-106 > Pep511-537 > Pep135-172,CD8⁺CD4^-T淋巴细胞数大小顺序依次为Pep403-421 > Pep76-106 > Pep511-537 > Pep240-257 > Pep135-172。合成的5条表位多肽中,Pep240-257、Pep76-106和Pep403-421可以诱导体液免疫,Pep403-421可以诱导细胞免疫,其中,Pep403-421可以同时诱导细胞免疫及体液免疫。本研究结果为深入了解S1蛋白的免疫学特性以及研发诊断试剂和有效表位疫苗奠定了基础。     

Prediction,Identification and Preliminary Study on Immune Efficacy of S1 Protein Epitope-polypeptides of Avian Infectious Bronchitis Virus

For the sake of verifying the immunogenicity of candidate epitope-polypeptide, the B and T cell epitopes of S1 protein of avian infectious bronchitis virus (IBV) H120 strain were predicted and the corresponding epitope-polypeptides were synthesized, and then were used to immunize mice, the immune effect was analyzed. Five epitope-polypeptides against S1 protein of IBV H120 strain were selected by epitope prediction software and acquired by chemical synthesis, then were immunized to mice. The specific antibodies, neutralizing antibodies and T lymphocyte subsets induced by each epitope-polypeptides were analyzed by indirect ELISA, neutralization test and flow cytometry. The ELISA results showed that the five epitope-polypeptides had good reactivity. The antibody titers of antisera induced by the five epitope-polypeptides sorted from high to low as follows: Pep76-106, Pep240-257, Pep511-537, Pep403-421, Pep135-172. The neutralization test results showed that the neutralization titers of antisera induced by the five epitope-polypeptides groups in mice were higher than that of the blank control group, and the order of neutralization titers was Pep240-257 = Pep403-421 = Pep511-537 > Pep76-106 = Pep135-172. The flow cytometry results showed that the percentages of CD3⁺, CD4⁺CD8^- and CD8⁺CD4^- T lymphocytes in all the five epitope-polypeptides groups were significantly higher (P<0.01) than those in the blank control group. The number of the CD3⁺ and CD4⁺CD8^- T lymphocytes sorted from large to small as follows: Pep403-421, Pep240-257, Pep76-106, Pep511-537 and Pep135-172. The number of the CD8⁺CD4^- T lymphocytes sorted from large to small as follows: Pep403-421, Pep76-106, Pep511-537, Pep240-257 and Pep135-172. In conclusion, Pep240-257, Pep76-106 and Pep403-421 could induce humoral immunity among the five epitope-polypeptides, while Pep403-421 could induce cellular immunity. Thus, peptide of Pep403-421 could induce cellular immunity and humoral immunity. This study laid a foundation for further understanding the immunological characteristics of the S1 protein and the development of diagnostic reagents and effective epitope vaccines.     

The Localization Changes of CD4+ and CD8+ T Lymphocytes in Chicken Lung at Different Ages

The aim of this study was to investigate the immune state of chicken lung in different periods.With lung tissue of Hy-line White chicken at different ages,the distribution and quantity changes of CD4⁺ and CD8⁺T lymphocytes in lung were studied using immunohistochemistry staining.The results showed that CD8⁺T lymphocytes appeared firstly in embryonic at 18 d,while CD4⁺T lymphocytes appeared at 1-day-old chicken.At 4-day-old,there were aggregates of lymphocytes at the junction of the primary and secondary bronchi,which formed obvious broncho-associated lymphoid tissue (BALT).CD4⁺T lymphocytes of each age mainly occupied the central area of BALT,while CD8⁺T lymphocytes mainly surrounded the periphery.Since 56 days old,CD8⁺T lymphocytes are distributed in the inner wall of third-order bronchial airway,atrial septum,gas exchange area and interlobular connective tissue,and are distributed throughout the lung.In terms of quantity change,with the growth of daily age,the number of CD4⁺T lymphocytes and CD8⁺T lymphocytes gradually increased,and the number of CD4⁺ T lymphocytes was more than that of CD8⁺ T lymphocytes before 35 days of age,while the number of CD8⁺ T lymphocytes was significantly more than that of CD4⁺ T lymphocytes at the same age thereafter.The results showed that the distribution and number of CD4⁺ and CD8⁺T lymphocytes in the lungs of chickens were correlated with the age,and the changes could reflect that the lungs before the age of 35 days were dominated by humoral immunity,while the lungs after that tended to be cellular immunity.     

松针对雏鸡免疫功能和血清胆固醇的影响

试验旨在研究松针对雏鸡免疫功能和血清胆固醇的影响。选用1日龄健康罗曼公鸡240只,随机分为4组,每组3个重复,每个重复20只鸡,对照组直接饲喂基础日粮和正常饮水,Ⅰ、Ⅱ组分别在基础日粮中以0.5、1.0 g/只剂量的松针粉拌料饲喂,Ⅲ组以0.5 g/只剂量的松针水煎液饮服。预试期4 d,正试期70 d。结果表明,Ⅰ、Ⅱ、Ⅲ组CD3⁺CD4⁺T淋巴细胞百分率在首免后第15天时高于对照组,但Ⅰ、Ⅱ组CD3⁺CD4⁺T淋巴细胞百分率在首免后第30、45、60天时则低于对照组,Ⅲ组在首免后第45天时则低于对照组;Ⅰ、Ⅱ、Ⅲ组CD3⁺CD8⁺T淋巴细胞百分率在首免后第15、30、45天时高于对照组,但Ⅰ、Ⅲ组在首免后第60天则低于对照组。Ⅰ、Ⅱ组新城疫抗体水平在首免后第14和21天时低于对照组,但在第28、35天时则高于对照组;Ⅲ组新城疫抗体水平在首免后第7、35天时低于对照组,但在第14、21天时则高于对照组。Ⅰ、Ⅱ、Ⅲ组血清总胆固醇和甘油三酯水平与对照组相比均有降低趋势,其中Ⅰ、Ⅱ组血清甘油三酯水平显著低于对照组和Ⅲ组（P<0.05）;Ⅱ、Ⅲ组血清低密度脂蛋白水平低于对照组,Ⅱ组血清高密度脂蛋白水平高于对照组,但差异均不显著（P>0.05）。综上所述,松针粉和水煎剂可促进T淋巴细胞的增殖与分化,提高CD3⁺CD4⁺和CD3⁺CD8⁺T淋巴细胞含量,协同疫苗增强特异性免疫,不同程度的降低血清胆固醇水平。     

Report on the analyses of mAb reactive with porcine CD8 for the second international swine CD workshop

Based on an analysis of their reactivity with porcine peripheral blood lymphocytes (PBL), only three of the 57 mAbs assigned to the T cell/activation marker group were grouped into cluster T9 along with the two wCD8 workshop standard mAbs 76-2-11 (CD8a) and 11/295/33 (CD8b). Their placement was verified through the use of two-color cytofluorometry which established that all three mAbs (STH101, #090; UCP1H12-2, #139; and PG164A, #051) bind exclusively to CD8⁺ cells. Moreover, like the CD8 standard mAbs, these three mAbs reacted with two proteins with a MW of 33 and 35 kDa from lymphocyte lysates and were, thus, given the wCD8 designation. Because the mAb STH101 inhibited the binding of mAb 76-2-11 but not of 11/295/33, it was given the wCD8a designation. The reactivity of the other two new mAbs in the T9 cluster with the various subsets of CD8⁺ lymphocytes were distinct from that of the other members in this cluster including the standards. Although the characteristic porcine CD8 staining pattern consisting of CD8^low and CD8^high cells was obtained with the mAb UCP1H12-2, a wider gap between the fluorescence intensity of the CD8^low and CD8^high lymphocytes was observed. In contrast, the mAb PG164A, not only exclusively reacted with CD4⁻/CD8^high lymphocytes, but it also failed to recognize CD4/CD8 double positive lymphocytes. It was concluded that this mAb is specific for a previously unrecognized CD8 epitope, and was, thus, given the wCD8c designation. A very similar reactivity pattern to that of PG164A was observed for two other mAbs (STH106, #094; and SwNL554.1, #009). Although these two mAbs were not originally positioned in the T cell subgroup because of their reactivity and their ability to inhibit the binding of PG164A, they were given the wCD8c designation. Overall, five new wCD8 mAbs were identified. Although the molecular basis for the differences in PBL recognition by these mAbs is not yet understood, they will be important in defining the role of CD8⁺ lymphocyte subsets in health and disease.     

Killing of Brucella antigen-sensitized macrophages by T lymphocytes in bovine brucellosis

The present study was an investigation into the role of T lymphocytes in the killing of antigen-sensitized macrophages (MΦ) in bovine brucellosis. Following confirmation of bovine T lymphocyte cell lines derived from Brucella abortus Strain 19 vaccinated steers as antigen-specific in proliferation studies using various antigens, we adapted an apoptosis assay for evaluation of cytotoxicity by these bovine T cells against autologous monocyte-derived macrophages (MDMΦ) as target cells. Various B. abortus antigen preparations were tested including whole γ-irradiated B. abortus bacteria (γBA), a soluble cytosolic protein fraction and a membrane-associated protein fraction. Both polyclonal and cloned T lymphocyte cell lines exhibited cytotoxicity against MDMΦ targets in an antigen-specific fashion. Polyclonal and cloned T lymphocyte cell lines demonstrated cytotoxic responses to varying degrees against B. abortus antigens regardless of whether the antigen used was whole nonviable bacteria, a soluble protein extract or a membrane-associated fraction of extracted bacteria. To further develop correlation of these responses to an in vivo host defense mechanism, cytotoxicity was evaluated using target cells that had been infected with live B. abortus S19 or B. abortus Strain 2308. Cytotoxic responses were also demonstrated consistently against infected targets with either strain of B. abortus although in most cases, cytotoxicity was higher against target cells sensitized with γBA compared to those infected with live bacteria. Cloned T lymphocyte cell lines were all CD4⁺, CD8⁻ cells indicating that the observed cytotoxic responses were most likely due to an inflammatory T_h1 response and may represent an important host defense mechanism induced by vaccination with live attenuated strains of B. abortus in cattle.     

Effect of a nonsteroidal aromatase inhibitor on in vitro and in vivo secretion of estradiol and on the estrous cycle in ewes.

Three experiments were performed to study effects of decreased concentrations of estradiol-17β (E₂) on lifespan and function of ensuing ovine corpora lutea (CL). In experiment 1, 52 follicles were collected from 10 ewes and placed into individual culture with 0 or .01 μCi ³H-androstenedione (10 ng; ³H-A) and 0, 10⁻¹¹, 10⁻⁹, 10⁻⁷, or 10⁻⁵ M of a nonsteroidal aromatase inhibitor, CGS16949A (CGS). Concentrations of E₂ secreted into the medium, and synthesis of estrogens as estimated by formation of ³H-water from ³H-A were decreased by 10⁻⁵ and 10⁻⁷ (P<.01), but not 10⁻⁹ or 10⁻¹¹ M CGS. In experiment 2, luteolysis was induced in 24 ewes by injection of PGF₂ on days 5 to 10 of the estrous cycle (0 hr). Ewes received 0, 0.5, 1.0, 2.0 or 4.0 mg CGS per kg BW i.v. at −12, 0, 12 and 24 hr, and an ovulatory dose of hCG at 36 hr. Jugular (P<.001) and vena caval (P<.001) concentrations of E₂ were decreased by CGS at all doses tested for 8 to 10 hr, but had returned to levels similar to control ewes by the time of the next injection. Concentrations of E₂ around the time of the LH surge were similar in control and treated ewes. During the subsequent luteal phase, concentrations of progesterone (P₄) were similar in control and treated ewes. Thus, transient decreases in E₂ during the follicular phase were not deleterious to the subsequent luteal phase. In experiment 3, luteolysis was induced in 18 ewes by injection of PGF₂ on days 6 or 7 (0 hr) of the estrous cycle. Ewes received 0 or 1 mg CGS per kg BW i.v. every 8 hr from 0 to 40 hr. Ovulation was induced with hCG at 36 hr. CGS reduced jugular (P<.001) and vena caval (P<.001) concentrations of E₂, prevented an endogenous surge of LH (P<.05) and increased (P<.001) concentrations of FSH. All ewes had ovulated a marked follicle by 72 hr, but onset of the luteal phase, as assessed by concentrations of P₄, was delayed (P<.01) in ewes receiving CGS. Delayed luteal phases were not solely attributable to the presence of new CL or to luteinization of follicular cysts. When data were aligned according to the day ewes were observed in estrus, profiles of P₄ did not differ with treatment. Therefore, normal luteal function ensued following estrus whether or not ewes re-ovulated. In conclusion, decreased secretion of E₂ by the preovulatory follicle was not involved in the ontogeny of CL of short lifespan or subnormal function. Instead, adequate production of E₂ or precisely timed E₂ secretion may be required during follicular development for subsequent functional luteinization.     

The immune response of sheep infected with larvae of the sheep blowfly Lucilia cuprina monitored via efferent lymph

Changes in lymphocyte traffic in efferent lymph from the prescapular lymph node of sheep were monitored during local primary and secondary infection with blowfly, Lucilia cuprina. During primary infections the response was characterised by an increase in the output of CD4⁺ T cells over CD8⁺ T cells for the first 48 h after wound initiation. By 72 h the output of CD8⁺ T cells exceeded that of CD4⁺ T cells. During secondary infections the increased output of CD8⁺ T cells was more pronounced and occurred earlier at approximately 48 h. The percentage of B lymphocytes as measured by sIg, CD45R and MHC class II expression increased at approximately 96–120 h after both primary and secondary infections, with the secondary response being greater than the primary. This increase in B cells corresponded with peak antibody titres recorded in the efferent lymph to a first instar antigen preparation as measured by ELISA. An increase in IFN-γ and soluble IL-2 receptor was recorded after both primary and secondary infections, with the response after secondary infection being greater than that recorded after primary larval infections.     

Quantitative analysis of the antigen-specific IFNgamma+ T cell-mediated immune response in conventional outbred pigs: kinetics and duration of the DNA-induced IFNgamma+ CD8+ T cell response

It is now well established that antigen-specific CD8⁺ T cells play a major role in vaccine-induced immunity against intracellular pathogens and tumor cells. The detection of these immune cells in outbred animals has been hampered mainly by the need to generate individual autologous antigen-presenting cells (APCs) due to the high degree of polymorphism of the major histocompatibility complex (MHC) Class I loci. We used individually derived immature porcine dendritic cells infected with a pox-based recombinant viral vector to ex vivo stimulate PBMCs from vaccinated conventional pigs. The frequencies of antigen-specific T cells was determined by the number of IFNγ-secreting cells in a quantitative enzyme-linked immune spot (ELISPOT) assay. Using this approach we were able to rank different pseudorabies virus (PRV) vaccines strategies for their ability to prime viral-specific IFNγ⁺ T cells. Plasmid DNA has recently emerged as a promising tool with multiple applications in the field of infectious diseases, allergy and cancer. We showed for the first time in this study that DNA immunization induced a long-lived antigen-specific IFNγ⁺ T cells response in conventional pigs. Additional studies allowed us to show that these virus-specific IFNγ⁺ responding cells detected in this ELISPOT assay were MHC-restricted and comprised in the CD8^bright pig T cell subset. These new data confirm the usefulness of DNA vaccines to control diseases requiring cellular immunity in pigs.     

板蓝根多糖对缺乳仔鼠免疫器官发育及T淋巴细胞亚群的影响

试验应用流式细胞术检测缺乳仔鼠CD3⁺、CD4⁺和CD8⁺ T淋巴细胞含量来研究添加不同剂量的板蓝根多糖(RIP)对缺乳仔鼠免疫器官及T淋巴细胞亚群的影响。结果表明:①RIP可增加缺乳仔鼠胸腺指数和脾脏指数。对胸腺指数和脾脏指数效果最好的RIP剂量随日龄增大而减小。②RIP可以增加缺乳仔鼠外周血CD3⁺、 CD4⁺ 、CD8⁺ T淋巴细胞数量。7~28日龄时初乳组(A组)、缺乳+中RIP组(C组)和缺乳+高RIP组(D组)3组CD3⁺ T淋巴细胞含量与缺乳组(B组)相比差异显著(P<0.05)。7~21日龄时初乳组(A组)、缺乳+中RIP组(C组)和缺乳+高RIP组(D组)3组CD4⁺ T淋巴细胞含量与缺乳组(B组)相比差异显著(P<0.05)。各处理组CD3⁺、 CD4⁺、CD8⁺ T淋巴细胞含量及CD4⁺/CD8⁺比值随着日龄的增加有所下降。适宜剂量的RIP可促进缺乳仔鼠免疫器官发育,提高T淋巴细胞亚群的水平。     

Production of interleukin-2 mRNA by bovine lymph node lymphocytes in response to concanavalin A, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin

Interleukin-2 (IL-2) is a lymphokine which, upon binding to its receptor, leads to the proliferation and differentiation of T-cells (helper, suppressor, and cytotoxic) and B-cells. While human and murine IL-2 have been extensively studied, less is known about bovine IL-2. In order to understand the induction of bovine IL-2 at the molecular level, we have examined IL-2 mRNA induction. The dose-responses and time courses of the production of IL-2 mRNA in response to Concanavalin A (ConA), 12-O-tetradecanoylphorbol-13-acetate (TPA), and ionomycin in lymph node lymphocytes (LNC) were determined. We found that high levels of IL-2 mRNA were produced in response to 1 μg ml⁻¹ ConA plus 10⁻⁸ M TPA, but that even higher levels were produced in response to 1 μM ionomycin plus 10⁻⁸ M TPA. We also found that LNC stimulated with ConA displayed two phases of IL-2 mRNA production, one occurring approximately 2–4 h after stimulation and one occurring approximately 10 h after stimulation. However, in the presence of ConA plus TPA or ionomycin plus TPA the response was monophasic. IL-2 mRNA was detected within 2 h of addition of ConA plus TPA (the earliest time examined), reached maximum levels within 6 h, and declined to low levels after 12 h. IL-2 mRNA from LNC incubated with ionomycin plus TPA appeared within 2 h, and reached maximum levels at about 9 h. In contrast to the decrease seen after 12 h with ConA plus TPA, IL-2 mRNA from these cells remained high for 18 h and declined to low levels after 24 h.     

玉屏风多糖对小鼠派氏结形态结构及其T细胞亚群的影响

通过研究玉屏风多糖（YPF-P）对小鼠派氏结（Peyer's patches,PPs）形态结构及其T细胞亚群的影响,探讨其对小鼠肠黏膜的免疫调节作用。选取96只SPF小鼠随机分为6组,分别为空白对照组（0.2 mL生理盐水）、YPF-P阳性对照组（YPF-P 200 mg/kg）、环磷酰胺（cyclophosvnamide,Cy）免疫抑制组（80 mg/kg Cy）及YPF-P低、中、高剂量组（100、200、400 mg/kg YPF-P）,饲喂1周,取小肠PPs,常规切片HE染色后应用图像分析技术检测PPs形态结构的变化;体外培养小鼠PPs淋巴细胞,采用流式细胞术研究PPs中T淋巴细胞亚群的变化。结果表明,YPF-P对小鼠小肠PPs的生长发育具有促进作用,Cy可极显著降低小鼠小肠PPs面积、小肠纵切面面积及PPs面积和小肠纵切面面积的比值（P < 0.01）,低、中、高剂量YPF-P可在一定程度上缓解Cy对小鼠小肠的损伤作用。同时,YPF-P可显著或极显著提高PPs中CD3⁺、CD4⁺ T淋巴细胞及CD4⁺/CD8⁺（P < 0.05; P < 0.01）。结果显示,YPF-P能提高Cy诱导的免疫抑制小鼠的肠黏膜免疫功能,并能促进PPs中相关T淋巴细胞增殖,对小鼠肠黏膜功能具有增强作用。     

Effects of a second mutant allele (V199I) at the PRKAG3 (RN) locus on carcass composition in pigs

The effect of a second mutant allele (V199I, here denoted rn*) at the PRKAG3 (RN) locus on carcass composition was determined in 334 pigs, entire males and females, from crosses between Swedish Hampshire (H) and Finnish Landrace (L) (H × LH; LH × H; LH × LH). Pigs were classified according to DNA test into the following PRKAG3 genotypes: RN⁻/RN⁻ (23%), RN⁻/rn⁺ (24%), RN⁻/rn* (33%), rn⁺/rn⁺ (8%), rn⁺/rn* (9%) and rn*/rn* (2%). The pigs were slaughtered at a commercial slaughterhouse and assessed 24 h postmortem. Right sides were fabricated into primary wholesale cuts, then further processed into defatted hams and loins, and a subset of hams (n = 122) was dissected into the five major individual muscles. The genotype frequencies for the subsample were RN⁻/RN⁻ (27%), RN⁻/rn⁺ (20%), RN⁻/rn* (35%), rn⁺/rn⁺ (9%), rn⁺/rn* (8%) and rn*/rn* (1%). Weights were recorded for meat and bone in ham and loin, fat in ham, back and shoulder and the individual dissected muscles. The genotype effect was significant (P < 0.05) for estimated lean meat content and the proportions of meat and bone and fat in ham and loin (of carcass weight). Also, the content of meat and bone in ham and loin, in proportion of whole ham and loin, respectively, differed significantly (P < 0.01) between genotypes. Estimated lean meat content was highest for RN⁻/RN⁻ (63.0%) and RN⁻/rn⁺ (63.1%) and lowest in the combined group rn*/⁻ (rn⁺/rn* and rn*/rn*, 61.7%); RN⁻/rn* (62.5%) and rn⁺/rn⁺(62.1%) were intermediate. The same results were found for meat and bone in ham and loin, as a proportion of whole ham and loin, respectively. RN⁻/RN⁻ and RN⁻/rn⁺ did not differ in any trait; however, they produced carcasses with the lowest proportions of fat within loin and the major wholesale cuts (ham, loin and shoulder). The carcass percentage of meat and bone in ham was higher in the three RN⁻/ genotypes (RN⁻/RN⁻, RN⁻/rn⁺ and RN⁻/rn*, P < 0.05) than in the rn*/⁻ group, whereas rn⁺/rn⁺ did not (P > 0.05) differ from any of the other genotypes. RN⁻/rn⁺ and RN⁻/rn* had higher (P < 0.05) proportion of meat and bone in loin compared to the rn*/⁻ group. We conclude that the second mutant allele found at the PRKAG3 (RN) locus, rn*, decreased the lean meat content compared with the two other alleles (RN⁻, rn⁺). The RN⁻/RN⁻ and RN⁻/rn⁺ genotypes were leanest, followed by RN⁻/rn* and rn⁺/rn⁺, and rn⁺/rn* and rn*/rn* were the fattest.     

禽网状内皮组织增生病病毒感染对SPF雏鸡血液和局部淋巴组织CD4+/CD8+细胞及相关细胞因子表达的影响

旨在探讨禽网状内皮组织增生病病毒（reticuloendotheliosis virus,REV）对SPF雏鸡血液和局部淋巴组织中T淋巴细胞数量以及相关细胞因子表达的影响。将96只1日龄SPF雏鸡随机分为REV感染组和对照组,应用流式细胞术、酸性α-醋酸萘酯酶染色（acid α-naphthyl acetate esterase,ANAE）、荧光定量PCR等方法对上述指标进行检测。试验数据表明,与对照组相比,感染组雏鸡血液CD4⁺T淋巴细胞数量在第7~35天显著降低、CD8⁺T淋巴细胞数量在第7~28天显著升高,CD4⁺/CD8⁺比值在第7~28天显著降低（均P<0.05或P<0.01）;局部淋巴组织哈德尔腺（Hader’s gland,HG）、派伊尔结（Peyer’s patch,PP）和盲肠扁桃体（caecal tonsil,CT）中ANAE⁺T淋巴细胞数量均显著降低（均P<0.05或P<0.01）;GH、PP和CT中细胞因子IL-2、IFN-γ和TNF-α 转录量都有不同程度升高。本研究表明,REV感染引起雏鸡血液中CD4⁺ T淋巴细胞数量降低、CD4⁺/CD8⁺T淋巴细胞比例失衡、局部淋巴组织中T淋巴细胞数量相对减少及细胞因子TNF-α转录持续升高与REV造成感染雏鸡细胞免疫功能显著降低密切相关。