穿孔素的研究进展

穿孔素（PFP）是主要由CTL和NK细胞产生的一种糖蛋白，通过在靶细胞膜上形成活性孔道使靶细胞渗透压改变而溶解，或者与颗粒酶协同作用而主秀导靶细胞凋亡，生物学功能以及其在抗感染免疫，免疫应答，疾病（如自身免疫疫病，肿瘤）和移植排斥反应等中所发挥的作用作一综述。     

热休克蛋白70在细胞凋亡途径中的作用研究进展

细胞凋亡或程序性细胞死亡是发育和衰老必不可少的程序。细胞凋亡受到促凋亡因子和抗凋亡因子的调控,而细胞凋亡的失控可导致许多疾病的发生。热休克蛋白(HSPs)是原核生物和真核生物中一种高度保守蛋白,HSP70是HSP家族普通的一员。HSP70通过多种方式干扰凋亡机制防止细胞凋亡。细胞凋亡的机制复杂,主要的凋亡途径包括外在(死亡受体)途径和内在(线粒体)途径、穿孔素/颗粒酶途径、内质网途径和溶酶体途径。本文综述了HSP70对细胞凋亡不同途径的研究进展。     

DEC205免疫应答机制及其分子疫苗的研究进展

DEC205是一种能够呈递抗原并介导树突状细胞内吞作用的受体。在免疫机制方面,DEC205具有抗原识别性好、抗原呈递速率高、参与细胞信号传递、调控免疫功能等作用。由于其在介导宿主抗肿瘤及在细菌、病毒和寄生虫免疫方面表现出的免疫调控功能及疫苗增强效果被免疫学、医学和兽医学等领域研究者所关注。文章综述了DEC205受体在体内分布情况、免疫应答机制及其在动物源性病原疫苗实验室试验及临床中的相关应用,展望了其未来在分子疫苗研发方面的应用前景,以期为相关的免疫学研究及生物制品的研发提供理论方面的借鉴。     

多胺与细胞凋亡

多胺与细胞生长及凋亡有密切的关系。多胺与细胞凋亡关系的研究将为细胞生长及抑癌研究提供科学的基础资料。文章主要从多胺的合成及分解 ,腐胺、精胺、ODC和 SSAT在细胞凋亡过程中的作用及 Caspase-3、P53、MAPK /JNK和原癌基因等参于多胺介导的细胞凋亡的调节机理等方面 ,对多胺与细胞凋亡间的关系进行了较为全面的介绍     

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。     

NOD-1在介导固有免疫中的作用

机体对微生物入侵的免疫炎症应答过程中模式识别受体(pattern recognition receptors,PRRs)是启动机体天然免疫应答机制的关键,主要在获得性免疫系统被活化之前发挥抗感染作用。NODl作为一种新的胞内识别受体参与了细胞凋亡和核因子NF-KB的活化,并与一些炎症性疾病密切相关,在天然免疫中发挥了重要的作用。     

锌与动物免疫细胞凋亡关系的研究进展

锌是机体必需的微量元素,它对免疫功能有很大影响,其在维持免疫系统的正常发育和功能方面有重要作用.锌与免疫系统的多种功能有关,对免疫系统的发育、稳定、调节有重要作用.缺锌将导致免疫器官萎缩、免疫细胞减少和抗体水平下降.研究表明,低锌能诱导淋巴细胞凋亡,而高锌抑制淋巴细胞凋亡;当锌浓度超过一定水平时,可产生神经毒性而诱导细胞凋亡.锌对细胞凋亡调控的可能机制和途径的研究有待进一步的研究.     

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。     

黄芪多糖和香菇多糖对雏鸡MΦ活性与IL-1体外诱生活性的影响

诸多研究表明,黄芪多糖(APS)和香菇多糖(LNT)均具有免疫促进作用,并在抗病毒、抗肿瘤方面日益受到重视,但两种多糖本身对靶细胞没有直接细胞毒作用,而是通过宿主介导发挥其抗感染和抗肿瘤作用[1-3].为了探索宿主介导的两种多糖免疫促进作用的生理学机理,特进行了此项研究.     

牛支原体致病机制研究进展

牛支原体是引起牛呼吸系统疾病的主要病原之一,给养牛业造成了严重的经济损失。论文对近期国内外关于其致病机制的研究进展进行梳理,以期为牛支原体肺炎的防控提供参考。对牛支原体致病机制的研究可为研制有效的疫苗和新型绿色药物及其他防控途径提供理论依据,进而在临床上有效控制牛支原体引发的疾病。论文涉及三方面内容:一是牛支原体黏附蛋白的黏附作用;二是牛支原体在黏附基础上其代谢产物对宿主细胞的损伤和相关蛋白介导的宿主细胞凋亡;三是免疫学致病机制,包括黏附蛋白的免疫原性及免疫逃避机制、宿主的免疫应答及炎症损伤和宿主的抗炎反应及免疫抑制机制。     

Perforin and granzymes in neurological infections: From humans to cattle

Perforin and granzymes are essential components of the cytotoxic granules present in cytotoxic T lymphocytes and natural killer cells. These proteins play a crucial role in a variety of conditions, including viral infections, tumor immune surveillance, and tissue rejection. Besides their beneficial effect in most of these situations, perforin and granzymes have also been associated with tissue damage and immune diseases. Moreover, it has been reported that perforin and granzymes released during viral infections could contribute to the pathogenesis of diseases. In this review, we summarize the information available on human perforin and granzymes and their relationship with neurological infections and immune disorders. Furthermore, we compare this information with that available for bovine and present data on perforin and granzymes expression in cattle infected with bovine alphaherpesvirus types1 and -5. To our knowledge, this is the first review analyzing the impact of perforin and granzymes on neurological infections caused by bovine herpesviruses.     

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3⁺CD5⁺CD6⁺ T-cells (−40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8 and CD8β lymphocytes, but was not expressed in γδ T-cells or monocyte/macrophages. The perforin positive CD3⁻ subset was phenotypically homogeneous and defined as CD5⁻CD6⁻CD8β⁻CD16⁺CD11b⁺. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3⁺) could be divided into at least three subsets. The first subset was CD4⁻CD5⁺CD6⁺CD11b⁻CD16⁻ most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8β. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4⁺CD5⁺CD6⁺CD8β⁺CD16⁻CD11b⁻ and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3⁺CD4⁻CD5⁺CD6⁻CD8β^±CD11b⁺CD16⁺, and possessed MHC-unrestricted cytotoxicity and LAK activity.     

Sheep perforin: identification and expression by gammadelta T cells from pregnant sheep uterine epithelium.

GammadeltaTCR-positive intraepithelial lymphocytes (IEL) in sheep uterus have been shown in previous studies to express CD8 and to contain prominent intracytoplasmic granules, indicating that they may be cytotoxic. Sheep perforin, which has not previously been described, was identified in the present study using RT-PCR based on primers from human and mouse perforin sequences. A 290 base pair (bp), partial sheep perforin sequence was obtained which showed 80.3% and 66.2% nucleotide identity with human and mouse perforin, respectively. GammadeltaTCR+ IEL from sheep pregnant uteri were found to express perforin mRNA providing further evidence that these cells are cytotoxic.     

Detection of intracellular antigens in porcine PBMC by flow cytometry: A comparison of fixation and permeabilisation reagents

The staining of intracellular antigens is a standard method in flow cytometry but still only occasionally used for immunologic studies in veterinary species. In order to improve information about suitable fixation/permeabilisation protocols in porcine lymphocytes we tested six fixation/permeabilisation variants for the staining of different intracellular antigens: CD79alpha, perforin, interferon-gamma and the cell cycle associated protein Ki-67. For the fixation/permeabilisation procedure, four commercial kits from BD Biosciences, ADG Bio Research, Dako and AbD Serotec were tested in comparison to non-commercial fixation solutions of formaldehyde together with either saponin or Tween-20 for permeabilisation. Perforin and Ki-67 expressing cells were optimally stained only with permeabilisation reagents containing saponin, which includes the BD kit. In contrast, labelling of CD79alpha and IFN-gamma was possible with all investigated fixation/permeabilisation variants; however, here saponin and Tween protocols induced a higher degree of unspecific binding. Also, scatter properties of cells treated with saponin were worse compared to samples prepared with the kits from ADG, Dako and Serotec. Simultaneous staining of cell surface antigens was not negatively affected by any of the fixation/permeabilisation variants. A universal recommendation for a single fixation/permeabilisation strategy could not be deduced from our data but our work provides a useful guideline for optimal staining of common intracellular antigens analyzed in swine lymphocytes.     

Granzyme B-mRNA expression by equine lymphokine activated killer (LAK) cells is associated with the induction of apoptosis in target cells

Lymphokine-activated killer (LAK) cells are a subset of cytotoxic cells capable of lysing freshly isolated tumor cells. While LAK activity is typically measured using the (51)Cr-release assay, here we used a non-radioactive flow cytometric method to demonstrate equine LAK activity. Equine peripheral blood mononuclear cells (PBMC) were stimulated in vitro with recombinant human interleukin 2 (hIL-2) to generate LAK cells. An equine tumor cell line, EqT8888, labeled with carboxyfluorescein succinimidyl ester (CFSE) was used as target cells. Following incubation of the targets with different concentrations of LAK cells, Annexin V was added to identify the early apoptotic cells. With increasing effector to target cell ratios, EqT8888 apoptosis was increased. We also measured interferon-gamma, granzyme B and perforin mRNA expression in the LAK cell cultures as possible surrogate markers for cytotoxic cell activity and found granzyme B mRNA expression correlated best with LAK activity. Also, we found that the reduced LAK activity of young horses was associated with decreased granzyme B mRNA expression. Our results indicate that fluorescence-based detection of LAK cell activity provides a suitable non-radioactive alternative to (51)Cr-release assays and mRNA expression of granzyme B can be used as surrogate marker for these cytotoxic cells.     

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.     

Structure and sequence variation of the canine perforin gene

Lymphocyte-mediated cytotoxicity is essential to control viral infections, limit lymphocyte expansion and activation, and survey for malignant cells. Humans with defects in lymphocyte cytotoxicity have reduced perforin function resulting in uncontrolled lymphocyte expansion, leading to excessive histiocyte activation and a hemophagocytic disorder. Dog breeds such as Bernese mountain dogs (BMD) have a high incidence of reactive and malignant diseases affecting histiocytes. This study addressed the hypothesis that changes in the perforin gene contribute to the development of hemophagocytic histiocytic sarcoma (HHS) in BMD. Canine perforin DNA was amplified and sequenced through multiple PCR assays from healthy and diseased dogs, and the gene structure determined by rapid amplification of cDNA ends. The coding component of the gene consists of 1679 bp, with two exons of 536 bp and 1143 bp separated by an intron of 865 bp. Gene configuration and location differ from that in other species although the coding sequence is highly conserved. Three silent single nucleotide polymorphisms (SNP) were identified. Analysis of their distribution indicated a consistent genotype among 6 middle-aged to older BMD without histiocytic diseases. Among samples from 10 dogs with HHS and 10 without histiocytic diseases SNP occurred with variable frequency. It was concluded that changes in the amino acid sequence of perforin were not associated with HHS but that a constellation of SNP may characterize BMD without histiocytic disease. Investigation of more dogs is required to confirm a specific genotype. Future studies should focus on the potential contribution of reduced perforin expression and/or function to HHS in dogs.     

Cell death of uterine natural killer cells in murine placenta during placentation and preterm periods

In the murine uterus granulated metrial gland (GMG) cells appear only during normal pregnancy. GMG cells belong to a member of natural killer (NK) cells and play an important role in fetus survival and placental growth. Our previous study revealed that mouse GMG/uterine NK (uNK) cells in the late pregnancy rapidly disappear from the uterus, due to the degenerative change classified as necrosis. But there are few reports regarding appearance and morphology of uNK cells during late pregnancy. We examined histologically and histochemically how and when uNK cells undergo cell death. The uNK cells in the metrial gland increased in number and reached maximum until day 12 of pregnancy. Sudden disappearance, however, occurred after day 15 and the granules reduced in both number and size. In situ DNA fragmentation detection revealed that DNA fragmented uNK cells increased in number during days 13 to 15 and reached 70.2% at day 15 of pregnancy. From days 13 to 17, uNK cells were positive against anti-perforin antibody. Ultrastructurally, uNK cells at day 15 showed poor organelles and unusual granules in structure. In uNK cells at day 17, condensation of nucleus chromatin, reduction in size and phagocytosis into other uNK cells were observed. These results suggested that uNK cells undergo at least two types of cell death, classified as necrosis and apoptosis, at the different stages of pregnancy, and that perforin is not a mediator for cell death.     

Characterization of age-related changes in bovine CD8+ T-cells

Evaluation of the changes induced by immunological interventions requires a baseline against which to compare those changes. The age-related changes in the CD8(+) T-cell population of cattle were studied. The results indicate that CD8(+) T-cells could be divided into γ/δ TCR1(+) and γ/δ TCR1(-) according to their expression of the γ/δ T-cell receptor. As a proportion, the CD8(+) γ/δ TCR1(+) population appears to increase with age. Within the CD8(+)γ/δ TCR1(-) a population of cells expressing a profile of surface molecules previously associated with effector memory T cells (CD45RO(+), CD62L(-), CD27(-), CD45RA(-) and CD28(-)) increases with age. Furthermore, a parallel increase with age in the proportion of CD8(+)CD45RO(+) T cells that express the cytotoxic granule protein perforin was observed. In peripheral tissues, namely lungs, it was found that the majority of CD8(+) T cells present expressed a phenotype indicative of previously primed T cells (high expression of CD45RO and perforin). In contrast, only a small population of memory CD8(+) T cells was present in lymphoid tissue where most of the CD8(+) T cells expressed a na?ve phenotype. In conclusion, in cattle, like in human, CD8(+) T cells that express a phenotype associated with antigen experience accumulate with age that may play a role in immunocompetence as the individual ages.     

Tumor necrosis factor-alpha induces apoptosis in cultured porcine luteal cells

Some studies in mammalian species recently demonstrated that tumor necrosis factor (TNF)-alpha plays an important role for corpus luteum (CL) function by way of apoptosis during the estrous cycle. The objectives of this study were to clarify the induction of apoptosis in cultured porcine luteal cells by TNF-alpha treatment. Luteal cells prepared from porcine ovaries collected from crossbred mature gilts on Days 10-14 of the estrous cycle were isolated and examined as follows: 1) Flow cytometric analysis was carried out to determine apoptosis in cultured luteal cells. 2) Single-cell gel electrophoresis (comet assay) was performed to investigate apoptotic DNA fragmentation in luteal cells. The results of the flow cytometric analysis and comet assay demonstrated coincidentally that TNF-alpha induces DNA fragmentation in luteal cells causing apoptosis. These results revealed that TNF-alpha is an inducing factor of apoptosis in luteal cells.