猪中性粒细胞中抗菌肽的提取及体外抗菌活性研究

抗菌肽是生物体内诱导表达具有广谱抗菌活性的小分子多肽。这些多肽是生物体非专一性的免疫应答产物,是生物体内免疫系统的重要成分。目前国内关于猪源抗菌肽的研究,主要针对于猪的小肠中的抗菌肽,而猪血中抗菌肽的提取及抗菌活性研究在国外虽已有少量报道,在国内却未见报道。由于猪血液中白细胞是免疫系统中起主要作用的免疫细胞,而其中性粒细胞约占白细胞总量的70%,从猪血液中性粒细胞中分离提取抗菌肽也就比较合理。因此本实验采用从猪血液中性粒细胞中分离提取小分子多肽,根据小分子多肽抗菌活性特点进而分离鉴定出抗菌多肽。同时,通过优化提取工艺,提高抗菌肽的得率和含量。     

育成猪免疫学指标的测定

6头健康长大杂交猪 ,体重 (2 5 .5± 1.4 )kg ,用于研究 7～ 11周龄猪免疫学特征。于 7、8、9、10、11周龄时 ,自前腔静脉采血 ,用显微镜法计数红、白细胞总数 ,经姬姆萨染色后作白细胞分类计数 ,用硝基蓝四氮唑(NBT)还原试验定量检测嗜中性白细胞吞噬功能 ,噻唑蓝 (MTT)比色法检测外周血中淋巴细胞转化 ,单向免疫扩散法测定血清IgG浓度 ,5 0 %溶血试验测定血清中补体总活性 ,同时 ,用植物血凝素 (PHA)皮肤试验检测迟发型超敏 (DTH)反应。结果明示 ,7～ 11周龄猪免疫反应差异不显著 (P >0 .0 5 ) ,即猪免疫系统已发育完全 ,适于用作免疫学研究的动物模型     

中草药制剂对泌乳奶牛机体免疫性能的影响

试验采用大青叶、大黄、党参等19味中草药分别组成内服和子宫灌注制剂治疗奶牛子宫内膜炎，并采集颈静脉血测定淋巴细胞刺激指数（SI）和中性粒细胞吞噬力，以观察中草药制剂对奶牛免疫性能的影响。结果显示，中草药制剂均可提高奶牛免疫力，内服中草药制剂应用效果优于子宫灌注制剂（P〈0．05）；复方制剂优于单味中草药制剂（P〈0．05）；复方中草药内服制剂可使淋巴细胞刺激指数（SI）和中性粒细胞吞噬力分别提高53．76％和4．58％（P〈0．01）；复方子宫灌注制剂可使淋巴细胞刺激指数（SI）和中性粒细胞吞噬力分别提高39．58％和1．98％（P〈0．01）。     

犬瘟热患犬中性粒细胞cAMP PDE活性变化及中药作用研究

研究犬瘟热患犬中性粒细胞(PMN)中环磷酸腺苷-磷酸二酯酶（cAMP-PDE）的活性变化及中药对其活性的体外调节作用，为抗炎药研究和病毒性炎症的中药治疗积累资料。提取犬瘟热患犬（n=9）和正常犬(n=6)的中性粒细胞制备cAMP-PDE样品，通过高效液相色谱(HPLC)法测定其活性及中药对其活性的影响。结果显示，犬瘟热患犬组中性粒细〖JP2〗胞cAMP-PDE活性（18.55%±2.66%）显著（P<0.05）高于正常组（11.54%±5.16%）。解表药等5类114味中药中芦根（77.27%）、胡黄连（70.41%）、女贞子（65.50%）、香薷（65.16%）、贝母（61.43%）、黄芩（60.67%）、淫羊藿（60.34%）、黄芪（59.13%）、青蒿（56.93%）、秦皮（55.55%）、辛夷（55.03%）、甘草（54.67%）、牛蒡子（53.40%）、麻黄（53.32%）14味对cAMP-PDE活性具有明显抑制作用。结果表明，中性粒细胞cAMP-PDE活性在犬瘟热患犬炎症过程中具有重要作用，对cAMP-PDE活性有明显抑制作用的中药对其炎症可能具有疗效，同时，中药抑制中性粒细胞中cAMP-PDE活性可能是其抗炎作用机理之一。     

Histopathological changes in canine allergic contact dermatitis patch test reactions. A study on spontaneously hypersensitive dogs

The histopathology of allergic patch test reactions in dogs with spontaneous allergic contact dermatitis (ACD) was investigated. Epidermal necrosis was present in half of the biopsies and neutrophilic granulocytes were present within epidermis in these biopsies as well as in some of the biopsies without necrosis. Spongiosis was only occasionally observed. Dermal infiltration with neutrophilic granulocytes and oedema was found in all biopsies while the number of lymphocytes was small. The results may indicate a different secretion of or responsiveness towards cytokines in canine ACD compared to ACD in man where epidermal necrosis is not a characteristic feature and where the predominant inflammatory cell is the lymphocyte. It is anticipated that histopathological investigation of patch test reactions in dogs will not be helpful in discriminating between allergic and toxic patch test reactions.     

Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 10⁴ clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.     

Detection of calprotectin and its correlation to the accumulation of neutrophils within equine large colon during ischaemia and reperfusion

REASON FOR PERFORMING STUDY: The cytosolic protein complex, calprotectin, is abundant in neutrophils and could be used to improve the ability to localise and assess neutrophil infiltration in the equine intestine during ischaemia and reperfusion (I/R), but further study is required. OBJECTIVES: To assess the number of calprotectin-containing cells by immunohistochemistry in correlation with direct counting and scoring of neutrophils in the equine colon during I/R. METHODS: One and 2 h ischaemia of the left dorsal colon were induced, followed by 30 min reperfusion under general anaesthesia or by 18 h reperfusion after anaesthetic recovery. Biopsies were processed for light microscopy and stained with H/E for detection of neutrophils. To identify calprotectin-containing cells, immunohistochemistry was performed on formalin-fixed tissues with the murine MAC 387 antibody and a biotin-free peroxidase staining procedure. The number of neutrophils within submucosal venules and the colonic mucosa were calculated and compared with the number of calprotectin-positive cells. RESULTS: The number of calprotectin-positive cells within submucosal venules and within the colonic mucosa correlated significantly with the accumulation of neutrophils within the corresponding tissue segments. Within the submucosal venules, both calprotectin-positive cells and H/E-stained neutrophils increased with duration of ischaemia and peaked after 30 min of reperfusion. After 18 h reperfusion the number of these cells declined within the vessels. After 2 h ischaemia, neutrophils started to migrate into the mucosa towards the epithelium, with a significant increase over time during reperfusion, and peak infiltration after 18 h reperfusion. CONCLUSIONS: Neutrophil infiltration into the colon after I/R is a time-dependent process, involving migration through the submucosa towards the epithelium.     

Evaluation of biomarkers of kidney injury following 4% succinylated gelatin and 6% hydroxyethyl starch 130/0.4 administration in a canine hemorrhagic shock model

Objective : To investigate the association between synthetic colloids and biomarkers of acute kidney injury (AKI) in dogs with hemorrhagic shock. Design : Experimental interventional study. Setting : University. Animals : Twenty‐four healthy ex‐racing Greyhounds. Interventions : Anesthetized Greyhounds subjected to hemorrhage for 60 min were resuscitated with 20 mL/kg of fresh whole blood (FWB), 6% hydroxyethyl starch (HES) 130/0.4, 4% succinylated gelatin (GELO), or 80 mL/kg of isotonic crystalloid (CRYST) over 20 min (n = 6 per treatment). Concentrations of biomarkers of AKI were measured at baseline, end of hemorrhage, and at 40 (T60), 100 (T120), and 160 (T180) min after fluid bolus. Biomarkers included neutrophil gelatinase‐associated lipocalin in urine and serum (uNGAL; sNGAL), and urine cystatin C (uCYSC), kidney injury molecule‐1 (uKIM), clusterin (uCLUST), osteopontin, gamma‐glutamyl transferase, monocyte chemoattractant protein‐1 (uMCP), interleukin‐6, interleukin‐8, protein (uPROT), hyaluronan, and F₂‐isoprostanes. Renal histology was scored for tubular injury and microvesiculation. Biomarker fold‐change from baseline was compared between groups using mixed effects models (Bonferroni–Holm corrected P<0.05). Frequencies of histology scores were compared by Fisher's exact test. Measurements and main results : In dogs treated with GELO, uNGAL fold‐change was markedly greater compared with all other groups at T60, T120, and T180 (all P<0.001), and uCYSC was greater at T60 compared with CRYST (P<0.001), and at T120 and T180 compared with all other groups (all P<0.001). Smaller, albeit significant, between‐group differences in uKIM, uCLUST, uMCP, and urine protein concentration were observed across the FWB, GELO, and HES groups, compared with CRYST. The GELO group more frequently had marked tubular microvesiculation than the other groups (P = 0.015) although tubular injury scores were comparable. Conclusion : In dogs with hemorrhagic shock, GELO was associated with greater magnitude increases in urine biomarkers of AKI and more frequent marked tubular microvesiculation, compared with FWB, CRYST, and HES.