Neutrophil Phagocytosis Following Inoculation of Salmonella choleraesuis into Swine

Neutrophils are an important mediator of host defence, especially in early stages of infection. A major function of neutrophils is the uptake and killing of invading microbes. Little is known about the effect of neutrophil activity on the pathogenesis and development of the carrier state in swine following infection with Salmonella choleraesuis. A human whole-blood microassay using flow cytometry was modified to measure the effect of S. choleraesuis infection in vivo on the rate of ingestion, or rate of uptake, of homologous bacteria by porcine neutrophils. Pigs were inoculated intranasally with 5–8×10⁸ CFU S. choleraesuis and blood was collected in heparinized tubes at –5, 0, 1, 2, 3 and 4 days post inoculation (PI). Heat-killed S. choleraesuis were labelled with fluorescein isothiocyanate and incubated for various times with diluted whole blood. Red blood cells were lysed, external non-phagocytized bacteria were quenched with a commercially available lysing solution, and fluorescence from internalized bacteria labelled with fluorescein isothiocyanate was detected by flow cytometry. The rate of uptake by neutrophils did not increase until 2 days PI and then remained elevated to 4 days PI. The minimal uptake of S. choleraesuis early after exposure to these organisms may provide an opportunity for the pathogen to colonize and/or replicate to levels that facilitate establishment of a carrier state or clinical infection in swine.     

Neutrophil adherence,phagocytic-nitroblue tetrazolium reduction and chemiluminescence in canine whole blood

Methods for measuring neutrophil adherence, phagocytic-nitroblue tetrazolium (NBT) reducing activity and chemiluminescence were applied to canine whole blood as means for routine assessment of neutrophil functions. The phagocytic-NBT reduction test appeared to be useful for monitoring the NBT reducing activity of phagocytic cells associated with phagocytic functions. Ethylene diamine tetraacetic acid suppressed both the adherence and the phagocytic-NBT reducing activity of neutrophils. Increased phagocytic-NBT reduction and an enhanced chemiluminescence response were observed in dogs with neutrophilia. These methods provide a rapid and practical screening procedure for measuring selected phagocytic functions in canine whole blood.     

β-羟丁酸抑制脂多糖诱导奶牛中性粒细胞核因子-κB信号通路的激活

高酮血症造成奶牛中性粒细胞先天免疫机能受到抑制,本研究探讨β-羟丁酸(BHBA)是否抑制脂多糖(LPS)诱导的奶牛中性粒细胞核因子-κB(NF-κB)信号通路的激活。分离健康奶牛中性粒细胞,采用LPS(100 ng/mL)和不同浓度(0.5、1.0、2.0和4.0 mmol/L)BHBA作用于中性粒细胞,收集细胞,应用实时荧光定量PCR(qRT-PCR)检测中性粒细胞中白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)和NF-κBp65 mRNA表达水平,Western blot检测NF-κBp65蛋白表达水平,比色法检测核因子-κB抑制物激酶β(IKKβ)激酶活性,酶联免疫吸附试验(ELISA)法检测促炎细胞因子TNF-α、IL-6和IL-1β的分泌量。结果表明：与对照组(不进行BHBA和LPS处理)相比较,LPS组(单独LPS处理)中IL-1β、IL-6、TNF-α和NF-κBp65 mRNA表达水平和NF-κBp65蛋白表达水平极显著增加(P <0.01),IKKβ激酶活性极显著增强(P<0.01),IL-1β和TNF-α的分泌...     

Intramammary infusion of an Enterococcus faecium SF68 preparation promoted the involution of drying off Holstein cows partly related to neutrophil‐associated matrix metalloproteinase 9

A problem for dairy cows following milk stasis is to cope with a high risk of intramammary infection and there is a need to initiate an extensive renewal of secretory modules in mammary glands so that milk production in next lactation may be optimized. We recently reported that ultrasonicated Enterococcus faecium SF68 (SF68) is compatible with cow mammary glands and an enhancer of innate immunity during the immediate post‐milk stasis period. The current study further examines the concomitant effect of ultrasonicated SF68 on mammary tissue remodeling. Four Holstein cows each received intramammary infusions of regular antibiotic dry‐cow formula (positive control) and two different doses of SF68 in different quarters. Analyses of individual quarter secretion samples showed faster neutrophil infiltration, earlier modifications in protein composition, including caseins and lactoferrins, as well as more prompt elevation of the specific unit of 92‐kDa matrix metalloproteinase 9 (MMP9) in SF68‐infused quarters compared to the positive controls. Intramammary infusion of ultrasonicated SF68 seems able to accelerate the regression of mammary synthetic capacity and potentiate the breakdown of glandular extracellular matrix, indicating a more efficient mammary gland involution. Correlation analyses imply that the ability of ultrasonicated SF68 to induce faster neutrophil chemotaxis and the associated MMP9 release is partly responsible.     

不同中药对中性粒细胞迁移微血管内皮细胞后溶菌酶释放量的影响

试验旨在探讨脂多糖（LPS）刺激大鼠肠黏膜微血管内皮细胞（RIMVECs）对跨内皮迁移中性粒细胞（PMNs）溶菌酶释放量的影响及白头翁、黄连、黄柏、秦皮、马齿苋的干预作用。选取0、0.1、1、10、100 μg/mL LPS刺激RIMVECs,采用ELISA法检测12和24 h细胞培养上清液中IL-6水平以确定后续试验所需LPS浓度。建立PMNs跨RIMVECs黏附模型和迁移的Transwell模型,采用细胞计数法、染色法、ELISA方法评价5种中药对LPS刺激RIMVECs后PMNs的黏附情况、迁移率及其溶菌酶释放量的影响。结果显示,与对照组相比,10 μg/mL LPS作用RIMVECs后24 h时的IL-6水平极显著升高（P<0.001）;与LPS组相比,黄连、白头翁组的PMNs黏附率显著降低（P<0.05）,溶菌酶释放量极显著升高（P<0.001）,秦皮、白头翁组迁移率显著降低（P<0.05）。提示LPS刺激内皮细胞显著抑制了PMNs杀菌酶的释放,中药黄连、白头翁可以显著减轻这种抑制作用,PMNs杀菌酶的释放可能与内皮细胞的功能完整性有关,本试验为进一步筛选有效中药水溶性成分提供了理论依据。     

Single oral β-cryptoxanthin administration increases its serum concentration and enhances peripheral blood neutrophil function in Holstein cattle

We investigated the effect of oral administration of β-cryptoxanthin (β-CRX) on its serum concentration and peripheral neutrophil functions by the chemiluminescence (CL) response in Holstein cattle. A single oral administration of β-CRX was performed for serum β-CRX concentration (0, 0.05, 0.1, or 0.2 mg/kg body weight [BW]) and for peak CL response of peripheral neutrophils (0.2 mg/kg BW). The serum β-CRX concentration was peaked on 2 days after, similar to peak CL response on 3 days after β-CRX administration. Therefore, a single oral administration of β-CRX (0.2 mg/kg BW) induces higher serum concentration and concurrently enhances bactericidal ability of peripheral neutrophils in Holstein cattle.     

Immunoexpression of cytokine tumour necrosis factor-α suggesting its role in formation and regression of corpus luteum in Indian buffalo

Tumour necrosis factor-α (TNF-α) is a cytokine that plays multiple important roles in corpus luteum (CL). Immunolocalization of expression of TNF-α in CL of buffalo was studied in different stages of its development and regression. Corpus luteum of healthy buffaloes (24) was collected from local slaughterhouses and categorized into early (stage I, 1–5 days, n = 6), mid (stage II, 6–11 days, n = 6), late luteal (stage III, 12–16 days, n = 6) and regressing phase (stage IV, 17–20 days, n = 6). In earliest phase of cyclic CL, per cent immunoexpression of TNF-α was significantly (p < .05) lower as compared to all phases with its expression being restricted to few developing luteal cells, usually in neutrophils. A significantly (p < .05) higher number of neutrophils with TNF-α immunoexpression were observed as compared to mid-luteal phase that indicated its role in initiation of angiogenesis at this stage. TNF-α immunoexpression almost doubled in mid-luteal phase, but the number of neutrophils exhibiting TNF-α was significantly (p < .05) lower with respect to all phases of CL. Immunoexpression percentage in late luteal phase increased sharply being significantly (p < .05) higher than earlier two phases of CL. In regressing phase, per cent immunostaining was maximum with highly significant (p < .05) difference as compared to all other stages, observed in all degrading luteal cells, abundant immune cells, that is neutrophils and macrophages which finally led to apoptosis and phagocytosis. Immunoexpression of TNF-α in early luteal phases served its role in initiation of angiogenesis, and its intense expression in regressing phase of CL suggested a shift in its role to apoptosis and structural luteal regression signifying both luteotropic and luteolytic roles in buffalo. This is probably the first study of its kind in buffaloes.