Optimization and validation of a flow cytometric method for immunophenotyping peripheral blood lymphocytes from cynomolgus monkeys (Macaca fascicularis)

BACKGROUND: Guidelines published by the Food and Drug Administration and Center for Human Medicinal Products describe the need to assess immunotoxic effects in nonclinical studies that evaluate drug toxicity, including the use of immunophenotyping to measure immunotoxicity. We are not aware of previous studies, however, that have validated methods for immunophenotyping peripheral blood lymphocyte subsets in whole blood samples from cynomolgus monkeys. OBJECTIVE: The purpose of this study was to optimize and validate a flow cytometric assay for immunophenotyping lymphocytes in the peripheral blood of cynomolgus monkeys. METHODS: A series of prevalidation experiments were done to determine optimal reagents, volumes, timing, and other procedural details of the flow cytometric assay. Using the optimized method, we then determined precision, interindividual variation, laboratory-to-laboratory variability, and sample stability. Stabilized human blood was used as a positive control for staining, processing, and analysis. The percentage and number of pan-T cells (CD3+), T-helper cells (CD3+4+), T cytotoxic/suppressor cells (CD3+8+), natural killer cells (CD3-16+), and B-cells (CD3-20+) were determined in 146 male and 140 female, clinically healthy monkeys and reference intervals were calculated. RESULTS: By doing 4-color staining with a lyse-wash method, intra- and interassay precision were <5% for all lymphocyte subsets. Variability between technicians and laboratories was minimal (CVs<3%). Samples were stable for up to 24 hours after staining and fixing. CONCLUSIONS: The validated method is extremely robust and can be performed under good laboratory practice conditions to support nonclinical studies. Reference intervals for lymphocyte subsets were similar to those previously reported.     

兰州地区胡麻田杂草消长动态及群落生态位研究

采用田间调查和室内测定、相对丰度和生态位计算与分析相结合的方法,对兰州地区胡麻田杂草群落进行了研究。初步明确了兰州地区胡麻田间杂草种类共有11科23种,其中主要科为:禾本科、菊科、藜科、苋科和旋花科;优势种群为:地肤、狗尾草、藜、苣荬菜、稗草和打碗花。同时明确了优势杂草的消长变化规律,即4月中旬为杂草始发期,5月中旬和6月中旬为2个出草高峰期。优势杂草生态位的研究结果表明,地肤的综合生态位宽度值最大,对胡麻的危害最大,其次为狗尾草和藜;地肤与狗尾草的时间生态位重叠值最大,与苣荬菜的水平生态位重叠值最大;而苣荬菜和藜的垂直生态位重叠值最大,它们相互之间利用资源的相似性较高。利用相对丰度和生态位宽度均能确定优势杂草的种类,反映杂草对作物危害程度的大小。     

DOPPLER MEASUREMENT OF SPLANCHNIC BLOOD FLOW DURING DIGESTION IN UNSEDATED NORMAL DOGS

The purpose of this study was to measure splanchnic blood flow during digestion in unsedated dogs by using duplex Doppler sonography. The study population consisted of 12 healthy dogs. Blood flow in the cranial mesenteric artery, the celiac artery, and the aorta was measured before a test meal and at 20, 60, and 90 minutes after eating. The following measurements were made or calculated: vessel diameter, peak systolic velocity, end diastolic velocity, mean velocity, resistive index, pulsatility index, and flow volume. There was a significant postprandial decrease in the resistive and pulsatility indices in both the cranial mesenteric (preprandial RI = 0.867, postprandial RI = 0.796, preprandial PI = 3.033, postprandial PI = 2.173) and the celiac (preprandial RI = 0.854, postprandial RI = 0.769, preprandial PI = 2.639, postprandial PI = 1.930) arteries. In both vessels the end diastolic velocity, the mean velocity, and the flow volume increased significantly postprandially. These changes occurred significantly earlier in the celiac artery than in the cranial mesenteric artery. The findings most likely correspond to postprandial splanchnic vasodilation. Doppler ultrasound provide a good methode of detecting changes in postprandial splanchnic blood flow in the dog.     

兰州百合同源四倍体诱导及其基因组大小估算

兰州百合(2n=2x=24)是重要的“药食同源”植物,进行其种质创新和基因组大小分析对于丰富其种质资源和生物学信息具有重要意义。以兰州百合的种子和试管小鳞茎为试验材料,通过秋水仙素浸泡诱导染色体加倍,经流式细胞仪倍性检测获得的同源四倍体植株分别有10和4株,四倍体诱导率分别为21.11%和33.27%。为了估算兰州百合基因组大小,以小麦品种“中国春”为内标,通过流式细胞仪测算兰州百合二倍体和四倍体植株中的DNA含量,得出兰州百合基因组为64.9 Gbp,是“中国春”核基因组的4倍。     

基于玉米种植的生长育肥猪粪污农田承载力

本研究旨在以生长育肥猪和玉米种植农田为模型,准确评定生长育肥猪粪肥养分的产生量和不同施肥量下玉米的生物产量及养分需求量,以此为基础建立基于粪肥还田利用的玉米种植农田粪肥承载系数。试验选取8头13周龄胎次和体重（（33.2±3.5）kg）相近的"杜×长×大"生长育肥猪进行饲养试验,分别于16和25周龄各进行一次消化试验,测定其粪、尿产量及粪肥氮、磷产生量。玉米种植试验设6个处理组,对照组施用化肥,负对照组不施肥,4个试验组分别按玉米种植全期氮标准需要量的100%、130%、160%和磷标准需要量的100%施用猪粪有机肥,分别在乳熟后期和完熟期测定玉米种植的氮、磷需求量,与猪粪肥氮、磷产生量拟合,测算粪肥农田承载系数。结果表明,按130%氮需求量施用猪粪有机肥,一季玉米种植乳熟后期和完熟期收获,对氮、磷的需求量分别为122.1、53.0 kg·hm^-2和190.2、62.8 kg·hm^-2,按130%氮施肥量下乳熟后期和完熟期收获的玉米农田承载系数,以N为基础为61.1头·hm^-2和95.2头·hm^-2,以P为基础为90.5头·hm^-2和107.3头·hm^-2,而且施用猪粪有机肥还可增加玉米生物产量,一定程度上增加玉米中氮、磷含量。因此,分别基于玉米对氮和磷的需求,单季玉米种植每公顷土地生长育肥猪的承载参数分别为95.2头和107.3头。     

The Standardization of an Apparatus for the Mixing of Soil Samples for Nematode Extractions

Quantitative determination of nematode populations in soils frequently necessitates the mixing of representative soil samples to form a homogeneous, compound sample from which the nematodes are extracted. A mixing apparatus was developed and standardized with the aid of a spectrophotometric technique by which the dispersion of a dye through the soil during mixing could be determined. It was found that the mixer is most suitable for use on sandy loam soils. Optimal mixing is obtained at a speed of 15 rpm for 2 minutes. Under these conditions mechanical damage to nematodes during mixing is minimal.     

猪精子质量的流式细胞检测分析

本试验将流式细胞术应用于猪精子分析,同时检测精子功能的多个特性,以保证精子具备多种特性且功能完整,顺利完成授精过程。分别采用SYBR-14/PI（Propidium Iodide）、YO-PRO-1/PI、PNA/PI及Mito Tracker/YO-PRO-1 4种染色方法对荣昌猪精子的质膜完整性、细胞凋亡、顶体完整性及线粒体功能进行了检测和分析;以SYBR-14⁺/PI^-表示质膜完整的活精子,YO-PRO-1^-/PI^-表示活精子,PNA^-/PI^-表示未发生顶体反应的活精子,Mito Tracker⁺/YO-PRO-1^-表示线粒体活性高的活精子,以上述检测指标的综合结果判定精子功能的完整性。本试验通过流式细胞术对荣昌猪精子进行质量检测和分析,结果表明,该方法可为临床诊断种猪不孕症提供理论依据,对有效治疗不孕症有重要意义。     

Flow cytometric evaluation of ki67 for the determination of malignancy grade in canine lymphoma

Ki67 is a nuclear antigen significantly correlated with degree of malignancy in human non‐Hodgkin lymphomas. We wanted to assess the ability of flow cytometric evaluation of Ki67 index (Ki67I) in differentiating the grade of malignancy in canine lymphomas. Ki67I was determined on lymph node aspirates of 90 immunophenotyped lymphomas classified according to the updated Kiel classification: 80 high grade (HG, 62 B cell and 18 T cell) and 10 low grade (LG, 3 B cell and 7 T cell) lymphomas. HG lymphomas showed significantly higher Ki67I compared with LG lymphomas (P < 0.0001). A significant difference in HG lymphomas was detected between B‐ and T‐immunophenotypes. Receiver operating characteristic (ROC) curve highlighted a high accuracy of Ki67I in recognizing HG lymphomas [area under the curve (AUC) = 99.4] and a cut‐off value of 12.2% was established (sensitivity = 96.3% and specificity = 100%). Thus, we suggest the combination of Ki67I flow cytometric determination and immunophenotype as a reliable tool to classify canine lymphomas.