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.     

川楝素对妊娠小鼠子宫组织中CD4+/CD8+T细胞和F4/80+巨噬细胞的影响

为探讨CD4+、CD8+ T淋巴细胞和F4/80+巨噬细胞在流产发生机制中的意义,研究中药单体成分川楝素诱导小鼠流产的作用及机理,本试验给妊娠5 d小鼠连续3 d腹腔注射不同剂量的川楝素溶液,对照组以等量的蒸馏水代替,于妊娠9 d处死.在给药后发现随着注射川楝素剂量的增加,小鼠的流产率逐渐上升,CD4+、CD8+ T淋...     

Pharmacokinetics and dynamics of mycophenolate mofetil after single‐dose oral administration in juvenile dachshunds

Mycophenolate mofetil (MMF) is recommended as an alternative/complementary immunosuppressant. Pharmacokinetic and dynamic effects of MMF are unknown in young‐aged dogs. We investigated the pharmacokinetics and pharmacodynamics of single oral dose MMF metabolite, mycophenolic acid (MPA), in healthy juvenile dogs purpose‐bred for the tripeptidyl peptidase 1 gene (TPP1) mutation. The dogs were heterozygous for the mutation (nonaffected carriers). Six dogs received 13 mg/kg oral MMF and two placebo. Pharmacokinetic parameters derived from plasma MPA were evaluated. Whole‐blood mitogen‐stimulated T‐cell proliferation was determined using a flow cytometric assay. Plasma MPA C_max (mean ± SD, 9.33 ± 7.04 μg/ml) occurred at <1 hr. The AUC_0–∞ (mean ± SD, 12.84±6.62 hr*μg/ml), MRT_inf (mean ± SD, 11.09 ± 9.63 min), T1/2 (harmonic mean ± PseudoSD 5.50 ± 3.80 min), and k/d (mean ± SD, 0.002 ± 0.001 1/min). Significant differences could not be detected between % inhibition of proliferating CD5+ T lymphocytes at any time point (p = .380). No relationship was observed between MPA concentration and % inhibition of proliferating CD5+ T lymphocytes (R = .148, p = .324). Pharmacodynamics do not support the use of MMF in juvenile dogs at the administered dose based on existing therapeutic targets.     

禽呼肠病毒感染对SPF鸡外周血T细胞亚型变化和细胞因子转录的影响

为探讨禽呼肠病毒(ARV)对SPF鸡外周血淋巴细胞中CD4+、CD8+T细胞数量变化和细胞因子mRNA转录水平的影响,利用流式细胞术和实时荧光定量PCR方法分别测定了ARV感染后1、7、14、21、28、35d感染组和对照组SPF鸡外周血淋巴细胞中CD4+、CD8+T细胞含量和细胞因子IL-1β、IL-6、IL-17、IL-18、IFN-γ、TNF-α基因mRNA相对转录时相。流式细胞术检测结果表明,SPF鸡感染ARV后7d和14dCD4+、CD8+T细胞比值高于对照组,其中感染7d,CD8+T细胞含量差异显著(P0.05);感染后1、21、28、35d感染组CD4+、CD8+T细胞比值均低于对照组,感染1d后CD4+、CD8+T细胞含量均差异显著(P0.05),说明外周血T细胞亚型变化是ARV感染的重要表现之一。实时荧光定量PCR结果表明,与对照组相比,感染组外周血淋巴细胞中IL-1β、IL-6(除7d外)、IL-18(除14d外)和TNF-α在整个感染过程中表达上调,IL-17和IFN-γ除感染1d外,均表达下调,说明IL-1β、IL-6、IL-17、IL-18、IFN-γ和TNF-α均参与了ARV的感染进程。     

玉屏风散对鸡脾脏淋巴细胞体外增殖的影响

将不同浓度的玉屏风散加入到鸡脾脏淋巴细胞中,培养24、48、72 h,采用MTT法观察其对鸡脾脏淋巴细胞增殖的影响.结果表明,不同浓度的玉屏风散均能协同ConA促进鸡脾脏淋巴细胞增殖的作用,并以500 mg/mL的玉屏风散培养48 h增殖作用最强.     

SARS灭活疫苗的实验免疫初步研究

观察 SARS灭活疫苗在实验动物体内的免疫效果 ,初步探讨 SARS灭活疫苗的免疫机理。方法 :利用 Vero E6细胞培养 SARS病毒 ,加入甲醛将其灭活 ,以此为抗原 ,筛选合适佐剂 ,制定合理的免疫程序 ,分别免疫 BAL B/ c小鼠、C57BL/ 6J小鼠及 SD大鼠 ,免疫后每两周采外周血一次 ,用流式细胞仪测外周血 CD4 、CD8 ,计算淋巴细胞总数。同时用 EL ISA法及中和抗体法测定抗 SARS冠状病毒 Ig G抗体的水平。结果显示 :与对照组比较 ,SARS灭活疫苗进行免疫后的实验动物外周血淋巴细胞的百分比计数、CD4 / CD8 T淋巴细胞之间的比值随着时间的变化均有不同程度的增长 ;Ig G中和抗体水平达到 1∶ 2 560 ,EL ISA抗体水平达到 1∶40 960。表明 SARS灭活疫苗可以有效刺激实验动物体内 T淋巴细胞、B淋巴细胞的活化过程 ,能成功诱导细胞免疫和体液免疫 ;SARS灭活疫苗同时还具有较强的免疫原性 ,可刺激实验动物产生具有免疫保护作用的特异性 Ig G抗体     

氟对牛脾淋巴细胞膜ATP酶活性的影响

取健康牛脾脏,常规方法分离培养淋巴细胞,用不同浓度氟化钠（NaF）染毒24h。低渗溶胞法提取细胞膜,检测膜上ATP酶活性。结果显示,与对照组相比较,染氟组淋巴细胞膜ATP酶活性降低,组间差异显著或极显著。表明：氟能抑制牛脾淋巴细胞膜ATP酶活性。     

Identification of CD3+ T lymphocytes in the green turtle Chelonia mydas

To understand the role of the immune system with respect to disease in reptiles, there is the need to develop tools to assess the host's immune response. An important tool is the development of molecular markers to identify immune cells, and these are limited for reptiles. We developed a technique for the cryopreservation of peripheral blood mononuclear cells and showed that a commercially available anti-CD3 epsilon chain antibody detects a subpopulation of CD3 positive peripheral blood lymphocytes in the marine turtle Chelonia mydas. In the thymus and in skin inoculated with phytohemagglutinin, the same antibody showed the classical staining pattern observed in mammals and birds. For Western blot, the anti-CD3 antibodies identified a 17.6 kDa band in membrane proteins of peripheral blood mononuclear cell compatible in weight to previously described CD3 molecules. This is the first demostration of CD3+ cells in reptiles using specific antibodies.     

重组红火蚁毒素蛋白SoliⅣ致病机理的初步研究

作者通过研究重组红火蚁毒素蛋白Solenopsis invictaⅣ (SoliⅣ)对兔及外周血淋巴细胞的影响,探讨红火蚁毒素蛋白致病机制。通过分离兔外周血淋巴细胞进行培养,经不同浓度的重组红火蚁毒素蛋白SoliⅣ分别和细菌脂多糖(LPS)、刀豆蛋白(ConA)共同刺激后,MTT法测定淋巴细胞的增殖情况;体内试验：用重组蛋白SoliⅣ从兔背部皮下注入,观察兔的临床反应,定期采血,用ELISA方法测定兔血清中白介素 4（IL-4）和总IgE的变化。重组红火蚁毒素蛋白SoliⅣ浓度为25、50、75 μg/ml和LPS共刺激时,与单独LPS刺激对照组相比,淋巴细胞增殖活性显著增高（P<0.05）;浓度为15、25、50、75 μg/ml和ConA共刺激时,与单独ConA刺激对照组比较,淋巴细胞增殖活性显著增高（P<0.05）;重组SoliⅣ过敏兔的血清中IL-4和总IgE的水平升高,在20 h左右达到最高值。试验结果表明,一定浓度的重组红火蚁毒素蛋白SoliⅣ能引起体外培养的T、B淋巴细胞增殖,过敏体质兔在重组红火蚁毒素蛋白SoliⅣ刺激后能引起Ⅰ型变态反应。     

B‐cell lymphoma with plasmacytoid differentiation,atypical cytoplasmic inclusions,and secondary leukemia in a dog

A 7‐year‐old male castrated Jack Russell Terrier was presented to the oncology service at the University of California–Davis Veterinary Medical Teaching Hospital for evaluation of suspected lymphoma. The dog had several enlarged lymph nodes and moderate lymphocytosis. Aspirates of an enlarged inguinal lymph node contained a bimorphic population of large immature lymphocytes and smaller cells with plasmacytoid features. Both cell types often contained a single large cytoplasmic inclusion that varied from clear to pale pink to sky blue. Cytologic changes were interpreted as most consistent with lymphoid neoplasia. Based on the predominantly mature cell morphology and some morphologic heterogeneity, the peripheral lymphocytosis was interpreted as most likely reactive in nature. However, the immunophenotype of the cells (CD20+, CD21+, CD79a+, MUM‐1+, and MHCII+) and clonality assays showed that tissue and blood lymphocytes were neoplastic B cells with clonal identity despite their different morphologic appearances. The cytoplasmic inclusions were positive with periodic acid‐Schiff and were immunoreactive for IgM and IgG. By transmission electron microscopy, inclusions consisted of aberrant rough endoplasmic reticulum; a few small Russell bodies were also noted. A final diagnosis of high‐grade B‐cell lymphoma with plasmacytoid differentiation, atypical cytoplasmic inclusions, and secondary leukemia was made. Chemotherapy was initiated, but the dog was euthanized due to severe and uncontrolled seizures 9 months after the initial diagnosis. This case extends the morphologic repertoire of canine plasmacytoid neoplasms and emphasizes their continuum with multicentric lymphoma. This case also demonstrates the need for advanced diagnostic techniques in establishing blood involvement in lymphoma in some instances.