Cyclosporin A improves murine pregnancy outcome in abortion-prone matings: involvement of CD80/86 and CD28/CTLA-4

Immune regulation during pregnancy is complex, and thus an optimal therapy for pregnancy complications is always a big challenge to reproductive medicine. Cyclosporin A (CsA), a potent immunosuppressant, prevents rejection of allografts by hosts, but little is known about the modulating effect of CsA on the materno-fetal relationship. Here, pregnant CBA/J females mated with DBA/2 males as an abortion-prone model were administered with CsA on day 4.5 of gestation, and the pregnant CBA/J females mated with BALB/c males were established as successful pregnancy control. It was demonstrated that administration of CsA at the window of implantation significantly up-regulated the expression of CTLA-4, while down-regulating the levels of CD80, CD86, and CD28 at the materno-fetal interface in the CBA/J x DBA/2 abortion-prone matings, and the embryo resorption rate of the abortion-prone matings reduced significantly after CsA treatment, implying that modulation of costimulatory molecule expression by CsA might contribute to preventing the fetus from maternal immune attack. In addition, treatment with CsA induced enhanced growth and reduced cell apoptosis of the murine trophoblast cells. Together, these findings indicate that CsA has a beneficial effect on the materno-fetal interface in abortion-prone matings, leading to a pregnancy outcome improvement, which might provide new therapeutics for spontaneous pregnancy wastage.     

Deoxynivalenol suppresses circulating and splenic leukocyte subpopulations in BALB/c mice: dose response,time course and sex differences

It was hypothesized that suppression of peripheral blood leukocyte subsets were markers of exposure to dietary deoxynivalenol (DON), a Fusarium graminearum mycotoxin in grain, at 1.0 mg kg^?1 but not at lesser doses in BALB/c mice. Groups of 10 female and 10 male BALB/c mice were fed 0, 0.25, 0.5, 1.0, and 2.0 mg kg^?1 DON for 14 and 28 days. Using flow cytometry with staining for leukocyte surface markers, the percentage of CD19⁺ leukocytes (B cells) in peripheral blood was decreased in both sexes of BALB/c mice after 14 days of exposure to 1.0 or 2.0 mg kg^?1 DON, whereas exposure to DON over 28 days did not inhibit B cells compared to the control diet. The percentage of mononuclear cells in peripheral blood was decreased in female BALB/c mice fed 1 and 2 mg kg^?1 DON after 14 days compared with control diet. The percentage of CD11b⁺ leukocytes (monocytes) in peripheral blood and total CD11b⁺ splenic leukocytes were decreased only in female mice fed 1.0 and 2.0 mg kg^?1 DON after 28 days compared with control diet, which shows the greater sensitivity to DON in females compared to males. It was concluded that BALB/c mice adapted to DON exposure because peripheral blood cellular effects of DON at 14 days disappeared by 28 days with the exception of monocyte changes in females. This suggests that female sex hormones potentiate one potential marker of DON immunotoxicity in BALB/c mice.     

酪蛋白糖巨肽对小鼠肠道免疫系统的影响

研究酪蛋白糖巨肽(CGMP)对正常小鼠肠相关淋巴组织的免疫应答反应的影响,对阐述CGMP 的生物学功能具有重要意义。本实验将BALB/c 雌性小鼠28 只随机分为4 组,每组7 只,分别为0.9% 生理盐水对照组(0.2mL/d),低、中、高3 种剂量组(30、120、300μg/d)。连续灌胃16d 后,取脾脏、PP 结(peyer s patches)和肠系膜淋巴结(mesenteric lymph node,MLN),制备细胞悬液,进行三色荧光标记后,用流式细胞仪检测CD3+、CD3+CD4+ 和CD3+CD8+T 淋巴细胞亚群的变化。结果表明：CGMP 各剂量组均能够促进脾脏和PP 结中CD3+ 和CD3+CD4+ 细胞的显著增加(P ＜ 0.05),高剂量能引起脾脏CD3+CD8+ 淋巴细胞的显著增多(P ＜ 0.05),低剂量能引起脾脏CD3+CD4+/ CD3+CD8+ 的显著增加(P ＜ 0.05);低剂量CGMP 能使MLN 中CD3+CD8+ 淋巴细胞显著增减少(P ＜ 0.01)。研究证实长期灌胃CGMP 会诱导肠黏膜产生获得性的免疫应答,并且不会引起机体产生口服耐受。     

嗜酸乳酸杆菌对自主活动受限型应激小鼠肠道黏膜免疫状态的影响

目的：探讨嗜酸乳酸杆菌（Lactobacillus acidophilus,LA）对自主活动受限型应激小鼠肠道黏膜免疫状态的影响。方法：以健康雌性BALB/c小鼠为受试动物,分为常规饲养组（NC组）、常规饲养条件灌胃LA组（NC＋LA组）、应激组（S组）、应激条件灌胃LA组（S＋LA组）,小鼠的LA灌胃剂量为108 CFU/d;应激组小鼠每天被限制自主活动3 h;15 d后取材,采用流式细胞术、酶联免疫吸附（enzyme-linked immunosorbent assay,ELISA）等方法,对肠系膜淋巴结（mesenteric lymph nodes,MLN）中CD4＋T细胞、CD8＋T细胞和CD11c＋树突细胞（CD11c＋ dendritic cell,CD11c＋ DC）比例,结肠组织白细胞介素-10（interleukin-10,IL-10）和白细胞介素-17（interleukin-17,IL-17）水平,以及小肠总分泌型免疫球蛋白A（secreted immunoglobulin A,sIgA）水平进行检测。结果：常规饲养条件下,LA能极显著提高小鼠MLN中CD4＋T细胞比例（P＜0.01）,显著提高MLN中CD11c＋ DC比例、小肠总sIgA水平和结肠组织IL-10水平（P＜0.05）,显著降低结肠组织IL-17水平（P＜0.05）,而对MLN中CD8＋T细胞比例无显著影响（P＞0.05）;应激条件能极显著降低小鼠MLN中CD4＋T细胞比例和CD11c＋ DC比例（P＜0.01）,显著降低CD8＋T细胞比例（P＜0.05）,极显著降低小鼠结肠组织IL-10水平和小肠总sIgA水平（P＜0.01）,极显著提高结肠组织IL-17水平（P＜0.01）;应激条件下,LA能显著提高小鼠MLN中CD4＋T细胞比例和CD11c＋ DC比例（P＜0.05）,显著提高小鼠结肠组织IL-10水平和小肠总sIgA水平（P＜0.05）,并显著降低结肠组织IL-17水平（P＜0.05）,而对小鼠MLN中CD8＋T细胞比例无显著影响（P＞0.05）。结论：给予自主活动受限型应激小鼠LA干预,可提高小鼠MLN中CD4＋T细胞和CD11c＋ DC比例,上调结肠IL-10和小肠总sIgA分泌水平,下调结肠IL-17分泌水平,调节应激小鼠的肠道黏膜免疫状态。     

Kinetics of murine decidual dendritic cells

Dendritic cells (DCs) are professional antigen presenting cells (APC) capable of induction of primary immune responses as well as immunologic tolerance. Myeloid and lymphoid subsets of murine DCs are able to shift cytokine responses of T cells toward Th2 and Th1 profiles respectively. Thus, DCs would be suitable candidates to mediate the balance of maternal immune responses to conception. We analyzed pregnancy-related variations in uterus and splenic DCs in a murine model. C57BL/6-mated Balb/c female mice with vaginal plugs were scarified at early, middle, and late pregnancy. Frozen sections of uterus and spleen at each stage of pregnancy were immunostained with CD11c- and MHC-II-specific antibodies. Two-color immunohistochemistry was also carried out using anti-CD11c and one of the antibodies against CD11b, CD8alpha, CD86, and DEC-205. Using morphometric analysis, the average density of DCs and relative percentage of myeloid (CD11c+, CD11b+) and lymphoid DCs (CD11c+, CD8a+) were determined at each stage. Our results showed that DCs are present throughout the pregnancy in decidua. The average density of decidual DCs at early pregnancy was significantly higher relative to middle and late gestation or to those of endometrial DCs of non-pregnant mice. Interestingly, the average density of decidual and splenic DCs, followed the same variations at different stages of pregnancy. The relative percentage of decidual lymphoid DCs (LDC) was significantly higher at mid-gestation when compared with other stages of pregnancy or non-pregnant mice. Inversely, the frequency of myeloid DCs (MDC) and the MDC/LDC ratio were statistically lower at the middle stage of pregnancy. A majority of decidual DCs expressed MHC-II and CD86. At early pregnancy, DCs were more concentrated subadjacent to the luminal epithelial layers, whereas at mid-or late gestation, DCs were randomly distributed in the stroma and around the epithelium. Mid-pregnancy period was a critical point with regard to splenic DCs kinetics, as both the average density of DCs and the frequency of MDCs decreased significantly when compared with early or late pregnancy, although the relative percentage of splenic LDCs did not change. Our data suggest that the balance of MDC and LDC is finely tuned throughout pregnancy, pointing an eminent immunoregulatory role of DCs in the maintenance of pregnancy.     

Biochemical characterization of a proteoglycan complex from an edible mushroom Ganoderma lucidum fruiting bodies and its immunoregulatory activity

A fraction, LZ-D-7, was isolated from the fruiting body of the edible mushroom Ganoderma lucidum using a series of chromatographic steps. Biochemical experiments showed that the complex is a proteoglycan and has a carbohydrate percentage of 95.3%, along with a protein percentage of 3.3%. Methylation analysis showed the polysaccharide moiety of LZ-D-7 had a backbone of 1,4-disubsituted-glucopyranosyl and 1,2,6-trisubstituted-glucopyranosyl residues. The branches were mainly composed of 1-substituted-glucopyranosyl residue, which was attached to 1,2,6-trisubstituted-glucopyranosyl residue via 2 or 6 position. Investigation of modulation of LZ-D-7 on BALB/c mouse spleen lymphocytes cells resulted in a three to four-fold increase of MSLs percentage. Analysis of activation and proliferation of MSLs indicated that LZ-D-7 activating most of the activated cells were B cells. The B cells were enlarged, expressed CD⁷¹⁺ and CD²⁵⁺ on the cell surface.     

菠萝蜜低聚肽的免疫调节作用研究

目的:探究菠萝蜜低聚肽(jackfruit oligopeptides,JOPs)免疫调节功能。方法:采用SPF级雌性BALB/c小鼠为实验对象,进行8项免疫实验测定,系统评价不同干预剂量(0.20、0.40、0.80 g/kg·bw)JOPs对小鼠免疫器官相对重量、细胞免疫功能、体液免疫功能、单核-巨噬细胞功能、自然杀伤细胞(nature killer cell,NK细胞)活性以及T淋巴细胞亚群的影响。结果:与空白对照组相比,JOPs 0.40、0.80 g/kg·bw剂量组脾脏指数显著提高(P<0.05),且JOPs 0.40 g/kg·bw剂量组脾脏指数显著高于乳清蛋白组(P<0.05);与空白对照组及乳清蛋白组相比,JOPs 0.40、0.80 g/kg·bw剂量组ConA诱导的小鼠脾淋巴细胞增殖能力、足跖增厚度、溶血空斑数、碳廓清吞噬指数a、巨噬细胞吞噬率与吞噬指数及NK细胞活性显著提高(P<0.05),JOPs 0.80 g/kg·bw剂量组血清溶血素含量、CD3⁺T细胞数量、CD4⁺T细胞百分比及CD4⁺CD25⁺/CD4⁺、CD4⁺/CD8⁺比值显著提高(P<0.05);各JOPs剂量组对小鼠体重增长、胸腺指数均无显著性影响(P>0.05)。结论:JOPs可提高机体适应性免疫及固有免疫功能,起到免疫增强作用。     

Contraception in mice immunized with recombinant zona pellucida subunit 3 proteins correlates with Th2 responses and the levels of interleukin 4 expressed by CD4+ cells

The immune responses and contraceptive effect in mice were tested following immunization with purified recombinant zona pellucida (ZP) proteins produced using a vaccinia (v) virus T7 mammalian expression system. Female BALB/c and CBA mice were immunized with recombinant mouse (m) ZP3 (vmZP3) or pig (p) ZPC (vpZPC) using Freund's adjuvants and boosted three times. Fertility and mean litter size were significantly reduced in groups of BALB/c mice immunized with recombinant vmZP3 and vpZPC compared with controls treated with Freund's adjuvants alone. In CBA mice, fertility and mean litter size were significantly reduced in groups of animals immunized with vmZP3 but not with vpZPC compared with the controls. Most infertile animals treated with vmZP3 and a single infertile BALB/c mouse treated with vpZPC lacked mature follicles in the ovaries, whilst no abnormalities were detected in the remaining vpZPC treated, fertile vmZP3 treated and control mice. All mice (both fertile and infertile) immunized with vmZP3 and vpZPC produced IgG antibodies, but the levels of total IgG, IgG1 and IgG2a did not correlate with infertility. All BALB/c and CBA mice immunized with vmZP3 and vpZPC showed greater delayed type hypersensitivity responses in the footpads after challenge with their respective antigens than controls, but these did not differ between the fertile and infertile mice. There was, however, a significant correlation between infertility and the levels of the Type 2 T helper cell (Th2) cytokine interleukin 4 produced by CD4+ cells from vmZP3 immunized mice in response to stimulation with vmZP3 and this did not apply to the levels of the Type 1 T helper cell (Th1) cytokine interferon gamma or the general proliferation response. The results support the conclusion that induction of Th2 responses in individual mice determines whether infertility develops in response to immunization with zona pellucida proteins.     

Chemokine response induced by Chlamydia trachomatis in prostate derived CD45+ and CD45- cells

The role of innate cells and their receptors within the male genital tract remains poorly understood. Much less is known about the relative contribution of different genital tract cells such as epithelial/stromal cells and resident leucocytes. In this study, we examined innate immune responses to Chlamydia trachomatis by prostate epithelial/stromal cells and prostate resident leucocytes. Murine prostate primary cultures were performed and leucocyte and epithelial/stromal cells were sorted based on surface protein expression of CD45 by magnetism-activated cell sorting or fluorescence-activated cell sorting. Prostate derived CD45- and CD45+ cells were infected with C. trachomatis and chemokine secretion assayed by ELISA. Similar experiments were performed using prostate CD45+ and CD45- cells from myeloid differentiation factor 88 (Myd88(-/-)) mice or toll-like receptor (Tlr2(-/-)) and Tlr4(mutant) double-deficient mice. Moreover, a TLR-signalling pathway array was used to screen changes in different genes involved in TLR-signalling pathways by real-time PCR. Prostate derived CD45- and CD45+ cells responded to chlamydial infection with the production of different chemokines. Both populations expressed genes involved in TLR signalling and required to respond to pathogen-associated molecular patterns and to C. trachomatis infection. Both populations required the adaptor molecule MYD88 to elicit chemokine response against C. trachomatis. TLR2-TLR4 was essential for chemokine production by CD45+ prostate derived cells, but in their absence, CD45- cells still produced significant levels of chemokines. We demonstrate that C. trachomatis is differentially recognised by prostate derived CD45+ and CD45- cells and suggest that diverse strategies are taking place in the local microenvironment of the host in response to the infection.     

Phagocytosis as a potential mechanism for microbial defense of mouse placental trophoblast cells

Trophoblast giant cells are active phagocytes during implantation and post-implantation. Phagocytosis decreases during placental maturation as the phagocytic function of nutrition is gradually replaced by the direct uptake of nutrients by the labyrinth zone trophoblast. We hypothesize that, after placental maturation, trophoblast cells maintain phagocytic functions for purposes other than nutrition. This study employs histological techniques to examine the ability of trophoblast cells to phagocytose microorganisms (yeast or bacteria)--in vivo in females receiving thioglycolate to activate macrophages and in vitro in the presence of phagocytic promoters such as interferon-gamma and complement component C3. Placental trophoblast cells from the second half of gestation show basal phagocytosis that can be dramatically up-regulated by these promoters when microorganisms are inoculated into pregnant animals or introduced into culture systems. Stimulated trophoblast cells phagocytosed organisms more rapidly and in greater numbers than non-stimulated trophoblast exposed to the same numbers of organisms. Taken together, our results indicate that trophoblast cells do not lose their ability to phagocytose during the placentation process, which may imply that trophoblast cells participate in embryonic and fetal innate immune defense through elimination of microorganisms present at the maternal-fetal interface.     

Bovine mononuclear leukocyte subpopulations in peripheral blood and mammary gland secretions during lactation.

Florescence flow cytometry and monoclonal antibodies were used to analyze the composition of leukocytes from peripheral blood and mammary gland secretions. Samples were obtained from Holstein dairy cows, free of IMI, at six time points during the lactation cycle. The percentage of monocytes in peripheral blood mononuclear cells varied from 15 to 31%; the lowest percentage occurred at the early nonlactation period. The percentage of T lymphocytes from mammary gland secretions varied from 16% during the periparturient period to 62% at late lactation. The B lymphocytes varied from 7% at late lactation to 25% during the periparturient period. Macrophages varied from 21 to 69%; the highest percentage occurred during the periparturient period. The mean ratio of CD4+:CD8+ T lymphocytes in the peripheral blood and mammary gland secretions was 1.53 and .85, respectively. A subpopulation of activated CD8+ T lymphocytes present in mammary gland secretions throughout the lactation cycle coexpressed a new activation molecule, ACT2. Increases in the proportion of CD8+ T lymphocytes were associated with an increase in the percentage of CD8+, ACT2+ T lymphocytes. Activated CD8+ T lymphocytes may play an important role in the regulation and expression of the local immune response to pathogens.     

车前子多糖对骨髓来源树突状细胞表型和吞噬功能的影响

目的:探讨车前子多糖对小鼠骨髓来源树突状细胞(BMDCs)表型和功能的影响。方法:从小鼠骨髓中分离单核细胞,加入细胞因子rmGM-CSF、rmIL-4诱导分化成未成熟树突状细胞,收集细胞加入促成熟刺激剂LPS(阳性对照组)或车前子多糖。倒置显微镜观察树突状细胞的形态,流式细胞术检测成熟树突状细胞的表面标志CD11c和MHCII的表达及吞噬FITC-dextran的变化。结果:经4种不同的车前子多糖作用40h后,显著促进CD11c+MHCII+双阳性细胞的比率,在浓度(0～50μg/ml)范围内呈现剂量依赖性,当浓度高于50μg/ml时,双阳性细胞表达率呈现降低趋势。经多糖作用后吞噬FITC-dextran的能力降低,其中PS3的作用效果最明显,表达PE-CD11c×FITC-dextran的细胞比率只有11.53%。结论:车前子多糖促进CD11c+MHCII+双阳性细胞的比率,降低对FITC-dextran的吞噬能力,初步表明车前子多糖可以促进树突状细胞的成熟。     

应用PG-100智能化猪胴体分级仪和人工分级方法建立猪胴体瘦肉率预测模型的研究

应用PG-100智能化猪胴体分级仪和人工分级方法对556头商品肉猪进行了相关性状的测定与分析,分别建立了智能分级方法和人工分级方法的最优猪胴体瘦肉率预测模型(智能分级:y=60.325-0.578x1+0.168x2,x1为倒数3～4肋骨间离背中线6～7cm处的脂肪厚度,x2为倒数3～4肋骨间离背中线6～7cm处的肌肉厚度,方程R2=0.864,RSD=2.33%;人工分级:y=57.235-0.575x7+0.207x10,x7为腰荐膘厚,x10为三角肌的肌肉厚度,方程R2=0.859,RSD=2.4%),从而为制定符合我国国情的《猪胴体分级标准》建立了良好的技术操作平台。     

纳豆激酶对大鼠酒精性肝损伤的改善效果及免疫调节作用

目的：探讨纳豆激酶对大鼠酒精性肝损伤的改善效果及免疫调节作用。方法：雄性Wistar大鼠随机分为5 组,正常对照组（生理盐水灌胃）、酒精模型组（酒精体积分数56%的白酒灌胃：第1周灌胃6 mL/（kg?d mb）,第2周灌胃8 mL/（kg?d mb）,第3周灌胃10 mL/（kg?d mb）,第4～10周灌胃11 mL/（kg?d mb））、纳豆激酶对照组（灌胃530.39 FU/（kg?d mb）纳豆激酶）、纳豆激酶干预组（灌胃530.39 FU/（kg?d mb）纳豆激酶＋酒精）、甘利欣干预组（灌胃200 mg/（kg?d mb）甘利欣＋酒精）;后两组酒精剂量同酒精模型组,实验持续10 周。苏木精-伊红染色观察肝脏组织形态结构,透射电子显微镜观察肝细胞超微结构;测定血清谷丙转氨酶（alanine aminotransferase,ALT）、谷草转氨酶（aspartate aminotransferase,AST）、γ-谷氨酰转肽酶（gamma-glutamyl transpeptidase,GGT）、胆碱酯酶（cholinesterase,CHE）活力;计算脾指数,流式细胞术检测大鼠外周血中CD4+、CD8+ T淋巴细胞亚群和自然杀伤（natural killer,NK）细胞比例。结果：酒精模型组大鼠肝组织形态结构和肝细胞超微结构均出现明显的病变损伤,纳豆激酶和甘利欣干预后得到显著改善。与酒精模型组相比,纳豆激酶干预组和甘利欣干预组大鼠血清ALT、AST、GGT活力显著下降（P＜0.05）,CHE活力有一定上升趋势。与酒精模型组相比,纳豆激酶干预组和甘利欣干预组大鼠脾指数、CD3+CD4+ T淋巴细胞比例、CD3+CD8+ T淋巴细胞比例均显著升高（P＜0.05）,TCRαβ+CD161a+ NK细胞比例有不同程度的上升趋势。结论：纳豆激酶对大鼠酒精性肝损伤有一定改善效果,其机制可能与调节CD4+、CD8+ T淋巴细胞、NK细胞等免疫细胞比例,提高机体免疫调节作用有关。     

茶树菇凝集素AAL、AAL-2检测小鼠CD8~+T淋巴细胞表面糖基化研究

本研究使用两种大型真菌茶树菇新型凝集素AAL(Agrocybe aegerita lectin)和AAL2(Agrocybe aegerita lectin 2),以检测小鼠CD8~+T淋巴细胞表面糖基化。用生物素标记的AAL和AAL2结合流式细胞仪,分别分析从C57BL/6小鼠脾脏和腹股沟淋巴结分离出来的CD8~+T淋巴细胞表面的糖基化水平,及其细胞因子表达水平。结果发现脾脏中AAL~+CD8~+,AAL2~+CD8~+T淋巴细胞所占的比例(%)分别为6.72±3.00和12.18±5.28,而腹股沟淋巴结的AAL~+CD8~+,AAL2~+CD8~+T淋巴细胞所占的比例(%)分别为12.18±5.28和10.15±3.46。细胞因子表达水平结果发现小鼠脾脏AAL2~+CD8~+T细胞表达perforin的细胞比例低于AAL2-CD8~+T细胞(p0.05,有统计学意义),腹股沟淋巴结中AAL~+CD8~+T细胞表达perforin的细胞比例则高于AAL-CD8~+T细胞(p0.05,有统计学意义)。该数据为研究小鼠脾脏和腹股沟淋巴结CD8~+T细胞表面糖基化提供了新的数据,也为两种新型凝集素AAL和AAL2的应用提供理论依据。     

Induction of mud crab (Scylla paramamosain) tropomyosin and arginine kinase specific hypersensitivity in BALB/c mice

BACKGROUND: Shellfish hypersensitivity is among the most common food allergies. The allergens tropomyosin (TM) and arginine kinase (AK) from mud crab (Scylla paramamosain) were purified to homogeneity. BALB/c female mice were sensitized with TM and AK by intragastric administration. Mice treated with normal saline served as the negative control (NC) group. RESULTS: Compared with NC group, mice that were treated with TM and AK developed reduced activity; meanwhile, their scratching behavior and specific‐IgE level were increased. Specific‐CD4 + T cells were significantly elevated in the splenocyte cultures of the mice upon TM and AK stimulation. However, compared with the positive control group (ovalbumin, OVA), there was no significant difference. The expression of IL‐4 in culture cells stimulated by TM, AK, and OVA group showed significant differences from the NC group, respectively. CONCLUSION: These results indicated that a BALB/c mouse model for sensitization to TM and AK from mud crab was successfully established, and the Th2 response was observed, displaying increased immunoglobulin E levels, together with the production of interleukin 4 and allergic symptoms. Copyright © 2011 Society of Chemical Industry     

Differentiation of viable and dead Escherichia coli O157:H7 cells on and in apple structures and tissues following chlorine treatment

Confocal scanning laser microscopy (CSLM) was used to differentiate viable and nonviable cells of Escherichia coli O157:H7 on and in raw apple tissues following treatment with water and 200 or 2,000 ppm active chlorine solution. Whole unwaxed Red Delicious cultivar apples at 25 degrees C were inoculated by dipping in a suspension of E. coli O157:H7 (8.48 log10 CFU/ml) at 4 degrees C, followed by treatment in water or chlorine solution at 21 degrees C for 2 min. The dead cells on and in apples were distinguished from live cells by treating tissue samples with SYTOX green nucleic acid stain. Viable and dead cells were then labeled with an antibody conjugated with a fluorescent dye (Alexa Fluor 594). The percentage of viable cells on the apple surface, as well as at various depths in surface and internal structures, was determined. The mean percentages of viable cells located at the sites after treatment with water or chlorinated water were in the following order, which also reflects the order of protection against inactivation: floral tube wall (20.5%) > lenticels (15.0%) > damaged cuticle surrounding puncture wounds (13.0%) > intact cuticle (8.1%). The location of viable cells within tissues was dependent on the structure. Except for lenticels, the percentage of viable cells increased as depth into the CSLM stacks increased, indicating that cells attached to subsurface structures were better protected against inactivation with chlorine than were cells located on exposed surfaces. Further research is warranted to investigate the efficacy of other chemical sanitizers. as well as that of surfactants and solvents in combination with sanitizers, in removing or killing E. coli O157:H7 lodgedin protective structures on the surface and within tissues of apples.     

Deviations in populations of peripheral blood mononuclear cells and endometrial macrophages in the cow during pregnancy

The presence of conceptus alloantigens necessitates changes in maternal immune function. Here, we used the cow to evaluate whether species with epitheliochorial placentation have changes in specific leukocyte populations during pregnancy similar to those reported in species with hemotropic placentae. At days 33-34 of pregnancy, there was no effect of pregnancy status on the number of cells positive for CD8, CD4, gammadeltaT cell receptor, or the monocyte marker CD68 in the peripheral blood mononuclear cell (PBMC) population. There was, however, an increase in the proportion of CD4(+) cells that were positive for CD25. There was no effect of status on the proportion of PBMCs that were CD8(+) when comparing preparturient cows to nonpregnant cows. However, preparturient cows had an increased percentage of PBMCs that were gammadeltaT cells and CD4(+)CD25(+) and a tendency for a lower percentage that were CD68(+) cells. Using immunolocalization with anti-CD68, it was found that pregnant cows had increased numbers of CD68(+) cells in the endometrial stroma as early as days 54-100 of gestation; this increase persisted through the last time examined (day 240 of gestation). Cells positive for CD68 were also positive for another macrophage/monocyte marker, CD14. In conclusion, pregnancy in the cow is associated with changes in peripheral and endometrial leukocyte numbers, which are similar to patterns observed in other species.     

Involvement of CD44 in leukocyte recruitment to the rat testis in experimental autoimmune orchitis

Experimental autoimmune orchitis (EAO) is characterized by an interstitial mononuclear cell infiltrate and a severe lesion of the seminiferous tubules with germ cells that undergo apoptosis and sloughing. The aim of this study was to determine the role of CD44 in testicular leukocyte recruitment in EAO. The biological functions of CD44 have been attributed to the generation of a functionally active hyaluronan-binding phenotype. Orchitis was induced in Sprague-Dawley adult rats by active immunization with an emulsion of testicular homogenate and complete Freund's adjuvant using Bordetella pertussis as co-adjuvant. Control rats (C) injected with saline and adjuvants and normal (N) untreated rats were also studied. CD44 expression was analyzed by flow cytometry in peripheral blood mononuclear cells (PBMC) and lymph node cells isolated from rats at different times after the first immunization. We observed an increase in the mean fluorescence intensity of both samples in the C and experimental (E) groups only after the immunization period. A significant decrease in percentage of CD44+PBMC and in mean fluorescence intensity was observed in rats with orchitis compared with the C group. By in vitro hyaluronic acid-binding assay we demonstrated that the percentage of PBMC adhesion was higher in the E group compared with the C and N groups. By immunohistochemistry, we observed a significant increase in the number of CD44+cells in the testicular interstitium of rats with severe orchitis compared with the N and C groups. These results suggested that the CD44 molecule is involved in the homing of lymphomonocytes into the testes of rats with autoimmune orchitis.     

紫薯花色苷对小鼠淋巴细胞免疫功能的调节作用

为探讨紫薯花色苷(PSPA)免疫调节作用,制备小鼠脾淋巴细胞,MTT法检测不同浓度PSPA对淋巴细胞增殖的影响;采用流式细胞术检测不同浓度PSPA对淋巴细胞膜表面标志和细胞周期的影响。结果:PSPA各浓度组淋巴细胞刺激指数均极显著(p<0.01)高于空白对照组;PSPA 20 mg/mL浓度组淋巴细胞刺激指数极显著(p<0.01)高于阳性对照组。PSPA 2.5 mg/mL浓度组淋巴细胞亚群CD4⁺/CD8⁺比值显著(p<0.05)高于空白对照组,PSPA 5、10和20 mg/mL浓度组淋巴细胞亚群CD4⁺/CD8⁺比值均极显著(p<0.01)高于空白对照组;PSPA 2.0浓度组淋巴细胞亚群CD4⁺CD8⁺百分率均极显著(p<0.01)高于阳性对照组。PSPA各浓度组处于S期的淋巴细胞的百分率均极显著(p<0.01)高于空白对照组。PSPA 20 mg/mL浓度组处于S期的淋巴细胞的百分率均极显著(p<0.01)高于阳性对照组。表明PSPA通过促进淋巴细胞增殖、提高CD4⁺/CD8⁺亚群比值、增加进入S期细胞的百分率,从而调节淋巴细胞的免疫功能。结论:PSPA具有明显的免疫调节活性。