嗜酸粒细胞过氧化物酶快速染色的研究

目的建立嗜酸粒细胞过氧化物酶快速染色方法,完善包括嗜酸粒细胞脱颗粒或中性粒细胞粗颗粒等各种情况下嗜酸粒细胞准确并快速计数的质量控制。方法随机选取75例血液病患者的骨髓涂片标本,要求常规瑞-姬染色骨髓分类嗜酸粒细胞≥3.5%,取材3d内。每份标本中选取两张,分别划入实验组和对照组。实验组标本进行嗜酸粒细胞过氧化物酶快速染色,对照组标本进行常规瑞-姬染色。分别计数嗜酸粒细胞百分数。结果实验组嗜酸粒细胞颗粒染成黑色,对包括中性粒细胞在内的其他细胞染色效果同瑞-姬染色,显微镜下嗜酸粒细胞显示醒目,可快速准确计数。结果与对照组比较,经t检验,P0.05,无统计学差异。结论嗜酸粒细胞过氧化物酶快速染色方法比较常规瑞-姬染色具有快速染色,对嗜酸粒细胞的显示更加醒目的优点,且对包括中性粒细胞在内的其他细胞染色兼有瑞-姬染色的效果。该方法值得推广用于快速骨髓细胞染色,且适用于包括嗜酸粒细胞形态不典型及中性粒细胞颗粒粗大等各种情况下的嗜酸细胞计数的质量控制,从而准确有效地服务于临床诊治。     

Eosinophil migration induced by mast cell chymase is mediated by extracellular signal-regulated kinase pathway

Mast cell chymase is known to induce eosinophil migration in vivo and in vitro. In the present study, we investigated possible involvement of mitogen-activated protein (MAP) kinases; extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, in the chymase-induced eosinophil migration. Human chymase induced a rapid phosphorylation of ERK1/2 and p38 in human eosinophilic leukemia EoL-1 cells, while no phosphorylation was detected in JNK. The chymase-induced phosphorylation of ERK and p38 was inhibited by pertussis toxin. Similar results were obtained in the experiments using mouse chymase and eosinophils. U0126 (the inhibitor for MAP/ERK kinase) suppressed chymase-induced migration of EoL-1 cells and mouse eosinophils. However, SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) showed little effect on the migration. It is suggested therefore that chymase activates ERK and p38 probably through G-protein-coupled receptor, and that ERK but not p38 cascade may have a crucial role in chymase-induced migration of eosinophils.     

Immunohistochemical artifact for nitrotyrosine in eosinophils or eosinophil containing tissue

Immunohistochemical artifacts for nitrotyrosine were investigated in eosinophils with regard to fixatives. Immunoreactivity for nitrotyrosine was revealed in separated eosinophils and in gastric mucosa fixed with periodate, lysine-paraformaldehyde (PLP). The increase in immunoreactivity by PLP was due to periodate itself, a component of PLP. Nitrotyrosine formed by peroxidase using NO 2 - and H 2 O 2 or by peroxynitrite was not completely inhibited by 100 mM dithionite but the immunoreactivity for nitrotyrosine antibodies by PLP was completely inhibited by 5.7 mM dithionite. Although untreated eosinophils or ovalbumin (OVA) did not show protein tyrosine nitration in a standard Western blot, the treatment of the blotted membrane with PLP increased the reactivities of proteins from eosinophils with anti-nitrotyrosine antibodies. The increase in immunoreactivity of OVA with anti-nitrotyrosine antibodies by PLP did not change with pre-treatment with dithionite but was abolished by treatment with dithionite after PLP fixation. In HPLC assays, periodate did not generate nitrotyrosine from l -tyrosine and aminotyrosine. These results suggest that the treatment of eosinophils or eosinophil-containing tissues with PLP fixative augments the immunoreactivity of nitrotyrosine antibodies with eosinophils due to the formation of epitopes similar to nitrotyrosine by an oxidation reaction of periodate, which evokes an artifact in nitrotyrosine immunohistochemistry.     

Effects of dexamethasone on estrogen- and pregnancy-induced plasticity in rat uterine sympathetic nerves

Estrogen and glucocorticoids are known to evoke opposing effects on the uterus. We analyzed the effects of dexamethasone (DEX) on uterine sympathetic denervation elicited by short- and long-term exposure to estrogen of intact prepubertal rats. We also studied the effects of DEX on the physiological degeneration of uterine sympathetic nerves at term pregnancy. Changes in innervation were assessed quantitatively by using computer-assisted methods on uterine cryostat tissue sections stained for tyrosine hydroxylase. At 24 h following treatment of prepubertal rats (25 days of age) with 1 μg or 2.5 μg estrogen, marked increases in uterine size and reductions in the percentage nerve area were observed. Co-administration of DEX (4 mg/kg) attenuated both these short-term estrogen-induced effects. Treatment of 19-day-old rats with a single dose of 25 μg estrogen provoked, at 26 days of age, a 54% reduction in the total nerve area. This reduction was abolished by the co-administration of nine doses of DEX (0.5 mg/kg) at 18–26 days of age. Treatment of rats with the same regime of DEX alone increased the total nerve area by 46% of the control values. Studies of control pregnant rats revealed the unexpected presence of intrauterine nerve fibers at term. Treatment of pregnant rats with six doses of DEX (4 mg/kg) at 16–21 days of age had no effects on the density of uterine sympathetic nerves. These results suggest that DEX has growth-promoting effects on immature uterine sympathetic nerves and may antagonize the degenerative effects elicited by long-term exposure to estrogen. This work was partially supported by PEDECIBA, Universidad de la República, Montevideo, Uruguay. The Third World Academy of Sciences (TWAS) supported the visit of A.I. Frías to the Laboratorio de Biología Celular (IIBCE, Montevideo, Uruguay).     

Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells

Eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 bind specifically and exclusively to CC chemokine receptor (CCR) 3, which is a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Bronchial epithelial cells represent an important source of chemokines, and thus we investigated in vitro and in vivo expression of eotaxin-2 and eotaxin-3 in bronchial epithelial cells in comparison with that of eotaxin-1. Immunohistochemistry showed increased expression of both eotaxin-2 and eotaxin-3 in addition to eotaxin-1 in asthmatics. Considerable amounts of eotaxins were secreted by bronchial epithelial lineage. As with eotaxin-1 production, generation of eotaxin-2 and eotaxin-3 by bronchial epithelial cells was up-regulated by IL-4 and IL-13, and attenuated by IFN-gamma and glucocorticoids. In addition to eotaxin-1 expression, but also eotaxin-2 and eotaxin-3 expression in the bronchial epithelium should be taken into consideration when developing the therapeutic strategies to treat eosinophilic airway diseases.     

Immunohistochemical localization of the eosinophil major basic protein in the uterus horn and cervix of the rat at term and after parturition

Summary Distribution of the eosinophil major basic protein (MBP) was studied in the rat uterus horn and cervix by means of immunohistochemistry using an antiserum raised against rat MBP. Various hormonal contexts were investigated: pre- and post-parturition, the estrous cycle, and ovariectomy followed by hormonal treatment or without treatment. MBP was detectable in the cervix as early as 12 h post-partum, appearing in the stroma close to the myometrium. The MBP had spread throughout the stroma toward the luminal epithelium after a few days. In contrast, no MBP was seen in sections of the corresponding pre- and post-partum uteri and in the pre-partum cervix. In cycling rats, MBP was distributed equally in the cervix and uterus and was more abundant during proestrus and estrus. In ovariectomized rats and in ovariectomized rats subsequently treated with progesterone, no MBP was detected in the cervix or uterus. In the cervix of ovariectomized rats treated with estradiol, MBP first appeared in the muscle layer situated between the two cervical lumina and then reached the stroma; within a few days only the stroma was stained. Inversely, in the uterus MBP-staining first appeared in the stroma. In conclusion, analysis of the distribution of MBP in rat uterus revealed a marked difference in the response of the cervix and horn to a hormonal environment.     

Ozone-induced inflammation assessed in sputum and bronchial lavage fluid from asthmatics: a new noninvasive tool in epidemiologic studies on air pollution and asthma

We investigated correlations between ozone-induced increases in inflammatory markers in induced sputum and in bronchial lavage fluid. Sixteen volunteers with intermittent asthma participated in a placebo-controlled parallel study with two exposures. Six days before and 16 h after the first exposure to ozone (0.4 ppm during 2 h) sputum was induced with hypertonic saline. This resulted in a significant increase in the sputum levels of eosinophil cationic protein (ECP; 1.8-fold; p = .03), neutrophil elastase (5.0-fold; p = .005) and the total cell number (1.6-fold; p = .02). After 4 weeks, a second exposure was randomized for air or ozone. Six days before and 16 h after the second exposure a bronchial lavage was performed. ECP values in sputum and in bronchial lavage fluid obtained after ozone correlated significantly (Rs = .79; p = .04), as did interleukin-8 (IL-8) values (Rs = .86; p = .01), and the percentage eosinophils (Rs = .89; p = .007). Moreover, the ozone-induced changes in percentage eosinophils observed in sputum and lavage fluid were highly correlated (Rs = .93; p = .003). In conclusion, changes in eosinophils, IL-8, and ECP markers induced by ozone and measured in sputum reflect the inflammatory responses in the lower airways of asthmatics, and may provide a noninvasive tool in epidemiologic studies on air pollution and asthma.     

Eosinophils attenuate arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway

Rheumatoid arthritis (RA) represents a type of autoimmune disease that mainly affect the joints due to persistent synovitis. Eosinophils were Th2 effector cells that have been shown to have anti-inflammatory role recently. In this study, we aimed to investigate the effects of eosinophils transfer on arthritis and underlying mechanisms. DBA/1 mice were induced with collagen-induced arthritis (CIA) and treated with purified eosinophils at different time points. We showed that eosinophils transfer attenuated arthritis in CIA mice. Meanwhile, TNF-α, IL-6, IL-12 and iNOS levels were decreased whereas TGF-β, IL-10, IL-13 and Arg1 levels were increased after eosinophil transfer. In vitro stimulation of bone marrow-derived macrophage (BMDM) with LPS and IFN-γ induced high expression of CD68, iNOS, TNF-α, IL-6, and IL-12, while treatment with eosinophils downregulated their expression levels. Furthermore, high levels of p-IκB and p-P38 expression in BMDM induced by LPS and IFN-γ could be suppressed by eosinophil treatment, and a P38 or IκB inhibitor accelerated the effect of eosinophils on macrophage polarization. Our results demonstrate that eosinophils exert anti-inflammatory effects in arthritis by inducing M2 macrophage polarization via inhibiting the IκB/P38 MAPK signaling pathway.     

A new stain for identification of avian leukocytes

Differential staining of avian leukocytes was achieved within 6 min following brief fixation in a methanolic solution of C.I. acid red 360 followed by immersion in a mixture containing C.I. basic blue 41, C.I. basic blue 141, and C.I. acid red 52. Heterophils contained black angular and punctate granules. Eosinophils contained bright purple granules. Lymphocytes displayed red nuclei and blue cytoplasm. Monocytes contained red-brown nuclei and lavender cytoplasm. Basophils showed red-orange granules. Thrombocytes stained deep purple. Compared to traditional panoptic stains like Wright's or Giemsa's, the new staining method provides brighter colors, more precise details of cellular structures, and shorter staining time. Significantly, it facilitates identification of avian leukocyte species based on differences in color as well as differences in size and shape.     

AMCase、Eotaxin-3及IL-13在动物模型中的研究进展

人类疾病的动物模型（AnimalModelofHumanDiseases）是生物医学科学研究中所建立的动物实验对象和材料。近年来,随着对动物模型的研究,各种人类疾病的实验模型得到广泛应用。酸性哺乳动物壳多糖酶（AMCase）作为壳多糖酶家族重要成员之一,与其下游信号分子嗜酸性粒细胞趋化因子（eotaxin．3）以及白细胞介素13（IL-3）的级联免疫反应,近年来成为了动物模型研究中的热点。本文总结了AMCase、eotaxin．3及IL-13在动物模型中的研究进展及其临床意义。