Constitutive expression of interleukin 4 in vivo does not lead to the development of T helper 2 type CD8+ T cells secreting interleukin 4 or interleukin 5.

Interleukin 4 (IL-4) has been shown to commit CD8+ T cells to a T helper (Th) 2 functional phenotype in vitro. To study the effects of IL-4 on CD8+ T cell development in vivo we analysed the CD8+ T cell phenotype in mice constitutively expressing IL-4. Purified CD8+ T cells from uninfected or flu infected IL-4 transgenic (tg) animals produced no detectable IL-4 or IL-5 after in vitro stimulation on anti-CD3 coated plates. However, CD8+ T cells from IL-4 tg mice could be converted into IL-4 and IL-5 producers in vitro in the presence of exogenous added IL-4, showing that these cells were still responsive to IL-4. IL-4 tg mice also showed a delay in influenza virus clearance from the lung, which was probably due to the observed reduction of total CD8+ T cell numbers in the IL-4 tg animals since IL-4 tg CD8+ T cells showed normal levels of influenza-specific cytotoxicity in comparison to controls. Taken together these results suggest that CD8+ T cells are not necessarily switched to a Th2 phenotype by the presence of IL-4 and that some other factor(s) may be important in the switch process of CD8+ T cells in vivo, since the addition of IL-4 during CD8+ T cell activation in vitro leads to Th2 type CD8+ T cells secreting IL-4 and IL-5.     

脑发育不同阶段慢性铅暴露对大鼠在体海马不同时间一氧化氮含量及合酶活性的影响

目的：比较脑发育不同阶段低浓度慢性铅暴露对大鼠在体海马一氧化氮含量及合酶活性的差异。方法：分别在仔鼠出生21天、42天、63天时测定对照组、断乳后暴露组、母体暴露组和持续暴露组仔鼠血铅含量和海马NO含量和NOS活性,并与对照组进行比较。结果：各暴露组在不同时间血铅含量均高于对照组（P〈0.05）。断乳后铅暴露组和持续铅暴露组NOS活性与对照组相比均有显著性差异（P〈0.05）,出生21天时明显高于对照组,而42天和63天明显低于对照组。持续暴露组NO含量与对照组相比有明显差异,出生21天时明显高于对照组,而42天和63天明显低于对照组。结论：脑发育任一阶段的慢性铅暴露均对海马NO含量和NOS活性有影响。与发育成熟海马相比,未成熟期海马对铅的神经毒性更为敏感,突触可塑性更易受损。     

Smad4蛋白及mRNA在卵巢不同发育阶段的表达

目的：探讨Smad4在不同发育阶段大鼠卵巢中蛋白及mRNA的表达。 方法： 选择不同发育时期大鼠卵巢，运用免疫组化方法检测卵巢中Smad4蛋白表达，并进行图像分析；采用半定量逆转录聚合酶链反应(RT-PCR)方法检测Smad4 mRNA在卵巢中的表达。 结果： 免疫组化结果显示Smad4主要表达在各级卵泡中，在卵巢发育早期，Smad4主要在原始卵泡和窦前卵泡中表达；随着卵巢的发育成熟，Smad4在窦状及成熟卵泡颗粒细胞和卵泡膜细胞的表达与间质细胞比较无显著差异（P>0.05）。Smad4在卵泡中的表达强度也发生了变化：随着卵泡的发育，Smad4在窦状及成熟卵泡卵母细胞的表达与窦前卵泡卵母细胞比较明显减弱（P<0.05，P<0.01）；在卵泡膜细胞的表达逐渐增强（P<0.01），而在各级卵泡颗粒细胞中的表达无显著差异（P>0.05）。RT-PCR结果显示各阶段卵巢均有mRNA的表达，从第3周起Smad4 mRNA的表达明显增强，与生后1 d比较差异显著（P<0.05）。 结论： 卵巢内存在Smad4，提示TGF-β家族对卵泡发育的调节很可能是通过Smad信号转导模式实现的。     

Analysis of ecdysteroids in different developmental stages of Hymenolepis diminuta

Prepatent and patent adult Hymenolepis diminuta from the intestines of rats, H. diminuta eggs recovered from the faeces of rats harbouring patent infections, and infective cysticercoids from the beetle intermediate host were analysed for free and conjugated ecdysteroids. Adult worms and eggs contained both free ecdysteroids and hydrolysable polar conjugated ecdysteroids, with comparatively large amounts of immunoreactive material also being detected following hydrolysis of the possible apolar conjugated ecdysteroid fraction. Free ecdysteroids were not detected in the cysticercoid sample. The concentration of free ecdysteroids in H. diminuta eggs was higher than that detected in the tissues of the adult worms. Ecdysone and 20-hydroxyecdysone were the major identified compounds of the free ecdysteroid fraction, whereas in the hydrolysed polar conjugated ecdysteroid fraction these two compounds were accompanied by 20,26-dihydroxyecdysone. The free ecdysteroid fraction also contained comparatively large amounts of unidentified immunoreactive material.     

T cells of different developmental stages differ in sensitivity to apoptosis induced by extracellular NAD

Extracellular nucleotides such as ATP and NAD can profoundly affect the functions of lymphocytes, macrophages, and other cells. We have recently shown that extracellular NAD induces rapid apoptosis in naive T cells by a mechanism involving the ADP-ribosylation of cell surface molecules. In the present paper, we describe that T cells of different developmental stages differ in their sensitivity to NAD-induced apoptosis. Thymocytes were less susceptible than peripheral lymph node T cells, and freshly activated cells were more resistant than resting cells. Sensitivity to NAD-induced apoptosis generally correlated with expression of the ADP-ribosyltransferase ART2.2, which is not expressed on thymocytes and shed from peripheral T cells upon activation. Our findings suggest that NAD-induced apoptosis does not play a role during thymic selection of T cells, but rather may play a role by preventing the activation of unwanted bystander T cells during an immune response, and thus may participate in the control of autoimmunity.     

A new mechanism for IL-5-dependent helminth control: neutrophil accumulation and neutrophil-mediated worm encapsulation in murine filariasis are abolished in the absence of IL-5

IL-5 production and eosinophilia are features of helminth infections, but results concerning the role of IL-5 and eosinophils (EP) in worm control are contradictory. We describe here a novel, IL-5-dependent mechanism of helminth control in vivo, using a fully permissive murine filariasis model, i.e. infection of BALB/c mice with Litomosoides sigmodontis. Worm control was exerted by the formation of inflammatory nodules around adult filariae which initially remained alive but were eventually killed within several weeks. The cell population essential for inflammatory nodule formation was found to be neutrophils (NP) but not EP. Neutralization of IL-5 led to a failure of both EP and NP accumulation at the site of infection (i.e. the thoracic cavity), resulting in cessation of inflammatory nodule formation around worms and in their survival. The role of NP in this process was confirmed by treatment of mice with anti-granulocyte colony stimulating factor (G-CSF) which also resulted in a lack of inflammatory nodule formation and worm killing albeit in the presence of EP. Since IL-5, due to the absence of IL-5 receptors on NP, does not act on these cells directly, it was investigated if anti-IL-5 altered the production of NP-chemotactic cytokines. In anti-IL-5-treated mice, cytokines known to promote NP accumulation like tumor necrosis factor-alpha, G-CSF and KC (IL-8) were found to be strongly reduced, while NP-deactivating cytokines like IL-10 were increased. In conclusion, IL-5 constitutes a cytokine essential for NP-mediated worm control in filarial infection.     

Characterization of fat body cells at different developmental stages of Culex pipiens

The fat body, originates from mesoderm, has many metabolic functions which changes as the embryonic development of the insect progresses. It plays an important role in the intermediate metabolism and in the metabolism of proteins, lipids and carbohydrates. It has roles in synthesis, absorption and storage of nutrients from hemolymph. It is also responsible for the production of immunological system components, antibacterial compounds and blood clotting proteins. The most common type of fat body cells are trophocytes (the basic cells of the fat body) and oenocytes are found associated with the fat body. In this study, it is aimed at determining the cell types contained in the fat body of Culex pipiens at different developmental stages as well as identifying the molecules such as carbohydrate, protein and lipid contained in each of these cells. Knowing the regional distribution of the fat body cells and the concentration of its content at each developmental stage is important in understanding the process related to its physiology and it may help in fighting against the pest C. pipiens, which is a vector species for many contagious diseases observed in humans and other species.To achieve our goal, we have employed different histochemical techniques (fixatives and staining methods) for staining C. pipiens preparates of different developmental stages and analyzed the structure of the fat body, its distribution, its cell types and the macromolecular contents of the cells. We only observed trophocytes and oenocytes as fat body components in C. pipiens. The trophocytes had all the three macromolecules (lipids, proteins, carbohydrates) in the cytoplasm varying in concentration between the different regions and different stages. The oenocytes were observed below the integument as well as between the muscles in the larvae of Culex pipiens. They were present either as single cells or in clusters and also varied in size. Their cytoplasm was stained strongly for proteins when bromophenol blue staining was applied, but it was rather heterogeneous due to the lipid inclusions. On the contrary, oenocytes were not observed among the adult C. pipiens preparations.     

OPS法玻璃化冷冻小鼠不同成熟时期卵母细胞的研究

目的比较OPS法玻璃化冷冻小鼠不同成熟时期卵母细胞解冻后体外成熟、体外受精及胚胎发育能力。方法收集ICR小鼠生殖泡期（GV）未成熟卵和成熟卵,分五组行OPS（open pulled straw,开放式拉细麦管）法玻璃化冷冻,Ⅰ组：直接冷冻GV期卵;Ⅱ组：GV期卵在体外培养8h后冷冻;Ⅲ组：GV期卵在体外培养16h后冷冻;Ⅳ组：GV期卵体外培养成熟（IVM）后冷冻;Ⅴ组：直接冷冻体内成熟卵。解冻后比较各组卵受精率、卵裂率及8细胞胚胎形成率。结果Ⅰ组（GV期卵）冷冻解冻后存活率达57．5％,显著高于Ⅱ组（34．1％）,Ⅲ组（32．0％）,Ⅳ组（29．6％）及Ⅴ组（31．0％）（P〈0．01）。各组体外受精率、卵裂率及8细胞胚胎形成率比较,差异不显著（P〉0．05）。结论采用OPS法玻璃化冷冻小鼠卵母细胞,冷冻GV期卵较冷冻其它成熟时期卵效果好。     

Partial denervation of the rat soleus muscle at two different developmental stages

Rat soleus muscles were partially denervated at two developmental stages. The L5 ventral ramus was sectioned in rats which were 4-6 days old, when the motor unit size of soleus muscles was still large, and at 17-19 days, when motor unit territory reached its adult value. The response of axons in the L4 ventral ramus to this procedure was then investigated. The removal of the L5 ventral ramus at 4-6 days results in an initial brief increase of motor unit size, after which the motor units retain the territory they occupied at 4-6 days. After removal of the L5 ventral ramus at 17-19 days, the L4 ventral ramus is able to expand to occupy a territory comparable in size to that of animals operated at 4-6 days. In both cases the final percentage of mean motor unit tension is two- to three-fold greater than that in normal muscles. Although the final motor unit territory is similar for both groups, it is achieved by different mechanisms. In animals operated on at 4-6 days the normal elimination of terminals does not occur, and the large neonatal motor units are retained, whereas in animals operated on at 17-19 days the peripheral field of L4 axons expands by axonal sprouting.     

不同发育时段大鼠心肌组织中超极化激活环核苷酸门控阳离子通道4阳性细胞的分布特征

目的 探讨超极化激活环核苷酸门控阳离子通道4(HCN4)在不同发育时期大鼠心肌组织中各类细胞的表达规律,为进一步研究HCN4对心肌的作用提供形态学依据。方法 出生1 d、1月、3月、6月健康SD大鼠各15只。取新鲜心脏,石蜡切片和冷冻切片,利用免疫组织化学和荧光技术观察不同发育时段大鼠心肌组织HCN4阳性细胞的表达分布情况及形态学特征。利用Western blotting 技术观察左右心室HCN4的蛋白含量。 结果 免疫组织化学和荧光染色结果显示,HCN4阳性细胞在出生后1 d、1月、3月、6月的血管平滑肌细胞,心内膜和血管的内皮细胞,心外膜的间皮细胞、成纤维细胞,大量心房肌细胞和少量心室肌细胞、传导组织细胞均有表达,但不同部位的表达细胞数量不均;Western blotting结果显示,心室HCN4蛋白随着月龄的增加表达逐渐降低。 结论 HCN4在大鼠心肌组织中的表达随年龄的增加逐渐下降,但增殖能力较强的细胞持续存在,提示HCN4可能对心肌组织中的多种细胞的生存起调控作用。     

Differential bioassay of interleukin 2 and interleukin 4

The T cell-derived lymphokines interleukin 2 (IL-2) and interleukin 4 (IL-4, originally BSF-1) both exhibit T cell growth-promoting activity. Recent observations that T cell lines commonly used as indicator cells in IL-2 bioassays also proliferate in response to IL-4 demonstrate the lack of specificity of these bioassays for IL-2. In this report we describe a bioassay method to differentiate IL-2 and IL-4 through the parallel use of two T cell lines with defined lymphokine specificity. The IL-2-responsive CT6 cell line, when used in conjunction with the IL-2- and IL-4-responsive HT-2 cell line, allows for the differentiation of IL-2 and IL-4 in conditioned media.     

Schlemm's canal and the collector channels at different developmental stages in the human eye

We studied Schlemm's canal (SC) and other posttrabecular structures of aqueous humor outflow in eyes from human fetuses (at 24, 26, 33 and 36 weeks of gestation), eyes from normal children (2 months and 8 years old) and normal adult eyes, using mesoscopy/diaphanization and light microscopy. This study points to the presence of a collector canal parallel to the outer wall of SC (the outer collector). At 24 weeks of gestation, the SC was observable throughout the entire circumference (360 degrees) and the anlage of the outer collector was present. At 26 weeks of gestation, the outer collector was still growing. At 33 weeks of gestation, the outer collector was parallel to the SC and had become visible in some sectors. At 36 weeks, the SC and the outer collector were clearly defined and connected by intercanal links. The intrascleral plexus was growing. At 2 months, the deep intrascleral plexus and the episcleral plexus continued to grow, developing by sectors. At 8 years of age, the same structures corresponded to those in adults. The outer collector and the intrascleral plexuses were divided into sectors not greater than 120 degrees.     

Establishment of different T cell sublines using either interleukin 2 or interleukin 4 as growth factors

Purified protein derivative reactive T cell lines were established under identical conditions with the exception that different lymphokines, namely interleukin (IL) 2 and IL 4 were employed as growth factors. IL 2 favored the development of T cell lines (LNC.2) which upon activation by concanavalin A (Con A) secreted predominantly lymphokines characteristic of T_H1 cells. By contrast, T cell lines established with the aid of IL4 as growth factor (LNC.4) produced mainly lymphokines representative of T_H2 cells. Apart from their pattern of lymphokine secretion LNC.2 and LNC.4 T cells were found to differ in their proliferative response to lymphokines and Con A. LNC.2 T cells proliferated only marginally in the presence of IL 4, Con A or a combination of Con A and IL 1. Furthermore, the IL 2-dependent proliferation of LNC.2 T cells was slightly but significantly diminished by IL 4. In contrast, LNC.4 T cells showed a substantial IL 4-induced proliferative response which was on the one hand synergistically enhanced by minimal amounts of IL 2 and, on the other hand, strongly inhibited by interferon-γ. In addition, LNC.4 T cells displayed a strong proliferation when stimulated by low concentrations of Con A in the presence of IL 1 as co-stimulator.     

不同发育阶段大鼠膀胱组织结构及Cx26表达变化的观察

目的 观察不同发育阶段大鼠膀胱组织结构的变化，及连接蛋白connexin26(Cx26)随组织发育的不同阶段而表达的变化。方法 分别取幼年、成年和老年三组大鼠膀胱，测量排空状态下膀胱壁厚度，用HE和PAS法染色观察膀胱壁组织结构变化，同时用免疫组织化学评价Cx26表达情况。结果 随着膀胱的发育成熟，膀胱壁组织显著增厚，逼尿肌肌纤维增粗，固有层结缔组织增加。早期Cx26在移行细胞层表达较弱，在成年期后稳定高表达。结论 在膀胱的发育过程中，组织结构发生显著变化，成年后移行细胞表达Cx26显著增强，与膀胱的发育和功能密切相关。     

Regulation of interleukin 4 production and of interleukin 4-producing cells.

Interleukin 4 (IL-4) is a cytokine with a broad range of functions on immune cells including the stimulation of IgE synthesis by B cells. IL-4 is synthesized by freshly isolated T cells following appropriate stimulation with either lectins or antibodies against selected surface antigens. IL-4 production has also been observed by freshly isolated FceR+ non-T, non-B cells. With T cell clones, IL-4 is produced by clones of the Th2 and Th0, but not of the Th1 type. The molecular basis for restricted cytokine production by such clones as well as the signals responsible for differentiation into either phenotype are only at the beginning to be understood.     

Cytoarchitectonic investigation of the rat spinal cord following ethylnitrosourea administration at different developmental stages

Summary We examined the histological findings and cytoarchitectonic alterations in the rat spinal cord following matrix cell degeneration caused at different developmental stages, from neural plate formation through neuroblast generation. Ethylnitrosourea (ENU) 20 mg/kg body weight was administered transplacentally to the fetuses on the 10th embryonic day (E10) to 14th. The observations were made until the 21st postnatal day. Normally, mitoses were present scatteredly in the matrix cell layer of the neural plate or neural tube on El0 or E11, and gradually restricted to the dorsal portion of the alar plate as development occurred. The localization and number of degenerative cells as well as the site and degree of neuronal decrease in the completed dysgenetic spinal cord seemed to correlate with the topography and frequency of the mitoses in the matrix cell layer at the time of ENU administration. Disorder in the pattern of cytoarchitecture of neurons was not observed. The degree of hypoplasia of the white matter was proportional to the intensity of decrease of the spinal neurons. Aberrant myelinated fibers were not seen. No reactive gliosis, fibrosis or abnormal vascularization was observed at any time.This study was supported by a Grant-in-Aid for Scientific Research (A) No. 60440046 from the Ministry of Education, Science and Culture, Japan