Increased lymphocyte trafficking to colonic microvessels is dependent on MAdCAM-1 and C-C chemokine mLARC/CCL20 in DSS-induced mice colitis

Although enhanced lymphocyte trafficking is associated with colitis formation, little information about its regulation is available. The aim of this study was to examine how the murine liver and activation-regulated chemokine (mLARC/CCL20) contributes to lymphocyte recruitment in concert with vascular adhesion molecules in murine chronic experimental colitis. T and B lymphocytes isolated from the spleen were fluorescence-labelled and administered to recipient mice. Lymphocyte adhesion to microvessels of the colonic mucosa and submucosa was observed with an intravital microscope. To induce colitis, the mice received two cycles of treatment with 2% dextran sodium sulphate (DSS). In some of the experiments antibodies against the adhesion molecules or anti-mLARC/CCL20 were administered, or CC chemokine receptor 6 (CCR6) of the lymphocytes was desensitized with excess amounts of mLARC/CCL20. Significant increases in T and B cell adhesion to the microvessels of the DSS-treated mucosa and submucosa were observed. In chronic colitis, the accumulation of lymphocytes was significantly inhibited by anti-mucosal addressin cell adhesion molecule (MAdCAM)-1 mAb, but not by anti-vascular cell adhesion molecule-1. In DSS-treated colonic tissue, the expression of mLARC/CCL20 was significantly increased, the blocking of mLARC/CCL20 by monoclonal antibody or the desensitization of CCR6 with mLARC/CCL20 significantly attenuated the DSS-induced T and B cell accumulation. However, the combination of blocking CCR6 with MAdCAM-1 did not further inhibit these accumulations. These results suggest that in chronic DSS-induced colitis, both MAdCAM-1 and mLARC/CCL20 may play important roles in T and B lymphocyte adhesion in the inflamed colon under flow conditions.     

Increased CD8+ T Cell Apoptosis in Scleroderma Is Associated with Low Levels of NF-κB

Our objectives were (1) to compare lymphocyte subpopulation apoptosis rates in SSc patients versus healthy controls and (2) to compare Bcl-2 and NF-kappa B expression in cultured CD8 lymphocytes of SSc patients versus controls. Peripheral blood samples were obtained from 27 SSc patients meeting the American College of Rheumatology criteria for SSc and 28 healthy individuals. Mononuclear cells were isolated by Ficoll-Hypaque density gradient separation and cultured for 48 hr. For determination of apoptosis within specific cell populations, samples were labeled with PE-conjugated monoclonal antibody to CD8, CD4, and a FITC-conjugated monoclonal antibody to Annexin V. Flow cytometry was carried out with a FACS operating with Cellquest software. CD8+ lymphocytes were positively selected with magnetic microbeads conjugated to antihuman CD8. CD8 T cells were separated, then incubated with activation for 48 hr, and NF-kappa B and Bcl-2 analysis was carried out using Western immunoblotting. The CD4:CD8 ratio was increased in SSc compared to controls (2.6 +/- 1.13 vs.1.87 +/- 0.76; P = 0.018). The spontaneous apoptosis rate of SSc CD8 lymphocytes was increased compared to that of controls of (21.9 +/- 13.7 vs. 13.3 +/- 9.9; P = 0.019). No difference was found in the rate of CD4 apoptosis of SSc patients versus controls (9.8 +/- 5.2 vs. 7.18 +/- 4.89%; P = ns). The expression of NF-kappa B in SSc CD8 lymphocytes was decreased compared with that of CD8 lymphocytes from healthy controls (144 +/- 13 vs. 188 +/- 11; P = 0.018). Whereas expression of Bcl-2 was similar in activated CD8+ T cells of SSc patients and healthy controls, CD8+ T cell apoptosis rate was found to be in reverse correlation with expression of NF-kappa B in these cells ( r = - 0.53, P = 0.029). The increased CD4:CD8 ratio in SSC may result from increased CD8+ T cell apoptosis. Increased SSc CD8 T cell apoptosis is associated with low levels of NF-kappa B.     

Isolation and partial characterisation of neuronal growth cones from neonatal rat forebrain

We have devised a method for the isolation of viable neuronal growth cones from neonatal rat forebrain. The method involves differential and density gradient centrifugation and exploits the relatively low buoyant density (approximately 1.018 g/cm3) of growth cones. There are no known biochemical markers for growth cones and it was necessary therefore to monitor for their presence during the isolation using transmission electron microscopy. Several criteria were used to identify isolated growth cones including the presence of filopodia, an extensive system of branching, tubular smooth endoplasmic reticulum and a region rich in microfilaments subjacent to the plasma membrane. These morphological features are similar to those of growth cones identified unequivocally in intact developing brain and in tissue culture. Electron microscopical analysis showed that greater than 90% of membrane-bound, identifiable objects in one fraction were growth cones by these criteria. The major contaminant consisted of membrane sacs and vesicles of unidentified origin. There were only small amounts of isolated rough endoplasmic reticulum and mitochondria. Isolated growth cones were roughly spherical in shape with a diameter of 1.9 +/- 0.5 micron (mean +/- 1 SD). They usually contained mitochondria, large granular vesicles and small vesicles, and occasionally contained coated vesicles, lysosomes, lamellar bodies and multivesicular bodies, and only very rarely, intermediate filaments. Occasionally, growth cones had rudimentary synapses on them. The viability of isolated growth cones was investigated by observing their behaviour in short-term culture. After a few hours in culture on poly-D-lysine-coated coverslips, growth cones flattened down and extended filopodia-like processes. This behaviour was inhibited by cytochalasin B and reversibly by cold (4 degrees C). We conclude that physiologically active growth cones can be isolated rapidly and in large numbers by the method described here.     

Time course of the increase in airway responsiveness associated with late asthmatic reactions to toluene diisocyanate in sensitized subjects

To understand better the mechanism of the increase in airway responsiveness associated with late asthmatic reactions, we determined the time course of toluene diisocyanate (TDI) effect on airway responsiveness in six sensitized subjects who exhibited a late asthmatic response after TDI exposure (0.018 +/- 0.005 ppm, 30 min) in the laboratory. Airway responsiveness was assessed before TDI exposure and then at 8 hr, 1 day, 1 wk, and 1 mo after TDI exposure. To assess responsiveness we determined the provocative dose of methacholine causing a decrease in FEV1 of 20% (PD20FEV1). The methacholine PD20 decreased from 0.50 mg geometric standard error of the mean (GSEM = 1.54) to 0.06 mg (GSEM = 1.55) (p less than 0.001) at 8 hr after exposure to TDI, was still decreased to 0.15 mg (GSEM = 1.93) (p less than 0.05) at 1 day, returned to 0.26 mg (GSEM = 1.91) (p greater than 0.05) at 1 wk, and returned to 0.43 mg (GSEM = 1.71) at 1 mo, indicating that full recovery occurred within 1 to 4 wk. These results demonstrate that TDI-induced late asthmatic response is associated with a reversible increase in airway responsiveness to methacholine and suggest that the TDI effect is linked to an acute inflammatory response in the airways.     

The influence of molecular structure on the affinity and efficacy of some β-adrenoceptor agonists

Summary The affinity and efficacy of a number of sympathomimetic amines structurally related to prenalterol and the selective ₁-adrenoceptor agonist RO 363 were determined using a combination of radioligand binding and organ bath techniques. Affinity of the molecules (pK _D) was calculated from their ability to displace the radioligand [¹²⁵I]iodocyanopindolol ([¹²⁵I]CYP) from -adrenoceptor sites in left atrial (₁) and uterine (₂) membrane homogenates. These pK _D values were used to calculate efficacy from the positive inotropic and uterine relaxant responses elicited by the drugs in organ bath experiments. The drugs studied were either arylethanolamines i.e., (–)-isoprenaline (ISO), p-hydroxyisoprenaline (pOH-ISO), compounds XIV and XVI or aryloxypropanolamine-derivatives, i.e., oxymethylene-isoprenaline (OM-ISO), prenalterol and Compound XI which possessed ap-phenol or catechol ring and an isopropyl or a homoveratryl amine substituent. Only ISO, OM-ISO, pOH-ISO and Compound XVI were active as agonists in both tissue preparations. These drugs were partial agonists which exhibited a wide range of pD₂ values and did not display any marked selectivity for either -adrenoceptor subtype. Compound XI and prenalterol were inactive as agonists and together with the partial agonists behaved as competitive antagonists to ISO in the two preparations. All drugs tested displaced [¹²⁵I]CYP from -adrenoceptor sites, however, there was also a wide range of potency amongst the drugs.Analysis of the structure-affinity and structure-efficacy relationships indicated that removal of the 3-hydroxyl group from the catechol ring reduces both affinity and efficacy without altering the selectivity of the drug for either -adrenoceptor subtype. While aryloxypropanolamine derivatives have generally higher affinities than arylethanol-amines, especially at -adrenoceptor sites, their efficacies are generally reduced at both -adrenoceptors. The presence of a homoveratryl group in aryloxypropanolamines enhances slightly the affinity for ₁- and reduces affinity for ₂-adrenoceptors. With this amine group, efficacy is markedly reduced at ₂- as opposed to ₂-adrenoceptor sites.Thus for prenalterol, the small degree of cardioselectivity can be attributed to the oxymethylene group whilst its lack of agonist activity (i.e., efficacy) reflects a combined action of this group and the absence of the 3-hydroxyl group on the phenyl ring. In RO363 it can be deduced that the oxymethylene group, together with the homoveratryl substituent are responsible for the observed selective affinity of the drug for ₁- as opposed to ₂-adrenoceptors.     

A qualitative exploration of the mechanisms,pathways and public health outcomes of a city centre 20mph speed limit intervention: The case of Belfast,United Kingdom

Twenty miles per hour (mph) speed limits can impact the health of the public (e.g., road safety, active travel). However, a better understanding of how individuals experience 20mph limits is required, to ensure interventions are cognisant of perceptions and potential un/intended outcomes. Focus groups (n = 9, 60 participants) to explore the Belfast 20mph intervention highlighted divergent perspectives and experiences including: 12 mechanisms (e.g., limited awareness), 15 pathways (e.g., reduced driving speed→improved liveability) and 10 public health outcomes (e.g., increased cyclist safety). Future interventions should consider un/intended outcomes and implement strategies to enhance effectiveness and mitigate harms (e.g., through training, enforcement).     

炎症细胞与淋巴细胞在鼻息肉发病机理中的相关性研究

目的：探讨炎症细胞、淋巴细胞及浆细胞在鼻息肉发病中的作用;方法：采用免疫组化SP法及HE、甲苯胺兰染色对34例鼻息肉和30例正常中鼻甲粘膜进行研究;结果：鼻息肉中嗜酸性粒细胞阳性率显著高于对照组织（P＜0.01）;鼻息肉中肥大细胞数量显著多于对照组织（P＜0.01）,肥大细胞数量在吸入性变应原皮肤试验阳性组与阴性组间无显著性差异;鼻息肉中T淋巴细胞阳性细胞（CD43）和B淋巴细胞阳性细胞（CD20）、浆细胞数量显著多于对照组织（P＜0.01）,鼻息肉中T淋巴细胞与B淋巴细胞之间无显著性差异;结论：鼻息肉中存在活跃的细胞免疫和体液免疫,与嗜酸性粒细胞、肥大细胞及中性粒细胞共同参与鼻息肉的发病。     

不同烧结温度对双相钙磷陶瓷颗粒材料介孔结构及其成骨性能的影响

目的通过体内外实验检测不同烧结温度下制备的不同介孔直径双相钙磷陶瓷（biphasic calcium phosphate，BCP）颗粒材料成骨能力差异，为筛选具备更好临床应用参数的 BCP 材料提供依据。方法将羟基磷灰石（hydroxyapatite，HA）及 β-磷酸三钙（β-tricalcium phosphate，β-TCP）以 8∶2 比例混合后，分别在 1 050、1 150 及 1 250℃ 下烧制 3 h 制备 3 种 BCP 材料（分别设为材料1、2、3），比表面积测试法（Brunauer-Emmett-Teller test，BET）测量材料的颗粒内部孔隙率及介孔直径、体积、面积，X 线衍射（X-ray diffraction，XRD）评估材料组成成份，扫描电镜观察材料微观表面形态。体外将第 3 代 SD 大鼠 BMSCs 与各材料共培养 7 d（分别设为 A、B、C 组），扫描电镜观察细胞黏附情况，鬼笔环肽染色观察 BMSCs 贴附于材料表面后的形态，细胞计数试剂盒 8 法检测细胞增殖活性。体内建立比格犬异位成骨模型：取 9 只比格犬，于每只犬双侧竖脊肌内制作 9 个肌袋，将肌袋随机分为 3 组（每组 3 个/只），A、B、C 组分别置入材料 1、2、3。术后 1、2、3 个月分别麻醉 3 只比格犬取材行 HE、Masson 及番红固绿染色，计算 BCP 间隙中的成骨面积比；行实时荧光定量 PCR（real-time fluorescence quantitative PCR，qRT-PCR）检测成骨相关基因 ALP、骨桥蛋白（osteopontin，OPN）、骨钙素（osteocalcin，OC）的表达。结果BET 检测示随烧结温度增加，颗粒内部孔隙率无明显变化，但介孔直径、体积及面积逐渐减小；XRD 检测示 3 种材料均可见 HA 及 β-TCP 两种 X 线衍射波；扫描电镜观察示 3 种材料表面有广泛分布的微孔，孔间有空隙相连。体外实验示 BMSCs 在 3 种材料表面黏附、增殖，B、C 组材料的细胞生物相容性优于 A 组。体内实验结果示，术后 2 个月开始 3 种材料颗粒孔隙内即可见明显的骨样组织沉积。各组成骨面积比随时间延长均增加，术后 2、3 个月 A 组成骨面积比显著高于 B、C 组，1 个月时显著高于 B 组（P<0.05）。qRT-PCR 检测示，A 组成骨相关基因表达在 2 个月时出现峰值，B、C 组各成骨相关基因表达随时间延长逐渐增加。术后 1 个月 A 组 ALP 和 OPN mRNA 相对表达量显著高于 B、C 组，术后 2 个月 A 组 OC mRNA 相对表达量显著高于 B、C 组，术后 3 个月 B、C 组 ALP mRNA 相对表达量及 B 组 OPN mRNA 相对表达量显著高于 A 组（P<0.05）；其余各时间点各组间比较各基因 mRNA 相对表达量差异均无统计学意义（P>0.05）。 结论不同烧结温度下制备的 BCP 材料，其介孔直径随温度增加而减小。不同介孔直径的 BCP 材料异位成骨能力存在差异，其中直径为 12.57 nm 的 BCP 材料能更早激活成骨基因，具备更强的成骨能力。介孔直径可作为一个优化 BCP 材料成骨能力的指标。     

锌指蛋白 A20 对兔腰椎间盘退变影响的实验研究

目的探讨锌指蛋白 A20 对兔腰椎间盘退变的影响。方法取 3 月龄新西兰大白兔 26 只，体质量 2.0～2.5 kg，经腹细针穿刺法制备 L_3、4、L_4、5、L_5、6 椎间盘退变模型，其中 24 只术后 4 周 MRI 检查明确造模成功，随机分为 4 组（n=6），于目标椎间盘中分别注射锌指蛋白 A20 过表达腺病毒（过表达 A20 组）、空载体腺病毒（空载体组）、PBS 液（对照组）、锌指蛋白 A20干扰腺病毒（干扰 A20 组）。于注射前 1 d 及注射后 1、2、3、6 d 行生物反应综合评分；注射后 2、4、8 周，各组行 MRI 检查并测量 T2 弛豫时间（T2 信号值）后，取材行阿利辛蓝染色观察椎间盘髓核细胞退变情况，免疫组织化学染色检测锌指蛋白 A20 以及椎间盘退变相关指标（Ⅱ型胶原、蛋白聚糖）的表达，Western blot 检测锌指蛋白 A20、NF-κB 结合蛋白［P65、磷酸化 P65（phosphate P65，P-P65）、Ⅱ型胶原、蛋白聚糖］、自噬相关蛋白［LC3 （LC3Ⅱ/LC3Ⅰ）、P62］以及炎症因子（TNF-α、IL-1β）的表达。 结果各组注射后各时间点生物反应综合评分均明显低于注射前 1 d（P<0.05）；注射后 6 d 干扰 A20 组评分明显低于其他组（P<0.05），其他组间比较差异均无统计学意义（P>0.05）。MRI 检测提示，注射后 2、4、8 周过表达 A20 组 T2 信号值均最高（P<0.05），2、4 周时干扰 A20 组最低（P<0.05），其余组间差异均无统计学意义（P>0.05）。阿利辛蓝染色显示，注射后 4 周过表达 A20 组蛋白聚糖含量最高（P<0.05）、干扰 A20 组最低（P<0.05）；8 周时过表达 A20 组蛋白聚糖含量显著高于其他组（P<0.05），其他组间比较差异无统计学意义（P>0.05）。免疫组织化学染色示，锌指蛋白 A20、Ⅱ型胶原、蛋白聚糖表达过表达 A20 组最高（P<0.05），干扰 A20 组上述蛋白表达最低（P<0.05）。Western blot 检测示锌指蛋白 A20、蛋白聚糖、Ⅱ型胶原、LC3 （LC3Ⅱ/LC3Ⅰ）蛋白相对表达量过表达 A20 组最高、干扰 A20 组最低，而 P-P65、TNF-α、IL-1β、P62 蛋白相对表达量过表达 A20 组最低、干扰 A20 组最高，与其他组比较差异均有统计学意义（P<0.05）；各组 P65 蛋白相对表达量差异均无统计学意义（P>0.05）。 结论锌指蛋白 A20 能通过抑制炎症反应，有效延缓兔腰椎间盘退变的进程。