Piecemeal degranulation of mast cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overexpress interleukin-4 (IL-4; Tepper et al, Cell 62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med 132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem 41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.     

Three‐dimensional cultures of mouse submandibular and parotid glands: a comparative study

Freshly isolated salivary cells can be plated on an extracellular matrix, such as growth factor‐reduced Matrigel (GFR‐MG), to induce the formation of three‐dimensional (3D) structures. Cells grown on GFR‐MG are able to form round structures with hollow lumina, capable of sustaining amylase expression. In contrast, cells grown on plastic do not exhibit these features. Our recent studies have used mouse parotid gland (PG) cells, grown on different extracellular matrices, as a model for acinar formation. However, PG cells were not able to respond to the secretory agonist carbachol beyond 5 days and did not sustain polarity over time, regardless of the substratum. An alternative option relies in the use of mouse submandibular glands (SMG), which are more anatomically accessible and yield a larger number of cells. We compared SMG and PG cell clusters (partially dissociated glands) for their ability to form hollow round structures, sustain amylase and maintain secretory function when grown on GFR‐MG. The results were as follows: (a) SMG cell clusters formed more organized and larger structures than PG cell clusters; (b) both SMG and PG cell clusters maintained α‐amylase expression over time; (c) SMG cell clusters maintained agonist‐induced secretory responses over time; and (d) SMG cell clusters maintained secretory granules and cell–cell junctions. These results indicate that mouse SMG cell clusters are more amenable for the development of a bioengineered salivary gland than PG cell clusters, as they form more organized and functional structures. Copyright © 2014 John Wiley & Sons, Ltd.     

The epidemiology of multiple sclerosis in Queensland, Australia

An epidemiological survey of multiple sclerosis (MS) in the State of Queensland was undertaken with its prevalence day being the national census day on June 30th, 1981, 20 years after a regional survey within the State. The relationship between increasing prevalence of MS and increasing south latitude within the State of Queensland which was suggested by the 1961 study was confirmed in the present study. The prevalence rate had increased significantly over the 20-year period between the studies but the State remained a medium frequency zone for MS (prevalence rate between 5 and 29 per 100 000 of population). Although a real increase in disease frequency could not be excluded as a contributing factor to the rise in prevalence, it was most likely due predominantly to an increase in life expectancy amongst the MS population and also in differential migration of a population at a greater risk of developing MS than the indigenous population. The proportions of Australian-born patients who had migrated to Queensland from the higher risk southern regions of Australia or travelled overseas to countries known to be high-risk for MS prior to disease onset, had fallen between the two surveys thus exerting, if anything, a negative influence on the change in prevalence. Analysis of MS prevalence rates amongst migrant populations in Queensland as compared to the more southerly city of Perth in Western Australia, suggested that the risk of acquisition of MS may extend over a wider age range than is generally accepted. Finally, there was an absence of MS cases amongst the Aboriginal population in Queensland but it can only cautiously be concluded from this study that the disease is rare in these peoples.     

Oxidation of LDL by myeloperoxidase and reactive nitrogen species: reaction pathways and antioxidant protection

Oxidative modification of low density lipoprotein (LDL) appears to play an important role in atherogenesis. Although the precise mechanisms of LDL oxidation in vivo are unknown, several lines of evidence implicate myeloperoxidase and reactive nitrogen species, in addition to ceruloplasmin and 15-lipoxygenase. Myeloperoxidase generates a number of reactive species, including hypochlorous acid, chloramines, tyrosyl radicals, and nitrogen dioxide. These reactive species oxidize the protein, lipid, and antioxidant components of LDL. Modification of apolipoprotein B results in enhanced uptake of LDL by macrophages with subsequent formation of lipid-laden foam cells. Nitric oxide synthases produce nitric oxide and, under certain conditions, superoxide radicals. Numerous other sources of superoxide radicals have been identified in the arterial wall, including NAD(P)H oxidases and xanthine oxidase. Nitric oxide and superoxide readily combine to form peroxynitrite, a reactive nitrogen species capable of modifying LDL. In this review, we examine the reaction pathways involved in LDL oxidation by myeloperoxidase and reactive nitrogen species and the potential protective effects of the antioxidant vitamins C and E.     

差速贴壁技术对大鼠脑皮质星形胶质细胞纯化率的影响

目的:观察差速贴壁技术对星形胶质细胞纯化率的影响,旨在建立一套可靠的大鼠脑皮质星形胶质细胞的取材分离、纯化培养技术。方法:实验于2006-06/08在泰山医学院生命科学研究所完成。实验材料:出生2～3d的Wistar大鼠,雌雄不拘,由泰山医学院生命科学研究所实验动物中心提供。实验方法:选用出生二三天的Wistar大鼠进行脑皮质星形胶质细胞原代培养。实验分两组培养:常规培养组和差速贴壁培养组。差速贴壁培养组分别于15,30min取出,轻轻翻转培养瓶,将上清液移至另一培养瓶中,放入培养箱中继续培养。7～10d后传代,待细胞分层生长后,置于37℃摇床中250r/min振荡18h,倒掉上清液,D-Hank’s液洗3次后,加入0.25%胰酶消化,倒置显微镜下观察,待细胞突起回缩后加入含血清的培养基终止消化,用吸管反复吹打使细胞从瓶壁上脱落,细胞悬液1000r/min离心5min后,弃上清液,加入含体积分数为0.2血清的DMEM培养基混悬沉淀,接种入预先涂有L-多聚赖氨酸的培养瓶中继续培养。采用双重免疫荧光法鉴定星形胶质细胞纯度,测定积分吸光度值判断星形胶质细胞的生长状况。结果:①应用差速贴壁技术培养星形胶质细胞可明显提高星形胶质细胞纯度[常规培养组:(82±3)%,差速贴壁培养组15min:(94±2)%,差速贴壁培养组30min:(95±2)%,P<0.01]。差速贴壁需要充分的时间,15min组和30min组在提高星形胶质细胞纯度方面无明显差别。②差速贴壁培养组星形胶质细胞积分吸光度值高于常规培养组(常规培养组:528±25,差速贴壁培养组15min:972±17,差速贴壁培养组30min:996±35,P<0.05)。结论:①差速贴壁技术可明显提高星形胶质细胞纯化度,并且星形胶质细胞生长状态明显优于常规培养方法。②最佳差速贴壁时间为15min,过长差速贴壁时间对提高星形胶质细胞纯度无明显影响。     

小鼠皮肤超氧化物歧化酶活性与枸杞多糖的干预

目的:观察枸杞多糖对皮肤胶原代谢和自由基产生的影响,探讨其抗皮肤衰老的作用。方法:实验于2005-06/2006-05在广东医学院整形外科研究所完成。①实验材料:清洁级昆明小鼠60只,月龄2个月,体质量16～24g,雌雄各半。②实验分组:将小鼠随机分为正常对照组、衰老模型组和抗衰老模型组,每组20只。③实验干预:模型组每日用D-半乳糖溶液皮下注射制造衰老模型,用量和时间为80mg/(kg·d)7d,120mg/(kg·d)14d,140mg/(kg·d)14d,180mg/(kg·d)7d。正常对照组每日注射同体积的生理盐水。抗衰老模型组在注射D-半乳糖期间以枸杞多糖灌胃,剂量为20mg/(kg·d),正常对照组和衰老组则以同体积的生理盐水代之灌胃。④实验评估:42d后切取小鼠颈背部皮肤,测定超氧化物歧化酶活力、羟脯氨酸和丙二醛含量。结果:56只小鼠进入结果分析(4只死亡)。①小鼠皮肤超氧化物歧化酶活力:与正常对照组相比,衰老组和抗衰老组小鼠皮肤超氧化物歧化酶活力降低,差异有显著性意义(P<0.01);抗衰老组与衰老模型组比较,超氧化物歧化酶活力增加,差异有显著性意义(P<0.01)。②与正常对照组相比,衰老组和抗衰老组小鼠皮肤羟脯氨酸和丙二醛含量增加,差异有显著性意义(P<0.01);抗衰老组与衰老组比较,羟脯氨酸和丙二醛含量均降低,差异有显著性意义(P<0.01)。结论:枸杞多糖改善皮肤老化的作用与提高小鼠皮肤超氧化物歧化酶活力,降低羟脯氨酸、丙二醛含量,影响胶原代谢有关。     

Provocation discography as a guide to planning operations on the spine

In a prospective study we attempted to define the role of lumbar discography in the investigation of patients with low back pain with or without non-dermatomal pain in the lower limb. The records of 195 patients were studied at least two years after a technically successful operation. Of 137 patients in whom discography had revealed disc disease and provoked symptoms, 89% derived significant and sustained clinical benefit from operation. Of 25 patients whose discs showed morphological abnormality but had no provocation of symptoms on discography only 52% had clinical success. These findings support the continued use of lumbar discography for the investigation of this particular group of patients.     

Loss of heterozygosity of M6P/IGF2R gene is an early event in the development of prostate cancer

BACKGROUND: The genetic events leading to initiation and/or progression of prostate cancer are not well characterized. The gene coding for the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) has recently been identified as a tumor suppressor in several types of cancer. The purpose of the present study is to determine whether the M6P/IGF2R gene is inactivated in human prostate cancer, and if so, whether this is an early or late transformational event. METHODS: In total, 43 patients with prostate cancer treated by radical prostatectomy, with archival material available for analysis, were assessed for loss of heterozygosity (LOH) in the M6P/IGF2R gene using six different gene-specific nucleotide polymorphisms. Regions of tumor, normal prostate and premalignant high-grade prostate intraepithelial neoplasia (PIN) were identified and cells were excised by laser capture microdissection (LCM). DNA segments were amplified using polymerase chain reaction (PCR). RESULTS: The M6P/IGF2R gene was polymorphic in 83.7% (36/43) of patients, and 41.7% (15/36) of these informative patients had LOH in the tumor tissue. In 11/15 patients with LOH in malignant tissue, high-grade PIN could be identified, and 63.6% (7/11) also had LOH in this premalignant tissue. CONCLUSIONS: This study is the first to find that the M6P/IGF2R gene is inactivated in prostate cancer. LOH in premalignant tissue as well suggests that mutation in the M6P/IGF2R gene is an early event in the development of prostate cancer, supporting the conclusion that it functions as a tumor suppressor gene in this disease.