粪肠球菌心内膜炎抗原EfaA在生物膜形成中的作用

目的 探讨粪肠球菌心内膜炎抗原EfaA在生物膜形成中的作用。方法 微量滴定板法测定生物膜形成能力,将efaA⁺、efaA^- 粪肠球菌S₁₄、S₁₄-pAT28、S₁₄-pAT28-efaA分别接种到96孔板,37 ℃培养24 h形成生物膜,结晶紫染色,测定孔板底部生物膜的OD₅₇₀ 值;MTT法比较S₁₄、S₁₄-pAT28、S₁₄-pAT28-efaA所形成的生物膜的活菌含量OD₄₉₂;共聚焦激光扫描显微镜(CLSM)观察比较efaA⁺、efaA^-肠球菌形成的生物膜的平均厚度、最大厚度。结果 efaA⁺ 转化株在各培养时间点微量滴定板法测定的生物膜形成能力OD₅₇₀ 值、MTT法测定的OD₄₉₂活菌含量、CLSM观察的生物膜的平均厚度、最大厚度均大于野生株及空质粒转化株,P<0.05;空质粒转化株与野生株比较无显著性差别,P>0.05。结论 粪肠球菌心内膜炎抗原efaA基因有利于肠球菌生物膜的形成。     

应用CLSM对自体表皮移植治疗白癜风的疗效观察

目的应用共聚焦激光扫描显微镜（CLSM）观察自体表皮移植治疗白癜风后的黑素细胞恢复状况。方法采用CLSM对白癜风皮损进行检测,选择镜下显示黑素细胞完全消失的皮损进行自体表皮移植,移植后1、3、6个月动态观察黑素细胞恢复状况。结果 20例白癜风患者中,28块皮损进行了自体表皮移植,临床总成活率为96.43%,移植后CLSM成像显示黑素细胞完全恢复率为82.14%。结论 CLSM检测可作为白癜风自体表皮移植治疗适应证的选择方法,也可作为移植治疗后连续动态观察黑素细胞恢复状况的可靠手段。     

Artificial Pseudomonas aeruginosa biofilms and confocal laser scanning microscopic analysis

Bacterial biofilms may be formed at various sites, including mucous membranes, teeth, and infectious lesions. To elucidate the structure and the function of biofilms, artificial biofilms of mucoid-type Pseudomonas aeruginosa organisms (strain PT1252) were made by centrifuging the organisms onto the surface of a coverglass and culturing further in broth media supplied continuously (45 ml/h). The biofilm structure at 4, 8, 12, and 24 h was visualized with fluorescent staining (SYTO9, propidium iodide [PI], and/or fluorescein isothiocyanate-concanavalin A [FITC-ConA]) by confocal laser scanning microscopy (CLSM). It was clearly demonstrated that the number of bacteria (10⁴–10⁶/ml) could be estimated by their fluorescence intensity. Sectional analysis of each biofilm layer (1-μm thickness) made it possible to demonstrate the three-dimensional development of biofilms, and revealed that the biofilms were 9 μm in height after 12 h. The live and dead organisms were differentiated by SYTO9 and PI, respectively, in situ in biofilms, and about 13% of the organisms were dead in 12-h-old biofilms. When 12-h-old biofilms were exposed to ciprofloxacin at minimum bactericidal concentration (6.26 μg/ml) for 90 min, all the organisms were killed, but some organisms (11 ± 1.3%; n = 3) in 24-h-old biofilms with thicker and denser structure were still alive after exposure for 120 min. These results indicate that the CLSM analysis of artificial biofilms was useful for elucidating bacterial functions in biofilms, and may lead to a new quantitative system for estimating the bactericidal efficacy of antibacterial drugs in biofilms. Received: October 23, 2000 / Accepted: February 5, 2001     

Dynamic MR imaging and tumor angiogenesis in DMBA-induced rat breast cancer: Three dimensional (3D) reconstructed image analysis of tumor microvessels by confocal laser scanning microscopy

We investigated the correlation between dynamic magnetic resonance imaging (MRI) findings in breast cancer and tumor angiogenesis in a dimethylbenz(a)anthracene (DMBA)-induced rat breast cancer model. In this study, we clearly demonstrated the three-dimensional (3D) architecture of tumor microvessel networks (MN) by confocal laser scanning microscopy (CLSM) and also investigated the hyperpermeability of tumor microvessels. Dynamic MRI findings were closely related to tumor angiogenesis. Three-dimensional reconstructed image analysis by CLSM revealed that the depicted images were dependent on microvessel density as well as microvessel permeability. It should be emphasized that dynamic MRI may have the potential to evaluate the tumor angiogenic activity in human breast carcinoma.     

Fabrication,in-vitro characterization,and enhanced in-vivo evaluation of carbopol-based nanoemulsion gel of apigenin for UV-induced skin carcinoma

The aim of this study was to develop a potential novel formulation of carbopol-based nanoemulsion gel containing apigenin using tamarind gum emulsifier which was having the smallest droplet size, the highest drug content, and a good physical stability for Skin delivery. Apigenin loaded nanoemulsion was prepared by high speed homogenization method and they were characterized with respect to morphology, zeta potential, differential scanning calorimeter study, and penetration studies. In-vitro release studies and skin permeation of apigenin loaded nanoemulsion by goat abdominal skin was determined using Franz diffusion cell and confocal laser scanning microscope (CLSM). The cytotoxicity of the reported formulation was evaluated in HaCaT Cells (A) and A431 cells (B) by MTT assay. The nanoemulsion formulation showed droplet size, polydispersity index, and zeta potential of 183.31?nm, 0.532, and 31.9?mV, respectively. The nanoemulsions were characterized by TEM demonstrated spherical droplets and FTIR to ensure the compatibility among its ingredients. CLSM showed uniform fluorescence intensity across the entire depth of skin in nanocarriers treatment, indicating high penetrability of nanoemulsion gel through goatskin. The nanoemulsion gel showed toxicity on melanoma (A341) in a concentration range of 0.4–2.0?mg/ml, but less toxicity toward HaCaT cells. The carbopol-based nanoemulsion gel formulation of apigenin possesses better penetrability across goatskin as compared to marketed formulation. Hence, the study postulates that the novel nanoemulsion gel of apigenin can be proved fruitful for the treatment of skin cancer in near future.     

Whole inactivated avian Influenza H9N2 viruses induce nasal submucosal dendritic cells to sample luminal viruses via transepithelial dendrites and trigger subsequent DC maturation

Nasal mucosal barrier is a key impediment for the absorption of influenza whole inactivated virus (WIV) intranasal vaccine. Yet it is still unclear how WIV cross the epithelial cells (ECs) in nasal cavity. Here, in vitro, a coculture system was well established, consisting of surrogate nasal ECs (Calu-3) and dendritic cells (DCs). After adding H9N2 WIV on the apical side of ECs, we found that submucosal DCs extended their transepithelial dendrites (TEDs) and sampled luminal viruses. However, ECs were not involved in the transepithelial transport of viruses. Subsequently, the phenotypic and functional maturation of DCs were also enhanced, whereas they were attenuated after blocking of TED formation by anti-JAM1 antibody. In vivo, we confirmed that H9N2 WIV were capable of inducing nasal submucosal DCs to sample luminal viruses via TEDs in the nasal passage but not nasal-associated lymphoid tissue (NALT). CD103⁺ and CD103⁻ DC subsets participated in this process. Of note, chemokine CCL20, released from the H9N2 WIV-induced ECs, played a vital role in DC recruitment and TED formation. Taken together, our findings indicated that TEDs played a critical role in facilitating viral transport across the epithelial barrier, which may guide the design of novel nasal mucosal vaccine strategies.     

Quantification of microparticle coating quality by confocal laser scanning microscopy (CLSM)

In this work, a novel protocol was developed for determining film coating thickness and coating quality of microparticles, based on the use of confocal laser scanning microscopy (CLSM). CLSM was found to be an adequate non-destructive technique for the quantification of the coating thickness and coating quality of individual thin-coated small particles. Combined with image analysis, it was possible to derive with high accuracy the coating thickness distribution of a representative number of microparticles. The performance of the novel methodology was assessed by the quantification of the coating thickness and coating quality of protein-coated microparticles produced by fluidized bed coating. It was found that the CLSM data on coating layer thickness were generally in good agreement with the results from chemical analysis, down to a thickness of 1–1.5 μm. Using CLSM the importance of setting up the appropriate distance between the coating nozzle and the powder bed with respect to microparticle coating quality in fluidized bed processing was illustrated. Coating quality was found to decrease with increasing distance the coating droplets have to travel before impinging onto the core particles as a result of spray-drying of the coating droplets. Also, coating quality decreased with increasing viscosity of the coating droplets, resulting in reduced spreading on the cores.     

Enhancement of Topical Delivery from Biodegradable Nanoparticles

PURPOSE: To determine whether and how encapsulation of lipophilic compounds in polymeric nanoparticles is able to improve topical delivery to the skin. METHODS: The penetration of octyl methoxycinnamate (OMC; Parsol MCX), a highly lipophilic sunscreen, into and across porcine ear skin in vitro was investigated, subsequent to encapsulation in poly(epsilon-caprolactone) nanoparticles, using tape-stripping. Confocal laser scanning microscopy (CLSM) was used to visualize the distribution of nanoparticles, charged with Nile red (NR), a lipophilic and fluorescent dye. RESULTS: Quantification of OMC in the skin using tape-stripping demonstrated that nanoparticulate encapsulation produced a 3.4-fold increase in the level of OMC within the stratum corneum (SC), although the use of nanoparticles did not appear to increase skin permeation (it was not possible to detect OMC in the receiver compartment after 6 h). The confocal images showed that the fluorescence profile observed in the skin after application of NR-containing nanoparticles was clearly different from that seen following application of NR dissolved in propylene glycol. Two hours postapplication of NR-containing nanoparticles, fluorescence was perceptible at greater depths (up to 60 microm) within the skin. CONCLUSIONS: i) Nanoparticulate encapsulation of OMC increased its "availability" with the SC. ii) The altered distribution of NR when delivered via nanoparticles was due, at least in part, to its altered thermodynamic activity (relative to that in propylene glycol) and, as a result, an increase in its partition coefficient into the SC.     

一种小鼠胸腺基质细胞和活化胸腺细胞共表达分子的研究

目的：研究抗小鼠胸腺基质细胞（ＭＴＳＣ）Ｐｆ１８－３单抗识别分子的表达特性。方法：采用流式细胞（ＦＡＣＳ）和激光扫描共焦显微镜（ＣＬＳＭ）分析ＴＳＣ单抗Ｐｆ１８－３染色特征。结果 ：ＦＡＣＳ分析发现Ｐｆ１８－３单抗识别分子可表达于胸腺、胎肝和骨髓等不同来源的基质细胞系和腹腔巨噬细胞表面。新鲜分离的胸腺细胞Ｐｆ１８－３识别分子表达阴性，但ＣｏｎＡ活化可诱导并上调其表达，随活化时间延长表达增高，且主要