Application of CEC procedures for the analysis of synthetic peptides: characterization of linear immunogenic peptides that mimic a HIV‐1 gp120 epitope

Abstract: In this study, we describe the application of a new analytical procedure based on capillary electrochromatographic (CEC) techniques for the characterization of different basic and acidic peptides using isocratic eluent conditions containing acetonitrile and ammonium acetate buffers of different molarities between pH 3.8 and 5.2. In particular, 10 immunogenic peptide analogs with isoelectric points ranging from 3.7 to 10.1 were investigated; nine of these peptides, 1 – 9 , were truncated analogs of the parent peptide, 10 , which is a peptidomimetic related to a HIV‐1 gp120 epitope. Several of these peptides have the propensity to form α‐helical secondary structures in solution. Electrochromatographic separations of these peptides were achieved with packed fused silica capillaries (25 cm packed length, 100 µm i.d.) containing 3 µm n‐octadecylsilica particles. The influence of temperature on the CEC elution behavior of these peptides, as well as the impact of changes in the eluent composition, e.g. pH, buffer concentration and acetonitrile content, were examined. The results confirm that improvements in the resolution and analysis of synthetic peptides by CEC procedures result from the increase in electro‐osmotic flow (EOF) as the temperature is increased. These findings emphasize the dominant influence of the temperature‐dependent viscosity parameter, η, on the EOF and thus on peptide resolution in CEC. Moreover, these investigations have shown that eluent properties can be specifically chosen to favor either electrophoretic mobility or chromatographic retention, with the overall CEC selectivity peptides of different sequence or composition reflecting the summated contributions from both separation mechanisms. Over the pH range 4.0–5.0, and using eluents with ionic strengths ranging from 6.2 to 15 mm ammonium acetate but containing a fixed volume fraction, ψ, of acetonitrile above ψ=0.40, the CEC retention behavior of peptides 1 – 10 correlated with a linear relationship linking the retention coefficient, κ_cec, and the differential frictional size‐to‐mass ratio parameter, ξ_fric, of these peptides. However, using eluents with a low acetonitrile content and low pH values, linear correlations were also observed between the incremental retention coefficient, Δκ_cec, and the product term [? 0.66(Δ∑χ_n) log(M_i/M_j)], which links the difference in intrinsic hydrophobicities and molecular masses of two peptides, P_i and P_j. This study thus demonstrates the power of CEC procedures in the analysis of synthetic bioactive peptides and provides a general experimental framework to evaluate, using CEC procedures, the influence of the key molecular attributes of peptides on their structure?retention dependencies. Finally, these studies provide additional, practical insights into the use of CEC procedures for the analysis, resolution and biophysical characterization of closely related peptide analogs derived from solid‐state peptide synthesis under conditions of different eluent composition or temperature.     

ACOP对大鼠血中NO、ET-1和CEC的影响及高压氧的作用

目的 探讨急性一氧化碳中毒（ACOP）对大鼠血管内皮及其细胞功能的影响和高压氧治疗（HBOT）的作用。方法 参照Jiang式染毒法制成ACOP大鼠模型,有相应对照地、分别对中毒后即刻和次日起每日1次HBOT的第1,3,7,14天后,血中一氧化氮（NO）、内皮素－1（ET－1）水平和循环内皮细胞（CEC）计数进行测定。结果（1）ACOP后即刻机体血中NO水平显著降低,ET－1水平和CEC计数显著升高（P＜0．05）;（2）HBOT后中毒大鼠血中NO水平显著回升（P＜0．05）,ET－1水平和CEC计数显著回降;其后上述指标都趋于恢复到正常水平。结论 （1）ACOP可导致机体严重的血管内皮及其细胞功能的损伤;（2）0．2MPa（绝对压）HBOT对ACOP导致的血管内皮及其功能损伤具有显著的改善和作用;（3）血中NO、ET－1水平和CEC计数指标的单独或联合检测,可作为临床上判断ACOP患者血管内皮及其功能损伤程度、评估疗效、预测预后的重要手段。     

Evaluation of proton beam radiation sensitivity of proliferating choroidal endothelial and retinal ganglion cells with clonogenic assay

Context: Proton beam therapy offers the advantage of precise delivery with limited damage to the healthy tissue and is being tested in the management of exudative age-related macular degeneration (AMD). However, the dosages tested are empirical and not based on preclinical studies.

Objective: In this study we evaluated the effects of varying doses of proton beam radiation on choroidal endothelial cells (CECs) and retinal ganglion cells (RGCs) using clonogenic assay to determine differential sensitivity.

Materials and methods: Each cell type has different efficiency to replicate (plating efficiency (PE)). PE of CEC (RF/6A) and RGC (RGC-5) grown in culture flasks was determined by plating 250 cells each (without any treatment) and counting the number of colonies after 13 days. Radiation induced sensitivity was determined by exposing the semi-confluent RF/6A and RGC-5 cells to proton beam at the doses of 0 (control), 2, 4, 8 and 12 cobalt gray equivalent (CGE). The ability of the cells to repair and replicate to form colonies were analyzed 13 days after radiation with crystal violet stain and the survival ratio was calculated. The significance of survival was analyzed using ANOVA (Graphpad Instat.3).

Results: The PE of CEC and RGC was 12.96?±?0.29% and 40.7?±?1.48%, respectively. A survival ratio of CEC at 2, 4, 8 and 12 CGE proton radiation was 66.0?±?8.6%, 44.3?±?6.5%, 7.6?±?0.3% and 1.14?±?0.06% on exposure to 2, 4, 8 and 12 CGE proton radiation, respectively, p?<?0.01). Survival ratio of RGC was 71.1?±?22.4% (p?=?0.05), 40.2?±?7.9%, 8.89?±?2.6% and 0.78?±?0.31% at 2, 4, 8 and 12 CGE dosages (p?<?0.001).

Discussion: CEC showed dose-dependent decrease in survival rate with values attaining significance at all radiation dosages. In contrast, RGC was comparatively radio resistant and were able to replicate at lower doses and sensitive at higher doses after proton beam radiation.

Conclusion: Since CECs proliferate during neovascularization, this clonogenic assay is a useful assay to assess the sensitivity of CEC to radiation. This study identified that CEC were more sensitive to proton beam radiation than RGC at all doses. This may provide a therapeutic window for administration of proton beam radiation in the management of AMD.     

复方降脂胶囊对实验性动脉粥样硬化鹌鹑血液中炎症因子的影响

目的观察复方降脂胶囊对实验性动脉粥样硬化(AS)鹌鹑血液中炎症因子的影响。方法采用高脂饲养法复制鹌鹑高脂血症模型,并诱发AS模型。采用复方降脂胶囊及洛伐他汀分组治疗;于第12周经右侧颈静脉采血,测定血浆中循环内皮细胞(CEC)数,酶联免疫吸附法检测血清中CD40、CD40L、转化生长因子(TGF)-β、血管细胞间黏附分子(sVCAM-1)、细胞间黏附分子(sICAM-1)的含量。结果 实验动物经复方降脂胶囊干预治疗后,可明显降低血液中循环内皮细胞的数量,降低血清中CD40、CD40L、TGF-β、sVCAM-1及sICAM-1的含量,与模型组对比有显著差异。结论复方降脂胶囊可降低血管内皮的损伤,抑制AS斑块发生发展过程中的炎症反应过程,可能是其治疗AS的机制。     

老年高血压病患者血管内皮功能与氧化低密度脂蛋白变化关系的探讨

目的探讨老年高血压患者血管内皮功能与氧化低密度脂蛋白变化关系，寻求较好的治疗方法。方法按WHO高血压诊断标准，选择年龄为60—72岁的Ⅱ期老年高血压患者60例，与30例正常健康者进行对照，比较两组一般特点，同时检测氧化低密度脂蛋白（OX—LDL）、内皮依赖舒张因子（NO），血管内皮素（ET），血管内皮细胞计数（CEC）。结果两组结果显示，正常对照组与高血压组在年龄，性别组成方面无显著差异，但两组的血压比较则有显著差异（P〈0．001），高血压组的OX—LDL、ET、CEC结果均高于正常对照组，NO结果则低于正常对照组。结论本研究结果提示与Ⅱ期老年高血压发生发展有肯定关系，提示临床在治疗中不仅注重血管内皮功能保护，同时加强抗氧化治疗，延缓高血压发展。     

Role of tumor necrosis factor receptor 2 (TNFR2) in colonic epithelial hyperplasia and chronic intestinal inflammation in mice.

BACKGROUND & AIMS: Tumor necrosis factor (TNF) induces multiple effects including cell proliferation and death by ligation with TNF receptor type II (TNFR2). We studied the role of TNFR2 in chronic inflammation-induced colonic epithelial alteration. METHODS: TNFR2 expression in colonic epithelial cells (CECs) was assessed by ribonuclease protection assay (RPA) and immunohistochemistry (IHC) in patients with inflammatory bowel disease (IBD) and murine colitis models. TNFR2 expression was also analyzed using COLO205 cells. The role of TNFR2 in colonic epithelial homeostasis was examined by generating interleukin 6-deficient TCR alpha KO (alpha IL-6DKO) or TNFR2-deficient TCR alpha (alpha TNFR2DKO) mice. RESULTS: TNFR2 expression was up-regulated in CEC in both human ulcerative colitis and Crohn's disease. In vitro studies showed that TNFR2 expression was up-regulated by a cooperative effect of key proinflammatory cytokines. By RPA, the increased expression of TNFR2 was detectable in TCR alpha KO mice with colitis compared with TCR alpha KO mice without colitis or wild-type mice. In alpha IL-6DKO mice, TNFR2 expression, proliferation, and nuclear factor kappa B activation of CECs were markedly reduced compared with TCR alpha KO mice. alpha TNFR2 mice also showed significantly less colonic epithelial proliferation compared with TCR alpha KO mice. CONCLUSIONS: Expression of TNFR2 is consistently increased on CECs in both murine colitis models as well as patients with IBD. TNFR2 may play an important role in colonic inflammation-associated alteration in the intestinal epithelium.     

Comparison of 2 Different Drug-Coated Balloons in In-Stent Restenosis: The RESTORE ISR China Randomized Trial

Objectives

The aim of the present study was to evaluate the angiographic efficacy, clinical safety, and effectiveness of the Restore paclitaxel-coated balloon in a randomized trial designed to enable the approval of the new device in China.

The DENALI Trial: An Interim Analysis of a Prospective,Multicenter Study of the Denali Retrievable Inferior Vena Cava Filter

PurposeTo assess safety and effectiveness of a nitinol retrievable inferior vena cava (IVC) filter in patients who require caval interruption to protect against pulmonary embolism (PE).Materials and MethodsTwo hundred patients with temporary indications for an IVC filter were enrolled in this prospective, multicenter clinical study. Patients undergoing filter implantation were to be followed for 2 years or for 30 days after filter retrieval. At the time of the present interim report, all 200 patients had been enrolled in the study, and 160 had undergone a retrieval attempt or been followed to 6 months with their filter in place. Primary study endpoints included technical and clinical success of filter placement and retrieval. Patients were also evaluated for recurrent PE, new or worsening deep vein thrombosis, and filter migration, fracture, penetration, and tilt.ResultsClinical success of placement was achieved in 94.5% of patients (172 of 182), with a one-sided lower limit of the 95% confidence interval of 90.1%. Technical success rate of filter placement was 99.5%. Technical success rate of retrieval was 97.3%; 108 filters were retrieved in 111 attempts. In two cases, the filter apex could not be engaged with a snare, and one device was engaged but could not be removed. Filter retrievals occurred at a mean indwell time of 165 days (range, 5–632 d). There were no instances of filter fracture, migration, or tilt greater than 15° at the time of retrieval or 6-month follow-up.ConclusionsIn this interim report, the nitinol retrievable IVC filter provided protection against pulmonary embolism, and the device could be retrieved with a low rate of complications.     

The analysis of DNA adducts: The transition from P-postlabeling to mass spectrometry

The technique of ³²P-postlabeling, which was introduced in 1982 for the analysis of DNA adducts, has long been the method of choice for in vivo studies because of its high sensitivity as it requires only <10 μg DNA to achieve the detection of 1 adduct in 10¹⁰ normal bases. ³²P-postlabeling has therefore been utilized in numerous human and animal studies of DNA adduct formation. Like all techniques ³²P-postlabeling does have several disadvantages including the use of radioactive phosphorus, lack of internal standards, and perhaps most significantly does not provide any structural information for positive identification of unknown adducts, a shortcoming that could significantly hamper progress in the field. Structural methods have since been developed to allow for positive identification of DNA adducts, but to this day, the same level of sensitivity and low sample requirements provided by ³²P-postlabeling have not been matched. In this mini review we will discuss the ³²P-postlabeling method and chronicle the transition to mass spectrometry via the hyphenation of gas chromatography, capillary electrophoresis, and ultimately liquid chromatography which, some 30 years later, is only just starting to approach the sensitivity and low sample requirements of ³²P-postlabeling.