耐碳青霉烯类肺炎克雷伯菌分子流行病学研究

目的分析武汉两所医院耐碳青霉烯类肺炎克雷伯菌(CRKP)临床分离株产碳青霉烯酶的情况,以及分子流行病学特征。方法收集武汉两所医院2018年1—10月临床分离的42株非重复CRKP,采用Carba NP试验方法对菌株产碳青霉烯酶情况进行初筛,聚合酶链反应(PCR)检测碳青霉烯酶基因携带情况,采用质粒接合试验分析耐药基因的水平转移情况,应用脉冲场凝胶电泳(PFGE)进行亲缘关系分析。结果 42株CRKP菌株中14株Carba NP试验阳性,其中有10株扩增出NDM-1基因,3株扩增出KPC-2基因。共13株CRKP碳青霉烯基因检测阳性,其中12株质粒接合试验成功。PFGE分析结果显示,携带NDM-1基因的CRKP菌株共分成6型,无明显优势型;携带KPC-2基因的CRKP菌株为同一型别。结论检出产NDM-1和KPC-2的CRKP菌株,质粒接合试验提示质粒介导的水平传播在碳青霉烯酶基因的播散过程中可能起重要作用。     

NP方案联合三维适形放疗治疗局部晚期非小细胞肺癌的疗效及安全性观察

目的：观察NP方案联合三维适形放疗治疗局部晚期非小细胞肺癌的疗效及安全性。方法：56例局部晚期非小细胞肺癌患者根据治疗方法不同随机分为三维适形放疗联合NP方案化疗组（观察组,28例）与单纯放疗组（对照组,28例）,比较两组疗效及毒副反应情况。结果：观察组的疗效明显高于对照组（P〈0.05）。两组的1年生存率、2年生存率比较,P〈0.05,差异有统计学意义。结论：NP方案联合三维适形放疗治疗局部晚期非小细胞肺癌的疗效确切,有效率高,近期疗效理想,提高了患者的生存率。     

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods.It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm²) was higher than in A549 (172 ± 59 Ω cm²) but similar to 16HBE14o- cells (1469 ± 156 Ω cm²). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm²) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm²) and 16HBE14o- (558 ± 267 Ω cm²). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549.The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.     

Influence of polymer hydrolysis on adjuvant effect of Gantrez®AN nanoparticles: Implications for oral vaccination

The adjuvant effect of methylvinylether-co-maleic anhydride (Gantrez®AN) nanoparticles was investigated during oral vaccination of mice with F4 adhesins of F4-positive Escherichia coli. To differentiate whether the adjuvant effect originated from a nanoparticle effect or a polymer effect, 20 μg F4 was administered as slightly crosslinked F4-containing nanoparticles (g(F4)_0.01) or as F4 mixed with slightly crosslinked pure nanoparticles (F4 + g_0.01).The F4-specific immune response was reduced using F4-containing nanoparticles due to complete shielding of F4, whereas oral administration of F4 + g_0.01 increased the level of F4-specific antibody-secreting cells (ASC) in the spleen. When repeating the vaccination study after 6 months using freshly prepared nanoparticles, the adjuvant effect of F4 + g_0.01 was lost due to an altered polymer reactivity caused by partial hydrolysis of anhydride groups of Gantrez®AN. Combining F4 with nanoparticles stabilised with a higher crosslinker amount during nanoparticle synthesis (F4 + g_0.22) could overcome the effect of partial polymer hydrolysis, as higher levels of ASC were detected. Hence, an in-depth characterisation of the Gantrez®AN polymer is required as stability issues can alter its biological effect during oral vaccination.     

Cerium oxide nanoparticle uptake kinetics from the gas-phase into lung cells in vitro is transport limited

Nowadays, aerosol processes are widely used for the manufacture of nanoparticles (NPs), creating an increased occupational exposure risk of workers, laboratory personnel and scientists to airborne particles. There is evidence that possible adverse effects are linked with the accumulation of NPs in target cells, pointing out the importance of understanding the kinetics of particle internalization.In this context, the uptake kinetics of representative airborne NPs over 30 min and their internalization after 24 h post-exposure were investigated by the use of a recently established exposure system. This system combines the production of aerosolized cerium oxide (CeO₂) NPs by flame spray synthesis with its simultaneous particle deposition from the gas-phase onto A549 lung cells, cultivated at the air-liquid interface.Particle uptake was quantified by mass spectrometry after several exposure times (0, 5, 10, 20 and 30 min). Over 35% of the deposited mass was found internalized after 10 min exposure, a value that increased to 60% after 30 min exposure. Following an additional 24 h post-incubation, a time span, after which adverse biological effects were observed in previous experiments, over 80% of total CeO₂ could be detected intracellularly.On the ultrastructural level, focal cerium aggregates were present on the apical surface of A549 cells and could also be localized intracellularly in vesicular structures. The uptake behaviour of aerosolized CeO₂ is in line with observations on cerium suspensions, where particle mass transport was identified as the rate-limiting factor for NP internalization.     

Toxicity of silver nanoparticles - nanoparticle or silver ion?

The toxicity of silver nanoparticles (AgNPs) has been shown in many publications. Here we investigated to which degree the silver ion fraction of AgNP suspensions, contribute to the toxicity of AgNPs in A549 lung cells. Cell viability assays revealed that AgNP suspensions were more toxic when the initial silver ion fraction was higher. At 1.5 μg/ml total silver, A549 cells exposed to an AgNP suspension containing 39% silver ion fraction showed a cell viability of 92%, whereas cells exposed to an AgNP suspension containing 69% silver ion fraction had a cell viability of 54% as measured by the MTT assay. In addition, at initial silver ion fractions of 5.5% and above, AgNP-free supernatant had the same toxicity as AgNP suspensions. Flow-cytometric analyses of cell cycle and apoptosis confirmed that there is no significant difference between the treatment with AgNP suspension and AgNP supernatant. Only AgNP suspensions with silver ion fraction of 2.6% or less were significantly more toxic than their supernatant as measured by MTT assays. From our data we conclude that at high silver ion fractions (≥5.5%) the AgNPs did not add measurable additional toxicity to the AgNP suspension, whereas at low silver ion fractions (≤2.6%) AgNP suspensions are more toxic than their supernatant.     

足月新生儿缺氧缺血性脑损伤大鼠模型的制作与鉴定

目的检测狂犬病毒NP蛋白的免疫原性。方法利用RT-PCR扩增狂犬病毒核蛋白（NP）基因,测序后将其克隆到PVAXl真核表达载体上：将PVAX1-NP转染Vero细胞,进行SDS．PAGE,TLWestern—blot分析;以重组质粒PVAX1-NP对小鼠进行免疫试验。结果经酶切分析、鉴定和测序验证,得到重组表达质粒PVAX1-NP,NP蛋白在Vero细胞中获得了表达,且表达产物具有免疫学活性：免疫小鼠抗体水平显著升高。结论狂犬病毒NP蛋白具有免疫原性。     

Evaluation of a new silica clotting time in the diagnosis of lupus anticoagulants

INTRODUCTION: A new commercial silica clotting time (SCT), the HemosIL SCT assay (Instrumentation Laboratory, Milan, Italy) was evaluated in the laboratory diagnosis of lupus anticoagulants (LAC). This integrated test system for screening and confirmation was compared with the frequently used aPTT-based PTT-LA and Staclot-LA (Diagnostica Stago, Asnières, France) in a patient population investigated for LAC and in a subpopulation who met the clinical criteria for antiphospholipid syndrome (APS). MATERIALS AND METHODS: 201 samples were analysed with the HemosIL SCT assay. Own reference values were calculated. Results are expressed as measured clotting times in seconds or as normalised ratios. RESULTS: SCT screen and PTT-LA had a sensitivity of, 61.1% and 63.8%, respectively. Normalising the results gained sensitivity up to 72.2% and 90%, respectively. The confirmation SCT and the Staclot-LA had a sensitivity of 30.6% and 63.9% with a specificity of 86.7% and 100%, respectively. Sensitivity of SCT for detecting LAC in clinical criteria positive patients was lower compared to aPTT and dRVVT (45.8% versus 66.7% and 65%). Combination of SCT/dRVVT and aPTT/dRVVT gave a sensitivity of 51.2% and 63.6%, with a specificity of 50.0% and 52.3%, respectively. CONCLUSIONS: In comparison with PTT-LA as screening test, the SCT screen shows an acceptable sensitivity. However, the HemosIL SCT assay including the confirmation step, has a much lower sensitivity in the diagnosis of LAC in comparison with the Staclot-LA test. Combining the HemosIL SCT assay with dRVVT results in a better sensitivity, although lower than the combined aPTT/dRVVT based method as usually performed in our lab.     

Proline-rich polypeptides in Alzheimer's disease and neurodegenerative disorders - Therapeutic potential or a mirage?

The development of effective and safe drugs for a growing Alzheimer disease population is an increasing need at present. Both experimental and clinical evidence support a beneficial effect of proline-rich polypeptides in a number of neurodegenerative diseases, including Alzheimer disease. Experimental data have shown that proline-rich polypeptides isolated from bovine neurohypophisis possess neuroprotective and neuromodulatory properties in mice with aluminum neurotoxicosis or neuronal damage caused by venoms and toxins. Proline-rich polypeptides from ovine colostrums, so called Colostrinin, have been shown to produce cognitive improvement in an experimental model and in patients with Alzheimer disease. However, the precise mechanism underlying the neuroprotective action of proline-rich polypeptides is not very well established. Moreover, studies pointing at a neuroprotective effect of proline-rich polypeptides from bovine neurohypophisis in humans have not been reported thus far. The authors conclude that more detailed information on the mode of action of proline-rich polypeptides is needed as well as confirmation of their efficacy in broad clinical trials before this approach can really show its potential in the treatment of neurodegenerative disorders.