Apoptotic primary normal human dermal fibroblasts for in vitro models of fibrosis

Recent studies show that apoptosis affects surrounding tissue, playing a role in diseases such as fibrosis, a significant global disease burden. Elucidating the mechanisms by which the different apoptotic cells present during fibrotic wound healing affect their environment would enable development of new therapies. We describe here a simple, rapid, and cost-effective method for inducing apoptosis of primary normal human dermal fibroblasts without affecting the overall cell viability of the population. Such population could be used for in vitro models of fibrotic wound healing in co-culture with other cells involved in this process to study events such as apoptosis-induced proliferation.     

Expression of calcineurin,calpastatin and heat shock proteins during ischemia and reperfusion

ObjectiveCalcineurin (CaN) interacts with calpains (Calpn) and causes cellular damage eventually leading to cell death. Calpastatin (Calp) is a specific Calpn inhibitor, along with CaN stimulation has been implicated in reduced cell death and self-repair. Molecular chaperones, heat shock proteins (Hsp70 and Hsp90) acts as regulators in Calpn signaling. This study aims to elucidate the role of CaN, Calp and Hsps during induced ischemia and reperfusion in primary cardiomyocyte cultures (murine).Methods and resultsProtein expression was analyzed concurrently with viability using flow cytometry (FACS) in ischemia- and reperfusion-induced murine cardiomyocyte cultures. The expression of Hsp70 and Hsp90, both being molecular chaperones, increased during ischemia with a concurrent increase in death of cells expressing these proteins. The relative expression of Hsp70 and Hsp90 during ischemia with respect to CaN was enhanced in comparison to Calp. Reperfusion slightly decreased the number of cells expressing these chaperones. There was no increase in death of cells co-expressing Hsp70 and Hsp90 along with CaN and Calp. CaN expression peaked during ischemia and subsequent reperfusion reduced its expression and cell death. Calp expression increased both during ischemia and subsequent reperfusion but cell death decreased during reperfusion.ConclusionThe present study adds to the existing knowledge that Hsp70, Hsp90, CaN and Calp interact with each other and play significant role in cardio protection.     

Long-term ischaemic and bleeding outcomes after primary percutaneous coronary intervention for ST-elevation myocardial infarction in the elderly

Background

The population is ageing rapidly and the proportion of patients aged ≥ 80 years undergoing primary percutaneous coronary intervention (PCI) is rising, but clinical trials have primarily been performed in younger patients.

Methods

Patients undergoing primary PCI between 2003 and 2008 were subdivided into 3 groups: < 60, 60-79, and ≥ 80 years. Endpoints at 3-year follow-up included all-cause mortality, recurrent myocardial infarction (reMI), stent thrombosis, target lesion revascularisation (TLR), bleeding (BARC bleeding ≥ 3), stroke, and major adverse cardiovascular events (MACE, a composite of cardiac mortality, reMI, stroke and TLR).

Results

2002 patients with ST-segment elevation myocardial infarction (STEMI) were included, 885 (44.2 %) aged < 60, 921 (46.0 %) 60–79, and 196 (9.7 %) ≥ 80 years. Comorbidities such as diabetes mellitus, prior stroke, malignant disease, anaemia, and chronic kidney disease were more prevalent in patients ≥ 80 years. The incidence of both ischaemic and bleeding events strongly increased with age. Age ≥ 80 years was an independent predictor of mortality (HR 2.56, 95 % CI1.69–3.87, p < 0.001), a borderline non-significant predictor of overall bleeding (HR 1.38, 95 %CI 0.95–2.00, p = 0.088), and a significant predictor of non-access site bleeding (HR 2.26, 95 %CI 1.46–3.51, p < 0.001).

Conclusion

Patients ≥ 80 years experienced high rates of ischaemic and bleeding complications; especially in this high-risk patient group individualised therapy is needed to optimise clinical outcomes.

Electronic Supplementary Material

The online version of this article (doi:10.1007/s12471-015-0733-2 contains supplementary material, which is available to authorized users.     

GCaMP as an indirect measure of electrical activity in rat trigeminal ganglion neurons

While debate continues over whether somatosensory information is transmitted via labeled line, population coding, frequency coding, or some combination therein, researchers have begun to address this question at the level of the primary afferent by using optical approaches that enable the assessment of neural activity in hundreds to even thousands of neurons simultaneously. However, with limited availability of tools to optically assess electrical activity in large populations of neurons, researchers have turned to genetically encoded Ca²⁺ indicators (GECIs) including GCaMP to enable the detection of increases in cytosolic Ca²⁺ concentrations as a correlate for neuronal activity. One of the most widely used GECIs is GCaMP6, which is available in three different versions tuned for sensitivity (GCaMP6s), speed (GCaMP6f), or a balance of the two (GCaMP6m). In order to determine if these issues were unique to GCaMP6 itself, or if they were inherent to more than one generation of GCaMP, we also characterized jGCaMP7. In the present study, we sought to determine the utility of the three GCaMP6 isoforms to detect changes in activity in primary afferents at frequencies ranging from 0.1–30 Hz. Given the heterogeneity of sensory neurons, we also compared the performance of each GCaMP6 isoform in subpopulations of neurons defined by properties used to identify putative nociceptive afferents: cell body size, isolectin B4 (IB4) binding, and capsaicin sensitivity. Finally, we compared results generated with GCaMP6 with that generated from neurons expressing the next generation of GCaMP, jGCaMP7s and jGCaMP7f. A viral approach, with AAV9-CAG-GCaMP6s/m/f, was used to drive GECI expression in acutely dissociated rat trigeminal ganglion (TG) neurons, and neural activity was driven by electrical field stimulation. Infection efficiency with the AAV serotype was high >95 %, and the impact of GCaMP6 expression in TG neurons over the period of study (<10 days) on the regulation of intracellular Ca²⁺, as assessed with fura-2, was minimal. Having confirmed that the field stimulation evoked Ca²⁺ transients were dependent on Ca²⁺ influx secondary to the activation of action potentials and voltage-gated Ca²⁺ channels, we also confirmed that the signal-to-noise ratio for each of the isoforms was excellent, enabling detection of a single spike in>90% of neurons. However, the utility of the GCaMP6 isoforms to enable an assessment of the firing frequency let alone changes in firing frequency of each neuron was relatively limited and isoform specific: GCaMP6s and 6m had the lowest resolution, enabling detection of spikes at 3 Hz in 15% and 32% of neurons respectively, but it was possible to resolve discrete single spikes up to 10 Hz in 36% of GCaMP6f neurons. Unfortunately, using other parameters of the Ca²⁺ transient, such as magnitude of the transient or the rate of rise, did not improve the range over which these indicators could be used to assess changes in spike number or firing frequency. Furthermore, in the presence of ongoing neural activity, it was even more difficult to detect a change in firing frequency. The frequency response relationship for the increase in Ca²⁺ was highly heterogeneous among sensory neurons and was influenced by both the GCaMP6 isoform used to assess it, the timing between the delivery of stimulation trains (inter-burst interval), and afferent subpopulation. Notably, the same deficiencies were observed with jGCaMP7s and 7f in resolving the degree of activity as were present for the GCaMP6 isoforms. Together, these data suggest that while both GCaMP6 and jGCaMP7 are potentially useful tools in sensory neurons to determine the presence or absence of neural activity, the ability to discriminate changes in firing frequency ≥ 3 Hz is extremely limited. As a result, GECIs should probably not be used in sensory neurons to assess changes in activity within or between subpopulations of neurons.     

原发性自身免疫性肝硬化的超声图征象及肝脏弹性的分析

摘要 目的：探讨与分析原发性自身免疫性肝硬化的超声图征象及肝脏弹性特征。方法：研究时间为2017年2月至2019年12月,选择原发性自身免疫性肝硬化86例作为病例组,同期选择正常志愿者86例作为对照组,所有入选者都给予常规超声与弹性成像,记录成像特征并判断相关性。结果：病例组的全血谷草转氨酶(aspartate aminotransferase,AST)、谷丙转氨酶(alanine transarninase,ALT)、直接胆红素(direct bilirubin,DBIL)、总胆红素(total bilirubin,TBIL)与碱性磷酸酶(Alkaline phosphatase,ALP值都显著高于对照组(P<0.05)。病例组的肝脏回声、边界、内回声、血流等超声特征与对照组对比差异都有统计学意义(P<0.05)。病例组肝脏组织剪切波速度(shear wave velocity,SWV)、肝硬度高于对照组,应变率比值(Strain rate, SR)值低于对照组,对比差异都有统计学意义(P<0.05)。病例组的肝脏弹性硬度分级与对照组对比差异有统计学意义(P<0.05)。在病例组中,Pearson分析显示肝脏弹性硬度分级与肝脏回声、边界、内回声、血流等存在相关性(P<0.05)。结论：常规超声及肝脏弹性可反映原发性自身免疫性肝硬化的临床特征,两者存在相关性,可提高该病的鉴别诊断水平。     

胸阻抗法心排血量比较直接PCI和延迟PCI对急性ST段抬高型心肌梗死患者心功能的影响

目的:采用胸阻抗法心排血量检测和比较直接和延迟经皮冠状动脉介入术(PCI)对急性ST段抬高型心肌梗死(STEMI)患者心功能的影响。方法:收集2016年1月-2018年6月于解放军南部战区海军第一医院收治的114例STEMI患者完整资料,分为直接PCI组48例,延迟PCI组40例,对照组(未行任何再灌注治疗)26例。采用胸阻抗法心排血量检测各组患者治疗后3天、6个月的每搏输出量(SV)、心指数(CI)、左心收缩力指数(CTI)、射血分数(EF)等心功能参数,并随访患者6个月内因心力衰竭再住院的情况。结果:治疗后6个月,直接PCI组和延迟PCI组SV、CI、CTI、EF均显著高于对照组,且直接PCI组SV、CTI、EF明显高于延迟PCI组,各组间差异具有统计学意义(P<0.05)。直接PCI组和延迟PCI组6个月内因心力衰竭再住院分别为2.08%和5%,均显著低于对照组(19.23%),差异具有统计学意义(P<0.05)。结论:直接和延迟PCI均能改善STEMI患者中远期心功能,且直接PCI效果更为显著。     

原发性肾小球肾炎患者血浆高同型半胱氨酸水平的变化及与钙磷乘积和血尿酸的相关性分析

目的:分析原发性肾小球肾炎患者血浆高同型半胱氨酸(Hcy)水平的变化及其与钙磷乘积、血尿酸(UA)的相关性。方法:选择2016年3月至2018年3月在我院就诊的80例原发性肾小球肾炎患者作为观察组,另选取同期于我院接受体检的50例健康者作为对照组。采用循环酶法测定受检者血浆Hcy水平,通过比色法测定血清UA,采用离子选择电极法测定血清钙离子(Ca^2+)、磷离子(P3+),对比两组各项血液指标水平及阳性检出率,并通过Pearson分析法分析血浆Hcy与钙磷乘积、UA的相关性。结果:观察组血浆Hcy、P^3+、钙磷乘积及UA水平高于对照组,Ca^2+低于对照组,差异有统计学意义(P<0.05)。观察组Hcy、Ca^2+、P^3+、UA阳性检出率高于对照组,差异有统计学意义(P<0.05)。血浆Hcy水平与Ca^2+呈负相关(r=-2.208,P<0.05),与P3+、钙磷乘积、UA呈正相关(r=0.102、0.592、0.703,P均<0.05)。结论:原发性肾小球肾炎患者血浆Hcy水平较高,且其水平与钙磷乘积、UA水平呈正相关,故监测三项指标变化可为临床防治该病提供科学依据。     

慢性牙周炎与原发性肝硬化的相关性分析

目的:探究慢性牙周炎与原发性肝硬化之间的相关性。方法:选择2017年1月至2019年1月于我院接受治疗的60例原发性肝硬化患者为研究组,选择同期于我院接受体格检查的100例健康个体为对照组,对两组患者分别实施牙周检查,对比两组患者牙周炎发生率,分析吸烟、饮酒对牙周炎发生率影响,按照Child-改良分级法对肝硬化患者进行评分并分级,对比各级原发性肝硬化患者慢性牙周亚发生率,并就慢性牙周炎与肝硬化相关性进行分析。结果:(1)对照组慢性牙周炎发生率显著低于研究组慢性牙周炎发病率(44.00%vs. 71.67%,P0.05);(2)研究组和对照组中吸烟者的慢性牙周炎患病率显著高于不吸烟者(P0.05),研究组中吸烟者发生慢性牙周炎患病率高于对照组,对比无统计学意义(P0.05);研究组和对照组饮酒者的慢性牙周炎患病率显著高于不饮酒者(P0.05),研究组中饮酒者发生慢性牙周炎患病率显著高于对照组(P0.05);(3)随着原发性肝硬化患者评分的升高,患者牙周炎患病率也随之上升,牙周附着丧失程度也出现加剧(P0.05)。结论:原发性肝硬化患者慢性牙周炎患病率高于健康个体,随着肝硬化患者病情的加剧,患者慢性牙周炎发生率也随之上升;另外,吸烟及饮酒会增加健康个体慢性牙周炎患病率。     

自噬参与原发性胆汁性胆管炎患者Th17/ Treg免疫失衡的研究

目的:Th17/Treg免疫失衡在原发性胆汁性胆管炎(PBC)的发病机制中起到关键作用。自噬是调节T细胞稳态的重要环节。本研究旨在探索自噬对PBC患者Th17/Treg免疫失衡的影响。方法:选取20例初次诊断的PBC患者,20例健康对照者,收集其外周血单个核细胞(PBMCs)。利用流式细胞术检测PBC患者与健康对照者PBMCs中Th17和Treg细胞分布及其胞内自噬的水平,进一步体外利用氯喹抑制自噬,证实自噬对PBC患者Th17/Treg平衡的影响。结果:相较于健康对照者,PBC患者Th17细胞占CD4~+T比例上升(P0.05),且血清中IL-17含量较高(P0.01);Treg细胞比例下降(P0.05),且血清中TGF-β含量较低(P0.01)。而PBC患者Th17细胞和Treg细胞内自噬水平均升高。另外,体外实验结果证实,自噬抑制剂氯喹能够有效地恢复PBC患者PBMCs中Th17/Treg平衡。结论:异常活化的自噬可导致PBC患者Th17/Treg免疫失衡,抑制自噬能够在体外恢复此平衡。因此,对异常自噬的干预可能将成为PBC疾病治疗的新方法。     

超声造影对原发性肝癌数字减影血管造影引导下肝动脉化疗栓塞治疗的疗效评估价值

目的:探讨超声造影评估原发性肝癌(HCC)数字减影血管造影(DSA)引导下肝动脉化疗栓塞(TACE)治疗疗效的价值。方法:选择2016年10月至2018年10月我院收治的77例HCC患者,均在DSA引导下行TACE治疗,治疗前后采用超声造影评价疗效。结果:77例HCC患者均顺利完成TACE治疗,治疗后始增强度、增强速率降低(P0.05),始增时间、达峰时间、峰值加速时间增加(P0.05)。治疗后完全缓解(CR)+部分缓解(PR)为有效共46例(有效组),稳定(SD)+进展(PD)为无效共31例(无效组),有效组治疗后始增强度、增强速率低于无效组(P0.05),始增时间、达峰时间、峰值加速时间多于无效组(P0.05)。受试者工作特征曲线(ROC)分析结果显示,始增时间、达峰时间、峰值加速时间、始增强度、增强速率预测HCC患者DSA引导下TACE治疗疗效的曲线下面积(AUC)分别为0.658、0.717、0.64、0.668、0.713。结论:超声造影定量参数在HCC患者DSA引导下TACE治疗前后有着较为明显的变化,其超声参数达峰时间、增强速率对DSA引导下TACE治疗疗效的预测效能较好。