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The decision for surgical intervention in the treatment of stenosis and for regurgitation of the mitral valve demands an objective and quantitative evaluation of the severity of mitral valve disease. The availability of ultrasound techniques capable of analysing flow velocities across valves and to produce representative images of valve orifices has increased the interest in the hydraulics of cardiac valves. To isolate and study the determinants of transmitral flow, an in vitro model of the human left heart was built. From the model it is possible to differentiate the influence of the different determinants of left heart performance on transmitral flow: preload, compliance of the left atrium and ventricle, peripheral resistance (afterload) and heart rate. The mechanical part of the model consists of a reservoir connected to an elastic closed circuit (Latex pulmonary veins, left atrium, left ventricle and aortic arch) with replaceable mitral and aortic valves. The electronic part of the model drives and controls the hydraulic part, allowing the independent regulation and monitoring of left atrial and left ventricular pressures p, volumes V and 'pV-loops' throughout the cardiac cycle at different cardiac rhythms. Left atrial filling pressure and aortic resistance are variable in a controlled fashion. Echo-Doppler study of the mitral valve and the transmitral valve flow is possible both from an atrial and a ventricular window in the model. This technical note describes the model.  相似文献   

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Endotoxin (lipopolysaccharide, LPS) inducible cytokine release by human whole blood is increasingly used to model inflammatory responses in vitro, to detect the presence of pyrogenic contaminations as well as to monitor disease states or immunomodulatory treatments ex vivo. However, the LPS-stimulated blood model primarily allows the assessment of monocyte responses. Here, a whole blood model was established which allows assessment of lymphocyte responses. Four different superantigens, namely staphylococcal enterotoxin A and B (SEA, SEB), toxic shock syndrome toxin-1 (TSST-1) or streptococcal exotoxin A (SPEA) were tested with respect to the induction of lymphokine release. All superantigens were capable of inducing significant amounts of the lymphokines interferon-gamma (IFNgamma), interleukin 2 (IL-2), IL-4, IL-5, IL-13 and tumor necrosis factor beta (TNFbeta) after 72 h of incubation. Concentration-dependencies and kinetics were determined. Blood from 160 healthy donors was used to assess the variability of SEB-inducible lymphokine release. Interindividual differences were more pronounced compared to LPS-inducible monokine release. However, the individual response was maintained when blood from six donors was tested once a week for 8 weeks, suggesting that the individual response represents a donor characteristic. The model appears to be suitable for the evaluation of immunomodulatory agents in vitro as well as ex vivo.  相似文献   

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The in vitro development of human mast cells from fetal liver cells with recombinant human stem cell factor in serum-containing RPMI was compared to that in AIM-V media with and without serum. Compared to serum-containing media, AIM-V medium caused mast cells to develop earlier and in greater numbers. By 2 weeks, about 60% of cells in serum-free AIM-V medium were phenotypic mast cells, 2 times the percentages in serum-containing media. By 6 weeks the percentages of mast cells were ≥80% under all conditions, but the number of mast cells was 3–4-fold greater in serum-free AIM-V medium than in serum-supplemented media. Mast cells obtained in serum-free AIM-V medium exhibited rounded nuclei, like tissue-derived mast cells; mast cells obtained in serum-supplemented media had segmented nuclei. By 10–12 weeks of culture about 40% of the AIM-V-derived cells showed strong chymase immunocytochemical staining, a pattern observed for only 14% of the cells in serum-containing media. AIM-V medium is a suitable medium for the development of human mast cells in vitro, and permits an earlier, more selective and greater expansion of mast cells than serum-containing media.  相似文献   

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Infections caused by Staphylococcus aureus are prevalent. The dramatically reduced discovery of new antibiotics, as well as the persistent emergence of resistant bacteria, represents a major health problem in both hospital and community settings. Using antibiotic enhancers to rescue existing classes of antibiotics is an attractive strategy. In this study, 16-aldehyde tanshinone I (ALT) was synthesized and bacteriostatic activity was explored. In addition, synergistic or additive activity between ALT and aminoglycoside antibiotics or β-lactam antibiotics in vitro was identified. Moreover, ALT was documented to augment clearance of streptomycin (STR) and ampicillin (AMP) against S. aureus in a murine infection model. Primary mechanistic insight indicated that ALT could damage the bacterial cell membrane, leading to accumulation of antibiotics inside bacterial cells. This finding might be useful for treating infections caused by S. aureus and expand the scope of application of tanshinones.  相似文献   

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An in vitro whole smoke (WS) exposure method was established to evaluate the toxicological effects of fresh cigarette smoke using the VITROCELL® system associated with the neutral red uptake (NRU) cytotoxicity assay. The VITROCELL® system is a newly representative culture and exposure system for in vitro studies of gases or complex mixtures. The impacts of two factors on cytotoxicity measurements of cigarette smoke were investigated using this WS exposure system. The factors include synthetic air exposure and optimal time to perform the NRU assay after smoke exposure. Results showed that synthetic air exposure used in the system did not significantly alter cell survival; 24 h after smoke exposure appeared to be an optimal time-point to assess the cytotoxicity of cigarette smoke. A clear dose–response relationship between smoke exposure and cell viability was demonstrated using this system, and the evaluation method was sensitive to distinguish the differences in smoke-induced cytotoxic effects from different cigarettes. In addition, we tried converting the values of EC50 from WS exposure testing into the values in unit used in total particulate matter (TPM) testing for a purpose of comparison, and the data indicate that the cytotoxicity of smoke measured by WS exposure is greater than that measured by TPM exposure.  相似文献   

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The blood–brain barrier (BBB) plays a crucial role in brain homeostasis, thereby maintaining the brain environment precise for optimal neuronal function. Its dysfunction is an intriguing complication of systemic lupus erythematosus (SLE). SLE is a systemic autoimmune disorder where neurological complications occur in 5–50% of cases and is associated with impaired BBB integrity. Complement activation occurs in SLE and is an important part of the clinical profile. Our earlier studies demonstrated that C5a generated by complement activation caused the loss of brain endothelial layer integrity in rodents. The goal of the current study was to determine the translational potential of these studies to a human system. To assess this, we used a two dimensional in vitro BBB model constructed using primary human brain microvascular endothelial cells and astroglial cells, which closely emulates the in vivo BBB allowing the assessment of BBB integrity. Increased permeability monitored by changes in transendothelial electrical resistance and cytoskeletal remodelling caused by actin fiber rearrangement were observed when the cells were exposed to lupus serum and C5a, similar to the observations in mice. In addition, our data show that C5a/C5aR1 signalling alters nuclear factor‐κB translocation into nucleus and regulates the expression of the tight junction proteins, claudin‐5 and zonula occludens 1 in this setting. Our results demonstrate for the first time that C5a regulates BBB integrity in a neuroinflammatory setting where it affects both endothelial and astroglial cells. In addition, we also demonstrate that our previous findings in a mouse model, were emulated in human cells in vitro, bringing the studies one step closer to understanding the translational potential of C5a/C5aR1 blockade as a promising therapeutic strategy in SLE and other neurodegenerative diseases.  相似文献   

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ObjectivesNew drugs and methods to efficiently fight carbapenem-resistant gram-negative pathogens are sorely needed. In this study, we characterized the preclinical pharmacokinetics (PK) and pharmacodynamics of the clinical stage drug candidate apramycin in time kill and mouse lung infection models. Based on in vitro and in vivo data, we developed a mathematical model to predict human efficacy.MethodsThree pneumonia-inducing gram-negative species Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were studied. Bactericidal kinetics were evaluated with time-kill curves; in vivo PK were studied in healthy and infected mice, with sampling in plasma and epithelial lining fluid after subcutaneous administration; in vivo efficacy was measured in a neutropenic mouse pneumonia model. A pharmacokinetic-pharmacodynamic model, integrating all the data, was developed and simulations were performed.ResultsGood lung penetration of apramycin in epithelial lining fluid (ELF) was shown (area under the curve (AUC)ELF/AUCplasma = 88%). Plasma clearance was 48% lower in lung infected mice compared to healthy mice. For two out of five strains studied, a delay in growth (~5 h) was observed in vivo but not in vitro. The mathematical model enabled integration of lung PK to drive mouse PK and pharmacodynamics. Simulations predicted that 30 mg/kg of apramycin once daily would result in bacteriostasis in patients.DiscussionApramycin is a candidate for treatment of carbapenem-resistant gram-negative pneumonia as demonstrated in an integrated modeling framework for three bacterial species. We show that mathematical modelling is a useful tool for simultaneous inclusion of multiple data sources, notably plasma and lung in vivo PK and simulation of expected scenarios in a clinical setting, notably lung infections.  相似文献   

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Paracrine signals produced from stem cells influence tissue regeneration by inducing the differentiation of endogenous stem or progenitor cells. However, many recent studies that have investigated paracrine signaling of stem cells have relied on either two-dimensional transwell systems or conditioned medium culture, neither of which provide optimal culture microenvironments for elucidating the effects of paracrine signals in vivo. In this study, we recapitulated in vivo-like paracrine signaling of human mesenchymal stem cells (hMSCs) to enhance functional neuronal differentiation of human neural stem cells (hNSCs) in three-dimensional (3D) extracellular matrices (ECMs) within a microfluidic array platform. In order to amplify paracrine signaling, hMSCs were genetically engineered using cationic polymer nanoparticles to overexpress glial cell-derived neurotrophic factor (GDNF). hNSCs were cultured in 3D ECM hydrogel used to fill central channels of the microfluidic device, while GDNF-overexpressing hMSCs (GDNF-hMSCs) were cultured in channels located on both sides of the central channel. This setup allowed for mimicking of paracrine signaling between genetically engineered hMSCs and endogenous hNSCs in the brain. Co-culture of hNSCs with GDNF-hMSCs in the 3D microfluidic system yielded reduced glial differentiation of hNSCs while significantly enhancing differentiation into neuronal cells including dopaminergic neurons. Neuronal cells produced from hNSCs differentiating in the presence of GDNF-hMSCs exhibited functional neuron-like electrophysiological features. The enhanced paracrine ability of GDNF-hMSCs was finally confirmed using an animal model of hypoxic-ischemic brain injury. This study demonstrates the presented 3D microfluidic array device can provide an efficient co-culture platform and provide an environment for paracrine signals from transplanted stem cells to control endogenous neuronal behaviors in vivo.  相似文献   

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《Research in microbiology》2016,167(2):114-125
The aim of this study was to screen how rapidly the human gut microbiota responds to diet in an in vitro model of the proximal colon (TIM-2 system). Two experimental diets were provided to the gut bacteria: a high carbohydrate and a high protein diet. The metabolic response and the composition of the microbiota were compared to a control diet simulating an average western meal. Short-chain and branched-chain fatty acids (SCFA and BCFA, respectively) production, in addition to changes in the community composition (profiling), were measured. The activity of the microbiota reflected differences between diets, exhibiting a trade-off between saccharolytic and proteolytic fermentation when compared to the control. Diversity analysis revealed a phylum-specific response depending on the diet tested. Most changes in the microbiome composition occurred during the first 24 h of the experiment. The outcome of this study elucidates the fact that human gut bacteria quickly respond to changes in diet. In addition, it confirms that variations in the concentration of carbohydrates and proteins modify the activity and composition of the microbiota, and these changes can potentially have an impact on the health of the host.  相似文献   

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A simple assay was developed to estimate functional mannose-binding lectin (MBL) levels in serum based on the principle of yeast-induced bystander lysis of chicken erythrocytes (ChE). The assay is sensitive to inhibition by ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (which allows alternative pathway activation), ethylene diamine tetraacetic acid (EDTA), mannose, N-acetylglucosamine and C1 esterase inhibitor (C1-INH), whereas it was not inhibited by galactose. A high-titer human anti-mannan antibody-containing serum with 0.06 microg MBL/ml gave a functional signal corresponding to 0.12 microg equivalents MBL/ml, indicating that anti-mannan antibodies are poorly hemolytic in the assay. The assay is well suited for the large-scale testing of patient samples for a functional MBL pathway of complement activation.  相似文献   

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We present a detailed analysis of the sensitivity of simulated Compound Action Current (CAC) and Compound Action Potential (CAP) recordings to specific model parameters, including the Single Fiber Action Currents (SFACs) and Single Fiber Action Potentials (SFAPs) that represent the contributions of each axon in the nerve bundle. In the preceding paper, we described a general method for simulating CACs and CAPs. This method uses a volume conduction model that incorporates the effects of the nerve bundle and other anisotropic properties of the region of the bundle that surrounds an individual nerve axon. In this paper, we present a complete analysis of the effects of incorrectly assigned model parameters on the simulated CAC and CAP. We also investigate the effects of incorrectly assigned parameters, recording noise, and data smoothing on the Conduction Velocity Distributions (CVDs) predicted from the CAC and CAP. We find that the simulated CAC is less sensitive to most of the parameters than is the CAP.  相似文献   

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目的研究结核分枝杆菌分泌抗原85A(antigen85A,Ag85A)重组真核表达质粒pcDNA3.1-Ag85A转染人外周血单核细胞的特性,探讨目的基因Ag85A能否在单核细胞表达,从而制备具有免疫活性的人外周血单核细胞。方法分离人外周血单核细胞并进行原代培养,流式细胞仪鉴定单核细胞纯度;Ag85A重组真核表达质粒pcDNA3.1-Ag85A转染单核细胞;RT-PCR检测目的基因Ag85A在单核细胞中的表达,目的蛋白Ag85A的表达采用Western blot鉴定。从外周血分离得到T淋巴细胞,与转染后的单核细胞共同孵育,并设空质粒转染组、重组质粒+单核细胞为对照组,3 d后用MTT法检测T淋巴细胞增殖情况。结果人外周血单核细胞经原代培养6 d后,获得大量高纯度的单核细胞,流式细胞术鉴定人单核细胞表面特异性标记CD14阳性的细胞高表达(98.35%);RT-PCR法鉴定表明Ag85A基因在转染单核细胞中成功转录,Western blot证实转染后的单核细胞可合成目的蛋白Ag85A。与外周血T淋巴细胞共同孵育后,经转染重组质粒的单核细胞相对对照组,有更强的刺激T淋巴细胞增殖的能力。结论 Ag85A重组真核表达质粒pcDNA3.1-Ag85A成功转染人外周血单核细胞,目的基因Ag85A能在人单核细胞中的表达。经转染的外周血单核细胞有刺激外周血T淋巴细胞增殖能力,从而为结核病的疫苗的研制以及为患获得性免疫缺陷病的结核病人的治疗提供新途径。  相似文献   

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The lack of a reliable mammalian neutrophil in vitro culture system has restricted our ability to examine their precise roles in mycobacterial infections. Previously, we developed the procedures for the isolation and culture of primary kidney-derived neutrophil-like cells from goldfish that are functionally and morphologically similar to mammalian neutrophils. The cultured primary goldfish neutrophils exhibited prolonged viability and functional effector responses. In this study, we demonstrate that when exposed to live or heat-killed Mycobacterium marinum, goldfish neutrophils increased their mRNA levels for several pro-inflammatory cytokines (il-1β1, il-1β2, tnfα-1, tnfα-2) and the cytokine receptors (ifngr1-1, tnfr1, tnfr2). These neutrophils also exhibited chemotaxis toward live mycobacteria, internalized the bacilli, and produced reactive oxygen intermediates (ROI) in response to pathogen exposure. The survival of intracellular mycobacteria was significantly reduced in activated neutrophils, indicating a robust killing response by these teleost granulocytes. We suggest that this goldfish primary neutrophil in vitro model system will provide important information regarding neutrophil-mediated host defense mechanisms against mycobacteria in teleosts as well as in higher vertebrates.  相似文献   

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CD31, a trans-homophilic inhibitory receptor expressed on both T- and B-lymphocytes, drives the mutual detachment of interacting leukocytes. Intriguingly, T cell CD31 molecules relocate to the immunological synapse (IS), where the T and B cells establish a stable interaction.Here, we show that intact CD31 molecules, which are able to drive an inhibitory signal, are concentrated at the periphery of the IS but are excluded from the center of the IS. At this site, were the cells establish the closest contact, the CD31 molecules are cleaved, and most of the extracellular portion of the protein, including the trans-homophilic binding sites, is shed from the cell surface.T cells lacking CD31 trans-homophilic binding sites easily establish stable interactions with B cells; at the opposite, CD31 signaling agonists inhibit T/B IS formation as well as the ensuing helper T cell activation and function. Confocal microscopy and flow cytometry analysis of experimental T/B IS shows that the T cell inhibitory effects of CD31 agonists depend on SHP-2 signaling, which reduces the phosphorylation of ZAP70.The analysis of synovial tissue biopsies from patients affected by rheumatoid arthritis showed that T cell CD31 molecules are excluded from the center of the T/B cell synapses in vivo. Interestingly, the administration of CD31 agonists in vivo significantly attenuated the development of the clinical signs of collagen-induced arthritis in DBA1/J mice.Altogether, our data indicate that the T cell co-inhibitory receptor CD31 prevents the formation of functional T/B immunological synapses and that therapeutic strategies aimed at sustaining CD31 signaling will attenuate the development of autoimmune responses in vivo.  相似文献   

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A new convenient method for the measurement of platelet-activating factor produced in vitro has been developed. This method involves incubation of neutrophils with stimuli, lipid extraction, purification of lipid extracts by normal phase high performance liquid chromatography (HPLC) and quantification of platelet-activating factor (PAF) by a competitive radioreceptor binding assay. The recovery of PAF from the extraction and purification procedures is 94.5 ± 0.9%. The sensitivity of the assay is 10 pg/tube. Human neutrophils stimulated with 0, 0.25, 0.5 1 and 2 μM A 23187 will produce ND, ND, 720, 840 and 900 pg of PAF, respectively (ND = not detectable). The values obtained for PAF produced by human neutrophils in the present assay are comparable to those obtained with the rabbit platelet aggregation assay.  相似文献   

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The beneficial health‐related effects of exercise are well recognized, and numerous studies have investigated underlying mechanism using various in vivo and in vitro models. Although electrical pulse stimulation (EPS) for the induction of muscle contraction has been used for quite some time, its application on cultured skeletal muscle cells of animal or human origin as a model of in vitro exercise is a more recent development. In this review, we compare in vivo exercise and in vitro EPS with regard to effects on signalling, expression level and metabolism. We provide a comprehensive overview of different EPS protocols and their applications, discuss technical aspects of this model including critical controls and the importance of a proper maintenance procedure and finally discuss the limitations of the EPS model.  相似文献   

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林伟  夏剑岚  王毅 《解剖学报》2014,45(2):216-220
目的探讨尿毒症毒素硫酸盐对甲酚(PCS)对人脐静脉内皮细胞(HUVECs)的毒性作用及机制。方法体外培养HUVECs,不同浓度对甲酚硫酸钠盐作用HUVECs,WST-1法检测细胞增殖;Matrigel法检测细胞成血管能力;特异性荧光探针2’,7’-二氯荧光素二乙酸酯(DCFH-DA)检测细胞活性氧自由基(ROS)水平;实时定量聚合酶链反应(Real-time PCR)及免疫印迹法(Western blotting)检测细胞中炎症因子及黏附分子表达情况。结果硫酸盐对甲酚对HUVECs增殖及成血管能力未有显著影响,分子机制研究发现,其能显著诱导细胞中ROS水平上调并促进其分泌炎症因子白细胞介素(IL)-1β、肿瘤坏死因子-a(TNF-a)及细胞间黏附分子-1(ICAM-1)。结论尿毒症毒素硫酸盐对甲酚可以加剧血管内皮细胞氧化应激及炎症反应状态,提示其可能参与慢性肾病患者动脉粥样硬化的发生发展。  相似文献   

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