RESUMO
Background and objectives: Smartphones are playing a pivotal role in everyday life, due to the opportunity they grant in terms of simplifying communication, entertainment, education and many other daily activities. Against such positive characteristics, smartphone interaction can result, in particular cases, in dangerous smartphone addiction patterns, possibly leading to several long-term detrimental psychophysiological conditions. Therefore, this pilot aims at assessing the feasibility of using an innovative approach, based on unobtrusive wearable sensors, used for the first time in this specific topic, and psychological questionnaires, to investigate the links between stress and emotions in a group of young, nonaddicted individuals performing smartphone interaction. Materials and methods: 17 volunteers were enrolled for the present study. The study protocol was divided into three phases, with an initial resting state (baseline) of three minutes, a smartphone interaction session (task) of the same length, and a final resting state (recovery), lasting three minutes. In the overall procedure, electrocardiogram (ECG) and galvanic skin response (GSR) measurements, both monitored by wearable sensors, were acquired in order to assess the functioning of the autonomic nervous system (ANS). Results: A significant decrease was seen in pNN50 during the smartphone interaction with respect to the baseline (Z = -2.675, p = 0.007), whereas the Low-to-High Frequency (LF/HF) ratio at task was somewhat correlated with phubbing behaviors (r = 0.655, p = 0.029), assessed through dedicated questionnaires. Conclusions: Taken together with the slight changes in GSR data, such results suggest the feasibility of this approach to characterize the ANS activation during smartphone interaction among young individuals. Further studies should enlarge the study population and involve smartphone-addicted subjects in order to increase the scientific and clinical relevance of such findings.
Assuntos
Sistema Nervoso Autônomo/fisiologia , Comportamento Aditivo/fisiopatologia , Comportamento Aditivo/psicologia , Monitorização Neurofisiológica/instrumentação , Smartphone , Estresse Psicológico/fisiopatologia , Estresse Psicológico/psicologia , Dispositivos Eletrônicos Vestíveis , Adulto , Eletrocardiografia , Emoções/fisiologia , Estudos de Viabilidade , Feminino , Resposta Galvânica da Pele/fisiologia , Determinação da Frequência Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Inquéritos e QuestionáriosRESUMO
Cardiac hypertrophy, in its aspects of localized thickening of the interventricular septum and concentric increase of the left ventricle, constitutes a risk factor of heart failure. Myocardial hypertrophy, in the presence of different degree of myocardial fibrosis, is paralleled by significant molecular, cellular, and histological changes inducing alteration of cardiac extracellular matrix composition as well as sarcomeres and cytoskeleton remodeling. Previous studies indicate osteopontin (OPN) and more recently survivin (SURV) overexpression as the hallmarks of heart failure although SURV function in the heart is not completely clarified. In this study, we investigated the involvement of SURV in intracellular signaling of hypertrophic cardiomyocytes and the impact of its transcriptional silencing, laying the foundation for novel target gene therapy in cardiac hypertrophy. Oligonucleotide-based molecules, like theranostic optical nanosensors (molecular beacons) and siRNAs, targeting SURV and OPN mRNAs, were developed. Their diagnostic and therapeutic potential was evaluated in vitro in hypertrophic FGF23-induced human cardiomyocytes and in vivo in transverse aortic constriction hypertrophic mouse model. Engineered erythrocyte was used as shuttle to selectively target and transfer siRNA molecules into unhealthy cardiac cells in vivo. The results highlight how the SURV knockdown could negatively influence the expression of genes involved in myocardial fibrosis in vitro and restores structural, functional, and morphometric features in vivo. Together, these data suggested that SURV is a key factor in inducing cardiomyocytes hypertrophy, and its shutdown is crucial in slowing disease progression as well as reversing cardiac hypertrophy. In the perspective, targeted delivery of siRNAs through engineered erythrocytes can represent a promising therapeutic strategy to treat cardiac hypertrophy. Theranostic SURV molecular beacon (MB-SURV), transfected into FGF23-induced hypertrophic human cardiomyocytes, significantly dampened SURV overexpression. SURV down-regulation determines the tuning down of MMP9, TIMP1 and TIMP4 extracellular matrix remodeling factors while induces the overexpression of the cardioprotective MCAD factor, which counterbalance the absence of pro-survival and anti-apoptotic SURV activity to protect cardiomyocytes from death. In transverse aortic constriction (TAC) mouse model, the SURV silencing restores the LV mass levels to values not different from the sham group and counteracts the progressive decline of EF, maintaining its values always higher with respect to TAC group. These data demonstrate the central role of SURV in the cardiac reverse remodeling and its therapeutic potential to reverse cardiac hypertrophy.
Assuntos
Cardiomegalia , Insuficiência Cardíaca , Animais , Cardiomegalia/genética , Cardiomegalia/terapia , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Survivina/genética , Survivina/metabolismo , Survivina/uso terapêutico , Remodelação VentricularRESUMO
In this work a continuum model for high frequency poroelastic longitudinal waves in hydrogels is presented. A viscoelastic force describing the interaction between the polymer network and the bounded water present in such materials is introduced. The model is tested by means of ultrasound wave speed and attenuation measurements in polyvinylalcohol hydrogel samples. The theory and experiments show that ultrasound attenuation decreases linearly with the increase in the water volume fraction beta of the hydrogel. The introduction of the viscoelastic force between the bounded water and the polymer network leads to a bi-phasic theory, showing an ultrasonic fast wave attenuation that can vary as a function of the frequency with a non-integer exponent in agreement with the experimental data in literature. When beta tends to 1 (100% of interstitial water) due to the presence of bounded water in the hydrogel, the ultrasound phase velocity acquires higher value than that of pure water. The ultrasound speed gap at beta=1 is confirmed by the experimental results, showing that it increases in less cross-linked gel samples which own a higher concentration of bounded water.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Modelos Teóricos , Álcool de Polivinil/química , Ultrassom , Elasticidade , Viscosidade , Água/químicaRESUMO
Cancer is fast becoming the most important cause of death worldwide, its mortality being mostly caused by late or wrong diagnosis. Novel strategies have been developed to identify early signs of cancer in a minimally obtrusive way, including the Electronic Nose (E-Nose) technology, user-friendly, cost- and time-saving alternative to classical approaches. This systematic review, conducted under the PRISMA guidelines, identified 60 articles directly dealing with the E-Nose application in cancer research published up to 31 January 2020. Among these works, the vast majority reported successful E-Nose use for diagnosing Lung Cancer, showing promising results especially when employing the Aeonose tool, discriminating subjects with Lung Cancer from controls in more than 80% of individuals, in most studies. In order to tailor the main limitations of the proposed approach, including the application of the protocol to advanced stage of cancer, sample heterogeneity and massive confounders, future studies should be conducted on early stage patients, and on larger cohorts, as to better characterize the specific breathprint associated with the various subtypes of cancer. This would ultimately lead to a better and faster diagnosis and to earlier treatment, possibly reducing the burden associated to such conditions.
Assuntos
Nariz Eletrônico , Neoplasias Pulmonares/diagnóstico , Testes Respiratórios , HumanosRESUMO
Melt-extruded guides for peripheral nerve repair based on poly(epsilon-caprolactone) (PCL) were realised and their physico-chemical properties were evaluated. Preliminarily, PCL cast films were found to support the attachment and proliferation of Neonatal Olfactory Bulb Ensheating Cells (NOBEC). S5Y5 neuroblastoma cells were cultured inside PCL guides in their uncoated form or coated with a non-specific adhesion protein (gelatin) and a specific peptide for nerve regeneration (poly(L-lysine)). Coating increased cell density (gelatin) and/or the cell density rate on substrates (poly(L-lysine); gelatin) as compared to uncoated guides. Various in vivo tests were carried out for the repair of small (0.5 cm), medium (1.5 cm) and long (4.5 cm) size defects in the peripheral nerves of Wistar rats. For the small nerve defects, uncoated and coated PCL guides were tested. Results from in vivo tests were subjected to histological examination after 45 days, 6 and 8 months postoperative for small, medium and large defects, respectively. Regeneration was found for small and medium size defects. For 0.5 cm defects, the coating did not affect regeneration significantly. Grip-tests also evidenced functional recovery for the 1.5 cm-long defects treated with PCL guides, after 6 months from implantation. On the other hand, mechanical stiffness of PCL conduits impaired the repair of 4.5 cm-long defects in 8-month period: the lack of flexibility of the guide to rat movements caused its detachment from the implant site. The research showed that PCL guides can be used for the successful repair of small and medium size nerve defects, with possible improvements by suitable bio-mimetic coatings.
Assuntos
Regeneração Tecidual Guiada/métodos , Regeneração Nervosa/efeitos dos fármacos , Nervos Periféricos/efeitos dos fármacos , Nervos Periféricos/fisiologia , Transição de Fase , Poliésteres/química , Poliésteres/farmacologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Fenômenos Mecânicos , Nervo Mediano/efeitos dos fármacos , Nervo Mediano/fisiologia , Camundongos , Nervo Fibular/efeitos dos fármacos , Nervo Fibular/fisiologia , Ratos , Ratos Wistar , TermogravimetriaRESUMO
Electronic nose (eNose) systems are particularly appreciated for their portability, usability, relative low cost, and real-time or near real-time response. Their application finds space in several domains, including environmental monitoring. Within this field, marine monitoring is of particular scientific relevance due to the fragility of this specific environment, daily threatened by human activities that can potentially bring to catastrophic and irreversible consequences on marine wildlife. Under such considerations, a systematic review, complying with the PRISMA guidelines, was conducted covering the period up to 15 October 2018, in PubMed, ScienceDirect, and Google Scholar. Despite the relatively low number of articles published on this specific topic and the heterogeneity of the technological approaches employed, the results obtained by the various groups highlight the positive contribution eNose has given and can provide in near future for the monitoring and safeguarding of this delicate environment.
Assuntos
Nariz Eletrônico/normas , Monitoramento Ambiental/métodos , Água do Mar/química , HumanosRESUMO
BACKGROUND: In the pathogenesis of atherosclerosis, a central role is represented by endothelial inflammation with influx of chemokine-mediated leukocytes in the vascular wall. Aim of this study was to analyze the effect of different shear stresses on endothelial gene expression and compute gene network involved in atherosclerotic disease, in particular to homeostasis, inflammatory cell migration, and apoptotic processes. METHODS: HUVECs were subjected to shear stress of 1, 5, and 10 dyne/cm2 in a Flow Bioreactor for 24 hours to compare gene expression modulation. Total RNA was analyzed by Affymetrix technology and the expression of two specific genes (CXCR4 and ICAM-1) was validated by RT-PCR. To highlight possible regulations between genes and as further validation, a bioinformatics analysis was performed. RESULTS: At low shear stress (1 dyne/cm2) we observed the following: (a) strong upregulation of CXCR4; (b) mild upregulation of Caspase-8; (c) mild downregulation of ICAM-1; (d) marked downexpression of TNFAIP3. Bioinformatics analysis showed the presence of network composed by 59 new interactors (14 transcription factors and 45 microRNAs) appearing strongly related to shear stress. CONCLUSIONS: The significant modulation of these genes at low shear stress and their close relationships through transcription factors and microRNAs suggest that all may promote an initial inflamed endothelial cell phenotype, favoring the atherosclerotic disease.
Assuntos
Aterosclerose/metabolismo , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células Endoteliais da Veia Umbilical Humana/metabolismo , Modelos Cardiovasculares , Resistência ao Cisalhamento , Estresse Fisiológico , Aterosclerose/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , HumanosRESUMO
The aim of this study was set-up and test of gelatin and carbon nanotubes scaffolds. Gelatin-based (5%) genipin cross-linked (0.2%) scaffolds embedding single-walled carbon nanotubes (SWCNTs, 0.3, 0.5, 0.7, 0.9, and 1.3% w/w) were prepared and mechanically/electrically characterized. For biological evaluation, H9c2 cell line was cultured for 10 days. Cytotoxicity, cell growth and differentiation, immunohistochemistry, and real-time PCR analysis were performed. Myoblast and cardiac differentiation were obtained by serum reduction to 1% (C1% ) and stimulation with 50 nM all trans-retinoic acid (CRA ), respectively. Immunohistochemistry showed elongated myotubes in C1% while round and multinucleated cells in CRA with also a significantly increased expression of natriuretic peptides (NP) and ET-1 receptors in parallel with a decreased ET-1. On scaffolds, cell viability was similar for Gel-SWCNT0.3%/0.9% ; NP and ET systems expression decreased in both concentrations with respect to control and CX-43, mainly due to a lacking of complete differentiation in cardiac phenotype during that time. Although further analyses on novel biomaterials are necessary, these results represent a useful starting point to develop new biomaterial-based scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2750-2762, 2018.
Assuntos
Diferenciação Celular , Gelatina/química , Coração/fisiologia , Mioblastos Cardíacos/metabolismo , Nanotubos de Carbono/química , Regeneração , Alicerces Teciduais/química , Animais , Linhagem Celular , Regulação da Expressão Gênica , Teste de Materiais , Proteínas Musculares/biossíntese , Mioblastos Cardíacos/citologia , RatosRESUMO
Polymethylmethacrylate core-shell fluorescent nanoparticles promote, in human lung A549 cancer cells, the internalization of a molecular beacon (MB) specific for survivin mRNA, an anti-apoptotic protein overexpressed in cancer cells. AIMS: To design an effective drug delivery system, the knowledge of the uptake mechanism and of the nanoparticles (NPs) and MB fate is required. MATERIALS AND METHODS AND KEY FINDINGS: Experiments with dextran as marker for endocytosis showed that in the presence of NPs the number of endocytic vesicles per cell doubled and their mean size significantly (pâ¯<â¯0.001) increased with respect to controls in absence of NPs, indicating an involvement of NPs in the endocytotic process. By using LysoTracker™ Deep Red, as marker of lysosomes, we found that nanoparticles co-localize with lysosomes. Moreover, a cellular release of nanoparticles detected in the culture medium, suggested a role of lysosomal exocytosis in nanoparticle elimination. The MB fluorescence in proximity of the labeled Endoplasmic Reticulum was indicative that the opening of the MB occurs in proximity of its target mRNA. SIGNIFICANCE: The results show the involvement of endocytotic pathway in the uptake of NPs, which are an appropriate delivery system capable of being eliminated by cells. Furthermore the data confirm that the MB can be considered an effective tool for the intracellular sensing.
Assuntos
Sistemas de Liberação de Medicamentos , Endocitose/efeitos dos fármacos , Nanopartículas/administração & dosagem , Polímeros/química , Survivina/metabolismo , Células A549 , Dextranos/administração & dosagem , Dextranos/metabolismo , Retículo Endoplasmático/metabolismo , Fluorescência , Humanos , Neoplasias Pulmonares/metabolismo , Lisossomos/metabolismo , Nanopartículas/metabolismo , Polimetil Metacrilato/química , RNA Mensageiro/metabolismo , Survivina/genéticaRESUMO
Three-dimensional (3D) tissue models offer new tools in the study of diseases. In the case of the engineering of cardiac muscle, a realistic goal would be the design of a scaffold able to replicate the tissue-specific architecture, mechanical properties, and chemical composition, so that it recapitulates the main functions of the tissue. This work is focused on the design and preliminary biological validation of an innovative polyester urethane (PUR) scaffold mimicking cardiac tissue properties. The porous scaffold was fabricated by thermally induced phase separation (TIPS) from poly(ε-caprolactone) diol, 1,4-butanediisocyanate, and l-lysine ethyl ester. Morphological and mechanical scaffolds characterization was accomplished by confocal microscopy, and micro-tensile and compression techniques. Scaffolds were then functionalized with fibronectin by plasma treatment, and the surface treatment was studied by x-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectra, and contact angle measurements. Primary rat neonatal cardiomyocytes were seeded on scaffolds, and their colonization, survival, and beating activity were analyzed for 14 days. Signal transduction pathways and apoptosis involved in cells, the structural development of the heart, and its metabolism were analyzed. PUR scaffolds showed a porous-aligned structure and mechanical properties consistent with that of the myocardial tissue. Cardiomyocytes plated on the scaffolds showed a high survival rate and a stable beating activity. Serine/threonine kinase (AKT) and extracellular signal-regulated kinases (ERK) phosphorylation was higher in cardiomyocytes cultured on the PUR scaffold compared to those on tissue culture plates. Real-time polymerase chain reaction analysis showed a significant modulation at 14 days of cardiac muscle (MYH7, prepro-ET-1), hypertrophy-specific (CTGF), and metabolism-related (SLC2a1, PFKL) genes in PUR scaffolds.
Assuntos
Biomimética , Butanos/química , Lisina/química , Miócitos Cardíacos/metabolismo , Nitrilas/química , Poliésteres/química , Poliuretanos/química , Animais , Apoptose , Células Cultivadas , Força Compressiva , Fibronectinas/metabolismo , Humanos , Imageamento Tridimensional , Microscopia Confocal , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Nanofibras/química , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Espectrofotometria Infravermelho , Espectroscopia de Infravermelho com Transformada de Fourier , Resistência à Tração , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
BACKGROUND: Autism Spectrum Disorders (ASD) represent a heterogeneous set of neurodevelopmental disorders characterized by impairments in social domain, where the autonomic nervous system (ANS) plays an important role. Several researchers have studied the ANS in ASD, during specific cognitive or sensory stimuli while few studies have examined response during social interactions. Wearable technologies can be very helpful in monitoring autonomic response in children with ASD in semi-naturalistic setting. The novelty of this study is to use such technologies to acquire physiological signals during therapeutic sessions supported by interactive "serious games" and to correlate the ANS response to the engagement of the child during sociocognitive tasks for an evaluation of the treatment effect and for the personalization of the therapy. METHOD: A wearable chest belt for electrocardiographic (ECG) signal recording was used and specific algorithms for the extraction of clinically relevant features (Heart Rate - HR, Root Mean Square of the Successive Differences - RMSSD and Respiratory Sinus Arrhythmia - RSA) were developed. Sociocognitive tasks were mediated by "serious games" implemented on two tablets, which allowed a precise coding of the behaviors of the children. A longitudinal assessment of the physiological response of the children during six months of treatment was performed. RESULTS: A link between physiological response, i.e. decrease in RMSSD and RSA, and engagement of the children during sociocognitive tasks was found. Longitudinal changes in the children's autonomic response, including a decrease of RSA during the engagement throughout the therapeutic sessions, were found. CONCLUSIONS: These results foster the feasibility of this methodology to be applied in a clinical setting for the monitoring of the ANS response of children with ASD during treatment. A larger sample of patients is needed to confirm these preliminary findings.
Assuntos
Transtorno do Espectro Autista , Eletrocardiografia Ambulatorial , Comportamento Social , Dispositivos Eletrônicos Vestíveis , Arritmia Sinusal/fisiopatologia , Transtorno do Espectro Autista/fisiopatologia , Transtorno do Espectro Autista/psicologia , Criança , Eletrocardiografia Ambulatorial/instrumentação , Eletrocardiografia Ambulatorial/métodos , Estudos de Viabilidade , Frequência Cardíaca/fisiologia , Humanos , Masculino , Processamento de Sinais Assistido por Computador/instrumentação , Análise e Desempenho de TarefasRESUMO
AIMS: The industrial development of a product requires performing a deep analysis to highlight its characteristics useful for future design. The clinical use of a product stimulates knowledge improvement about it in a constant effort of progress. This work shows the biological characterization of CMC composite mesh. CMC polypropylene prosthesis was seeded with Human fibroblast BJ. Samples (cells and medium) were collected at different time points in order to perform different analysis: inflammatory markers quantification; collagens immunohistochemistry; matrix metalloproteinases zimography; extracellular matrix proteomic profile. FINDINGS: CMC presented a good cell viability rate and cell growth during the 21 days. The inflammatory profile showed an initial secretion of anti-inflammatory IL-10 and a final increase of pro-inflammatory IL-6. Immunocytochemistry highlighted a similar Collagen type I/type III ratio. The proteomic analysis evidenced the ECM protein content profile composed, mainly, by collagens, fibronectin, laminin. MMPs resulted both expressed when in contact to mesh. CONCLUSIONS: CMC shows a good cell biocompatibility and growth. The increase of pro-inflammatory markers could stimulate proliferation, influencing the integration process in human body. Proteomics highlights the ECM modulation by CMC. An integrated investigation of these biological analyses with mechanical data should improve the design process of a new product. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2045-2052, 2017.
Assuntos
Hérnia Abdominal/metabolismo , Teste de Materiais , Proteômica , Telas Cirúrgicas , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Hérnia Abdominal/cirurgia , Herniorrafia , Humanos , Imuno-Histoquímica , Interleucina-10/metabolismo , Interleucina-6/metabolismoRESUMO
This study describes an actuated bioreactor which mimics the pulsatile contractile motion of the intestinal barrier using electro-responsive elastomers as smart materials that undergo deformation upon electrical stimulation. The device consists of an annular dielectric elastomer actuator working as a radial artificial muscle able to rhythmically contract and relax a central cell culture well. The bioreactor maintained up to 4 h of actuation at a frequency of 0.15 Hz and a strain of 8%-10%, to those of the cyclic contraction and relaxation of the small intestine. In vitro tests demonstrated that the device was biocompatible and cell-adhesive for Caco-2 cells, which formed a confluent monolayer following 21 days of culture in the central well. In addition, cellular adhesion and cohesion were maintained after 4 h of continuous cyclic strain. These preliminary results encourage further investigations on the use of dielectric elastomer actuation as a versatile technology that might overcome the limitations of commercially available pneumatic driving systems to obtain bioreactors that can cyclically deform cell cultures in a biomimetic fashion.
Assuntos
Materiais Biomiméticos , Biomimética/instrumentação , Reatores Biológicos , Elastômeros , Peristaltismo/fisiologia , Materiais Biocompatíveis , Células CACO-2 , Adesão Celular , Técnicas de Cultura de Células , Estimulação Elétrica , Humanos , Intestino Delgado/fisiologia , PolímerosRESUMO
The quantitative determination of specific cellular messenger-RNA is extremely important both in basic and applied research, especially in diagnostic and pharmacological fields. In order to perform a direct and easy quantification of transcripts on cell extracts, the feasibility of an analytical device able to selectively detect a defined target RNA in a complex mixture while avoiding labelling, retrotranscription and amplification steps, has been explored. In particular, several aspects necessary to obtain good selectivity in target recognition, stability, reusability and sensitivity of a gene specific biosensor were considered. For the development of suitable probe-receptors, analysis of the nucleotide sequence of the target mRNA was carried out to localise the preferred binding regions. As criteria for optimisation, we selected accessibility and uniqueness. Oligonucleotide probes, designed to specifically bind these sequences, were synthesised by using particular monomers producing nuclease-resistant RNA strands with high affinity towards the target. Quartz crystal microbalance (QCM) technology was selected to realise a microgravimetric sensor able to bind the RNA under investigation through a complementary oligonucleotide probe. Covalent immobilisation of bioreceptor molecules to the transducer sensitive surface ensured a stable integration between the two. The binding ability of immobilised probes was tested evaluating their annealing behaviour with both complementary oligonucleotides and full-length target mRNA. The conditions necessary for the regeneration of biosensor were also assessed. Measurements of shift in QCM resonant frequency, performed by hybridisation experiments in liquido, demonstrate that a label-free RNA-biosensor with high specificity, reusability and the ability to give quantitative information, can be realised.
Assuntos
Técnicas Biossensoriais/instrumentação , Eletroquímica/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Sondas de Oligonucleotídeos/síntese química , Sondas de Oligonucleotídeos/genética , RNA/análise , RNA/genética , Técnicas Biossensoriais/métodos , Eletroquímica/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Reutilização de Equipamento , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Integração de SistemasRESUMO
An electrochemical immunoassay for neopterin was developed using recently produced specific antibodies immobilized to protein A-coated magnetic beads in combination with differential pulse voltammetry and screen-printed array of electrodes. Neopterin-alkaline phosphatase conjugate was used as label in a competitive assay format. Multiplexed analysis of neopterin was demonstrated by replacing the traditional ELISA with electrochemical detection and the traditional plastic wells with screen-printed array of electrodes. The optimized electrochemical method, based on polyclonal antibodies, reached a limit of detection of 0.008 ng/mL with an average RSD %=10. Serum samples collected from patients with sepsis, healthy volunteers and other patients without a confirmed clinical diagnosis were also analyzed. The obtained results, compared with those of a commercial ELISA kit, had a significant correlation, showing the possibility to distinguish among the serum samples from ill or healthy subjects.
Assuntos
Técnicas Biossensoriais , Neopterina/análise , Anticorpos/imunologia , Biomarcadores , Técnicas Eletroquímicas , Eletrodos , Humanos , Proteínas Imobilizadas/imunologia , Imunoensaio , Inflamação , Neopterina/sangue , Neopterina/imunologia , Sepse/sangueRESUMO
A study of antibody immobilisation techniques on quartz and fibre optic surfaces for immunosensors has been carried out. Methods of covalent antibody immobilisation which have not previously been applied to optical fibres were investigated, and compared with classical methods found in the literature. Preliminary experiments on covalent immobilisation methods on planar quartz surfaces were conducted to enable us to choose the most suitable protein immobilisation technique for sensor applications. The immobilisation studies were directed in particular towards obtaining a high density of binding sites for the analyte of interest. Two of the most promising methods, antibody immobilisation on surfaces coated with dextran based hydrogel and F(ab')-SH fragments bound to silanised glass, which resulted in surface densities of active sites of above 0.45 pmol/cm2, were selected for further experiments on a fibre optic total internal reflection fluorescence immunosensor and gave satisfactory responses to changes in analyte concentrations of the order of 10(-8) M. The efficiency of polar organic solvents, such as dimethylsulfoxide, in dissociating the antigen-antibody complex and hence to regenerate the immunosensor surface was also evaluated.
Assuntos
Anticorpos/química , Técnicas Biossensoriais/instrumentação , Eletrodos , Tecnologia de Fibra Óptica/instrumentação , Imunoensaio/métodos , Imunoglobulina G/análise , Albumina Sérica/análise , Espectrometria de Fluorescência/instrumentação , Adsorção , Animais , Anticorpos/análise , Anticorpos/imunologia , Complexo Antígeno-Anticorpo/análise , Complexo Antígeno-Anticorpo/imunologia , Técnicas Biossensoriais/métodos , Materiais Revestidos Biocompatíveis/síntese química , Materiais Revestidos Biocompatíveis/química , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Imunoensaio/instrumentação , Imunoglobulina G/imunologia , Coelhos , Albumina Sérica/imunologia , Espectrometria de Fluorescência/métodosRESUMO
Therapeutic applications of ultrasound are currently limited to dental plaque removal, physiotherapy and lithotripsy. However, several in vitro and experimental studies have shown the ability of ultrasound to accelerate clot dissolution. This effect is mainly influenced by the intensity and frequency of the beam. High ultrasound energies, although effective, can induce early reocclusion, while moderate intensities and low ultrasound frequencies are better tolerated and equally effective. So far, few patients with acute myocardial infarction have been treated by ultrasound catheters. In patients with ischemic stroke, transcranial Doppler was monitored during the venous administration of recombinant tissue-type plasminogen activator (rt-PA) and the occluded vessel was recanalyzed earlier than in other studies by rt-PA alone (without Doppler monitoring). Ultrasound-accelerated thrombolysis is caused by a strengthening of the enzymatic action, favored by acoustic cavitation. As the microbubbles of echocontrast agents lower the cavitation threshold, they can further enhance the thrombolytic process. New generation microbubbles, able to bind to the thrombus surface, could facilitate thrombus-microbubbles interaction. The combination of ultrasound, microbubbles and fibrinolytic agents could benefit the treatment of a variety of cardiovascular diseases.
Assuntos
Terapia Trombolítica/métodos , Gases , Humanos , Fatores de Tempo , UltrassonografiaRESUMO
Abundant experimental evidence demonstrates that endothelial cells are sensitive to flow; however, the effect of fluid pressure or pressure gradients that are used to drive viscous flow is not well understood. There are two principal physical forces exerted on the blood vessel wall by the passage of intra-luminal blood: pressure and shear. To analyze the effects of pressure and shear independently, these two stresses were applied to cultured cells in two different types of bioreactors: a pressure-controlled bioreactor and a laminar flow bioreactor, in which controlled levels of pressure or shear stress, respectively, can be generated. Using these bioreactor systems, endothelin-1 (ET-1) and nitric oxide (NO) release from human umbilical vein endothelial cells were measured under various shear stress and pressure conditions. Compared to the controls, a decrease of ET-1 production by the cells cultured in both bioreactors was observed, whereas NO synthesis was up-regulated in cells under shear stress, but was not modulated by hydrostatic pressure. These results show that the two hemodynamic forces acting on blood vessels affect endothelial cell function in different ways, and that both should be considered when planning in vitro experiments in the presence of flow. Understanding the individual and synergic effects of the two forces could provide important insights into physiological and pathological processes involved in vascular remodeling and adaptation.
Assuntos
Reatores Biológicos , Endotelina-1/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Óxido Nítrico/biossíntese , Fenômenos Biomecânicos , Endotelina-1/metabolismo , Regulação da Expressão Gênica , Hemodinâmica/fisiologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Pressão Hidrostática/efeitos adversos , Estresse Mecânico , Engenharia TecidualRESUMO
After passage through biological barriers, nanomaterials inevitably end up in contact with the vascular endothelium and can induce cardiovascular damage. In this study the toxicity and sub-lethal effects of six types of nanoparticle, including four of industrial and biomedical importance, on human endothelial cells were investigated using different in vitro assays. The results show that all the particles investigated induce some level of damage to the cells and that silver particles were most toxic, followed by titanium dioxide. Furthermore, endothelial cells were shown to be more susceptible when exposed to silver nanoparticles under flow conditions in a bioreactor. The study underlines that although simple in vitro tests are useful to screen compounds and to identify the type of effect induced on cells, they may not be sufficient to define safe exposure limits. Therefore, once initial toxicity screening has been conducted on nanomaterials, it is necessary to develop more physiologically relevant in vitro models to better understand how nanomaterials can impact on human health.
Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Nanopartículas/toxicidade , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/análise , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Humanos , Nanopartículas/química , Poliestirenos/química , Poliestirenos/toxicidade , Prata/química , Prata/toxicidade , Titânio/química , Titânio/toxicidade , Fator de von Willebrand/análise , Fator de von Willebrand/metabolismoRESUMO
Permeability studies across epithelial barriers are of primary importance in drug delivery as well as in toxicology. However, traditional in vitro models do not adequately mimic the dynamic environment of physiological barriers. Here, we describe a novel two-chamber modular bioreactor for dynamic in vitro studies of epithelial cells. The fluid dynamic environment of the bioreactor was characterized using computational fluid dynamic models and measurements of pressure gradients for different combinations of flow rates in the apical and basal chambers. Cell culture experiments were then performed with fully differentiated Caco-2 cells as a model of the intestinal epithelium, comparing the effect of media flow applied in the bioreactor with traditional static transwells. The flow increases barrier integrity and tight junction expression of Caco-2 cells with respect to the static controls. Fluorescein permeability increased threefold in the dynamic system, indicating that the stimulus induced by flow increases transport across the barrier, closely mimicking the in vivo situation. The results are of interest for studying the influence of mechanical stimuli on cells, and underline the importance of developing more physiologically relevant in vitro tissue models. The bioreactor can be used to study drug delivery, chemical, or nanomaterial toxicity and to engineer barrier tissues.