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Fucoidans from brown algae are described as anti-inflammatory, antioxidative, and antiangiogenic. We tested two Saccharina latissima fucoidans (SL-FRO and SL-NOR) regarding their potential biological effects against age-related macular degeneration (AMD). Primary porcine retinal pigment epithelium (RPE), human RPE cell line ARPE-19, and human uveal melanoma cell line OMM-1 were used. Cell survival was assessed in tetrazolium assay (MTT). Oxidative stress assays were induced with erastin or H2O2. Supernatants were harvested to assess secreted vascular endothelial growth factor A (VEGF-A) in ELISA. Barrier function was assessed by measurement of trans-epithelial electrical resistance (TEER). Protectin (CD59) and retinal pigment epithelium-specific 65 kDa protein (RPE65) were evaluated in western blot. Polymorphonuclear elastase and complement inhibition assays were performed. Phagocytosis of photoreceptor outer segments was tested in a fluorescence assay. Secretion and expression of proinflammatory cytokines were assessed with ELISA and real-time PCR. Fucoidans were chemically analyzed. Neither toxic nor antioxidative effects were detected in ARPE-19 or OMM-1. Interleukin 8 gene expression was slightly reduced by SL-NOR but induced by SL-FRO in RPE. VEGF secretion was reduced in ARPE-19 by SL-FRO and in RPE by both fucoidans. Polyinosinic:polycytidylic acid induced interleukin 6 and interleukin 8 secretion was reduced by both fucoidans in RPE. CD59 expression was positively influenced by fucoidans, and they exhibited a complement and elastase inhibitory effect in cell-free assay. RPE65 expression was reduced by SL-NOR in RPE. Barrier function of RPE was transiently reduced. Phagocytosis ability was slightly reduced by both fucoidans in primary RPE but not in ARPE-19. Fucoidans from Saccharina latissima, especially SL-FRO, are promising agents against AMD, as they reduce angiogenic cytokines and show anti-inflammatory and complement inhibiting properties; however, potential effects on gene expression and RPE functions need to be considered for further research.
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Laminaria , Degeneração Macular , Humanos , Animais , Suínos , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Laminaria/metabolismo , Peróxido de Hidrogênio/metabolismo , Interleucina-8/metabolismo , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismoRESUMO
Cellular tight junctions play a key role in establishing a barrier between different compartments of the body by regulating the selective passage of different solutes across epithelial and endothelial tissues. Over the past decade, significant efforts have been conducted to develop more clinically relevant "organ-on-a-chip" models with integrated trans-epithelial electrical resistance (TEER) monitoring systems to help better understand the fundamental underpinnings of epithelial tissue physiology upon exposure to different substances. However, most of these platforms require the use of high-cost and time-consuming photolithography processes, which limits their scalability and practical implementation in clinical research. To address this need, we have developed a low-cost microfluidic platform with an integrated electrode array that allows continuous real-time monitoring of TEER and the risk of bubble formation in the microfluidic system by using scalable manufacturing technologies such as screen printing and laser processing. The integrated printed electrode array exhibited excellent stability (with less than â¼0.02 Ω change in resistance) even after long-term exposure to a complex culture medium. As a proof of concept, the fully integrated platform was tested with HMT3522 S1 epithelial cells to evaluate the tight barrier junction formation through TEER measurement and validated with standard immunostaining procedures for Zonula occludens-1 protein. This platform could be regarded as a stepping stone for the fabrication of disposable and low-cost organ and tissue-on-a-chip models with integrated sensors to facilitate studying the dynamic response of epithelial tissues to different substances in more physiologically relevant conditions.
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Células Epiteliais , Dispositivos Lab-On-A-Chip , Células Epiteliais/fisiologia , Linhagem Celular , Eletrodos , Impedância ElétricaRESUMO
The kidney proximal tubule (PT) mediates renal drug elimination in vivo and is a major site of drug-induced toxicity. To reliably assess drug efficacy, it is crucial to construct a model in which PT functions are replicated. Current animal studies have proven poorly predictive of human outcome. To address this, we developed a physiologically relevant micro-physiological system (MPS) model of the human PT, the aProximate MPS Flow platform (Patent No: G001336.GB). In this model, primary human PT cells (hPTCs) are subjected to fluidic media flow and a shear stress of 0.01-0.2 Pa. We observe that these cells replicate the polarity of hPTCs and exhibit a higher expression of all the key transporters of SLC22A6 (OAT1), SLC22A8 (OAT3), SLC22A2 (OCT2), SLC47A1 (MATE1), SLC22A12 (URAT1), SLC2A9 (GLUT9), ABCB1 (MDR1), ABCC2 (MRP2), LRP2 (megalin), CUBN (cubilin), compared with cells grown under static conditions. Immunofluorescence microscopy confirmed an increase in OAT1, OAT3, and cilia protein expression. Increased sensitivity to nephrotoxic protein cisplatin was observed; creatinine and FITC-albumin uptake was significantly increased under fluidic shear stress conditions. Taken together, these data suggest that growing human PT cells under media flow significantly improves the phenotype and function of hPTC monolayers and has benefits to the utility and near-physiology of the model.
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Intercellular contacts between epithelial cells are established and maintained by the apical junctional complexes (AJCs). AJCs conserve cell polarity and build epithelial barriers to pathogens, inhaled allergens, and environmental particles in the respiratory tract. AJCs consist of tight junctions (TJs) and adherens junctions (AJs), which play a key role in maintaining the integrity of the airway barrier. Emerging evidence has shown that different microorganisms cause airway barrier dysfunction by targeting TJ and AJ proteins. This review discusses the pathophysiologic mechanisms by which several microorganisms (bacteria and viruses) lead to the disruption of AJCs in airway epithelial cells. We present recent progress in understanding signaling pathways involved in the formation and regulation of cell junctions. We also summarize the potential chemical inhibitors and pharmacological approaches to restore the integrity of the airway epithelial barrier. Understanding the AJCs-pathogen interactions and mechanisms by which microorganisms target the AJC and impair barrier function may further help design therapeutic innovations to treat these infections.
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The presence of protein corona on the surface of nanoparticles modulates their physiological interactions such as cellular association and targeting property. It has been shown that α-mangostin (αM)-loaded poly(ethylene glycol)-poly(l-lactide) (PEG-PLA) nanoparticles (NP-αM) specifically increased low density lipoprotein receptor (LDLR) expression in microglia and improved clearance of amyloid beta (Aß) after multiple administration. However, how do the nanoparticles cross the bloodâbrain barrier and access microglia remain unknown. Here, we studied the brain delivery property of PEG-PLA nanoparticles under different conditions, finding that the nanoparticles exhibited higher brain transport efficiency and microglia uptake efficiency after αM loading and multiple administration. To reveal the mechanism, we performed proteomic analysis to characterize the composition of protein corona formed under various conditions, finding that both drug loading and multiple dosing affect the composition of protein corona and subsequently influence the cellular uptake of nanoparticles in b.End3 and BV-2 cells. Complement proteins, immunoglobulins, RAB5A and CD36 were found to be enriched in the corona and associated with the process of nanoparticles uptake. Collectively, we bring a mechanistic understanding about the modulator role of protein corona on targeted drug delivery, and provide theoretical basis for engineering brain or microglia-specific targeted delivery system.
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Determining the amount of a drug transferred into breast milk is critical for benefit-risk analysis of breastfeeding when a lactating mother takes medications. In this study, we developed a human mammary epithelial cell (MEC)-based permeability assay to assess drug permeability across the mammary epithelium. Human MEC cell MCF10F formed tight junctions when cultured on Transwells with culture medium containing insulin, hydrocortisone and epidermal growth factor (EGF). Formation of integral cell barrier and morphology of the cells were confirmed by assessing trans-epithelial electrical resistance (TEER), flux of fluorescent tracers and imaging with transmission electron microscopy (TEM). MCF10F cells showed consistent P-glycoprotein (P-gp) transporter expression when culturing on Transwell inserts versus on petri dish. A few P-gp transporter drug substrates were used to estimate the permeability from this assay. Human plasma and breast milk were used as incubation medium in basolateral and apical chambers respectively to mimic physiological conditions. The predicted milk to plasma (M/P) ratios were reasonably good. The current effort to develop the MEC-based permeability assay to facilitate M/P ratio prediction showed promising results. This assay may have a potential to be developed as a useful in vitro technique for determining the transfer of small-molecule therapeutic drugs into breast milk.
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Inhaled substances, such as consumer products, chemicals at the workplace, and nanoparticles, can affect the lung function in several ways. In this paper, we explore the adverse outcome pathway (AOP) that starts when inhaled substances that reach the alveoli inhibit the function of the lung surfactant, and leads to decreased lung function. Lung surfactant covers the inner surface of the alveoli, and regulates the surface tension at the air-liquid interface during breathing. The inhibition of the lung surfactant function leads to alveolar collapse because of the resulting high surface tension at the end of expiration. The collapsed alveoli can be re-opened by inspiration, but this re-opening causes shear stress on cells covering the alveoli. This can damage the alveolar-capillary membrane integrity, allowing blood components to enter the alveolar airspace. Blood components, such as albumin, can interact with the lung surfactant and further inhibit its function. The collapse of the alveoli is responsible for a decrease in the surface area available for blood oxygenation, and it reduces the volume of air that can be inhaled and exhaled. These different key events lead to decreased lung function, characterized by clinical signs of respiratory toxicity and reduced blood oxygenation. Here we present the weight of evidence that supports the AOP, and we give an overview of the methods available in vitro and in vivo to measure each key event of the pathway, and how this AOP can potentially be used in screening for inhalation toxicity.
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This paper reports a transwell insert-embedded microfluidic device capable of culturing cells at an air-liquid interface (ALI), mimicking the in vivo alveolar epithelium microenvironment. Integration of a commercially available transwell insert makes the device fabrication straightforward and eliminates the tedious device assembly processes. The transwell insert can later be detached from the device for high-resolution imaging of the cells. In the experiments, the cells showing type-I pneumocyte markers are exploited to construct an in vitro alveolar epithelium model, and four culture conditions including conventional liquid/liquid culture (LLC) and air-liquid interface (ALI) cell culture in normal growth medium, and ALI cell culture with inflammatory cytokine (TNF-α) stimulation and ethanol vapor exposure are applied to investigate their effects on the alveolar epithelium barrier function. The barrier permeability is time-lapse monitored using trans-epithelial electrical resistance (TEER) measurement and immunofluorescence staining of the tight junction protein (ZO-1). The results demonstrate the functionalities of the device, and further show the applications and advantages of the constructed in vitro cell models for the lung studies.
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The apical junctional complexes (AJCs) of airway epithelial cells are a key component of the innate immune system by creating barriers to pathogens, inhaled allergens, and environmental particles. AJCs form between adjacent cells and consist of tight junctions (TJs) and adherens junctions (AJs). Respiratory viruses have been shown to target various components of the AJCs, leading to airway epithelial barrier dysfunction by different mechanisms. Virus-induced epithelial permeability may allow for allergens and bacterial pathogens to subsequently invade. In this review, we discuss the pathophysiologic mechanisms leading to disruption of AJCs and the potential ensuing ramifications. We focus on the following viruses that affect the pulmonary system: respiratory syncytial virus, rhinovirus, influenza viruses, immunodeficiency virus, and other viruses such as coxsackievirus, adenovirus, coronaviruses, measles, parainfluenza virus, bocavirus, and vaccinia virus. Understanding the mechanisms by which viruses target the AJC and impair barrier function may help design therapeutic innovations to treat these infections.
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Junções Íntimas/virologia , Viroses/fisiopatologia , Animais , Humanos , CamundongosRESUMO
One of the commonly used techniques for drug delivery is to temporarily increase the permeability of tissue barriers. Acoustic energies such as ultrasound and shock waves are known to modulate tissue permeability. Recently, it was found that shock waves modulate the blood-brain barrier in the rat brain without injection of contrast agents such as microbubbles. This finding implies that modulation of other tissue barriers by shock wave exposure without contrast agents may be possible. To examine whether the modulation is also possible with other tissue barriers, we here investigated whether shock waves would modulate an in vitro tissue barrier model consisting of epithelial cells cultured on culture inserts. The permeability of the epithelium sheets evaluated by trans-epithelial electrical resistance (TEER) was increased following shock waves at a peak pressure of 11 MPa. The increased permeability recovered within 2 h. This enhancement was realized with one-shot low-energy shock waves having an acoustic energy of 0.013 mJ/mm2. Monitoring the peak pressures in every exposure revealed that the minimum peak pressure required for the enhancement is 2.9 MPa. These results indicate that shock wave exposure has the potential to temporarily increase the permeability of epithelium barriers to enhance drug delivery without contrast agents. Graphical abstract Enhancements of epithelial barrier permeability were evaluated by trans-epithelial electrical resistance (TEER) before and after shock wave exposures.
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Sistemas de Liberação de Medicamentos , Epitélio/metabolismo , Permeabilidade , Som , Animais , Engenharia Biomédica , Cães , Impedância Elétrica , Humanos , Técnicas In Vitro , Células Madin Darby de Rim Canino , RatosRESUMO
Microfluidic devices are gaining increasing popularity due to their wide applications in various research areas. Herein, we propose a two-layer multi-channel microfluidic device allowing for direct-contact cell-vessel co-culture. Using the device, we built a co-culture model of the outer blood-retina barrier (oBRB), mimicking the in vivo retinal pigment epithelial cells-Bruch membrane-fenestrated choroids. To demonstrate the versatility of the design, we further modified the device by inserting platinum electrodes for trans-epithelial electrical resistance (TEER) measurement, demonstrating the feasibility of on-chip assessment of the epithelial barrier integrity. Our proposed design allows for direct-contact co-culture of cell-cell or cell-vessel, modifiable for real-time evaluation of the state of the epithelial monolayers.
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Mannan oligosaccharide (MOS) is well-known as an effective fed supplement for livestock to increase their nutrients absorption and health status. Pentasaccharide of mannan (MOS5) was reported as a molecule that possesses the ability to increase tight junction of epithelial tissue, but the structure and mechanism of action remains undetermined. In this study, the mechanism of action and structure of MOS5 were investigated. T84 cells were cultured and treated with MOS5 compared with vehicle and compound C, a 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibitor. The results demonstrated that the ability of MOS5 to increase tight junction integration was inhibited in the presence of dorsomorphine (compound C). Phosphorylation level of AMPK was elevated in MOS5 treated group as determined by Western blot analysis. Determination of MOS5 structure was performed using enzymatic mapping together with 1H, 13C NMR, and 2D-NMR analysis. The results demonstrated that the structure of MOS5 is a ß-(1,4)-mannotetraose with α-(1,6)-galactose attached at the second mannose unit from non-reducing end.
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The AMP-activated protein kinase (AMPK) is principally known as a major regulator of cellular energy status, but it has been recently shown to play a key structural role in cell-cell junctions. The aim of this study was to evaluate the impact of AMPK activation on the reassembly of tight junctions in intestinal epithelial Caco-2 cells. We generated Caco-2 cells invalidated for AMPK α1/α2 (AMPK dKO) by CRISPR/Cas9 technology and evaluated the effect of the direct AMPK activator 991 on the reassembly of tight junctions following a calcium switch assay. We analyzed the integrity of the epithelial barrier by measuring the trans-epithelial electrical resistance (TEER), the paracellular permeability, and quantification of zonula occludens 1 (ZO-1) deposit at plasma membrane by immunofluorescence. Here, we demonstrated that AMPK deletion induced a delay in tight junction reassembly and relocalization at the plasma membrane during calcium switch, leading to impairments in the establishment of TEER and paracellular permeability. We also showed that 991-induced AMPK activation accelerated the reassembly and reorganization of tight junctions, improved the development of TEER and paracellular permeability after calcium switch. Thus, our results show that AMPK activation ensures a better recovery of epithelial barrier function following injury.
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Proteínas Quinases Ativadas por AMP/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Junções Íntimas/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Células CACO-2 , Cálcio/metabolismo , Permeabilidade da Membrana Celular , Análise Mutacional de DNA , Humanos , Sistema de Sinalização das MAP Quinases , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
The possibility of counteracting inflammation-related barrier defects with dietary compounds such as (poly)phenols has raised much interest, but information is still scarce. We have investigated here if (+)-catechin (CAT) and procyanidin B2 (PB2), two main dietary polyphenols, protect the barrier function of intestinal cells undergoing inflammatory stress. The cell model adopted consisted of co-cultured Caco-2 and HT29-MTX cells, while inflammatory conditions were mimicked through the incubation of epithelial cells with the conditioned medium of activated macrophages (MCM). The epithelial barrier function was monitored through trans-epithelial electrical resistance (TEER), and ROS production was assessed with dichlorofluorescein, while the expression of tight-junctional proteins and signal transduction pathways were evaluated with Western blot. The results indicated that MCM produced significant oxidative stress, the activation of NF-κB and MAPK pathways, a decrease in occludin and ZO-1 expression, and an increase in claudin-7 (CL-7) expression, while TEER was markedly lowered. Neither CAT nor PB2 prevented oxidative stress, transduction pathways activation, ZO-1 suppression, or TEER decrease. However, PB2 prevented the decrease in occludin expression and both polyphenols produced a huge increase in CL-7 abundance. It is concluded that, under the conditions adopted, CAT and PB2 do not prevent inflammation-dependent impairment of the epithelial barrier function of intestinal cell monolayers. However, the two compounds modify the expression of tight-junctional proteins and, in particular, markedly increase the expression of CL-7. These insights add to a better understanding of the potential biological activity of these major dietary flavan-3-ols at intestinal level.
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Biflavonoides/farmacologia , Catequina/farmacologia , Permeabilidade/efeitos dos fármacos , Proantocianidinas/farmacologia , Substâncias Protetoras/farmacologia , Proteínas de Junções Íntimas/metabolismo , Células CACO-2 , Técnicas de Cultura de Células , Células Epiteliais , Células HT29 , Humanos , Intestinos/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Ocludina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
BACKGROUND: Inflammatory Bowel Diseases (IBD), are considered a growing global disease, with about ten million people being affected worldwide. Maintenance of intestinal barrier integrity is crucial for preventing IBD onset and exacerbations. Some recent patents regarding oily formulations containing probiotics (WO2010122107A1 and WO2010103374A9) and the use of probiotics for gastrointestinal complaints (US20110110905A1 and US9057112B2) exist, or are pending application. OBJECTIVE: In this work, we studied the effect of a fixed combination of registered Lactobacillus reuteri and Lactobacillus acidophilus strains and herbal extracts in an in vitro inflammation experimental model. METHODS: Caco-2 cell monolayer was exposed to INF-γ+TNF-α or to LPS; Trans Epithelial Electrical Resistance (TEER) and paracellular permeability were investigated. ZO-1 and occludin Tight Junctions (TJs) were also investigated by mean of immunofluorescence. RESULTS: Pre-treatment with the fixed combination of probiotics and herbal extracts prevented the inflammation-induced TEER decrease, paracellular permeability increase and TJs translocation. CONCLUSIONS: In summary, the fixed combination of probiotics and herbal extracts investigated in this research was found to be an interesting candidate for targeting the re-establishment of intestinal barrier function in IBD conditions.
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Inflamação/tratamento farmacológico , Extratos Vegetais/farmacologia , Probióticos/farmacologia , Células CACO-2 , Humanos , Estresse FisiológicoRESUMO
BACKGROUND: VSL#3 has been extensively investigated and is currently recommended for the prevention and treatment of chronic pouchitis and ulcerative colitis. Nonetheless, in vitro and in vivo studies have recently shown variability in the VSL#3 efficacy often attributed to the manufacturing process. OBJECTIVE: The aim was to comparatively study the in vitro effects of two VSL#3 preparations produced in different sites (named US- and Italy-made VSL#3) on CaCo-2 epithelial barrier model in terms of trans-epithelial electrical resistance (TEER), dextran flux and expression of Tight Junctions (TJ) proteins i.e. zonulin-1 (ZO-1) and occludin, in the absence or presence of a heat stress-related damage of monolayer. METHODS: TEER was evaluated on CaCo-2 differentiated monolayers. Epithelial permeability of polarized monolayers was assessed by measuring the FITC-labeled dextran flux from the apical to basolateral chambers. ZO-1/occludin levels were analyzed by western blot analysis. A set of experiments was performed to compare the effects of both VSL#3 on TEER values, dextran flux and ZO-1/occludin expression in CaCo-2 monolayers after heat stress exposure. RESULTS: US- and Italy-made VSL#3 have opposing effects on TEER values, dextran flux, and ZO- 1/occludin expression, being all these parameters negatively influenced just by Italy-made product. US-made probiotic did not affect baseline TEER, dextran flux and ZO-1 expression and strongly increased occludin levels. Of note, pre-treatment of monolayer with US-made VSL#3, but not Italy-made product, totally prevented the heat-induced epithelial barrier integrity loss. CONCLUSION: Our data trigger the need for reassessing efficacy or safety of the Italy-made VSL#3 considering intestinal epithelial barrier plays an important role in maintaining host health.
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Permeabilidade da Membrana Celular/fisiologia , Mucosa Intestinal/metabolismo , Modelos Biológicos , Probióticos/farmacocinética , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Composição de Medicamentos , Impedância Elétrica , Humanos , Mucosa Intestinal/efeitos dos fármacos , Itália , Permeabilidade/efeitos dos fármacos , Probióticos/classificação , Probióticos/uso terapêutico , Estados UnidosRESUMO
Inflammatory bowel diseases, which consist of chronic inflammatory conditions of the colon and the small intestine, are considered a global disease of our modern society. Recently, the interest toward the use of herbal therapies for the management of inflammatory bowel diseases has increased because of their effectiveness and favourable safety profile, compared to conventional drugs. Boswellia serrata Roxb. and Curcuma longa L. are amongst the most promising herbal drugs, however, their clinical use in inflammatory bowel diseases is limited and little is known on their mechanism of action. The aim of this work was to investigate the effects of two phytochemically characterized extracts of B. serrata and C. longa in an in vitro model of intestinal inflammation. Their impact on cytokine release and reactive oxygen species production, as well as the maintenance of the intestinal barrier function and on intestinal mucosa immune cells infiltration, has been evaluated. The extracts showed a good protective effect on the intestinal epithelium at 1 µg/mL, with TEER values increasing by approximately 1.5 fold, compared to LPS-stimulated cells. C. longa showed an anti-inflammatory mechanism of action, reducing IL-8, TNF-α and IL-6 production by approximately 30%, 25% and 40%, respectively, compared to the inflammatory stimuli. B. serrata action was linked to its antioxidant effect, with ROS production being reduced by 25%, compared to H2O2-stimulated Caco-2 cells. C. longa and B. serrata resulted to be promising agents for the management of inflammatory bowel diseases by modulating in vitro parameters which have been identified in the clinical conditions.
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Epithelial cell lines are often used to evaluate the effect of exogenous/endogenous stimuli on epithelial barrier function and innate immune responses in allergic airway diseases, without clear view on differences between epithelial cell lines and primary nasal epithelial cell responses. In this observational study, we compared the response of Calu-3 and primary nasal epithelial cells to two relevant exogenous stimuli: i.e. Staphylococcus aureus enterotoxin B (SEB) and house dust mite (HDM). Stimulation of Calu-3 cells with SEB decreased epithelial integrity in a dose dependent manner, which was associated with a significant increase in IL-6 and IL-8 production. In contrast, no alteration in barrier integrity or IL-6 and IL-8 production was seen when primary nasal epithelial cells were stimulated with SEB. HDM extract altered the integrity of primary nasal epithelial cells, but not of Calu-3 epithelial cells. Increased IL-8 production was seen after stimulation with HDM in primary nasal epithelial cells and not in Calu-3 epithelial cells. In conclusion, immune and barrier function differ between different epithelial cell types studied. As a consequence, care must be taken when interpreting data using different epithelial cell types.
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Lipid-based drug delivery systems, a well-tolerated class of formulations, have been evaluated extensively to enhance the bioavailability of poorly soluble drugs. However, it has been difficult to predict the in vivo performance of lipid dosage forms based on conventional in vitro techniques such as cell monolayer permeability studies because of the complexity of the gastrointestinal processing of lipid formulations. In the current study, we explored the feasibility of coupling Caco-2 and Madin-Darby canine kidney monolayer permeability studies with lipolysis, a promising in vitro technique to evaluate lipid systems. A self-emulsifying lipid delivery system was formulated using a blend of oil (castor oil), surfactant (Labrasol® or PL497), and co-surfactant (lecithin). Formulations demonstrating high drug solubility and rapid self-emulsification were selected to study the effect of lipolysis on in vitro cell permeability. Lipolysis of the formulations was carried out using pancreatin as the digestive enzyme. All the digested formulations compromised monolayer integrity as indicated by lowered trans-epithelial electrical resistance (TEER) and enhanced Lucifer yellow (LY) permeability. Further, the changes in TEER value and LY permeability were attributable to the digestion products of the formulation rather than the individual lipid excipients, drug, digestion enzyme, or the digestion buffer. The digested formulations were fractionated into pellet, oily phase, and aqueous phase, and the effect of each of these on cell viability was examined. Interestingly, the aqueous phase, which is considered important for in vivo drug absorption, was responsible for cytotoxicity. Because lipid digestion products lead to disruption of cell monolayer, it may not be appropriate to combine lipolysis with cell monolayer permeability studies. Additional in vivo studies are needed to determine any potential side effects of the lipolysis products on the intestinal permeability barrier, which could determine the suitability of lipid-based systems for oral drug delivery.
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Sistemas de Liberação de Medicamentos , Acridinas/administração & dosagem , Acridinas/química , Administração Oral , Animais , Células CACO-2 , Óleo de Rícino/administração & dosagem , Óleo de Rícino/química , Sobrevivência Celular/efeitos dos fármacos , Cães , Excipientes/administração & dosagem , Excipientes/química , Humanos , Lecitinas/administração & dosagem , Lecitinas/química , Lipólise , Células Madin Darby de Rim Canino , Permeabilidade , Tensoativos/administração & dosagem , Tensoativos/química , Tetra-Hidroisoquinolinas/administração & dosagem , Tetra-Hidroisoquinolinas/químicaRESUMO
Nanofibrous scaffolds provide high surface area for cell attachment, and resemble the structure of the collagen fibers which naturally occur in the basement membrane and extracellular matrix. A label free and non-destructive method of assessing the interaction of cell tissue and scaffolds aids in the ability to discern the effective quality and magnitude of any scaffold modifications. Impedance cell spectroscopy is a biosensing method that employs a functional approach to assessing the cell monolayer. The electrical impedance barrier function of a cell monolayer represents the level of restriction to diffusion of charged species between all adjacent cells across an entire contiguous cellular monolayer. The impedance signals from many individual paracellular pathways contribute to the bulk measurement of the whole monolayer barrier function. However, the scaffold substrate must be entirely porous in order to be used with electrochemical cell impedance spectroscopy (ECIS) and cells must be closely situated to the electrodes. For purposes of evaluating cell-scaffold constructs for tissue engineering, non-invasive evaluation of cell properties while seeded on scaffolds is critical. A Transwell-type assay makes a measurement across a semi-permeable membrane, using electrodes placed on opposing sides of the membrane immersed in fluid. It was found that by suspending a nanofiber scaffold across a Transwell aperture, it is possible to integrate a fully functional nanofiber tissue scaffold with the ECIS Transwell apparatus. Salivary epithelial cells were grown on the nanofiber scaffolds and tight junction formation was evaluated using ECIS measurements in parallel with immunostaining and confocal imaging. The trans-epithelial resistance increased coordinate with cell coverage, culminating with a cell monolayer, at which point the tight junction proteins assemble and strengthen, reaching the peak signal. These studies demonstrate that ECIS can be used to evaluate tight junction formation in cells grown on nanofiber scaffolds and on effects of scaffold conditions on cells, thus providing useful biological feedback to inform superior scaffold designs.