RESUMEN
Bacteria can cause infectious diseases even at ultralow concentrations (<1 CFU/mL). It is important to rapidly identify bacterial contamination at ultralow concentrations. Herein, FITC-labeled gelatinase-sensitive nanoparticles (GNPs@FITCs) and NFM@GNP@FITCs are designed and fabricated as ultralow concentration bacteria detection platforms based on an enzymatic cascade reaction-amplifying strategy. Bacterial secretions could trigger the dissociation of GNPs@FITCs to release FITC, with gelatinase used as the model secretion. The detectable signal of ultralow concentration bacteria could be amplified effectively by the gelatinase-triggered cascade reaction. Bacterial concentration was evaluated by the change in fluorescence density. The results showed that the GNPs@FITCs and NFM@GNP@FITCs could be used for identifying bacterial contamination qualitatively, even when the bacterial contamination is lower than 1 CFU/mL. Moreover, the method has better timeliness and convenience, when compared with national standards. As solid films, NFM@GNP@FITCs have better long-term storage stability than GNPs@FITCs. The potential applications of GNPs@FITC and NFM@GNP@FITCs were proved by detecting pathogenic bacteria in food. All the results showed that the method has great potential for screening pathogenic bacterial contamination qualitatively.
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Bacterias , Bacterias/aislamiento & purificación , Bacterias/enzimología , Fluoresceína-5-Isotiocianato/química , Microbiología de Alimentos , Nanopartículas del Metal/química , Colorantes Fluorescentes/química , Nanopartículas/químicaRESUMEN
A novel type of colorimetric/fluorescent nanopaper indicator has been developed from the melt-extruded poly (vinyl alcohol-co-ethylene) nanofibers with surface anchored metal-organic frameworks (MOFs) by an interfacial coordination strategy. Specifically, the fluorescein isothiocyanate molecules could be anchored to the nanofiber surface by nickel ions and co-assembled into a hydrophilic nanocoating via a dynamic water/alcohol solvent evaporation method. Interestingly, this hydrophilic surface enables fast adsorption of moistures and interaction with biological amine vapors, resulting a saffron cake-layer of MOF nanocrystals with ultra-sensitive colorimetric/fluorescent responses based on an alkaline pH/ammonia induced competitive coordination mechanism. Finally, these porous nanofibrous matrix and active nanocoating make the nano-paper an ultra-sensitive optical platform for in-situ monitoring of the shrimp freshness from mins to weeks. Therefore, this composite film shows great potential into advanced paper-based indicators for food quality control and safety in processing industry.
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Colorimetría , Fluoresceína-5-Isotiocianato , Estructuras Metalorgánicas , Nanofibras , Níquel , Papel , Colorimetría/métodos , Nanofibras/química , Animales , Estructuras Metalorgánicas/química , Níquel/química , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Penaeidae/química , Mariscos/análisisRESUMEN
Theranostic sutures are derived from innovative ideas to enhance wound healing results by adding wound diagnostics and therapeutics to typical sutures by functionalizing them with additional materials. Here, we present a new direct electrospinning method for the fast, continuous, inexpensive, and high-throughput production of versatile nanofibrous-coated suture threads, with precise control over various essential microstructural and physical characteristics. The thickness of the coating layer and the alignment of nanofibers with the thread's direction can be adjusted by the user by varying the spooling speed and the displacement between the spinneret needle and thread. To show the flexibility of our method for a range of different materials selected, gelatin, polycaprolactone, silk fibroin, and PEDOT:PSS (poly(3,4-ethylene dioxythiophene):poly(styrene sulfonate)) were the resultant nanofibers characterized by scanning electron microscopy (SEM) imaging and conductivity tests. In a series of in vitro and ex vivo tests (pig skin), sutures were successfully tested for their flexibility and mechanical properties when used as weaving and knotting sutures, and their biocompatibility with a keratinocyte cell line. For temperature-based drug-releasing tests, two fluorescent molecules as drug models with high and low molecular weight, namely fluorescein isothiocyanate-dextran (20 kDa) and rhodamine B (470 Da), were used, and their steady release with incremental increase of temperature to 37 °C over 120 min was seen, which is appropriate for bacterial treatment drugs. Given the advantages of the presented technique, it seems to have promising potential to be used in future medical applications for wound closure and bacterial infection treatment via a temperature-triggered drug release strategy.
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Nanofibras , Rodaminas , Suturas , Cicatrización de Heridas , Nanofibras/química , Animales , Cicatrización de Heridas/efectos de los fármacos , Humanos , Rodaminas/química , Porcinos , Poliésteres/química , Dextranos/química , Gelatina/química , Nanoporos , Fluoresceína-5-Isotiocianato/química , Materiales Biocompatibles Revestidos/química , Queratinocitos/citología , Queratinocitos/metabolismo , Fibroínas/química , Línea CelularRESUMEN
Tight junction disruption can lead to pathogenesis of various diseases without therapeutic strategy to recover intestinal barrier integrity. The main objective of this study is to demonstrate the effect of Solanum melongena L. extract (SMLE) on intestinal tight junction recovery and its underlying mechanism. Intestinal barrier function is attenuated by Ca2+ depletion. SMLE treatment increased TER value across T84 cell monolayers. Permeability assay reveals that Ca2+ depletion promotes 4-kDa FITC-dextran permeability, but not 70-kDa FITC-dextran. SMLE suppresses the rate of 4-kDa FITC-dextran permeability, indicating that SMLE inhibits paracellular leak pathway permeability. SMLE-mediated TER increase and leak pathway suppression are abolished by neither calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor nor AMP-activated protein kinase (AMPK) inhibitor. Furthermore, mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) inhibitors have no effects on SMLE-mediated TER increase and leak pathway suppression. Interestingly, SMLE is unable to enhance TER value and diminish leak pathway permeability in T84 cell monolayers pre-treated with sirtuin-1 (SIRT-1) inhibitor. Immunofluorescence staining reveals that SMLE enhances re-assembly of tight junction proteins, including occludin and ZO-1 to intercellular space but this effect is abolished by SIRT-1 inhibitor. These data suggest that SMLE promotes intestinal tight junction re-assembly via SIRT-1-dependent manner.
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Extractos Vegetales , Sirtuina 1 , Uniones Estrechas , Sirtuina 1/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Extractos Vegetales/farmacología , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ocludina/metabolismo , Permeabilidad/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Proteína de la Zonula Occludens-1/metabolismo , Dextranos , Serina-Treonina Quinasas TOR/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivadosRESUMEN
Taylor dispersion analysis (TDA) is a rapid and precise method for determining the hydrodynamic radius (RH) of various substances. We present a versatile TDA system with a flow-through sample injection device, two compact 3-in-1 detectors, and a high-voltage power supply. The 3D-printed detectors combine fluorimetry (FD), photometry (AD@255 nm), and contactless conductometry (C4D) in a single head, enabling simultaneous detection at one capillary window. Using bovine serum albumin (BSA) as a model analyte, we compare TDA with different detection methods. BSA labeled with fluorescein isothiocyanate (FITC) is analyzed in both pulse mode and capillary electrophoresis (CE) TDA. FD and AD detection yield similar RH values, except when FITC binds with small ions in the buffer. In phosphate buffer, C4D underestimates RH values by approximately 18 % due to BSA self-association. In Tris-based buffers, C4D values are 87%-96 % of AD values in pulse mode. With CE-TDA using Tris-CHES buffer, no statistical difference is found across all detections. The system is also applied to CE-TDA of various compounds, particularly charged saccharides. CE-TDA improves the accuracy of TDA results from C4D. We demonstrate the resolution of mixed C4D-TDA signals with assistance from FD and AD signals, successfully resolving gluconate peaks fully covered by another compound. The versatile system with 3-in-1 detection offers a powerful tool for TDA of mixtures and enhances sample throughput.
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Fluoresceína-5-Isotiocianato , Fluorometría , Fotometría , Albúmina Sérica Bovina , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/análisis , Fluorometría/métodos , Bovinos , Fotometría/métodos , Fluoresceína-5-Isotiocianato/química , Animales , Hidrodinámica , Electroforesis Capilar/métodosRESUMEN
Diacylglycerol O-acyltransferase 1 (DGAT1) is a key enzyme for fat absorption step in the enterocytes. We previously reported that DGAT1 inhibition increased plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in corn oil-loaded rats via protein kinase C (PKC) activation. In the present study, we investigated the mechanism with respect to the morphology and permeability of the small intestine, focusing on PKC function, and found that shortening of the intestinal villi and a decrease in the number of tdT-mediated dUTP-biotin nick-end labeling-positive cells in the tips of the villi were observed in the jejunum of DGAT1 inhibitor-treated rats loaded with corn oil. These results suggested that the tips of the villi were shed into the intestinal lumen. Next, fluorescein isothiocyanate-dextran, 110â¯kDa (FD-110) was administered intraduodenally to DGAT1 inhibitor-treated rats loaded with corn oil and we found that plasma FD-110 concentrations increased, indicating that the intestinal permeability to molecules with a molecular weight of approximately 110,000 (e.g., ALT and AST) increased. Taken together, the present results suggested that DGAT1 inhibitor-treatment in combination with corn oil causes ALT and AST to leak from the enterocytes into the blood by shedding the tips of the intestinal villi and increasing intestinal permeability.
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Alanina Transaminasa , Aspartato Aminotransferasas , Aceite de Maíz , Diacilglicerol O-Acetiltransferasa , Mucosa Intestinal , Permeabilidad , Animales , Alanina Transaminasa/sangre , Masculino , Aspartato Aminotransferasas/sangre , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Diacilglicerol O-Acetiltransferasa/metabolismo , Permeabilidad/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratas , Dextranos , Proteína Quinasa C/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fluoresceína-5-Isotiocianato/análogos & derivados , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Absorción Intestinal/efectos de los fármacos , Ratas Sprague-Dawley , Ratas Wistar , Funcion de la Barrera IntestinalRESUMEN
Subcutaneous (SC) injection is a common route of administration for drug compounds with poor oral bioavailability. However, bioavailability is often variable and incomplete, and there is as yet no standard accepted medium for simulation of the human SC environment. In this work we evaluate a FRAP based method for quantitative determination of local self-diffusion coefficients within extracellular matrix (ECM) mimetic hydrogels, potentially useful as in vitro models for drug transport in the ECM after SC injection. Gels were made consisting of either agarose, cross-linked collagen (COL) and hyaluronic acid (HA) or cross-linked HA. The diffusivities of uncharged FITC-dextran (FD4), the highly charged poly-lysine (PLK20) and poly-glutamic acid (PLE20) as well as the GLP-1 analogue exenatide were determined within the gels using FRAP. The diffusion coefficients in uncharged agarose gels were in the range of free diffusion in PBS. The diffusivity of cationic PLK20 in gels containing anionic HA was substantially decreased due to strong electrostatic interactions. Peptide aggregation could be observed as immobile fractions in experiments with exenatide. We conclude that the FRAP method provides useful information of peptides' interactions and transport properties in hydrogel networks, giving insight into the mechanisms affecting absorption of drug compounds after subcutaneous injection.
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Dextranos , Exenatida , Matriz Extracelular , Ácido Hialurónico , Hidrogeles , Péptidos , Hidrogeles/química , Difusión , Matriz Extracelular/metabolismo , Inyecciones Subcutáneas , Exenatida/farmacocinética , Exenatida/química , Exenatida/administración & dosificación , Ácido Hialurónico/química , Dextranos/química , Dextranos/farmacocinética , Péptidos/química , Péptidos/farmacocinética , Péptidos/administración & dosificación , Ácido Poliglutámico/química , Ácido Poliglutámico/análogos & derivados , Polilisina/química , Colágeno/química , Sefarosa/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/administración & dosificación , Fluoresceína-5-Isotiocianato/farmacocinética , HumanosRESUMEN
Aim: Lysosomal pH changes are associated with drug resistance, cell growth and invasion of tumors, but effective and specific real-time monitoring of lysosomal pH compounds for cancer therapy is lacking. Materials & methods: Here, based on the covalent linkage of the anticancer drug palbociclib and fluorescent dye fluorescein isothiocyanate (FITC), we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Results & discussion: Pal-FITC fluoresces is 20-fold stronger than that of FITC and shows a linear response in the pH range of 4.0-8.2 (R2 = 0.9901). Pal-FITC blocks cells in G1 phase via Cyclin D-CDK4/6-Rb. Conclusion: Our study provides new strategies for tumor-targeted imaging and personalized therapy.
Based on the covalent linkage of the anticancer drug and the fluorescent dye, we designed and developed a novel palbociclib-derived multifunctional molecule (Pal-FITC) for lysosomal targeting and diagnostic therapeutic integration. Pal-FITC responded linearly in the pH range of 4.08.2. In addition, Pal-FITC was able to effectively treat lung cancer without toxic side effects on normal cells. It has a significant cell cycle blocking phenomenon and blocks G1 phase cells via Cyclin D-CDK4/6-Rb. Our study provides a new strategy for tumor-targeted imaging and personalized therapy.
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Antineoplásicos , Lisosomas , Piperazinas , Piridinas , Humanos , Piridinas/química , Piridinas/farmacología , Lisosomas/metabolismo , Piperazinas/química , Piperazinas/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Colorantes Fluorescentes/síntesis química , Fluoresceína-5-Isotiocianato/química , Proliferación Celular/efectos de los fármacos , Concentración de Iones de Hidrógeno , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Estructura MolecularRESUMEN
The perivascular space (PVS) surrounds cerebral blood vessels and plays an important role in clearing waste products from the brain. Their anatomy and function have been described for arteries, but PVS around veins remain poorly characterized. Using in vivo 2-photon imaging in mice, we determined the size of the PVS around arteries and veins, and their connection with the subarachnoid space. After infusion of 70 kD FITC-dextran into the cerebrospinal fluid via the cisterna magna, labeled PVS were evident around arteries, but veins showed less frequent labeling of the PVS. The size of the PVS correlated with blood vessel size for both pial arteries and veins, but not for penetrating vessels. The PVS around pial arteries and veins was separated from the subarachnoid space by a thin meningeal layer, which did not form a barrier for the tracer. In vivo, FITC-dextran signal was observed adjacent to the vessel wall, but minimally within the wall itself. Post-mortem, there was a significant shift in the tracer's location within the arterial wall, extending into the smooth muscle layer. Taken together, these findings suggest that the PVS around veins has a limited role in the exchange of solutes between CSF and brain parenchyma.
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Encéfalo , Arterias Cerebrales , Animales , Ratones , Encéfalo/irrigación sanguínea , Arterias Cerebrales/anatomía & histología , Sistema Glinfático , Fluoresceína-5-Isotiocianato/análogos & derivados , Dextranos , Masculino , Venas Cerebrales/anatomía & histología , Ratones Endogámicos C57BL , Espacio SubaracnoideoRESUMEN
Amide bonds are one of the most prevalent phenomena in nature and are utilized frequently in drug and material design. However, forming amide bonds is not always efficient or high yielding, particularly when the amine used to conjugate to a carboxylic acid is a weak nucleophile. This limitation precludes many useful amino compounds from participating in conjugation reactions to form amides. A particularly valuable amino compound, which is also a very weak nucleophile, is the amino porphyrin, valued for its role as a photosensitizer, fluorescent agent, catalyst, or, upon metalation, even a very efficient contrast agent for magnetic resonance imaging (MRI). In this work, we propose fast and high-yield coupling of an unreactive amine - the amino porphyrin - to carboxylic acid via isothiocyanate conjugation. Reactions can be achieved in one step at room temperature in one hour, achieving quantitative conversion and near perfect selectivity. Both metalated and unmetalated porphyrin, as well as fluorescein isothiocyanate (FITC), demonstrated efficient conjugation. To illustrate the value of the proposed method, we created a new blood-pool MRI contrast agent that reversibly binds to serum albumin. This new blood-pool agent, known as MITC-Deox (MRI isothiocyanate that links with deoxycholic acid), substantially reduced T1 relaxation times in blood vessels in mice, remained stable for 1 hour, cleared from blood by 24 hours, and was eliminated from the body after 4 days. The proposed method for efficient amide formation is a superior alternative to existing coupling methods, opening a door to novel synthesis of MRI contrast agents and beyond.
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Amidas , Medios de Contraste , Porfirinas , Porfirinas/química , Porfirinas/síntesis química , Amidas/química , Amidas/síntesis química , Animales , Ratones , Medios de Contraste/química , Medios de Contraste/síntesis química , Imagen por Resonancia Magnética , Estructura Molecular , Isotiocianatos/química , Fluoresceína-5-Isotiocianato/químicaRESUMEN
Labrafac™ MC60 (glycerol monocaprylocaprate) is a lipid-based excipient used in oral formulations as a solubiliser. Due to the high proportions of established permeability enhancers, caprylate (C8) and caprate (C10), in Labrafac™ MC60, we hypothesised that it might behave as an intestinal permeation enhancer. We therefore evaluated this using two paracellular markers (ex vivo) and insulin (in vivo) as model molecules. Ex vivo studies were conducted in isolated muscle-stripped rat colonic mucosae mounted in Ussing chambers. Apical addition of Labrafac™ MC60 (8, 12, and 16 mg/ml) enhanced the apparent permeability coefficients (Papp) of [14C] mannitol and FITC-dextran 4 kDa (FD4) across colonic mucosae. Similar effects were observed in isolated jejunal mucosae, but at higher concentrations (40 mg/ml). The enhancing capacity of Labrafac™ MC60 was transient due to reversibility of reductions in transepithelial electrical resistance (TEER) upon wash-out and effects on fluxes were molecular weight-dependent (MW) as suggested by fluxes of a set of high MW FITC-dextrans. The permeability enhancing effects of Labrafac™ MC60 ex vivo were maintained in the presence of simulated intestinal fluids, FaSSIF and FaSSCoF, in both jejunal and colonic mucosae, respectively. Following intra-intestinal regional instillations to rats, the relative bioavailability of 50 IU/kg insulin ad-mixed with Labrafac™ MC60 was 5 % in jejunum (40 mg/ml) and 6 % in colon (8 mg/ml). When Labrafac™ MC60 was combined with PEG-60 hydrogenated castor oil (1 % v/v), this further increased the bioavailability of insulin to 8 % in jejunum. Absorption enhancement was also maintained in the presence of FaSSIF in jejunal instillations. Histology after 120 min exposure to Labrafac™ MC60 in vivo for both jejunum and colon was similar to untreated control. Labrafac™ MC60 therefore acts as a non-damaging intestinal permeation enhancer for macromolecules and can be considered as another excipient in screening programmes to develop orally administered macromolecules.
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Dextranos , Excipientes , Fluoresceína-5-Isotiocianato , Glicéridos , Absorción Intestinal , Mucosa Intestinal , Permeabilidad , Animales , Masculino , Absorción Intestinal/efectos de los fármacos , Dextranos/farmacocinética , Dextranos/administración & dosificación , Excipientes/química , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Glicéridos/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Fluoresceína-5-Isotiocianato/administración & dosificación , Insulina , Ratas , Manitol , Ratas Wistar , Colon/metabolismo , Colon/efectos de los fármacos , Yeyuno/metabolismo , Yeyuno/efectos de los fármacosRESUMEN
Here, a novel porous microneedle (PMN) device with bilaterally aligned electroosmotic flow (EOF) enabling controllable dual-mode delivery of molecules is developed. The PMNs placed at anode and cathode compartments are modified with anionic poly-2-acrylamido-2-methyl-1-propanesulfonic acid and cationic poly-(3-acrylamidopropyl) trimethylammonium, respectively. The direction of EOF generated by PMN at the cathode compartment is, therefore, reversed from cathode to anode, countering the unwanted cathodal suctioning of interstitial fluid caused by reverse iontophoresis. With the bilateral alignment of EOF, the versatility of the proposed device is evaluated by delivering molecules with different charges and sizes using Franz cell. In addition, a 3D printed probe device is developed to ease practical handling and minimize electrical stimulation by integrating two PMNs in closed proximity. Finally, the performance of the integrated probe device is demonstrated by dual delivery of a variety of molecules (methylene blue, rhodamine B, and fluorescein isothiocyanate-dextran) using pig skin and vaccination using mice with delivered ovalbumin.
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Electroósmosis , Agujas , Rodaminas , Animales , Porcinos , Ratones , Electroósmosis/instrumentación , Rodaminas/química , Porosidad , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/química , Dextranos/química , Azul de Metileno/química , Ovalbúmina/administración & dosificación , Ovalbúmina/química , Sistemas de Liberación de Medicamentos/instrumentación , Sistemas de Liberación de Medicamentos/métodos , Piel/metabolismo , Microinyecciones/instrumentación , Microinyecciones/métodos , Impresión TridimensionalRESUMEN
While ketogenic diets (KDs) may have potential as adjunct treatments for gastrointestinal diseases, there is little knowledge on how the fat source of these diets impacts intestinal health. The objective of this study was to investigate how the source of dietary fat of KD influences experimental colitis. We fed nine-week-old male C57BL/6J mice (n = 36) with a low-fat control diet or KD high either in saturated fatty acids (SFA-KD) or polyunsaturated linoleic acid (LA-KD) for four weeks and then induced colitis with dextran sodium sulfate (DSS). To compare the diets, we analyzed macroscopic and histological changes in the colon, intestinal permeability to fluorescein isothiocyanate-dextran (FITC-dextran), and the colonic expression of tight junction proteins and inflammatory markers. While the effects were more pronounced with LA-KD, both KDs markedly alleviated DSS-induced histological lesions. LA-KD prevented inflammation-related weight loss and the shortening of the colon, as well as preserved Il1b and Tnf expression at a healthy level. Despite no significant between-group differences in permeability to FITC-dextran, LA-KD mitigated changes in tight junction protein expression. Thus, KDs may have preventive potential against intestinal inflammation, with the level of the effect being dependent on the dietary fat source.
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Colitis , Colon , Sulfato de Dextran , Dieta Cetogénica , Grasas de la Dieta , Modelos Animales de Enfermedad , Fluoresceína-5-Isotiocianato/análogos & derivados , Ratones Endogámicos C57BL , Animales , Colitis/inducido químicamente , Colitis/dietoterapia , Masculino , Ratones , Grasas de la Dieta/efectos adversos , Colon/patología , Colon/metabolismo , Permeabilidad , Proteínas de Uniones Estrechas/metabolismo , Interleucina-1beta/metabolismo , Mucosa Intestinal/patología , Mucosa Intestinal/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ácidos Grasos , DextranosRESUMEN
The integrity of the intestinal barrier is crucial for regulating the passage of pathogens and toxins, while facilitating nutrient absorption. The everted gut sac technique, an ex-vivo technique, can be used to study interventions on barrier function. This cost-effective approach utilizes relatively large gut segments to study specific intestinal regions. Typically, intact (non-stripped) intestinal segments are used, but their use may underestimate permeability due to the medial positioning of blood vessels relative to the seromuscular layer and serosa. However, removing these layers risks physical damage, resulting in an overestimation of intestinal permeability. Therefore, we investigated the impact of stripping jejunal segments on permeability to fluorescein isothiocyanate-dextran (FITC, 4 kDa) and tetramethylrhodamine isothiocyanate-dextran (TRITC, 40 kDa), and on the absorption of glucose, lysine, and methionine in jejunal segments from 80 piglets at 8 d postweaning. Piglets were subjected to either high or low sanitary housing conditions and diets provoking intestinal protein fermentation or not, expected to influence intestinal permeability. Stripping of the seromuscular layer and serosa increased the passage of 4 kDa FITC-dextran (stripped vs. non-stripped; 1.1 vs. 0.9 pmol/cm2/min, Pâ <â 0.001), glucose (40.0 vs. 19.1 pmol/cm2/min, Pâ <â 0.001), lysine (2.5 vs. 2.0 nmol/cm2/min, Pâ <â 0.001), and methionine (4.1 vs. 2.7 pmol/cm2/min, Pâ <â 0.001). As permeability increased, the differences in methionine passage between stripped and non-stripped intestinal segments also increased (slopeâ =â 1.30, Pâ =â 0.009). The coefficients of variation were comparable between stripped and non-stripped intestines (over all treatments, stripped vs. non-stripped 38% vs. 40%). Stripping, by isolating mucosal processes without introducing additional variation, is thus recommended for studies on intestinal permeability or absorption.
The intestinal barrier is vital for nutrient passage, while impeding pathogen and toxin translocation. The everted gut sac technique is used to study intestinal permeability, incubating an isolated, everted, intestinal segment filled with buffer solution in a medium containing the substances of interest. After incubation, the translocation of the substances into the created intestinal sac can be measured. Typically, intact intestinal segments are used, but under physiological conditions, nutrients do not need to pass the seromuscular layer and serosa to enter the blood flow. Therefore, removing these layers may be preferable, but, on the other hand, also risks physical damage. This study compared the use of non-stripped vs. stripped intestinal segments. Permeability to two markers (FITC-dextran, 4kDa and TRITC-dextran, 40 kDa), and absorption of glucose, lysine, and methionine were measured in non-stripped and stripped jejunal segments obtained from 80 piglets at 8 d postweaning. The piglets were housed under different hygiene and dietary conditions, which were anticipated to alter intestinal permeability. Stripping the seromuscular layer and serosa increased the passage of FITC-dextran, glucose, lysine, and methionine, without reducing assay precision due to physical damage. Thus, removal of the seromuscular layer and serosa is preferred for studying intestinal permeability or absorption.
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Permeabilidad , Animales , Porcinos/fisiología , Mucosa Intestinal/metabolismo , Destete , Yeyuno , Dextranos/metabolismo , Fluoresceína-5-Isotiocianato/análogos & derivados , Absorción Intestinal , Masculino , Femenino , Funcion de la Barrera IntestinalRESUMEN
BACKGROUND: Caenorhabditis elegans (C. elegans) has been recognized for being a useful model organism in small-molecule drug screens and drug efficacy investigation. However, there remain bottlenecks in evaluating such processes as drug uptake and distribution due to a lack of appropriate chemical tools. PURPOSE: This study aims to prepare fluorescence-labeled leonurine as an example to monitor drug uptake and distribution of small molecule in C. elegans and living cells. METHODS: FITC-conjugated leonurine (leonurine-P) was synthesized and characterized by LC/MS, NMR, UV absorption and fluorescence intensity. Leonurine-P was used to stain C. elegans and various mammalian cell lines. Different concentrations of leonurine were tested in conjunction with a competing parent molecule to determine whether leonurine-P and leonurine shared the same biological targets. Drug distribution was analyzed by imaging. Fluorometry in microplates and flow cytometry were performed for quantitative measurements of drug uptake. RESULTS: The UV absorption peak of leonurine-P was 490â¼495 nm and emission peak was 520 nm. Leonurine-P specifically bound to endogenous protein targets in C. elegans and mammalian cells, which was competitively blocked by leonurine. The highest enrichment levels of leonurine-P were observed around 72 h following exposure in C. elegans. Leonurine-P can be used in a variety of cells to observe drug distribution dynamics. Flow cytometry of stained cells can be facilely carried out to quantitatively detect probe signals. CONCLUSIONS: The strategy of fluorescein-labeled drugs reported herein allows quantification of drug enrichment and visualization of drug distribution, thus illustrates a convenient approach to study phytodrugs in pharmacological contexts.
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Caenorhabditis elegans , Ácido Gálico , Animales , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacocinética , Ácido Gálico/metabolismo , Humanos , Fluoresceína-5-Isotiocianato/análogos & derivados , Citometría de Flujo , Fluorescencia , Colorantes FluorescentesRESUMEN
Chimeric antigen receptor T-cell (CAR-T cell) therapy has become a promising treatment option for B-cell hematological tumors. However, few optional target antigens and disease relapse due to loss of target antigens limit the broad clinical applicability of CAR-T cells. Here, we conjugated an antibody (Ab) fusion protein, consisting of an Ab domain and a SpyCatcher domain, with the FITC-SpyTag (FITC-ST) peptide to form a bispecific safety switch module using a site-specific conjugation system. We applied the safety switch module to target CD19, PDL1, or Her2-expressing tumor cells by constructing FMC63 (anti-CD19), antiPDL1, or ZHER (anti-Her2)-FITC-ST, respectively. Those switch modules significantly improved the cytotoxic effects of anti-FITC CAR-T cells on tumor cells. Additionally, we obtained the purified CD8+ T cells by optimizing a shorter version of the CD8-binding aptamer to generate anti-FITC CD8-CAR-T cells, which combined with the CD4-FITC-ST switch module (anti-CD4) to eliminate the CD4-positive tumor cells in vitro and in vivo. Overall, we established a novel safety switch module by site-specific conjugation to enhance the antitumor function of universal CAR-T cells, thereby expanding the application scope of CAR-T therapy and improving its safety and efficacy.
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Diseño de Fármacos , Inmunoterapia Adoptiva , Linfoma de Células B , Inmunoterapia Adoptiva/métodos , Linfoma de Células B/tratamiento farmacológico , Humanos , Fluoresceína-5-Isotiocianato/química , Péptidos/química , Dominios Proteicos , Receptores Quiméricos de Antígenos/química , Receptores Quiméricos de Antígenos/genética , Anticuerpos/química , Anticuerpos/genéticaRESUMEN
The goblet cells of the gastrointestinal tract (GIT) produce glycoproteins called mucins that form a protective barrier from digestive contents and external stimuli. Recent evidence suggests that the milk fat globule membrane (MFGM) and its milk phospholipid component (MPL) can benefit the GIT through improving barrier function. Our objective was to compare the effects of two digested MFGM ingredients with or without dextran sodium sulfate (DSS)-induced barrier stress on mucin proteins. Co-cultured Caco-2/HT29-MTX intestinal cells were treated with in vitro digests of 2%, 5%, and 10% (w/v) MFGM or MPL alone for 6 h or followed by challenge with 2.5% DSS (6 h). Transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran (FD4) permeability measurements were used to measure changes in barrier integrity. Mucin characterization was performed using a combination of slot blotting techniques for secreted (MUC5AC, MUC2) and transmembrane (MUC3A, MUC1) mucins, scanning electron microscopy (SEM), and periodic acid Schiff (PAS)/Alcian blue staining. Digested MFGM and MPL prevented a DSS-induced reduction in secreted mucins, which corresponded to the prevention of DSS-induced increases in FD4 permeability. SEM and PAS/Alcian blue staining showed similar visual trends for secreted mucin production. A predictive bioinformatic approach was also used to identify potential KEGG pathways involved in MFGM-mediated mucosal maintenance under colitis conditions. This preliminary in silico evidence, combined with our in vitro findings, suggests the role of MFGM in inducing repair and maintenance of the mucosal barrier.
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Dextranos , Fluoresceína-5-Isotiocianato/análogos & derivados , Glucolípidos , Glicoproteínas , Gotas Lipídicas , Humanos , Células CACO-2 , Azul Alcián , Glicoproteínas/farmacología , Células Epiteliales , MucinasRESUMEN
Introduction: The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. Methods: We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. Results: The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CAR-T cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of co-stimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CAR-T cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. Conclusion: CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach.
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Neuroblastoma , Receptores Quiméricos de Antígenos , Humanos , Linfocitos T , Interleucina-15/metabolismo , Interleucina-7/metabolismo , Fluoresceína-5-Isotiocianato , Citocinas/metabolismoRESUMEN
AIMS: In patients with ulcerative colitis (UC), no biomarker is available to help the physician to choose the most suitable biotherapy. The primary objective of this pilot study was to assess the feasibility of identification of α4ß7- and TNF-expressing cells, to predict the response to vedolizumab using confocal laser endoscopy (CLE). METHODS: Patients with moderate-to-severe UC, naïve of biotherapy, received vedolizumab. Clinical evaluation was performed at each infusion. Endoscopic evaluation was performed before inclusion and at week 22. Fresh colonic biopsies were stained using FITC-labelled vedolizumab and Alexa fluor-labelled adalimumab and ex vivo dual-band CLE images were acquired. Blood samples were collected to measure trough concentrations of vedolizumab and to determine absolute counts of T and B cells subpopulations, NK cells and monocytes. RESULTS: Nineteen patients were enrolled in the study and received at least one dose of vedolizumab. Clinical remission and endoscopic improvement were observed in 58% of whom 5 patients (45%) had an endoscopic subscore of 0. In terms of clinical response and remission, endoscopic improvement and histologic response, FITC-conjugated vedolizumab staining tended to be higher in responder patients compared to non-responders at week 22. A threshold value of 6 positive FITC-vedolizumab staining areas detected by CLE seemed informative to discriminate the responders and non-responders. The results were similar in terms of clinical remission and endoscopic improvement with a sensitivity of 78% and a specificity of 85% (p = 0.05). Trough concentrations and blood immune cells were not associated with responses to vedolizumab. CONCLUSION: This pilot study demonstrate that dual-band CLE is feasible to detect α4ß7- and TNF-expressing cells. Positive α4ß7 staining seems to be associated with clinical and endoscopic remission in UC patients treated by anti-α4ß7-integrin, subject to validation by larger-scale studies. Clinical-trial.gov: NCT02878083.
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Anticuerpos Monoclonales Humanizados , Colitis Ulcerosa , Humanos , Proyectos Piloto , Fluoresceína-5-Isotiocianato , Biomarcadores , Endoscopía Gastrointestinal , Fármacos Gastrointestinales/uso terapéutico , Resultado del Tratamiento , Inducción de RemisiónRESUMEN
BACKGROUND: Intestinal barrier dysfunction in acute pancreatitis (AP) may progress to systemic inflammatory response syndrome (SIRS) and multi-organ failures by causing bacterial translocation. Larazotide acetate (LA) is a molecule that acts as a tight junction (TJ) regulator by blocking zonulin (Zo) receptors in the intestine. AIMS: In our study, we aimed to investigate the effects of LA on intestinal barrier dysfunction and bacterial translocation in the AP model in rats. METHODS: Thirty-two male Sprague-Dawley rats were divided into 4 groups; control, larazotide (LAR), AP, and AP + LAR. The AP model was created by administering 250 mg/100 g bm L-Arginine intraperitoneally 2 times with an hour interval. AP + LAR group received prophylactic 0.01 mg/mL LA orally for 7 days before the first dose of L-Arginine. For intestinal permeability analysis, fluorescein isothiocyanate-dextran (FITC-Dextran) was applied to rats by gavage. The positivity of any of the liver, small intestine mesentery, and spleen cultures were defined as bacterial translocation. Histopathologically damage and zonulin immunoreactivity in the intestine were investigated. RESULTS: Compared to the control group, the intestinal damage scores, anti-Zo-1 immunoreactivity H-Score, serum FITC-Dextran levels and bacterial translocation frequency (100% versus 0%) in the AP group were significantly higher (all p < 0.01). Intestinal damage scores, anti-Zo-1 immunoreactivity H-score, serum FITC-Dextran levels, and bacterial translocation frequency (50% versus 100%) were significantly lower in the AP + LAR group compared to the AP group (all p < 0.01). CONCLUSIONS: Our findings show that LA reduces the increased intestinal permeability and intestinal damage by its effect on Zo in the AP model in rats, and decreases the frequency of bacterial translocation as a result of these positive effects.