ABSTRACT
The number of allergy sufferers has been increasing with the increase in chemicals to which we are potentially exposed. We have discovered that tributyrin, a short-chain triacylglycerol (TAG), enhanced fluorescein isothiocyanate (FITC)-induced contact hypersensitivity in a mouse model. Medium-chain triacylglycerols (MCTs) are used in cosmetics, with which we come into direct contact frequently, to maintain skin conditions and as a thickening agent for cosmetics. In this study, we examined whether MCTs with different side chain lengths enhanced skin sensitization to FITC in the mouse model. During skin sensitization to FITC, the presence of tributyrin (side chain carbon number, 4; C4) as well as that of each MCT, tricaproin (C6), tricaprylin (C8), or tricaprin (C10), resulted in enhanced skin sensitization, whereas that of trilaurin (C12) did not. As to the mechanism underlying the enhanced sensitization, three MCTs (C6, C8 and C10) facilitated migration of FTIC-presenting CD11c+ dendritic cells to draining lymph nodes. These results indicated that not only tributyrin but also MCTs, up to side chain carbon number 10, have an adjuvant effect on FITC-induced skin hypersensitivity in mice.
Subject(s)
Dermatitis, Contact , Animals , Mice , Adjuvants, Immunologic/pharmacology , Dendritic Cells , Dermatitis, Contact/etiology , Fluorescein/pharmacology , Fluorescein-5-isothiocyanate/toxicity , Isothiocyanates/pharmacology , Lymph Nodes , Mice, Inbred BALB C , Triglycerides/toxicityABSTRACT
The present study shows that a dual-signal nanoprobe consisting of DNAzyme-functionalized porous carbon nanospheres (PCNs) responds to microRNA-21 and zinc ion (Zn2+). The fluorescent probe undergoes an increase in the fluorescence intensity of fluorescein isothiocyanate (FITC) (with excitation/emission wavelengths at 488/517 nm) and the fluorescence intensity of cyanine-5 (Cy5) (with excitation/emission wavelengths at 633/670 nm) in the presence of microRNA-21 and Zn2+. The recognition between microRNA-21 and its complementary strand in the PCNs induces the separation of Zn2+-specific DNAzyme from PCNs, thus resulting in the increase of green fluorescence, and the exogenous Zn2+ triggers the rupture of cleavage strand of DNAzyme and recovery of red fluorescence. This nanoprobe allows us to acquire in vitro the determination of microRNA-21 in the range of 2-300 nM with a detection limit of 0.57 nM and the determination of Zn2+ in the range 2-100 nM with a detection limit of 0.43 nM, and in situ simultaneous imaging in MCF-7 breast cancer cells. Therefore, this strategy permits to obtain the expression levels of different biomarkers in living cells, providing a useful tool for diagnosis of cancers and understanding their biological process. Graphical abstract Schematic representation of the DNAzyme-functionalized porous carbon nanospheres for the imaging analysis of microRNA-21 and Zn2+ in living cells.
Subject(s)
DNA, Catalytic/chemistry , Fluorescent Dyes/chemistry , MicroRNAs/analysis , Nanospheres/chemistry , Zinc/analysis , Animals , Carbon/chemistry , Carbon/toxicity , DNA, Catalytic/toxicity , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/toxicity , Fluorescent Dyes/toxicity , Human Umbilical Vein Endothelial Cells , Humans , Limit of Detection , MCF-7 Cells , MicroRNAs/metabolism , Microscopy, Confocal , Microscopy, Fluorescence , Nanospheres/toxicity , Porosity , Rats , Spectrometry, Fluorescence , Zinc/metabolismABSTRACT
pH regulates the function of many organelles and plays a pivotal role in requiring multitud cellular behaviors. Compared with single fluorescent probes, ratio fluorescent probes have higher sensitivity and immunity to interference. Herein, a novel Janus ratio nanoprobe was developed for intracellular pH detection. Modified rhodamine B probe and fluorescein isothiocyanate (FITC) were individually encapsulated in the independent hemispheres of Janus microparticles fabricated via Pickering emulsion. Moreover, it exhibits a satasified ratiometric detection of pH compared to the previous core-shell structure and organic small molecule probe. Accordingly, the Janus nanoprobe possesses many important features as an attractive sensor, including high anti-jamming capability, excellent stability, good reversibility and low cytotoxicity. Variations of the two fluorescence intensities (Fgreen/Fred) resulted in a ratiometric pH fluorescent sensor, which can respond to wide range of pH values from 3 to 8. To be more specific, with a single excitation wavelength of 488â¯nm, there are dual emission bands centered at 538â¯nm and 590â¯nm. Also the Janus nanoprobe displays a excellent linear relationship in the physiologically relevant pH range of 4.0-6.0. Consequently, detecting of pH and imaging was successfully achieved in living cells, which provides a simple and reliable method for detecting intracelluar pH and other similar substances.
Subject(s)
Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes/chemistry , Nanoparticles/chemistry , Rhodamines/chemistry , Cell Line, Tumor , Cell Survival , Fluorescein-5-isothiocyanate/toxicity , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/toxicity , Humans , Hydrogen-Ion Concentration , Microscopy, Fluorescence , Nanoparticles/toxicity , Rhodamines/chemical synthesis , Rhodamines/toxicity , Silicon Dioxide/chemistry , Spectrometry, FluorescenceABSTRACT
The goal of this study was to elucidate the anti-pruritic and anti-inflammatory efficacy of ruxolitinib cream in experimentally-induced dermatitis. Atopic dermatitis (AD), the most common chronic relapsing inflammatory skin disease, significantly impairs patients' quality of life, with pruritus being a common complaint. The sensation of itch results from the interplay between epidermal barrier dysfunction, upregulated immune signaling and the activation of the central nervous system. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway plays a central role in pro-inflammatory cytokine signaling in AD. Ruxolitinib cream is a potent and selective JAK1/2 inhibitor currently undergoing clinical evaluation in adults with mild-to-moderate AD (NCT03745638, NCT03920852 and NCT03745651). The efficacy of ruxolitinib cream was tested in murine models of acute and chronic dermatitis and was also characterized in an ex vivo human skin dermatitis model. Ruxolitinib cream was highly effective at ameliorating disease symptoms in multiple murine dermatitis models through downregulation of T helper (Th)2-driven inflammation, resulting in reduced skin thickening and decreased itch. Pathway analysis of mouse ear tissue and human skin explants underscored the role for ruxolitinib in ameliorating inflammation and reducing itch via modulation of the JAK-STAT pathway. Together, the data offer a strong rationale for the use of ruxolitinib cream as a potent therapeutic agent for the clinical management of atopic dermatitis.
Subject(s)
Dermatitis/drug therapy , Janus Kinase Inhibitors/therapeutic use , Pruritus/drug therapy , Pyrazoles/therapeutic use , Administration, Cutaneous , Animals , Betamethasone/administration & dosage , Betamethasone/therapeutic use , Clobetasol/administration & dosage , Clobetasol/therapeutic use , Cytokines/biosynthesis , Cytokines/genetics , Cytokines/toxicity , Disease Models, Animal , Drug Eruptions/drug therapy , Drug Eruptions/immunology , Drug Evaluation, Preclinical , Female , Fluorescein-5-isothiocyanate/toxicity , Grooming/drug effects , Humans , In Vitro Techniques , Interleukin-33/genetics , Janus Kinase Inhibitors/administration & dosage , Lymphocyte Subsets/drug effects , Lymphocyte Subsets/immunology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Nitriles , Ointments , Organ Culture Techniques , Pyrazoles/administration & dosage , Pyrimidines , Random Allocation , Signal Transduction/drug effects , Skin/drug effects , Specific Pathogen-Free Organisms , T-Lymphocytes, Helper-Inducer/drug effects , T-Lymphocytes, Helper-Inducer/immunology , Transcriptome , Thymic Stromal LymphopoietinABSTRACT
This study aimed to investigate the mechanisms of Yu-Ping-Feng-San (YPFS) on attenuating allergic inflammation in the initial stage of atopic dermatitis (AD). AD mouse model was established with fluorescein isothiocyanate (FITC) sensitization and elicitation. Epithelial barrier structure was observed with transmission electron microscope. The populations of dendritic cells (DCs) and group 2 innate lymphoid cells (ILC2s) were detected by flow cytometry. Human immortalized keratinocyte (HaCaT) cells were stimulated with Poly(I:C)/TNF-α in vitro to assessthymic stromal lymphopoietin (TSLP), interleukin (IL)-33 and nuclear factor-κB (NF-κB) levels or expressions by immunofluorescence, enzyme linked immunosorbent assay (ELISA) and western blot. In the initial stage of AD, ear swelling and infiltration of inflammatory cells in ear tissues were markedly attenuated with YPFS treatments. The damaged structures of ear epithelium and the increased levels of Th2-cytokines induced by FITC were significantly rescued in YPFS-treated mice. The production of pro-allergic cytokines, TSLP and IL-33, as well as the cell populations of their target cells DCs and ILC2s were decreased in AD model, respectively. Likewise, the levels of TSLP and IL-33 in Poly(I:C)/TNF-α-stimulated HaCaT cells showed the same results. Lower levels of p-NF-κB were detected with YPFS treatment, and the expressions of TSLP and IL-33 could be further decreased with inhibiting of NF-κB. Therefore, YPFS attenuates allergic inflammation in the initial stage of AD probably through regulating NF-κB-TSLP/IL-33 pathway, which may provide a novel effective target for the prevention and treatment of allergic diseases.
Subject(s)
Anti-Allergic Agents/therapeutic use , Cytokines/metabolism , Dermatitis, Atopic/drug therapy , Drugs, Chinese Herbal/therapeutic use , Inflammation/prevention & control , Animals , Anti-Allergic Agents/pharmacology , Cell Line , Dendritic Cells/pathology , Dermatitis, Atopic/chemically induced , Dermatitis, Atopic/metabolism , Disease Models, Animal , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Epithelial Cells/metabolism , Fluorescein-5-isothiocyanate/toxicity , Inflammation/metabolism , Inflammation/pathology , Keratinocytes/drug effects , Keratinocytes/metabolism , Lymphocytes/metabolism , Lymphocytes/pathology , Mice, Inbred BALB C , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plant Extracts/therapeutic useABSTRACT
The prevalence of skin allergies could be partly due to the increased exposure to chemicals from consumer products. Chemicals that can enhance hypersensitivity caused by other chemicals are the focus of this study. We have demonstrated that phthalate esters with short chain alcohols enhance fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) in a mouse model. We have also found that tributyrin, a triacylglycerol (TAG) with three butyric acids, enhances sensitization to FITC. To elucidate such an enhanced skin sensitization might be based on a general feature of TAG, we compared tributyrin and triolein, a natural TAG, as to an adjuvant effect on FITC-CHS. Triolein is the dominant TAG in olive oil and contains long chain mono-unsaturated fatty acids. Unlike tributyrin and dibutyl phthalate (DBP), triolein did not exhibit an adjuvant effect. With triolein, enhancement of FITC-presenting CD11c+ dendritic cell trafficking to draining lymph nodes was weak, and the activation status of DC, as revealed as CD86 expression, was low. We found a difference in the pattern of skin cytokine production, i.e., that thymic stromal lymphopoietin was produced with DBP and interleukin-1ß with tributyrin. Triolein did not induce either of these cytokines. This illustrates that the adjuvant effect of tributyrin on FITC-CHS is not a general phenomenon for TAGs. Although beneficial effects may be expected through oral administration of tributyrin, the effect on skin immune systems should be considered.
Subject(s)
Adjuvants, Immunologic/pharmacology , Allergens/toxicity , Dermatitis, Contact/immunology , Dibutyl Phthalate/pharmacology , Fluorescein-5-isothiocyanate/toxicity , Triglycerides/pharmacology , Animals , Cell Movement/drug effects , Cytokines/immunology , Dendritic Cells/drug effects , Dendritic Cells/physiology , Female , Lymph Nodes/immunology , Mice, Inbred BALB C , Skin/drug effects , Skin/immunologyABSTRACT
Extracellular acidity is correlated with the development of various pathological states and bulk pH measurements could not report surface acidity. In this study, we have developed a ratiometric fluorescent probe that aggregates upon interaction with cells, allowing persistent labeling of cells and in situ measurement of cell surface pH. The ternary nanoplatform is constructed by a convenient noncovalent combination of bovine serum albumin protected gold nanoclusters (BSA-AuNCs), fluorescein isothiocyanate (FITC) labeled cationic peptides (CPs), and FITC-free CPs. The red fluorescent AuNCs serve as reference fluorophore, while FITC labeled peptides act as specific recognition element for H+ and FITC unlabeled peptides are used for delivery. The probe displays a sensitive fluorescence ratiometric response for pH in the range of 5.0-9.5 with calculated p Ka of 7.2. Further studies have demonstrated that this nanosensor also has properties of high selectivity, reversibility to pH fluctuations, as well as low cytotoxicity. The new surface pH-measurement tool was validated in mapping extracellular pH and monitoring acidification regarding cell metabolism, demonstrating its potential for bioimaging and biosensing.
Subject(s)
Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes/chemistry , Metal Nanoparticles/chemistry , Animals , Biosensing Techniques/methods , Cattle , Cell Line, Tumor , Cell Membrane/metabolism , Fluorescein-5-isothiocyanate/toxicity , Fluorescence , Fluorescent Dyes/toxicity , Gold/chemistry , Gold/toxicity , Humans , Hydrogen-Ion Concentration , Metal Nanoparticles/toxicity , Microscopy, Confocal/methods , Microscopy, Fluorescence/methods , Peptides/chemistry , Peptides/toxicity , Serum Albumin, Bovine/chemistry , Serum Albumin, Bovine/toxicityABSTRACT
Contact hypersensitivity (CHS) to preservatives is receiving increased attention. Parabens are widely used in foods, pharmaceutics and cosmetics as preservatives. The skin sensitizing activity of parabens remains controversial but a few investigations have been made as to whether parabens could facilitate sensitization to other chemicals. We have shown that di-n-butyl phthalate (DBP), a phthalate ester, has an adjuvant effect in a fluorescein isothiocyanate (FITC)-induced CHS mouse model. We have also demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. Comparative studies of phthalate esters revealed that TRPA1 agonistic activity and the adjuvant effect on FITC-CHS coincide. Here we focused on two commonly used parabens, butyl paraben (BP) and ethyl paraben (EP), as to their adjuvant effects. BALB/c mice were epicutneously sensitized with FITC in acetone in the presence or absence of a paraben. Sensitization to FITC was evaluated as the ear-swelling response after FITC challenge. BP but not EP enhanced skin sensitization to FITC, but the effect of BP was much weaker than that of DBP. Mechanistically, BP enhanced the trafficking of FITC-presenting CD11c+ dendritic cells (DCs) from the skin to draining lymph nodes as well as cytokine production by draining lymph nodes. When the TRPA1 agonistic activity was measured with a cell line expressing TRPA1, BP exhibited higher activity than EP. The present study provides direct in vivo evidence that BP causes sensitization to other chemicals by means of a mouse FITC-CHS model.
Subject(s)
Dermatitis, Contact/metabolism , Dextrans/toxicity , Disease Models, Animal , Fluorescein-5-isothiocyanate/analogs & derivatives , Parabens/toxicity , Animals , CHO Cells , Cricetinae , Cricetulus , Dermatitis, Contact/pathology , Dose-Response Relationship, Drug , Female , Fluorescein-5-isothiocyanate/toxicity , Mice , Mice, Inbred BALB CABSTRACT
IL-21 is mainly produced by activated CD4+ T cells and is involved in the activation of immune cells such as T cells and macrophages. In contrast, IL-21 suppresses dendritic cell maturation. We studied the effect of IL-21 in a mouse model of FITC-induced contact hypersensitivity using IL-21 isoform transgenic (IL-21iso-Tg) mice. Tissue inflammation at 24 hours after elicitation in IL-21iso-Tg mice was significantly weaker than that in wild-type mice. In agreement with tissue inflammation, recruitment of CD4+ and CD8+ T cells, neutrophils, and macrophages into the inflamed tissue was decreased in IL-21iso-Tg mice. In addition, both mRNA expression and protein production of inflammatory cytokines were lower in IL-21iso-Tg mice. In the skin, T cells were activated at inducible skin-associated lymphoid tissue, which is likely a gut-associated lymphoid tissue. The mRNA level of CXCL2, an essential chemokine for inducible skin-associated lymphoid tissue formation, was significantly lower in IL-21iso-Tg mice, and histological analysis showed that dendritic cell clustering, a preliminary step in inducible skin-associated lymphoid tissue formation, was impaired. Our study showed that IL-21 down-regulated inducible skin-associated lymphoid tissue formation and reduced contact hypersensitivity response.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/immunology , Dermatitis, Contact/immunology , Interleukins/metabolism , Animals , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Dendritic Cells/metabolism , Dermatitis, Contact/etiology , Dermatitis, Contact/pathology , Disease Models, Animal , Flow Cytometry , Fluorescein-5-isothiocyanate/toxicity , Mice, Inbred BALB C , Mice, Inbred C57BL , Skin/pathologyABSTRACT
Little attention has been paid to chemicals that can enhance hypersensitivity caused by other chemicals. We have demonstrated that phthalate esters with short chain alcohols enhance fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) in a mouse model. Furthermore, phthalate esters with such an enhancing effect were found to activate transient receptor potential ankyrin 1 (TRPA1) cation channels, which are expressed on a part of sensory neurons, using a TRPA1-expressing cell line. In this study, we examined these activities of esters comprising glycerol and a short chain fatty acid, i.e. dibutyrin and tributyrin. We carried out chemical synthesis of dibutyrin isomers. Each dibutyrin isomer weakly activated TRPA1 and slightly enhanced skin sensitization to FITC. Unexpectedly, TRPA1 activation and enhancement of FITC-CHS were much more evident in the presence of tributyrin. Mechanistically, tributyrin induced increased dendritic cell trafficking from the skin to draining lymph nodes. Tributyrin enhanced interferon-γ (IFN-γ) production by draining lymph nodes, while its effect on interleukin-4 (IL-4) production was relatively less prominent. These results suggested that tributyrin concomitantly caused TRPA1 activation and an adjuvant effect on FITC-CHS.
Subject(s)
Adjuvants, Immunologic/toxicity , Dermatitis, Contact/immunology , TRPA1 Cation Channel/metabolism , Triglycerides/toxicity , Adjuvants, Immunologic/administration & dosage , Animals , CHO Cells , Cell Movement/drug effects , Cricetulus , Dendritic Cells/drug effects , Dermatitis, Contact/metabolism , Disease Models, Animal , Female , Fluorescein-5-isothiocyanate/administration & dosage , Fluorescein-5-isothiocyanate/toxicity , Humans , Interferon-gamma/metabolism , Interleukin-4/metabolism , Lymph Nodes/drug effects , Lymph Nodes/immunology , Mice, Inbred BALB C , Skin/drug effects , Skin/immunology , Triglycerides/administration & dosageABSTRACT
Labeling of aerosol particles with a radioactive, magnetic, or optical tracer has been employed to confirm particle localization in cell compartments, which has provided useful evidence for correlating toxic effects of inhaled particles. However, labeling requires several physicochemical steps to identify functionalities of the inner or outer surfaces of particles, and moreover, these steps can cause changes in size, surface charge, and bioactivity of the particles, resulting in misinterpretations regarding their toxic effects. This study addresses this challenging issue with a goal of introducing an efficient strategy for constantly supplying labeled aerosol particles in a single-pass configuration without any pre- or post-physicochemical batch treatments of aerosol particles. Carbon black (CB, simulating combustion-generated soot) or calcium carbonate (CC, simulating brake-wear fragments) particles were constantly produced via spark ablation or bubble bursting, respectively. These minute particles were incorporated with fluorescein isothiocyanate-poly(ethylene glycol) 2-aminoethyl ether acetic acid solution at the orifice of a collison atomizer to fabricate hybrid droplets. The droplets successively entered a diffusion dryer containing 254-nm UV irradiation; therefore, the droplets were dynamically stiffened by UV to form fluorescent probes on particles during solvent extraction in the dryer. Particle size distributions, morphologies, and surface charges before and after labeling were measured to confirm fluorescence labeling without significant changes in the properties. In vitro assays, including confocal imaging, were conducted to confirm the feasibility of the labeling approach without inducing significant differences in bioactivity compared with untreated CB or CC particles.
Subject(s)
Environmental Monitoring/methods , Inhalation Exposure/analysis , Models, Biological , Particulate Matter/analysis , Staining and Labeling/methods , A549 Cells , Aerosols , Calcium Carbonate/analysis , Cell Survival/drug effects , Environmental Monitoring/instrumentation , Equipment Design , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/toxicity , Fluorescent Dyes/chemistry , Fluorescent Dyes/toxicity , Humans , Microscopy, Electron, Transmission , Particle Size , Particulate Matter/toxicity , Soot/analysis , Staining and Labeling/instrumentation , Surface PropertiesABSTRACT
Cholesterol-containing molecules or nanoparticles play a significant role in achieving favorable plasma membrane imaging and efficient cellular uptake of drugs by the excellent membrane anchoring capability of the cholesterol moiety. By linking cholesterol to a water-soluble component (such as poly(ethylene glycol), PEG), the resulting cholesterol-PEG conjugate can form micelles in aqueous solution through self-assembly, and such a micellar structure represents an important drug delivery vehicle in which hydrophobic drugs can be encapsulated. However, the understanding of the subcellular fate and cytotoxicity of cholesterol-PEG conjugates themselves remains elusive. Herein, by using cholesterol-PEG2000-fluorescein isothiocyanate (Chol-PEG-FITC) as a model system, we found that the Chol-PEG-FITC molecules could attach to the plasma membranes of mammalian cells within 10 min and such a firm membrane attachment could last at least 1 h, displaying excellent plasma membrane staining performance that surpassed that of commonly used commercial membrane dyes such as DiD and CellMask. Besides, we systematically studied the endocytosis pathway and intracellular distribution of Chol-PEG-FITC and found that the cell surface adsorption and endocytosis processes of Chol-PEG-FITC molecules were lipid-raft-dependent. After internalization, the Chol-PEG-FITC molecules gradually reached many organelles with membrane structures. At 5 h, they were mainly distributed in lysosomes and the Golgi apparatus, with some in the endoplasmic reticulum (ER) and very few in the mitochondrion. At 12 h, the Chol-PEG-FITC molecules mostly aggregated in the Golgi apparatus and ER close to the nucleus. Finally, we demonstrated that Chol-PEG-FITC was toxic to mammalian cells only at concentrations above 50 µM. In summary, Chol-PEG-FITC can be a promising plasma membrane imaging reagent to avoid the fast cellular internalization and quick membrane detachment problems faced by commercial membrane dyes. We believe that the investigation of the dynamic subcellular fate of Chol-PEG-FITC can provide important knowledge to facilitate the use of cholesterol-PEG conjugates in fields such as cell surface engineering and drug delivery.
Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/metabolism , Polyethylene Glycols/metabolism , Cholesterol/analogs & derivatives , Cholesterol/chemistry , Cholesterol/toxicity , Endocytosis/drug effects , Flow Cytometry , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/toxicity , Fluorescent Dyes/chemistry , Fluorescent Dyes/toxicity , Humans , MCF-7 Cells , Membrane Microdomains/metabolism , Micelles , Microscopy, Confocal , Particle Size , Polyethylene Glycols/chemistry , Polyethylene Glycols/toxicityABSTRACT
Di-n-butyl phthalate (DBP), a phthalate ester, has been shown to have an adjuvant effect on fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse models. Di-n-butyl maleate (DBM), widely used as a plasticizer for industrial application, has been reported to cause dermatitis in humans. DBM is a butyl alcohol ester of di-carboxylic acid that represents a part of the DBP structure, while di-n-butyl fumarate (DBF) is a trans isomer of DBM. We examined whether DBM or DBF exhibits an adjuvant effect like DBP does. When BALB/c mice were epicutaneously sensitized with FITC in the presence of DBM or DBF, the FITC-specific CHS response was enhanced, as we have observed for DBP. As to underlying mechanisms, DBM and DBF facilitated the trafficking of FITC-presenting CD11c(+) dendritic cells (DCs) from skin to draining lymph nodes and increased the cytokine production by draining lymph nodes. In conclusion, DBM and DBF may have an effect that aggravates contact dermatitis through a skin sensitization process.
Subject(s)
Dermatitis, Contact , Drug Hypersensitivity , Fluorescein-5-isothiocyanate/toxicity , Fumarates/toxicity , Maleates/toxicity , Animals , Cytokines/genetics , Cytokines/metabolism , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Lymph Nodes/metabolism , Mice , Mice, Inbred BALB C , Molecular StructureABSTRACT
We demonstrate that the incorporation of one or two amino acids of phenylalanine (F) or 4-fluoro phenylalanine ((f)F) will greatly lower the background fluorescence intensities of conventional quenched probes with quenchers. This enhanced quenching effect was due to the synergetic effect of the aggregation caused quenching and the presence of a quencher. Such strategy will not greatly affect the enzyme recognition properties to the probes. We also demonstrated that our self-assembled nanoprobe with the enhanced quenching effect showed a better performance in cells for the detection of cell apoptosis than the unassembled probes. Our study demonstrates that using molecular self-assembly can optimize and improve the performance of molecular probes and it provides a simple but very useful strategy to boost the signal-to-noise ratios of fluorescence probes.
Subject(s)
Molecular Probes/chemistry , Amino Acid Sequence , Apoptosis/drug effects , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/metabolism , Fluorescein-5-isothiocyanate/toxicity , Fluorescence Resonance Energy Transfer , Fluorescent Dyes/chemistry , Fluorescent Dyes/metabolism , Fluorescent Dyes/toxicity , HeLa Cells , Humans , Microscopy, Confocal , Molecular Probes/metabolism , Molecular Probes/toxicity , Peptides/chemical synthesis , Peptides/chemistry , Peptides/metabolism , Rhodamines/chemistry , Rhodamines/metabolism , Rhodamines/toxicity , Signal-To-Noise RatioABSTRACT
Macrophages are becoming increasingly significant in the progression of atherosclerosis (AS). Molecular imaging of macrophages may improve the detection and characterization of AS. In this study, dendrimer-entrapped gold nanoparticles (Au DENPs) with polyethylene glycol (PEG) and fluorescein isothiocyanate (FI) coatings were designed, tested, and applied as contrast agents for the enhanced computed tomography (CT) imaging of macrophages in atherosclerotic lesions. Cell counting kit-8 assay, fluorescence microscopy, silver staining, and transmission electron microscopy revealed that the FI-functionalized Au DENPs are noncytotoxic at high concentrations (3.0 µM) and can be efficiently taken up by murine macrophages in vitro. These nanoparticles were administered to apolipoprotein E knockout mice as AS models, which demonstrated that the macrophage burden in atherosclerotic areas can be tracked noninvasively and dynamically three-dimensionally in live animals using micro-CT. Our findings suggest that the designed PEGylated gold nanoparticles are promising biocompatible nanoprobes for the CT imaging of macrophages in atherosclerotic lesions and will provide new insights into the pathophysiology of AS and other concerned inflammatory diseases.
Subject(s)
Atherosclerosis/pathology , Gold/chemistry , Macrophages/chemistry , Metal Nanoparticles/chemistry , Molecular Imaging/methods , Tomography, X-Ray Computed/methods , Animals , Cell Line , Erythrocytes/drug effects , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/pharmacokinetics , Fluorescein-5-isothiocyanate/toxicity , Gold/pharmacokinetics , Gold/toxicity , Hemolysis/drug effects , Humans , Macrophages/cytology , Metal Nanoparticles/toxicity , Mice , Polyethylene Glycols/chemistry , Tissue DistributionABSTRACT
OBJECTIVE: To explore the use of oleic acid (OA) in ocular drug delivery. METHODS: Six compounds, namely rhodamine B, sodium-fluorescein, fluorescein isothiocyanate (FITC) dextrans of 4, 10, 20 and 40 kDa were selected as model drugs. The effect of OA on the corneal permeability of drugs was evaluated in vitro, using isolated rabbit corneas by a Franz diffusion cell. The safety of OA was assessed on the basis of corneal hydration level. The ocular irritation of OA was also tested in rabbits in vivo using the Draize eye test. RESULTS: In the presence of OA, at a concentration of 0.02-0.1%, the maximum increase in the apparent permeability coefficient (Papp) was 3.21-, 1.76- and 1.57-fold for rhodamine B, sodium-fluorescein and FITC-dextran of 4 kDa, respectively. However, no significant permeability enhancement of FITC-dextrans of 4, 10, 20 and 40 kDa was found in the presence of OA. It enhanced the corneal penetration of model compounds in a concentration-dependent manner. The Papp values of rhodamine B decreased with increasing concentration of OA, while the Papp values of sodium-fluorescein and FITC-dextrans of 4 kDa increased. The Papp enhanced by 0.1% OA was logarithmically correlated to the molecular weight of model drugs (R(2) = 0.9991). With the 0.02%, 0.05% and 0.1% oleic application, the corneal hydration values were <83%, and Draize scores were <4. CONCLUSION: OA may have potential clinical benefits in improving the ocular drug delivery of both hydrophilic and lipophilic compounds.
Subject(s)
Cell Membrane Permeability/physiology , Cornea/drug effects , Fluorescent Dyes/pharmacokinetics , Oleic Acid/pharmacology , Animals , Cornea/metabolism , Dextrans/pharmacokinetics , Dextrans/toxicity , Drug Carriers , Fluorescein/pharmacokinetics , Fluorescein/toxicity , Fluorescein-5-isothiocyanate/analogs & derivatives , Fluorescein-5-isothiocyanate/pharmacokinetics , Fluorescein-5-isothiocyanate/toxicity , Fluorescent Dyes/toxicity , Irritants/toxicity , Ophthalmic Solutions/pharmacokinetics , Ophthalmic Solutions/toxicity , Rabbits , Rhodamines/pharmacokinetics , Rhodamines/toxicityABSTRACT
Recently, a transglutaminase 3 knockout (TGM3/KO) mouse was generated that showed impaired hair development, but no gross defects in the epidermal barrier, although increased fragility of isolated corneocytes was demonstrated. Here we investigated the functionality of skin barrier in vivo by percutaneous sensitization to FITC in TGM3/KO (n=64) and C57BL/6 wild-type (WT) mice (n=36). Cutaneous inflammation was evaluated by mouse ear swelling test (MEST), histology, serum IgE levels, and by flow cytometry from draining lymph nodes. Inflammation-induced significant MEST difference (P<0.0001) was detected between KO and WT mice and was supported also by histopathology. A significant increase of CD4+ CD25+-activated T cells (P<0.01) and elevated serum IgE levels (P<0.05) in KO mice indicated more the development of FITC sensitization than an irritative reaction. Propionibacter acnes-induced intracutaneous inflammation showed no difference (P=0.2254) between the reactivity of WT and KO immune system. As in vivo tracer, FITC penetration from skin surface followed by two-photon microscopy demonstrated a more invasive percutaneous penetration in KO mice. The clinically uninvolved skin in TGM3/KO mice showed impaired barrier function and higher susceptibility to FITC sensitization indicating that TGM3 has a significant contribution to the functionally intact cutaneous barrier.
Subject(s)
Dermatitis, Contact/immunology , Dermatitis, Contact/microbiology , Gram-Positive Bacterial Infections/immunology , Propionibacterium acnes/immunology , Transglutaminases/immunology , Animals , Dermatitis, Contact/etiology , Edema/immunology , Edema/metabolism , Female , Flow Cytometry , Fluorescein-5-isothiocyanate/toxicity , Gram-Positive Bacterial Infections/metabolism , Immunoglobulin E/immunology , Lymph Nodes/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Propionibacterium acnes/metabolism , Skin/immunology , Skin/metabolism , Skin/microbiology , Transglutaminases/geneticsABSTRACT
Sensitization and challenge using dinitrofluorobenzene (DNFB) induce contact hypersensitivity (CHS) with Th1 cell infiltration, whereas those using FITC generate CHS with Th2 cell infiltration. In this study, we attempted to determine the role of CXCR3, a chemokine receptor, in Th1- and Th2-type CHS induced by DNFB or FITC using CXCR3-deficient (CXCR3(-/-)) mice. Ear swelling was prolonged after DNFB challenge in CXCR3(-/-) mice, which was accompanied by increased Th1 cytokines and decreased TGF-ß and IL-10 expression at a late time point of CHS, whereas there was no significant difference between wild-type and CXCR3(-/-) mice in FITC-induced CHS. In Th1-type CHS, the number of regulatory T cells (Tregs) was decreased in the challenged ear of CXCR3(-/-) mice compared with that of wild-type mice, suggesting that CXCR3 would be important in migration of Tregs into the site of inflammation. Moreover, we examined the characteristics of CXCR3(+) Tregs both in vitro and in vivo, revealing that CXCR3(+) Tregs expressed high levels of TGF-ß and IL-10 as well as IFN-γ compared with CXCR3(-) Tregs. When CXCR3(-/-) mice were injected with CXCR3(+) Tregs, the prolonged ear swelling induced by DNFB was normalized. Taken together, our results suggest that CXCR3(+) Tregs play a key role for quenching Th1-type CHS.
Subject(s)
Dermatitis, Contact/immunology , Receptors, CXCR3/immunology , Th1 Cells/immunology , Adoptive Transfer , Animals , Dinitrofluorobenzene/immunology , Fluorescein-5-isothiocyanate/toxicity , Fluorescent Antibody Technique , Immunohistochemistry , Irritants/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, CXCR3/deficiency , Reverse Transcriptase Polymerase Chain Reaction , Th2 Cells/immunologyABSTRACT
Magnetic nanoparticles (MNPs) functionalized with specific ligands are emerging as a highly integrated platform for cancer targeting, drug delivery, and magnetic resonance imaging applications. In this study, we describe a multifunctional magnetic nanoparticle system (FITC-Tat MNPs) consisting of a fluorescently labeled cell penetrating peptide (FITC-Tat peptide), a biocompatible block copolymer PEG(600)-b-poly(glycerol monoacrylate) (PEG(600)-b-PGA), and a superparamagnetic iron oxide (SPIO) nanoparticle core. The particles were prepared by direct chemisorption of PEG(600)-b-PGA conjugated with FITC-Tat peptide on the SPIO nanoparticles. FITC-MNPs without Tat were prepared for comparison. Flow cytometry assays revealed significantly higher uptake of FITC-Tat MNPs compared to FITC-MNPs in Caco-2 cells. These results were confirmed using confocal laser scanning microscopy (LSCM), which further demonstrated that the FITC-Tat MNPs accumulated in the cytoplasm and nucleus while the FITC-MNPs were localized in the cell membrane compartments. The FITC-Tat MNPs did not exhibit observable cytotoxicity in MTS assays.
Subject(s)
Acrylates/chemistry , Fluorescein-5-isothiocyanate/metabolism , Glycerol/chemistry , Magnetite Nanoparticles , Peptide Fragments/metabolism , Polyethylene Glycols/chemistry , Polymers/chemistry , tat Gene Products, Human Immunodeficiency Virus/metabolism , Acrylates/toxicity , Biological Transport , Caco-2 Cells , Flow Cytometry , Fluorescein-5-isothiocyanate/chemistry , Fluorescein-5-isothiocyanate/toxicity , Glycerol/toxicity , Humans , Kinetics , Microscopy, Confocal , Particle Size , Peptide Fragments/chemistry , Peptide Fragments/toxicity , Polyethylene Glycols/toxicity , Polymers/toxicity , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/toxicityABSTRACT
Fluorescent dyes (e.g., dansyl, fluoresceine isothiocyanate, or naphthalimide groups) are widely used as markers to study biological properties of drugs. In order to evaluate possible mediated cytotoxicity, we attached three molecules each to 1,3,5-tris(3-propylamino)benzene initially synthesized as core molecule for the design of dendrimers. Cytotoxic effects were only observed for the NO(2)-substituted naphthalimide conjugate. The intracellular distribution was visualized via confocal fluorescence microscopy and pointed to an accumulation in the endosome or nucleus, dependent on the cell line used.