RESUMEN
We designed a simple, inexpensive, and user-friendly assay using mesoporous silica nanoparticles to detect analytes. Highly stable and uniform palladium nanoparticles covered with mesoporous silica (Pd@mSiO2) were generated and characterized extensively using physical methods. Human Serum Albumin (HSA) protein or ssDNA specific to the HIV gag region was capped onto the Pd@mSiO2 electrostatically. This "cap" prevented the Pd(0) inside the mesoporous silica nanoparticles from catalyzing the conversion of non-fluorescent molecules to fluorescent molecules. In the presence of target anti-HSA antibodies or complementary sequence (HIV gag), HSA protein or DNA cap dissociated from the surface of Pd@mSiO2-NH2 through the specific antigen-antibody reaction or DNA hybridization, allowing Pd(0) to convert the non-fluorescent molecules to fluorescent molecules. The limit and range of detection of anti-HSA antibodies were 3.8 nM and 3.8 nM to 133.3 nM, respectively. The limit and range of detection of HIV gag were 1.6 nM and 1.6 nM to 15 nM, respectively. This simple, inexpensive, "add sample and measure" diagnostic assay could potentially be incorporated into point of care diagnostics for low-resource settings.
Asunto(s)
Infecciones por VIH , Nanopartículas del Metal , Nanopartículas , Humanos , Paladio , Dióxido de Silicio , ADNRESUMEN
Enteropathogenic bacterial infections are a global health issue associated with high mortality, particularly in developing countries. Efficient host protection against enteropathogenic bacterial infection is characterized by coordinated responses between immune and nonimmune cells. In response to infection in mice, innate immune cells are activated to produce interleukin (IL)-23 and IL-22, which promote antimicrobial peptide (AMP) production and bacterial clearance. IL-36 cytokines are proinflammatory IL-1 superfamily members, yet their role in enteropathogenic bacterial infection remains poorly defined. Using the enteric mouse pathogen, C.rodentium, we demonstrate that signaling via IL-36 receptor (IL-36R) orchestrates a crucial innate-adaptive immune link to control bacterial infection. IL-36R-deficient mice (Il1rl2-/- ) exhibited significant impairment in expression of IL-22 and AMPs, increased intestinal damage, and failed to contain C. rodentium compared to controls. These defects were associated with failure to induce IL-23 and IL-6, two key IL-22 inducers in the early and late phases of infection, respectively. Treatment of Il1rl2-/- mice with IL-23 during the early phase of C. rodentium infection rescued IL-22 production from group 3 innate lymphoid cells (ILCs), whereas IL-6 administration during the late phase rescued IL-22-mediated production from CD4+ T cell, and both treatments protected Il1rl2-/- mice from uncontained infection. Furthermore, IL-36R-mediated IL-22 production by CD4+ T cells was dependent upon NFκB-p65 and IL-6 expression in dendritic cells (DCs), as well as aryl hydrocarbon receptor (AhR) expression by CD4+ T cells. Collectively, these data demonstrate that the IL-36 signaling pathway integrates innate and adaptive immunity leading to host defense against enteropathogenic bacterial infection.
Asunto(s)
Inmunidad Adaptativa , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Inmunidad Innata , Receptores de Interleucina-1/metabolismo , Animales , Citrobacter rodentium/patogenicidad , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/microbiología , Interleucina-1/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Noqueados , Receptores de Interleucina-1/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Inflammatory bowel disease (IBD) is characterized as chronic inflammation in the gastrointestinal tract, which includes two main subtypes, Crohn's disease and ulcerative colitis. Endoscopy combined with biopsy is the most effective way to establish IBD diagnosis and disease management. Imaging techniques have also been developed to monitor IBD. Although effective, the methods are expensive and invasive, which leads to pain and discomfort. Alternative noninvasive biomarkers are being explored as tools for IBD prognosis and disease management. This review focuses on novel biomarkers that have emerged in recent years. These serological biomarkers and microRNAs could potentially be used for disease management in IBD, thereby decreasing patient discomfort and morbidity.
Asunto(s)
Colitis Ulcerosa , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Biomarcadores , Colitis Ulcerosa/diagnóstico , Endoscopía Gastrointestinal , Humanos , Enfermedades Inflamatorias del Intestino/diagnósticoRESUMEN
Genetic knockout (KO) of peptide transporter-1 (PepT1) protein is known to provide resistance to acute colitis and colitis-associated cancer (CAC) in mouse models. However, it was unclear which molecule(s) or pathway(s) formed the basis for these protective effects. Recently, we demonstrated that the PepT1-/- microbiota is sufficient to protect against colitis and CAC. Given that PepT1 KO alters the gut microbiome and thereby changes the intestinal metabolites that are ultimately reflected in the feces, we investigated the fecal metabolites of our PepT1 KO mice. Using a liquid chromatography-mass spectrometry (LC-MS)-based untargeted-metabolomics technique, we found that the fecal metabolites were significantly different between the KO and normal wild-type (WT) mice. Among the altered fecal metabolites, tuberonic acid (TA) was sevenfold higher in KO mouse feces than in WT mouse feces. Accordingly, we studied whether the increased TA could direct an anti-inflammatory effect. Using in vitro models, we discovered that TA not only prevented lipopolysaccharide (LPS)-induced inflammation in macrophages but also improved the epithelial cell healing processes. Our results suggest that TA, and possibly other fecal metabolites, play a crucial role in the pathway(s) associated with the anticolitis effects of PepT1 KO.NEW & NOTEWORTHY Fecal metabolites were significantly different between the KO and normal wild-type (WT) mice. One fecal metabolite, tuberonic acid (TA), was sevenfold higher in KO mouse feces than in WT mouse feces. TA prevented lipopolysaccharide (LPS)-induced inflammation in macrophages and improved the epithelial cell healing process.
Asunto(s)
Microbioma Gastrointestinal/fisiología , Mucosa Intestinal/metabolismo , Metaboloma/fisiología , Transportador de Péptidos 1/metabolismo , Cicatrización de Heridas/fisiología , Animales , Modelos Animales de Enfermedad , Inflamación/metabolismo , Mucosa Intestinal/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Metabolómica , Ratones , Ratones Noqueados , Transportador de Péptidos 1/genéticaRESUMEN
The gut epithelium acts to separate host immune cells from unrestricted interactions with the microbiota and other environmental stimuli. In response to epithelial damage or dysfunction, immune cells are activated to produce interleukin (IL)-22, which is involved in repair and protection of barrier surfaces. However, the specific pathways leading to IL-22 and associated antimicrobial peptide (AMP) production in response to intestinal tissue damage remain incompletely understood. Here, we define a critical IL-36/IL-23/IL-22 cytokine network that is instrumental for AMP production and host defense. Using a murine model of intestinal damage and repair, we show that IL-36γ is a potent inducer of IL-23 both in vitro and in vivo. IL-36γ-induced IL-23 required Notch2-dependent (CD11b+CD103+) dendritic cells (DCs), but not Batf3-dependent (CD11b-CD103+) DCs or CSF1R-dependent macrophages. The intracellular signaling cascade linking IL-36 receptor (IL-36R) to IL-23 production by DCs involved MyD88 and the NF-κB subunits c-Rel and p50. Consistent with in vitro observations, IL-36R- and IL-36γ-deficient mice exhibited dramatically reduced IL-23, IL-22, and AMP levels, and consequently failed to recover from acute intestinal damage. Interestingly, impaired recovery of mice deficient in IL-36R or IL-36γ could be rescued by treatment with exogenous IL-23. This recovery was accompanied by a restoration of IL-22 and AMP expression in the colon. Collectively, these data define a cytokine network involving IL-36γ, IL-23, and IL-22 that is activated in response to intestinal barrier damage and involved in providing critical host defense.
Asunto(s)
Inmunidad Innata/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Interleucinas/inmunología , Cicatrización de Heridas/inmunología , Animales , Enfermedades Inflamatorias del Intestino/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Ratones , Ratones TransgénicosRESUMEN
Nanoparticles (NPs) have been utilized to deliver drugs to the intestinal epithelium in vivo. Moreover, NPs derived from edible plants are less toxic than synthetic NPs. Here, we utilized ginger NP-derived lipid vectors (GDLVs) in a proof-of-concept investigation to test the hypothesis that inhibiting expression of divalent metal-ion transporter 1 (Dmt1) would attenuate iron loading in a mouse model of hereditary hemochromatosis (HH). Initial experiments using duodenal epithelial organ cultures from intestine-specific Dmt1 knockout (KO) (Dmt1int/int) mice in the Ussing chamber established that Dmt1 is the only active iron importer during iron-deficiency anemia. Further, when Dmt1int/int mice were crossed with mice lacking the iron-regulatory hormone, hepcidin (Hepc-/-), iron loading was abolished. Hence, intestinal Dmt1 is required for the excessive iron absorption that typifies HH. Additional experiments established a protocol to produce GDLVs carrying functional Dmt1 small interfering RNAs (siRNAs) and to target these gene delivery vehicles to the duodenal epithelium in vivo (by incorporating folic acid [FA]). When FA-GDLVs carrying Dmt1 siRNA were administered to weanling Hepc-/- mice for 16 days, intestinal Dmt1 mRNA expression was attenuated and tissue iron accumulation was blunted. Oral delivery of functional siRNAs by FA-GDLVs is a suitable therapeutic approach to mitigate iron loading in murine HH.
Asunto(s)
Hemocromatosis/metabolismo , Hepcidinas/metabolismo , Nanopartículas/química , Factores de Transcripción/metabolismo , Zingiber officinale , Animales , Femenino , Células HEK293 , Hemocromatosis/genética , Hepcidinas/genética , Humanos , Hierro/metabolismo , Hierro de la Dieta , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/genéticaRESUMEN
Overcoming adverse effects and selectively delivering drug to target cells are two major challenges in the treatment of ulcerative colitis (UC). Lysine-proline-valine (KPV), a naturally occurring tripeptide, has been shown to attenuate the inflammatory responses of colonic cells. Here, we loaded KPV into hyaluronic acid (HA)-functionalized polymeric nanoparticles (NPs). The resultant HA-KPV-NPs had a desirable particle size (â¼272.3 nm) and a slightly negative zeta potential (â¼-5.3 mV). These NPs successfully mediated the targeted delivery of KPV to key UC therapy-related cells (colonic epithelial cells and macrophages). In addition, these KPV-loaded NPs appear to be nontoxic and biocompatible with intestinal cells. Intriguingly, we found that HA-KPV-NPs exert combined effects against UC by both accelerating mucosal healing and alleviating inflammation. Oral administration of HA-KPV-NPs encapsulated in a hydrogel (chitosan/alginate) exhibited a much stronger capacity to prevent mucosa damage and downregulate TNF-α, thus they showed a much better therapeutic efficacy against UC in a mouse model, compared with a KPV-NP/hydrogel system. These results collectively demonstrate that our HA-KPV-NP/hydrogel system has the capacity to release HA-KPV-NPs in the colonic lumen and that these NPs subsequently penetrate into colitis tissues and enable KPV to be internalized into target cells, thereby alleviating UC.
Asunto(s)
Antiinflamatorios/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Portadores de Fármacos , Ácido Hialurónico/química , Hormonas Estimuladoras de los Melanocitos/farmacología , Nanopartículas/química , Fragmentos de Péptidos/farmacología , Administración Oral , Alginatos/química , Animales , Antiinflamatorios/química , Antiinflamatorios/metabolismo , Quitosano/química , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Composición de Medicamentos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Expresión Génica , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Humanos , Hidrogeles/química , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Hormonas Estimuladoras de los Melanocitos/química , Hormonas Estimuladoras de los Melanocitos/metabolismo , Ratones , Terapia Molecular Dirigida , Nanopartículas/administración & dosificación , Tamaño de la Partícula , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Células RAW 264.7 , Dodecil Sulfato de Sodio , Electricidad Estática , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The use of nanotechnology for drug delivery has shown great promise for improving cancer treatment. However, potential toxicity, hazardous environmental effects, issues with large-scale production, and potential excessive costs are challenges that confront their further clinical applications. Here, we describe a nanovector made from ginger-derived lipids that can serve as a delivery platform for the therapeutic agent doxorubicin (Dox) to treat colon cancer. We created nanoparticles from ginger and reassembled their lipids into ginger-derived nanovectors (GDNVs). A subsequent characterization showed that GDNVs were efficiently taken up by colon cancer cells. Viability and apoptosis assays and electric cell-substrate impedance-sensing technology revealed that GDNVs exhibited excellent biocompatibility up to 200 µmol/l; by contrast, cationic liposomes at the same concentrations decreased cell proliferation and increased apoptosis. GDNVs were capable of loading Dox with high efficiency and showed a better pH-dependent drug-release profile than commercially available liposomal-Dox. Modified GDNVs conjugated with the targeting ligand folic acid mediated targeted delivery of Dox to Colon-26 tumors in vivo and enhanced the chemotherapeutic inhibition of tumor growth compared with free drug. Current experiments explore the feasibility of producing nature-derived nanoparticles that are effective as a treatment vehicle while potentially attenuating the issues related to traditional synthetic nanoparticles.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Doxorrubicina/administración & dosificación , Lípidos/química , Nanopartículas/química , Zingiber officinale/química , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/química , Doxorrubicina/farmacología , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Células HT29 , Humanos , Ratones , Nanopartículas/administración & dosificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.
Asunto(s)
Proteínas Bacterianas/inmunología , Farmacorresistencia Bacteriana/inmunología , Francisella/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Evasión Inmune/inmunología , Inflamasomas/inmunología , Lipoproteínas/inmunología , Animales , Membrana Celular/genética , Membrana Celular/inmunología , Farmacorresistencia Bacteriana/genética , Francisella/genética , Infecciones por Bacterias Gramnegativas/genética , Evasión Inmune/genética , Inflamasomas/genética , Secuencias Invertidas Repetidas/inmunología , Lipoproteínas/genética , Ratones , Ratones NoqueadosRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) protects against high-fat diet (HFD)-induced hepatic steatosis in mice, however, the mechanisms involved are not known. In this study we investigated the effects of GDNF overexpression and nanoparticle delivery of GDNF in mice on hepatic steatosis and fibrosis and the expression of genes involved in the regulation of hepatic lipid uptake and de novo lipogenesis. Transgenic overexpression of GDNF in liver and other metabolically active tissues was protective against HFD-induced hepatic steatosis. Mice overexpressing GDNF had significantly reduced P62/sequestosome 1 protein levels suggestive of accelerated autophagic clearance. They also had significantly reduced peroxisome proliferator-activated receptor-γ (PPAR-γ) and CD36 gene expression and protein levels, and lower expression of mRNA coding for enzymes involved in de novo lipogenesis. GDNF-loaded nanoparticles were protective against short-term HFD-induced hepatic steatosis and attenuated liver fibrosis in mice with long-standing HFD-induced hepatic steatosis. They also suppressed the liver expression of steatosis-associated genes. In vitro, GDNF suppressed triglyceride accumulation in Hep G2 cells through enhanced p38 mitogen-activated protein kinase-dependent signaling and inhibition of PPAR-γ gene promoter activity. These results show that GDNF acts directly in the liver to protect against HFD-induced cellular stress and that GDNF may have a role in the treatment of nonalcoholic fatty liver disease.
Asunto(s)
Dieta Alta en Grasa , Hígado Graso/metabolismo , Hígado Graso/prevención & control , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Hígado/metabolismo , PPAR gamma/metabolismo , Animales , Antígenos CD36/genética , Antígenos CD36/metabolismo , Hígado Graso/etiología , Hígado Graso/patología , Factor Neurotrófico Derivado de la Línea Celular Glial/administración & dosificación , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/uso terapéutico , Células Hep G2 , Humanos , Hígado/patología , Ratones , Ratones Transgénicos , Nanopartículas/administración & dosificación , Nanopartículas/uso terapéutico , PPAR gamma/genética , Transducción de Señal/fisiología , Triglicéridos/metabolismoRESUMEN
BACKGROUND & AIMS: Nanoparticles have been explored as carriers of small interfering RNAs (siRNAs) and might be developed to treat patients with inflammatory bowel disease (IBD). Overexpression of CD98 on the surface of colonic epithelial cells and macrophages promotes the development and progression of IBD. We developed an orally delivered hydrogel that releases nanoparticles with single-chain CD98 antibodies on their surface (scCD98 functionalized) and loaded with CD98 siRNA (siCD98). We tested the ability of the nanoparticles to reduce levels of CD98 in the colons of mice with colitis. METHODS: scCD98-functionalized siCD98-loaded nanoparticles were fabricated using a complex coacervation technique. We investigated the cellular uptake and lysosome escape profiles of the nanoparticles in Colon-26 cells and RAW 264.7 macrophages using fluorescence microscopy. Colitis was induced by transfer of CD4(+)CD45RB(high) T cells to Rag(-/-) mice or administration of dextran sodium sulfate to C57BL/6 mice. Mice were then given hydrogel (chitosan and alginate) containing scCD98-functionalized nanoparticles loaded with siCD98 or scrambled siRNA (control) via gavage. RESULTS: The scCD98-functionalized nanoparticles were approximately 200 nm in size and had high affinity for CD98-overexpressing cells. The scCD98-functionalized siCD98-loaded nanoparticles significantly reduced levels of CD98 in Colon-26 cells and RAW 264.7 macrophages, along with production of inflammatory cytokines (tumor necrosis factor α, interleukin-6, and interleukin-12). In mice with colitis, administration of the scCD98-functionalized siCD98-loaded nanoparticles reduced colon expression of CD98. Importantly, the severity of colitis was also reduced compared with controls (based on loss of body weight, myeloperoxidase activity, inflammatory cytokine production, and histological analysis). Approximately 24.1% of colonic macrophages (CD11b(+)CD11c(-)F4/80(+)) in the mice had taken up fluorescently labeled siRNA-loaded nanoparticles within 12 hours of administration. CONCLUSIONS: Nanoparticles containing surface CD98 antibody and loaded with siCD98 reduce expression of this protein by colonic epithelial cells and macrophages, and oral administration decreases the severity of colitis in mice. This nanoparticle in hydrogel (chitosan/alginate) formulation might be developed to treat patients with IBD.
Asunto(s)
Colitis/prevención & control , Colon/metabolismo , Proteína-1 Reguladora de Fusión/genética , Proteína-1 Reguladora de Fusión/inmunología , Terapia Genética/métodos , Nanomedicina/métodos , Nanopartículas , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Anticuerpos de Cadena Única/administración & dosificación , Administración Oral , Alginatos/química , Animales , Línea Celular , Quitosano/química , Colitis/inducido químicamente , Colitis/genética , Colitis/inmunología , Colitis/metabolismo , Colitis/patología , Colon/inmunología , Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hidrogeles , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/metabolismo , Índice de Severidad de la Enfermedad , Factores de Tiempo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Better detections of circulating microRNAs (miRNAs) as disease biomarkers could advance diseases diagnosis and treatment. Current analysis methods or sensors for research and applications are challenged by the low concentrations and wide dynamic range (from aM to nM) of miRNAs in a physiological sample. Here, we report a one-step label-free electrochemical sensor comprising a triple-stem DNA-redox probe structure on a gold microelectrode. A new signal amplification mechanism without the need of a redox enzyme is introduced. The novel strategy overcomes the fundamental limitations of microelectrode DNA sensors that fail to generate detectable current, which is primarily due to the limited amount of redox probes in response to the target analyte binding. By employing a reductant, tris(2-carboxyethyl) phosphine hydrochloride (TCEP) in the detection buffer solution, each redox molecule on the detection probe is cyclically oxidized at the electrode and reduced by the reductant; thus, the signal is amplified in situ during the detection period. The combined merits in the diagnosis power of cyclic voltammetry and the high sensitivity of pulse voltammetry enable parallel analysis for method validation and optimization previously inaccessible. As such, the detection limit of miRNA-122 was 0.1 fM via direct readout, with a wide detection range from sub fM to nM. The detection time is within minutes, which is a significant improvement over other macroscopic sensors and other relevant techniques such as quantitative reverse transcription polymerase chain reaction (qRT-PCR). The high selectivity of the developed sensors is demonstrated by the discrimination against two most similar family sequences: miR-122-3p present in serum and 2-mismatch synthetic RNA sequence. Interference such as nonspecific adsorption, a common concern in sensor development, is reduced to a negligible amount by adopting a multistep surface modification strategy. Importantly, unlike qRT-PCR, the microelectrochemical sensor offers direct absolute quantitative readout that is amenable to clinical and in-home point-of-care (POC) applications. The sensor design is flexible, capable of being tailored for detection of different miRNAs of interest. Combined with the fact that the sensor was constructed at microscale, the method can be generalized for high throughput detection of miRNA signatures as disease biomarkers.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , MicroARNs/análisis , MicroARNs/química , Microelectrodos , Procesamiento de Señales Asistido por ComputadorRESUMEN
Intestinal CD98 expression plays a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in intestinal cells therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as inflammatory bowel disease. Here, the advantages of nanoparticles (NPs) are used, including their ability to easily pass through physiological barriers and evade phagocytosis, high loading concentration, rapid kinetics of mixing and resistance to degradation. Using physical chemistry characterizations techniques, CD98 siRNA/polyethyleneimine (PEI)-loaded NPs was characterized (diameter of ~480 nm and a zeta potential of -5.26 mV). Interestingly, CD98 siRNA can be electrostatically complexed by PEI and thus protected from RNase. In addition, CD98 siRNA/PEI-loaded NPs are nontoxic and biocompatible with intestinal cells. Oral administration of CD98/PEI-loaded NPs encapsulated in a hydrogel reduced CD98 expression in mouse colonic tissues and decreased dextran sodium sulfate-induced colitis in a mouse model. Finally, flow cytometry showed that CD98 was effectively downregulated in the intestinal epithelial cells and intestinal macrophages of treated mice. Finally, the results collectively demonstrated the therapeutic effect of "hierarchical nano-micro particles" with colon-homing capabilities and the ability to directly release "molecularly specific" CD98 siRNA in colonic cells, thereby decreasing colitis.
Asunto(s)
Proteína-1 Reguladora de Fusión/química , Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Nanopartículas/química , Polietileneimina/química , ARN Interferente Pequeño/química , Animales , Línea Celular , Supervivencia Celular , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Células Dendríticas/metabolismo , Sulfato de Dextran/efectos adversos , Proteína-1 Reguladora de Fusión/genética , Técnicas de Transferencia de Gen , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/patología , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Intestinos/efectos de los fármacos , Intestinos/patología , Macrófagos/metabolismo , Ratones , Nanopartículas/administración & dosificación , Nanopartículas/ultraestructura , ARN Interferente Pequeño/genéticaRESUMEN
Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal (GI) tract associated with an increased risk of colorectal cancer (CRC). Current treatments for both IBD and colitis-associated CRC suffer from numerous side effects. Parthenolide (PTL) is a sesquiterpene lactone with anti-inflammatory activity, and previous studies have demonstrated that PTL is a potent inhibitor of the NF-κB pathway. Micheliolide (MCL), substantially more stable than PTL in vivo, was recently developed, and this study aimed to decipher its suitability as therapeutic tool for IBD and IBD-associated diseases. Similar to PTL, MCL inhibited NF-κB activation and subsequent pro-inflammatory pathways activation in vitro. Pro-drug forms of both compounds inhibited the DSS-induced colitis when administrated intraperitoneally or encapsulated in a polysaccharide gel designed to release drugs in the colon. Interestingly, MCL was found to attenuate carcinogenesis in AOM/DSS-induced CRC, thus providing new candidate for the treatment of inflammatory bowel disease and CRC.
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Colitis/prevención & control , Neoplasias Colorrectales/prevención & control , FN-kappa B/antagonistas & inhibidores , Sesquiterpenos de Guayano/farmacología , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Colitis/complicaciones , Neoplasias Colorrectales/complicaciones , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Lipopolisacáridos/farmacología , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Animal models of inflammatory bowel disease (IBD) are valuable tools for investigating the factors involved in IBD pathogenesis and evaluating new therapeutic options. The dextran sodium sulfate (DSS)-induced model of colitis is arguably the most widely used animal model for studying the pathogenesis of and potential treatments for ulcerative colitis (UC), which is a primary form of IBD. This model offers several advantages as a research tool: it is highly reproducible, relatively easy to generate and maintain, and mimics many critical features of human IBD. Recently, it has also been used to study the role of gut microbiota in the development and progression of IBD and to investigate the effects of other factors, such as diet and genetics, on colitis severity. However, although DSS-induced colitis is the most popular and flexible model for preclinical IBD research, it is not an exact replica of human colitis, and some results obtained from this model cannot be directly applied to humans. This review aims to comprehensively discuss different factors that may be involved in the pathogenesis of DSS-induced colitis and the issues that should be considered when using this model for translational purposes.
This review discusses different factors that may be involved in the pathogenesis of DSS-induced colitis and the issues that should be considered when using this model for translational purposes.
Asunto(s)
Colitis , Sulfato de Dextran , Modelos Animales de Enfermedad , Sulfato de Dextran/toxicidad , Animales , Humanos , Colitis/inducido químicamente , Colitis/patología , Microbioma Gastrointestinal , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/microbiología , Enfermedades Inflamatorias del Intestino/microbiologíaRESUMEN
Here, we describe immunofluorescent (IF) staining assay of 3D cell culture colonoids isolated from mice colon as described previously. Primary cultures developed from isolated colonic stem cells are called colonoids. Immunofluorescence can be used to analyze the distribution of proteins, glycans, and small molecules-both biological and non-biological ones. Four-day-old colonoid cell cultures grown on Lab-Tek 8-well plate are fixed by paraformaldehyde. Fixed colonoids are then subjected to antigen retrieval and blocking followed by incubation with primary antibody. A corresponding secondary antibody tagged with desired fluorescence is used to visualize primary antibody-marked protein. Counter staining to stain actin filaments and nucleus to assess cell structure and DNA in nucleus is performed by choosing the other two contrasting fluorescences. IF staining of colonoids can be utilized to visualize molecular markers of cell behavior. This technique can be used for translation research by isolating colonoids from colitis patients' colons, monitoring the biomarkers, and customizing their treatments. Key features ⢠Analysis of molecular markers of cell behavior. ⢠Protocol to visualize proteins in 3D cell culture. ⢠This protocol requires colonoids isolated from mice colon grown on matrigel support. ⢠Protocol requires at least eight days to complete.
RESUMEN
Lipid nanoparticle (LNP)-based drug delivery systems (DDSs) are widely recognized for their ability to enhance efficient and precise delivery of therapeutic agents, including nucleic acids like DNA and mRNA. Despite this acknowledgment, there is a notable knowledge gap regarding the systemic biodistribution and organ accumulation of these nanoparticles. The ability to track LNPs in vivo is crucial for understanding their fate within biological systems. Fluorescent labeling of LNPs facilitates real-time tracking, quantification, and visualization of their behavior within biological systems, providing valuable insights into biodistribution, cellular uptake, and the optimization of drug delivery strategies. Our prior research established reversely engineered LNPs as an exceptional mRNA delivery platform for treating ulcerative colitis. This study presents a detailed protocol for labeling interleukin-22 (IL-22) mRNA-loaded LNPs, their oral administration to mice, and visualization of DiR-labeled LNPs biodistribution in the gastrointestinal tract using IVIS spectrum. This fluorescence-based approach will assist researchers in gaining a dynamic understanding of nanoparticle fate in other models of interest. Key features ⢠This protocol is developed to assess the delivery of IL-22 mRNA to ulcerative colitis sites using lipid nanoparticles. ⢠This protocol uses fluorescent DiR dye for imaging of IL-22 mRNA-loaded lipid nanoparticles in the gastrointestinal tract of mice. ⢠This protocol employs the IVIS spectrum for imaging.
RESUMEN
Inflammatory bowel disease (IBD), marked by chronic gastrointestinal tract inflammation, poses a significant global medical challenge. Current treatments for IBD, including corticosteroids, immunomodulators, and biologics, often require frequent systemic administration through parenteral delivery, leading to nonspecific drug distribution, suboptimal therapeutic outcomes, and adverse effects. There is a pressing need for a targeted drug delivery system to enhance drug efficacy and minimize its systemic impact. Nanotechnology emerges as a transformative solution, enabling precise oral drug delivery to inflamed intestinal tissues, reducing off-target effects, and enhancing therapeutic efficiency. The advantages include heightened bioavailability, sustained drug release, and improved cellular uptake. Additionally, the nano-based approach allows for the integration of theranostic elements, enabling simultaneous diagnosis and treatment. Recent preclinical advances in oral IBD treatments, particularly with nanoformulations such as functionalized polymeric and lipid nanoparticles, demonstrate remarkable cell-targeting ability and biosafety, promising to overcome the limitations of conventional therapies. These developments signify a paradigm shift toward personalized and effective oral IBD management. This review explores the potential of oral nanomedicine to enhance IBD treatment significantly, focusing specifically on cell-targeting oral drug delivery system for potential use in IBD management. We also examine emerging technologies such as theranostic nanoparticles and artificial intelligence, identifying avenues for the practical translation of nanomedicines into clinical applications.
Asunto(s)
Sistemas de Liberación de Medicamentos , Enfermedades Inflamatorias del Intestino , Nanomedicina , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Administración Oral , Nanomedicina/métodos , Animales , Nanopartículas/administración & dosificación , Nanopartículas/químicaRESUMEN
CD98 is a type II transmembrane glycoprotein whose expression increases in intestinal epithelial cells (IECs) during intestinal inflammation. Enteropathogenic Escherichia coli (EPEC) is a food-borne human pathogen that attaches to IECs and injects effector proteins directly into the host cells, thus provoking an inflammatory response. In the present study, we investigated CD98 and EPEC interactions in vitro and ex vivo and examined FVB wild-type (WT) and villin-CD98 transgenic mice overexpressing human CD98 in IECs (hCD98 Tg mice) and infected with Citrobacter rodentium as an in vivo model. In vivo studies indicated that CD98 overexpression, localized to the apical domain of colonic cells, increased the attachment of C. rodentium in mouse colons and resulted in increased expression of proinflammatory markers and decreased expression of anti-inflammatory markers. The proliferative markers Ki-67 and cyclin D1 were significantly increased in the colonic tissue of C. rodentium-infected hCD98 Tg mice compared to that of WT mice. Ex vivo studies correlate with the in vivo data. Small interfering RNA (siRNA) studies with Caco2-BBE cells showed a decrease in adherence of EPEC to Caco2 cells in which CD98 expression was knocked down. In vitro surface plasmon resonance (SPR) experiments showed direct binding between recombinant hCD98 and EPEC/C. rodentium proteins. We also demonstrated that the partial extracellular loop of hCD98 was sufficient for direct binding to EPEC/C. rodentium. These findings demonstrate the importance of the extracellular loop of CD98 in the innate host defense response to intestinal infection by attaching and effacing (A/E) pathogens.
Asunto(s)
Infecciones por Enterobacteriaceae/inmunología , Proteína-1 Reguladora de Fusión/metabolismo , Inmunidad Innata , Mucosa Intestinal/metabolismo , Animales , Células CACO-2 , Citrobacter rodentium , Colon , Infecciones por Enterobacteriaceae/microbiología , Escherichia coli Enteropatógena , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Proteína-1 Reguladora de Fusión/genética , Regulación de la Expresión Génica/inmunología , Humanos , Masculino , Ratones , Ratones Transgénicos , Peroxidasa , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
We and others have shown that the dipeptide cotransporter PepT1 is expressed in immune cells, including macrophages that are in close contact with the lamina propria of the small and large intestines. In the present study, we used PepT1-knockout (KO) mice to explore the role played by PepT1 in immune cells during dextran sodium sulfate (DSS)-induced colitis. DSS treatment caused less severe body weight loss, diminished rectal bleeding, and less diarrhea in PepT1-KO mice than in wild-type (WT) animals. A histological examination of colonic sections revealed that the colonic architecture was less disrupted and the extent of immune cell infiltration into the mucosa and submucosa following DSS treatment was reduced in PepT1-KO mice compared with WT animals. Consistent with these results, the DSS-induced colitis increase in colonic myeloperoxidase activity was significantly less in PepT1-KO mice than in WT littermates. The colonic levels of mRNAs encoding the inflammatory cytokines CXCL1, interleukin (IL)-6, monocyte chemotactic protein-1, IL-12, and interferon-γ were significantly lower in DSS-treated PepT1-KO mice than in DSS-treated WT animals. Colonic immune cells from WT had significantly higher level of proinflammatory cytokines then PepT1 KO. In addition, we observed that knocking down the PepT1 expression decreases chemotaxis of immune cells recruited during intestinal inflammation. Antibiotic treatment before DSS-induced colitis eliminated the differential expression of inflammatory cytokines between WT and PepT1-KO mice. In conclusion, PepT1 in immune cells regulates the secretion of proinflammatory cytokines triggered by bacteria and/or bacterial products, and thus has an important role in the induction of colitis. PepT1 may transport small bacterial products, such as muramyl dipeptide and the tripeptide L-Ala-gamma-D-Glu-meso-DAP, into macrophages. These materials may be sensed by members of the nucleotide-binding site-leucine-rich repeat family of intracellular receptors, ultimately resulting in altered homeostasis of the intestinal microbiota.