RESUMEN
The modern nanomedicine incorporates the multimodal treatments into a single formulation, offering innovative cancer therapy options. Nanosheets function as carriers, altering the solubility, biodistribution, and effectiveness of medicinal compounds, resulting in more efficient cancer treatments and reduced side effects. The non-toxic nature of fluorinated graphene oxide (FGO) nanosheets and their potential applications in medication delivery, medical diagnostics, and biomedicine distinguish them from others. Leveraging the unique properties of Lissachatina fulica snail mucus (LfSM), FGO nanosheets were developed to reveal the novel characteristics. Consequently, LfSM was utilized to create non-toxic, environmentally friendly, and long-lasting FGO nanosheets. Ultraviolet-visible (UV-vis) spectroscopy revealed a prominent absorbance peak at 235 nm. The characterization of the synthesized FGO nanosheets involved X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), high-resolution transmission electron microscopy (HR-TEM), and atomic force microscopy (AFM) analyses. The antimicrobial activity data demonstrated a broad spectrum of antibacterial effects against Escherichia coli, Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The cytotoxicity efficacy of LfSM-FGO nanosheets against pancreatic cancer cell line (PANC1) showed promising results at low concentrations. The study suggests that FGO nanosheets made from LfSM could serve as alternate factors for in biomedical applications in the future.
Asunto(s)
Grafito , Nanoestructuras , Caracoles , Grafito/química , Grafito/farmacología , Animales , Caracoles/química , Humanos , Nanoestructuras/química , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Pruebas de Sensibilidad Microbiana , Moco/química , Moco/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Halogenación , Supervivencia Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Línea Celular Tumoral , Tamaño de la PartículaRESUMEN
Antibiotic use is a risk factor for development of inflammatory bowel diseases (IBDs). IBDs are characterized by a damaged mucus layer, which does not separate the intestinal epithelium from the microbiota. Here, we hypothesized that antibiotics affect the integrity of the mucus barrier, which allows bacterial penetrance and predisposes to intestinal inflammation. We found that antibiotic treatment led to breakdown of the colonic mucus barrier and penetration of bacteria into the mucus layer. Using fecal microbiota transplant, RNA sequencing followed by machine learning, ex vivo mucus secretion measurements, and antibiotic treatment of germ-free mice, we determined that antibiotics induce endoplasmic reticulum stress in the colon that inhibits colonic mucus secretion in a microbiota-independent manner. This antibiotic-induced mucus secretion flaw led to penetration of bacteria into the colonic mucus layer, translocation of microbial antigens into circulation, and exacerbation of ulcerations in a mouse model of IBD. Thus, antibiotic use might predispose to intestinal inflammation by impeding mucus production.
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Antibacterianos , Colon , Microbioma Gastrointestinal , Mucosa Intestinal , Moco , Animales , Antibacterianos/farmacología , Antibacterianos/efectos adversos , Ratones , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Microbioma Gastrointestinal/efectos de los fármacos , Colon/metabolismo , Colon/efectos de los fármacos , Colon/patología , Colon/microbiología , Moco/metabolismo , Enfermedades Inflamatorias del Intestino/inducido químicamente , Enfermedades Inflamatorias del Intestino/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/microbiología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Modelos Animales de Enfermedad , Trasplante de Microbiota Fecal , Ratones Endogámicos C57BL , HumanosRESUMEN
Purpose: Effective mucosal delivery of drugs continues to pose a significant challenge owing to the formidable barrier presented by the respiratory tract mucus, which efficiently traps and clears foreign particulates. The surface characteristics of micelles dictate their ability to penetrate the respiratory tract mucus. In this study, polymeric micelles loaded with insulin (INS) were modified using mucus-penetrative polymers. Methods: We prepared and compared polyethylene glycol (PEG)-coated micelles with micelles where cell-penetrating peptide (CPP) is conjugated to PEG. Systematic investigations of the physicochemical and aerosolization properties, performance, in vitro release, mucus and cell penetration, lung function, and pharmacokinetics/pharmacodynamics (PK/PD) of polymeric micelles were performed to evaluate their interaction with the respiratory tract. Results: The nano-micelles, with a particle size of <100 nm, exhibited a sustained-release profile. Interestingly, PEG-coated micelles exhibited higher diffusion and deeper penetration across the mucus layer. In addition, CPP-modified micelles showed enhanced in vitro cell penetration. Finally, in the PK/PD studies, the micellar solution demonstrated higher maximum concentration (Cmax) and AUC0-8h values than subcutaneously administered INS solution, along with a sustained blood glucose-lowering effect that lasted for more than 8 h. Conclusion: This study proposes the use of mucus-penetrating micelle formulations as prospective inhalation nano-carriers capable of efficiently transporting peptides to the respiratory tract.
Asunto(s)
Péptidos de Penetración Celular , Insulina , Micelas , Polietilenglicoles , Insulina/administración & dosificación , Insulina/farmacocinética , Insulina/química , Polietilenglicoles/química , Polietilenglicoles/farmacocinética , Animales , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/farmacocinética , Humanos , Tamaño de la Partícula , Administración por Inhalación , Masculino , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Ratas Sprague-Dawley , Moco/química , Moco/metabolismo , Moco/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/efectos de los fármacos , Glucemia/efectos de los fármacos , Glucemia/análisisRESUMEN
OBJECTIVE: Aim: The purpose was to identify the morphological and functional features of the colonic mucus barrier in patients with symptomatic uncomplicated diverticular disease and acute uncomplicated diverticulitis. PATIENTS AND METHODS: Materials and Methods: In the research, three groups were formed. Group 1 included fragments of the mucous membrane of the large intestine, which were collected from 12 people during autopsies. The results of autopsies and histological examination of the material did not reveal any gastrointestinal pathology. Group 2 included biopsies of the mucous membrane of the large intestine from the area of the diverticulum of 34 patients with symptomatic uncomplicated diverticular disease. Group 3 included biopsies of the mucous membrane of the large intestine of 26 patients with acute uncomplicated diverticulitis. Histological (hematoxylin and eosin staining), histochemical (PAS reaction) and immunohistochemical (mouse monoclonal antibodies to Mucin 2 (MUC2) and Mucin 4 (MUC4)) staining methods were used. A morphometric study was also carried out. RESULTS: Results: In patients with diverticular disease, the authors identified disturbances in the morphofunctional state of the mucus barrier of the colon, the structure and function of goblet cells contained in its mucous membrane, characterized by a decrease in the thickness of the mucus layer covering the surface of the mucous membrane; a decrease in the size and number of goblet cells with a decrease in their mucus-producing ability; a change in the mucin profile, characterized by a violation of the content of MUC2 and MUC4. These changes were greatest in patients with acute uncomplicated diverticulitis compared with patients with symptomatic uncomplicated diverticular disease. CONCLUSION: Conclusions: The identified disturbances in the morphofunctional state of the mucus barrier of the colon, structural and functional changes in goblet cells may be one of the mechanisms for the development of acute uncomplicated diverticulitis and symptomatic uncomplicated diverticular disease.
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Mucosa Intestinal , Humanos , Masculino , Femenino , Mucosa Intestinal/patología , Mucosa Intestinal/metabolismo , Persona de Mediana Edad , Anciano , Moco/metabolismo , Colon/patología , Colon/metabolismo , Diverticulitis del Colon/patología , Diverticulitis del Colon/metabolismo , Enfermedad Aguda , Adulto , Mucina 2/metabolismo , Células Caliciformes/patología , Células Caliciformes/metabolismoRESUMEN
Oral administration of peptide represents a promising delivery route, however, it is hindered by the harsh gastrointestinal environment, leading to low in vivo absorption. In this study, auto-adaptive protein corona-AT 1002-cationic liposomes (Pc-AT-CLs) are constructed with the characteristic of hydrophilic and electrically neutral surface properties for the encapsulation of liraglutide. BSA protein corona is used to coat AT-CLs reducing the adherence of mucus, and may fall off after penetrating the mucus layer. Transmucus transport experiment demonstrated that the mucus penetration amount of Pc-AT-CLs are 1.45 times that of AT-CLs. After penetrating the mucus layer, AT-CLs complete transmembrane transport by the dual action of AT and cationic surface properties. Transmembrane transport experiment demonstrated that the apparent permeability coefficient (Papp) of AT-CLs is 2.03 times that of CLs. In vivo tests demonstrated that Pc-AT-CLs exhibited a significant hypoglycemic effect and enhanced the relative bioavailability comparing to free liraglutide. Pc-AT-CLs protect liraglutide from degradation, facilitate its absorption, and ultimately improve its oral bioavailability.
Asunto(s)
Sistemas de Liberación de Medicamentos , Hipoglucemiantes , Liposomas , Liraglutida , Moco , Animales , Liraglutida/administración & dosificación , Liraglutida/farmacocinética , Liraglutida/farmacología , Moco/metabolismo , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/química , Humanos , Disponibilidad Biológica , Administración Oral , Masculino , Ratas Sprague-Dawley , Ratas , Absorción Intestinal/efectos de los fármacosRESUMEN
The mucus serves as a protective barrier in the gastrointestinal tract against microbial attacks. While its role extends beyond merely being a physical barrier, the extent of its active bactericidal properties remains unclear, and the mechanisms regulating these properties are not yet understood. We propose that inflammation induces epithelial cells to secrete antimicrobial peptides, transforming mucus into an active bactericidal agent. To investigate the properties of mucus, we previously developed mucosoid culture models that mimic the healthy human stomach epithelium. Similar to organoids, mucosoids are stem cell-driven cultures; however, the cells are cultivated on transwells at air-liquid interface. The epithelial cells of mucosoids form a polarized monolayer, allowing differentiation into all stomach lineages, including mucus-secreting cells. This setup facilitates the secretion and accumulation of mucus on the apical side of the mucosoids, enabling analysis of its bactericidal effects and protein composition, including antimicrobial peptides. Our findings show that TNFα, IL1ß, and IFNγ induce the secretion of antimicrobials such as lactotransferrin, lipocalin2, complement component 3, and CXCL9 into the mucus. This antimicrobial-enriched mucus can partially eliminate Helicobacter pylori, a key stomach pathogen. The bactericidal activity depends on the concentration of each antimicrobial and their gene expression is higher in patients with inflammation and H.pylori-associated chronic gastritis. However, we also find that H. pylori infection can reduce the expression of antimicrobial encoding genes promoted by inflammation. These findings suggest that controlling antimicrobial secretion in the mucus is a critical component of epithelial immunity. However, pathogens like H. pylori can overcome these defenses and survive in the mucosa.
Asunto(s)
Péptidos Antimicrobianos , Mucosa Gástrica , Helicobacter pylori , Inflamación , Moco , Humanos , Moco/metabolismo , Moco/microbiología , Péptidos Antimicrobianos/metabolismo , Mucosa Gástrica/microbiología , Mucosa Gástrica/metabolismo , Mucosa Gástrica/inmunología , Inflamación/metabolismo , Células Epiteliales/microbiología , Células Epiteliales/metabolismo , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/metabolismo , Infecciones por Helicobacter/inmunología , Estómago/microbiología , Organoides/metabolismo , Organoides/microbiologíaRESUMEN
Colibactin is a recently characterized pro-carcinogenic genotoxin produced by pks+ Escherichia coli. We hypothesized that cystic fibrosis (CF)-associated dysfunctional mucus structure increases the vulnerability of host mucosa to colibactin-induced DNA damage. In this pilot study, we tested healthy-appearing mucosal biopsy samples obtained during screening and surveillance colonoscopies of adult CF and non-CF patients for the presence of pks+ E. coli, and we investigated the possibility of detecting a novel colibactin-specific DNA adduct that has not been yet been demonstrated in humans. While CF patients had a lower incidence of pks+ E. coli carriage (~8% vs 29%, p = 0.0015), colibactin-induced DNA adduct formation was detected, but only in CF patients and only in those who were not taking CFTR modulator medications. Moreover, the only patient found to have colon cancer during this study had CF, harbored pks+ E. coli, and had colibactin-induced DNA adducts in the mucosal samples. Larger studies with longitudinal follow-up should be done to extend these initial results and further support the development of colibactin-derived DNA adducts to stratify patients and their risk.
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Colon , Fibrosis Quística , Aductos de ADN , Escherichia coli , Mucosa Intestinal , Moco , Péptidos , Policétidos , Fibrosis Quística/microbiología , Fibrosis Quística/metabolismo , Humanos , Policétidos/metabolismo , Aductos de ADN/metabolismo , Adulto , Escherichia coli/genética , Escherichia coli/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Péptidos/metabolismo , Masculino , Colon/microbiología , Colon/patología , Colon/metabolismo , Femenino , Proyectos Piloto , Moco/metabolismo , Moco/microbiología , Persona de Mediana Edad , Adulto Joven , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patologíaRESUMEN
Dendritic cells (DCs) are pivotal in regulating allergic asthma. Our research has shown that the absence of Sema3E worsens asthma symptoms in acute and chronic asthma models. However, the specific role of PlexinD1 in these processes, particularly in DCs, remains unclear. This study investigates the role of PlexinD1 in CD11c+ DCs using a house dust mite (HDM) model of asthma. We generated CD11c+ DC-specific PlexinD1 knockout (CD11cPLXND1 KO) mice and subjected them, alongside wild-type controls (PLXND1fl/fl), to an HDM allergen protocol. Airway hyperresponsiveness (AHR) was measured using FlexiVent, and immune cell populations were analyzed via flow cytometry. Cytokine levels and immunoglobulin concentrations were assessed using mesoscale and ELISA, while collagen deposition and mucus production were examined through Sirius-red and periodic acid Schiff (PAS) staining respectively. Our results indicate that CD11cPLXND1 KO mice exhibit significantly exacerbated AHR, characterized by increased airway resistance and tissue elastance. Enhanced mucus production and collagen gene expression were observed in these mice compared to wild-type counterparts. Flow cytometry revealed higher CD11c+ MHCIIhigh CD11b+ cell recruitment into the lungs, and elevated total and HDM-specific serum IgE levels in CD11cPLXND1 KO mice. Mechanistically, co-cultures of B cells with DCs from CD11cPLXND1 KO mice showed significantly increased IgE production compared to wild-type mice.These findings highlight the critical regulatory role of the plexinD1 signaling pathway in CD11c+ DCs in modulating asthma features.
Asunto(s)
Asma , Antígeno CD11c , Células Dendríticas , Modelos Animales de Enfermedad , Inmunoglobulina E , Ratones Noqueados , Moco , Animales , Asma/inmunología , Asma/metabolismo , Asma/patología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Ratones , Antígeno CD11c/metabolismo , Moco/metabolismo , Pyroglyphidae/inmunología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismo , Alérgenos/inmunología , Ratones Endogámicos C57BL , Glicoproteínas de Membrana , Péptidos y Proteínas de Señalización IntracelularRESUMEN
Current prophylactic and disease control measures in aquaculture highlight the need of alternative strategies to prevent disease and reduce antibiotic use. Mucus covered mucosal surfaces are the first barriers pathogens encounter. Mucus, which is mainly composed of highly glycosylated mucins, has the potential to contribute to disease prevention if we can strengthen this barrier. Therefore, aim of this study was to develop and characterize fish in vitro mucosal surface models based on commercially available cell lines that are functionally relevant for studies on mucin regulation and host-pathogen interactions. The rainbow trout (Oncorhynchus mykiss) gill epithelial cell line RTgill-W1 and the embryonic cell line from Chinook salmon (Oncorhynchus tshawytscha) CHSE-214 were grown on polycarbonate membrane inserts and chemically treated to differentiate the cells into mucus producing cells. RTGill-W1 and CHSE-214 formed an adherent layer at two weeks post-confluence, which further responded to treatment with the γ-secretase inhibitor DAPT and prolonged culture by increasing the mucin production. Mucins were metabolically labelled with N-azidoacetylgalactosamine 6 h post addition to the in vitro membranes. The level of incorporated label was relatively similar between membranes based on RTgill-W1, while larger interindividual variation was observed among the CHSE in vitro membranes. Furthermore, O-glycomics of RTgill-W1 cell lysates identified three sialylated O-glycans, namely Galß1-3(NeuAcα2-6)GalNAcol, NeuAcα-Galß1-3GalNAcol and NeuAcα-Galß1-3(NeuAcα2-6)GalNAcol, resembling the glycosylation present in rainbow trout gill mucin. These glycans were also present in CHSE-214. Additionally, we demonstrated binding of the fish pathogen A. salmonicida to RTgill-W1 and CHSE-214 cell lysates. Thus, these models have similarities to in vivo mucosal surfaces and can be used to investigate the effect of pathogens and modulatory components on mucin production.
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Interacciones Huésped-Patógeno , Mucinas , Oncorhynchus mykiss , Animales , Mucinas/metabolismo , Oncorhynchus mykiss/metabolismo , Línea Celular , Membrana Mucosa/metabolismo , Salmón/metabolismo , Branquias/metabolismo , Células Epiteliales/metabolismo , Moco/metabolismoAsunto(s)
Vesículas Extracelulares , Interleucina-4 , MicroARNs , Mucina 5AC , Moco , Mucina 5AC/metabolismo , Mucina 5AC/genética , Vesículas Extracelulares/metabolismo , Humanos , Moco/metabolismo , Interleucina-4/metabolismo , MicroARNs/metabolismo , MicroARNs/genética , Lavado Nasal (Proceso)/métodos , AnimalesRESUMEN
Endoscopic lung volume reduction (ELVR) is an established treatment option for patients with severe emphysema. Not all patients are candidates for this type of intervention, and in the context of significant airway secretions, they may be excluded from treatment. Bronchial Rheoplasty (BR) was developed to treat mucus hypersecretion by delivering nonthermal pulsed electric fields to the airway epithelium and submucosa. The literature to date demonstrates that patients treated with BR in clinical studies have a reduction in airway goblet cell hyperplasia as well as substantive clinical improvement in the setting of chronic bronchitis (CB). In this case series, we present four patients treated at three different institutions who had previously undergone ELVR with beneficial outcome. However, over time, these patients subsequently developed worsening clinical issues, including complaints of increased and thickened mucus, along with exacerbations in the setting of a loss of some ELVR-associated benefits. These patients then underwent exploratory treatment with BR with the intent of reducing their secretion burden and potentially restoring the efficacy associated with the initial placement of the airway valves. All BR procedures were well tolerated, and three of the four patients showed substantial improvement in their symptom burden. Airway examinations during the second of the two BR procedures also revealed what appeared to be less airway mucosal inflammation and a decrease in the quantity of airway secretions. Therefore, treatment with BR may have the potential to improve and restore the initial benefits associated with ELVR, thus enhancing long-term outcomes. Further clinical studies with sufficient follow-up are warranted to assess this in a larger cohort of patients, and to determine whether treatment with BR prior to ELVR may make more patients eligible for this treatment through reduction in their secretions and/or symptoms.
Asunto(s)
Broncoscopía , Pulmón , Neumonectomía , Enfisema Pulmonar , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Broncoscopía/métodos , Volumen Espiratorio Forzado , Pulmón/fisiopatología , Pulmón/cirugía , Moco/metabolismo , Neumonectomía/efectos adversos , Neumonectomía/métodos , Enfisema Pulmonar/cirugía , Enfisema Pulmonar/fisiopatología , Enfisema Pulmonar/diagnóstico , Recuperación de la Función , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Inhalation injuries arising from exposure to toxic gases or smoke in fires or industrial accidents pose grave risks and significant respiratory complications. The limited efficacy of current treatment strategies stems from challenges in delivering therapeutic agents across the mucus barrier to the damaged trachea and bronchus. This research explores the reparative potential and underlying mechanisms of sputum-penetrable magnetic nanoparticles (MNPs) coated with poly(N-isopropylacrylamide) (PNIPAM), combined with polyethylene glycol (PEG), and loaded with ambroxol hydrochloride (AH) (MNPs@PNIPAM-AH@PEG) as an innovative therapeutic approach for inhalation injuries. The PNIPAM coating, a thermo-responsive polymer, aims to enhance targeted drug release under an external stimulus. The PEG component is designed to mitigate hydrophobic repulsion and electrostatic forces, facilitating nanoagent penetration of the mucus barrier-an obstacle in inhalation injury treatment. PEG's hydrophilicity, combined with the magnetically attracted NPs, enables deep penetration through the mucus layer adhering to the mucus epithelium. Thermal effects break the outer thermal shell of MNPs, accelerating drug release, resolving sputum, and reducing inflammation. The results showed improved therapeutic impact by significantly reducing inflammation, enhancing mucociliary clearance, and promoting tissue repair. Moreover, the MNPs@PNIPAM-AH@PEG NPs showed good biocompatibility and biosafety both in vitro and in vivo. This research underscores the potential of MNPs@PNIPAM-AH@PEG NPs as a novel therapeutic strategy for inhalation injuries, paving the way for innovative treatments in emergency medicine and respiratory care.
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Resinas Acrílicas , Ambroxol , Moco , Polietilenglicoles , Animales , Polietilenglicoles/química , Resinas Acrílicas/química , Moco/metabolismo , Moco/efectos de los fármacos , Ambroxol/química , Ambroxol/administración & dosificación , Ambroxol/farmacología , Ratones , Liberación de Fármacos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/administración & dosificación , Masculino , HumanosRESUMEN
Increased disulfide crosslinking of secreted mucins causes elevated viscoelasticity of mucus and is a key determinant of mucus dysfunction in patients with cystic fibrosis (CF) and other muco-obstructive lung diseases. In this study, we describe the synthesis of a novel thiol-containing, sulfated dendritic polyglycerol (dPGS-SH), designed to chemically reduce these abnormal crosslinks, which we demonstrate with mucolytic activity assays in sputum from patients with CF. This mucolytic polymer, which is based on a reportedly anti-inflammatory polysulfate scaffold, additionally carries multiple thiol groups for mucolytic activity and can be produced on a gram-scale. After a physicochemical compound characterization, we compare the mucolytic activity of dPGS-SH to the clinically approved N-acetylcysteine (NAC) using western blot studies and investigate the effect of dPGS-SH on the viscoelastic properties of sputum samples from CF patients by oscillatory rheology. We show that dPGS-SH is more effective than NAC in reducing multimer intensity of the secreted mucins MUC5B and MUC5AC and demonstrate significant mucolytic activity by rheology. In addition, we provide data for dPGS-SH demonstrating a high compound stability, low cytotoxicity, and superior reaction kinetics over NAC at different pH levels. Our data support further development of the novel reducing polymer system dPGS-SH as a potential mucolytic to improve mucus function and clearance in patients with CF as well as other muco-obstructive lung diseases.
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Glicerol , Polímeros , Esputo , Compuestos de Sulfhidrilo , Humanos , Glicerol/química , Polímeros/química , Polímeros/farmacología , Esputo/metabolismo , Esputo/química , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacología , Fibrosis Quística/metabolismo , Fibrosis Quística/tratamiento farmacológico , Mucina 5AC/metabolismo , Enfermedades Pulmonares Obstructivas/tratamiento farmacológico , Enfermedades Pulmonares Obstructivas/metabolismo , Mucina 5B/metabolismo , Sulfatos/química , Sulfatos/farmacología , Expectorantes/farmacología , Expectorantes/química , Moco/metabolismo , Moco/química , Reología , Acetilcisteína/farmacología , Acetilcisteína/química , ViscosidadRESUMEN
Mucins secreted by mucous cells constitute a core part of the defense line against the invasion of pathogens. However, mucins' structure and immunological functions remain largely unknown in teleost fish. In this study, two typical mucins, Muc2 and Muc5ac of flounder (Paralichthys olivaceus), were cloned and their physicochemical properties, structure and conservation were analyzed. Notably, specific antibodies against flounder Muc2 and Muc5ac were developed. It was verified at the gene and protein level that Muc2 was expressed in the hindgut and gills but not in the skin, while Muc5ac was expressed in the skin and gills but not in the hindgut. After flounders were immunized by immersion with inactivated Edwardsiella tarda, Muc2 and Muc5ac were significantly upregulated at both the gene expression and protein levels, and Muc2+/Muc5ac+ mucous cells proliferated and increased secretion of Muc2 and Muc5ac. Moreover, Muc2 and Muc5ac exerted retention and clearance effects on E. tarda in a short period (within 1 dpi). These results revealed the characterization of fish mucins Muc2 and Muc5ac at the protein level and clarified the role of mucins as key guardians to maintain the mucus barrier, which advanced our understanding of teleost mucosal barrier.
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Edwardsiella tarda , Lenguado , Inmunidad Mucosa , Mucina 5AC , Mucina 2 , Animales , Lenguado/inmunología , Lenguado/microbiología , Lenguado/metabolismo , Mucina 2/metabolismo , Mucina 2/genética , Mucina 5AC/metabolismo , Mucina 5AC/genética , Edwardsiella tarda/inmunología , Proteínas de Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Secuencia de Aminoácidos , Branquias/metabolismo , Branquias/inmunología , Moco/metabolismo , Moco/inmunología , Clonación Molecular , FilogeniaRESUMEN
Recent evidence indicates that repeated antibiotic usage lowers microbial diversity and ultimately changes the gut microbiota community. However, the physiological effects of repeated - but not recent - antibiotic usage on microbiota-mediated mucosal barrier function are largely unknown. By selecting human individuals from the deeply phenotyped Estonian Microbiome Cohort (EstMB), we here utilized human-to-mouse fecal microbiota transplantation to explore long-term impacts of repeated antibiotic use on intestinal mucus function. While a healthy mucus layer protects the intestinal epithelium against infection and inflammation, using ex vivo mucus function analyses of viable colonic tissue explants, we show that microbiota from humans with a history of repeated antibiotic use causes reduced mucus growth rate and increased mucus penetrability compared to healthy controls in the transplanted mice. Moreover, shotgun metagenomic sequencing identified a significantly altered microbiota composition in the antibiotic-shaped microbial community, with known mucus-utilizing bacteria, including Akkermansia muciniphila and Bacteroides fragilis, dominating in the gut. The altered microbiota composition was further characterized by a distinct metabolite profile, which may be caused by differential mucus degradation capacity. Consequently, our proof-of-concept study suggests that long-term antibiotic use in humans can result in an altered microbial community that has reduced capacity to maintain proper mucus function in the gut.
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Antibacterianos , Bacterias , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal , Moco , Humanos , Microbioma Gastrointestinal/efectos de los fármacos , Animales , Antibacterianos/farmacología , Ratones , Moco/metabolismo , Moco/microbiología , Bacterias/clasificación , Bacterias/genética , Bacterias/efectos de los fármacos , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Mucosa Intestinal/microbiología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Masculino , Femenino , Heces/microbiología , Adulto , Persona de Mediana Edad , Akkermansia , Ratones Endogámicos C57BL , Colon/microbiología , Bacteroides fragilis/efectos de los fármacosRESUMEN
This study investigates Cystobasidium benthicum (Cb) probiotic yeast and Cyrtocarpa edulis (Ce) fruit dietary effects, single (0.5 %) or combined (Cb:Ce, 0.25:0.25 %), on growth performance, humoral immunity in serum and skin mucus, and intestinal morphology of Nile tilapia (Oreochromis niloticus) after 14 and 28 days. The Cb group presented the highest (P < 0.05) specific growth rate, weight gain, and absolute growth rate with respect to the control group. Immunological assays indicated that Cb, Ce and Cb:Ce groups increased serum nitric oxide concentration compared to the control group (P < 0.05). Cb and Cb:Ce groups showed the highest serum myeloperoxidase enzyme activity at day 14 and 28, respectively (P < 0.05); whereas, Cb:Ce group had the highest (P < 0.05) myeloperoxidase activity in skin mucus. The superoxide dismutase enzyme activity was unaffected. On day 28, Cb, Ce, and Cb:Ce groups showed higher and lower (P < 0.05) catalase enzyme activity in serum and skin mucus, respectively, compared with the control group. Only the Cb group had higher (P < 0.05) total protein concentration in serum (day 14) and skin mucus (day 14 and 28) with respect to the control group. The lysozyme activity in serum (day 28) and skin mucus (day 14) was higher (P < 0.05) in the Cb group compared to the control group. Only the skin mucus of Ce group showed bactericidal activity against Aeromonas dhakensis (P < 0.05). Histological studies indicated that Cb and Cb:Ce groups increased microvilli height, and Cb, Ce and Cb:Ce augmented goblet cell area at day 14 compared to the control group (P < 0.05). At day 28, microvilli height was higher in all groups and the number of intraepithelial leukocytes increased in Cb and Ce groups with respect to the control group (P < 0.05). The ex vivo assay revealed that A. dhakensis in leukocytes decreased cell viability similar to the control group (P < 0.05). A principal component analysis (PCA) confirmed the results. In conclusion, C. benthicum in the diet was the best supplement to improve the growth and immunity of Nile tilapia.
Asunto(s)
Alimentación Animal , Cíclidos , Dieta , Frutas , Probióticos , Animales , Probióticos/administración & dosificación , Cíclidos/crecimiento & desarrollo , Cíclidos/inmunología , Dieta/veterinaria , Peroxidasa/metabolismo , Óxido Nítrico/metabolismo , Intestinos/microbiología , Intestinos/inmunología , Piel , Inmunidad Humoral , Moco/metabolismo , Superóxido Dismutasa/metabolismo , Catalasa/metabolismoRESUMEN
Traditionally, developing inhaled drug formulations relied on trial and error, yet recent technological advancements have deepened the understanding of 'inhalation biopharmaceutics' i.e. the processes that occur to influence the rate and extent of drug exposure in the lungs. This knowledge has led to the development of new in vitro models that predict the in vivo behavior of drugs, facilitating the enhancement of existing formulation and the development of novel ones. Our prior research examined how simulated lung fluid (SLF) affects the solubility of inhaled drugs. Building on this, we aimed to explore drug dissolution and permeability in lung mucosa models containing mucus. Thus, the permeation of four active pharmaceutical ingredients (APIs), salbutamol sulphate (SS), tiotropium bromide (TioBr), formoterol fumarate (FF) and budesonide (BUD), was assayed in porcine mucus covered Calu-3 cell layers, cultivated at an air liquid interface (ALI) or submerged in a liquid covered (LC) culture system. Further analysis on BUD and FF involved their transport in a mucus-covered PAMPA system. Finally, their dissolution post-aerosolization from Symbicort® was compared using 'simple' Transwell and complex DissolvIt® apparatuses, alone or in presence of porcine mucus or polymer-lipid mucus simulant. The presence of porcine mucus impacted both permeability and dissolution of inhaled drugs. For instance, permeability of SS was reduced by a factor of ten in the Calu-3 ALI model while the permeability of BUD was reduced by factor of two in LC and ALI setups. The comparison of dissolution methodologies indicated that drug dissolution performance was highly dependent on the setup, observing decreased release efficiency and higher variability in Transwell system compared to DissolvIt®. Overall, results demonstrate that relatively simple methodologies can be used to discriminate between formulations in early phase drug product development. However, for more advanced stages complex methods are required. Crucially, it was clear that the impact of mucus and selection of its composition in in vitro testing of dissolution and permeability should not be neglected when developing drugs and formulations intended for inhalation.