RESUMO
BACKGROUND: Parkinson's disease (PD) is one of the most prevalent neurodegenerative diseases, following Alzheimer's disease. The onset of PD is characterized by the loss of dopaminergic neurons in the substantia nigra. Stem cell therapy has great potential for the treatment of neurodegenerative diseases, and human nasal turbinate-derived stem cells (hNTSCs) have been found to share some characteristics with mesenchymal stem cells. Although the Hippo signaling pathway was originally thought to regulate cell size in organs, recent studies have shown that it can also control inflammation in neural cells. METHODS: Dopaminergic neuron-like cells were differentiated from SH-SY5Y cells (DA-Like cells) and treated with 1-Methyl-4-phenylpyridinium iodide to stimulate Reactive oxidative species (ROS) production. A transwell assay was conducted to validate the effect of hNTSCs on the Hippo pathway. We generated an MPTP-induced PD mouse model and transplanted hNTSCs into the substantia nigra of PD mice via stereotaxic surgery. After five weeks of behavioral testing, the brain samples were validated by immunoblotting and immunostaining to confirm the niche control of hNTSCs. RESULTS: In-vitro experiments showed that hNTSCs significantly increased cell survival and exerted anti-inflammatory effects by controlling ROS-mediated ER stress and hippocampal signaling pathway factors. Similarly, the in-vivo experiments demonstrated an increase in anti-inflammatory effects and cell survival rate. After transplantation of hNTSCs, the PD mouse model showed improved mobility and relief from PD symptoms. CONCLUSION: hNTSCs improved the survival rate of dopaminergic neurons by manipulating the hippocampal pathway through Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding motif (TAZ) by reducing inflammatory cytokines. In this study, we found that controlling the niche of hNTSCs had a therapeutic effect on PD lesions.
Assuntos
Modelos Animais de Doenças , Via de Sinalização Hippo , Células-Tronco Neurais , Doença de Parkinson , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Conchas Nasais , Humanos , Animais , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Doença de Parkinson/terapia , Doença de Parkinson/metabolismo , Camundongos , Conchas Nasais/metabolismo , Neurônios Dopaminérgicos/metabolismo , Parte Compacta da Substância Negra/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Substância Negra/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Diferenciação CelularRESUMO
This study investigated the influence of hypoxic culture conditions on human nasal inferior turbinate-derived stem cells (hNTSCs), a subtype of mesenchymal stem cells (MSCs). It aimed to discern how hypoxia affected hNTSC characteristics, proliferation, and differentiation potential compared to hNTSCs cultured under normal oxygen levels. After obtaining hNTSCs from five patients, the samples were divided into hypoxic and normoxic groups. The investigation utilized fluorescence-activated cell sorting (FACS) for surface marker analysis, cell counting kit-8 assays for proliferation assessment, and multiplex immunoassays for cytokine secretion study. Differentiation potential-osteogenic, chondrogenic, and adipogenic-was evaluated via histological examination and gene expression analysis. Results indicated that hNTSCs under hypoxic conditions preserved their characteristic MSC phenotype, as confirmed by FACS analysis demonstrating the absence of hematopoietic markers and presence of MSC markers. Proliferation of hNTSCs remained unaffected by hypoxia. Cytokine expression showed similarity between hypoxic and normoxic groups throughout cultivation. Nevertheless, hypoxic conditions reduced the osteogenic and promoted adipogenic differentiation potential, while chondrogenic differentiation was relatively unchanged. These insights contribute to understanding hNTSC behavior in hypoxic environments, advancing the development of protocols for stem cell therapies and tissue engineering.
Assuntos
Células-Tronco Mesenquimais , Conchas Nasais , Humanos , Conchas Nasais/metabolismo , Conchas Nasais/patologia , Células Cultivadas , Hipóxia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Citocinas/metabolismoRESUMO
OBJECTIVE: Contagious ecthyma is a severe and highly contagious disease caused by an orf virus (ORFV). The virus is responsible for substantial economic losses in the goat industry and threatens humans. We previously determined the role of ORFV129 protein, one of the five ankyrin-repeat proteins coded by the orf genome, in suppressing the transcription of pro-inflammatory cytokines IL-6, IL-1ß and IFN-γ. In the present study, we identified 14 cellular proteins (complement C1q binding protein [C1QBP], MCM7, EIF5A, PKM, SLC6A, TSPAN6, ATP6AP2, GPS1, MMADHC, HSPB6, SLC35B1, MTF1, P3H4, and IL15RA) that interact with ORFV129 using a yeast two-hybrid system in goat turbinate bone cells (GFTCs). The interaction between ORFV129 and (C1QBP), an immune-related protein, was confirmed using immunofluorescence co-localization and co-immunoprecipitation assays. C1QBP overexpression inhibited ORFV replication, whereas the knockdown of C1QBP promoted ORFV replication in GFTCs. Furthermore, ORFV or ORFV129 increased C1QBP expression in GFTCs, indicated that ORFV129-C1QBP interaction might contribute to the ORFV-induced host immune process. In addition, our research showed that ORFV increased the expression of ORFV129, cytokine IL-6, IL-1ß and IFN-γ. C1QBP overexpression induced IFN-γ production and reduced IL-6 and IL-1ß production. Conversely, C1QBP knockdown induced IL-1ß production and reduced IFN-γ and IL-1ß production. Moreover, augmentation of ORFV129 expression enhanced the inhibition of the secretion of cytokines IL-6, IL-1ß, and IFN-γ induced by the altered expression of C1QBP. These findings suggest different downstream pathways might be involved in regulating different cytokines induced by ORFV129 expression in GFTCs.
Assuntos
Ectima Contagioso , Doenças das Cabras , Vírus do Orf , Doenças dos Ovinos , Humanos , Ovinos , Animais , Vírus do Orf/genética , Complemento C1q/metabolismo , Interleucina-6/metabolismo , Cabras , Conchas Nasais/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Citocinas/genética , Citocinas/metabolismo , Imunidade , Tetraspaninas/metabolismo , Receptor de Pró-Renina , Proteínas de Transporte/metabolismoRESUMO
The aim of this study was to validate the use of human brain organoids (hBOs) to investigate the therapeutic potential and mechanism of human-neural-crest-derived nasal turbinate stem cells (hNTSCs) in models of Alzheimer's disease (AD). We generated hBOs from human induced pluripotent stem cells, investigated their characteristics according to neuronal markers and electrophysiological features, and then evaluated the protective effect of hNTSCs against amyloid-ß peptide (Aß1-42) neurotoxic activity in vitro in hBOs and in vivo in a mouse model of AD. Treatment of hBOs with Aß1-42 induced neuronal cell death concomitant with decreased expression of neuronal markers, which was suppressed by hNTSCs cocultured under Aß1-42 exposure. Cytokine array showed a significantly decreased level of osteopontin (OPN) in hBOs with hNTSC coculture compared with hBOs only in the presence of Aß1-42. Silencing OPN via siRNA suppressed Aß-induced neuronal cell death in cell culture. Notably, compared with PBS, hNTSC transplantation significantly enhanced performance on the Morris water maze, with reduced levels of OPN after transplantation in a mouse model of AD. These findings reveal that hBO models are useful to evaluate the therapeutic effect and mechanism of stem cells for application in treating AD.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Síndromes Neurotóxicas , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Organoides/metabolismo , Osteopontina , Conchas Nasais/metabolismoRESUMO
Neural crest-derived cells (NCDCs), a class of adult stem cells not restricted to embryonic tissues, are attractive tissue regenerative therapy candidates because of their ease of isolation, self-renewing properties, and multipotency. Although adult NCDCs can undergo osteogenic differentiation in vitro, whether they induce bone formation in vivo remains unclear. Previously, our group reported findings showing high amounts of NCDCs scattered throughout nasal concha tissues of adult mice. In the present study, NCDCs in nasal conchae labeled with enhanced green fluorescent protein (EGFP) were collected from adult P0-Cre/CAG-CAT-EGFP double transgenic mice, then cultured in serum-free medium to increase the number. Subsequently, NCDCs were harvested and suspended in type I atelocollagen gel, then an atelocollagen sponge was used as a scaffold for the cell suspension. Atelocollagen scaffolds with NCDCs were placed on bone defects created in a mouse calvarial bone defect model. Over the ensuing 12 weeks, micro-CT and histological analysis findings showed that mice with scaffolds containing NCDCs had slightly greater bone formation as compared to those with a scaffold alone. Furthermore, Raman spectroscopy revealed spectral properties of bone in mice that received scaffolds with NCDCs similar to those of native calvarial bone. Bone regeneration is important not only for gaining bone mass but also chemical properties. These results are the first to show the validity of biomolecule-free adult nasal concha-derived NCDCs for bone regeneration, including the chemical properties of regenerated bone tissue.
Assuntos
Células-Tronco Adultas/citologia , Regeneração Óssea/fisiologia , Crista Neural/citologia , Transplante de Células-Tronco/métodos , Conchas Nasais/citologia , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Crista Neural/metabolismo , Conchas Nasais/metabolismoRESUMO
Objective:To investigate the expression and clinical significance of TET gene and 5hmC in chronic sinusitis. Methods:Acquiring 20 cases of nasal polyps from chronic sinusitis with polypsï¼CWï¼, 20 cases of uncinate process tissues from chronic sinusitis without polypsï¼CSï¼, 10 cases of middle turbinate tissues from patients with nasal septum deviated as normal groupï¼Nï¼.The expression of TET gene and 5hmC in chronic sinusitis was measured by immunofluorescence, Western-Blot and Quantitative real-time PCR. Results:Both TET1/2/3 gene and 5 hmC were highly expressed in the CS group, but were lower in the CW group and the N group.There were statistically significant differences between the CS group and the CW groupï¼P<0.05ï¼, and between the CS group and the N groupï¼P<0.05ï¼.The expressions of TET1 and TET3 in the N group were higher than those in the CW group, and the difference was statistically significantï¼P<0.05ï¼. Conclusion:DNA demethylation plays an important role in the formation of nasal polyps.High expression of TET1, TET2, TET3 and 5hmC May reduce the risk of nasal polyps.Increased DNA demethylation in chronic sinusitis may reduce the risk of nasal polyp formation.When the degree of DNA methylation in chronic sinusitis is high and the degree of DNA demethylation is low, the disease may develop to CRSwNP, and when the degree of DNA methylation is low and the degree of DNA demethylation is high, the disease may develop to CRSsNP.
Assuntos
Pólipos Nasais , Rinite , Sinusite , Doença Crônica , Metilação de DNA , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Pólipos Nasais/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Rinite/genética , Sinusite/genética , Conchas Nasais/metabolismoRESUMO
OBJECTIVE: Sinonasal inverted papilloma (SNIP) is a benign tumor characterized by an aggressive growth, a tendency to recur, and an association with malignancy. However, the precise etiology of SNIP is still unknown. The objective of this study was to identify the expression pattern of speckled protein 100 (Sp100) in the malignant transformation (MT) of SNIP and its correlation with human papillomavirus (HPV)-16 and HPV-18 infections and other clinical features. This would further help in understanding the possible mechanisms for the development of SNIP. METHODS: Individual nasal mucosa specimens from 40 patients (25 males and 15 females) and 10 inferior turbinate specimens as controls were included in the present study. The samples were divided into several sections for histopathological examination, HPV DNA detection, and immunohistochemical staining. RESULTS: We observed that as SNIP progressed, the Sp100 protein expression was gradually downregulated, and SP100 localization changed from nucleus to the cytoplasm. Positive rate of HPV infection in the SNIP with MT group was higher than that in the other groups, and Sp100 expression was correlated to HPV infections and SNIP with MT. However, no correlation was observed between Sp100 expression and clinical features, such as age, gender, and smoking. CONCLUSION: Positive rate of HPV infection is high in the SNIP with MT and has a correlation with Sp100 expression. In addition, the expression of Sp100 is downregulated in SNIP with MT, and Sp100 may play a role in the progression of SNIP.
Assuntos
Alphapapillomavirus/genética , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Papiloma Invertido/genética , Infecções por Papillomavirus/genética , Neoplasias dos Seios Paranasais/genética , Infecções Respiratórias/genética , Transformação Celular Neoplásica/genética , DNA Viral/metabolismo , Progressão da Doença , Regulação para Baixo/genética , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Mucosa Nasal/virologia , Papiloma Invertido/virologia , Infecções por Papillomavirus/virologia , Neoplasias dos Seios Paranasais/virologia , Infecções Respiratórias/virologia , Conchas Nasais/metabolismo , Conchas Nasais/virologiaRESUMO
BACKGROUND: Studies have reported that epithelial cell proliferation may be involved in the pathogenesis of nasal polyps (NPs). Estrogen receptor (ER)-α, one type of ER, is related to anti-inflammatory action and cell survival in certain tissues. In this study, we examined the presence or absence of ER-α in NPs and healthy inferior turbinate mucosae. We also investigated the effect of dexamethasone on ER-α expression, cell viability, and apoptosis in RPMI 2650 cells. METHODS: Immunohistochemical staining and Western blot analysis were conducted to determine the expression of ER-α in 15 NPs and 15 healthy inferior turbinate mucosae. After treating RPMI 2650 cells with dexamethasone, ER-α expression was analyzed using Western blot analysis and cell viability was determined using the MTT assay. Western blot analysis and annexin V-phycoerythrin (PE) staining were used to examine apoptotic cell death. RESULTS: Western blot analysis showed that ER-α expression was upregulated in 13 of the 15 NP tissues. Immunohistochemical staining for ER-α confirmed the results of the Western blot analysis. When RPMI 2650 cells were treated with dexamethasone, both ER-α expression and cell viability were decreased. Furthermore, the treatment of RPMI 2650 cells with dexamethasone increased apoptotic cell death, as shown by increased levels of BAX and cleaved caspase-3, decreased levels of Bcl-2, and an increased percentage of positive annexin V-PE stained cells. CONCLUSION: ER-α expression was higher in NPs than in healthy inferior turbinate mucosae. When RPMI 2650 cells were treated with dexamethasone, ER-α expression was downregulated, cell viability decreased, and apoptosis increased. The decreased cell viability may be related, at least in part, to the decreased ER-α protein levels, which likely contributed to the induction of apoptotic cell death in RPMI 2650 cells.
Assuntos
Dexametasona/farmacologia , Receptor alfa de Estrogênio/biossíntese , Pólipos Nasais/metabolismo , Anti-Inflamatórios/farmacologia , Apoptose , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular , Dexametasona/química , Endoscopia , Fulvestranto , Humanos , Imuno-Histoquímica , Queratinas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sais de Tetrazólio , Tiazóis , Conchas Nasais/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Recent studies have revealed that increased reactive oxidative stress (ROS) induced by particulate matter (PM) affects tight junction (TJ) functions; however, the molecular mechanisms underlying this effect have not been evaluated fully. Cultured human epithelial cells obtained from inferior turbinate tissues were exposed to an urban PM (UPM) standard reference material (SRM 1648a). Intracellular ROS level and expression of proinflammatory cytokines and TJ proteins were examined. Expression level of phosphorylated (p)-Akt, p38, p65 were compared between exposed and unexposed cells. Cells were pretreated with the ROS scavenger N-acetylcysteine (NAC) or Akt inhibitor MK-2206 before exposure to determine whether the changes in cellular ROS and TJ protein expression could be reversed. Exposure to UPM significantly increased ROS levels and inflammatory cytokine expression levels, and decreased expression of TJ proteins zonula occludins (ZO)-1, occludin, claudin-1, and E-cadherin. UPM exposure increased p-Akt, p-p38, and p65 expression levels, and NAC pretreatment reversed these effects. Akt inhibition decreased UPM-induced ROS formation and p38 and p65 protein phosphorylation, and restored the decreased ZO-1 and E-cadherin expression. Akt inhibition and ROS scavenging may provide targets for maintaining epithelial integrity by restoring decreased TJ protein expression during exposure to UPM.
Assuntos
Poluentes Atmosféricos/toxicidade , Células Epiteliais/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Mucosa Nasal/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Material Particulado/toxicidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Junções Íntimas/metabolismo , Acetilcisteína/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Estresse Oxidativo/genética , Transdução de Sinais , Proteínas de Junções Íntimas/genética , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Conchas Nasais/efeitos dos fármacos , Conchas Nasais/metabolismo , UrbanizaçãoRESUMO
Air-liquid interface (ALI) cultures are ex vivo models that are used extensively to study the epithelium of patients with chronic respiratory diseases. However, the in vitro conditions impose a milieu different from that encountered in the patient in vivo, and the degree to which this alters gene expression remains unclear. In this study we employed RNA sequencing to compare the transcriptome of fresh brushings of nasal epithelial cells with that of ALI-cultured epithelial cells from the same patients. We observed a strong correlation between cells cultured at the ALI and cells obtained from the brushed nasal epithelia: 96% of expressed genes showed similar expression profiles, although there was greater similarity between the brushed samples. We observed that while the ALI model provides an excellent representation of the in vivo airway epithelial transcriptome for mechanistic studies, several pathways are affected by the change in milieu.
Assuntos
Mucosa Nasal/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Mucosa Respiratória/metabolismo , Transcriptoma , Idoso , Ar , Fumar Cigarros/efeitos adversos , Meios de Cultura/química , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Anotação de Sequência Molecular , Mucosa Nasal/patologia , Cultura Primária de Células , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Mucosa Respiratória/patologia , Análise de Sequência de RNA , Conchas Nasais/metabolismo , Conchas Nasais/patologiaRESUMO
BACKGROUND: Although Pasteurella multocida is highly prevalent pathogen in animals and plays an important role in swine respiratory diseases, only a few studies on the use of bacteriophages specific to Pasteurella multocida disease have been reported. OBJECTIVE: The object of this study was to investigate the therapeutic effect of specific P. multocida bacteriophages and to identify genes related to bacteriophage signaling utilizing RNA microarrays in swine nasal turbinate cells. METHODS: Pas-MUP-1 phages were applied 24 h prior to P. multocida infection (1 × 107 cfu/ml) at several concentrations of bacterial infection. Cells were incubated to detect cytokines and 24 h to detect mucin production. And real-time quantitative PCR was performed to examine related genes expression. To determine the change of total gene expression based on P. multocida and Pas-MUP-1 treatment, we performed RNA sequencing experiments. RESULTS: We found that P. multocida-infected PT-K75 cells show increased gene expression of IL-1ß, IL-6, and Muc1 in a dose-dependent manner. Interestingly, these genes resulted in decreased expression in P. multocida pretreated with the P. multocida-specific Pas-MUP-1 bacteriophage. RNA sequencing analysis revealed that bacteriophage administration regulated genes associated with immune and inflammatory responses, and the regulated genes were dramatically concentrated in the cytokine/chemokine-based signaling pathways. Pas-MUP-1 treatment was shown to regulate P. multocida induced gene expression in the bacteria. CONCLUSION: These results suggest the specific bacteriophage has therapeutic potential as an alternative to antibiotic treatment to defend against P. multocida infection by altering inflammatory gene expression profiles.
Assuntos
Bacteriófagos/fisiologia , Pasteurella multocida/fisiologia , Pasteurella multocida/virologia , Suínos/microbiologia , Conchas Nasais/microbiologia , Animais , Células Cultivadas , Perfilação da Expressão Gênica , Ontologia Genética , Mediadores da Inflamação/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Mucina-1/genética , Mucina-1/metabolismo , RNA Mensageiro/metabolismo , Suínos/genética , Suínos/metabolismo , Conchas Nasais/metabolismoRESUMO
We have designed and characterized an injectable, electrostatically bonded, in situ-forming hydrogel system consisting of a cationic polyelectrolyte [(methoxy)polyethylene glycol-b-(poly(ε-caprolactone)-ran-poly(L-lactic acid)] (MP) copolymer derivatized with an amine group (MP-NH2) and anionic BMP2. To the best of our knowledge, there have been hardly any studies that have investigated electrostatically bonded, in situ-forming hydrogel systems consisting of MP-NH2 and BMP2, with respect to how they promote in vivo osteogenic differentiation of human turbinate mesenchymal stem cells (hTMSCs). Injectable formulations almost immediately formed an electrostatically loaded hydrogel depot containing BMP2, upon injection into mice. The hydrogel features and stability of BMP2 inside the hydrogel were significantly affected by the electrostatic attraction between BMP2 and MP-NH2. Additionally, the time BMP2 spent inside the hydrogel depot was prolonged in vivo, as evidenced by in vivo near-infrared fluorescence imaging. Biocompatibility was demonstrated by the fact that hTMSCs survived in vivo, even after 8â¯weeks and even though relatively few macrophages were in the hydrogel depot. The osteogenic capacity of the electrostatically loaded hydrogel implants containing BMP2 was higher than that of a hydrogel that was simply loaded with BMP2, as evidenced by Alizarin Red S, von Kossa, and hematoxylin and eosin staining as well as osteonectin, osteopontin, osteocalcin, and type 1α collagen mRNA expression. The results confirmed that our injectable, in situ-forming hydrogel system, electrostatically loaded with BMP2, can enhance in vivo osteogenic differentiation of hTMSCs.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Hidrogéis , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , Conchas Nasais/metabolismo , Adulto , Animais , Feminino , Xenoenxertos , Humanos , Hidrogéis/química , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/citologia , Camundongos , Eletricidade Estática , Transplante de Células-Tronco , Conchas Nasais/citologiaRESUMO
BACKGROUND: 15-Lipoxygenase 1 (15LO1) is expressed in airway epithelial cells in patients with type 2-high asthma in association with eosinophilia. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also associated with type 2 inflammation and eosinophilia. CCL26/eotaxin 3 has been reported to be regulated by 15LO1 in lower airway epithelial cells. However, its relation to 15LO1 in patients with CRSwNP or mechanisms for its activation are unclear. OBJECTIVE: We sought to evaluate 15LO1 and CCL26 expression in nasal epithelial cells (NECs) from patients with CRSwNP and healthy control subjects (HCs) and determine whether 15LO1 regulates CCL26 in NECs through extracellular signal-regulated kinase (ERK) activation. METHODS: 15LO1, CCL26, and phosphorylated ERK were evaluated in NECs from patients with CRSwNP and HCs. 15LO1/CCL26 and CCL26/cytokeratin 5 were colocalized by means of immunofluorescence. IL-13-stimulated NECs were cultured at an air-liquid interface with or without 15-lipoxygenase 1 gene (ALOX15) Dicer-substrate short interfering RNAs (DsiRNA) transfection, a specific 15LO1 enzymatic inhibitor, and 2 ERK inhibitors. Expression of 15LO1 and CCL26 mRNA and protein was analyzed by using quantitative RT-PCR, Western blotting, and ELISA. RESULTS: 15LO1 expression was increased in nasal polyp (NP) epithelial cells compared with middle turbinate epithelial cells from patients with CRSwNP and HCs. 15LO1 expression correlated with CCL26 expression and colocalized with CCL26 expression in basal cells of the middle turbinate and NPs from patients with CRSwNP. In primary NECs in vitro, IL-13 induced 15LO1 and CCL26 expression. 15LO1 knockdown and inhibition decreased IL-13-induced ERK phosphorylation and CCL26 expression. ERK inhibition (alone) similarly decreased IL-13-induced CCL26. Phosphorylated ERK expression was increased in NECs from CRSwNP subjects and positively correlated with both 15LO1 and CCL26 expression. CONCLUSIONS: 15LO1 expression is increased in NP epithelial cells and contributes to CCL26 expression through ERK activation. 15LO1 could be considered a novel therapeutic target for CRSwNP.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Pólipos Nasais/metabolismo , Mucosa Respiratória/metabolismo , Rinite/metabolismo , Sinusite/metabolismo , Conchas Nasais/metabolismo , Adulto , Araquidonato 15-Lipoxigenase/genética , Células Cultivadas , Quimiocina CCL26/metabolismo , Doença Crônica , Ativação Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pólipos Nasais/complicações , RNA Interferente Pequeno/genética , Mucosa Respiratória/patologia , Rinite/complicações , Sinusite/complicações , Regulação para CimaRESUMO
BACKGROUND: Allergic rhinitis is characterized by a remodeling of nasal epithelium. Since the Notch and TGF-ß signaling pathways are known to be involved in cell differentiation and remodeling processes and leptin adipokine has already been identified as a marker for homeostasis in human bronchial and nasal epithelial cells of asthmatics, roles played by these pathways have been investigated for chronic allergic rhinitis. METHODS: The leptin/leptin receptor expression has been investigated in a study with 40 biopsies from allergic (AR, nâ¯=â¯18) and non-allergic (C, nâ¯=â¯22) inferior turbinates, using immunohistochemistry, immunofluorescence staining and RT-PCR. In addition, extracts from in vitro samples prepared from primary cells of inferior turbinates as well as in vitro cultured human nasal epithelial RPMI 2650 cells (ATCC-CCL-30) were also tested for leptin expression and activation of the Notch-1 pathway. RESULTS: With regards to AR, in vivo expression levels of both leptin and its receptor significantly decreased in comparison to C. Furthermore, leptin receptor mRNA was significantly reduced in AR as compared to C. Immunofluorescence showed an apparent co-expression of leptin receptor with Notch-1, which was not seen with TGF-ß. In vitro, in primary turbinate epithelial cells, the expression of leptin receptor and Notch-1 significantly decreased in AR as compared to C. Moreover, in RPMI 2650 cells, leptin receptor expression was shown to be induced by Notch-1 ligand signaling. CONCLUSION: Thus, both the leptin and Notch-1 pathways appear to represent markers for epithelial homeostasis in allergic rhinitis.
Assuntos
Leptina/genética , Mucosa Nasal/metabolismo , Receptor Notch1/genética , Receptores para Leptina/genética , Rinite Alérgica/genética , Adulto , Biópsia , Estudos de Casos e Controles , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Leptina/metabolismo , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/patologia , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1/metabolismo , Receptores para Leptina/metabolismo , Rinite Alérgica/metabolismo , Rinite Alérgica/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Conchas Nasais/metabolismo , Conchas Nasais/patologiaRESUMO
OBJECTIVE: We investigated the difference in ciliary beat responsiveness to acetylcholine in ex vivo and the difference in the expressions of associated molecules (M1/M3 muscarinic receptors, pannexin-1 and P2X7 purinergic receptor) between the nasal polyp and turbinate mucosa. STUDY DESIGN: Laboratorial study. PARTICIPANTS: Nasal polyp and inferior turbinate were collected from patients with hypertrophic rhinitis and/or nasal polyp during endoscopic sinonasal surgery. MAIN OUTCOME MEASURES: The mucosa was cut into thin strips, and ciliary movement was observed under a phase-contrast light microscope equipped with a high-speed digital video camera. The samples were also examined by scanning electron microscopy, fluorescence immunohistochemistry, and quantitative reverse transcription-polymerase chain reaction. RESULTS: Cilia were well preserved in both tissues at the ultrastructural level. The baseline ciliary beat frequency (CBF) was not different between the two tissues. The CBF of the turbinate was significantly increased by stimulation with acetylcholine (P < 0.001), but that of the polyp was not. The ratio of the acetylcholine-stimulated CBF to the baseline CBF was significantly lower in the polyp than in the turbinate (P < 0.001). Immunohistochemical study revealed that immunoreactivities for M3, pannexin-1 and P2X7 were weaker in the polyp than in the turbinate. The mRNA expressions of M1, M3 and P2X7 were significantly lower and that of pannexin-1 tended to be lower in the polyp than in the turbinate. CONCLUSIONS: These results indicate that ciliary beat responsiveness to acetylcholine is decreased in the nasal polyp. This may be explained by the decreased expressions of M3, P2X7 and probably pannexin-1 in this tissue.
Assuntos
Acetilcolina/farmacologia , Cílios/efeitos dos fármacos , Mucosa Nasal/efeitos dos fármacos , Mucosa Nasal/metabolismo , Pólipos Nasais/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Cílios/ultraestrutura , Conexinas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Depuração Mucociliar/efeitos dos fármacos , Pólipos Nasais/cirurgia , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Rinite/cirurgia , Conchas Nasais/efeitos dos fármacos , Conchas Nasais/metabolismo , Conchas Nasais/ultraestruturaRESUMO
BACKGROUND AND AIMS: The roles of Fas in immune system are multifaceted, and the interaction between Fas receptor and Fas ligand is essential for maintaining the immune tolerance. We aimed to assess the level of the expression of Fas receptor on nasal inferior turbinate mucosa in patients with mild persistent allergic rhinitis (M PAR) and moderate to severe (M/S) PAR and determined the relationship between disease severity and production of Fas. METHODS: A total of 70 patients with M/S PAR, 70 patients with M PAR, and 70 healthy individuals were enrolled in this study. We obtained biopsies of nasal inferior turbinate mucosa from the participants. The expression of Fas mRNA was evaluated by real-time polymerase chain reaction. The presence and location of Fas were determined by immunohistochemistry. The number of eosinophils per field, blood eosinophils, total serum IgE levels, and specific serum IgE levels were measured. Clinical manifestations of patients were assessed by Total Nasal Syndrome Score (TNSS). RESULTS: The expression of Fas in patients with M/S PAR was decreased significantly compared to the control group and patients with M PAR. Local mucosal expression of Fas was correlated with specific IgE, nasal eosinophil count, and TNSS. CONCLUSION: According to the results of this study, there might be a relationship between the expression of Fas receptor on nasal turbinate mucosa and the severity of persistent allergic rhinitis.
Assuntos
Rinite Alérgica/genética , Rinite Alérgica/fisiopatologia , Receptor fas/genética , Receptor fas/metabolismo , Adulto , Animais , Eosinófilos/metabolismo , Feminino , Humanos , Imunoglobulina E/sangue , Imuno-Histoquímica , Contagem de Leucócitos , Masculino , Mucosa Nasal/metabolismo , Mucosa Nasal/patologia , RNA Mensageiro/metabolismo , Rinite Alérgica/metabolismo , Conchas Nasais/metabolismo , Conchas Nasais/patologia , Adulto JovemRESUMO
BACKGROUND: Although the role of neurotrophins such as nerve growth factor and brain-derived neurotrophic factor in nasal polyps development has been studied, the contribution of neurotrophin-3 has not been evaluated yet. This study aimed to investigate the possible role of neurotrophin-3 in nasal polyps pathogenesis. METHODS: The study group comprised 70 non-allergic nasal polyps patients and the control group consisted of 53 patients with middle turbinate concha bullosa. Specimens were taken, during surgery, from the ethmoid sinus nasal polyps in the nasal polyps group and from the lateral part of the middle turbinate concha bullosa in the control group. Tissue and serum levels of neurotrophin-3 were assessed by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. RESULTS: Nasal polyps patients had higher tissue neurotrophin-3 scores (p < 0.001). There was no statistically significant difference between groups regarding serum neurotrophin-3 levels (p = 0.417). Tissue neurotrophin-3 staining scores in the nasal polyps group had no statistically significant correlation with Lund-Mackay scores (p = 0.792). CONCLUSION: Neurotrophin-3 may have a local effect in nasal polyps pathogenesis, without joining systemic circulation.
Assuntos
Pólipos Nasais/etiologia , Pólipos Nasais/metabolismo , Fatores de Crescimento Neural/metabolismo , Adulto , Asma/complicações , Asma/metabolismo , Estudos de Casos e Controles , Seio Etmoidal/metabolismo , Feminino , Humanos , Masculino , Pólipos Nasais/patologia , Neurotrofina 3 , Conchas Nasais/metabolismoRESUMO
OBJECTIVE: Motile cilia impairment is a common condition in patients with chronically inflamed airways, such as is seen in nasal polyps (NPs). The mechanism underlying this pathogenic condition is complex and not fully understood. METHODS: We investigated the presence and localization of dynein axonemal heavy chain 5 (DNAH5) in motile cilia using immunofluorescence staining in paraffin-embedded nasal biopsies from NPs (n = 120) and inferior turbinate mucosa (n = 35) of healthy controls. We also performed single-cell staining on cytospin samples (NP = 5, control = 5). Three patterns of DNAH5 localization are defined, including pattern A (presence throughout the axoneme), pattern B (undetectable in the distal part of the axoneme), and pattern C (completely missing throughout the entire axoneme). We developed a semiquantitative scoring system for which 0 = (pattern A > 70%); 1 = (patterns A + B > 70%); and 2 = (pattern C ≥ 30%) in each high-power field (5 fields per sample). RESULTS: Based on our DNAH5 scoring system, the median (1st and 3rd quartile) score was 0.3 (0.2 and 0.4) for samples from controls, and 1.1 (0.6 and 1.6) for samples from NPs in paraffin specimens (P < 0.001). The DNAH5 score had a significant positive relationship with the Lund-Mackay computed tomography score (r = 0.329, P = 0.005) and was higher in patients with eosinophilic NPs (P = 0.006). For cytospin samples, the mean percentage of patterns A, B, and C were 74%, 14%, and 12% in controls, and 48%, 20%, and 32% in NPs, respectively. CONCLUSION: Our results suggest that the absence or mislocalization of DNAH5 from motile cilia is a common and potentially important pathological phenomenon in chronically inflamed airway epithelium. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E97-E104, 2018.
Assuntos
Dineínas do Axonema/análise , Transtornos da Motilidade Ciliar/metabolismo , Pólipos Nasais/química , Adulto , Biomarcadores/análise , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/metabolismo , Pólipos Nasais/complicações , Conchas Nasais/metabolismoRESUMO
Histamine is an important mediator of allergic reactions, and mucus hypersecretion is a major allergic symptom. However, the direct effect of histamine on mucus secretion from airway mucosal epithelia has not been clearly demonstrated. TMEM16A is a Ca2+-activated chloride channel, and it is closely related to fluid secretion in airway mucosal epithelia. We investigated whether histamine directly induces fluid secretion from epithelial cells or submucosal glands (SMG) and mechanisms related, therewith, in allergic airway diseases. In pig airway tissues from the nose or trachea, histamine was a potent secretagogue that directly induced strong responses. However, gland secretion from human nasal tissue was not induced by histamine, even in allergic rhinitis patients. Histamine type 1 receptor (H1R) and histamine type 2 receptor (H2R) were not noted in SMG by in situ hybridization. Cultured primary human nasal epithelial (NHE) cells were used for the measurement of short-circuit current changes with the Ussing chamber. Histamine-induced slight responses of anion secretions under normal conditions. The response was enhanced by IL-4 stimulation through TMEM16A, which might be related to fluid hypersecretion in allergic rhinitis. Pretreatment with IL-4 augmented the histamine response that was suppressed by a TMEM16A inhibitor. TMEM16A expression was enhanced by 24-h treatment of IL-4 in human nasal epithelial cells. The expression of TMEM16A was significantly elevated in an allergic rhinitis group, compared with a control group. We elucidated histamine-induced fluid secretions in synergy with IL-4 through TMEM16A in the human airway epithelium. In addition, we observed species differences between pigs and humans in terms of gland secretion of histamine.
Assuntos
Canais de Cloreto/metabolismo , Histamina/farmacologia , Interleucina-4/farmacologia , Muco/metabolismo , Proteínas de Neoplasias/metabolismo , Mucosa Respiratória/metabolismo , Adulto , Animais , Anoctamina-1 , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Biológicos , Mucosa Nasal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Histamínicos/genética , Receptores Histamínicos/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Rinite Alérgica/metabolismo , Rinite Alérgica/patologia , Sus scrofa , Traqueia/patologia , Conchas Nasais/metabolismoRESUMO
OBJECTIVE: The cysteinyl leukotrienes (CysLTs) are lipid mediators that have been implicated in the pathogenesis of allergic rhinitis. Pharmacological studies of CysLTs indicate that two classes of receptors, CysLT1R and CysLT2R exist. CysLT1R is a high affinity LTD4 receptor with lower affinity for LTC4, and a CysLT1R antagonist is currently used to treat asthma and allergic rhinitis. CysLT2R binds to LTC4 and LTD4 with equal affinity. GPR99 (also called GPR80), previously described as an oxoglutarate receptor (OXGR1), has recently emerged as a potential novel receptor with LTE4. The purpose of this study was to determine the expression and localization of GPR99 protein in the human nasal mucosa. METHODS: Human turbinates were obtained after turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. GPR99 protein expression was evaluated by western blotting, and the specific cells expressing GPR99 protein identified by immunostaining using a commercial anti-GPR99 (OXGR1) monoclonal antibody. RESULTS: A 38-kDa band was detected in the western blots of human nasal samples by using the anti-GPR99 monoclonal antibody. We did not find any differences in GPR99 protein levels between allergic and non-allergic nasal mucosa. The immunohistochemical studies revealed that the anti-GPR99 monoclonal antibody mainly labeled vascular smooth muscle cells in the nasal mucosa. CONCLUSION: These immunohistochemical results suggest that GPR99 may play some roles in the vascular response. Further functional studies will be necessary to clarify the biological significance of the GPR99 receptor in nasal vasculature.