RESUMEN
The types of interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses are not well-characterized due to the low number of co-infection cases described since the onset of the pandemic. We have evaluated the interactions between SARS-CoV-2 (D614G mutant) and influenza A(H1N1)pdm09 or respiratory syncytial virus (RSV) in the nasal human airway epithelium (HAE) infected simultaneously or sequentially (24 h apart) with virus combinations. The replication kinetics of each virus were determined by RT-qPCR at different post-infection times. Our results showed that during simultaneous infection, SARS-CoV-2 interferes with RSV-A2 but not with A(H1N1)pdm09 replication. The prior infection of nasal HAE with SARS-CoV-2 reduces the replication kinetics of both respiratory viruses. SARS-CoV-2 replication is decreased by a prior infection with A(H1N1)pdm09 but not with RSV-A2. The pretreatment of nasal HAE with BX795, a TANK-binding kinase 1 inhibitor, partially alleviates the reduced replication of SARS-CoV-2 or influenza A(H1N1)pdm09 during sequential infection with both virus combinations. Thus, a prior infection of nasal HAE with SARS-CoV-2 interferes with the replication kinetics of A(H1N1)pdm09 and RSV-A2, whereas only A(H1N1)pdm09 reduces the subsequent infection with SARS-CoV-2. The mechanism involved in the viral interference between SARS-CoV-2 and A(H1N1)pdm09 is mediated by the production of interferon.
Asunto(s)
Células Epiteliales/virología , Subtipo H1N1 del Virus de la Influenza A/fisiología , Nasofaringe/citología , Virus Sincitial Respiratorio Humano/fisiología , SARS-CoV-2/fisiología , Interferencia Viral , Replicación Viral , Coinfección , Humanos , Interacciones Microbianas , Nasofaringe/virologíaRESUMEN
Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Nasofaringe/inmunología , Nariz/citología , Mucosa Respiratoria/inmunología , SARS-CoV-2/inmunología , Anticuerpos Antivirales/sangre , COVID-19/inmunología , COVID-19/patología , Granulocitos/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Células Asesinas Naturales/inmunología , Células T de Memoria/inmunología , Monocitos/inmunología , Nasofaringe/citología , Nasofaringe/virología , Neutrófilos/inmunología , Nariz/inmunología , Nariz/virología , Estudios Prospectivos , Mucosa Respiratoria/citología , Mucosa Respiratoria/virologíaRESUMEN
B7 homolog 3 (B7-H3) is a recently found superfamily B7 molecule and therefore has significant involvement in immunological regulation. However, the relationships of B7-H3 expression with the tumor microenvironment (TME), response to immunotherapy, and prognosis in head and neck squamous cell carcinoma (HNSCC) are still unknown. In the present analysis, we determined B7-H3 as a novel biomarker that predicts the prognosis and response to immunotherapy in HNSCC. B7-H3 expression is enhanced in HNSCC compared to normal sample and is stably expressed in HNSCC cell line. Besides, high B7-H3 expression is correlated with a dismal prognosis and resistance to immunotherapy and contributes to an immunosuppressive microenvironment. Moreover, single-cell RNA sequencing (scRNA-seq) analysis shows that B7-H3 is mainly expressed in the stromal as well as malignant cells. In conclusion, the study provides insight in understanding the prognostic value of B7-H3 in HNSCC and highlights its involvement in promoting the immunosuppressive microenvironment, which presents an attractive strategy for antibody-based immunotherapy.
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Antígenos B7/biosíntesis , Genómica/métodos , Neoplasias de Cabeza y Cuello/metabolismo , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia , Proteínas de la Membrana/sangre , Proteínas de Neoplasias/sangre , Proteómica/métodos , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos B7/genética , Secuencia de Bases , Biomarcadores , Línea Celular Transformada , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/terapia , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Nasofaringe/citología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Pronóstico , ARN Neoplásico/biosíntesis , Análisis de la Célula Individual , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/terapia , Células del Estroma/metabolismo , Resultado del Tratamiento , Microambiente TumoralRESUMEN
The impact of prematurity on human development and neonatal diseases, such as bronchopulmonary dysplasia, has been widely reported. However, little is known about the effects of prematurity on the programs of stem cell self-renewal and differentiation of the upper respiratory epithelium, which is key for adaptation to neonatal life. We developed a minimally invasive methodology for isolation of neonatal basal cells from nasopharyngeal (NP) aspirates and performed functional analysis in organotypic cultures to address this issue. We show that preterm NP progenitors have a markedly distinct molecular signature of abnormal proliferation and mitochondria quality control compared to term progenitors. Preterm progenitors had lower oxygen consumption at baseline and were unable to ramp up consumption to the levels of term cells when challenged. Although they formed a mucociliary epithelium, ciliary function tended to decline in premature cells as they differentiated, compared to term cells. Together, these differences suggested increased sensitivity of preterm progenitors to environmental stressors under non-homeostatic conditions.
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Displasia Broncopulmonar/patología , Perfilación de la Expresión Génica/métodos , Nasofaringe/citología , Oxígeno/metabolismo , Células Madre/citología , Displasia Broncopulmonar/genética , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Recién Nacido , Recien Nacido Prematuro , Nasofaringe/metabolismo , Análisis de Secuencia de ARN , Células Madre/metabolismoRESUMEN
Imaging the Eustachian tube is challenging because of its complex anatomy and limited accessibility. This study fabricated a fiber-based optical coherence tomography (OCT) catheter and investigated its potential for assessing the Eustachian tube anatomy. A customized OCT system and an imaging catheter, termed the Eustachian OCT, were developed for visualizing the Eustachian tube. Three male swine cadaver heads were used to study OCT image acquisition and for subsequent histologic correlation. The imaging catheter was introduced through the nasopharyngeal opening and reached toward the middle ear. The OCT images were acquired from the superior to the nasopharyngeal opening before and after Eustachian tube balloon dilatation. The histological anatomy of the Eustachian tube was compared with corresponding OCT images, The new, Eustachian OCT catheter was successfully inserted in the tubal lumen without damage. Cross-sectional images of the tube were successfully obtained, and the margins of the anatomical structures including cartilage, mucosa lining, and fat could be successfully delineated. After balloon dilatation, the expansion of the cross-sectional area could be identified from the OCT images. Using the OCT technique to assess the Eustachian tube anatomy was shown to be feasible, and the fabricated OCT image catheter was determined to be suitable for Eustachian tube assessment.
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Cateterismo/métodos , Endoscopía/métodos , Trompa Auditiva/diagnóstico por imagen , Tomografía de Coherencia Óptica/métodos , Tejido Adiposo/citología , Tejido Adiposo/diagnóstico por imagen , Animales , Cartílago/citología , Cartílago/diagnóstico por imagen , Cateterismo/instrumentación , Dilatación , Endoscopía/instrumentación , Trompa Auditiva/anatomía & histología , Trompa Auditiva/citología , Masculino , Membrana Mucosa/citología , Membrana Mucosa/diagnóstico por imagen , Nasofaringe/citología , Nasofaringe/diagnóstico por imagen , Porcinos , Tomografía de Coherencia Óptica/instrumentaciónRESUMEN
Planar cell polarity (PCP) signalling specifies the orientation of epithelial cells and regulates directional beating of motile cilia of multiciliated epithelial cells. Clinically, defects in cilia function are associated with nasopharyngeal symptoms. The polarity of the nasopharyngeal epithelium is poorly understood. Here, we demonstrated PCP in the nasopharyngeal epithelium. Multiciliated cells (MCCs) were uniformly aligned with their long axis parallel to the tissue axis of the nasopharynx (NP). In addition, PCP proteins exhibited an asymmetrical localisation between adjacent cells. Motile cilia were uniformly aligned in the same direction within both individual cells and neighbouring cells, which manifested as cilial polarity in MCCs. Mutation of Vangl2, a mammalian homologue of the Drosophila PCP gene, resulted in significant disruption of the orientation of epithelial cells. Finally, keratin-5-positive basal cells constantly replenished the luminal ciliated cells; the new dynamic ciliated cells were also oriented parallel to the tissue axis. These results indicate a role for the PCP pathway in the uniform orientation of dynamically replenished epithelial cells in the NP.
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Polaridad Celular , Cilios/metabolismo , Células Epiteliales/metabolismo , Epitelio/metabolismo , Nasofaringe/metabolismo , Animales , Cilios/ultraestructura , Células Epiteliales/citología , Células Epiteliales/ultraestructura , Epitelio/ultraestructura , Proteínas con Dominio LIM/metabolismo , Mamíferos/metabolismo , Proteínas de la Membrana/metabolismo , Ratones Noqueados , Microscopía Electrónica de Transmisión , Nasofaringe/citología , Nasofaringe/ultraestructura , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was first detected in late December 2019 and has spread worldwide. Coronaviruses are enveloped, positive sense, single-stranded RNA viruses and employ a complicated pattern of virus genome length RNA replication as well as transcription of genome length and leader containing subgenomic RNAs. Although not fully understood, both replication and transcription are thought to take place in so-called double-membrane vesicles in the cytoplasm of infected cells. Here we show detection of SARS-CoV-2 subgenomic RNAs in diagnostic samples up to 17 days after initial detection of infection and provide evidence for their nuclease resistance and protection by cellular membranes suggesting that detection of subgenomic RNAs in such samples may not be a suitable indicator of active coronavirus replication/infection.
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Prueba de COVID-19/métodos , COVID-19/diagnóstico , Genoma Viral , ARN Viral/aislamiento & purificación , SARS-CoV-2/genética , Replicación Viral , Adulto , COVID-19/virología , Citoplasma/virología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno/genética , Humanos , Masculino , Persona de Mediana Edad , Nasofaringe/citología , Nasofaringe/virología , Orofaringe/citología , Orofaringe/virología , Reacción en Cadena de la Polimerasa , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/patogenicidad , Factores de Tiempo , Adulto JovenRESUMEN
OBJECTIVE: Nasopharyngeal swabs (NPS) are the collection modality of choice for reverse-transcription polymerase chain reaction (RT-PCR) multiplex array for respiratory viruses. NPS gather both extracellular material and human respiratory epithelial cells and, when used with RT-PCR, have reliable sensitivity for detection of viral infection. GOALS: At our institution, we identified a 1.7% re-order rate within 7-days for NPS destined for RT-PCR respiratory pathogen multiplex, which we hypothesize may be due in part to low confidence in adequate collection. We sought an inexpensive and accessible strategy for benchside quality assurance of NPS adequacy by observing microscopic content of viral transport media. PROCEDURE: For eight-hundred one NPS samples collected during routine clinical practice in November 2019, aliquots of viral transport media were air-dried and safranin-stained on glass slides under a fume hood. We then counted morphologically distinct ciliated columnar epithelial cells (CCEs), which are prevalent in the nasopharynx. RESULTS: Twenty percent of samples negative for respiratory pathogens by RT-PCR (BioFire FilmArray RP2, Cepheid GeneXpert) had no CCEs, while just seven percent of positive samples had no CCEs. Pearson's Chi-squared test was used to compare presence of CCEs between samples that were positive and negative for respiratory pathogens by RT-PCR (p=1.6×10-36). CONCLUSION: We posit that samples without identifiable CCEs have a greater likelihood of inadequate collection. The basic, benchside protocol that we describe here demonstrates potential to reduce unnecessary re-testing when deployed to confirm negative tests despite high clinical suspicion: a strategy which may help conserve NPS reagents.
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Nasofaringe/citología , Manejo de Especímenes/métodos , Pruebas Diagnósticas de Rutina , Humanos , Técnicas de Diagnóstico Molecular/métodos , Nasofaringe/virología , Control de Calidad , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Virosis/diagnóstico , Virosis/virología , Virus/genéticaRESUMEN
In the present study, 3D biomimetic platforms were fabricated with guiding grating to mimic extracellular matrix topography, porous membrane to resemble the epithelial porous interface and trenches below to represent blood vessels as an in vitro tissue microenvironment. Fabrication technologies were developed to integrate the transparent biocompatible polydimethylsiloxane platforms with preciously controlled dimensions. Cell migration behaviors of an immortalized nasopharyngeal epithelial cell line (NP460) and a nasopharyngeal carcinoma cell line (NPC43) were studied on the 2D and 3D platforms. The NP460 and NPC43 cells traversing through the porous membrane and migrating in the trenches below were studied by time-lapse imaging. Before traversing through the pores, NP460 and NPC43 cells migrated around the pores but NPC43 cells had a lower migration speed with less lamellipodia spreading. After traversing to trenches below, NPC43 cells moved faster with an alternated elongated morphology (mesenchymal migration mode) and round morphology (amoeboid migration mode) compared with only mesenchymal migration mode for NP460 cells. The cell traversing probability through porous membrane on platforms with 30 µm wide trenches below was found to be the highest when the guiding grating was perpendicular to the trenches below and the lowest when the guiding grating was parallel to the trenches below. The present study shows important information on cell migration in complex 3D microenvironment with various dimensions and could provide insight for pathology and treatment of nasopharyngeal carcinoma.
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Materiales Biomiméticos , Técnicas de Cultivo de Célula/instrumentación , Movimiento Celular , Células Epiteliales/fisiología , Membranas Artificiales , Carcinoma Nasofaríngeo/patología , Neoplasias Nasofaríngeas/patología , Nasofaringe/citología , Línea Celular Tumoral , Diseño de Equipo , Herpesvirus Humano 4/patogenicidad , Humanos , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/virología , Invasividad Neoplásica , Porosidad , Factores de Tiempo , Microambiente TumoralRESUMEN
M cells play a pivotal role in the induction of immune responses within the mucosa-associated lymphoid tissues. M cells exist principally in the follicle-associated epithelium (FAE) of the isolated solitary lymphoid follicles as well as in the lymphoid follicles of nasopharynx-associated lymphoid tissue and gut associated lymphoid tissue (GALT). Through lymphatic cannulation it is possible to investigate local immune responses induced following nasal Ag delivery in sheep. Hence, identifying sheep M cell markers would allow the targeting of M cells to offset the problem of trans-epithelial Ag delivery associated with inducing mucosal immunity. Sheep cDNA from the tonsils of the oropharynx and nasopharynx was PCR amplified using Glycoprotein-2 (GP2)-specific primers and expressed as a poly-His-tagged recombinant sheep GP2 (56 kDa) in HEK293 cells. The recombinant GP2 protein was purified using Ni-NTA affinity chromatography and polyclonal serum against the protein was raised in rats. The antiserum recognized the recombinant sheep GP2 and purified rat IgG against GP2 stained M cells in sections of sheep tonsils from nasopharynx and oropharynx. M cells were found to be present in epithelium of the palatine tonsils (oropharynx), pharyngeal tonsils as well as tubal tonsils (nasopharynx). They were also present in the FAE of the scattered lymphoid follicles over the base of the nasopharynx. Thus, GP2 has been identified to be an important M cell marker of nasopharynx and oropharynx-associated lymphoid tissues in sheep.
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Proteínas Ligadas a GPI/genética , Tejido Linfoide/inmunología , Nasofaringe/inmunología , Orofaringe/inmunología , Animales , Biomarcadores , Proteínas Ligadas a GPI/inmunología , Células HEK293 , Humanos , Inmunidad Mucosa/inmunología , Tejido Linfoide/citología , Nasofaringe/citología , Orofaringe/citología , Tonsila Palatina/citología , Tonsila Palatina/inmunología , Ovinos/inmunologíaRESUMEN
BACKGROUND: It has been found that chronic rhinosinusitis (CRS) increases the risk of developing nasopharyngeal carcinoma (NPC). CRS can be caused by gastro-oesophageal reflux (GOR) that may reach nasopharynx. The major component of refluxate, bile acid (BA) has been found to be carcinogenic and genotoxic. BA-induced apoptosis has been associated with various cancers. We have previously demonstrated that BA induced apoptosis and gene cleavages in nasopharyngeal epithelial cells. Chromosomal cleavage occurs at the early stage of both apoptosis and chromosome rearrangement. It was suggested that chromosome breaks tend to cluster in the region containing matrix association region/scaffold attachment region (MAR/SAR). This study hypothesised that BA may cause chromosome breaks at MAR/SAR leading to chromosome aberrations in NPC. This study targeted the AF9 gene located at 9p22 because 9p22 is a deletion hotspot in NPC. METHODS: Potential MAR/SAR sites were predicted in the AF9 gene by using MAR/SAR prediction tools. Normal nasopharyngeal epithelial cells (NP69) and NPC cells (TWO4) were treated with BA at neutral and acidic pH. Inverse-PCR (IPCR) was used to identify chromosome breaks in SAR region (contains MAR/SAR) and non-SAR region (does not contain MAR/SAR). To map the chromosomal breakpoints within the AF9 SAR and non-SAR regions, DNA sequencing was performed. RESULTS: In the AF9 SAR region, the gene cleavage frequencies of BA-treated NP69 and TWO4 cells were significantly higher than those of untreated control. As for the AF9 non-SAR region, no significant difference in cleavage frequency was detected between untreated and BA-treated cells. A few breakpoints detected in the SAR region were mapped within the AF9 region that was previously reported to translocate with the mixed lineage leukaemia (MLL) gene in an acute lymphoblastic leukaemia (ALL) patient. CONCLUSIONS: Our findings suggest that MAR/SAR may be involved in defining the positions of chromosomal breakages induced by BA. Our report here, for the first time, unravelled the relation of these BA-induced chromosomal breakages to the AF9 chromatin structure.
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Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/farmacología , Rotura Cromosómica , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Nasofaringe/citología , Matriz Nuclear/metabolismo , Apoptosis/genética , Línea Celular , Rotura Cromosómica/efectos de los fármacos , Simulación por Computador , ADN-Topoisomerasas de Tipo II/metabolismo , Células Epiteliales/metabolismo , Humanos , Matriz Nuclear/efectos de los fármacosRESUMEN
Neisseria meningitidis, a commensal ß-proteobacterium of the human nasopharynx, constitutes a worldwide leading cause of sepsis and epidemic meningitis. A recent genome-wide association study suggested an association of its type II-C CRISPR/Cas system with carriage and thus less invasive lineages. Here, we show that knock-out strains lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells which constitutes a central step in the pathogenesis of invasive meningococcal disease. Transcriptome sequencing data further suggest that meningococcal Cas9 does not affect the expression of surface adhesins but rather exerts its effect on cell adhesion in an indirect manner. Consequently, we speculate that the meningococcal CRISPR/Cas system exerts novel functions beyond its established role in defence against foreign DNA.
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Adhesión Bacteriana/genética , Sistemas CRISPR-Cas/genética , Células Epiteliales/microbiología , Nasofaringe/citología , Neisseria meningitidis/genética , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular , Regulación Bacteriana de la Expresión Génica , Humanos , Mutación/genética , Neisseria meningitidis/crecimiento & desarrollo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma/genéticaRESUMEN
BACKGROUND: The epithelium is the first line of defense against pathogens. Notably the epithelial cells lining the respiratory track are crucial in sensing airborne microbes and mounting an effective immune response via the expression of target genes such as cytokines and chemokines. Gene expression regulation following microbial recognition is partly regulated by chromatin re-organization and has been described in immune cells but data from epithelial cells is not as detailed. Here, we report genome-wide changes of the H3K27ac mark, characteristic of activated enhancers and promoters, after stimulation of nasopharyngeal epithelial cells with the bacterial endotoxin Lipopolysaccharide (LPS). RESULTS: In this study, we have identified 626 regions where the H3K27ac mark showed reproducible increase following LPS induction in epithelial cells. This indicated that sensing of LPS led to opening of the chromatin in our system. Moreover, this phenomenon seemed to happen extensively at enhancers regions and we could observe instances of Super-enhancer formation. As expected, LPS-increased H3K27ac regions were found in the vicinity of genes relevant for LPS response and these changes correlated with up-regulation of their expression. In addition, we found the induction of H3K27ac mark to overlap with the binding of one of the NF-kB members and key regulator of the innate immune response, RELA, following LPS sensing. Indeed, inhibiting the NF-kB pathway abolished the deposition of H3K27ac at the TNF locus, a target of RELA, suggesting that these two phenomena are associated. CONCLUSIONS: Enhancers' selection and activation following microbial or inflammatory stimuli has been described previously and shown to be mediated via the NF-kB pathway. Here, we demonstrate that this is also likely to occur in the case of LPS-sensing by nasopharyngeal epithelial cells as well. In addition to validating previous findings, we generated a valuable data set relevant to the host immune response to epithelial cell colonizing or infecting pathogens.
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Regulación de la Expresión Génica/efectos de los fármacos , Histonas/metabolismo , Lipopolisacáridos/farmacología , Línea Celular Tumoral , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Inmunidad Innata , Nasofaringe/citología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Respiratory tract infections have an enormous social economic impact, with high incidence of hospitalization and high costs. Adequate specimen collection is the first crucial step for the correct diagnosis of viral respiratory infections. OBJECTIVES: The present retrospective study aimed: i) to verify the cell yield obtained from sampling the nasal respiratory tract using mid-turbinate flocked swabs; ii) to evaluate the normalization of viral load, based on cell number; and iii) to compare the kinetics of viral infection obtained with normalized vs non-normalized viral load. STUDY DESIGN: The number of cells were quantified by real-time PCR in residual extract of nasal swabs tested for respiratory viruses detection and stored at -80 °C in a universal transport medium (UTM™). RESULTS: A total of 513 virus-positive and 226 virus-negative samples were analyzed. Overall, a median of 4.42 log10 ß2-microgolubin DNA copy number/ml of UTM™ (range 1.17-7.26) was detected. A significantly higher number of cells was observed in virus-positive as compared to virus-negative samples (4.75 vs 3.76; p < 0.001). Viral loads expressed as log10 RNA copies/ml of UTM™ and log10 RNA copies/median number of cells were compared in virus-positive samples and a strict correlation (r = 0.89, p < 0.001) and agreement (R2 = 0.82) were observed. In addition, infection kinetics were compared using the two methods with a follow-up series of eight episodes of viral infection and the mean difference was -0.57 log10 (range -1.99 to 0.40). CONCLUSIONS: The normalization of viral load using cellular load compliments the validation of real-time PCR results in the diagnosis of respiratory viruses but is not strictly needed.
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ADN/análisis , Nasofaringe/citología , Infecciones del Sistema Respiratorio/diagnóstico , Manejo de Especímenes/métodos , Carga Viral/métodos , Virus/aislamiento & purificación , Humanos , Cinética , Nasofaringe/virología , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones del Sistema Respiratorio/virología , Estudios Retrospectivos , Sensibilidad y Especificidad , Carga Viral/normas , Virosis/diagnóstico , Esparcimiento de Virus , Virus/genéticaRESUMEN
Deregulated microRNAs play an important role in the development and progression of various types of cancer. In our previous study, we observed that microRNA3423p (miR3423p) was one of the most markedly downregulated microRNAs in two nasopharyngeal carcinoma (NPC) cell lines compared to nonneoplastic cells by using whole genome small RNA sequencing. In the present study, we confirmed that the expression of miR3423p was significantly reduced in NPC tissues compared with normal nasopharyngeal epithelial tissues. Overexpression of miR3423p inhibited proliferation, epithelialmesenchymal transition (EMT), migration and invasiveness of NPC cells. In addition, we observed that Cdc42, a Rho GTPase family member involved in cell proliferation and metastasis, is a direct target of miR3423p. Additionally, ML141, a smallmolecule inhibitor of Cdc42, efficiently suppressed the invasion of NPC cells compared with the control cells. Finally, we analyzed NPC tissues derived from 10 NPC patients and subjected them to quantitative RTPCR and immunohistochemistry assays for concomitant determination of the expression levels of miR3423p and Cdc42. Our results revealed that miR3423p levels were significantly inversely correlated with the protein levels of its target Cdc42. The results of the present study indicated that miR3423p inhibited NPC tumor growth and invasion by directly targeting the Cdc42 pathway.
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Carcinoma/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Neoplasias Nasofaríngeas/genética , Proteína de Unión al GTP cdc42/genética , Biopsia , Carcinoma/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/genética , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/genética , Epitelio/patología , Humanos , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patología , Nasofaringitis/patología , Nasofaringe/citología , Nasofaringe/patología , Invasividad Neoplásica/genética , Pirazoles/farmacología , Sulfonamidas/farmacología , Proteína de Unión al GTP cdc42/antagonistas & inhibidores , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Inhibition of viral replication by icIgA antibodies has only been observed with in vitro studies using epithelial cell lines in transwell cultures. This effect appears to involve an interaction between polymeric immunoglobulin A (pIgA) and viral particles within an intracellular compartment, since IgA is transported across polarized cells. Polyclonal guinea pig antisera against purified influenza A virus and mouse antisera prepared against Influenza A/H3N2 hemagglutinin (HA0) cleavage loop peptides, were used in confocal fluorescence microscopy to show specific staining of wild-type influenza H1N1 and H3N2 viruses in clinical specimens. The HA0 cleavage loop peptides used for intranasal immunization of mice were designed and synthesized from specific conserved regions of influenza A/H1N1 & A/H3N2 viruses. Anti-human secretory IgA antibodies were used to show co-localisation of influenza A virus and icIgA. The results showed specific immunofluorescent staining of influenza A/H3N2 (X31) (HA0 uncleaved)-infected MDCK cells and the presence of icIgA in respiratory exudate cells of infected patients. Both results confirm specific co-localisation and suggest interaction between influenza A virus and icIgA in patients' respiratory exudate cells. Importantly, antisera to the mouse anti-HA0 cleavage site were specific for wild-type virus in clinical specimens, indicating that the conserved region of HA0 was present in the uncleaved form. Similar staining and colocalization patterns between icIgA and virus were observed with polyclonal guinea pig antisera against influenza A virus. These are the first observations of co-localization of influenza A virus and intracellular IgA in clinical specimens. Role of icIgA: This report shows the co-localization of influenza A virus HA0 and icIgA antibodies in respiratory exudate cells of patients who were culture and viral RNA positive, suggesting that icIgA directed against the conserved HA0 site may have a privileged and unique opportunity to act on immature virus and thus prevent HA0 cleavage, maturation and subsequent cycles of viral replication. The precise mechanism by which icIgA mediates intracellular viral neutralization remains to be fully elucidated. SIGNIFICANCE: The above findings in clinical specimens would contribute strongly to our understanding of the mechanisms and kinetics of icIgA neutralization in relation to viral entry and early replication steps of mucosal viral infections. A rapid, objective and sensitive assay - by ex vivo enumeration of respiratory epithelial cells that have co-localized influenza virus and icIgA - would contribute to further mucosal immunity studies and inform the design of more effective vaccines against influenza and other viral infections transmitted via the mucosal route e.g. respiratory syncytial virus, rotavirus.
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Anticuerpos Antivirales/inmunología , Citoplasma/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Inmunoglobulina A/inmunología , Virus de la Influenza A/inmunología , Nasofaringe/virología , Animales , Perros , Técnica del Anticuerpo Fluorescente , Cobayas , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H3N2 del Virus de la Influenza A/inmunología , Células de Riñón Canino Madin Darby , Ratones , Nasofaringe/citología , Replicación ViralRESUMEN
Neutrophil recruitment to the nasopharynx (NP) is a central event in resolution of NP-initiated microbial infections. A vigorous neutrophil response in infected tissues is also associated with the outcome of adverse tissue pathology. Therefore, differences in infection-induced tissue neutrophil numbers may correlate with pathogenesis events. Existing methods of quantifying neutrophils require evaluation of NP samples within hours of procurement as flow cytometry based cell quantification methods require live neutrophil cells. Therefore, we developed a novel RT-PCR method that could reliably quantify neutrophil counts in frozen NP wash samples. mRNA transcripts of the genes encoding CD16, CD18, and CD62L were identified as neutrophil-specific in NP samples and not significantly variable in response to stimulation by heat killed bacteria, and can be used to derive an accurate assessment of neutrophil content in a sample even in the presence of epithelial cells. Using flow cytometry as a comparator, the method was validated in human NP wash samples. We conclude that this PCR-based method should prove useful for providing a quantitative estimate of neutrophil recruitment to the NP during infection and pathogenesis.
Asunto(s)
Nasofaringe/inmunología , Nasofaringe/metabolismo , Neutrófilos , Reacción en Cadena de la Polimerasa/métodos , Recuento de Células , Células Epiteliales/inmunología , Citometría de Flujo/métodos , Humanos , Nasofaringe/citología , Infiltración NeutrófilaRESUMEN
Cancer initiation and progression follow complex changes of cellular architecture and biomechanical property. Cancer cells with more submissive (or "softer") than their healthy counterparts attributed to the reorganization of the complex cytoskeleton structure, may be considered as a potential anti-tumor therapeutic target. In this study, atomic force microscopy (AFM) was carried out to detect the topographical and biophysical changes of nasopharyngeal carcinoma CNE-2Z cells and normal nasopharyngeal epithelial cells NP69-SV40T by treating the Disulfiram chelated with Cu2+ (DSF-Cu). DSF-Cu induced the apoptotic population, ROS production and decreased the NF-κB-p65 expression of CNE-2Z cells, which was much higher than those of NP69-SV40T cells. DSF-Cu caused the obvious changes of cell morphology and membrane ultrastructure in CNE-2Z cells. The roughness decreased and stiffness increased significantly in CNE-2Z cells, which correlated with the rearrangement of intracellular F-actin, FLNa and α-tubulin structures in CNE-2Z cells. And the adhesion force of CNE-2Z cells was also increased accompanied with the increased E-cadherin expression. However, these results could not be observed in the NP69-SV40T cells even the concentration of DSF reached up to 400nM. Finally, the detection of cell wound scratch assay confirmed DSF-Cu could inhibit the migration of CNE-2Z cells, but no effect on NP69-SV40T cells. These findings demonstrated the selective cytotoxicity of DSF-Cu in CNE-2Z cells may attribute to the different mechanical properties and cytoskeleton rearrangement from the normal nasopharyngeal epithelial cells.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma/tratamiento farmacológico , Disulfiram/análogos & derivados , Neoplasias Nasofaríngeas/tratamiento farmacológico , Actinas/metabolismo , Apoptosis/efectos de los fármacos , Fenómenos Biomecánicos , Carcinoma/patología , Carcinoma/fisiopatología , Adhesión Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/patología , Citoesqueleto/fisiología , Disulfiram/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/fisiología , Filaminas/metabolismo , Humanos , Microscopía de Fuerza Atómica , Carcinoma Nasofaríngeo , Neoplasias Nasofaríngeas/patología , Neoplasias Nasofaríngeas/fisiopatología , Nasofaringe/citología , Nasofaringe/efectos de los fármacos , Nasofaringe/fisiología , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción ReIA/antagonistas & inhibidores , Tubulina (Proteína)/metabolismoRESUMEN
Many assays have been developed for the detection of influenza virus which is an important respiratory pathogen. Development of these assays commonly involves the use of human clinical samples for validation of their performance. However, clinical samples can be difficult to obtain, deteriorate over time, and be inconsistent in composition. The goal of this study was to develop a simulated respiratory secretion (SRS) that could act as a surrogate for clinical samples. To this end, we determined the effects major respiratory secretion components (Na+, K+, Ca2+, cells, albumin IgG, IgM, and mucin) have on the performance of influenza assays including both nucleic acid amplification and rapid antigen assays. Minimal effects on the molecular assays were observed for all of the components tested, except for serum derived human IgG, which suppressed the signal of the rapid antigen assays. Using dot blots we were able to show anti-influenza nucleoprotein IgG antibodies are common in human respiratory samples. We composed a SRS that contained mid-point levels of human respiratory sample components and studied its effect compared to phosphate buffered saline and virus negative clinical sample matrix on the Veritor, Sofia, CDC RT-PCR, Simplexa, cobas Liat, and Alere i influenza assays. Our results demonstrated that a SRS can interact with a variety of test methods in a similar manner to clinical samples with a similar impact on test performance.
Asunto(s)
Biomarcadores/análisis , Gripe Humana/diagnóstico , Nasofaringe/citología , Nasofaringe/metabolismo , Proteínas del Núcleo Viral/inmunología , Células A549 , Algoritmos , Diagnóstico Precoz , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/inmunología , Virus de la Influenza B/genética , Virus de la Influenza B/inmunología , Modelos Biológicos , Técnicas de Diagnóstico Molecular/métodos , Nasofaringe/inmunología , Nasofaringe/virología , Sensibilidad y EspecificidadRESUMEN
The auditory tube plays a fundamental role in regulating middle ear pressure. A "system" sensitive to a pressure gradient between the middle ear and the ambient environment is necessary. The presence of mechanoreceptors in the middle ear and the tympanic membrane has been studied, but the presence of these receptors in the nasopharyngeal region remains unclear. The aim of this study is to confirm the presence of pressure sensitive corpuscles in the nasopharynx. An experimental study was conducted on five fresh and unembalded human cadavers. The pharyngeal ostium of the auditory tube and its periphery was removed in one piece by video-assisted endonasal endoscopy. Samples were fixed in formaldehyde solution, embedded in paraffin, and cut. Slides were analyzed by HES (Hematoxyline Eosine Safran) coloration, by S100 protein and neurofilament protein immunostaining. Encapsulated nerve endings were researched and identified by slides analysis. Eight samples were included in our study. On seven samples, Ruffini corpuscles were identified in the mucosa of the posterior area of the pharyngeal ostium, with a higher concentration in the pharyngeal recess and in the posterior nasopharyngeal wall. Our study identified nasopharyngeal mechanoreceptors that could detect the nasopharyngeal pressure and, by extension, the atmospheric pressure. These findings support the theory of the neuronal reflex arc of isobaric system of the middle ear, based on the existence of a "system" sensitive to a pressure gradient between the middle ear and the ambient environment. Understanding of this system has been helpful in the diagnosis and management of middle ear diseases.