RESUMEN
Lung cancer diagnostics have progressed greatly in the previous decade. Development of molecular testing to identify an increasing number of potentially clinically actionable genetic variants, using smaller samples obtained via minimally invasive techniques, is a huge challenge. Tumour heterogeneity and cancer evolution in response to therapy means that repeat biopsies or circulating biomarkers are likely to be increasingly useful to adapt treatment as resistance develops. We highlight some of the current challenges faced in clinical practice for molecular testing of EGFR, ALK, and new biomarkers such as PDL1. Implementation of next generation sequencing platforms for molecular diagnostics in non-small-cell lung cancer is increasingly common, allowing testing of multiple genetic variants from a single sample. The use of next generation sequencing to recruit for molecularly stratified clinical trials is discussed in the context of the UK Stratified Medicine Programme and The UK National Lung Matrix Trial.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias Pulmonares/patología , Quinasa de Linfoma Anaplásico , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/análisis , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/genética , Mutación , Proteínas Tirosina Quinasas Receptoras/genética , Índice de Severidad de la EnfermedadRESUMEN
The LungPath project investigated differences in lung cancer diagnostic practice by following the diagnostic pathways of 1507 patients from 19 representative English lung cancer centres. We found large variation in the proportion of patients receiving positron emission tomography-CT scan (range 13%-64%) and endobronchial ultrasound (range 2%-31%). There was also wide variation in the proportion of patients with good performance status who had their tumours histologically confirmed (range 61%-100%). The variation is discussed with reference to current national guidelines and implications for patient care.
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Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Diagnóstico por Imagen/métodos , Diagnóstico por Imagen/normas , Neoplasias Pulmonares/diagnóstico , Estadificación de Neoplasias/métodos , Broncoscopía , Carcinoma de Pulmón de Células no Pequeñas/epidemiología , Endosonografía , Inglaterra/epidemiología , Humanos , Biopsia Guiada por Imagen , Neoplasias Pulmonares/epidemiología , Morbilidad/tendencias , Tomografía de Emisión de Positrones , Reproducibilidad de los Resultados , Tomografía Computarizada por Rayos XRESUMEN
RATIONALE: The current management of lymphoma requires accurate diagnosis and subtyping of de novo lymphoma and of relapsed or refractory lymphoma in known cases. The role of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the clinical management of lymphomas is unclear. OBJECTIVES: To investigate the use of EBUS-TBNA in the diagnosis of de novo and relapsed mediastinal lymphomas. METHODS: A total of 2,256 consecutive patients who underwent EBUS-TBNA in a tertiary center between February 2008 and April 2013 were prospectively evaluated. The diagnostic accuracy and clinical use of EBUS-TBNA in 100 cases of de novo or suspected relapsed mediastinal lymphoma was investigated by comparing EBUS-TBNA diagnosis with the final diagnosis. MEASUREMENTS AND MAIN RESULTS: De novo mediastinal lymphoma was correctly diagnosed by EBUS-TBNA in 45 (88%) of 51 and relapsed lymphoma in 15 (100%) of 15 lymphoma cases. EBUS-TBNA accurately established a diagnosis other than lymphoma in 32 (97%) of 33 patients with suspected lymphoma relapse. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of EBUS-TBNA in the diagnosis of mediastinal lymphoma were 89%, 97%, 98%, 83%, and 91%, respectively. Sensitivity of EBUS-TBNA in subtyping lymphomas into high-grade non-Hodgkin lymphoma, low-grade non-Hodgkin lymphoma, and Hodgkin lymphoma was 90%, 100%, and 79%, respectively. EBUS-TBNA diagnosis was adequate for clinical management in 84 (84%) of 100 cases. CONCLUSIONS: Multimodality evaluation of EBUS-TBNA can be successful in the diagnosis of de novo mediastinal lymphomas and is ideally suited in distinguishing lymphoma relapse from alternative pathologies; it is least sensitive in subtyping Hodgkin lymphoma.
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Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico , Linfoma/patología , Neoplasias del Mediastino/patología , Recurrencia Local de Neoplasia/patología , Adulto , Anciano , Anciano de 80 o más Años , Diagnóstico Diferencial , Femenino , Humanos , Linfoma/diagnóstico por imagen , Masculino , Neoplasias del Mediastino/diagnóstico por imagen , Persona de Mediana Edad , Recurrencia Local de Neoplasia/diagnóstico por imagen , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
Metaplastic thymoma is a rare thymic neoplasm that has generally been considered to follow a benign to indolent clinical course; however, 3 metaplastic thymomas with high-grade malignant transformation to sarcomatoid carcinoma have been reported. In recent years, both conventional metaplastic thymomas and this subset showing malignant transformation have been associated with recurrent YAP1::MAML2 fusions. We report a metaplastic thymoma showing transformation to squamous cell carcinoma, that to our knowledge is the fourth reported in the literature with transition to overtly malignant features and the first showing pure carcinomatous transformation, and in which YAP1::MAML2 fusion was demonstrated via next generation sequencing.
RESUMEN
The coxsackie and adenovirus receptor (CAR) is a member of the junctional adhesion molecule (JAM) family of adhesion receptors and is localised to epithelial cell tight and adherens junctions. CAR has been shown to be highly expressed in lung cancer where it is proposed to promote tumor growth and regulate epithelial mesenchymal transition (EMT), however the potential role of CAR in lung cancer metastasis remains poorly understood. To better understand the role of this receptor in tumor progression, we manipulated CAR expression in both epithelial-like and mesenchymal-like lung cancer cells. In both cases, CAR overexpression promoted tumor growth in vivo in immunocompetent mice and increased cell adhesion in the lung after intravenous injection without altering the EMT properties of each cell line. Overexpression of WTCAR resulted in increased invasion in 3D models and enhanced ß1 integrin activity in both cell lines, and this was dependent on phosphorylation of the CAR cytoplasmic tail. Furthermore, phosphorylation of CAR was enhanced by substrate stiffness in vitro, and CAR expression increased at the boundary of solid tumors in vivo. Moreover, CAR formed a complex with the focal adhesion proteins Src, Focal Adhesion Kinase (FAK) and paxillin and promoted activation of the Guanine Triphosphate (GTP)-ase Ras-related Protein 1 (Rap1), which in turn mediated enhanced integrin activation. Taken together, our data demonstrate that CAR contributes to lung cancer metastasis via promotion of cell-matrix adhesion, providing new insight into co-operation between cell-cell and cell-matrix proteins that regulate different steps of tumorigenesis.
RESUMEN
Airway inflammation and remodelling are important pathophysiologic features in asthma and other respiratory conditions. An intact epithelial cell layer is crucial to maintain lung homoeostasis, and this depends on intercellular adhesion, whilst damaged respiratory epithelium is the primary instigator of airway inflammation. The Coxsackievirus Adenovirus Receptor (CAR) is highly expressed in the epithelium where it modulates cell-cell adhesion stability and facilitates immune cell transepithelial migration. However, the contribution of CAR to lung inflammation remains unclear. Here we investigate the mechanistic contribution of CAR in mediating responses to the common aeroallergen, House Dust Mite (HDM). We demonstrate that administration of HDM in mice lacking CAR in the respiratory epithelium leads to loss of peri-bronchial inflammatory cell infiltration, fewer goblet-cells and decreased pro-inflammatory cytokine release. In vitro analysis in human lung epithelial cells confirms that loss of CAR leads to reduced HDM-dependent inflammatory cytokine release and neutrophil migration. Epithelial CAR depletion also promoted smooth muscle cell proliferation mediated by GSK3ß and TGF-ß, basal matrix production and airway hyperresponsiveness. Our data demonstrate that CAR coordinates lung inflammation through a dual function in leucocyte recruitment and tissue remodelling and may represent an important target for future therapeutic development in inflammatory lung diseases.
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Neumonía , Pyroglyphidae , Receptores Virales , Animales , Humanos , Ratones , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Pulmón/metabolismo , Neumonía/metabolismo , Mucosa Respiratoria/metabolismo , Receptores Virales/metabolismoRESUMEN
(1) Background: Non-small cell lung cancer (NSCLC) in young patients is uncommon. Real-world evidence on the outcomes of these patients is limited. (2) Methods: We conducted a retrospective cohort study of young NSCLC patients, age < 50 years at diagnosis, who were treated between 2011−2020 in South-East-London cancer centres. Clinicopathological characteristics, treatment and outcomes were analysed. (3) Results: Of 248 NSCLC patients, median age was 46 years, 50% (n = 125) female, 58% (n = 145) white, 18% (n = 45) black and 4% (n = 10) Asian ethnicity. Amongst patients with a documented smoking history, 30% (n = 64) were never-smokers. Most patients had adenocarcinoma (77%, n = 191) and presented with metastatic disease (67%, n = 166). Only 31% (n = 76) had treatment with curative intent. In patients who presented or developed metastatic non-squamous NSCLC (n = 179), EGFR mutation status was known in 88% (n = 157) and mutation present in 19% (n = 34), ALK was known in 66% (n = 118) with a translocation in 10% (n = 18), ROS1 status was known in 57% (n = 102) with a translocation in 4% (n = 8), and KRAS status was known in 66% (n = 119) with a mutation in 12% (n = 22). Overall, 76% (n = 152) patients with metastatic NSCLC received first-line systemic anti-cancer therapy. Median overall survival in metastatic NSCLC was 9.0 months (95% CI 6.5−11.6 months), with superior median overall survival in those with a targeted therapy option (28.7 months) compared to those without (6.6 months; p < 0.001). (4) Conclusion: Young patients contribute a significant proportion of those presenting with lung cancer. They present with advanced stage at diagnosis and have a poor prognosis. Identification of a targeted therapy option is associated with improved survival. However, most patients do not have a known genomic driver, which is in part due to limited testing, particularly in the early years of this study period. These findings highlight the particular importance of rapid-turnaround comprehensive genomic profiling in this age group and the need to identify strategies to facilitate earlier diagnosis in young NSCLC patients.
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Endosonografía/métodos , Neoplasias Pulmonares/patología , Neoplasias de Tejido Muscular/patología , Arteria Pulmonar/patología , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico/métodos , Humanos , Neoplasias Pulmonares/cirugía , Masculino , Neoplasias de Tejido Muscular/cirugía , Tomografía Computarizada por Tomografía de Emisión de Positrones , Adulto JovenRESUMEN
BACKGROUND: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has emerged as an important tool for the diagnosis and staging of lung cancer but its role in the diagnosis of tuberculous intrathoracic lymphadenopathy has not been established. The aim of this study was to describe the diagnostic utility of EBUS-TBNA in patients with intrathoracic lymphadenopathy due to tuberculosis (TB). METHODS: 156 consecutive patients with isolated intrathoracic TB lymphadenitis were studied across four centres over a 2-year period. Only patients with a confirmed diagnosis or unequivocal clinical and radiological response to antituberculous treatment during follow-up for a minimum of 6 months were included. All patients underwent routine clinical assessment and a CT scan prior to EBUS-TBNA. Demographic data, HIV status, pathological findings and microbiological results were recorded. RESULTS: EBUS-TBNA was diagnostic of TB in 146 patients (94%; 95% CI 88% to 97%). Pathological findings were consistent with TB in 134 patients (86%). Microbiological investigations yielded a positive culture of TB in 74 patients (47%) with a median time to positive culture of 16 days (range 3-84) and identified eight drug-resistant cases (5%). Ten patients (6%) did not have a specific diagnosis following EBUS; four underwent mediastinoscopy which confirmed the diagnosis of TB while six responded to empirical antituberculous therapy. There was one complication requiring an inpatient admission. CONCLUSIONS: EBUS-TBNA is a safe and effective first-line investigation in patients with tuberculous intrathoracic lymphadenopathy.
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Biopsia con Aguja Fina/estadística & datos numéricos , Endosonografía/estadística & datos numéricos , Tuberculosis Ganglionar/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biopsia con Aguja Fina/métodos , Broncoscopía , Diagnóstico Diferencial , Endosonografía/métodos , Femenino , Estudios de Seguimiento , Humanos , Ganglios Linfáticos/diagnóstico por imagen , Ganglios Linfáticos/microbiología , Ganglios Linfáticos/patología , Masculino , Persona de Mediana Edad , Mycobacterium tuberculosis/aislamiento & purificación , Reproducibilidad de los Resultados , Estudios Retrospectivos , Cavidad Torácica , Tuberculosis Ganglionar/diagnóstico por imagen , Adulto JovenRESUMEN
The coxsackie B virus and adenovirus receptor (CAR) is an attachment receptor for Adenovirus serotype 5 (Ad5) and in many cell types forms homodimers with neighbouring cells as part of a cell adhesion complex. CAR co-operates with cell surface integrin receptors to enable efficient viral entry, but little is known about the mechanism of crosstalk between these two receptor types. Here we show that overexpression of CAR in human epithelial cells leads to increased basal activation of p44/42 MAPK and this is required for efficient Ad5 infection. We demonstrate that CAR forms homodimers in cis and that this dimerisation is enhanced in the presence of Ad5 in a phospho-p44/42-dependent manner. CAR-induced p44/42 activation also leads to increased activation of beta1 and beta3 integrins. Analysis of CAR mutants demonstrates that the cyto domain of CAR is required for CAR-induced p44/42 activation, integrin activation and localisation to cell junctions. This data for the first time demonstrates that signalling downstream of CAR can have a dual effect on integrins and CAR itself in order to promote efficient viral binding to cell membranes.
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Integrina beta1/metabolismo , Integrina beta3/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Receptores Virales/metabolismo , Adenoviridae/metabolismo , Adenoviridae/patogenicidad , Secuencia de Aminoácidos , Animales , Adhesión Celular/fisiología , Línea Celular Tumoral , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Dimerización , Activación Enzimática , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Integrina beta1/genética , Integrina beta3/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Datos de Secuencia Molecular , Conformación Proteica , Receptores Virales/química , Receptores Virales/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transducción de Señal/fisiologíaRESUMEN
Tuberculosis (TB) remains a major global threat and diagnosis of active TB ((ATB) both extra-pulmonary (EPTB), pulmonary (PTB)) and latent TB (LTBI) infection remains challenging, particularly in high-burden countries which still rely heavily on conventional methods. Although molecular diagnostic methods are available, e.g., Cepheid GeneXpert, they are not universally available in all high TB burden countries. There is intense focus on immune biomarkers for use in TB diagnosis, which could provide alternative low-cost, rapid diagnostic solutions. In our previous gene expression studies, we identified peripheral blood leukocyte (PBL) mRNA biomarkers in a non-human primate TB aerosol-challenge model. Here, we describe a study to further validate select mRNA biomarkers from this prior study in new cohorts of patients and controls, as a prerequisite for further development. Whole blood mRNA was purified from ATB patients recruited in the UK and India, LTBI and two groups of controls from the UK (i) a low TB incidence region (CNTRLA) and (ii) individuals variably-domiciled in the UK and Asia ((CNTRLB), the latter TB high incidence regions). Seventy-two mRNA biomarker gene targets were analyzed by qPCR using the Roche Lightcycler 480 qPCR platform and data analyzed using GeneSpring™ 14.9 bioinformatics software. Differential expression of fifty-three biomarkers was confirmed between MTB infected, LTBI groups and controls, seventeen of which were significant using analysis of variance (ANOVA): CALCOCO2, CD52, GBP1, GBP2, GBP5, HLA-B, IFIT3, IFITM3, IRF1, LOC400759 (GBP1P1), NCF1C, PF4V1, SAMD9L, S100A11, TAF10, TAPBP, and TRIM25. These were analyzed using receiver operating characteristic (ROC) curve analysis. Single biomarkers and biomarker combinations were further assessed using simple arithmetic algorithms. Minimal combination biomarker panels were delineated for primary diagnosis of ATB (both PTB and EPTB), LTBI and identifying LTBI individuals at high risk of progression which showed good performance characteristics. These were assessed for suitability for progression against the standards for new TB diagnostic tests delineated in the published World Health Organization (WHO) technology product profiles (TPPs).
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Biomarcadores/metabolismo , Tuberculosis Latente/genética , Tuberculosis Latente/inmunología , Adolescente , Asia , Biología Computacional/métodos , Pruebas Diagnósticas de Rutina/métodos , Femenino , Humanos , Masculino , ARN Mensajero/genética , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Prueba de Tuberculina/métodos , Reino UnidoRESUMEN
The coxsackievirus and adenovirus receptor (CAR) is a transmembrane receptor that plays a key role in cell-cell adhesion. CAR is found in normal epithelial cells and is increased in abundance in various human tumors, including lung carcinomas. We investigated the potential mechanisms by which CAR contributes to cancer cell growth and found that depletion of CAR in human lung cancer cells reduced anchorage-independent growth, epidermal growth factor (EGF)-dependent proliferation, and tumor growth in vivo. EGF induced the phosphorylation of CAR and its subsequent relocalization to cell junctions through the activation of the kinase PKCδ. EGF promoted the binding of CAR to the chromokinesin KIF22. KIF22-dependent regulation of microtubule dynamics led to delayed EGFR internalization, enhanced EGFR signaling, and coordination of CAR dynamics at cell-cell junctions. These data suggest a role for KIF22 in the coordination of membrane receptors and provide potential new therapeutic strategies to combat lung tumor growth.
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Adenocarcinoma/patología , Proliferación Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Proteínas de Unión al ADN/metabolismo , Cinesinas/metabolismo , Neoplasias Pulmonares/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Apoptosis , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/genética , Proteínas de Unión al ADN/genética , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Cinesinas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.
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Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Multimerización de Proteína , Animales , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Matriz Extracelular/metabolismo , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Ligandos , Modelos Biológicos , Modelos Moleculares , Fotoblanqueo , Polímeros/química , Dominios Proteicos , Proteínas Quinasas/química , Proteínas Quinasas/metabolismoRESUMEN
The coxsackie and adenovirus receptor (CAR) is a transmembrane receptor that plays a key role in controlling adhesion between adjacent epithelial cells. CAR is highly expressed in epithelial cells and was originally identified as a primary receptor for adenovirus cell binding. However, studies over the last 10 years have demonstrated that CAR plays a key role in co-ordinating cell-cell adhesion under homeostatic conditions including neuronal and cardiac development and cell junction stability; it has also been implicated in pathological states such as cancer growth and leukocyte transmigration during inflammation. Here we provide an overview of the functions of CAR as an adhesion molecule and highlight the emerging important role for CAR in controlling both recruitment of immune cells and in tumorigenesis.
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Adhesión Celular , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Inflamación/metabolismo , Animales , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/química , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/genética , Regulación de la Expresión Génica , HumanosRESUMEN
Advanced lung cancer has poor survival with few therapies. EGFR tyrosine kinase inhibitors (TKIs) have high response rates in patients with activating EGFR mutations, but acquired resistance is inevitable. Acquisition of the EGFR T790M mutation causes over 50% of resistance; MET amplification is also common. Preclinical data suggest synergy between MET and EGFR inhibitors. We hypothesized that EGFR-MET dimerization determines response to MET inhibition, depending on EGFR mutation status, independently of MET copy number. We tested this hypothesis by generating isogenic cell lines from NCI-H1975 cells, which co-express L858R and T790M EGFR mutations, namely H1975L858R/T790M (EGFR TKI resistant); H1975L858R (sensitized) and H1975WT (wild-type). We assessed cell proliferation in vitro and tumor growth/stroma formation in derived xenograft models in response to a MET TKI (SGX523) and correlated with EGFR-MET dimerization assessed by Förster Resonance Energy Transfer (FRET). SGX523 significantly reduced H1975L858R/T790M cell proliferation, xenograft tumor growth and decreased ERK phosphorylation. The same was not seen in H1975L858R or H1975WT cells. SGX523 only reduced stroma formation in H1975L858R. SGX523 reduced EGFR-MET dimerization in H1975L858R/T790M but induced dimer formation in H1975L858R with no effect in H1975WT. Our data suggests that MET inhibition by SGX523 and EGFR-MET heterodimerisation are determined by EGFR genotype. As tumor behaviour is modulated by this interaction, this could determine treatment efficacy.
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Adenocarcinoma/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Células HEK293 , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNFα acts in a paracrine manner on epithelial cells via a TNFR1-PI3K-PKCδ pathway leading to CAR phosphorylation and subsequent transmigration across cell junctions. Moreover, we show that CAR is hyper-phosphorylated in vivo in acute and chronic lung inflammation models and this response is required to facilitate immune cell recruitment. This represents a novel mechanism of feedback between leukocytes and epithelial cells during TEpM and may be important in controlling responses to pro-inflammatory cytokines in pathological settings.
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Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Leucocitos/fisiología , Migración Transendotelial y Transepitelial/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Comunicación Celular , Línea Celular Transformada , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Humanos , Uniones Intercelulares/fisiología , Leucocitos/efectos de los fármacos , Ratones , Fosforilación , Neumonía , Migración Transendotelial y Transepitelial/fisiologíaRESUMEN
BACKGROUND: Mycobacterium tuberculosis infection is a leading cause of infectious death worldwide. Gene-expression microarray studies profiling the blood transcriptional response of tuberculosis (TB) patients have been undertaken in order to better understand the host immune response as well as to identify potential biomarkers of disease. To date most of these studies have focused on pulmonary TB patients with gene-expression profiles of extra-pulmonary TB patients yet to be compared to those of patients with pulmonary TB or sarcoidosis. METHODS: A novel cohort of patients with extra-pulmonary TB and sarcoidosis was recruited and the transcriptional response of these patients compared to those with pulmonary TB using a variety of transcriptomic approaches including testing a previously defined 380 gene meta-signature of active TB. RESULTS: The 380 meta-signature broadly differentiated active TB from healthy controls in this new dataset consisting of pulmonary and extra-pulmonary TB. The top 15 genes from this meta-signature had a lower sensitivity for differentiating extra-pulmonary TB from healthy controls as compared to pulmonary TB. We found the blood transcriptional responses in pulmonary and extra-pulmonary TB to be heterogeneous and to reflect the extent of symptoms of disease. CONCLUSIONS: The transcriptional signature in extra-pulmonary TB demonstrated heterogeneity of gene expression reflective of symptom status, while the signature of pulmonary TB was distinct, based on a higher proportion of symptomatic individuals. These findings are of importance for the rational design and implementation of mRNA based TB diagnostics.
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Transcriptoma , Tuberculosis Pulmonar/diagnóstico , Tuberculosis/diagnóstico , Recuento de Células Sanguíneas , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Mycobacterium tuberculosis/aislamiento & purificación , Sarcoidosis/diagnóstico , Sarcoidosis/genética , Sarcoidosis/metabolismo , Tuberculosis/genética , Tuberculosis/metabolismo , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/metabolismo , Regulación hacia ArribaRESUMEN
We present a novel integrated multimodal fluorescence microscopy technique for simultaneous fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM) and fluorescence anisotropy imaging (FAIM). This approach captures a series of polarization-resolved fluorescence lifetime images during a FRAP recovery, maximizing the information available from a limited photon budget. We have applied this method to analyse the behaviour of GFP-labelled coxsackievirus and adenovirus receptor (CAR) in living human epithelial cells. Our data reveal that CAR exists in oligomeric states throughout the cell, and that these complexes occur in conjunction with high immobile fractions of the receptor at cell-cell junctions. These findings shed light on previously unknown molecular associations between CAR receptors in intact cells and demonstrate the power of combined FRAP, FLIM and FAIM microscopy as a robust method to analyse complex multi-component dynamics in living cells.