NOD2 in monocytes negatively regulates macrophage development through TNFalpha.

Objective: It is believed that intestinal recruitment of monocytes from Crohn's Disease (CD) patients who carry NOD2 risk alleles may repeatedly give rise to recruitment of pathogenic macrophages. We investigated an alternative possibility that NOD2 may rather inhibit their differentiation from intravasating monocytes. Design: The monocyte fate decision was examined by using germ-free mice, mixed bone marrow chimeras and a culture system yielding macrophages and monocyte-derived dendritic cells (mo-DCs). Results: We observed a decrease in the frequency of mo-DCs in the colon of Nod2-deficient mice, despite a similar abundance of monocytes. This decrease was independent of the changes in the gut microbiota and dysbiosis caused by Nod2 deficiency. Similarly, the pool of mo-DCs was poorly reconstituted in a Nod2-deficient mixed bone marrow (BM) chimera. The use of pharmacological inhibitors revealed that activation of NOD2 during monocyte-derived cell development, dominantly inhibits mTOR-mediated macrophage differentiation in a TNFα-dependent manner. These observations were supported by the identification of a TNFα-dependent response to muramyl dipeptide (MDP) that is specifically lost when CD14-expressing blood cells bear a frameshift mutation in NOD2. Conclusion: NOD2 negatively regulates a macrophage developmental program through a feed-forward loop that could be exploited for overcoming resistance to anti-TNF therapy in CD.

NOD2 Signaling Circuitry during Allergen Sensitization Does Not Worsen Experimental Neutrophilic Asthma but Promotes a Th2/Th17 Profile in Asthma Patients but Not Healthy Subjects.

Nucleotide-binding oligomerization domain 2 (NOD2) recognizes pathogens associated with the development of asthma. Moreover, NOD2 adjuvants are used in vaccine design to boost immune responses. Muramyl di-peptide (MDP) is a NOD2 ligand, which is able to promote Th2/Th17 responses. Furthermore, polymorphisms of the NOD2 receptor are associated with allergy and asthma development. This study aimed to evaluate if MDP given as an adjuvant during allergen sensitization may worsen the development of Th2/Th17 responses. We used a mouse model of Th2/Th17-type allergic neutrophil airway inflammation (AAI) to dog allergen, with in vitro polarization of human naive T cells by dendritic cells (DC) from healthy and dog-allergic asthma subjects. In the mouse model, intranasal co-administration of MDP did not modify the AAI parameters, including Th2/Th17-type lung inflammation. In humans, MDP co-stimulation of allergen-primed DC did not change the polarization profile of T cells in healthy subjects but elicited a Th2/Th17 profile in asthma subjects, as compared with MDP alone. These results support the idea that NOD2 may not be involved in the infection-related development of asthma and that, while care has to be taken in asthma patients, NOD2 adjuvants might be used in non-sensitized individuals.

NOD-like receptors in asthma.

Asthma is an extremely prevalent chronic inflammatory disease of the airway where innate and adaptive immune systems participate collectively with epithelial and other structural cells to cause airway hyperresponsiveness, mucus overproduction, airway narrowing, and remodeling. The nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular innate immune sensors that detect microbe-associated molecular patterns and damage-associated molecular patterns, well-recognized for their central roles in the maintenance of tissue homeostasis and host defense against bacteria, viruses and fungi. In recent times, NLRs have been increasingly acknowledged as much more than innate sensors and have emerged also as relevant players in diseases classically defined by their adaptive immune responses such as asthma. In this review article, we discuss the current knowledge and recent developments about NLR expression, activation and function in relation to asthma and examine the potential interventions in NLR signaling as asthma immunomodulatory therapies.

A Notch/STAT3-driven Blimp-1/c-Maf-dependent molecular switch induces IL-10 expression in human CD4+ T cells and is defective in Crohn´s disease patients.

Immunosuppressive Interleukin (IL)-10 production by pro-inflammatory CD4+ T cells is a central self-regulatory function to limit aberrant inflammation. Still, the molecular mediators controlling IL-10 expression in human CD4+ T cells are largely undefined. Here, we identify a Notch/STAT3 signaling-module as a universal molecular switch to induce IL-10 expression across human naïve and major effector CD4+ T cell subsets. IL-10 induction was transient, jointly controlled by the transcription factors Blimp-1/c-Maf and accompanied by upregulation of several co-inhibitory receptors, including LAG-3, CD49b, PD-1, TIM-3 and TIGIT. Consistent with a protective role of IL-10 in inflammatory bowel diseases (IBD), effector CD4+ T cells from Crohn's disease patients were defective in Notch/STAT3-induced IL-10 production and skewed towards an inflammatory Th1/17 cell phenotype. Collectively, our data identify a Notch/STAT3-Blimp-1/c-Maf axis as a common anti-inflammatory pathway in human CD4+ T cells, which is defective in IBD and thus may represent an attractive therapeutic target.

A rapid test for the environmental detection of pigeon antigen.

INTRODUCTION: Avoidance of inhaled bird antigens is essential to prevent hypersensitivity pneumonitis disease progression. The aim of the present study was to develop a sandwich enzyme link immunoassay (ELISA) and an immunochromatographic test (ICT) and compare their ability to detect pigeon antigens in environmental samples. METHODS: An amplified sandwich ELISA using pigeon serum as a calibration standard and a ICT using gold-labeled anti-pigeon serum antibodies for the rapid detection of pigeon antigens in environmental samples were developed. Twenty-two different airborne samples were collected and analysed using both methods. Strip density values obtained with ICT were calculated and compared with the concentrations determined by the ELISA method for pigeon antigens. Strips results were also visually analysed by five independent evaluators. RESULTS: The ELISA method to quantify pigeon antigen had a broader range (58.4 and 10,112.2 ng/ml), compared to the ICT assay (420 to 3360 ng/ml). A kappa index of 0.736 (p < 0.0001) was obtained between the observers evaluating the ICT strips. The results of the ELISA and the relative density of the ICT showed a highly significant correlation (rs:0.935; p < 0.0001). Bland-Altman plot also confirmed excellent agreement between the two methods (mean difference: -1.626; p < 0.0001). CONCLUSIONS: Since there was a good correlation between both assays, we can conclude that the rapid and simple ICT assay is a good and valid alternative, which does not require expensive equipment, for the validated ELISA technique.

NOD1 sensing of house dust mite-derived microbiota promotes allergic experimental asthma.

BACKGROUND: Asthma severity has been linked to exposure to gram-negative bacteria from the environment that are recognized by NOD1 receptor and are present in house dust mite (HDM) extracts. NOD1 polymorphism has been associated with asthma. OBJECTIVE: We sought to evaluate whether either host or HDM-derived microbiota may contribute to NOD1-dependent disease severity. METHODS: A model of HDM-induced experimental asthma was used and the effect of NOD1 deficiency was evaluated. Contribution of host microbiota was evaluated by fecal transplantation. Contribution of HDM-derived microbiota was assessed by 16S ribosomal RNA sequencing, mass spectrometry analysis, and peptidoglycan depletion of the extracts. RESULTS: In this model, loss of the bacterial sensor NOD1 and its adaptor RIPK2 improved asthma features. Such inhibitory effect was not related to dysbiosis caused by NOD1 deficiency, as shown by fecal transplantation of Nod1-deficient microbiota to wild-type germ-free mice. The 16S ribosomal RNA gene sequencing and mass spectrometry analysis of HDM allergen, revealed the presence of some muropeptides from gram-negative bacteria that belong to the Bartonellaceae family. While such HDM-associated muropeptides were found to activate NOD1 signaling in epithelial cells, peptidoglycan-depleted HDM had a decreased ability to instigate asthma in vivo. CONCLUSIONS: These data show that NOD1-dependent sensing of HDM-associated gram-negative bacteria aggravates the severity of experimental asthma, suggesting that inhibiting the NOD1 signaling pathway may be a therapeutic approach to treating asthma.

Validation of an Asbestos Exposure Questionnaire (QEAS-7) for Clinical Practice.

INTRODUCTION: The seven-item QEAS-7 questionnaire (exposure to asbestos questionnaire) has been designed as a useful and simple tool to establish the probability of exposure to asbestos. The objective of the present study is to validate the QEAS-7 following the recommended methodology. METHODS: The QEAS-7 was prospectively administered to 90 subjects with and without asbestos-related disease (ARD), on two consecutive occasions by two independent researchers. Logical and content validity was evaluated by a committee of experts and construct validity through hypothesis testing. Intra- and interobserver reliability was assessed by calculating Cohen's Kappa index (κ), which was estimated as weak if below 0.40, moderate if between 0.41 and 0.60 and good/very good if above 0.60. The comparison between proportions was examined using Pearson's Chi-square test. RESULTS: The majority of participants (88.9%) were male. Mean age was 70.8 years (SD = 8.4) and most of the sample had completed primary education but had not progressed further (62.2%). Forty-three had ARD. The logical, content and construct validity of the QEAS-7 was considered adequate both by a committee of experts and by the users interviewed. The mean administration time was 9 min and 25 s (SD = 3 min and 49 s). The verification of the five hypotheses confirmed the construct validity and the intra- and interobserver reliability to be κ = 0.93 and κ = 0.50 respectively. The concordance in the estimation of asbestos exposure was κ = 0.65. CONCLUSIONS: The QEAS-7 is a simple, valid and reliable tool for estimating the probability of exposure to asbestos. Its application in clinical practice appears justified. What is already known about this subject? No studies have been published to date on the validation of specific questionnaires designed to determine asbestos exposure for routine use by healthcare staff in the clinical setting. What are the new findings? This questionnaire can be considered a comprehensible, viable, valid and reliable instrument for identifying exposure to asbestos. Its brevity and simplicity of administration make it ideally suited for use in daily clinical practice. How might this impact on policy or clinical practice in the foreseeable future? This questionnaire can be of help for physicians attending to patients with suspected asbestos-related diseases both in the hospital and in the primary care setting.

The Aryl Hydrocarbon Receptor in Asthma: Friend or Foe?

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has emerged as an important player in asthma control. AhR is responsive to environmental molecules and endogenous or dietary metabolites and regulates innate and adaptive immune responses. Binding of this receptor by different ligands has led to seemingly opposite responses in different asthma models. In this review, we present two sides of the same coin, with the beneficial and deleterious roles of AhR evaluated using known endogenous or exogenous ligands, deficient mice or antagonists. On one hand, AhR has an anti-inflammatory role since its activation in dendritic cells blocks the generation of pro-inflammatory T cells or shifts macrophages toward an anti-inflammatory M2 phenotype. On the other hand, AhR activation by particle-associated polycyclic aromatic hydrocarbons from the environment is pro-inflammatory, inducing mucus hypersecretion, airway remodelling, dysregulation of antigen presenting cells and exacerbates asthma features. Data concerning the role of AhR in cells from asthmatic patients are also reviewed, since AhR could represent a potential target for therapeutic immunomodulation.

Development of a Dipstick Assay for Soy Allergens: Inexpensive Detection to Control Allergen Exposure.

Allergen measurements are use in the food industry and are also routinely performed as part of indoor air quality investigations and occupational health monitoring. In this chapter we describe how to develop a simple, convenient, rapid test to analyze soy allergens that can be used in production environments by non-skilled staff to facilitate immediate corrective action and minimize risk and that can be produced in laboratories not equipped with sophisticated instruments.The strip assay described consists of a membrane that is bonded to an adhesive support where an absorbent pad is also attached to absorb excess reagents. The membrane is stripped with specific antibodies against soy allergens in this case (test line) and a goat anti-rabbit IgG antibody (control line). The strip is exposed to a mix of sample and gold-conjugated specific antibody. Colored bands are read out visually or by densitometry.

Effects of diesel exhaust particle exposure on a murine model of asthma due to soybean.

BACKGROUND: Exposure to soybean allergens has been linked to asthma outbreaks. Exposure to diesel exhaust particles (DEP) has been associated with an increase in the risk of asthma and asthma exacerbation; however, in both cases the underlying mechanisms remain poorly understood, as does the possible interaction between the two entities. OBJECTIVE: To investigate how the combination of soybean allergens and DEP can affect the induction or exacerbation of asthma in a murine model. METHODS: BALB/c mice received intranasal instillations of saline, 3 or 5 mg protein/ml soybean hull extract (SHE), or a combination of one of these three solutions with DEP. Airway hyperresponsiveness (AHR), pulmonary inflammation in bronchoalveolar lavage, total serum immunoglobulin E and histological studies were assessed. RESULTS: A 5 mg protein/ml SHE solution was able by itself to enhance AHR (p = 0.0033), increase eosinophilic inflammation (p = 0.0003), increase levels of IL-4, IL-5, IL-13, IL-17A, IL-17F and CCL20, and reduce levels of IFN-γ. The combination of 5 mg protein/ml SHE with DEP also produced an increase in AHR and eosinophilic inflammation, but presented a slightly different cytokine profile with higher levels of Th17-related cytokines. However, while the 3 mg protein/ml SHE solution did not induce asthma, co-exposure with DEP resulted in a markedly enhanced AHR (p = 0.002) and eosinophilic inflammation (p = 0.004), with increased levels of IL-5, IL-17F and CCL20 and decreased levels of IFN-γ. CONCLUSIONS & CLINICAL RELEVANCE: The combination of soybean allergens and DEP is capable of triggering an asthmatic response through a Th17-related mechanism when the soybean allergen concentration is too low to promote a response by itself. DEP monitoring may be a useful addition to allergen monitoring in order to prevent new asthma outbreaks.

Utilidad del lavado broncoalveolar en el diagnóstico de enfermedades relacionadas con el amianto / Utility of Bronchoalveolar Lavage for the Diagnosis of Asbestos-Related Diseases

Introducción: El análisis del lavado broncoalveolar (LBA) se ha propuesto como técnica objetiva para certificar la exposición a amianto. Sin embargo, la fiabilidad y rendimiento diagnóstico de este procedimiento diagnóstico no se han analizado en España. El propósito de este estudio fue evaluar la utilidad del análisis de cuerpos de amianto (CA) en el LBA para el diagnóstico de enfermedades relacionadas con el amianto (ERA). Métodos: Se analizaron muestras de LBA de 72 pacientes (66 varones, edad media de 66 años) sometidos a broncoscopia. También se analizó el tejido pulmonar de 23 de estos pacientes. La exposición al amianto se evaluó a partir de la anamnesis y la revisión de las historias clínicas de los pacientes. Las muestras de LBA y de tejido pulmonar se procesaron, y la cantidad de CA se determinó mediante microscopia óptica. El valor umbral aceptado para diagnosticar una enfermedad relacionada con el amianto fue de 1 CA/ml de LBA o 1.000 CA/g de tejido seco. Resultados: Treinta y nueve pacientes refirieron exposición a amianto. En 13 (33%) de estos pacientes, los niveles de CA fueron superiores a 1 CA/ml de LBA. De los 33 pacientes no expuestos, los valores de CA fueron superiores a 1 CA/ml de LBA en 5 casos (15%). La diferencia entre los niveles de CA de los pacientes expuestos y los no expuestos fue significativa (p = 0,006). La curva ROC indicó que el nivel de 0,5 CA/ml de LBA era el que alcanzaba mayor sensibilidad (46%), con un 83% de especificidad. El grado de correlación entre los niveles de CA en el LBA y el tejido pulmonar fue de 0,633 (p = 0,002). Conclusiones: El estudio del LBA ofrece una prueba objetiva de la exposición a amianto. La buena correlación observada entre los recuentos de CA en el LBA y en el tejido pulmonar indica la validez de ambas técnicas para analizar el contenido de amianto (AU)

Introduction: Bronchoalveolar lavage (BAL) analysis has been proposed as an objective technique for confirming asbestos exposure. However, the reliability and diagnostic yield of this procedure has not been studied in Spain. The aim of this study was to assess the usefulness of the analysis of asbestos bodies (AB) in bronchoalveolar lavage (BAL) for the diagnosis of asbestos-related diseases (ARD). Methods: BAL samples from 72 patients (66 male, mean age 66 years) undergoing bronchoscopy were analyzed. Lung tissue from 23 of these patients was also analyzed. Asbestos exposure was assessed by anamnesis and a review of the patient's medical records. BAL and lung samples were processed and AB count was determined by light microscopy. The accepted threshold value to diagnose asbestos-related diseases was 1 AB/ml BAL or 1000 AB/gr dry tissue. Results: Thirty-nine patients reported exposure to asbestos. Of these, 13 (33%) presented AB values above 1 AB/ml BAL. In the 33 non-exposed patients, 5 (15%) presented AB values above 1 AB/ml BAL. There was a significant difference between the AB levels of exposed and non-exposed patients (P = .006). The ROC curve showed that a value of 0.5 AB/ml BAL achieved the most satisfactory sensitivity, 46%, and a specificity of 83%. The correlation between AB levels in BAL and lung was 0.633 (P = .002). Conclusions: BAL study provides objective evidence of exposure to asbestos. The good correlation between the AB counts in BAL and lung tissue indicates that both techniques are valid for the analysis of asbestos content (AU)

Utility of Bronchoalveolar Lavage for the Diagnosis of Asbestos-Related Diseases. / Utilidad del lavado broncoalveolar en el diagnóstico de enfermedades relacionadas con el amianto.

INTRODUCTION: Bronchoalveolar lavage (BAL) analysis has been proposed as an objective technique for confirming asbestos exposure. However, the reliability and diagnostic yield of this procedure has not been studied in Spain. The aim of this study was to assess the usefulness of the analysis of asbestos bodies (AB) in bronchoalveolar lavage (BAL) for the diagnosis of asbestos-related diseases (ARD). METHODS: BAL samples from 72 patients (66 male, mean age 66 years) undergoing bronchoscopy were analyzed. Lung tissue from 23 of these patients was also analyzed. Asbestos exposure was assessed by anamnesis and a review of the patient's medical records. BAL and lung samples were processed and AB count was determined by light microscopy. The accepted threshold value to diagnose asbestos-related diseases was 1 AB/ml BAL or 1000 AB/gr dry tissue. RESULTS: Thirty-nine patients reported exposure to asbestos. Of these, 13 (33%) presented AB values above 1 AB/ml BAL. In the 33 non-exposed patients, 5 (15%) presented AB values above 1 AB/ml BAL. There was a significant difference between the AB levels of exposed and non-exposed patients (P=.006). The ROC curve showed that a value of 0.5 AB/ml BAL achieved the most satisfactory sensitivity, 46%, and a specificity of 83%. The correlation between AB levels in BAL and lung was 0.633 (P=.002). CONCLUSIONS: BAL study provides objective evidence of exposure to asbestos. The good correlation between the AB counts in BAL and lung tissue indicates that both techniques are valid for the analysis of asbestos content.

Estudios inmunológicos y pruebas de provocación bronquial específicas, técnicas esenciales para el diagnóstico de neumonitis por hipersensibilidad / Immunological studies and specific bronchial challenge tests, essential techniques for the diagnosis of hypersensitivity pneumonitis

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A rapid test for soy aeroallergens exposure assessment.

BACKGROUND: Determining soy aeroallergens levels is extremely important in the assessment of health risks due to these airborne substances. Currently, soy aeroallergens exposure in the environment is monitored using enzyme immunoassays (EIA) which must be evaluated in a specialized laboratory by skilled personnel. OBJECTIVE: To describe the development and performance of a rapid immunochromatography assay for the detection of soy aeroallergens in environmental samples. METHODS: A test strip using gold labeled anti-soy hull low molecular weight extract (SHLMWE) antibody for the rapid detection of soy aeroallergens in environmental samples was developed. One hundred nineteen airborne samples were analysed in parallel by the strip assay and the anti-SHLMWE sandwich EIA. The assay results were visually analysed by three independent observers who ranked samples as: -, + or ++. Strips were also scanned and analysed by densitometry. RESULTS: The rapid test detected a range of concentrations from 6.25 to 25 ng/mL. Agreement in strip assay interpretations between evaluators was substantial (Kappa = 0.63; CI 0.544-0.715). Visual interpretation also gave a good concordance with EIA results, with sensitivity ranging from 77.3 to 100 and specificity from 65 to 83.5 depending on the observer. Furthermore, a strong correlation was observed between densitometry results of strip assay and EIA determinations. CONCLUSIONS: The strip assay developed is rapid, simple, and sensitive and does not require expensive equipment or specific skills. It has considerable potential in the environmental monitoring field for screening soy aeroallergens levels in port cities where allergen measurements are not currently performed. Due to its simplicity, the test will improve the management of soy allergic patients by controlling environmental allergen exposure without the need for apparatus or skilled personnel.

Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells.

Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs.