Prevalence and Quantification of the Effects of Sexual Harassment Victimization of School-Aged Adolescents.

BACKGROUND/AIM: Sexual harassment has become a serious social and public health problem in adolescents, causing adverse effects on mental health. Nevertheless, some behaviours arise that, due to their characteristics, might be misinterpreted as sexual harassment. A field study using a survey with non-probabilistic accidental sampling was designed in order to estimate the prevalence of sexual harassment victimization in the Spanish adolescent population as well as to quantify the harms. METHOD: A total of 1028 Spanish adolescents, 54.3% females and 45.7% males aged 13-17 years (M = 15.21, SD = 1.03), responded to a diagnostic measure of sexual harassment victimization and an inventory measure of internalizing and externalizing mental health problems (MHPs). RESULTS: The results showed a significant prevalence of diagnosed sexual harassment victimization of school-aged adolescents, 24.1%, 95% CI [0.215, 0.267], with adverse effects on internalizing and externalizing MHPs. As for the internalizing MHPs, the results exhibited moderate adverse effects on depression, anxiety, somatic burns, posttraumatic symptoms, and obsessive-compulsive symptoms, as well as mild adverse effects on social anxiety. Regarding externalizing MHPs, the results revealed moderate adverse effects on hyperactivity-impulsivity, anger control, and antisocial behaviour, as well as mild adverse effects on attention problems, aggression, and defiant behaviour. In addition, it was confirmed that sexual harassment victimization affects adolescent females to a greater extent, with the effect being significantly greater in internalizing than in externalizing MHPs. CONCLUSIONS: The results obtained are discussed and future lines of research and intervention are proposed to promote the implementation of prevention and intervention programs that address this phenomenon and, in turn, improve the physical, psychological, and social well-being of adolescents.

Eficacia del tratamiento en la competencia cognitiva en sentenciados por violencia de género / Treatment efficacy on the cognitive competence of convicted intimate partner violence offenders

La evaluación de la eficacia del tratamiento de maltratadores en los Registros Oficiales o los Informes de las parejas no es válida, al tiempo que las intervenciones eficaces se dirigen a dotarlos de competencia cognitiva y conductual. Por ello diseñamos un estudio de campo para medir los efectos de la intervención en la competencia cognitiva. Un total de 100 condenados por violencia de género que completaron un programa penitenciario de tratamiento en la comunidad fueron evaluados pre- y post-tratamiento en la competencia cognitiva, esto es, auto-concepto, inteligencia emocional, atribución de responsabilidad, destrezas cognitivas para el afrontamiento de eventos estresantes en pareja, expresión y control de la ira, y creencias irracionales y pensamientos distorsionados. Los resultados mostraron un efecto significativo del tratamiento en la adquisición de las destrezas cognitivas. Sucintamente, el tratamiento potenció el auto-concepto general el 18.2% y las dimensiones internas del auto-concepto auto-satisfacción el 33.5% y comportamiento el 25.6%; y la claridad para discriminar las emociones el 31.8%. Además, el tratamiento incrementó la asunción interna de responsabilidad el 31.8%; el control de la ira el 19.1%; y el uso de estrategias adaptativas para el afrontamiento de eventos estresantes en pareja entre el 48.9% y el 61.3%. Finalmente, el tratamiento redujo el uso de estrategias desadaptativas entre el 25.6% y el 35.1%; y las creencias irracionales y pensamientos distorsionados relacionados con el uso de la violencia, rol de género y dependencia emocional, en el 78.2%, 48.2% y 63.6%, respectivamente. No obstante, no todos los maltratadores se beneficiaron del tratamiento

The evaluation of the efficacy of treatment of batterers in Official or Couple Records is not valid, whilst the efficient interventions are focused on the empowerment of cognitive and behavioural competence. A field study for measuring the effects of the treatment on the cognitive competence was designed. A total of 100 batterers who had completed a community penitentiary intervention programme, were assessed pre- and post-treatment in cognitive competence i.e., self-concept, emotional intelligence, attribution of responsivity, cognitive skills to cope intimate-partner-related stressful events, expression and control of anger, and irrational beliefs and distorted thoughts. The results showed a significant effect of the treatment in the acquisition of cognitive competence skills. Succinctly, treatment empowered the general self-concept in 18.2%, and the internal dimensions of self-concept self-satisfaction in 33.5% and behaviour in 25.6%; and clarity to discriminate among moods in 31.8%. Moreover, treatment increased the assumption of internal responsivity in 31.8%; anger control in 19.1%; and the use of adaptive strategies to cope intimate-partner-related stressful events between 48.9% and 61.3%. Finally, treatment involved a fall in the use of maladaptive strategies to cope intimate-partner-related stressful events between 25.6% and 35.1%; and in the irrational beliefs and distorted thoughts related with the use of violence, in 78.2%, 48.2% y 63.6%, for use of violence, gender roles and emotional dependence, respectively. Nonetheless, not all the batterers benefited from treatment

[Compliance of latent tuberculosis infection treatment in a cohort of TB contacts]. / Cumplimiento del tratamiento de la infección tuberculosa latente en una cohorte de contactos de enfermos de tuberculosis.

OBJECTIVE: Compliance with the treatment of Latent Tuberculous Infection (ITL) is a determining factor in the control of tuberculosis. The objective of this study was to estimate the acceptance and compliance of the ITL treatment, and associated factors in contacts of patients with tuberculosis in Lleida. METHODS: Epidemiological analytical observational study, of a retrospective cohort follow-up, since the 1 January 2015 to 31 December 2016. The Participants were contacts of patients with tuberculosis in Lleida. Variables of the index case and independent and dependent variables (acceptance and compliance of the treatment) of contacts were studied through univariate and bivariate analysis. The strength of association was studied with odds ratio (OR) and confidence interval (IC) of 95% they were adjusted by a multivariate regression model. RESULTS: 69.1% of tuberculosis cases had a contact study, and 47.5% had at least one contact with prescription of preventive treatment. The treatment was accepted by 94.5% of contacts, and only 70.3% finalized this treatment. The completion was more frequent in patients who knew the meaning of latent tuberculosis infection treatment (ORa: 2.0; CI: 95% 1.0-4.1).

OBJETIVO: El cumplimiento del tratamiento de la Infección Tuberculosa Latente (ITL) es un factor determinante del control de la tuberculosis. El objetivo de este trabajo fue estimar la aceptación y el cumplimiento del tratamiento de la ITL y factores asociados en contactos de enfermos con tuberculosis en Lleida. METODOS: Estudio epidemiológico analítico observacional de seguimiento de una cohorte retrospectiva, desde el 1 de enero de 2015 al 31 de diciembre de 2016. Los participantes fueron los contactos de enfermos con tuberculosis de Lleida. Se estudiaron variables del caso índice y variables independientes y dependientes (aceptación y cumplimiento de la ITL) de los contactos, a través del análisis univariado y multivariado. La fuerza de asociación se estudió con el odds ratio (OR) y su intervalo de confianza (IC) del 95% y se ajustaron mediante modelos de regresión logística. RESULTADOS: El 69,1% de los casos de tuberculosis poseían estudio de contactos, y el 47,5% tenían algún contacto con prescripción de tratamiento preventivo. El 94,5% de los contactos aceptó el tratamiento de la ITL, y éste fue finalizado por el 70,3%. El cumplimiento fue más frecuente en los pacientes que conocían el significado del tratamiento de la ITL (ORa: 2,0; IC: 95% 1,0-4,1). CONCLUSIONES: En relación a la aceptación/cumplimiento del tratamiento de la ITL, destaca la influencia positiva de poseer conocimientos sobre la infección y su tratamiento. Los profesionales de Atención Primaria deberían proporcionar mayor educación sanitaria con el fin de mejorar el cumplimiento terapéutico de la ITL.

Cumplimiento del tratamiento de la infección tuberculosa latente en una cohorte de contactos de enfermos de tuberculosis / Compliance of latent tuberculosis infection treatment in a cohort of TB contacts

Fundamentos: El cumplimiento del tratamiento de la Infección Tuberculosa Latente (ITL) es un factor determinante del control de la tuberculosis. El objetivo de este trabajo fue estimar la aceptación y el cumplimiento del tratamiento de la ITL y factores asociados en contactos de enfermos con tuberculosis en Lleida. Métodos: Estudio epidemiológico analítico observacional de seguimiento de una cohorte retrospectiva, desde el 1 de enero de 2015 al 31 de diciembre de 2016. Los participantes fueron los contactos de enfermos con tuberculosis de Lleida. Se estudiaron variables del caso índice y variables independientes y dependientes (aceptación y cumplimiento de la ITL) de los contactos, a través del análisis univariado y multivariado. La fuerza de asociación se estudió con el odds ratio (OR) y su intervalo de confianza (IC) del 95% y se ajustaron mediante modelos de regresión logística. Resultados: El 69,1% de los casos de tuberculosis poseían estudio de contactos, y el 47,5% tenían algún contacto con prescripción de tratamiento preventivo. El 94,5% de los contactos aceptó el tratamiento de la ITL, y éste fue finalizado por el 70,3%. El cumplimiento fue más frecuente en los pacientes que conocían el significado del tratamiento de la ITL (ORa: 2,0; IC: 95% 1,0-4,1). Conclusiones: En relación a la aceptación/cumplimiento del tratamiento de la ITL, destaca la influencia positiva de poseer conocimientos sobre la infección y su tratamiento. Los profesionales de Atención Primaria deberían proporcionar mayor educación sanitaria con el fin de mejorar el cumplimiento terapéutico de la ITL

Background: Compliance with the treatment of Latent Tuberculous Infection (ITL) is a determining factor in the control of tuberculosis. The objective of this study was to estimate the acceptance and compliance of the ITL treatment, and associated factors in contacts of patients with tuberculosis in Lleida. Design: Epidemiological analytical observational study, of a retrospective cohort follow-up, since the 1 January 2015 to 31 December 2016. The Participants were contacts of patients with tuberculosis in Lleida. Variables of the index case and independent and dependent variables (acceptance and compliance of the treatment) of contacts were studied through univariate and bivariate analysis. The strength of association was studied with odds ratio (OR) and confidence interval (IC) of 95% they were adjusted by a multivariate regression model. Results: 69.1% of tuberculosis cases had a contact study, and 47.5% had at least one contact with prescription of preventive treatment. The treatment was accepted by 94.5% of contacts, and only 70.3% finalized this treatment. The completion was more frequent in patients who knew the meaning of latent tuberculosis infection treatment (ORa: 2.0; CI: 95% 1.0-4.1). Conclusions: There was a positive influence of having knowledge of the meaning of the infection and its treatment. Primary Care should be involved in providing more and better health education in order to improve therapeutic compliance

Crioglobulina con propiedad crioaglutinina en un paciente con linfoma esplénico de zona marginal / Cryoglobulin with cold agglutinin properties in a patient with splenic marginal zone lymphoma / Crioglobulina com propriedade crioaglutinina em um paciente com linfoma esplênico de zona marginal

El objetivo del trabajo fue describir un caso de linfoma esplénico de zona marginal (LEZM) con un componente monoclonal que posee propiedades de crioglobulina y crioaglutinina, un hallazgo de muy baja frecuencia. Una paciente con LEZM padeció una anemia hemolítica autoinmune 5 años después del diagnóstico, con hematocrito de 0,15 L/L, hemoglobina 49 g/L, lactato deshidrogenasa 16,82 μkat/L, prueba de Coombs directa positiva con anti- IgG/C3d, bilirrubina total 90,6 μmol/L e indirecta de 58,1 μmol/L. No presentó evidencia clínica ni serológica de infección por VIH, hepatitis B ni C. El proteinograma sérico presentó un pico monoclonal de 14 g/L, con crioglobulinemia positiva a las 24 h, y un criocrito de 30%. La crioglobulina purificada fue de tipo I con un componente monoclonal IgM-lambda, coincidente al observado en suero. El suero, el eluato a 37 °C y la crioglobulina purificada de la paciente presentaron actividad de crioaglutinina con especificidad anti-I, fenómeno producido por la misma inmunoglobulina. El hallazgo de una crioglobulina con propiedades de crioaglutinina en pacientes con LEZM no ha sido descrito previamente en la bibliografía.

The aim of this paper is to describe a case of splenic marginal zone lymphoma (SMZL) with monoclonal component having properties of cryoglobulin and cold agglutinin, a finding of very low frequency. A patient with SMZL suffered autoimmune hemolytic anemia five years after diagnosis, with hematocrit 0.15 L/L, hemoglobin 49 g/L, lactate dehydrogenase 16.82 μkat/L, direct Coombs test with anti-IgG/C3d positive, total bilirubin 90.6 μmol/L and indirect 58.1 μmol/L. She presented no clinical or serological evidence of HIV, hepatitis B or C infection. Serum protein electrophoresis showed a monoclonal peak of 14 g/L, with positive cryoglobulinemia at 24 hours, and 30% cryocrit. Purified cryoglobulin was type I with a monoclonal IgM-lambda component coincident with that observed in serum. The patient serum, eluate at 37 °C and purified cryoglobulin showed cold agglutinin activity with anti-I specificity, phenomenon produced by the same immunoglobulin. The finding of a cryoglobulin with cold agglutinin properties in patients with SMZL has not been previously described in the literature.

O objetivo do trabalho foi descrever um caso de linfoma esplênico de zona marginal (LEZM) com um componente monoclonal com propriedades do crioglobulina e crioaglutinina, um achado de muito baixa frequência. Um doente com LEZM sofreu uma anemia hemolítica autoimune cinco anos após o diagnóstico, com hematócrito de 0,15 L/L, hemoglobina 49 g/L, lactato desidrogenase de 16,82 μkat/L, teste de Coombs direto positivo com anti-IgG/C3d, bilirrubina total 90,6 μmol/L e indireta 58,1 μmol/L. Não apresentou evidência clínica ou sorológica de infecção por HIV, hepatite B ou C. O proteinograma sérico mostrou um pico monoclonal de 14 g/L, com crioglobulinemia positiva 24 horas, e um criocrito de 30%. Crioglobulina purificada foi tipo I com o componente monoclonal IgM-lambda, coincidente com a observada no soro. O soro, o eluato a 37 ° C e a crioglobulina purificada do paciente mostraram atividade de crioaglutinina com especificidade anti-I, fenômeno produzido pela mesma imunoglobulina. O achado de uma crioglobulina com propriedades de crioaglutinina em pacientes com LEZM não foi previamente descrito na literatura.

Free DNA in cystic fibrosis airway fluids correlates with airflow obstruction.

Chronic obstructive lung disease determines morbidity and mortality of patients with cystic fibrosis (CF). CF airways are characterized by a nonresolving neutrophilic inflammation. After pathogen contact or prolonged activation, neutrophils release DNA fibres decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). NETs have been described to act in a beneficial way for innate host defense by bactericidal, fungicidal, and virucidal actions. On the other hand, excessive NET formation has been linked to the pathogenesis of autoinflammatory and autoimmune disease conditions. We quantified free DNA structures characteristic of NETs in airway fluids of CF patients and a mouse model with CF-like lung disease. Free DNA levels correlated with airflow obstruction, fungal colonization, and CXC chemokine levels in CF patients and CF-like mice. When viewed in combination, our results demonstrate that neutrophilic inflammation in CF airways is associated with abundant free DNA characteristic for NETosis, and suggest that free DNA may be implicated in lung function decline in patients with CF.

An interactive network of elastase, secretases, and PAR-2 protein regulates CXCR1 receptor surface expression on neutrophils.

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis.

Localization and functionality of the inflammasome in neutrophils.

Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein containing a CARD (ASC), AIM2, and caspase-1. Subcellular fractionation and microscopic analyses further showed that inflammasome components were localized in the cytoplasm and also noncanonically in secretory vesicle and tertiary granule compartments. Whereas IL-1ß and IL-18 were expressed at the mRNA level and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1ß protein compared with partially purified neutrophils. Serine proteases and caspases were differentially involved in IL-1ß release, depending on the stimulus. Spontaneous activation of the NLRP3 inflammasome in neutrophils in vivo affected IL-1ß, but not IL-18 release. In summary, these studies show that human neutrophils express key components of the inflammasome machinery in distinct intracellular compartments and release IL-1ß and IL-18, but not IL-1α or IL-33 protein. Targeting the neutrophil inflammasome may represent a future therapeutic strategy to modulate neutrophilic inflammatory diseases, such as cystic fibrosis, rheumatoid arthritis, or sepsis.

Expression and regulation of interferon-related development regulator-1 in cystic fibrosis neutrophils.

A genome-wide association study identified interferon-related development regulator-1 (IFRD1), a protein expressed by neutrophils, as a key modifier gene in cystic fibrosis (CF) lung disease. Here, we investigated the expression and regulation of IFRD1 in CF neutrophils. IFRD1 expression was quantified in peripheral blood and airway neutrophils from patients with CF, patients with non-CF lung disease, and healthy control subjects. The regulation of IFRD1 expression was analyzed using isolated neutrophils and ex vivo stimulation assays with CF airway fluids. IFRD1 single-nucleotide polymorphisms (SNPs) were analyzed in a CF cohort (n = 572) and correlated with longitudinal lung function and IFRD1 expression. Patients with CF expressed higher protein levels of IFRD1 in peripheral blood neutrophils compared with healthy or non-CF disease control subjects. Within patients with CF, IFRD1 protein expression levels in neutrophils were lower in airway fluids compared with peripheral blood. High IFRD1 expression was positively associated with the production of reactive oxygen species (ROS) in CF neutrophils. In vitro regulation studies showed that CF airway fluid and the CF-characteristic chemokines CXCL8 and CXCL2 down-regulated IFRD1 expression in neutrophils, an effect that was mediated through CXCR2. Genetic analyses showed that three IFRD1 SNPs were associated with longitudinal declines in lung function, and modulated IFRD1 expression. These studies demonstrate that IFRD1 expression is systemically up-regulated in human CF neutrophils, is linked to the production of ROS, and is modulated by chemokines in CF airway fluids, depending on the IFRD1 genotype. Understanding the regulation of IFRD1 may pave the way for novel therapeutic approaches to target neutrophilic inflammation in CF.

Neutrophils express distinct RNA receptors in a non-canonical way.

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics.

Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by perpetuated neutrophilic inflammation with progressive tissue destruction. Neutrophils represent the major cellular fraction in CF airway fluids and are known to form neutrophil extracellular traps (NETs) upon stimulation. Large amounts of extracellular DNA-NETs are present in CF airway fluids. However, the structural contribution of NETs to the matrix composition of CF airway fluid remains poorly understood. We hypothesized that CF airway fluids consist of distinct DNA-NETs that are associated to subcellular structures. METHODOLOGY/PRINCIPAL FINDINGS: We employed atomic force microcopy (AFM) and scanning electron microcopy to ultrastructurally characterize the nature of CF sputum and the role of NETs within the extracellular CF sputum matrix. These studies demonstrate that CF sputum is predominantly composed of a high-density meshwork of NETs and NETosis-derived material. Treatment of CF sputum with different DNases degraded CF NETs and efficiently liquefied the mucous-like structure of CF sputum. Quantitative analysis of AFM results showed the presence of three globular fractions within CF sputum and the larger two ones featured characteristics of neutrophil ectosomes. CONCLUSIONS/SIGNIFICANCE: These studies suggest that excessive NET formation represents the major factor underlying the gel-like structure of CF sputum and provide evidence that CF-NETs contain ectosome-like structures that could represent targets for future therapeutic approaches.

CXCR1 and CXCR2 haplotypes synergistically modulate cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease severity is largely independent on the CF transmembrane conductance regulator (CFTR) genotype, indicating the contribution of genetic modifiers. The chemokine receptors CXCR1 and CXCR2 have been found to play essential roles in the pathogenesis of CF lung disease. Here, we determine whether genetic variation of CXCR1 and CXCR2 influences CF lung disease severity. Genomic DNA of CF patients in Germany (n = 442) was analysed for common variations in CXCR1 and CXCR2 using a single-nucleotide polymorphism (SNP) tagging approach. Associations of CXCR1 and CXCR2 SNPs and haplotypes with CF lung disease severity, CXCR1 and CXCR2 expression, and neutrophil effector functions were assessed. Four SNPs in CXCR1 and three in CXCR2 strongly correlated with age-adjusted lung function in CF patients. SNPs comprising haplotypes CXCR1_Ha and CXCR2_Ha were in high linkage disequilibrium and patients heterozygous for the CXCR1-2 haplotype cluster (CXCR1-2_Ha) had lower lung function compared with patients with homozygous wild-type alleles (forced expiratory volume in 1 s ≤ 70% predicted, OR 7.24; p = 2.30 × 10(-5)). CF patients carrying CXCR1-2_Ha showed decreased CXCR1 combined with increased CXCR2 mRNA and protein expression, and displayed disturbed antibacterial effector functions. CXCR1 and CXCR2 genotypes modulate lung function and antibacterial host defence in CF lung disease.

CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation.

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein-coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase-independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

NETs formed by human neutrophils inhibit growth of the pathogenic mold Aspergillus fumigatus.

Neutrophil extracellular traps (NETs) represent a distinct mechanism to control and eliminate microbial infections. Our results show that conidia and germ tubes of the human pathogenic mold Aspergillus fumigatus are able to trigger the formation of NETs. Viable fungal cells are not essentially required for this host-pathogen interaction. Neutrophils engulf conidia and thereby inhibit their germination, a process that is independent of NETosis. In the experimental set-up used in this study neutrophils do not kill germ tubes, but reduce their polar growth and this inhibition depends on NETs as it can be overcome by the addition of DNase-1. The Zn(2+) chelator calprotectin is associated with the Aspergillus-induced NETs and addition of Zn(2+) abrogates the NET-mediated growth inhibition. In summary, our data provide evidence that NETs are not sufficient to kill A. fumigatus, but might be a valuable tool to confine infection.

Fibrin mimics neutrophil extracellular traps in SEM.

Neutrophil extracellular traps (NETs) are extracellular web-like structures produced by activated polymorphonuclear neutrophils. NETs kill bacteria extracellularly, but their role in human pathology remains largely unclear. One possible way of studying NETs is through the SEM approach. However, web-like structures observed with SEM in sites of inflammation have been interpreted either as NETs or as fibrin. Thus, the question arises whether a reliable SEM discrimination between NETs and fibrin is at all possible. NET samples were collected as purulent crevicular exudate from periodontal pockets. DNase-digested controls for SEM were employed to demonstrate the DNA backbone and immuno-staining for confocal laser scanning microscopy was used to show the citrullinated histones of NETs. Blood clot samples were treated in the same way as the exudate samples to demonstrate that fibrin and fibrinolysis can mimic NETs and DNA digestion, respectively. No discrimination between fibrin and NETs based on morphological criteria in SEM was possible. Furthermore, only a vague distinction between DNA digestion and fibrinolysis could be made. These findings unambiguously indicate that the discrimination between NETs and fibrin by means of SEM is untrustworthy for samples of inflammatory exudate.

Expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases.

BACKGROUND: Inflammatory lung diseases are a major morbidity factor in children. Therefore, novel strategies for early detection of inflammatory lung diseases are of high interest. Bacterial lipopolysaccharide (LPS) is recognized via Toll-like receptors and CD14. CD14 exists as a soluble (sCD14) and membrane-associated (mCD14) protein, present on the surface of leukocytes. Previous studies suggest sCD14 as potential marker for inflammatory diseases, but their potential role in pediatric lung diseases remained elusive. Therefore, we examined the expression, regulation and significance of sCD14 and mCD14 in pediatric lung diseases. METHODS: sCD14 levels were quantified in serum and bronchoalveolar lavage fluid (BALF) of children with infective (pneumonia, cystic fibrosis, CF) and non-infective (asthma) inflammatory lung diseases and healthy control subjects by ELISA. Membrane CD14 expression levels on monocytes in peripheral blood and on alveolar macrophages in BALF were quantified by flow cytometry. In vitro studies were performed to investigate which factors regulate sCD14 release and mCD14 expression. RESULTS: sCD14 serum levels were specifically increased in serum of children with pneumonia compared to CF, asthma and control subjects. In vitro, CpG induced the release of sCD14 levels in a protease-independent manner, whereas LPS-mediated mCD14 shedding was prevented by serine protease inhibition. CONCLUSIONS: This study demonstrates for the first time the expression, regulation and clinical significance of soluble and membrane CD14 receptors in pediatric inflammatory lung diseases and suggests sCD14 as potential marker for pneumonia in children.

Infiltrated neutrophils acquire novel chemokine receptor expression and chemokine responsiveness in chronic inflammatory lung diseases.

Various inflammatory diseases are characterized by tissue infiltration of neutrophils. Chemokines recruit and activate leukocytes, but neutrophils are traditionally known to be restricted in their chemokine receptor (CR) expression repertoire. Neutrophils undergo phenotypic and functional changes under inflammatory conditions, but the mechanisms regulating CR expression of infiltrated neutrophils at sites of chronic inflammation are poorly defined. Here we show that infiltrated neutrophils from patients with chronic inflammatory lung diseases and rheumatoid arthritis highly express CR on their surface that are absent or only marginally expressed on circulating neutrophils, i.e., CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4, as measured by flow cytometry, immunohistochemistry, and confocal microscopy. The induction of CR surface expression on infiltrated neutrophils was functionally relevant, because receptor activation by chemokine ligands ex vivo modulated neutrophil effector functions such as respiratory burst activity and bacterial killing. In vitro studies with isolated neutrophils demonstrated that the surface expression of CR was differentially induced in a cytokine-mediated, protein synthesis-dependent manner (CCR1, CCR3), through Toll-like (CXCR3) or NOD2 (CCR5) receptor engagement, through neutrophil apoptosis (CCR5, CXCR4), and/or via mobilization of intracellular CD63(+) granules (CXCR3). CR activation on infiltrated neutrophils may represent a key mechanism by which the local inflammatory microenvironment fine-tunes neutrophil effector functions in situ. Since the up-regulation of CR was exclusively found on infiltrated neutrophils at inflammatory sites in situ, the targeting of these G protein-coupled receptors may have the potential to site-specifically target neutrophilic inflammation.

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease.

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

Bronchoalveolar pepsin, bile acids, oxidation, and inflammation in children with gastroesophageal reflux disease.

INTRODUCTION: Gastroesophageal reflux has been suggested as an underlying cause of chronic lung disease. The aim of this study was to assess the value of pepsin and bile acids, both components of GI secretions, in the lungs of children with chronic lung diseases as possible markers for gastroesophageal reflux disease and their relation to oxidation and inflammation. MATERIALS AND METHODS: BAL was performed in 96 children with different chronic lung diseases. Gastroesophageal reflux was analyzed by two-channel, 24-h esophageal pH measurements. Lung pepsin and bile acids were measured in BAL enzymatically, interleukin (IL)-8 by enzyme-linked immunosorbent assay, and protein carbonyls by slot blot immunoassay. RESULTS: Sixty-five of the 96 children (68%) had an extensive proximal acidic reflux index. Children with reflux had higher pepsin concentrations in their BAL fluid (BALF), compared to children without reflux despite low specificity. No differences were observed for bile acids. Percentages of neutrophils, levels of protein carbonyls, and levels of IL-8 in BALF correlated with the number of proximal reflux events. CONCLUSIONS: Pulmonary microaspiration as demonstrated by pepsin detection in BALF is common in children with chronic lung diseases, suggesting that gastroesophageal reflux may contribute significantly to the disease pathogenesis. BALF pepsin concentration correlates positively with the number of proximal reflux events. Protein oxidation in BALF is higher in children with extensive proximal acidic reflux, suggesting that pulmonary microaspirations contribute to lung damage.