Search

Background. Both pandemic and interpandemic influenza is associated with high morbidity and mortality worldwide. Seasonal epidemics are caused by both influenza A and B virus strains that cocirculate with varying predominance and may give rise to severe illness equally. According to World Health Organization recommendations, current annual vaccines are composed of 2 type A and 1 type B virus-specific component. Methods. As a novel attenuated live vaccine against influenza B virus, we generated a hemagglutinin cleavage site mutant of strain B/Lee/40 by replacing the common monobasic cleavage site recognized by trypsinlike proteases with an elastase-sensitive site, and we investigated the in vitro properties, attenuation, humoral responses, and efficacy in mice. Results. This mutant virus replicated in cell culture equally well as the wild type but in a strictly elastasedependent manner. In contrast to the mouse-pathogenic parental virus, the cleavage site mutant was fully attenuated in mice and not detectable in their lungs. After 1 intranasal immunization, the animals survived lethal challenge with wild-type virus without weight loss or any other signs of disease. Furthermore, no challenge virus could be reisolated from the lungs of vaccinated mice. Conclusions. These findings demonstrate that proteolytic activation mutants can serve as live vaccine against influenza B virus. [ABSTRACT FROM AUTHOR]

Recent observations indicate that autoimmune disorders and allergies are more closely connected to each other than traditionally considered. Both diseases are characterized by a break of immunological tolerance and in both Th1- and Th2-driven mechanisms may be involved. Moreover, patients suffering from severe chronic atopic diseases have been shown to develop autoreactive antibodies and vice versa autoreactive IgE antibodies may contribute to the perpetuation of allergic immune responses. However, animal models for the investigation of the underlying mechanisms have not been available so far. Here we demonstrate that allergic sensitization and subsequent airway aerosol challenge of mice to a potential autoantigen leads to an allergic airway inflammation and chronic exposure of sensitized mice to a foreign allergen induces autoreactive antibodies. Thus, autoreactive immune mechanisms may be induced either by molecular mimicry or as a consequence of chronic allergic inflammatory processes and these reactivities may then possibly contribute to the perpetuation of allergic immune responses even in the absence of exogenous allergens and to the development of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Summary: Allergic diseases are inflammatory disorders that develop on the basis of complex gene–environment interactions. The prevalence of allergies is steadily increasing and seems to be associated with modern lifestyle. Therefore, it was hypothesized that high living standards and hygienic conditions are correlated with an increased risk for the development of an allergic disease. This so-called “hygiene hypothesis” states that due to reduced exposure to microbial components, the proposed allergy-preventing potential of these factors is no more present in sufficient qualities and/or quantities, which leads to an imbalance of the immune system with a predisposition to the development of allergic disorders. Meanwhile, several epidemiological studies were conducted supporting this concept and generating novel ideas for the underlying mechanisms that were then followed up by use of well-defined animal models and human studies. The current view of cellular and molecular mechanisms responsible for these phenomena includes changes in the fine balancing of T helper cell 1 (Th1), Th2 and regulatory T cell (Treg) responses which are triggered by altered or missing innate immune cell activation. In fact, proper activation of cells of the innate immune system via their so-called pattern recognition receptors has been demonstrated to play a crucial role in early shaping of the immune system and suppression of the development of Th2-driven allergic immune responses. These processes start already in utero and prenatal as well as early postnatal developmental stages seem to represent a certain “window of opportunity” for allergy-preventing environmental influences. [Copyright &y& Elsevier]

Abstract: Exogenous application of keratinocyte growth factor protects the lung against a variety of injurious stimuli. KGF-treatment leads to pronounced hyperplasia of alveolar epithelial type II cells and to stabilization of surfactant homeostasis after lung injury. Epidermal fatty acid-binding protein is involved in the synthesis of surfactant phospholipids and acts as an antioxidant scavenging reactive lipids. We treated adult rats with recombinant human keratinocyte growth factor (Palifermin) via intratracheal instillation and analyzed the expression of epidermal fatty acid-binding protein mRNA and protein by quantitative RT-PCR, immunoblotting as well as immunohistochemistry. Keratinocyte growth factor-treatment in vivo leads to an increased expression of epidermal fatty acid-binding protein mRNA and protein in the total lung. Epidermal fatty acid-binding protein mRNA expression per alveolar epithelial type II cell remains constant as shown in isolated type II cells. Epidermal fatty acid-binding protein immunoreactivity is seen in most if not all hyperplastic alveolar epithelial type II cells, and is mainly localized to the cytoplasm. The increase in epidermal fatty acid-binding protein gene expression associated with type II cell hyperplasia might contribute to the molecular mechanisms mediating lung protection by keratinocyte growth factor. [Copyright &y& Elsevier]

Background: Alveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2 has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-α and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations. Methods: AM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA. Results: In untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2 exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+ cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-α production was lower compared to ED7- AM. However, iNOS and IL-12 expression were also increased in the ED7+ subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12. Conclusion: NO2 exposure induces the infiltration of an AM subpopulation that, on the one hand may exert antiinflammatory functions by the production of high amounts of IL-10 but on the other hand may contribute to the pathology of NO2-induced lung damage by selective expression of certain matrix metalloproteinases. [ABSTRACT FROM AUTHOR]

Examines whether a shift to either a helper T cell (Th) type 1-like or a Th2-like profile could delineate the contribution of T cells to the development of silicosis. Th1/Th2 cytokine expression in lymph nodes of silicotic animals; Interleukin expresssion in lymph nodes of silicotic animals.

Provides information on a study which examined the T-cell activation by analysis of cytokine expression in the enlarged thoracic lymph nodes of rats after silica aerosol exposure. Materials and methods; Results; Discussion.

Background: Epigenetic modifications are known to mediate both beneficial and unfavorable effects of environmental exposures on the development and clinical course of asthma. On the molecular level, epigenetic mechanisms participate in multiple aspects of the emerging and ongoing asthma pathology. Summary: Studies performed in the last several years expand our knowledge on the role of histone acetylation, a classical epigenetic mark, in the regulation of (patho)physiological processes of diverse cells playing a central role in asthma, including those belonging to the immune system (e.g., CD4+ T cells, macrophages) and lung structure (e.g., airway epithelial cells, pulmonary fibroblasts). Those studies demonstrate a number of specific histone acetylation-associated mechanisms and pathways underlying pathological processes characteristic for asthma, as well as report their modification modalities. Key Messages: Dietary modulation of histone acetylation levels in the immune system might protect against the development of asthma and other allergies. Interfering with the enzymes controlling the histone acetylation status of structural lung and (local) immune cells might provide future therapeutic options for asthmatics. Despite some methodological obstacles, analysis of the histone acetylation levels might improve asthma diagnostics. [ABSTRACT FROM AUTHOR]

Background: IL‐17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation. Methods: Flow‐cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL‐17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses. Results: We identified Th17 cells as the main population of IL‐17A‐ and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL‐17A and its receptor subunit IL‐17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL‐17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro‐inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL‐17A and TNF synergistically induced the Th17‐promoting cytokines IL‐6 and IL‐1β as well as the Th17‐attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor‐promoting role of Th17 cells in OC. Conclusions: Our findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL‐17A/TNF‐mediated induction of mesothelial‐mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options. [ABSTRACT FROM AUTHOR]

Rationale and Objective: Plasma extracellular vesicles (EVs) represent a vital source of molecular information about health and disease states. Due to their heterogenous cellular sources, EVs and their cargo may predict specific pathomechanisms behind disease phenotypes. Here we aimed to utilize EV microRNA (miRNA) signatures to gain new insights into underlying molecular mechanisms of obesity‐associated low type‐2 asthma. Methods: Obese low type‐2 asthma (OA) and non‐obese low type‐2 asthma (NOA) patients were selected from an asthma cohort conjointly with healthy controls. Plasma EVs were isolated and characterised by nanoparticle tracking analysis. EV‐associated small RNAs were extracted, sequenced and bioinformatically analysed. Results: Based on EV miRNA expression profiles, a clear distinction between the three study groups could be established using a principal component analysis. Integrative pathway analysis of potential target genes of the differentially expressed miRNAs revealed inflammatory cytokines (e.g., interleukin‐6, transforming growth factor‐beta, interferons) and metabolic factors (e.g., insulin, leptin) signalling pathways to be specifically associated with OA. The miR‐17–92 and miR‐106a–363 clusters were significantly enriched only in OA. These miRNA clusters exhibited discrete bivariate correlations with several key laboratory (e.g., C‐reactive protein) and lung function parameters. Plasma EV miRNA signatures mirrored blood‐derived CD4+ T‐cell transcriptome data, but achieved an even higher sensitivity in identifying specifically affected biological pathways. Conclusion: The identified plasma EV miRNA signatures and particularly the miR‐17–92 and ‐106a–363 clusters were capable to disentangle specific mechanisms of the obesity‐associated low type‐2 asthma phenotype, which may serve as basis for stratified treatment development. [ABSTRACT FROM AUTHOR]

The global burden of respiratory diseases is very high and still on the rise, prompting the need for accurate models for basic and translational research. Several model systems are currently available ranging from simple airway cell cultures to complex tissue-engineered lungs. In recent years, human lung organoids have been established as highly transferrable three-dimensional in vitro model systems for lung research. For acute infectious and chronic inflammatory diseases as well as lung cancer, human lung organoids have opened possibilities for precise in vitro research and a deeper understanding of mechanisms underlying lung injury and regeneration. Human lung organoids from induced pluripotent stem cells or from adult stem cells of patients' samples introduce tools for understanding developmental processes and personalized medicine approaches. When further state-of-the-art technologies and protocols come into use, the full potential of human lung organoids can be harnessed. High-throughput assays in drug development, gene therapy, and organoid transplantation are current applications of organoids in translational research. In this review, we emphasize novel approaches in translational and personalized medicine in lung research focusing on the use of human lung organoids. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) gain increasing attention due to their (patho-)physiological role in intercellular signaling, specifically in the communication between distant organs. Recent studies highlight a connection between the adipose tissue (AT) and the lung via (immuno-)modulatory EVs in disorders such as obesity-associated asthma and lung cancer-associated cachexia. Although lung cancer-derived EVs induce lipolysis and myotube atrophy in vivo, pathogenic effects were also reported in the opposite direction with the involvement of AT-derived EVs in cancer-promoting responses and potentially in asthma development. In contrast, the majority of studies on AT-derived EVs demonstrate their protective influence on the asthmatic lung. Beneficial effects, such as induction of anti-inflammatory pathways in vitro and in ovalbumin (OVA)-induced asthma mouse models, were particularly conveyed by EVs enriched from AT-derived mesenchymal stem/stromal cells (AT-MSCs), which therefore pose an interesting subject in possible future therapeutic applications. Likewise, AT-MSC-derived EVs exerted beneficial effects in several other pulmonary abnormalities, such as different types of lung injury or pathological changes related to chronic obstructive pulmonary disease. These contradictory findings highlight the need for extensive research to widen the understanding of the role of EVs in the development of diseases and interconnectivity between organs. [ABSTRACT FROM AUTHOR]

Background: Early‐life exposure to certain environmental bacteria including Acinetobacter lwoffii (AL) has been implicated in protection from chronic inflammatory diseases including asthma later in life. However, the underlying mechanisms at the immune‐microbe interface remain largely unknown. Methods: The effects of repeated intranasal AL exposure on local and systemic innate immune responses were investigated in wild‐type and Il6−/−, Il10−/−, and Il17−/− mice exposed to ovalbumin‐induced allergic airway inflammation. Those investigations were expanded by microbiome analyses. To assess for AL‐associated changes in gene expression, the picture arising from animal data was supplemented by in vitro experiments of macrophage and T‐cell responses, yielding expression and epigenetic data. Results: The asthma preventive effect of AL was confirmed in the lung. Repeated intranasal AL administration triggered a proinflammatory immune response particularly characterized by elevated levels of IL‐6, and consequently, IL‐6 induced IL‐10 production in CD4+ T‐cells. Both IL‐6 and IL‐10, but not IL‐17, were required for asthma protection. AL had a profound impact on the gene regulatory landscape of CD4+ T‐cells which could be largely recapitulated by recombinant IL‐6. AL administration also induced marked changes in the gastrointestinal microbiome but not in the lung microbiome. By comparing the effects on the microbiota according to mouse genotype and AL‐treatment status, we have identified microbial taxa that were associated with either disease protection or activity. Conclusion: These experiments provide a novel mechanism of Acinetobacter lwoffii‐induced asthma protection operating through IL‐6‐mediated epigenetic activation of IL‐10 production and with associated effects on the intestinal microbiome. [ABSTRACT FROM AUTHOR]

This document is a reply to a correspondence letter regarding a scientific study on obese asthmatics. The authors clarify that their primary aim was to gain insights into the underlying mechanisms of obesity-associated low type-2 asthma, rather than establish diagnostic biomarkers. They mention that further validation is needed for the identified microRNA markers. The authors also address questions about the cell types responsible for the release of extracellular vesicles with different microRNA cargo in obese asthmatics. The study was supported by various funding sources and the authors declare no conflicts of interest. [Extracted from the article]

No abstract available [ABSTRACT FROM AUTHOR]

Asthma is among the most common chronic inflammatory diseases worldwide. Recent evidence indicates that the pathogenesis shows a high degree of heterogeneity. Patient subsets have been identified that exhibit different cellular and molecular patterns of dysregulation. A prominent example is eosinophilic Th2-driven asthma. These unique and molecular patterns are termed endotypes. Characterization of endotypes has broad implications for therapeutic interventions. Although ∼80% of asthmatic patients respond well to standard anti-inflammatory therapies, the remaining subset particularly consisting of severe patients requires a more specialized endotype-specific approach. This interrelationship between clinical phenotypes, molecular endotypes and endotype-specific therapies is the focus of this review. [ABSTRACT FROM AUTHOR]

Optimal pre-analytical conditions for blood sample processing and isolation of selected cell populations for subsequent transcriptomic and epigenomic studies are required to obtain robust and reproducible results. This pilot study was conducted to investigate the potential effects of timing of CD4+ T-cell processing from peripheral blood of atopic and non-atopic adults on their transcriptomic and epigenetic profiles. Two heparinized blood samples were drawn from each of three atopic and three healthy individuals. For each individual, CD4+ T-cells were isolated from the first blood sample within 2 h (immediate) or from the second blood sample after 24 h storage (delayed). RNA sequencing (RNA-Seq) and histone H3K27 acetylation chromatin immunoprecipitation sequencing (ChIP-Seq) analyses were performed. A multiplicity of genes was shown to be differentially expressed in immediately processed CD4+ T-cells from atopic versus healthy subjects. These differences disappeared when comparing delayed processed cells due to a drastic change in expression levels of atopy-related genes in delayed processed CD4+ T-cells from atopic donors. This finding was further validated on the epigenomic level by examining H3K27 acetylation profiles. In contrast, transcriptomic and epigenomic profiles of blood CD4+ T-cells of healthy donors remained rather unaffected. Taken together, for successful transcriptomics and epigenomics studies, detailed standard operation procedures developed on the basis of samples from both healthy and disease conditions are implicitly recommended. [ABSTRACT FROM AUTHOR]

Aspirin-exacerbated respiratory disease (AERD) is characterized by overproduction of the pro-inflammatory eicosanoids. Although immunoglobulin E-mediated sensitization to aeroallergens is common among AERD patients, it does not belong to the defining disease characteristics. In this study of 133 AERD patients, we sought to find a relationship between sensitization to aeroallergens and local (leukotriene E4, prostaglandin E2 and prostaglandin D2) and/or systemic (leukotriene E4) production of arachidonic acid metabolites. Interestingly, a negative association between pro-inflammatory eicosanoid levels in induced sputum supernatant or urine and sensitization to aeroallergens was observed. This inverse relationship might suggest the presence of a protective effect of atopic sensitization to aeroallergens against stronger local airway inflammation and higher systemic AERD-related inflammatory activity. [ABSTRACT FROM AUTHOR]

Background: While childhood asthma prevalence is rising in Westernized countries, farm children are protected. The mitogen‐activated protein kinase (MAPK) pathway with its negative regulator dual‐specificity phosphatase‐1 (DUSP1) is presumably associated with asthma development. Objectives: We aimed to investigate the role of MAPK signaling in childhood asthma and its environment‐mediated protection, including a representative selection of 232 out of 1062 children from two cross‐sectional cohorts and one birth cohort study. Methods: Peripheral blood mononuclear cells (PBMC) from asthmatic and healthy children were cultured upon stimulation with farm‐dust extracts or lipopolysaccharide. In subgroups, gene expression was analyzed by qPCR (PBMCs, cord blood) and NanoString technology (dendritic cells). Protein expression of phosphorylated MAPKs was measured by mass cytometry. Histone acetylation was investigated by chromatin immunoprecipitation. Results: Asthmatic children expressed significantly less DUSP1 (p =.006) with reduced acetylation at histone H4 (p =.012) compared with healthy controls. Farm‐dust stimulation upregulated DUSP1 expression reaching healthy levels and downregulated inflammatory MAPKs on gene and protein levels (PBMCs; p ≤.01). Single‐cell protein analysis revealed downregulated pMAPKs upon farm‐dust stimulation in B cells, NK cells, monocytes, and T‐cell subpopulations. Conclusion: Lower DUSP1 baseline levels in asthmatic children and anti‐inflammatory regulation of MAPK in several immune cell types by farm‐dust stimulation indicate a regulatory function for DUSP1 for future therapy contributing to anti‐inflammatory characteristics of farming environments. [ABSTRACT FROM AUTHOR]

Oligonucleotides (ONs) are an emerging class of drugs being developed for the treatment of a wide variety of diseases including the treatment of respiratory diseases by the inhalation route. As a class, their toxicity on human lungs has not been fully characterized, and predictive toxicity biomarkers have not been identified. To that end, identification of sensitive methods and biomarkers that can detect toxicity in humans before any long term and/or irreversible side effects occur would be helpful. In light of the public's greater interests, the Inhalation Subcommittee of the Oligonucleotide Safety Working Group (OSWG) held expert panel discussions focusing on the potential toxicity of inhaled ONs and assessing the strengths and weaknesses of different monitoring techniques for use during the clinical evaluation of inhaled ON candidates. This white paper summarizes the key discussions and captures the panelists' perspectives and recommendations which, we propose, could be used as a framework to guide both industry and regulatory scientists in future clinical research to characterize and monitor the short and long term lung response to inhaled ONs. [ABSTRACT FROM AUTHOR]

Holler J, Zakrzewicz A, Garn H, Hirschburger M, Kummer W, Padberg W, Grau V. Increased expression of epidermal fatty acid-binding protein by alveolar macrophages during acute rejection of rat lungs. APMIS 2010; 118: 791–800. In the lung, epidermal fatty acid-binding protein (E-FABP) is expressed by alveolar macrophages (AM) and alveolar epithelial cells type II (AEII). E-FABP may regulate macrophage activation and is involved in the metabolism of surfactant phospholipids. As macrophage activation and surfactant dysfunction are associated with rejection, we hypothesize that E-FABP expression is changed during acute rejection of pulmonary grafts. Orthotopic left lung transplantations were performed in the Dark Agouti to Lewis and in the isogeneic Lewis to Lewis rat strain combinations. E-FABP expression was analyzed in the lung by immunohistochemistry, immunoblotting and quantitative reverse transcription-polymerase chain reaction (RT-PCR). Alveolar leukocytes obtained by bronchoalveolar lavage were analyzed by RT-PCR. Immunohistochemistry of isografts revealed strong E-FABP immunoreactivity in AEII and a moderate immunoreactivity in AM. In allografts undergoing acute rejection, AM exhibiting increased E-FABP immunoreactivity accumulated. Immunoblots revealed a single band at 15 kDa, which corresponds to the expected molecular mass of E-FABP. The levels of E-FABP mRNA were higher in allografts than in isografts and control lungs. Furthermore, alveolar leukocytes isolated by bronchoalveolar lavage from allografts displayed higher E-FABP mRNA expression levels than leukocytes from isografts and controls. In conclusion, we demonstrate for the first time upregulation of E-FABP expression in AM during severe inflammation. [ABSTRACT FROM AUTHOR]

Background: While several systemic immunomodulatory effects of allergen‐specific immunotherapy (AIT) have been discovered, local anti‐inflammatory mechanisms in the respiratory tract are largely unknown. We sought to elucidate local and epithelial mechanisms underlying allergen‐specific immunotherapy in a genome‐wide approach. Methods: We induced sputum in hay fever patients and healthy controls during the pollen peak season and stratified patients by effective allergen immunotherapy or as untreated. Sputum was directly processed after induction and subjected to whole transcriptome RNA microarray analysis. Nasal secretions were analyzed for Secretoglobin1A1 (SCGB1A1) and IL‐24 protein levels in an additional validation cohort at three defined time points during the 3‐year course of AIT. Subsequently, RNA was extracted and subjected to an array‐based whole transcriptome analysis. Results: Allergen‐specific immunotherapy inhibited pro‐inflammatory CXCL8, IL24, and CCL26mRNA expression, while SCGB1A1, IL7, CCL5, CCL23, and WNT5BmRNAs were induced independently of the asthma status and allergen season. In our validation cohort, local increase of SCGB1A1 occurred concomitantly with the reduction of local IL‐24 in upper airways during the course of AIT. Additionally, SCGB1A1 was identified as a suppressor of epithelial gene expression. Conclusions: Allergen‐specific immunotherapy induces a yet unknown local gene expression footprint in the lower airways that on one hand appears to be a result of multiple regulatory pathways and on the other hand reveals SCGB1A1 as novel anti‐inflammatory mediator of long‐term allergen‐specific therapeutic intervention in the local environment. [ABSTRACT FROM AUTHOR]

Allergic diseases of the (upper and lower) airways, the skin and the gastrointestinal tract, are on the rise, resulting in impaired quality of life, decreased productivity, and increased healthcare costs. As allergic diseases are mostly tissue‐specific, local sampling methods for respective biomarkers offer the potential for increased sensitivity and specificity. Additionally, local sampling using noninvasive or minimally invasive methods can be cost‐effective and well tolerated, which may even be suitable for primary or home care sampling. Non‐ or minimally invasive local sampling and diagnostics may enable a more thorough endotyping, may help to avoid under‐ or overdiagnosis, and may provide the possibility to approach precision prevention, due to early diagnosis of these local diseases even before they get systemically manifested and detectable. At the same time, dried blood samples may help to facilitate minimal‐invasive primary or home care sampling for classical systemic diagnostic approaches. This EAACI position paper contains a thorough review of the various technologies in allergy diagnosis available on the market, which analytes or biomarkers are employed, and which samples or matrices can be used. Based on this assessment, EAACI position is to drive these developments to efficiently identify allergy and possibly later also viral epidemics and take advantage of comprehensive knowledge to initiate preventions and treatments. [ABSTRACT FROM AUTHOR]

Genetic (G) and environmental (E) factors are involved in the etiology and course of the major psychoses (MP), i.e. major depressive disorder (MDD), bipolar disorder (BD), schizoaffective disorder (SZA) and schizophrenia (SZ). The neurobiological correlates by which these predispositions exert their influence on brain structure, function and course of illness are poorly understood. In the FOR2107 consortium, animal models and humans are investigated. A human cohort of MP patients, healthy subjects at genetic and/or environmental risk, and control subjects (N = 2500) has been established. Participants are followed up after 2 years and twice underwent extensive deep phenotyping (MR imaging, clinical course, neuropsychology, personality, risk/protective factors, biomaterials: blood, stool, urine, hair, saliva). Methods for data reduction, quality assurance for longitudinal MRI data, and (deep) machine learning techniques are employed. In the parallelised animal cluster, genetic risk was introduced by a rodent model (Cacna1c deficiency) and its interactions with environmental risk and protective factors are studied. The animals are deeply phenotyped regarding cognition, emotion, and social function, paralleling the variables assessed in humans. A set of innovative experimental projects connect and integrate data from the human and animal parts, investigating the role of microRNA, neuroplasticity, immune signatures, (epi-)genetics and gene expression. Biomaterial from humans and animals are analyzed in parallel. The FOR2107 consortium will delineate pathophysiological entities with common neurobiological underpinnings ("biotypes") and pave the way for an etiologic understanding of the MP, potentially leading to their prevention, the prediction of individual disease courses, and novel therapies in the future. [ABSTRACT FROM AUTHOR]

The prevalence of allergic diseases such as allergic rhinitis, asthma, food allergy, and atopic dermatitis has increased dramatically during the last decades, which is associated with altered environmental exposures and lifestyle practices. The purpose of this review was to highlight the potential role for dietary fatty acids, in the prevention and management of these disorders. In addition to their nutritive value, fatty acids have important immunoregulatory effects. Fatty acid‐associated biological mechanisms, human epidemiology, and intervention studies are summarized in this review. The influence of genetics and the microbiome on fatty acid metabolism is also discussed. Despite critical gaps in our current knowledge, it is increasingly apparent that dietary intake of fatty acids may influence the development of inflammatory and tolerogenic immune responses. However, the lack of standardized formats (ie, food versus supplement) and standardized doses, and frequently a lack of prestudy serum fatty acid level assessments in clinical studies significantly limit our ability to compare allergy outcomes across studies and to provide clear recommendations at this time. Future studies must address these limitations and individualized medical approaches should consider the inclusion of specific dietary factors for the prevention and management of asthma, food allergy, and atopic dermatitis. [ABSTRACT FROM AUTHOR]

Aim: Non‐alcoholic fatty liver disease (NAFLD) patients benefit from physical exercise. This study aimed to investigate the effect of acute exercise on hepatic gene expression in different mouse models of NAFLD. Methods: C57BL/6J mice were fed with a control (CD) or a high fat (HFD) diet. AlbCrePtenflox/flox (Pten‐KO) and Fxr−/− mice, two genetic models of NAFLD with insulin hypersensitivity and resistance, respectively, were fed with CD. After 4 weeks, mice were randomly assigned to exercise or sedentariness. Mice were killed 15 min or 3 h after the running/sedentary period. Genome‐wide hepatic gene expression was evaluated with the Illumina Micro‐array platform. Quantitative polymerase chain reaction confirmed changes in gene expression. Results: Acute exercise transiently affected the expression of genes involved in the immune response in C57BL/6 mice fed with CD and this effect normalized in the recovery phase. Acute exercise affected genes involved in gluconeogenesis in the insulin resistant Fxr−/− model. Genes involved in lipid metabolism were affected in C57BL/6 mice fed with CD, but not in mouse models of NAFLD. Genes involved in DNA damage response pathways were deregulated only in C57BL/6 mice fed with CD and not in mouse models of NAFLD. Conclusion: The simultaneous analysis of different NAFLD models revealed that an acute exercise bout affects hepatic gene expression differentially according to animal models and that most of the differentially expressed genes are involved in glucose and fatty acid metabolism, immune regulation, and DNA damage response. [ABSTRACT FROM AUTHOR]

Background: Asthma is a chronic respiratory disease with marked clinical and pathophysiological heterogeneity. Specific pathways are thought to be involved in the pathomechanisms of different inflammatory phenotypes of asthma; however, direct in vivo comparison has not been performed. Methods: We developed mouse models representing three different phenotypes of allergic airway inflammation—eosinophilic, mixed, and neutrophilic asthma via different methods of house dust mite sensitization and challenge. Transcriptomic analysis of the lungs, followed by the RT‐PCR, western blot, and confocal microscopy, was performed. Primary human bronchial epithelial cells cultured in air‐liquid interface were used to study the mechanisms revealed in the in vivo models. Results: By whole‐genome transcriptome profiling of the lung, we found that airway tight junction (TJ), mucin, and inflammasome‐related genes are differentially expressed in these distinct phenotypes. Further analysis of proteins from these families revealed that Zo‐1 and Cldn18 were downregulated in all phenotypes, while increased Cldn4 expression was characteristic for neutrophilic airway inflammation. Mucins Clca1 (Gob5) and Muc5ac were upregulated in eosinophilic and even more in neutrophilic phenotype. Increased expression of inflammasome‐related molecules such as Nlrp3, Nlrc4, Casp‐1, and IL‐1β was characteristic for neutrophilic asthma. In addition, we showed that inflammasome/Th17/neutrophilic axis cytokine—IL‐1β—may transiently impair epithelial barrier function, while IL‐1β and IL‐17 increase mucin expressions in primary human bronchial epithelial cells. Conclusion: Our findings suggest that differential expression of TJ, mucin, and inflammasome‐related molecules in distinct inflammatory phenotypes of asthma may be linked to pathophysiology and might reflect the differences observed in the clinic. Tight junction, mucin, and inflammasome‐related molecules are differentially expressed in the lungs in distinct inflammatory phenotypes of allergic airway inflammation (AAI): eosinophilic, mixed, and neutrophilic AAI. Zo‐1 and Claudin‐18 are downregulated, whereas Clca1 (Gob5) and Muc5ac are upregulated in all phenotypes. Claudin‐4, Nlrp3, Nlrc4, Casp‐1, and IL‐1β are upregulated in neutrophilic AAI. IL‐1β impairs epithelial barrier function and increases MUC5AC expression in differentiated primary human bronchial epithelial cells (HBECs). IL‐17 also increases MUC5AC expression in HBECs. [ABSTRACT FROM AUTHOR]

Autism spectrum disorder (ASD) is a class of neurodevelopmental disorders characterized by persistent deficits in social communication/interaction, together with restricted/repetitive patterns of behavior. ASD is among the most heritable neuropsychiatric conditions, and while available evidence points to a complex set of genetic factors, the SHANK gene family has emerged as one of the most promising candidates. Here, we assessed ASD-related phenotypes with particular emphasis on social behavior and cognition in Shank1 mouse mutants in comparison to heterozygous and wildtype littermate controls across development in both sexes. While social approach behavior was evident in all experimental conditions and social recognition was only mildly affected by genotype, Shank1−/− null mutant mice were severely impaired in object recognition memory. This effect was particularly prominent in juveniles, not due to impairments in object discrimination, and replicated in independent mouse cohorts. At the neurobiological level, object recognition deficits were paralleled by increased brain-derived neurotrophic factor (BDNF) protein expression in the hippocampus of Shank1−/− mice; yet BDNF levels did not differ under baseline conditions. We therefore investigated changes in the epigenetic regulation of hippocampal BDNF expression and detected an enrichment of histone H3 acetylation at the Bdnf promoter1 in Shank1−/− mice, consistent with increased learning-associated BDNF. Together, our findings indicate that Shank1 deletions lead to an aberrant cognitive phenotype characterized by severe impairments in object recognition memory and increased hippocampal BDNF levels, possibly due to epigenetic modifications. This result supports the link between ASD and intellectual disability, and suggests epigenetic regulation as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Background: Evidence regarding sublingual immunotherapy (SLIT) efficacy and its good safety profile has been demonstrated with pollen and house dust mite (HDM) allergens in the treatment of airway allergies. In addition, the use of grass pollen presents a SLIT disease-modifying treatment for respiratory allergies. Objectives: The aim of this study was to demonstrate the efficacy of HDM-based SLIT in mouse models of allergic airway inflammation and to gain insights into the involved local immunological mechanisms. Methods: Balb/c mice were sensitized/challenged with Dermatophagoides farinae (Der f) extract and underwent Der f-SLIT in prophylactic and therapeutic settings. The SLIT efficacy was assessed using lung function measurements, analysis of local inflammatory responses by bronchoalveolar lavage cell differentiation and lung histology. Humoral and cellular responses were monitored by ELISA, cytokine bead array and flow cytometry analyses. Results: In a prophylactic setting, Der f-SLIT with 12 development units per dose reduced the eosinophil-dominated inflammatory response in the lung paralleled by a marked reduction in airway hyperresponsiveness. Local Th2 responses were prevented as demonstrated by significantly lower levels of IL-5 and IL-13. Additionally, SLIT-treated mice revealed a lower proportion of CD4-CD8- ?d cells and a higher frequency of CD8+CD25+IFN?+ T cells in the lungs compared to sham-treated mice. In a therapeutic setting, Der f-SLIT also resulted in reduced inflammatory responses in the lung. Conclusion: The efficacy of Der f-SLIT was demonstrated in prophylactic and therapeutic conditions using experimental mouse models of HDM-induced airway inflammation. A potential role of a so far underestimated lymphocyte subpopulation was also indicated. [ABSTRACT FROM AUTHOR]

Gastric mucosa-associated lymphoid tissue (MALT) lymphomas develop from a chronic Helicobacter infection. Phospholipase C gamma 2 (PLCG2) is important for B-cell survival and proliferation. We used BALB/c mice with a gain-of-function mutation in the Plcg2 gene (Ali5) to analyze its role in the development of gastric MALT lymphoma. Heterozygous BALB/c Plcg2Ali5/+ and wildtype (WT) mice were infected with Helicobacter felis (H. felis) and observed up to 16 months for development of gastric MALT lymphomas. In contrast to our initial hypothesis, Plcg2Ali5/+ mice developed MALT lymphomas less frequently than their WT littermates after long-term infection of 16 months. Infected Plcg2Ali5/+ mice showed downregulation of proinflammatory cytokines and decreased H. felis-specific IgG1 and IgG2a antibody responses. These results suggested a blunted immune response of Plcg2Ali5/+ mice towards H. felis infection. Intriguingly, Plcg2Ali5/+ mice harboured higher numbers of CD73 expressing regulatory T cells (Tregs), possibly responsible for impaired immune response towards Helicobacter infection. We suggest that Plcg2Ali5/+ mice may be protected from developing gastric MALT lymphomas as a result of elevated Treg numbers, reduced response to H. felis and decrease of proinflammatory cytokines. [ABSTRACT FROM AUTHOR]

The article discusses the clinical trial of SB010 novel DNA enzyme that involves patients with allergic asthma with sputum eosinophilia and had biphasic early and late asthmatic responses after allergen provocation. Topics covered include study design and screening and randomization, the use of a methacholine challenge to evaluate airway responsiveness, and graphs showing lung function after allergen challenge.

Background: Cross-sectional epidemiological studies have demonstrated that farm milk from traditional farm settings possesses allergoprotective properties. Up to now, it has not been clarified which milk ingredient is responsible for protection against allergic diseases. As farm milk is rich in conjugated linoleic acids (CLA), it is hypothesized that this n-3 polyunsaturated fatty acid family contributes to the allergoprotective capacity of farm milk. We aim to prove this hypothesis in a murine model of allergic airway inflammation. Methods: To prove the bioavailability and allergoprotective capacity of milk-associated CLA in a standardized protocol, milk batches that differed significantly in terms of their CLA content were spray dried and incorporated into a basic diet by substituting the regular sunflower fat fraction. Initially, the milk CLA uptake from the diet was monitored via measurement of the CLA content in plasma and erythrocyte membranes obtained from supplemented mice. To determine whether a milk CLA-enriched diet possesses allergoprotective properties, female Balb/c mice were fed the milk CLA-enriched diet ahead of sensitization and a challenge with ovalbumin (OVA) and the parameters of airway inflammation and eisosanoid pattern were measured. Results: In animals, supplementation with a diet rich in milk CLA resulted in elevated CLA levels in plasma and erythrocyte membranes, indicating bioavailability of milk fatty acids. Though membrane-associated phospholipid patterns were affected by supplementation with milk CLA, this application neither reduced the hallmarks of allergic airway inflammation in sensitized and OVA-challenged mice nor modified the eiconsanoid pattern in the bronchoalveolar lavage fluid of these animals. Conclusion: Milk-associated CLA was not capable of preventing murine allergic airway inflammation in an animal model of OVA-induced allergic airway inflammation. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Allergic asthma is associated with chronic airway inflammation and progressive airway remodelling. However, the dynamics of the development of these features and their spontaneous and pharmacological reversibility are still poorly understood. We have therefore investigated the dynamics of airway remodelling and repair in an experimental asthma model and studied how pharmacological intervention affects these processes. Methods: Using BALB/c mice, the kinetics of chronic asthma progression and resolution were characterised in absence and presence of inhaled corticosteroid (ICS) treatment. Airway inflammation and remodelling was assessed by the analysis of bronchoalveolar and peribronichal inflammatory cell infiltrate, goblet cell hyperplasia, collagen deposition and smooth muscle thickening. Results: Chronic allergen exposure resulted in early (goblet cell hyperplasia) and late remodelling (collagen deposition and smooth muscle thickening). After four weeks of allergen cessation eosinophilic inflammation, goblet cell hyperplasia and collagen deposition were resolved, full resolution of lymphocyte inflammation and smooth muscle thickening was only observed after eight weeks. ICS therapy when started before the full establishment of chronic asthma reduced the development of lung inflammation, decreased goblet cell hyperplasia and collagen deposition, but did not affect smooth muscle thickening. These effects of ICS on airway remodelling were maintained for a further four weeks even when therapy was discontinued. Conclusions: Utilising a chronic model of experimental asthma we have shown that repeated allergen exposure induces reversible airway remodelling and inflammation in mice. Therapeutic intervention with ICS was partially effective in inhibiting the transition from acute to chronic asthma by reducing airway inflammation and remodelling but was ineffective in preventing smooth muscle hypertrophy. [ABSTRACT FROM AUTHOR]

Abstract: The DNAzyme hgd40 was shown to effectively reduce expression of the transcription factor GATA-3 RNA which plays an important role in the regulation of Th2-mediated immune mechanisms such as in allergic bronchial asthma. However, uptake, biodistribution and pharmacokinetics of hgd40 have not been investigated yet. We examined local and systemic distribution of hgd40 in naive mice and mice suffering from experimental asthma. Furthermore, we evaluated the pharmacokinetics as a function of dose following single and repeated administration in rats and dogs. Using intranasal administration of fluorescently labeled hgd40 we demonstrated that the DNAzyme was evenly distributed in inflamed asthmatic mouse lungs within minutes after single dose application. Systemic distribution was investigated in mice using radioactive labeled hgd40. After intratracheal application, highest amounts of hgd40 were detected in the lungs. High amounts were also detected in the bladder indicating urinary excretion as a major elimination pathway. In serum, low systemic hgd40 levels were detected already at 5min post application (p.a.), subsequently decreasing over time to non-detectable levels at 2h p.a. As revealed by Single Photon Emission Computed Tomography, trace amounts of hgd40 were detectable in lungs up to 7days p.a. Also in the toxicologically relevant rats and dogs, hgd40 was detectable in blood only shortly after inhalative application. The plasma pharmacokinetic profile was dose and time dependent. Repeated administration did not lead to drug accumulation in plasma of dogs and rats. These pharmacokinetic of hgd40 provide guidance for clinical development, and support an infrequent and convenient dose administration regimen. [Copyright &y& Elsevier]

The continual rise of asthma in industrialised countries stands in strong contrast to the situation in developing lands. According to the modified Hygiene Hypothesis, helminths play a major role in suppressing bystander immune responses to allergens, and both epidemiological and experimental studies suggest that the tropical parasitic trematode Schistosoma mansoni elicits such effects. The focus of this study was to investigate which developmental stages of schistosome infection confer suppression of allergic airway inflammation (AAI) using ovalbumin (OVA) as a model allergen. Moreover, we assessed the functional role and localization of infection-induced CD4+Foxp3+ regulatory T cells (Treg) in mediating such suppressive effects. Therefore, AAI was elicited using OVA/adjuvant sensitizations with subsequent OVA aerosolic challenge and was induced during various stages of infection, as well as after successful anti-helminthic treatment with praziquantel. The role of Treg was determined by specifically depleting Treg in a genetically modified mouse model (DEREG) during schistosome infection. Alterations in AAI were determined by cell infiltration levels into the bronchial system, OVA-specific IgE and Th2 type responses, airway hyper-sensitivity and lung pathology. Our results demonstrate that schistosome infection leads to a suppression of OVA-induced AAI when mice are challenged during the patent phase of infection: production of eggs by fecund female worms. Moreover, this ameliorating effect does not persist after anti-helminthic treatment, and depletion of Treg reverts suppression, resulting in aggravated AAI responses. This is most likely due to a delayed reconstitution of Treg in infected-depleted animals which have strong ongoing immune responses. In summary, we conclude that schistosome-mediated suppression of AAI requires the presence of viable eggs and infection-driven Treg cells. These data provide evidence that helminth derived products could be incorporated into treatment strategies that specifically target suppression of immune responses in AAI by inducing Treg cells. Author Summary: Infections with schistosomes, such as S. mansoni, S. japonicum and S. haematobium, are considered a major public health concern. Morbidity arises through granulomatous responses to eggs that become trapped in infected tissues. Interestingly, schistosomes belong to the group of helminths that have been shown to reduce allergy or autoimmunity. Indeed, the evidence provided by epidemiological surveys and experimental animal models has been so overwhelming that such helminths are now included in the Hygiene Hypothesis. However, since helminths provoke immunological responses that are similar to those seen in allergy (increased eosinophilia and IgE) it is suggested that additional mechanisms dampen such allergic responses. Helminth-induced regulatory T cells (Treg) are considered a component of these modulatory networks. Using an allergic airway inflammation model, we have elucidated that schistosome-mediated protection requires patency, that is, active egg production from fecund female worms. In addition, protection was shown to be mediated by infection-induced Treg. Interestingly, in endemic countries it is usually individuals with strong patent infections that show reduced allergic prevalence. Thus, further research into the immunomodulatory capacity of schistosome-egg derived factors may elucidate novel drug candidates or enhance treatment strategies to reduce allergic responses on the cellular level. [ABSTRACT FROM AUTHOR]

The continual rise of asthma in industrialised countries stands in strong contrast to the situation in developing lands. According to the modified Hygiene Hypothesis, helminths play a major role in suppressing bystander immune responses to allergens, and both epidemiological and experimental studies suggest that the tropical parasitic trematode Schistosoma mansoni elicits such effects. The focus of this study was to investigate which developmental stages of schistosome infection confer suppression of allergic airway inflammation (AAI) using ovalbumin (OVA) as a model allergen. Moreover, we assessed the functional role and localization of infection-induced CD4+Foxp3+ regulatory T cells (Treg) in mediating such suppressive effects. Therefore, AAI was elicited using OVA/adjuvant sensitizations with subsequent OVA aerosolic challenge and was induced during various stages of infection, as well as after successful anti-helminthic treatment with praziquantel. The role of Treg was determined by specifically depleting Treg in a genetically modified mouse model (DEREG) during schistosome infection. Alterations in AAI were determined by cell infiltration levels into the bronchial system, OVA-specific IgE and Th2 type responses, airway hyper-sensitivity and lung pathology. Our results demonstrate that schistosome infection leads to a suppression of OVA-induced AAI when mice are challenged during the patent phase of infection: production of eggs by fecund female worms. Moreover, this ameliorating effect does not persist after anti-helminthic treatment, and depletion of Treg reverts suppression, resulting in aggravated AAI responses. This is most likely due to a delayed reconstitution of Treg in infected-depleted animals which have strong ongoing immune responses. In summary, we conclude that schistosome-mediated suppression of AAI requires the presence of viable eggs and infection-driven Treg cells. These data provide evidence that helminth derived products could be incorporated into treatment strategies that specifically target suppression of immune responses in AAI by inducing Treg cells. [ABSTRACT FROM AUTHOR]

Abstract: DNAzymes are single-stranded catalytic DNA molecules that bind and cleave specific sequences in a target mRNA molecule. Their potential as novel therapeutic agents has been demonstrated in a variety of disease models. However, no studies have yet addressed their toxicology and safety pharmacology profiles in detail. Here we describe a detailed toxicological analysis of inhaled hgd40, a GATA-3-specific DNAzyme designed for the treatment of allergic bronchial asthma. Subacute toxicity, immunotoxicity, and respiratory, cardiovascular, and CNS safety pharmacology were analyzed in rodents and non-rodents, and genotoxicity was assessed in human peripheral blood. Overall, hgd40 was very well tolerated when delivered by aerosol inhalation or slow intravenous infusion. Only marginal reversible histopathological changes were observed in the lungs of rats receiving the highest dose of inhaled hgd40. The changes consisted of slight mononuclear cell infiltration and alveolar histiocytosis, and moderate hyperplasia of bronchus-associated lymphoid tissue. No local or systemic adverse effects were observed in dogs. No compound-related respiratory, cardiovascular, or CNS adverse events were observed. The only relevant immunological findings were very slight dose-dependent changes in interleukin-10 and interferon-γ levels in bronchoalveolar lavage fluid. Taken together, these results support direct delivery of a DNAzyme via inhalation for the treatment of respiratory disease. [Copyright &y& Elsevier]

The immunoproteasome subunit β5i has been shown to play an important role in Th1/Th17 driven models of colitis and arthritis. However, the function of β5i in Th2 dependent diseases remains enigmatic. To study the role of β5i in Th2-driven pathology, β5i knockout (KO) and control mice were tested in different models of experimental allergic asthma. β5i-deficient mice showed reduced OVA/Alum- and subcutaneous/OVA-induced acute asthma with decreased eosinophilia in the bronchoalveolar lavage (BAL), low OVA-specific IgG1 and reduced local and systemic Th2 cytokines. While Th2 cells in the lungs were reduced, Tregs and Th1 cells were not affected. Attenuated asthma in β5i KO mice could not be attributed to defects in OVA uptake or maturation of dendritic cells in the lung. Surprisingly, β5i deficient mice developed HDM asthma which was comparable to control mice. Here, we present novel evidence for the requirement of the β5i immunosubunit to generate a strong Th2 response during OVA- but not HDM-induced acute asthma. The unexpected role of β5i in OVA asthma remains to be clarified. [ABSTRACT FROM AUTHOR]

Background: Lymphatic vessels play a pivotal role in fluid drainage and egress of immune cells from the lung. However, examining murine lung lymphatics is hampered by the expression of classical lymph endothelial markers on other cell types, which hinders the unambiguous identification of lymphatics. The expression of CD90/Thy-1 on lymph endothelium was recently described and we therefore examined its suitability to identify murine pulmonary lymph vessels under healthy and inflammatory conditions. Methodology/Principal Findings: Immunohistochemistry with a monoclonal antibody against CD90.2/Thy-1.2 on 200 µm thick precision cut lung slices labeled a vascular network that was distinct from blood vessels. Preembedding immunostaining and electron microscopy verified that the anti-CD90.2/Thy-1.2 antibody labeled lymphatic endothelium. Absence of staining in CD90.1/Thy-1.1 expressing FVB mice indicated that CD90/Thy-1 was expressed on lymph endothelium and labeling was not due to antibody cross reactivity. Double-labeling immunohistochemistry for CD90/Thy-1 and α-smooth muscle actin identified two routes for lymph vessel exit from the murine lung. One started in the parenchyma or around veins and left via venous blood vessels. The other began in the space around airways or in the space between airways and pulmonary arteries and left via the main bronchi. As expected from the pulmonary distribution of lymph vessels, intranasal application of house dust mite led to accumulation of T cells around veins and in the connective tissue between airways and pulmonary arteries. Surprisingly, increased numbers of T cells were also detected around intraacinar arteries that lack lymph vessels. This arterial T cell sheath extended to the pulmonary arteries where lymph vessels were located. Conclusions/Significance: These results indicate that CD90/Thy-1 is expressed on lymphatic endothelial cells and represents a suitable marker for murine lung lymph vessels. Combining CD90/Thy-1 labeling with precision cut lung slices allows visualizing the anatomy of the lymphatic system in normal and inflamed conditions. [ABSTRACT FROM AUTHOR]

Abstract: DNAzymes are potent novel drugs for the treatment of inflammatory diseases such as atopic dermatitis. DNAzymes represent a novel class of pharmaceuticals that fulfil a causal therapy by interruption of the inflammation cascade at its origin. There are two challenges regarding the dermal application of DNAzymes: the large molecular weight and the sensitivity to DNases as part of the natural skin flora. To overcome these limitations suitable carrier systems have to be considered. Nano-sized drug carrier systems (submicron emulsions, microemulsions) are known to improve the skin uptake of drugs due to their ability to interact with the skin''s lipids. To protect the drug against degradation, the hydrophilic drug may be incorporated into the inner aqueous phase of carrier systems, such as water-in-oil-in-water multiple emulsions. In the present study various emulsions of pharmaceutical grade were produced. Their physicochemical properties were determined and the influence of preservation systems on stability was tested. Drug release and skin uptake studies using various skin conditions and experimental set-ups were conducted. Furthermore, cellular uptake was determined by flow cytometric analysis. The investigations revealed that the developed multiple emulsion is a suitable and promising drug carrier system for the topical application of DNAzyme. [Copyright &y& Elsevier]

DNAzymes of the 10-23 family represent an important class of antisense molecules with implications for therapeutic treatment of diseases. These molecules are single-stranded oligodeoxynucleotides combining the high specificity of oligonucleotide base pairing with an inherent RNA-cleaving enzymatic activity. However, like other oligonucleotide-based molecules these substances might exert so-called off-target effects, which have not been investigated so far for this molecule class. Therefore, the present study investigates putative off-target effects of DNAzymes on innate immune mechanisms using GATA-3-specific DNAzymes that have recently been developed as novel therapeutic approach for the treatment of allergic diseases including allergic asthma. The conserved catalytic domain of 10-23 DNAzymes contains a CpG motif that may stimulate innate immune cells via Toll-like receptor 9 (TLR-9). Therefore, potential TLR-9-mediated as well as TLR-9 independent cell activation was investigated using TLR-9-transfected HEK293 cells, macrophage cell lines and primary innate immune cells. Furthermore, putative effects of GATA-3-specific DNAzymes on the activation of neutrophil granulocytes and degranulation of mast cells/basophils were analyzed. In summary, no innate immune cell-stimulating activities of the tested DNAzymes were observed in any of the systems. Consequently, use of GATA-3-specific DNAzymes may represent a novel and highly specific approach for the treatment of allergic diseases. [ABSTRACT FROM AUTHOR]

Summary: Chronic obstructive pulmonary disease (COPD) is one of the most common causes of death worldwide. We report in an emphysema model of mice chronically exposed to tobacco smoke that pulmonary vascular dysfunction, vascular remodeling, and pulmonary hypertension (PH) precede development of alveolar destruction. We provide evidence for a causative role of inducible nitric oxide synthase (iNOS) and peroxynitrite in this context. Mice lacking iNOS were protected against emphysema and PH. Treatment of wild-type mice with the iNOS inhibitor N6-(1-iminoethyl)-L-lysine (L-NIL) prevented structural and functional alterations of both the lung vasculature and alveoli and also reversed established disease. In chimeric mice lacking iNOS in bone marrow (BM)-derived cells, PH was dependent on iNOS from BM-derived cells, whereas emphysema development was dependent on iNOS from non-BM-derived cells. Similar regulatory and structural alterations as seen in mouse lungs were found in lung tissue from humans with end-stage COPD. [ABSTRACT FROM AUTHOR]

Background: We have previously shown that the allergic sensitization to ovalbumin does not represent a superantigen-like immune response. In gene-targeted mice (ΔD-iD) with a single modified Diversity gene segment (DH) of the immunoglobulin heavy chain, enriched for charged amino acids, the asthma phenotype in a murine model was markedly alleviated compared to wild-type animals. Objective: We now sought to determine whether the confinement to a single DH gene segment alone leads to a reduced allergic phenotype. Methods: We examined another gene-targeted mouse strain (ΔD-DFL) with a single DH gene segment which encodes for neutral amino acids, thus reflecting the preferential repertoire in wild-type mice. Mice were sensitized intraperitoneally to ovalbumin. Results: Despite the constraint to a single DH gene segment, ΔD-DFL mice mounted high total and allergen-specific IgG1 and IgE serum levels after sensitization to ovalbumin. The affinity constants of allergen-specific IgG1 antibodies did not differ between ΔD-DFL and wild type. Following challenge with aerosolized allergen, a marked local TH2 cytokine response and an eosinophilic airway inflammation developed. Quantitative histology revealed increased mucus production and intense goblet cell metaplasia which were identical to those in wild type. Moreover, ΔD-DFL mice developed an airway hyperreactivity to methacholine and to the specific allergen, which both did not differ from those in wild-type animals. Conclusion: A single DH gene segment is sufficient for the establishment of the asthma phenotype in a murine model of allergic airway inflammation. Thus, the allergic phenotype depends on the amino acid composition and not on the diversity of the classical antigen-binding site. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: DNAzymes are a group of RNA-cleaving DNA oligonucleotides that contain a catalytic domain and represent a novel class of antisense molecules. Although single-stranded DNAzymes may represent the most effective nucleic acid drug to date, the sensitivity to nuclease degradation is challenging. Therefore, it is important to develop a drug delivery system, which protects the molecule against degradation during dermal application. In the present study, the potential protective effect, regarding the dermal application of DNAzyme, of multiple (W/O/W) emulsions, W/O emulsions, submicron emulsion and microemulsions were investigated using a HPLC method. The HPLC method enables the quantitative analysis of DNAzyme as well as the detection of degradation products. The differences between the activity of DNase I and the activity of nucleases located in the porcine skin were compared. It was found that the degradation of an aqueous solution of DNAzyme is depending on the DNase I activity as well as on the incubation time. Furthermore, the activity of neutral and acid nucleases in skin tissue was determined to be 5.2 and 14.8U per 1g of porcine skin tissue, respectively. Investigation of the protective character of different delivery systems revealed that formulations containing DNAzyme in the outer water phase (submicron emulsion and microemulsion) did not exhibit any form of protective effect, whereas formulations containing DNAzyme in the inner water phase (multiple emulsion and W/O emulsion) were able to prevent the DNAzyme degradation to a considerable degree. Consequently, these formulations are promising candidates for the dermal drug delivery of oligonucleotides. [Copyright &y& Elsevier]

Type I allergy is a classical Th2-driven hypersensitivity disease based on IgE recognition of environmental allergens. Exposure of allergic individuals to exogenous allergens leads to immediate type inflammation caused by degranulation of mast cells via IgE-allergen immune complexes and the release of inflammatory mediators, proteases and pro-inflammatory cytokines. However, allergic inflammation can occur and persist in the absence of exposure to exogenous allergens and might paradoxically resemble a Th1-mediated chronic inflammatory reaction. We summarize evidence supporting the view that autoimmune mechanisms might contribute to these processes. IgE recognition of autoantigens might augment allergic inflammation in the absence of exogenous allergen exposure. Moreover, autoantigens that activate Th1-immune responses could contribute to chronic inflammation in allergy, thus linking allergy to autoimmunity. [Copyright &y& Elsevier]

In the era of personalized medicine, insights into the molecular mechanisms that differentially contribute to disease phenotypes, such as asthma phenotypes including obesity-associated asthma, are urgently needed. Peripheral blood was drawn from 10 obese, non-atopic asthmatic adults with a high body mass index (BMI; 36.67 ± 6.90); 10 non-obese, non-atopic asthmatic adults with normal BMI (23.88 ± 2.73); and 10 healthy controls with normal BMI (23.62 ± 3.74). All asthmatic patients were considered to represent a low type-2 asthma phenotype according to selective clinical parameters. RNA sequencing (RNA-Seq) was conducted on peripheral blood CD4+ T cells. Thousands of differentially expressed genes were identified in both asthma groups compared with heathy controls. The expression of interferon (IFN)-stimulated genes associated with IFN-related signaling pathways was specifically affected in obese asthmatics, while the gap junction and G protein-coupled receptor (GPCR) ligand binding pathways were enriched in both asthma groups. Furthermore, obesity gene markers were also upregulated in CD4+ T cells from obese asthmatics compared with the two other groups. Additionally, the enriched genes of the three abovementioned pathways showed a unique correlation pattern with various laboratory and clinical parameters. The specific activation of IFN-related signaling and viral infection pathways might provide a novel view of the molecular mechanisms associated with the development of the low type-2 obesity-associated asthma phenotype, which is a step ahead in the development of new stratified therapeutic approaches. [ABSTRACT FROM AUTHOR]

The treatment of dyspeptic disorders with anti-acids leads to an increased risk of sensitization against food allergens. As these drugs are taken by 30-50% of pregnant women due to reflux and heart-burn, we aimed here to investigate the impact of maternal therapy with anti-acids on the immune response in the offspring in a murine model. Codfish extract as model allergen was fed with or without sucralfate, an anti-acid drug, to pregnant BALB/c mice during pregnancy and lactation. These mothers developed a codfish-specific allergic response shown as high IgG1 and IgE antibody levels and positive skin tests. In the next step we analyzed whether this maternal sensitization impacts a subsequent sensitization in the offspring. Indeed, in stimulated splenocytes of these offspring we found a relative Th2-dominance, because the Th1- and T-regulatory cytokines were significantly suppressed. Our data provide evidence that the anti-acid drug sucralfate supports sensitization against food in pregnant mice and favors a Th2-milieu in their offspring. From these results we propose that anti-acid treatment during pregnancy could be responsible for the increasing number of sensitizations against food allergens in young infants. [ABSTRACT FROM AUTHOR]

Production of superoxide radicals is a central property of professional phagocytes used to combat invading microorganisms. Even though the number of macrophages and neutrophils is often increased in the lungs of patients with chronic lung diseases, these patients frequently suffer from bacterially induced exacerbations. To understand the underlying mechanisms, we investigated the production of superoxide radicals by bronchoalveolar lavage (BAL) cells in a rat NO2 exposure model (10 ppm NO2 for 1, 3, or 20 days). We showed that cells from NO2-exposed animals display a significantly impaired superoxide radical release after zymosan stimulation. The use of specific inhibitors (antimycin or diphenyleneiodonium [DPI]) revealed that the major enzyme systems, NADPH oxidase and complex III of the respiratory chain, are affected. In addition, we investigated gene expression and enzyme activities of antioxidant enzymes. mRNA expression was significantly enhanced for glutathione peroxidase (GPx)-3 and CuZn-superoxide dismutase (SOD) in BAL cells from animals exposed 3 and 20 days, and GPx and SOD enzyme activities were increased in BAL cells from rats exposed 20 days. In conclusion, concomitant occurrence of reduced production and increased scavenging of superoxide radicals resulted in the drastically impaired release of these radicals from BAL cells of NO2-exposed rats. [Copyright &y& Elsevier]

The lung is protected against oxidative stress by a variety of antioxidants and type II pneumocytes seem to play an important role in antioxidant defense. Previous studies have shown that inhalation of NO 2 results in acute and chronic lung injury. How the expression and enzyme activity of antioxidant enzymes are influenced in type II cells of different inflammatory stages has yet not been studied. To elucidate this question, we exposed rats to 10 ppm NO 2 for 3 or 20 days to induce acute or chronic lung injury. From these and air-breathing rats, type II pneumocytes were isolated. The mRNA expression and protein content of CuZnSOD and MnSOD as well as total SOD-specific enzyme activity were determined. For the acute lung injury (3 d NO 2 ), the expression of CuZnSOD mRNA was significantly increased, while MnSOD expression was significantly reduced after 3 days of NO 2 exposure. For the chronic lung injury (20 d NO 2 ), CuZnSOD expression was still enhanced, while MnSOD expression was comparable to control. In parallel to CuZnSOD mRNA expression, the protein amount was significantly increased in acute and chronic lung injury however MnSOD protein content exhibited no intergroup differences. Total SOD enzyme activity showed a significant decrease after 3 days of NO 2 exposure and was similar to control after 20 days. We conclude that during acute and chronic lung injury in type II pneumocytes expression and protein synthesis of CuZnSOD and MnSOD are regulated differently. [ABSTRACT FROM AUTHOR]