Marion Kauth

Respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study

Background: Acute exacerbations of chronic obstructive pulmonary disease (AE-COPD) are a common cause of hospital admission. Many exacerbations are believed to be due to upper and/or lower respiratory tract viral infections, but the incidence of these infections in patients with COPD is still undetermined. Methods: Respiratory syncytial virus (RSV), influenza A and B, parainfluenza 3, and picornaviruses were detected by nested reverse transcription polymerase chain reaction (RT-PCR) in upper (nasal lavage) and lower respiratory tract specimens (induced sputum). In a 2:1 case-control set up, 85 hospitalised patients with AE-COPD and 42 patients with stable COPD admitted for other medical reasons were studied. Results: Respiratory viruses were found more often in sputum and nasal lavage of patients with AE-COPD (48/85, 56%) than in patients with stable COPD (8/42, 19%, p<0.01). The most common viruses were picornaviruses (21/59, 36%), influenza A (15/59, 25%), and RSV (13/59, 22%). When specimens were analysed separately, this difference was seen in induced sputum (exacerbation 40/85 (47%) v stable 4/42 (10%), p<0.01) but was not significant in nasal lavage (exacerbation 26/85 (31%) v stable 7/42 (17%), p=0.14). In patients with AE-COPD, fever was more frequent in those in whom viruses were detected (12/48, 25%) than in those in whom viruses were not detected (2/37, 5%, p=0.03). Conclusion: Viral respiratory pathogens are found more often in respiratory specimens of hospitalised patients with AE-COPD than in control patients. Induced sputum detects respiratory viruses more frequently than nasal lavage in these patients. These data indicate that nasal lavage probably has no additional diagnostic value to induced sputum in cross-sectional studies on hospitalised patients with AE-COPD and that the role of viral infection in these patients is still underestimated.

Inhalation of stable dust extract prevents allergen induced airway inflammation and hyperresponsiveness

Background: Recent epidemiological studies have shown that growing up on a traditional farm provides protection from the development of allergic disorders such as hay fever and allergic asthma. We present experimental evidence that substances providing protection from the development of allergic diseases can be extracted from dust collected in stables of animal farms. Methods: Stable dust was collected from 30 randomly selected farms located in rural regions of the Alps (Austria, Germany and Switzerland). The dust was homogenised with glass beads and extracted with physiological sodium chloride solution. This extract was used to modulate immune response in a well established mouse model of allergic asthma. Results: Treatment of mice by inhalation of stable dust extract during sensitisation to ovalbumin inhibited the development of airway hyperresponsiveness and airway eosinophilia upon challenge, as well as the production of interleukin 5 by splenocytes and of antigen specific IgG1 and IgE. Dust extract also suppressed the generation of human dendritic cells in vitro. The biological activity of the dust extract was not exclusively mediated by lipopolysaccharide. Conclusions: Stable dust from animal farms contains strong immune modulating substances. These substances can interfere with the development of both cellular and humoral immunity against allergens, thus suppressing allergen sensitisation, airway inflammation, and airway hyperresponsiveness in a murine model of allergic asthma.

Arabinogalactan isolated from cowshed dust extract protects mice from allergic airway inflammation and sensitization

BACKGROUND Extract from cowshed dust (CDE) is a source of immunomodulating substances. We have previously shown that such substances protect from experimental allergic disorders in a mouse model of asthma. OBJECTIVE The objective of this study was to identify immunomodulatory molecules in extracts of dust from an allergy protective farming environment. METHODS Polysaccharides were isolated from CDE and plants by chromatography and precipitation with specific reagents. Polysaccharides were then characterized by nuclear magnetic resonance spectroscopy. Subsequently, the allergy-protective potential of isolated polysaccharides was tested in a mouse model of asthma. RESULTS The authors demonstrate that plant arabinogalactans are contained in CDE in high concentrations. The source of this arabinogalactan is fodder, in particular a prevalent grass species known as Alopecurus pratensis. Treatment of murine dendritic cells with grass arabinogalactan resulted in autocrine IL-10 production. Interestingly, these dendritic cells were not able to induce an allergic immune response. Furthermore, intranasal application of grass arabinogalactan protected mice from developing atopic sensitization, allergic airway inflammation and airway hyperreactivity in a mouse model of allergic asthma. This allergy-protective effect is specific for grass arabinogalactan because control experiments with arabinogalactan from gum arabic and larch revealed that these molecules do not show allergy-protective properties. This is likely because of structural differences because we were able to show by nuclear magnetic resonance spectroscopy that although they are predominantly composed of arabinose and galactose, the molecules differ in structure. CONCLUSIONS The authors conclude that grass arabinogalactans are important immunomodulatory substances that contribute to the protection from allergic airway inflammation, airway hyperresponsiveness, and atopic sensitization in a mouse model of asthma.

Inflammatory Response in Acute Viral Exacerbations of COPD

Background:Respiratory viruses are important triggers of acute exacerbations of COPD (AE-COPD). However, the inflammatory response in virus-positive exacerbations is still not fully understood.Methods:We investigated CRP, IL-6, IL-8, IL-10, IFN-γ, blood and sputum cells in patients with acute exacerbation (n = 36) and in stable disease (n = 20) and correlated these parameters to virus detection in respiratory secretions.Results:Similar to other studies we found a significant increase in systemic CRP and absolute numbers of blood leukocytes in AE-COPD patients. Sputum IL-6 levels and sputum eosinophils tended to be higher during exacerbation. In patients with detection of respiratory viruses in nasal lavage, local IL-6 production in sputum was significantly increased; FEV1 was significantly decreased and both parameters were inversely correlated to each other.Conclusion:This study supports previous findings of both, increased local and systemic inflammation in acute exacerbation of COPD. In virus-associated exacerbations, IL-6 is significantly increased and negatively correlated to FEV1 indicating a relation between virus-induced inflammation and airway obstruction. However, regarding our finding and previous data, it is becoming increasingly clear that the mediators investigated so far do not permit identifying the etiology of AE-COPD. Hence, further studies are needed to better define the inflammatory response in AE-COPD in general and in viral exacerbations in particular.

Anti-BDCA-4 (neuropilin-1) antibody can suppress virus-induced IFN-alpha production of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDC) in human blood are the main source of virus‐induced interferon (IFN)‐alpha. They exhibit a lineage‐negative phenotype but all express BDCA‐4, which is homologous to the neuronal receptor neuropilin‐1. Specific staining with anti‐BDCA‐4 antibody is used for positive isolation of PDC from blood by magnetic cells sorting. Here, it is demonstrated that these positively selected PDC showed reduced or completely abolished IFN‐alpha release compared to unstained PDC, which were negatively selected by magnetic depletion of lineage‐positive blood mononuclear cells. In addition, treatment of these unstained PDC with anti‐BDCA‐4 mAb also resulted in at least two‐fold lower or reduced virus‐induced IFN‐alpha production. It is shown that the antibody not only affects cell survival or block virus attachment but also reduces IFN‐alpha release induced by non‐viral CpG oligodeoxynucleotides. In conclusion, data suggest an immunoregulatory role for BDCA‐4 on PDC as demonstrated for IFN‐alpha response to virus.

A ternary complex consisting of AICD, FE65, and TIP60 down-regulates Stathmin1

The ternary complex consisting of AICD/FE65/TIP60 is thought to play a role in gene expression and was suggested to have a crucial impact in Alzheimers disease. AICD is the intracellular subdomain of the amyloid precursor protein (APP) and able to bind the adapter protein FE65 and the histone acetyltransferase TIP60 setting up a nuclear dot-like phenotype. Within this work we readdressed the generation of the complex as a function of its compartments. Subsequently, we studied the proteome of AFT expressing cells vs. controls and identified Stathmin1 significantly down-regulated in AFT cells. Stathmin1 functions as an important regulatory protein of microtubule dynamics and was found associated with neurofibrillary tangles in brains of Alzheimers disease patients. We validated our results using an independent label-free mass spectrometry based method using the same cell culture model. In a reversal model with diminished APP expression, caused by simultaneous knock-down of all three members of the APP family, we further confirmed our results, as Stathmin1 was regulated in an opposite fashion. We hypothesize that AICD-dependent deregulation of Stathmin1 causes microtubule disorganization, which might play an important role for the pathophysiology of Alzheimers disease.

Modulation of dendritic cell function by cowshed dust extract.

We have shown previously that inhalation of cowshed dust extract (CDE) resulted in decreased airway reactivity, eosinophilic inflammation and sensitization in a mouse model of allergic asthma. Our data suggested down-regulation of allergic immune response rather than activation of a Th1 response towards the model allergen. However, the precise mechanism of allergy protection is not yet understood in detail. To gain deeper insight into CDE-induced immune modulation, we have analysed the effects of CDE on dendritic cell biology. Dendritic cells were generated from murine bone marrow cells (BMDC). Cells were stimulated with CDE and subsequently used to sensitize mice via the airways. Our results showed that cells were not able to prime mice for allergic immune response when they were treated with CDE 2 days before pulsing with allergen, whereas cells that were stimulated with CDE simultaneously to OVA pulsing induced a fully developed allergic immune response. Surprisingly, CDE-treated cells that were not able to prime mice for allergic immune response exhibit an activated phenotype with high expression of the co-stimulatory surface molecule CD86. Moreover, CDE-treated cells transiently produced high amounts of cytokines such as IL-10, IL-12p70 and TNF-α. Interestingly, blocking of autocrine-produced IL-10 in vitro partially restored the allergy-inducing capacity of CDE-exposed cells. Thus, we conclude that prolonged exposure to CDE reduces the allergy-inducing capacity of dendritic cells. Furthermore, we present evidence that an autocrine IL-10 dependent mechanism seems to be involved in down-regulation of dendritic cell function due to stimulation with CDE.

Synergistically Upregulated Interleukin-10 Production in Cocultures of Monocytes and T Cells after Stimulation with Respiratory Syncytial Virus

Background: Respiratory syncytial virus (RSV) is known as a causal factor of severe bronchiolitis in young children. It has also been detected in patients with chronic obstructive pulmonary disease (COPD), a disease that is associated with an increased number of T cells in the bronchial mucosa. Here, we investigated the potential direct interaction between RSV and T cells and its impact on cytokine response. Methods: Purified human peripheral blood T cells were stimulated with RSV in vitro and analyzed by flow cytometry and fluorescence microscopy. Cytokine expression and release were measured in T cell cultures and in cocultures with peripheral blood monocytes as well as with alveolar macrophages from bronchoalveolar lavage fluid by quantitative real-time PCR and ELISA. Results: It was shown that RSV adhered to the surface of T cells. Stimulation of purified T cells with RSV led to a significant increase in interleukin (IL)-10 mRNA expression after 24 h. Moreover, in cocultures of T cells with monocytes or alveolar macrophages, IL-10 production was synergistically upregulated 24 h after stimulation with RSV. Conclusion: These results suggest that RSV can cause an excessive IL-10 response leading to downregulation of antiviral defense mechanisms and reduced elimination of respiratory pathogens when antigen-presenting cells and T cells are simultaneously present on the site of infection. This effect may possibly contribute to high frequencies of respiratory pathogens found in patients with chronic inflammatory airway diseases associated with increased local T cell influx such as COPD.

Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects

Background: Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. Objective: We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. Methods: L lactis G121–induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor‐treated (bafilomycin and cytochalasin D) human monocyte‐derived dendritic cells (moDCs), bone marrow–derived mouse dendritic cells (BMDCs), and moDC/naive CD4+ T‐cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin‐induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. Results: L lactis G121–treated murine BMDCs and human moDCs released TH1‐polarizing cytokines and induced TH1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of TH1‐polarizing cytokines, costimulatory molecule expression, and T‐cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll‐like receptor (Tlr) 13−/− BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The TH1‐polarizing activity of L lactis G121–treated human DCs was blocked by TLR8‐specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide‐binding oligomerization domain‐containing protein (NOD) 2. Conclusion: Bacterial RNA is the main driver of L lactis G121–mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8. GRAPHICAL ABSTRACT Figure. No caption available.

Stepwise isolation of human peripheral erythrocytes, T lymphocytes, and monocytes for blood cell proteomics.

Density gradient centrifugation and magnetic‐ or fluorescence‐activated cell sorting are common and robust techniques for the isolation of different types of blood cells. In this article, we give detailed description of a stepwise application of these methods as one isolation strategy for enrichment of different cell types from one blood sample.