Compartmental and temporal dynamics of chronic inflammation and airway remodelling in a chronic asthma mouse model.

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.

Biodistribution of the GATA-3-specific DNAzyme hgd40 after inhalative exposure in mice, rats and dogs.

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 5 min 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 7 days 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.

Absence of unspecific innate immune cell activation by GATA-3-specific DNAzymes.

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.

Epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes.

BACKGROUND: Bronchial asthma is a chronic inflammatory disease resulting from complex gene-environment interactions. Natural microbial exposure has been identified as an important environmental condition that provides asthma protection in a prenatal window of opportunity. Epigenetic regulation is an important mechanism by which environmental factors might interact with genes involved in allergy and asthma development. OBJECTIVE: This study was designed to test whether epigenetic mechanisms might contribute to asthma protection conferred by early microbial exposure. METHODS: Pregnant maternal mice were exposed to the farm-derived gram-negative bacterium Acinetobacter lwoffii F78. Epigenetic modifications in the offspring were analyzed in T(H)1- and T(H)2-relevant genes of CD4(+) T cells. RESULTS: Prenatal administration of A lwoffii F78 prevented the development of an asthmatic phenotype in the progeny, and this effect was IFN-γ dependent. Furthermore, the IFNG promoter of CD4(+) T cells in the offspring revealed a significant protection against loss of histone 4 (H4) acetylation, which was closely associated with IFN-γ expression. Pharmacologic inhibition of H4 acetylation in the offspring abolished the asthma-protective phenotype. Regarding T(H)2-relevant genes only at the IL4 promoter, a decrease could be detected for H4 acetylation but not at the IL5 promoter or the intergenic T(H)2 regulatory region conserved noncoding sequence 1 (CNS1). CONCLUSION: These data support the hygiene concept and indicate that microbes operate by means of epigenetic mechanisms. This provides a new mechanism in the understanding of gene-environment interactions in the context of allergy protection.

Triazine dendrimers as nonviral vectors for in vitro and in vivo RNAi: the effects of peripheral groups and core structure on biological activity.

A family of triazine dendrimers, differing in their core flexibility, generation number, and surface functionality, was prepared and evaluated for its ability to accomplish RNAi. The dendriplexes were analyzed with respect to their physicochemical and biological properties, including condensation of siRNA, complex size, surface charge, cellular uptake and subcellular distribution, their potential for reporter gene knockdown in HeLa/Luc cells, and ultimately their stability, biodistribution, pharmacokinetics and intracellular uptake in mice after intravenous (iv) administration. The structure of the backbone was found to significantly influence siRNA transfection efficiency, with rigid, second generation dendrimers displaying higher gene knockdown than the flexible analogues while maintaining less off-target effects than Lipofectamine. Additionally, among the rigid, second generation dendrimers, those with either arginine-like exteriors or peripheries containing hydrophobic functionalities mediated the most effective gene knockdown, thus showing that dendrimer surface groups also affect transfection efficiency. Moreover, these two most effective dendriplexes were stable in circulation upon intravenous administration and showed passive targeting to the lung. Both dendriplex formulations were taken up into the alveolar epithelium, making them promising candidates for RNAi in the lung. The ability to correlate the effects of triazine dendrimer core scaffolds, generation number, and surface functionality with siRNA transfection efficiency yields valuable information for further modifying this nonviral delivery system and stresses the importance of only loosely correlating effective gene delivery vectors with siRNA transfection agents.

Effective prevention and therapy of experimental allergic asthma using a GATA-3-specific DNAzyme.

BACKGROUND: Allergic bronchial asthma is a chronic inflammatory disease of the airways. The transcription factor GATA-3 was shown to play an important role in TH2 cell activation, but also in the regulation of other cell types involved in bronchial asthma including mast cells, eosinophils, and epithelial cells. DNAzymes represent a new class of antisense molecules that combines the specificity of DNA base pairing with an inherent RNA-cleaving enzymatic activity. OBJECTIVE: To develop a GATA-3 mRNA-specific DNAzyme and analyze its allergy-preventing activity in murine models of experimental allergic asthma. METHODS: The most active DNAzyme (termed gd21) was selected by in vitro cleavage assays. Allergic airway inflammation was assessed by inflammatory cell and cytokine analysis within bronchoalveolar lavage. Lung histology, including goblet cell hyperplasia and lung function, was analyzed using head-out body-plethysmography. RESULTS: Intranasal administration of gd21 prevented airway inflammation and mucus production and inhibited development of airway hyperresponsiveness to methacholine in models of acute allergic airway inflammation. Similar effects were also detected in a model of chronic experimental asthma. Interestingly, gd21 was at least as effective as other antisense molecules, and off-target effects were not detected. Further experiments indicated that pulmonary surfactant may facilitate the cellular uptake of gd21 by acting as an endogenous transfectant. CONCLUSION: These results indicate that topical application of the GATA-3-specific DNAzyme is a promising novel approach for the treatment of allergic bronchial asthma.

Anti-ulcer treatment during pregnancy induces food allergy in mouse mothers and a Th2-bias in their offspring.

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 heartburn, 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.