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SummaryBackground Targeting PD-1/PD-1 ligand signalling is an established treatment option for cancer. The role of these molecules in allergic asthma has been investigated in several mouse studies yielding conflicting results. However, human studies investigating the expression and regulation of PD-1 and its ligands in allergic inflammation are lacking. Objective To analyse the expression and regulation of PD-1 and its ligands in human allergic asthma. Methods The well-established human asthma model of segmental allergen challenge (SAC) was used to analyse the regulation of PD-1 and its ligands PD-L1 and PD-L2 on T lymphocytes and dendritic cells by flow cytometry. The impact of immunoglobulin E (IgE)-mediated signalling on PD-L1 expression was analysed on isolated plasmacytoid dendritic cells (pDCs). Results PD-1 expression by blood CD4+ T cells was negatively associated with total and specific (against the allergen used for provocation) IgE serum concentrations. Twenty-four hours after SAC, a small decrease in endobronchial PD-1+CD4+ T cells was accompanied by an increase in PD-L1 expression on endobronchial myeloid dendritic cells (mDCs) and pDCs. The PD-L1 up-regulation on pDCs was not induced by IgE-mediated mechanisms. In contrast, PD-L2 was only detected on endobronchial mDCs and was significantly down-regulated 24 hours after SAC. Conclusion and clinical relevance This study shows, for the first time, an association of a low PD-1 expression by circulating CD4+ T cells with high total and specific (against the allergen used for provocation) IgE concentrations in allergic asthma. In addition, we demonstrate a differential regulation of PD-1 ligands on endobronchial DCs after allergen challenge which may favour Th2 inflammation. Therefore, modulating PD-1 ligand-mediated pathways might be a promising target in allergic asthma.

SummaryBackground Plasmacytoid dendritic cells (pDCs) infiltrate sites of acute Th2-dominant inflammation, but their role in allergic asthma remains unclear. Objective To characterize circulating pDCs from patients with allergic asthma outside their respective allergen season. Methods Adhesion molecules, co-stimulatory molecules, immunoglobulin receptors and chemokine receptors were quantified on blood pDCs from 20 patients with allergic asthma and 18 healthy controls using flow cytometry. In addition, IL-6-, TNF-α- and IFN-α-secretion were analysed after stimulating isolated pDCs with TLR7- and TLR9-ligands. Results Plasmacytoid dendritic cells from patients with allergic asthma showed an increased expression of chemokine receptors involved in inflamed tissue homing such as CCR2, CCR4, CCR9, CCR10, CXCR2, CXCR5 and CXCR6, while the expression of the lymph node homing receptor CXCR3 was down-regulated. In addition, these pDCs exhibited a higher expression of activation markers and Th2-associated molecules such as CD40, CD62L, CD64 and FceRIα. In contrast, TLR7-mediated IL-6-, TNF-α- and IFN-α-secretion was significantly reduced in pDCs from patients with asthma. The TLR9-mediated cytokine response was only suppressed in those patients who were treated with inhaled corticosteroids (ICS) during previous allergen seasons. The same effect was observed for CD54 and OX40L expression. Conclusions We report an increased expression of activation markers, and Th2-associated molecules, and an increased migratory potential of circulating pDCs in allergic asthma. These changes are accompanied by a reduced TLR7-mediated cytokine response. In addition, our results suggest a longterm impact of ICS treatment on the characteristics of circulating pDCs.

The essential oils (EOs) of green seeds from Daucus carota subsp. maximus growing wild in Pantelleria Island (Sicily, Italy) were characterized. EOs were extracted by steam distillation, examined for their inhibitory properties against food-borne Gram-positive and Gram-negative bacteria and analyzed for the chemical composition by gas chromatography (GC) and mass spectrometry (MS). Undiluted EOs showed a large inhibition spectrum against Gram-positive strains and also vs. Acinetobacter spp. and Stenotrophomonas maltophilia. The minimum inhibition concentration (MIC) was in the range 1.25 – 2.50 μl/ml for the most sensitive strains. The chemical analysis indicated that D. carota subsp. maximus EOs included 34 compounds (five monoterpene hydrocarbons, six oxygenated monoterpenes, 14 sesquiterpene hydrocarbons, four oxygenated sesquiterpenes, camphorene and four other compounds), accounting for 95.48% of the total oil, and that the major chemicals were carotol, β-bisabolene, and isoelemicin.

To cite this article: Bratke K, Klein C, Kuepper M, Lommatzsch M, Christian Virchow J. Differential development of plasmacytoid dendritic cells in Th1- and Th2-like cytokine milieus. Allergy 2011; 66: 386–395. Abstract Background: Plasmacytoid dendritic cells (pDCs) infiltrate sites of Th1- and Th2-dominant inflammation and many studies have been performed to analyse their role in these immune responses. In contrast, much less is known about the effects of a Th1 or Th2 cytokine milieu on pDC function. Therefore, we investigated the impact of Th1- and Th2-like conditions during the development of pDCs on their antigen expression and function. Methods: PDCs were matured in vitro by the addition of IL-3 under Th1- or Th2-like conditions. Antigen expression and TLR7-ligand-induced cytokine secretion was analysed by flow cytometry and ELISA. Furthermore, the CD4+ T-cell polarizing capacity of pDCs was determined as well as their potential to induce CD4+ T-cell proliferation. Results: PDCs matured under Th1-like conditions showed a higher expression of antigens involved in T-cell co-stimulation and antigen presentation like CD40, CD80, CD83 and HLA-DR as well as a higher secretion of IL-6 and IFN-α in response to TLR7-ligation compared to Th2-pDCs. Furthermore, Th1-pDCs induced a significantly higher CD4+ T-cell proliferation and primed a higher percentage of CD4+ T cells to express IFN-γ and IL-2 after TLR7-ligation compared to Th2-pDCs. In contrast, Th2-pDCs were characterized by a significant upregulation of BDCA-3 and IL-4 expression following TLR7-ligation. Conclusion: This study is the first to demonstrate the crucial impact of a surrounding cytokine environment on the development of pDC function including antigen expression. Based on these findings, it can be speculated that antiviral/bacterial pDC functions could be impaired during acute allergic conditions.

Summary Background Plasmacytoid dendritic cells (pDCs) are involved in a variety of immune functions. However, the expression of cytotoxic granule proteins like granzymes and perforin in human pDCs is still poorly understood. Objective The aim of this study was to systematically analyse the expression and regulation of cytotoxic granule proteins in human pDCs. Methods The expression of cytotoxic proteins was analysed by RT-PCR, flow cytometry, and fluorescence microscopy. The functional expression of these proteins was confirmed in a flow-cytometry-based cytotoxicity assay using K562 cells as targets. In order to analyse the regulation of pDC-derived cytotoxic proteins in infectious and allergic diseases, human pDCs were analysed after stimulation with toll-like receptor (TLR)7/9 ligands and in the human asthma model of segmental allergen challenge. Results Granzyme B (GrB), but not the granzymes A, H, K, M or perforin, was specifically expressed by human pDCs and this GrB expression was up-regulated by IL-3 stimulation. In addition, IL-3-stimulated pDCs were found to kill K562 cells in a GrB- and caspase-dependent manner. TLR7/9 ligands significantly suppressed GrB expression in pDCs. In contrast, there was an up-regulation of GrB in endobronchial pDCs 24 h after allergen challenge, and this was accompanied by enhanced GrB concentrations in bronchoalveolar lavage fluid. Conclusion We report the selective expression of GrB in human pDCs and show for the first time pDC-mediated GrB- and caspase-dependent cytotoxicity against target cells. In addition, the regulation of GrB expression was investigated in vitro and in vivo providing an evidence for a specific role of pDC-derived GrB in allergic inflammation. Cite this as: K. Bratke, J. Nielsen, F. Manig, C. Klein, M. Kuepper, S. Geyer, P. Julius, M. Lommatzsch and J. C. Virchow, Clinical & Experimental Allergy, 2010 (40) 1015–1024.

Thin functional films were deposited on aluminum with an atmospheric pressure plasma jet using hexamethyldisiloxane (HMDSO) as precursor. A high dynamic deposition rate on the order of 450 nm . m . min(-1) was achieved. Composition and structure of the thin films show a strong dependence on the downstream location of the precursor injection. A 4 mm downstream shift of the precursor injection increases the carbon content in the thin film by a factor of 2.5, as indicated by XPS analysis and alters the degree of cross-linking according to the FTIR spectra. The coating with the low carbon content (17 at.-%) provides corrosion resistance for aluminum 2024 unclad exposed for 96 h to a neutral salt spray test. The coating with the high carbon content reduces the adhesion of an epoxy resin to the surface and may be used as a release coating.

Polyethylene (PE) samples were activated by an atmospheric pressure plasma jet. The improvement in adhesive bond strength is attributed to the incorporation of oxygen-containing functional groups into the PE surface. Optical emission spectroscopy in combination with XPS analysis shows differences in the surface reactions for a plasma jet operated with air or pure nitrogen. The results indicate that the surface modifications take place in two different environments with respect to location and time: (a) reactions while the substrate is hit by the plasma jet, and (b) reactions outside the plasma jet after the treatment.

There is growing evidence that target-derived neurotrophins regulate the function of visceral neurons after birth. However, the postnatal profile of neurotrophin supply from internal organs is poorly described. In this study, we compared neurotrophin concentrations in lysates of murine peripheral target tissues (lung, heart, liver, colon, spleen, thymus, kidney and urinary bladder) at different time points after birth. In most organs, there was a decrease of neurotrophin concentrations in the first weeks after birth. In contrast, there were characteristic increases of specific neurotrophins during adolescence or adulthood. These increases were found for nerve growth factor (NGF) in the heart, thymus, kidney and liver, for brain-derived neurotrophic factor (BDNF) in the lung, and for neurotrophin-3 (NT-3) in the colon. In conclusion, we show that neurotrophins display a very differential and dynamic profile in internal organs after birth.

Brain-derived neurotrophic factor (BDNF) is a candidate molecule for mediating functional neuronal changes in allergic bronchial asthma. Recently, enhanced production of BDNF during allergic airway inflammation caused by infiltrating T-cells and macrophages as well as by resident airway epithelial cells has been described. It was the aim of this study to investigate the effect of enhanced BDNF levels on lung function and airway inflammation in a mouse model of allergic inflammation. Ovalbumin-sensitised BALB/c mice were challenged in two consecutive allergen challenges. Prior to the challenge, the mice were treated with either anti-BDNF antibodies or isotype-matched control antibodies. Airway responsiveness to methacholine, capsaicin and electric field stimulation, as well as airway inflammation and chronic airway obstruction 1 week after the last allergen challenge were assessed. Anti-BDNF blocked enhanced reactivity in response to capsaicin, but not airway smooth muscle hyper-reactivity in vivo. Furthermore, persistent airway obstruction, as observed 1 week after the last allergen challenge, was to a large extent prevented by anti-BDNF treatment. In vitro, BDNF and anti-BDNF treatment had a profound effect on local neuronal hyper-reactivity, as shown by electric field stimulation experiments. In contrast, neither BDNF nor anti-BDNF treatment affected airway inflammation. Our data indicate that development of allergen-induced neuronal hyper-reactivity in mice is partially mediated by BDNF. British Journal of Pharmacology (2004) 141, 431-440. doi:10.1038/sj.bjp.0705638

Neurotrophins such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are potent mediators of neuronal plasticity in the adult. There is increasing evidence that they regulate a variety of immune functions as well. Thus, neurotrophins are candidate molecules for neuroimmune interactions in allergic bronchial asthma, where elevated neurotrophin levels have been reported. In a mouse model of allergic airway inflammation we have identified macrophages and lymphocytes as additional cellular sources of NGF and BDNF in the inflamed lung. There was an unusual time course of BDNF in bronchoalveolar lavage fluid. BDNF levels peaked 1 week after the last allergen challenge, and did not correlate with the time course of the inflammatory response. In a series of experiments using blocking anti-NGF and anti-BDNF antibodies, we have shown that NGF specifically enhances inflammation and the allergic early-phase response. In contrast, BDNF influenced chronic airway obstruction and local neuronal hyperreactivity without affecting inflammation. Using transgenic mice overexpressing NGF in the airway epithelium, we have confirmed the data obtained from anti-NGF experiments. Allergen-challenged NGF overexpressors displayed a markedly augmented airway inflammation, early-phase response, and sensory irritation compared to wild-type mice. Studies with p75-NTR (-/-) knockout mice showed that these NGF effects are at least in part mediated by the low-affinity neurotrophin receptor. Thus, our experiments suggest that NGF and BDNF have a profound, but differential impact on allergic airway dysfunction.

Neuronally differentiated PC12 cells undergo synchronous apoptosis when deprived of nerve growth factor (NGF). Here we show that NGF withdrawal induces actinomycin D- and cycloheximide-sensitive caspase (ICE-like) activity. The peptide inhibitor of caspase activity, N-acetyl-Asp-Glu-Val-Asp-aldehyde, was more potent than acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone in preventing NGF withdrawal-induced apoptosis, suggesting an important role for caspase-3 (CPP32)-like proteases. We observed a peak of reactive oxygen species (ROS) 6 h after NGF withdrawal. ROS appear to be required for apoptosis, because cell death is prevented by the free radical spin trap, N-tert-butyl-alpha-phenylnitrone, and the antioxidant, N-acetylcysteine. ROS production was blocked by actinomycin D, cycloheximide, and caspase protease inhibitors, suggesting that ROS generation is downstream of new mRNA and protein synthesis and activation of caspases. Forced expression of either BCL-2 or the BCL-2-binding protein BAG-1 blocked NGF withdrawal-induced apoptosis, activation of caspases, and ROS generation, showing that they function upstream of caspases. Coexpression of BCL-2 and BAG-1 was more protective than expression of either protein alone.

Neurotrophins are known as target-derived factors regulating the development, survival and function of distinct neuronal subsets. This family of structurally related growth factors includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4. Beyond their originally described effects on neurones, neurotrophins are involved in many other biological processes including immune regulation, smooth muscle differentiation and neuroendocrine functions. The pathogenesis of allergic bronchial asthma is characterized by a complex interaction between inflammatory, epithelial, smooth muscle cells and neurones. This results in the development of airway inflammation, airway obstruction, airway hyperresponsiveness and airway remodelling. The altered neuronal control of airway smooth muscle tone is a hallmark in bronchial asthma. Growing evidence suggests that T cells, B cells, macrophages and mast cells produce high quantities of neurotrophic factors during allergic inflammation. Therefore, they are likely candidates as mediators between allergic inflammation and airway hyperresponsiveness. Neurotrophins increase excitability and neurotransmitter synthesis in sensory neurones and can also affect motor neurones. They enhance local neuropeptide release and augment central reflexes. Additionally, neurotrophins can affect immune functions and smooth muscle physiology. Therefore, neurotrophins are involved in the cellular communication between different compartments of the asthmatic lung. Delineating the mechanisms of this cross-talk will give new insights into the pathogenesis of asthma.

Besides their recognized dependence on nerve growth factor (NGF) during development, the dependence of mature sympathetic ganglion neurons on other neurotrophins is still unclear. Here, we have investigated the sympathetic innervation of back skin in mice overexpressing brain-derived neurotrophic factor (BDNF) under the alpha-myosin heavy-chain promoter, as well as in BDNF knockout (-/-) mice. Compared with wild-type controls, the dorsal skin of BDNF overexpressing mice displayed a significantly enhanced number of adrenergic, tyrosine hydroxylase-immunoreactive (IR) nerve fibres, while cholinergic or peptidergic sensory nerve fibres appeared unaltered. The adrenergic hyperinnervation in dorsal skin of BDNF overexpressing mice was most pronounced in the arrector pili muscle of hair follicles, while no increase of tyrosine hydroxylase-or neuropeptide Y-IR fibres associated with subcutaneous blood vessels was found. Instead, back skin of BDNF knockout (-/-) mice contained significantly fewer tyrosine hydroxylase-IR dermal nerve fibres than wild-type animals. This suggests that BDNF plays an important role in the control of different subsets of adrenergic innervation in murine back skin, and indicates that paravertebral sympathetic ganglia display a previously unrecognized differential BDNF-dependence in vivo.

Purpose This retrospective study was designed to investigate the re-injection interval, efficacy and safety of OZURDEX® in routine clinical practice. Methods This analysis contains data from 87 patients from 10 sites in Germany who had received at least 2 OZURDEX® injections. Data was collected from the time of the patients’ first injection until 3 – 6 months following their latest OZURDEX® injection. Results The mean time to OZURDEX® re-injection between 1st and 2nd treatments was 141 days (5.03 months) in the overall population. Mean time intervals for the BRVO and CRVO sub-populations were 153 days (5.46 months) and 127 days (4.52 months) respectively. In the overall population, a mean LogMAR BCVA improvement from 0.68 to 0.51 was recorded following the last OZURDEX® injection (mean time of 11.0 weeks post-injection). For BRVO, mean improvement was from 0.54 to 0.43 (mean time of 10.5 weeks post injection) and for CRVO, 0.83 to 0.58 (mean time of 11.5 weeks post injection). Reductions in central retinal thickness were also observed. Intraocular pressure measurements over 25mmHg were reported in 19.5% of patients. No glaucoma surgeries were reported. 5 patients underwent cataract surgery during the course of the study (4 had known lens opacity at baseline and opacity status data from the 5th was missing). Conclusion In this real life study, OZURDEX® was found to be safe and effective with repeat treatments. The mean re-injection interval for RVO patients was 5.0 months.