- English (8) (remove)
- Radioligand binding studies : structure affinity relationships of antagonists atmuscarinic, serotonergic and histaminergic receptor subtypes (2003)
- GPCRs and ligand-gated ion channels mediate a great variety of physiological effects within the human brain and periphery. The search for selective ligands at these target sites as pharmacological tools or new drug candidates is of great interest. With increasing knowledge of the great diversity of some receptor families, compounds formerly considered to be selective, turned out to be non-selective with regard to recently identified subtypes, splice variants or additional receptor subunits. This work provides SAR studies by means of radioligand binding experiments at serotonergic h5-HT3A and h5-HT4(b) receptors, histamine hH1 receptors and muscarinic hM1-5 receptors. ...
- Pharmacological actions and targets of boswellic acids in human leukocytes and platelets (2006)
- Boswellia serrata gum resin extracts (frankincense) have been used for centuries in folk medicine in Asia and Africa. They have shown beneficial therapeutic effects, particularly in the treatment of chronic inflammatory diseases. Clinical studies on humans confirmed an anti-inflammatory and anti-cancer potential of Frankincense preparations. Boswellic acids (BAs) are the major ingredients, responsible for the pharmacological action of the extracts. Molecular and cellular studies with BAs revealed a number of targets including 5-lipoxygenase (LO), topoisomerases and the NF-κB pathway. Since there is little information on the modulation of cellular physiology by BAs, this work was designed to provide a detailed investigation of the cellular and molecular effects of BAs in several cell types related to inflammation. We report that 11-keto-BAs are potent activators of functional responses in human neutrophils, a type of leukocytes mediating acute inflammatory processes. Neutrophil activation by 11-keto-BAs is reflected by enhanced generation of oxygen radicals, release of arachidonic acid (AA) and the subsequent transformation of AA to pro-inflammatory eicosanoids. Investigation of the participating signalling pathways identified Ca2+, phosphoinositide-3 kinase, and members of the MAP kinase family (ERKs) as mediators. Second, we present a detailed study of the modulation of human platelet physiology and intracellular signalling events by BAs. Intriguingly, we discovered an inverse structure-activity relationship of BAs regarding platelet activation, with 11-methylene-BAs being superior over 11-keto-BAs. Thus, 11-methylene-BAs stimulated platelet Ca2+ mobilisation, MAP kinase and Akt activation, AA release, 12-LO and cyclooxygenase product formation, and thrombin generation. Novel Ca2+-independent activation pathways of platelet lipid metabolism were discovered. In contrast, 11-keto-BAs were inactive but found to inhibit platelet (p)12-LO directly. Interaction with p12-LO was confirmed in a pulldown assay using immobilised BAs as bait. Finally, BAs were shown to attenuate the activation of monocytes, a cell type responsible for the maintenance of chronic inflammatory states. Impairment of Ca2+ homeostasis is likely conferred by inhibition of Ca2+ influx channels. Taken together, our results shed light on the modulation of intracellular physiology of inflammatory cells by BAs, contributing to a better understanding of the anti-inflammatory effects exerted by frankincense preparations.
- Role of endothelial Cytochrome P450 epoxygenases in the regulation of angiogenesis (2008)
- Cytochrome P450 epoxygenases of the 2C family (CYP2C) are highly expressed in the endothelium and metabolize arachidonic acid to different regioisomers of epoxyeicosatrienoic acids (EET). They have a number of roles in the regulation of vascular tone and homeostasis by activating different signal transduction pathways and have recently been reported to be involved in proliferation and angiogenesis. However, the exact mechanisms by which epoxygenases regulate angiogenesis are still unclear. Therefore, the initial aim of the present study was to characterize the relevance of major signalling molecules that are involved in angiogenesis and to investigate possible signalling pathways involved. Initially the effect of CYP2C9 overexpression on expression levels of EphB4, a tyrosine kinase that plays a role in a number of developmental processes, was investigated. EphB4 protein expression was increased in CYP2C9 overexpressing cells without any effects on expression levels of its ligand ephrinB2. To clarify whether EphB4 is a critical determinant of CYP2C9-induced angiogenesis, endothelial cell sprouting was assessed using a collagen gel-based in vitro angiogenesis assay. Following transfection with EphB4 antisense or scrambled oligonucleotides, capillary-like structures were clearly present after 24 hours in cells overexpressing CYP2C9, while EphB4 downregulation abolished CYP2C9-induced sprouting. In addition stimulation of human umbilical vein endothelial cells with VEGF resulted in an increase in CYP2C expression and a subsequent increase of 11,12-EET production; an effect that was abolished by the CYP epoxygenases inhibitor MSPPOH as well as when cells were infected with a dominant negative mutant of AMPK. In vivo 11,12-EET treatment increased EphB4 expression in mesenteric arteries as well as in Matrigel plugs; an effect that was abolished when plugs were impregnated at the same time with small interfering RNA (siRNA) for EphB4. Furthermore, impregnation of Matrigel plugs with VEGF resulted in endothelial cell and smooth muscle cell recruitment into a Matrigel plug and this effect was mediated by CYP2C9-derived EETs as it was prevented by 14,15-EEZE. When infiltration of EET impregnated plugs with endothelial cells and pericytes/smooth muscle cells in vivo was compared to the effects seen in VEGF treated plugs, it was apparent that only EET treatment resulted in the formation of tube like structures that were covered by smooth muscle cells. Therefore, the final aim of the study was to further define the consequences of EET signalling in vivo as well as to characterize its physiological relevance. This hypothesis could be assessed by isolectin injection through the tail-vein where isolectin was taken up only by the EET-impregnated plug. Moreover ultrasound measurements revealed accumulation of contrast agent in EET impregnated plugs compared to control plugs. Taken together our findings emphasize that CYP2C plays a crucial role in the vessel formation process by modulating the effects mediated by two important control elements of the angiogenic response, namely VEGF and EphB4. CYP2C-derived EETs not only participate as second messengers in the angiogenic response, but have the potential to influence much more than angiogenesis by enhancing smooth muscle cell/pericyte recruitment to endothelial cell tubes to promote vascular maturation.
- Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection (2009)
- Autoimmune hepatitis (AIH) is a chronic liver disease of unknown etiology, characterized by a loss of tolerance against hepatocytes leading to the progressive destruction of hepatic parenchyma and cirrhosis. Clinical signs for AIH are interface hepatitis and portal plasma cell infiltration, hypergammaglobulinemia, and autoantibodies. Based on serological markers AIH is defined in subtypes. The hallmark of AIH type 2 are type 1 liver/kidney microsomal autoantibodies (LKM-1), whereas AIH type 1 is characterized by the presence of anti-nuclear (ANA) and/or anti-smooth muscular (SMA) autoantibodies. The major autoantigen recognized specifically by LKM-1 autoantibodies was identified as the 2D6 isoform of the cytochrome P450 enzyme family (CYP2D6). Not much is known about the etiology and pathogenic mechanisms of AIH so far and most animal models available result in only transient hepatic liver damage after a rather complex initiation method. It was the aim of my project to generate a novel animal model for AIH that reflects the chronic and progressive destruction of the liver characteristic for the human disease while using a defined and feasible initiating event to further analyze the pathogenic mechanisms leading to the autoimmune-mediated destruction of the liver. Therefore, mice transgenically expressing the human CYP2D6 in the liver and wild-type mice were infected with a liver-tropic adenovirus expressing the human CYP2D6 (Ad-2D6). Selftolerance to CYP2D6 was broken in Ad-2D6-infected mice, resulting in persistent autoimmune liver damage, apparent by cellular infiltration, hepatic fibrosis and necrosis. Similar to type 2 AIH patients, Ad-2D6-infected mice generated LKM-1-like antibodies recognizing the same immunodominant epitope of CYP2D6. Taken together, we could introduce a new animal model that reflects the persistent autoimmune-mediated liver damage as well as the serological marker characteristic for AIH type 2 and we could demonstrate that chronic autoimmune diseases targeting the liver can be triggered by molecular mimicry occurring in the context of a hepatotropic viral infection.
- GTPase activating protein Rap1GAP2 and synaptotagmin-like protein 1 interact and are involved in platelet dense granule secretion (2008)
- Platelets are anucleate cells that play a major role in hemostasis and thrombosis in the vasculature. During primary hemostasis platelets adhere to sites of vascular damage and the initial platelet coat is reinforced by additional platelets forming a stable aggregate. At the same time platelets secrete their intracellular granules containing substances that further activate platelets in an autocrine and paracrine fashion and affect local coagulation and endothelial smooth muscle cell function. The small guanine nucleotide binding protein Rap1 regulates the activity of the platelet integrin alphaIIbbeta3 and thus platelet aggregation. Rap1 activity is controlled by guanine nucleotide exchange factors and GTPase activating proteins. In platelets, Rap1GAP2 is the only GTPase activating protein of Rap1. In order to identify Rap1GAP2-associated proteins, a genetic two-hybrid screening in yeast was performed and synaptotagmin-like protein 1 (Slp1, also called JFC1) was found as a new putative binding partner of Rap1GAP2. Slp1 is a tandem C2 domain containing protein and is known to bind to Rab27, a small GTPase involved in platelet dense granule secretion. The direct interaction between Rap1GAP2 and Slp1 was confirmed in yeast and in transfected cells. More importantly, Slp1 is expressed in platelets and binding of endogenous Rap1GAP2 and Slp1 was verified in these cells. The Rap1GAP2 and Slp1 interaction sites were mapped by mutational analysis. Rap1GAP2 binds through the -TKXT- motif within its C-terminus to the C2A domain of Slp1. Moreover, the Slp1 binding -TKXT- motif of Rap1GAP2 was confirmed by complementary approaches using short synthetic Rap1GAP2 peptides. The C2A domain of Slp1 is a phospholipid binding domain and thus mediates binding of Slp1 to the plasma membrane. Phospholipid overlay assays revealed that simultaneous binding of Slp1 via its C2A domain to Rap1GAP2 and to phospholipids can occur. In addition, the interaction between Rap1GAP2 and Slp1 is regulated by cAMP-dependent protein kinase (cAK or PKA), and kinase activation in platelets enhanced binding of endogenous Rap1GAP2 to Slp1. In-vitro phosphorylation assays revealed that Slp1 is a substrate of PKA, and serine 111 was identified as phosphorylation site. Since Slp1 is a Rab27 binding protein, a trimeric complex of Slp1, Rab27 and Rap1GAP2 is conceivable. The association of Slp1, Rab27 and Rap1GAP2 was investigated by immunofluorescence and co-immuno-precipitation experiments in both, transfected cells and platelets. By Slp1 affinity chromatography and subsequent mass spectrometric analysis additional Slp1 binding proteins were identified in platelets, and binding of Slp1 to Rab8 was confirmed in pull-down assays. To investigate the functional significance of the interaction between Rap1GAP2 and Slp1, an assay system was established to determine serotonin secretion of streptolysin-O permeabilized platelets. Addition of recombinant Slp1 protein to permeabilized platelets strongly inhibited platelet dense granule secretion, whereas addition of recombinant Rap1GAP2 protein or synthetic Rap1GAP2 peptide enhanced secretion. Deleting the Slp1 binding -TKXT- motif abolished the stimulatory effect of Rap1GAP2 on secretion. Addition of Rap1 to permeabilized platelets had no effect on secretion. These findings indicate that the Rap1GAP2 effect on platelet secretion does not depend on the GTPase activating function of Rap1GAP2, but is rather dependent on the -TKXT- mediated interaction of Rap1GAP2 with Slp1. In addition, in-vitro GAP assays revealed that Slp1 binding to Rap1GAP2 does not affect the Rap1GAP activity of Rap1GAP2, and adhesion assays excluded a role for the Rap1GAP2/Slp1 interaction in cell adhesion. Altogether, the results of the present study demonstrate that besides its function in platelet aggregation by controlling the activity of the small guanine nucleotide binding protein Rap1, Rap1GAP2 is involved in platelet dense granule secretion by the new -TKXT- mediated interaction with the Rab27 and membrane binding protein Slp1. In addition, the interaction between Rap1GAP2 and Slp1 is embedded into an elaborate network of protein-protein interactions in platelets which appear to be regulated by phosphorylation. Future studies will in particular aim to dissect the molecular details of Rap1GAP2 and Slp1 action in platelet secretion and investigate the potential biochemical and pharmacological value of the unique protein binding -TKXT- motif of Rap1GAP2.
- The influence of the Maillard reaction on the immunogenic properties of food allergens (2010)
- Die Maillard-Reaktion findet während der Lagerung und thermischen Verarbeitung von Lebensmitteln zwischen den darin enthaltenen Proteinen und reduzierenden Kohlehydraten statt. Als Ergebnis der Reaktion entstehen sogenannte advanced glycation end products (AGEs), Protein-Derivate mit Glykierungs-Strukturen. Da Lebensmittel vor dem Verzehr häufig erhitzt werden, ist der Einfluss von AGEs auf die Pathogenese von Nahrungsmittelallergien von großem Interesse. Die Maillard-Reaktion könnte zur Bildung von neuen, für die Pathogenese der Nahrungsmittelallergie relevanten, Immunepitopen beitragen. Das Ziel dieser Arbeit war es, den Einfluss der Maillard-Reaktion auf die T-Zell-Immunogenität, die Antigenität und die von beiden Eigenschaften abhängige Allergenität von Nahrungsmittelallergenen zu untersuchen. Zunächst wurde der Einfluss der Maillard-Reaktion auf die T-Zell-Immunogenität von Ovalbumin (OVA), einem Allergen des Hühnereiweißes, untersucht. Dafür wurde glykiertes OVA (AGE-OVA) hergestellt indem das Protein zusammen mit Glukose erhitzt wurde. In dieser Arbeit konnte zum ersten Mal gezeigt werden, dass ein AGE-Derivat eines Lebensmittelallergens eine höhere T-Zellen-Immunogenität besitzt, als sein natives Gegenstück. Die Aktivierung und Proliferation von CD4+ T-Zellen durch AGE-OVA wurde in vitro durch Co-Kultivierung der T-Zellen mit dendritischen Zellen (DZ) untersucht. DZ sind professionelle Antigen- präsentierende Zellen, welche im Pathomechanismus der Allergie eine wichtige Rolle spielen. Im Vergleich zu nativen OVA und OVA welches ohne Glukose erhitzt wurde, führte die Stimulierung mit AGE-OVA zu einer deutlich erhöhten Aktivierung von OVA-spezifischen CD4+ T-Zellen. Damit DZ T-Zellen aktivieren können, muss das Allergen zunächst durch die DZ aufgenommen werden. In der vorliegenden Arbeit wurde gezeigt, dass die Aufnahme von AGE-OVA wesentlich höher war als die der Kontrollen. Außerdem konnte der scavenger receptor class A type I and II (SR-AI/II) als einer der hauptverantwortlichen Rezeptoren für die Aufnahme von AGE-OVA identifiziert werden. Zusammenfassend lässt sich aus den Ergebnissen dieser Arbeit die Hypothese aufstellen, dass die Glykierung von OVA eine erhöhte Assoziation des Allergens mit SR-AI/II ermöglicht, welche zu einer verstärkten Aufnahme des Allergens durch die DZ führt. Dadurch können mehr Peptide des Allergens an MHC II gebunden und auf der Zelloberfläche präsentiert werden. Das wiederum führt zur beobachteten stärkeren OVA-spezifischen CD4+ T-Zell-Aktivierung durch AGE-OVA. Als nächstes wurde die T-Zell-Immunogenität und Antigenität von AGE-OVA in vivo in einem Mausmodel untersucht. Es zeigte sich, dass AGE-OVA auch in vivo im Vergleich zu den nicht glykierten OVA-Formen eine erhöhte T-Zell-Immunogenität besitzt. Des weiteren führte die Immunisierung mit AGE-OVA zu einer erhöhten Produktion von IgE-Antikörpern. Somit wurde in dieser Arbeit gezeigt, dass AGE-OVA in vivo nicht nur eine erhöhte CD4+ T-Zell-Immunogenität besitzt, sondern auch eine höhere Antigenität hat als natives und ohne Glukose erhitztes OVA. Diese Ergebnisse harmonieren gut miteinander da CD4+ T-Zellen eine zentrale Rolle in der Aktivierung von B-Zellen und der IgE-Produktion durch selbige Zellen spielen. IgE-Antikörper besitzen eine essentielle Funktion beim Auslösen der klinischen Symptomatik der Allergie. Zusammenfassend lässt deshalb sagen, dass die Maillard-Reaktion die Allergenität von OVA erhöhen könnte. Zum Schluss wurden noch die immunstimulatorischen Eigenschaften des Erdnussallergens (AGE)-Ara h 2 untersucht. Da Erdnüsse häufig ernsthafte allergische Reaktionen hervorrufen und selten roh verzehrt werden, war es vom großen Interesse den Einfluss der Maillard-Reaktion auf Immunogenität und Antigenität von rekombinanten Ara h 2 (rAra h 2) zu untersuchen. Es zeigte sich, dass die Glykierung von rAra h 2 durch die Maillard-Reaktion die T-Zellen-Immunogenität, als auch die Antigenität des Allergens reduziert. Abschließend lässt sich sagen, dass die Maillard-Reaktion die allergenen Eigenschaften von Lebensmittelallergenen erheblich beeinflusst indem es die T-Zell-Immunogenität des Allergens verändert. Die Mechanismen welche die T-Zell-Immunogenität beeinflussen wurden hier näher untersucht. Wenn die Glykierung nicht die Bindung der T-Zellen- und/oder B-Zellen-Rezeptoren inhibiert, wird die Allergen-spezifische CD4+ T-Zell-Aktivierung und die davon abhängige IgE-Produktion dadurch erhöht, dass das glykierte Allergen durch DZ verstärkt über SR-AI/II aufgenommen wird. Die vorliegende Arbeit liefert wertvolle Information über die Allergenität von Proteinen die durch die Maillard-Reaktion modifiziert wurden and trägt dazu bei die Mechanismen von Nahrungsmittelallergien besser zu verstehen.
- Leukocyte attraction and transmigration in type 1 diabetes: neutralization of CXCR3 and JAM-C reduces the severity of disease (2010)
- Type 1 diabetes (T1D) is a chronic T cell-mediated autoimmune disorder that results in the destruction of insulin-producing pancreatic ß cells leading to life-long dependence on exogenous insulin. Attraction, activation and transmigration of inflammatory cells to the site of ß-cell injury depend on two major molecular interactions. First, interactions between chemokines and their receptors expressed on leukocytes result in the recruitment of circulating inflammatory cells to the site of injury. In this context, it has been demonstrated in various studies that the interaction of the chemokine CXCL10 with its receptor CXCR3 expressed on circulating cells plays a key role in the development of T1D. Second, once arrived at the site of inflammation adhesion molecules promote the extravasation of arrested cells through the endothelial cell layer to penetrate the site of injury. Here, the junctional adhesion molecule (JAM) JAM-C expressed on endothelial cells is involved in the process of leukocyte diabedesis. It was recently demonstrated that blocking of JAM-C efficiently attenuated cerulein-induced pancreatitis in mice. In my thesis I studied the influence of the CXCL10/CXCR3 interaction on the one hand, and of the adhesion molecule JAM-C on the other hand, on trafficking and transmigration of antigen-specific, autoaggressive T cells in the RIP-LCMV mouse model. RIP-LCMV mice express the glycoprotein (GP) or the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV) as a target autoantigen specifically in the ß cells of the islets of Langerhans and turn diabetic after LCMV-infection. In my first project I found that pharmacologic blockade of CXCR3 during development of virus-induced T1D results in a significant delay but not in an abrogation of overt disease. However, neither the frequency nor the migratory properties of islet-specific T cells was significantly changed during CXCR3 blockade. In the second project I was able to demonstrate that JAM-C was upregulated around the islets in RIP-LCMV mice after LCMV infection and its expression correlated with islet infiltration and functional ß-cell impairment. Blockade with a neutralizing anti-JAM-C antibody slightly reduced T1D incidence, whereas overexpression of JAM-C on endothelial cells did not accelerate virus-induced diabetes. In summary, our data suggest that both CXCR3 as well as JAM-C are involved in trafficking and transmigration of antigen-specific autoaggressive T cells to the islets of Langerhans. However, the detection of only a moderate influence on the onset of clinical disease during CXCR3 or JAM-C blockade reflects the complex pathogenesis of T1D and indicates that several different inflammatory factors need to be neutralized in order to achieve a stable and persistent protection from disease.
- Epicutanoeus immunotherapy as a novel prophylactic and therapeutic strategy for birch pollen allergy (2014)
- The development of a convenient, effective and safe allergen-specific immunotherapy (SIT) for birch pollen allergy, one of the most prevalent allergic diseases in Northern Europe, North America and Northern Japan, is of crucial importance. Epicutaneous immunotherapy (EPIT) has gained attention as a safe and non-invasive alternative for subcutaneous immunotherapy, a conventional SIT. However, clinical studies showed a limited effcacy of EPIT, indicating the necessity of improvement of the treatment regime. In this study, we hypothesized that a combination of a hypoallergen with an appropriate adjuvant could be a strategy to improve EPIT. To verify this hypothesis, we aimed at investigating the efficacy of epicutaneous treatment with rBet v 1, the major birch pollen allergen, plus Toll-like receptor (TLR) agonists for prophylaxis and therapy of birch pollen allergy using a murine model of birch pollen-induced allergic asthma. Furthermore, the efficacy of rBet v 1B2, a hypoallergenic variant of Bet v 1, as a therapeutic allergen in EPI was pre-clinically investigated. TLRs recognize conserved microbial molecules (like PAMPs), and are known to promote the counter-regulation of TH2 responses by the induction of TH1-type and/or regulatory cytokines by immune cells. The hypoallergen Bet v 1B2 is a folding-variant of the wild-type allergen rBet v 1 with reduced allergenicity, but retained T-cell immunogenicity. The low allergenicity, could allow the application of hypoallergens in higher doses, and therefore provide a safer and more effective treatment to regulate T-cell immune responses. First, the expression and purification of recombinant Bet v 1 and Bet v 1B2 was optimized. Compared to natural proteins, recombinant proteins offer the possibility to use well-defined molecules with a consistent pharmaceutical quality. Using optimal Escherichia coli expression strains in combination with immobilized metal chelate affinity chromatography (IMAC) and size exclusion chromatography (SEC), we successfully prepared a large amount of rBet v 1 and rBet v 1B2 with a high purity. The allergenic potency of rBet v 1 and the hypoallergenic characteristics of rBet v 1B2 were confirmed by measurement of IgE reactivity and mediator release capacity using ELISA and basophil activation tests, respectively. In a second part, a murine model of birch pollen-induced allergic asthma was established. It was shown that intraperetoneal sensitization with an optimal dose of rBet v 1 and intranasal challenge with birch pollen extract induced elevated IgE levels, airway eosinophilia and pulmonary inflammation in BALB/c mice. The clinical features are comparable to those in patients with allergic asthma, indicating that sensitized and challenged mice could be used for a pre-clinical study to assess the efficacy of the treatment for birch pollen allergy. Next, we investigated the adjuvant effects of Polyadenylic:polyuridylic acid (Poly(A:U)), a TLR3 agonist, and R848 (resiquimod), a TLR7 agonist, in prophylactic EPI with rBet v 1 to intervene with birch pollen allergy. Here, we hypothesized that TLR3 and TLR7 could be possible target receptors to induce adjuvant effects in EPI, since these receptors are expressed in Langerhans cells and dermal dendritic cells, persistent antigen presenting cells in the cutaneous tissues. BALB/c mice received EPI with rBet v 1 alone, or plus Poly(A:U), or R848 on their depilated back using patches. Mice treated epicutaneously were then sensitized with rBet v 1 plus ALUM and intranasally challenged with birch pollen extract. We found that prophylactic EPI with rBet v 1 plus R848 inhibited the production of Bet v 1-specific IgE antibodies in sensitization, suppressed pulmonary inflammation and airway hyperreactivity upon challenge. In contrast to R848, no adjuvant effect of Poly(A:U) on suppression of asthmatic features was observed. Our results indicated that R848, but not Poly(A:U), could be a potential adjuvant for prophylactic EPI of birch pollen induced allergic asthma. Finally, the therapeutic potency of EPI with rBet v 1, or rBet v 1B2 alone, or plus R848 was assessed. After sensitization and challenge, mice received therapeutic EPI with rBet v 1 alone, or plus R848, and re-challenge with birch pollen extract. We found that therapeutic treatment with Bet v 1B2 reduced established Bet v 1-specific IgE antibodies, pulmonary inflammation and airway hyperreactivity upon re-challenge. Therapeutic treatment with the recombinant wild-type allergen does not influence these key characteristics of allergic asthma. In contrast to the findings in the prophylactic treatment with rBet v 1 plus R848,no therapeutic benefit was found upon combination with R848. This could be due to the high number of treatment days. Reduction of this number may lead to a beneficial effect. However, these findings indicate that Bet v 1B2 could be a potential therapeutic agent for the treatment of established birch pollen induced allergic asthma. In conclusion, this study demonstrates for the first time that prophylactic EPI with the recombinant form of Bet v 1 in combination with R848 could prevent and suppress asthmatic features in an established birch pollen allergy. Not only therapeutic, but also prophylactic applications of EPI could be of importance to prevent allergic sensitization, considering the high prevalence of allergic diseases. R848 could be a potential adjuvant for enhancing the prophylactic potential of EPI for the treatment of birch pollen allergy. Furthermore, the beneficial use of the hypoallergen Bet v 1B2 in therapeutic EPI was demonstrated by intervention of established asthmatic features. In the future, a combination of hypoallergens alone or together with adjuvants in EPIT could lead to a more convenient and effective therapeutic treatment of established birch pollen induced allergic asthma.