[Show abstract][Hide abstract] ABSTRACT: Background:
We revealed in previous studies that nitration of food proteins reduces the risk of de novo sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, in vitro experiments suggested an increased capacity of effector cell activation by nitrated food proteins.
The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy.
BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen β-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood.
A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation.
Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.
[Show abstract][Hide abstract] ABSTRACT: Papain is commonly used in food, pharmaceutical, textile and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Der p 1 from house dust mite per se exhibits percutaneous sensitization potential.
We aimed here to investigate the potential of papain itself in epicutaneous sensitization.
The effects of papain on tight junction proteins were tested in-vitro in human primary keratinocytes. Using C57BL/6 WT and TLR4-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier and immune cell recruitment.
Our results show, that papain affects the skin barrier by increasing transepidermal water loss, degrading tight junction proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4-independent and, in spite of its capacity to degrade epidermal tight junction proteins, does not rely on its enzymatic function.
From our data we conclude that papain has all features to act as a strong allergen via the skin.
[Show abstract][Hide abstract] ABSTRACT: Papain is commonly used in food, pharmaceutical, textile and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Der p 1 from house dust mite per se exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction proteins were tested in-vitro in human primary keratinocytes. Using C57BL/6 WT and TLR4-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier and immune cell recruitment. Our results show, that papain affects the skin barrier by increasing transepidermal water loss, degrading tight junction proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was independent of its enzymatic function. The mechanism for specific sensitization was TLR4-independent and, in spite of its capacity to degrade epidermal tight junction proteins, does not rely on its enzymatic function. From our data we conclude that papain has all features to act as a strong allergen via the skin.Journal of Investigative Dermatology accepted article preview online, 23 February 2015. doi:10.1038/jid.2015.58.
Full-text · Article · Feb 2015 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: Abstract Adeno-associated viruses (AAVs) are established vectors for gene therapy of different human diseases. AAVs are assembled of 60 capsomers, which can be genetically modified, allowing high-density display of short peptide sequences at their surface. The aim of our study was to evaluate the immunogenicity and safety of an adeno-associated virus-like particle (AAVLP)-displayed B-cell peptide epitope taking ovalbumin (OVA) as a model antigen or allergen from egg, respectively. An OVA-derived B-cell epitope was expressed as fusion protein with the AAV-2 capsid protein of VP3 (AAVLP-OVA) and for control, with the nonrelated peptide TP18 (AAVLP-TP18). Cellular internalization studies revealed an impaired uptake of AAVLP-OVA by mouse BMDC, macrophages, and human HeLa cells. Nevertheless, BALB/c mice immunized subcutaneously with AAVLP-OVA formed similarly high titers of OVA-specific IgG1 compared to mice immunized with the native OVA. The extent of the immune response was independent whether aluminum hydroxide or water in oil emulsion was used as adjuvant. Furthermore, in mice immunized with native OVA, high OVA-specific IgE levels were observed, which permitted OVA-specific mast-cell degranulation in a β-hexosaminidase release assay, whereas immunizations with AAVLP-OVA rendered background IgE levels only. Accordingly, OVA-immunized mice, but not AAVLP-OVA immunized mice, displayed an anaphylactic reaction with a significant drop of body temperature upon intravenous OVA challenge. From this mouse model, we conclude that AAVLPs that display B-cell epitope peptides on their surface are suitable vaccine candidates, especially in the field of allergy.
[Show abstract][Hide abstract] ABSTRACT: Background
The major house dust mite allergen Der p 2 is a structural and functional homologue of MD-2 within the TLR4–CD14–MD-2 complex. An asthma mouse model in TLR4-deficient mice recently suggested that the allergic immune response against Der p 2 is solely dependent on TLR4 signaling. We investigated whether similar mechanisms are important for Der p 2 sensitization via the skin.Methods
In an epicutaneous sensitization model, the response to recombinant Der p 2 in combination with or without lipopolysaccharide (LPS) was compared between C57BL/6 WT and TLR4-deficient mice. We further analyzed possible adjuvant function of exogenous cysteine proteases.ResultsSensitization with rDer p 2 induced similar levels of allergen-specific IgG1 and IgE antibodies in both mouse strains. LPS increased the systemic (antibody levels, cytokine release by restimulated splenocytes) and local (infiltration of immune cells into the skin) Th2 immune responses, which against our expectations were stronger in the absence of functional TLR4 expression. Barrier disruption by papain, a protease with structural homology to Der p 1, did not enhance the sensitization capacity of rDer p 2. However, the presence of LPS increased the stability of rDer p 2 against the protease.Conclusion
Our data suggest that rDer p 2 alone can cause a strong TH2-biased response via the skin being enhanced in the presence of LPS. This response is not reliant on functional TLR4, but vice versa TLR4 expression rather protects against epicutaneous sensitization to house dust mite allergen Der p 2.
[Show abstract][Hide abstract] ABSTRACT: The currently applied immunotherapy of type I allergy with aluminium hydroxide (alum) as adjuvant elicits -among other side effects- an initial IgE-boost. In contrast, CpG-oligodeoxynucleotides (ODNs) drive the immune response towards Th1. The biodegradable material protamine can spontaneously form nanoparticles together with such ODNs. Our aim was to investigate the immune response induced by protamine-based nanoparticles (proticles) with CpG-ODN as an allergen delivery system. Proticles complexed with Ara h 2 extracted from raw peanuts as model allergen were injected subcutaneously into naïve BALB/c mice. Ara h 2-specific antibodies were analyzed by ELISA and rat basophilic leukemia (RBL) cell assay. Cytokine levels were investigated in supernatants of stimulated splenocytes. The in vivo distribution after subcutaneous injection was examined via fluorescence imaging. BMDCs were stimulated with proticles and expression of stimulation and maturation markers as well as cytokines in supernatants were investigated. A favorable increase of Ara h 2-specific IgG2a antibodies was found after immunization with proticles-Ara h 2, whereas Ara h 2-specific IgE was not detectable. Accordingly, the ratio of IL-5/IFN-gamma was low in this group. Granuloma formation was completely absent at injection sites of proticles. The distribution of Ara h 2 after subcutaneous injection was markedly decelerated when complexed to proticles. Stimulation of BMDCs with proticles-Ara h 2 caused upregulation of CD11c and CD80 as well as an increased IL-6 production. Our data suggest that biodegradable protamine-based nanoparticles with CpG-ODN counteract the Th2-dominated immune response induced by an allergen and therefore are suitable as novel carrier system for immunotherapy of allergy.
No preview · Article · Mar 2013 · European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V
[Show abstract][Hide abstract] ABSTRACT: Peanut allergy causes severe type 1 hypersensitivity reactions and conventional immunotherapy against peanut allergy is associated with a high risk of anaphylaxis.
Our current study reports proof of concept experiments on the safety of a stably denatured variant of the major peanut allergen Ara h 2 for immunotherapy. We determined the impact of structure loss of Ara h 2 on its IgE binding and basophil degranulation capacity, T cell reactivity as well as anaphylactic potential.
The secondary structure of untreated and reduced/alkylated Ara h 2 variants was determined by circular dichroism spectroscopy. We addressed human patient IgE binding to Ara h 2 by ELISA and Western blot experiments. RBL-SX38 cells were used to test the degranulation induced by untreated and reduced/alkylated Ara h 2. We assessed the anaphylactic potential of Ara h 2 variants by challenge of sensitized BALB/c mice. T cell reactivity was investigated using human Ara h 2-specific T cell lines and splenocytes isolated from sensitized mice.
Reduction/alkylation of Ara h 2 caused a decrease in IgE binding capacity, basophil degranulation and anaphylactic potential in vivo. However, the human T cell response to reduced/alkylated and untreated Ara h 2 was comparable. Mouse splenocytes showed higher metabolic activity upon stimulation with reduced/alkylated Ara h 2 and released similar IL-4, IL-13 and IFNγ levels upon treatment with either Ara h 2 variant.
Reduced/alkylated Ara h 2 might be a safer alternative than native Ara h 2 for immunotherapeutic treatment of peanut allergic patients.
No preview · Article · Dec 2012 · Clinical & Experimental Allergy
[Show abstract][Hide abstract] ABSTRACT: The major house dust mite allergens Der p 1 and Der p 2 are prevalent inducers of eczema. Der p 1 is a cysteine protease disrupting epithelial barriers, whereas Der p 2 functionally mimics the LPS-binding compound MD-2 within the TLR4 complex. In this work, we tested the percutaneous sensitizing capacity of recombinant (r) Der p 1 and Der p 2 in BALB/c mice. Mice were sensitized by percutaneous application of low (10 μg/application) and high dose (100 μg) rDer p 1 or rDer p 2, or with rDer p 1 followed by rDer p 2. Allergen-specific and total IgE antibodies were determined by ELISA. Eczema of BALB/c was classified by the itching score and corresponded to erosions. Infiltrating immune cells were identified by haematoxylin/eosin and Giemsa staining for eosinophils or mast cells, CD3 staining for T lymphocytes. Percutaneous treatments with rDer p 1, but not rDer p 2-induced specific IgG1. However, cotreatment with rDer p 1 led to increase in anti-Der p 2 IgG titres. Both allergens elicited skin erosions because of scratching, thickening of the epidermis, and eosinophil and T-cell infiltration. Our data indicate that recombinant mite allergens in the absence of adjuvant are sufficient for inducing eczema in BALB/c mice. As the enzymatic activity of an allergen might be an important cofactor for specific sensitization via the skin, Der p 1 may act as adjuvant for other allergens too. The presented mouse model is suitable for investigating the mechanisms of allergic eczema.
Full-text · Article · Nov 2012 · Experimental Dermatology
[Show abstract][Hide abstract] ABSTRACT: Colorectal cancer (CRC) is one of the leading causes of cancer morbidity and mortality in Western countries. One of the risk factors for colorectal tumorigenesis is vitamin D insufficiency.
The aim of this study was to establish whether increasing dietary vitamin D intake can prevent or delay development of chemically induced preneoplastic lesions in the colon of mice.
We fed six weeks old female C57BL/6 J mice (n = 28) with increasing vitamin D3 concentrations (100, 400, 1000, 2500, 5000 IU/kg diet). To induce dysplasia, a preneoplastic lesion, we injected mice with the carcinogen azoxymethane (10 mg/kg) intraperitoneally, followed by three cycles of 2% dextran sodium sulfate salt, a tumor promoter, in the drinking water.
To test our hypothesis that high vitamin D intake prevents formation of preneoplastic lesions, we have investigated the effect of increasing dietary vitamin D on development of premalignant colorectal lesions, serum 25-hydroxyvitamin D3 (25-D3) levels, and expression of renal vitamin D system genes.
Dietary vitamin D concentration correlated inversely with dysplasia score (Spearman's correlation coefficient, ρ: −0.579, p = 0.002) and positively with serum 25-D3 levels (ρ: 0.752, p = 0.001). Increasing dietary vitamin D concentration beyond 1000 IU/kg led to no further increase in circulating 25-D3 levels, while the dysplasia score leveled out at ≥2500 IU/kg vitamin D. High dietary vitamin D intake led to increased renal mRNA expression of the vitamin D catabolizing enzyme cyp24a1 (ρ: 0.518, p = 0.005) and decreased expression of the vitamin D activating enzyme cyp27b1 (ρ: −0.452, p = 0.016), protecting the body from toxic serum levels of the active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25-D3).
Our data showed that increasing dietary vitamin D intake is able to prevent chemically induced preneoplastic lesions. The maximum impact was achieved when the mice consumed more than 2500 IU vitamin D/kg diet.
This article is part of a Special Issue entitled ‘Vitamin D Workshop’.
Full-text · Article · Sep 2012 · The Journal of steroid biochemistry and molecular biology
[Show abstract][Hide abstract] ABSTRACT: Background
Adeno-associated viruses (AAV) are non-human pathogenic and replication defective ssDNA viruses. The surface of AAV consists of 60 capsomers, which can be exploited for high density display of recombinant peptides. AAV-like particles (AAVLP) can be generated via assembly of the recombinant capsid protein VP3. The aim of this study was to evaluate the uptake mechanism, immunogenicity and safety aspects of an AAVLP-displayed B-cell epitope, taking ovalbumin (OVA) as a model antigen/allergen.
An OVA derived linear B-cell epitope and for control purposes OVA-non related peptide TP18 (cholesterol-ester transfer protein 18) were inserted into capsid protein VP3 of AAVLPs.
Life cell microscopy indicated that AAVLP internalized into HeLa epithelial cells and remained in intracellular vesicles up to 18 hours. When we immunized BALB/c subcutaneously, sera of AAVLP-OVA immunized mice showed similar titres of OVA-specific IgG1 compared to mice immunized with OVA protein. However, in OVA immunized mice high OVA-specific IgE levels could be recorded, whereas immunizations with OVA-AAVLP rendered background IgE levels only. In accordance, sera of OVA mice which permitted mast cell degranulation upon OVA trigger in a specific β-hexosaminidase release assay, whereas sera of OVA-AAVLP mice did not contain anaphylactogenic antibodies. In an in vivo anaphylaxis experiment, upon intravenous OVA challenge OVA-immunized mice presented significant drop of body temperature, whereas AAVLP-OVA mice remained unaffected.
Our study demonstrates the immunogenicity, safety and efficacy of AAVLP as display system of B-cell epitopes for vaccination.
Full-text · Article · Feb 2012 · World Allergy Organization Journal
[Show abstract][Hide abstract] ABSTRACT: Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250 mJ cm(-2) UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.
Full-text · Article · Jan 2011 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.
Full-text · Article · May 2010 · Journal of Investigative Dermatology