Anna Lukschal

Protamine nanoparticles with CpG-oligodeoxynucleotide prevent an allergen-induced Th2-response in BALB/c mice

The currently applied immunotherapy of type I allergy with aluminum hydroxide (alum) as adjuvant elicits - among other side effects - an initial IgE-boost. In contrast, CpG-oligodeoxynucleotides (ODNs) drive the immune response toward 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 was investigated. A favorable increase in 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.

Interleukin-10: An Anti-Inflammatory Marker To Target Atherosclerotic Lesions via PEGylated Liposomes

Atherosclerosis (AS) causes cardiovascular disease, which leads to fatal clinical end points like myocardial infarction or stroke, the most prevalent causes of death in developed countries. An early, noninvasive method of detection and diagnosis of atherosclerotic lesions is necessary to prevent and treat these clinical end points. Working toward this goal, we examined recombinant interleukin-10 (IL-10), stealth liposomes with nanocargo potency for NMRI relevant contrast agents, and IL-10 coupled to stealth liposomes in an ApoE-deficient mouse model using confocal laser-scanning microscopy (CLSM). Through ex vivo incubation and imaging with CLSM, we showed that fluorescently labeled IL-10 is internalized by AS plaques, and a low signal is detected in both the less injured aortic surfaces and the arteries of wild-type mice. In vivo experiments included intravenous injections of (i) fluorescent IL-10, (ii) IL-10 targeted carboxyfluorescin (CF−) labeled stealth liposomes, and (iii) untargeted CF-labeled stealth liposomes. Twenty-four hours after injection the arteries were dissected and imaged ex vivo. Compared to free IL-10, we observed a markedly stronger fluorescence intensity with IL-10 targeted liposomes at AS plaque regions. Moreover, untargeted CF-labeled liposomes showed only weak, unspecific binding. Neither free IL-10 nor IL-10 targeted liposomes showed significant immune reaction when injected into wild-type mice. Thus, the combined use of specific anti-inflammatory proteins, high payloads of contrast agents, and liposome particles should enable current imaging techniques to better recognize and visualize AS plaques for research and prospective therapeutic strategies.

Heating Affects Structure, Enterocyte Adsorption and Signalling, As Well as Immunogenicity of the Peanut Allergen Ara h 2

Previous studies have indicated that specific molecular properties of proteins may determine their allergenicity. Allergen interaction with epithelia as the first contact site could be decisive for a resulting immune response. We investigate here for the major peanut allergen Ara h 2 whether thermal processing results in structural changes which may impact the proteins molecular interactions with enterocytes, subsequent cellular signalling response, and immunogenicity.Ara h 2 was heat processed and analyzed in terms of patient IgE binding, structural alterations, interaction with human enterocytes and associated signalling as well as immunogenicity in a food allergy mouse model.Heating of Ara h 2 led to significantly enhanced binding to Caco-2/TC7 human intestinal epithelial cells. Structural analyses indicated that heating caused persistent structural changes and led to the formation of Ara h 2 oligomers in solution. Heated protein exhibited a significantly higher immunogenic potential in vivo as determined by IgG and IgE serum antibody levels as well as IL-2 and IL-6 release by splenocytes. In human Caco-2/TC7 cells, Ara h 2 incubation led to a response in immune- and stress signalling related pathway components at the RNA level, whereas heated allergen induced a stress-response only.We suggest from this peanut allergen example that food processing may change the molecular immunogenicity and modulate the interaction capacity of food allergens with the intestinal epithelium. Increased binding behaviour to enterocytes and initiation of signalling pathways could trigger the epimmunome and influence the sensitization capacity of food proteins.

Characterization of intrinsic and extrinsic risk factors for celery allergy in immunosenescence

Recent studies indicated an underestimation of allergies in elderly. In our experimental food allergy model of protein feeding under acid-suppression we aimed to assess whether food allergy can be induced in immunosenescent mice. Furthermore, the impact of gastric digestion on celery allergenicity was evaluated in aged patients. Measurements of serum zinc and iron levels in senescent and adult BALB/c mice for definition of the nutritional status indicated a possible alteration of the immune response in the aged animals due to reduced zinc and iron levels. Feedings of mice with digestion-sensitive celery proteins under physiological gastric conditions induced IgG1 and IgG2a in the aged and preferentially IgG1 in the adult animals. In contrast, incomplete digestion due to acid-suppression rendered celery-specific IgE, positive skin tests and elevated IL-5 levels in both age groups. Also in aged celery allergic patients (mean age 72 years) properly digested celery showed decreased capacity to bind and crosslink IgE as evaluated by skin tests and IgE immunoblot. Thus, in the geriatric murine model, celery allergy was induced only if gastric digestion was hindered. Accordingly, gastric proteolysis decreased in vitro and in vivo IgE-reactivity against celery proteins in aged allergic patients.

Establishing an allergic eczema model employing recombinant house dust mite allergens Der p 1 and Der p 2 in BALB/c mice

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.

Myocardial lipofuscin-laden lysosomes contain the apoptosis marker caspase-cleaved cytokeratin-18

Background  Acute coronary syndrome is related to increased circulatory concentration of soluble apoptosis specific caspase‐cleaved cytokeratin‐18 (ccCK‐18). Potential cardiac sources of this intermediate filament derivative have not been investigated to date.

Do hypoallergenic cats exist? -- Determination of major cat allergen Fel d 1 production in normal and hypoallergenic cat breeds

Background and aims Due to the increasing prevalence of cat allergy the demand for so-called hypoallergenic cats and the number of respective breeders is rising. The aim of our study was to examine the hypoallergenicity of these cats, taking the major allergen Fel d 1 as a marker molecule. This molecule is an important asthma inducer and primarily produced in the sebaceous, salivary and anal glands and is distributed on the fur by licking.

Anti-idiotypic Fab Fragments Image a Conserved N-terminal Epitope Patch of Grass Pollen Allergen Phl p 1

BACKGROUND AND AIMS: Naturally occurring anti-idiotypic antibodies structurally mimic the original antibody epitope. Anti-idiotypes, therefore, are interesting tools for the portrayal of conformational B-cell epitopes of allergens. In this study we used this strategy particularly for major timothy grass pollen (Phleum pratense) allergen Phl p 1. METHODS AND RESULTS: We used a combinatorial phage display library constructed from the peripheral IgG repertoire of a grass pollen allergic patient which was supposed to contain anti-idiotypic Fab specificities. Using purified anti-Phl p 1 IgG for biopanning, several Fab displaying phage clones could be isolated. 100 amplified colonies were screened for their binding capacity to anti-Phl p 1-specific antibodies, finally resulting in four distinct Fab clones according to sequence analysis. Interestingly, heavy chains of all clones derived from the same germ line sequence and showed high homology in their CDRs. Projecting their sequence information on the surface of the natural allergen Phl p 1 (PDB ID: 1N10) indicated matches on the N-terminal domain of the homo-dimeric allergen, including the bridging region between the two monomers. The resulting epitope patches were formed by spatially distant sections of the primary allergen sequence. CONCLUSION: In this study we report that anti-idiotypic specificities towards anti-Phl p 1 IgG, selected from a Fab library of a grass pollen allergic patient, mimic a conformational epitope patch being distinct from a previously reported IgE epitope area.