Guro Gafvelin

Topological Rules for Membrane Protein Assembly in Eukaryotic Cells

Insertion into the endoplasmic reticulum membrane of model proteins with one, two, and four transmembrane segments and different distributions of positively charged residues in the N-terminal tail and the polar loops has been studied both in vitro and in vivo Membrane insertion of these same constructs has previously been analyzed in Escherichia coli, thus making possible a detailed comparison between the topological rules for membrane protein assembly in prokaryotic and eukaryotic cells. In general, we find that positively charged residues have similar effects on the membrane topology in both systems when they are placed in the N-terminal tail but that the effects of charged residues in internal loops clearly differ. Our results rule out a sequential start-stop transfer model where successive hydrophobic segments insert with alternating orientations starting from the most N-terminal one as the only mechanism for membrane protein insertion in eukaryotic cells.

Cytokine and Antibody Responses in Birch-Pollen-Allergic Patients Treated with Genetically Modified Derivatives of the Major Birch Pollen Allergen Bet v 1

Background: Recently, recombinant hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, were used to treat birch-pollen-allergic patients in a double-blind, placebo-controlled, multi-centre immunotherapy study. The aim of this study was to evaluate the effects of vaccination with aluminium-hydroxide-adsorbed recombinant Bet v 1 derivatives versus placebo on T-cell, cytokine and antibody responses in a subgroup of patients. Methods: Blood was drawn from patients of the Swedish centre (n = 27; rBet v 1 fragments: n = 10; rBet v 1 trimer: n = 8, and placebo-aluminium hydroxide: n = 9) before the start and after completion of the treatment. PBMC were stimulated with rBet v 1 and analysed for cytokine (IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-γ)-secreting cells by ELISpot. Bet v 1-specific antibody levels in serum (IgG1–4, IgE and IgA) were measured by ELISA. Skin prick tests with defined Bet v 1 concentrations were performed before and 10–11 months after the beginning of the study. Results: Bet v 1-specific IgG levels, consisting of IgG1, IgG2 and IgG4, were significantly increased after treatment with recombinant allergen derivatives. Treatment with rBet v 1 trimer led to a significant (p < 0.05) reduction of Bet v 1-reactive IL-5- and IL-13-producing cells, reflecting a reduced Th2 response. In addition, a decreased number of Bet v 1-reactive IL-4 producing (p = 0.07) and an increase of IL-12-producing (p = 0.06) cells was noted in the trimer-treated patients. In contrast to placebo, active treatment resulted in significantly reduced immediate-type skin reactions to Bet v 1 even 10–11 months after treatment. Conclusion: Vaccination with recombinant hypoallergenic Bet v 1 derivatives induces a Bet v 1-specific IgG response and leads to reduced skin reactivity in allergic patients. A reduction of Bet v 1-specific Th2 responses was observed in trimer-treated patients, which may reflect the intrinsic property of this allergen derivative.

Exploiting the 21st amino acid—purifying and labeling proteins by selenolate targeting

Selenium is essential to human life and occurs in selenoproteins as selenocysteine (Sec), the 21st amino acid. The selenium atom endows selenocysteine with unique biochemical properties, including a low pKa and a high reactivity with many electrophilic agents. Here we describe the introduction of selenocysteine into recombinant non-selenoproteins produced in Escherichia coli, as part of a small tetrapeptide motif at the C terminus. This selenocysteine-containing motif could subsequently be used as a protein tag for purification of the recombinant protein, selenolate-targeted labeling with fluorescent compounds or radiolabeling with either γ-emitting 75Se or short-lived positron emitters such as 11C. The results presented here thus show how a wide range of biotechnological applications can be developed starting from the insertion of selenocysteine into proteins.

The non-proteolytic house dust mite allergen Der p 2 induce NF-κB and MAPK dependent activation of bronchial epithelial cells

Background House dust mites (HDM) are well‐known as a source of indoor aeroallergens and for causing allergic airway diseases. Some proteolytic HDM allergens are known to activate respiratory epithelial cells to produce pro‐inflammatory mediators, while there is limited knowledge regarding such activity among non‐proteolytic HDM allergens.

Immune regulation by CD4+CD25+ T cells and interleukin‐10 in birch pollen‐allergic patients and non‐allergic controls

Background CD4+CD25+ regulatory T (Treg) cells and the cytokines IL‐10 or TGF‐β play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non‐infectious antigens such as allergens.

Allergen provocation increases TH2-cytokines and FOXP3 expression in the asthmatic lung.

To cite this article: Thunberg S, Gafvelin G, Nord M, Grönneberg R, Grunewald J, Eklund A, van Hage M. Allergen provocation increases TH2‐cytokines and FOXP3 expression in the asthmatic lung. Allergy 2010; 65: 311–318.

The Major Cat Allergen, Fel d 1, in Diagnosis and Therapy

Sensitization to cat is a common cause of allergic disease all over the world. Symptoms range from mild rhinoconjunctivitis to potentially life-threatening asthmatic exacerbations. In vivo and in vitro diagnostics of cat allergy is currently based on cat dander extract. As allergen extracts from natural sources are heterogeneous in composition, the allergen content may vary. With the introduction of allergens produced by recombinant techniques, a large panel of recombinant allergenic molecules including the major cat allergen, recombinant Fel d 1, has become available for immunological investigations, diagnosis and treatment. Studies have shown that this single allergen, which belongs to the uteroglobin protein family, is at least as good as cat dander extract in identifying cat-allergic patients. The introduction of recombinant Fel d 1-based tests into clinical practice will increase our knowledge of this single allergen molecule as a diagnostic tool and improve the selection for therapy of cat allergy. Several different modes for allergen-specific immunotherapy of cat allergy based on Fel d 1 have been developed. These include Fel d 1 hypoallergens and allergen constructs where Fel d 1 is coupled to immunomodulatory proteins or carriers. The approaches have been evaluated in experimental in vitro and in vivo model systems with promising results. In addition, immunotherapy with Fel d 1 peptides containing T-cell epitopes has been tested in clinical trials. After initial problems with adverse reactions, more recent data show that peptide immunotherapy modulates the immune response to Fel d 1 and reduces early- and late-phase effector reactions in cat-allergic patients.

Rational design of hypoallergens applied to the major cat allergen Fel d 1

Background Allergen‐specific immunotherapy is the only treatment for allergic disease providing long‐lasting symptom relief. Currently, it is mainly based on the use of crude allergen extracts. The treatment may be improved by the use of genetically engineered allergens, hypoallergens, aiming at a more effective and safer therapy.

Carbohydrate-based particles reduce allergic inflammation in a mouse model for cat allergy.

Background:  Allergen‐specific immunotherapy (ASIT) is the only treatment of allergic disease that gives long‐lasting relief of symptoms. However, concerns for safety and efficiency have highlighted the need for improvement of the therapy. We have previously suggested carbohydrate‐based particles (CBPs) as a novel adjuvant and allergen carrier for ASIT. Our aim of this study was to evaluate the therapeutic potential of CBPs in ASIT, employing a mouse model for cat allergy.

Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens.

Allergen-specific immunotherapy is the only treatment that provides long lasting relief of allergic symptoms. Currently, it is based on repeated administration of allergen extracts. To improve the safety and efficacy of allergen extract-based immunotherapy, application of hypoallergens, i.e. modified allergens with reduced IgE binding capacity but retained T-cell reactivity, has been proposed. It may, however, be difficult to predict how to modify an allergen to create a hypoallergen. Directed molecular evolution by DNA shuffling and screening provides a means by which to evolve proteins having novel or improved functional properties without knowledge of structure-function relationships of the target molecules. With the aim to generate hypoallergens we applied multigene DNA shuffling on three group 2 dust mite allergen genes, two isoforms of Lep d 2 and Gly d 2. DNA shuffling yielded a library of genes from which encoded shuffled allergens were expressed and screened. A positive selection was made for full-length, high-expressing clones, and screening for low binding to IgE from mite allergic patients was performed using an IgE bead-based binding assay. Nine selected shuffled allergens revealed 80-fold reduced to completely abolished IgE binding compared with the parental allergens in IgE binding competition experiments. Two hypoallergen candidates stimulated allergen-specific T-cell proliferation and cytokine production at comparable levels as the wild-type allergens in patient peripheral blood mononuclear cell cultures. The two candidates also induced blocking Lep d 2-specific IgG antibodies in immunized mice. We conclude that directed molecular evolution is a powerful approach to generate hypoallergens for potential use in allergen-specific immunotherapy.