Biomedical subjects

Sabine Flückiger

Publications and source records attributed to Sabine Flückiger.

11 recordsLinked to original sources

PubMed2006

Sensitization to the yeast Malassezia sympodialis is specific for extrinsic and intrinsic atopic eczema.

The opportunistic yeast Malassezia sympodialis belongs to the normal cutaneous flora but can also cause IgE-mediated sensitization in patients suffering from atopic eczema (AE). We investigated 706 individuals by ImmunoCAPm70 and skin-prick tests with a crude M. sympodialis extract. In AE patients, we further performed skin prick tests, atopy patch tests, ELISA, and peripheral blood mononuclear cells proliferation assays with recombinant M. sympodialis allergens (rMala s 1 and 5-9). In 52/97 patients with AE-specific IgE against M. sympodialis was detectable. Almost no reactivity to M. sympodialis was seen in patients suffering from other allergic diseases (4/571) and no reactivity at all was seen in healthy controls (0/38). Skin tests showed variable recognition patterns against the different molecular structures with a predominant sensitization to rMala s 1, 5, 6, and 9, confirmed also by specific serum IgE to these allergens. Interestingly, IgE- and T-cell-mediated reactivity against M. sympodialis was also found in patients with the intrinsic form of AE. Thus, sensitization to M. sympodialis is specific for AE patients and occurs in both the extrinsic and intrinsic variant of eczema. Recombinant yeast allergens represent a useful tool to study molecular structures and differential sensitization patterns in the pathogenesis of AE.

Adolescent↗

PubMed2006

Analysis of the cross-reactivity and of the 1.5 A crystal structure of the Malassezia sympodialis Mala s 6 allergen, a member of the cyclophilin pan-allergen family.

Cyclophilins constitute a family of proteins involved in many essential cellular functions. They have also been identified as a panallergen family able to elicit IgE-mediated hypersensitivity reactions. Moreover, it has been shown that human cyclophilins are recognized by serum IgE from patients sensitized to environmental cyclophilins. IgE-mediated autoreactivity to self-antigens that have similarity to environmental allergens is often observed in atopic disorders. Therefore comparison of the crystal structure of human proteins with similarity to allergens should allow the identification of structural similarities to rationally explain autoreactivity. A new cyclophilin from Aspergillus fumigatus (Asp f 27) has been cloned, expressed and showed to exhibit cross-reactivity in vitro and in vivo. The three-dimensional structure of cyclophilin from the yeast Malassezia sympodialis (Mala s 6) has been determined at 1.5 A (1 A=0.1 nm) by X-ray diffraction. Crystals belong to space group P4(1)2(1)2 with unit cell dimensions of a=b=71.99 A and c=106.18 A. The structure was solved by molecular replacement using the structure of human cyclophilin A as the search model. The refined structure includes all 162 amino acids of Mala s 6, an active-site-bound Ala-Pro dipeptide and 173 water molecules, with a crystallographic R- and free R-factor of 14.3% and 14.9% respectively. The overall structure consists of an eight-stranded antiparallel beta-barrel and two alpha-helices covering the top and bottom of the barrel, typical for cyclophilins. We identified conserved solvent-exposed residues in the fungal and human structures that are potentially involved in the IgE-mediated cross-reactivity.

Allergens↗

PubMed2006

The crystal structure of Aspergillus fumigatus cyclophilin reveals 3D domain swapping of a central element.

The crystal structure of Aspergillus fumigatus cyclophilin (Asp f 11) was solved by the multiwavelength anomalous dispersion method and was refined to a resolution of 1.85 A with R and R(free) values of 18.9% and 21.4%, respectively. Many cyclophilin structures have been solved to date, all showing the same monomeric conformation. In contrast, the structure of A. fumigatus cyclophilin reveals dimerization by 3D domain swapping and represents one of the first proteins with a swapped central domain. The domain-swapped element consists of two beta strands and a subsequent loop carrying a conserved tryptophan. The tryptophan binds into the active site, inactivating cis-trans isomerization. This might be a means of biological regulation. The two hinge loops leave the protein prone to misfolding. In this context, alternative forms of 3D domain swapping that can lead to N- or C-terminally swapped dimers, oligomers, and aggregates are discussed.

Amino Acid Sequence↗

PubMed2006

Fungal allergies: a yet unsolved problem.

Airborne fungal spores have been implicated as causative factors in respiratory allergy, particularly asthma. However, the prevalence of fungal sensitization is not known mainly due to the lack of standardized fungal extracts and to the overwhelming number of fungal species able to elicit IgE-mediated reactions. Recent work based on high-throughput cloning of fungal allergens revealed that fungi are able to produce extremely complex repertoires of species-specific and cross-reactive allergens. There is evidence that fungal sensitization also contributes to auto-reactivity against self-antigens due to shared epitopes with homologous fungal allergens. Detailed studies at structural and immunological level indicate molecular mimicry as a basic mechanism involved in perpetuation of severe chronic allergic diseases. The real challenge at present is not related to cloning or production of a large number of different fungal allergens but rather to the assessment of the clinical relevance of each single structure. To date, substitution of complex extracts presently used in the diagnosis of fungal allergy by single, perfectly standardized components seems feasible in contrast to specific immunotherapy which is still not developed. Recombinant fungal allergens might create new perspectives in diagnosis and therapy of fungal allergy.

Animals↗

PubMed2005

IgE-mediated and T cell-mediated autoimmunity against manganese superoxide dismutase in atopic dermatitis.

BACKGROUND: Autoreactivity of patients with atopic dermatitis (AD) to human proteins has been postulated as a decisive pathogenetic factor for AD. OBJECTIVE: In this study, it was investigated whether the stress-inducible enzyme manganese superoxide dismutase (MnSOD) of human and fungal origin might act as an autoallergen in atopic dermatitis. METHODS: Patients with AD (n = 69; mean SCORAD [SCORing Atopic Dermatitis], 27) and other inflammatory skin diseases as well as with inhalant allergies were investigated. The presence of specific IgE against recombinant MnSOD of fungal and human origin and the fungal extracts of Aspergillus fumigatus and Malassezia sympodialis was measured by CAP, ELISA, skin prick test, and in subset of patients also by atopy patch tests (APTs) and PBMC proliferation assays. Cross-reactivity between allergens was determined by CAP inhibition. The presence of MnSOD in human skin in various inflammatory skin conditions was investigated by immunohistochemistry. RESULTS: Specific IgE antibodies against human MnSOD correlating with the disease activity were found in 29 out of 67 patients with AD. The human protein was able to induce in vitro T-cell reactivity and eczematous reactions in APT in MnSOD-sensitized patients with AD. MnSOD was upregulated in various inflammatory skin reactions and APT skin specimens. Cosensitization to structurally related and cross-reacting fungal MnSOD and the skin-colonizing yeast M sympodialis was observed in all patients sensitized against human MnSOD. CONCLUSION: Human MnSOD may play a role as an autoallergen in a subset of patients with AD, including nonatopic eczema. By molecular mimicry leading to cross-reactivity such sensitization might be induced primarily by exposure to environmental fungal MnSOD of M sympodialis .

Adult↗

PubMed2005

Time course of antibody response to recombinant Aspergillus fumigatus antigens in cystic fibrosis with and without ABPA.

We determined follow-up levels of specific serum IgE to the recombinant Aspergillus fumigatus (A. fumigatus) allergens rAsp f 1, 3, 4 and 6 in patients suffering from cystic fibrosis (CF) with and without allergic bronchopulmonary aspergillosis (ABPA). Over a 32-month period follow-up data of 74 patients were collected. According to serology, 11 CF patients were not sensitized (CF controls), 40 were sensitized to A. fumigatus (Asp. f-sens.) and 23 patients fulfilled the serologic criteria for ABPA. Of these 23 ABPA patients 11 expressed the full clinical ABPA picture (classicABPA) and 12 failed to show sufficient relevant clinical signs (seroABPA), despite positive serology. The 23 ABPA patients had 16-18 times higher serum levels of specific IgE to rAsp f 4 and/or rAsp f 6 than those of Asp. f-sens. patients (rAsp f 4: 31.3 +/- 45 EU/ml vs. 1.9 +/- 2.2 EU/ml and rAsp f 6: 39.0 +/- 44.3 EU/ml vs 2.1 +/- 1.7 EU/ml). The combination of increased total serum IgE (>1000 IU/l) and increased specific IgE to rAsp f 4 and/or rAsp f 6 allowed to diagnose classicABPA with 100% specificity and 64% sensitivity and with a high predicted positive (100%) and a high predicted negative (94%) value. During a combined treatment (seven patients) with oral corticosteroid and itraconazole, itraconazole alone (two patients) or neither oral corticosteroid nor itraconazole therapy (two patients) total serum IgE and specific IgE to rAsp f 4 and/or rAsp f 6 did decrease but did not normalize. Over the observation period, lung function remained unchanged, independent of whether oral steroids and/or concomitant itraconazole were either given or not given. In the follow-up of CF patients with ABPA under therapy the determination of total or specific IgE serum levels were of limited value to guide therapy.

Adolescent↗

PubMed2004

Cloning allergens via phage display.

Although an impressive list of allergenic structures has been elucidated during the last decade by classical cloning methods, the size of the repertoire of molecular structures able to elicit allergic reactions is still unknown. Selective enrichment of cDNA libraries displayed on phage surface with serum IgE from allergic individuals combined with robotic-based high-throughput screening technology has proved to be extremely successful for the rapid isolation of allergens. The basic concept of linking the phenotype, expressed as gene product displayed on the phage coat, to its genetic information integrated into the phage genome, creates fusion proteins covalently associated with the infectious particle itself. Therefore, cDNA libraries displayed on phage surface can be screened for the presence of specific clones using the discriminative power of affinity purification. The selection of IgE-binding clones involves the enrichment of phage binding to serum IgE immobilised to a solid phase during consecutive rounds of affinity selection. As a consequence of the physical linkage between genotype and phenotype, sequencing of the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of the surface-displayed allergen. In spite of some biological limitations imposed by Escherichia coli as expression host, phage surface display technology has strongly contributed to the rapid isolation of a vast variety of IgE-binding structures.

Allergens↗

PubMed2004

Cloning, expression and characterization of two new IgE-binding proteins from the yeast Malassezia sympodialis with sequence similarities to heat shock proteins and manganese superoxide dismutase.

Malassezia sympodialis is an opportunistic yeast that colonizes human skin and may induce IgE and T cell reactivity in patients with atopic eczema/dermatitis syndrome (AEDS). Previously, we have cloned and expressed six recombinant allergens (rMala s 1 and rMala s 5 to rMala s 9) from this yeast. By combining high throughput screening and phage surface display techniques, 27 complete and partial IgE-binding clones of M. sympodialis have been identified. Here we enlarged the panel of recombinant M. sympodialis allergens by RACE-PCR, cloning and nucleotide sequencing to obtain the coding sequences of two new IgE-binding clones. The coding sequences of one of the clones showed similarity to the heat shock protein (HSP) family and the other to manganese superoxide dismutase (MnSOD), and both had a high degree of homology to human counterparts. The coding sequences were expressed in Escherichia coli as six-histidine tagged recombinant proteins and generated products with molecular masses of 86.1 kDa for HSP and 22.4 kDa for MnSOD. Their IgE-binding frequencies were shown to be 69% and 75%, respectively, to 28 sera from AEDS patients with serum IgE to M. sympodialis extract, indicating that HSP and MnSOD are major M. sympodialis allergens. In inhibition immunoblotting, M. sympodialis extract could inhibit the binding of serum IgE from AEDS patients to rHSP and rMnSOD in a concentration-dependent manner. The high frequency of sera from AEDS patients, showing IgE binding to both HSP and MnSOD, indicates that these allergens, designated Mala s 10 and Mala s 11, could play a role in AEDS.

Adult↗

PubMed2002

Comparison of the crystal structures of the human manganese superoxide dismutase and the homologous Aspergillus fumigatus allergen at 2-A resolution.

Manganese superoxide dismutase (MnSOD) of Aspergillus fumigatus, a fungus involved in many pulmonary complications, has been identified as IgE-binding protein. It has been shown also that MnSODs from other organisms, including human, are recognized by IgE Abs from individuals sensitized to A. fumigatus MnSOD. Comparison of the fungal and the human crystal structure should allow the identification of structural similarities responsible for IgE-mediated cross-reactivity. The three-dimensional structure of A. fumigatus MnSOD has been determined at 2-A resolution by x-ray diffraction analysis. Crystals belonged to space group P2(1)2(1)2(1) with unit cell dimensions of a = 65.88 A, b = 98.7 A, and c = 139.28 A. The structure was solved by molecular replacement using the structure of the human MnSOD as a search model. The final refined model included four chains of 199-200 amino acids, four manganese ions, and 745 water molecules, with a crystallographic R-factor of 19.4% and a free R-factor of 23.3%. Like MnSODs of other eukaryotic organisms, A. fumigatus MnSOD forms a homotetramer with the manganese ions coordinated by three histidines, one aspartic acid, and one water molecule. The fungal and the human MnSOD share high similarity on the level of both primary and tertiary structure. We identified conserved amino acids that are solvent exposed in the fungal and the human crystal structure and are therefore potentially involved in IgE-mediated cross-reactivity.

Allergens↗

PubMed2002

Cyclophilins, a new family of cross-reactive allergens.

Type I allergic reactions occur by immediate release of anaphylactogenic mediators due to cross-linking of IgE bound to the high-affinity Fc(epsilon)RI on the surface of effector cells of sensitized individuals after allergen exposure. IgE-mediated hypersensitivity against normally innocuous environmental antigens is of clinical importance because of an increasing incidence of asthma and severe atopic diseases causing raising health care burdens to the society. A vast variety of different molecular structures has been shown to be able to induce hypersensitivity reactions. However, the high structural homology between phylogenetically conserved allergenic proteins present in different, apparently unrelated sources of exposure seems to play an important role in IgE-mediated poly-sensitization. These allergen families, formally termed pan-allergens, represent proteins sharing a high degree of sequence homology. Here we report cloning, production and serological investigations of a new pan-allergen family, the cyclophilins, found to be cross-reactive across species including humans. IgE-mediated cross-reactivity against autoantigens may contribute to perpetuation of severe atopic disorders even in the absence of exogenous allergen exposure. The molecular definition of pan-allergen families may substantially contribute to reduce the number of structures needed for diagnosis and therapy of allergic diseases based on highly pure, standardized recombinant allergens.

Allergens↗

PubMed2002

Immunological and structural analysis of IgE-mediated cross-reactivity between manganese superoxide dismutases.

BACKGROUND: Allergy results from inappropriate immune responses to normally innocuous proteins. More than 300 IgE-binding proteins have been cloned and shown to cover structurally and functionally heterogeneous protein families including enzymes, backbone and storage proteins as well as proteins with unknown function. Structurally related cross-reactive allergens are involved in the pathogenesis of important clinical syndromes. We found that phylogenetically highly conserved proteins, including human manganese superoxide dismutase (MnSOD), are involved in IgE-mediated autoreactivity. METHODS: MnSOD cloned from different phylogenetically distant species were produced as recombinant proteins in Escherichia coli and used to study IgE-mediated cross-reactivity and proliferative responses in peripheral blood mononuclear cells (PBMC) of individuals sensitised to Aspergillus fumigatus MnSOD. Homology models of the three-dimensional structures of MnSOD from A. fumigatus, Drosophila melanogaster and Saccharomyces cerevisiae using the human MnSOD structure refined at 2.2 A as template were constructed to identify conserved amino acid residues exposed to the solvent. RESULTS: Cross-reactivity between the MnSOD at B and T cell level was demonstrated by inhibition experiments showing shared B cell epitopes and by the capability of the different MnSODs to induce proliferative responses in PBMCs of sensitised individuals. Structural modelling allowed to identify conserved residues exposed to the solvent. The identified residues are scattered over the sequence of the enzyme indicating putative conformational IgE-binding epitopes. CONCLUSIONS: The results obtained corroborate molecular mimicry as a plausible mechanism to explain autoreactivity to human MnSOD. Moreover, homology modelling provides a rational tool to identify conserved residues involved in defining cross-reactive B cell epitopes.

Adult↗