Biomedical subjects

Reto Crameri

Publications and source records attributed to Reto Crameri.

At least 19 recordsLinked to original sources

PubMed2007

Cross-reactivity and 1.4-A crystal structure of Malassezia sympodialis thioredoxin (Mala s 13), a member of a new pan-allergen family.

We have identified thioredoxins (Trx) of Malassezia sympodialis, a yeast involved in the pathogenesis of atopic eczema, and of Aspergillus fumigatus, a fungus involved in pulmonary complications, as novel IgE-binding proteins. We show that these Trx, including the human enzyme, represent cross-reactive structures recognized by serum IgE from individuals sensitized to M. sympodialis Trx. Moreover, all three proteins were able to elicit immediate-type allergic skin reactions in sensitized individuals, indicating a humoral immune response based on molecular mimicry. To analyze structural elements involved in these reactions, the three-dimensional structure of M. sympodialis Trx (Mala s 13) has been determined at 1.4-A resolution by x-ray diffraction analysis. The structure was solved by molecular replacement and refined to a crystallographic R factor of 14.0% and a free R factor of 16.8% and shows the typical Trx fold. Mala s 13 shares 45% sequence identity with human Trx and superposition of the solved Mala s 13 structure with those of human Trx reveals a high similarity with a root mean square deviation of 1.11 A for all Calpha atoms. In a detailed analysis of the molecular surface in combination with sequence alignment, we identified conserved solvent-exposed amino acids scattered over the surface in both structures which cluster to patches, thus forming putative conformational B cell epitopes potentially involved in IgE-mediated cross- and autoreactivity.

Adult↗

PubMed2006

Novel vaccines and adjuvants for allergen-specific immunotherapy.

Advances in genetic engineering and biotechnology, in parallel to increased understanding of disease processes and mechanisms of protective immunity, have facilitated the development of novel rational vaccination concepts for allergy. The spectrum ranges from utilizing recombinant proteins, peptides, new adjuvants, immunomodulatory therapy and DNA vaccines, to use of new application routes for both prophylaxis and treatment of allergic disease. New therapeutic concepts based on patient-tailored immunotherapy using recombinant allergens, anti-IgE and sublingual immunotherapies are slowly moving from the bench to the clinics. The additive value of new treatments above existing therapies must be, however, focused on the improvement of efficacy for long-term cure and increased patient compliance to warrant a broad applicability that has to compete with the symptomatic control of allergic and asthmatic diseases of currently available drugs.

Adjuvants, Immunologic↗

PubMed2006

Effect of allergic bronchopulmonary aspergillosis on lung function in children with cystic fibrosis.

RATIONALE: The relationship between sensitization to Aspergillus fumigatus and progression of pulmonary function is not yet established in cystic fibrosis (CF). OBJECTIVES: We aimed to evaluate onset of A. fumigatus sensitization and development of allergic bronchopulmonary aspergillosis (ABPA), as well as to determine the physiologic factors of lung function influencing these mechanisms in CF. METHODS: Serial annual lung function tests performed in 122 children with CF (62 males; 60 females; age: 6-18 yr) provided data pertaining to FRC measured by plethysmography, lung clearance index, volume of trapped gas, effective specific airway resistance, and forced expiratory indices (FEV1, FEF at 50% VC). Specific IgE to recombinant A. fumigatus allergens, rAspf1 and rAspf3, served as marker for sensitization, and to rAspf4 and rAspf6 as indications for a serologic ABPA, were clinically diagnosed (Nelson criteria). By linear mixed-effect model analysis, five patient groups, (1) not sensitized and free from Pseudomonas aeruginosa, (2) intermittently P. aeruginosa colonized, (3) chronically P. aeruginosa infected, (4) sensitized, and (5) with ABPA, were retrospectively evaluated. MEASUREMENTS AND MAIN RESULTS: A. fumigatus sensitization was best reflected by increased rAspf1+3-specific IgE levels, whereas, in most patients, sensitization was preceded by P. aeruginosa infection. Patients with ABPA demonstrated the most severe progression in all lung function parameters, and FEF at 50% VC, volume of trapped gas, and effective specific airway resistance were the best predictors (p < 0.0001). However, regarding distinction between sensitization to A. fumigatus and development of ABPA in the course of CF, chronic P. aeruginosa infection has to be taken into account. CONCLUSIONS: Airway narrowing, gas trapping, and small airway disease are the major targets for functional derangement in ABPA.

Adolescent↗

PubMed2006

HS1-associated protein X-1 interacts with membrane-bound IgE: impact on receptor-mediated internalization.

Engagement of the BCR triggers signals that control affinity maturation, memory induction, differentiation, and various other physiological processes in B cells. In previous work, we showed that truncation of the cytoplasmic tail of membrane-bound Ig (mIg)E in vivo resulted in lower serum IgE levels, decreased numbers of IgE-secreting plasma cells, and the abrogation of specific secondary responses correlating with a defect in the selection of high-affinity Abs during the germinal center reaction. We concluded that the Ag receptor is necessary at all times during Ab responses not only for the maturation process, but also for the expansion of Ag-specific B cells. Based on these results, we asked whether the cytoplasmic tail of mIgE, or specific proteins binding the cytoplasmic tail in vivo commit a signal transduction accompanying the B cell along its differentiation process. In this study, we present the identification of HS1-associated protein X-1 as a novel protein interacting with the cytoplasmic tail of mIgE. ELISA, surface plasmon resonance analysis, and coimmunoprecipitation experiments confirmed the specific interaction in vitro. In functional assays, we clearly showed that HS1-associated protein X-1 expression levels influence the efficiency of BCR-mediated Ag internalization.

Amino Acid Sequence↗

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

NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum.

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.

Alcohol Oxidoreductases↗

PubMed2006

Wheat and maize thioredoxins: a novel cross-reactive cereal allergen family related to baker's asthma.

BACKGROUND: Baker's asthma is a serious problem for a significant proportion of workers in bakeries, confectionaries, and the food industry. Although several wheat allergens related to baker's asthma have been described, standardized reagents for a reliable diagnosis are not yet available. OBJECTIVE: To clone novel wheat allergens related to baker's asthma and investigate the cross-reactive potential of their maize and human homologues. METHODS: A wheat cDNA phage display library was screened with sera from bakers with occupational asthma for IgE-binding structures. Homologous sequences from maize and human thioredoxins were amplified from corresponding cDNA libraries. RESULTS: Within the enriched wheat cDNA repertoire we identified, among others, the sequence encoding wheat thioredoxin-hB (Triticum aestivum allergen 25 [Tri a 25]). The recombinant protein displayed enzymatic activity, and we observed a sensitization rate of 47% among bakers with occupational asthma and of 35% among patients with grass pollen allergy, but without a clinical history of cereal allergy. Furthermore, the previously characterized maize thioredoxin-h1 (Zea mays allergen 25 [Zea m 25]), sharing 74% identity with Tri a 25, exhibited distinct IgE cross-reactivity with its wheat homologue. Two bakers also showed sensitization to human thioredoxin, which shares 29% identity with Tri a 25. In a comparative study, we included recombinant alpha-amylase inhibitor 0.19, showing a sensitization rate of 65% in individuals with baker's asthma. CONCLUSION: Thioredoxins represent a novel family of cross-reactive allergens that might contribute to the symptoms of baker's asthma and might in addition be related to grass pollen allergy, as indicated by the reactivity of grass pollen allergic patients to cereal thioredoxins. CLINICAL IMPLICATIONS: The recombinant cereal thioredoxins will, together with the already reported wheat allergens, contribute to a more reliable diagnosis of baker's asthma and, perhaps, become a tool for the development of component-resolved immunotherapy.

Allergens↗

PubMed2006

Allergy diagnosis, allergen repertoires, and their implications for allergen-specific immunotherapy.

Immunotherapy for allergic diseases represents an important but largely unmet medical need. Conventional immunotherapy suffers from several breakdowns related to the quality of the extracts used, the risk of inducing anaphylactic reactions, and the extremely long treatment time. Many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome using genetically engineered recombinant allergens. New therapeutic strategies based on recombinant technology include peptide-based vaccines, engineered hypoallergens with reduced IgE-binding properties, nucleotide-conjugated vaccines that promote Th1 responses, and the possibility of developing prophylactic allergen vaccines.

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

Identification of Borrelia burgdorferi ribosomal protein L25 by the phage surface display method and evaluation of the protein's value for serodiagnosis.

The phage surface display technique was used to identify Borrelia burgdorferi antigens. By affinity selection with immunoglobulin G from pooled sera of six Lyme borreliosis (LB) patients, the ribosomal protein L25 was identified. The diagnostic value of L25 was investigated by an enzyme-linked immunosorbent assay, using sera from 80 LB patients and 75 controls, and the use of the protein resulted in a specificity of 99% and a 23% sensitivity, which qualify L25 as a useful antigen when combined with others.

Adult↗

PubMed2006

The role of sensitization to Malassezia sympodialis in atopic eczema.

Atopic eczema (AE) is a chronic relapsing, highly pruritic inflammation of the skin with a worldwide prevalence of 10-20% in children and of 1-3% in adults. Malassezia sympodialis has been reported as the most frequent skin-colonizing yeast in both AE patients and healthy individuals. Approximately 50% of the AE patients show immediate-type skin reactions or have specific serum IgE against M. sympodialis. Sensitization to the yeast occurs almost exclusively in AE patients. The main cause for this specific sensitization may be the disrupted skin barrier facilitating allergen uptake. So far thirteen allergens of Malassezia have been cloned produced, characterized and partly studied in vitro and in vivo. Phylogenetically conserved allergen structures, such as manganese superoxide dismutase, may play a role as cross-reactive allergens in a subset of AE patients as a result of molecular mimicry and cross-reactivity with structurally related human proteins and might contribute to the perpetuation of the inflammatory skin reactions. The use of recombinant Malassezia allergens will contribute to elucidate the pathways of sensitization occurring in AE, the underlying immunological mechanisms governing IgE- and T-cell-mediated responses and may provide new therapeutic options to alleviate Malassezia-related symptoms in AE.

Allergens↗

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↗

PubMed2006

Regulation of the IgE response at the molecular level: impact on the development of systemic anti IgE therapeutic strategies.

The classical allergic reaction starts within seconds or minutes after antigen contact and is induced by antibodies produced by a special subset of B lymphocytes. These antibodies belong to the IgE subclass and are responsible for Type I hyper-reactivity reactions. IgE plays a minor role in healthy individuals. In allergic individuals, however, IgE antibodies trigger allergic responses through allergen-mediated cross-linking on effector cells followed by mediator release. The mechanisms inducing a switch to IgE production are not fully understood with the consequence that allergies are mainly treated with antisymptomatic drugs. To develop basic therapies, many questions concerning the very complex regulation of IgE expression have to be understood. Positive and negative regulators influence the synthesis of IgE. Experiments in our laboratory could show that not only regulatory molecules, but also the membrane bound IgE itself controls the quantity and quality of the IgE produced. This fact becomes more and more interesting, because the signals generated by the B-cell receptor may be important targets for interference in allergic patients, in whom the titer and the affinity of the IgE antibodies for the allergen are directly related to disease activity.

Adenine↗

PubMed2005

Correlation-associated peptide networks of human cerebrospinal fluid.

Profiling of peptides and small proteins from either human body fluids or tissues by chromatography and subsequent mass spectrometry reveals several thousand individual peptide signals per sample. Any peptide is an intermediate in the course of biosynthesis, post-translational modification (PTM), proteolytic processing and degradation. Changes in the concentration of one peptide often affects the concentration of the other, hence a challenge consists in the development of suitable tools to turn this large amount of data into biologically relevant information. Comprehensive statistical analysis of the peptide profiling data allows associating peptides, which are closely related in terms of peptide biochemistry. Here, the bioinformatic concept of peptide networks, correlation-associated peptide networks (CANs), is introduced. Peptides with statistical similarity of their concentrations are grouped in form of networks, and these networks are interpreted in terms of peptide biochemistry. The spectrum of functional relationships found in cerebrospinal fluid CAN covers PTM and proteolytic degradation of peptides, clearance processing in the complement cascade, common secretion of peptides by neuroendocrine cells as well as ubiquitin-mediated degradation. Our results indicate that CAN is a powerful bioinformatic tool for the systematic analysis and interpretation of large peptidomics and proteomics data and helps to discover novel bioactive and diagnostic peptides.

Chemical Fractionation↗

PubMed2005

A major allergen gene-fusion protein for potential usage in allergen-specific immunotherapy.

BACKGROUND: Specific immunotherapy is a common treatment of allergic diseases and could potentially be applied to other immunologic disorders. Despite its use in clinical practice, more defined and safer allergy vaccine preparations are required. Differences between epitopes of IgE that recognize the 3-dimensional structure of allergens and T cells that recognize linear amino acid sequences provide a suitable tool for novel vaccine development for specific immunotherapy. OBJECTIVE: The aim of the study was to delete B-cell epitopes and prevent IgE crosslinking, but to preserve T-cell epitopes by fusion of 2 major allergens of bee venom because of a change in the conformation. METHODS: By genetic engineering, we produced a fusion protein composed of the 2 major bee venom allergens: phospholipase A 2 (Api m 1) and hyaluronidase (Api m 2). RESULTS: The Api m [1/2] fusion protein induced T-cell proliferation and both T H 1-type and T H 2-type cytokine responses. In contrast, IgE reactivity was abolished, and profoundly reduced basophil degranulation and type 1 skin test reactivity was observed. Pretreatment of mice with Api m [1/2] fusion protein significantly suppressed the development of specific IgE as well as other antibody isotypes after immunization with the native allergen. CONCLUSION: The novel fusion protein of 2 major allergens bypasses IgE binding and mast cell/basophil IgE FcepsilonRI crosslinking and protects from IgE development.

Adult↗

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↗