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R Crameri

Publications and source records attributed to R Crameri.

At least 55 records · Page 3Linked to original sources
PubMed1999

Selective enrichment and high-throughput screening of phage surface-displayed cDNA libraries from complex allergenic systems.

Phage surface display technology, first described in 1985, enables the construction of large combinatorial peptide and antibody libraries. The basic concept of linking the phenotype, expressed as gene product displayed on the phage surface, to its genetic information integrated into the phage genome, allows the survey of large libraries for the presence of specific clones using the discriminative power of affinity purification. The selection procedure involves the enrichment of phage by binding to an immobilized target molecule. As a consequence of the physical linkage between genotype and phenotype, sequencing the DNA of the integrated section of the phage genome can readily elucidate the amino acid sequence of a displayed gene product. Phage surface display technology has revolutionized our ability to select agonist and antagonist peptides, antibodies with desired specificities, and DNA-binding molecules. We have extended phage surface display to access cDNA libraries with this powerful screening technology based on affinity selection of desired clones. Here we discuss construction of cDNA libraries displayed on the phage surface, selective enrichment of clones and robotics-based high-throughput screening of enriched libraries.

Allergens↗
PubMed1998

Studies on pulmonary and systemic Aspergillus fumigatus-specific IgE and IgG antibodies in horses affected with chronic obstructive pulmonary disease (COPD).

Inhalant exposure to Aspergillus fumigatus (Asp. f.) antigens induces marked inflammatory and immunological alterations in the lungs of horses affected with chronic obstructive pulmonary disease (COPD). In this study we investigated the role of specific allergen(s) present in Asp. f. on systemic and pulmonary IgE and IgG responses in control and COPD-affected horses, using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting techniques. Compared with controls, horses affected with COPD had significantly higher levels of BALF IgE and IgG to somatic Asp. f. antigens as well as to the allergen 1/a (Asp. f. 1/a). Serum levels of IgE and IgG against these antigens did not differ between control and COPD-affected horses. Antigen specific IgE and IgG levels did not correlate between BALF and serum. Scanning of Asp. f. and IgE and IgG blots revealed bands that are recognised by both IgE- and IgG-specific antibodies. Additionally, all horses responded with BALF IgE- and IgG-specific for 93, 35, 31 and 23 kDa allergens, suggesting that these antigens are involved in the induction of airway IgE and IgG responses. These allergens may have the potential to be used as biomarkers for the diagnosis of Asp. f. related exacerbations of equine COPD.

Animals↗
PubMed1998

Characterization of two dog IgE-specific antibodies elicited by different recombinant fragments of the epsilon chain in hens.

Two recombinant [His]6-tagged fragments of the canine immunoglobulin E (IgE) heavy chain (second domain: IgEf2 and third and fourth domains: IgEf3/4) were cloned, expressed in Escherichia coli (E. coli) as [His]6-tagged proteins, and affinity-purified over nickel-nitrilotriacetic acid columns. The recombinant proteins were used to immunize hens. The raised and affinity-purified chicken antibodies (Ab) isolated from egg yolk exhibited specific binding to the respective recombinant canine IgE fragment (IgEf) on immunoblots and displayed high titers against the IgEf in ELISA. Immunoblotting of canine serum separated by PAGE under native conditions with the IgEf2- and IgEf3/4-specific Ab resulted in staining of a protein of approximately 180 kilodaltons (kD). The IgEf3/4-specific Ab further recognized an 80 kD protein in IgEf3/4-specific Ab affinity-enriched dog serum separated under denaturing conditions. In an ELISA for the detection of antigen-specific IgE in dog serum, reduced binding of the IgEf-specific Ab was observed after heat treatment of the dog serum. The reactivity of both of the raised chicken Ab was only present in postimmune reagents and could only be inhibited by preincubation with the IgEf used for immunization and not with dog immunoglobulin G, E. coli extract, or with a nonrelevant recombinant [His]6-tagged protein. In immunohistochemistry, the IgEf3/4-specific Ab specifically recognized cells in paraffin-embedded tissue sections of lymph nodes. Furthermore, both of the IgEf-specific Ab elicited positive immediate type 1 skin reactions in dogs. Semiquantitative assessment of total serum IgE in dogs was developed using IgEf2-specific Ab as coating reagent and the biotinylated IgEf3/4-specific Ab as developing Ab in ELISA. In conclusion, both IgEf-specific Ab recognize native dog IgE with the advantages that they are directed against different and known constant domains of the IgE molecule, and that they can be used for immunohistochemistry on paraffin-embedded tissue. The two dog IgE-specific Ab could initiate clinical research on the involvement of immediate-type hypersensitivity reactions in dogs.

Animals↗
PubMed1998

Differential IgE recognition of recombinant Aspergillus fumigatus allergens by cystic fibrosis patients with allergic bronchopulmonary aspergillosis or Aspergillus allergy.

Allergic bronchopulmonary aspergillosis (ABPA), an intense inflammatory reaction to Aspergillus in the lung, is recognized as a severe complication in patients with cystic fibrosis (CF). The diagnosis of ABPA in CF patients sensitized to Aspergillus fumigatus is complicated by interfering laboratory and clinical findings shared by the diseases. We have used cDNA encoding A. fumigatus allergens which were cloned from a cDNA library displayed on phage surface to produce recombinant proteins in Escherichia coli. Differential IgE responses to the allergens in A. fumigatus-sensitized CF patients with or without ABPA and CF controls without sensitization to A. fumigatus were demonstrated. A secreted ribotoxin (rAsp f 1) and a peroxisomal protein (rAsp f 3) were recognized by sera from A. fumigatus-sensitized CF-patients with or without ABPA. An intracellular manganese superoxide dismutase (rAsp f 6) and rAsp f 4, a protein with unknown function, were recognized exclusively by IgE from sera of CF patients with ABPA. Therefore, Asp f 4 and Asp f 6 represent specific markers for ABPA and allow a sensitive, fully specific diagnosis of the disease. The data suggest distinct IgE responses to colonization of the bronchial tree in CF patients with ABPA or A. fumigatus allergy and therefore a differential recognition of the pathogen in the two IgE-related inflammatory diseases.

Adolescent↗
PubMed1998

Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli.

BACKGROUND: Hyaluronidase (Hya) is one of several allergens in honeybee venom. Its cDNA sequence was recently described. OBJECTIVE: We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity. METHODS: In Escherichia coli Hya was produced as inclusion body 6 x His-fusion protein. In baculovirus-infected insect cells expression was obtained by cotransfection of linearized Bac-N-Blue DNA and pMelBac transfer vector into Spodoptera frugiperda cells. RESULTS: Enzymatic activity of Hya from the baculovirus system was equal to nHya, and that of the enzyme expressed in E. coli was only 20% to 30% of nHya. In vitro IgE binding was similar in nHya and the enzyme from baculovirus but markedly lower in Hya expressed in E. coli. CONCLUSIONS: Biologic activity of Hya expressed in baculovirus-infected insect cells was comparable with that of the natural enzyme, indicating a native-like conformation of the recombinant protein. In contrast, the enzyme expressed in E. coli as an inclusion-body protein and reconstituted in vitro reached only 20% to 30% of the activity of nHya.

Allergens↗
PubMed1998

Enzymatic activity of soluble phospholipase A2 does not affect the specific IgE, IgG4 and cytokine responses in bee sting allergy.

BACKGROUND: The soluble bee venom phospholipase A2 (PLA) represents the major allergen/antigen for allergic and hyperimmune individuals following bee sting. A number of studies implicate enzymes, and PLA in particular, as potent allergens. We have studied specific activation of T cells by enzymatically active and inactive mutants of PLA, and secretion of cytokines regulating IgE and IgG4 antibody formation. METHODS: Recombinant (r) wild type PLA (rPLA-WT) and an enzymatically inactive rPLA (rPLA-H34Q) were produced in Escherichia coli. Eleven bee venom allergic patients and three hyperimmune, healthy individuals were included in the study. After specific stimulation of PBMC with the rPLA variants, proliferative response, IFNgamma, IL-2, IL-4, IL-5, and IL-13 production, as well as total and PLA-specific IgE and IgG4 production, were analysed. RESULTS: Similar levels of specific B cell recognition, proliferative and cytokine responses were observed after stimulation with either enzymatically active or inactive rPLA. In addition, equal amounts of antigen-specific and total IgE and IgG4 antibodies were produced by stimulation with both forms of rPLA. CONCLUSIONS: The enzymatic activity of PLA does not influence the specific activation and cytokine production by T cells from bee venom-sensitized or hyperimmune individuals, or the IgE/IgG4 antibodies synthesis by B cells in vitro.

Adult↗
PubMed1998

Skin-test reactivity and isotype-specific immune responses to recombinant Asp f 3, a major allergen of Aspergillus fumigatus.

BACKGROUND: The in vitro diagnostic value of recombinant allergens depends on the correlation between allergen specific IgE in serum and ability of the protein to elicit immediate type allergic reactions in sensitized individuals. Objective The study was carried out to evaluate the reliability of recombinant Asp f 3 (rAsp f 3)-based serological determinations in ELISA and ImmunoCAP compared to skin-test responses in A. fumigatus sensitized subjects. METHODS: Patients suffering from allergic bronchopulmonary aspergillosis (ABPA, n=11) or allergic asthma with A. fumigatus sensitization (n =8) and healthy control subjects (n=4) were investigated. Intradermal skin tests (IDT) and allergen-specific serology were performed using rAsp f 3 protein. RESULTS: All 11 patients with ABPA and five out of eight A. fimigatus-sensitized asthmatics without ABPA exhibited a type I skin reaction. All rAsp f 3 skin test positive subjects showed relevant rAsp f 3-specific serum IgE levels compared with IDT-negative individuals who scored below the rAsp f 3-ImmunoCAP cut-off values of < 0.35 kU(A)/L. Analyses of rAsp f 3-specific immunoglobulins revealed significantly higher serum levels of IgG, IgG1, IgG4 and IgE antibodies in patients with ABPA compared with A. fumigatus-sensitized asthmatics and healthy controls. CONCLUSION: rAsp f 3 represents a major allergen of A. fumigatus. It is recognized by 84% of asthmatic individuals sensitized to the fungus and elicits specific type I skin reactions. The skin test outcome strictly depends on the presence of rAsp f 3-specific IgE and the lack of false positive or false negative results comparing skin test outcome with specific serum IgE levels determined by ELISA or rAsp f 3-ImmunoCAP suggests the possibility to rely on serological data obtained with recombinant allergens to diagnose sensitization to A. fumigatus.

Adult↗
PubMed1998

Disease-specific recombinant allergens for the diagnosis of allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA), a severe pulmonary complication caused by Aspergillus fumigatus, is considered a complex clinical syndrome with defined serological, pathological radiological and clinical features. The diagnosis of ABPA is often difficult because of several overlapping clinical and laboratory findings shared between asthma with sensitization to A. fumigatus and ABPA, but essential for treatment to prevent severe deterioration of pulmonary function. We have cloned A. fumigatus allergens from a cDNA library displayed on phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. The single recombinant allergens were used to assess the immunological response in representative groups of A. fumigatus-sensitized asthmatics with or without ABPA and healthy controls. The allergens rAsp f 1, a 16.9 kDa ribotoxin, rAsp f 3, a 18.5 kDa peroxisomal protein, and rAsp f 6, a 23 kDa manganese superoxide dismutase, were identified as proteins with known biological function and rAsp f 4, a 30 kDa allergen, lacks sequence homology to known proteins. The secreted ribotoxin rAsp f 1 and rAsp f 3 are recognized by serum IgE of A. fumigatus-sensitized asthmatics with or without ABPA, whereas the non-secreted manganese superoxide dismutase rAsp f 6 and the rAsp f 4 allergen are exclusively recognized by serum IgE of ABPA patients. The dissection of IgE-mediated immune responses to single recombinant A. fumigatus allergens in asthmatic patients allow a discrimination between ABPA and A. fumigatus sensitization with high specificity (100%) and sensitivity (90%).

Adult↗
PubMed1998

[Molecular aspects and diagnostic value of fungal allergens].

Cloning, sequencing and production of highly pure recombinant allergens allows to produce perfectly standardised allergen preparations. The development of a new cloning system based on filamentous phage allowed the fast isolation and characterisation of allergens from the fungus Aspergillus fumigatus. The produced recombinant allergens were tested in serological and clinical studies as well as for their performance for routine assessments in the ImmunoCAP-system. Thereby, a perfect correlation between skin test results and serology was found showing the potential of recombinant allergens for the diagnosis of allergic diseases. Moreover, the characterisation of fungal allergens substantially contributes to our understanding of the molecular nature of proteins involved in the elicitation of allergic reactions. Apart from allergenic proteins with unknown biological function, fungal allergens can be subdivided into two classes: 1. Species-specific, secreted proteins and 2. cytoplasmic, even in phylogenetically distant organisms, well conserved proteins. These fungal allergens show extended sequence similarity, a high level of IgE cross-reactivity and in some cases also cross-reactivity with homologous human proteins indicating autoimmune reactions involved in fungal allergy.

Allergens↗
PubMed1998

Recombinant Aspergillus fumigatus allergens: from the nucleotide sequences to clinical applications.

Members of the genus Aspergillus are opportunistic pathogens for cold- and warm-blooded animals. They are associated with an impressive spectrum of diseases in humans, ranging from benign colonization of the lung to life-threatening diseases such as invasive aspergillosis or allergic bronchopulmonary aspergillosis (ABPA). Aspergillus fumigatus is the etiological agent identified in most of the Aspergillus-related human diseases and is therefore of particular clinical relevance. A major problem in the diagnosis of A. fumigatus-related complications arises from the lack of standardized fungal extracts. The allergenicity of commercial A. fumigatus extracts depends on many factors including the strain used, growth conditions, harvesting and extraction procedures hampering the development of diagnostic reagents suitable for the discrimination between A. fumigatus-related diseases. Molecular DNA/RNA technologies and biotechnological procedures allow cloning, characterization and production of large amounts of single, highly pure allergens. The development of phage surface display technologies for cloning cDNAs has speeded up the isolation of candidate cDNAs encoding allergens. Screening of an A. fumigatus cDNA library displayed on the surface of phage M13 allowed characterization and production of a panel of allergens identified as proteins with known biological function or as allergens without sequence homology to known proteins. They belong to two functional categories: secreted and cytoplasmic proteins. Secreted allergens are recognized by serum IgE of A. fumigatus-sensitized individuals with or without ABPA, whereas nonsecreted allergens are exclusively recognized by serum IgE of ABPA patients. The dissection of IgE-mediated immune responses to single A. fumigatus allergens allows a discrimination between ABPA and A. fumigatus sensitization with high specificity and sensitivity demonstrating the power of recombinant allergens for the differential diagnosis of A. fumigatus-related pulmonary complications.

Adolescent↗
PubMed1998

Determinants and mechanisms of human immune responses to bee venom phospholipase A2.

The elicitation of an immune response to protein antigens depends on the specific recognition of antigenic determinants (epitopes) by T and B lymphocytes. Bee venom phospholipase A2 (PLA) represents the major antigen/allergen of honey bee venom. It displays three dominant immunogenic peptide and one glycopeptide T cell recognition sites. These epitopes are equally recognized by both allergic and nonallergic individuals. A mixture of the three epitope containing peptides was successfully used in specific immunotherapy of bee venom-allergic patients. Both peptide and whole bee venom immunotherapy induced a state of specific anergy in T cells. The production of specific IgE and IgG4 antibodies directly correlated with the secreted interleukin-4:gamma-interferon (IL-4:IFNgamma) ratio, which itself depended on the concentration of available antigen and the strength of the T cell-activating signal. This signal comprises accumulated molecular interactions delivered by engagement of the antigenic peptide/MHC class II complex with the T cell receptor (TcR). Indeed the thermodynamic laws of chemical equilibrium reactions reveal that the antigen concentration, together with the equilibration constant Ki and the related Gibbs standard free energy DeltaG degrees of the MHC-II/Ag/TcR complex reaction, may govern the secreted IL-4:IFNgamma ratio, and in consequence, differential IgE and IgG4 antibody formation. Ki includes epitope and MHC-II haplotype variability and therefore represents a measure of immunological individuality. A major B cell epitope was determined by using point-mutated PLA. Specific antigen recognition by B cells can trigger distinct cytokine profiles in T cells and contribute to the differential regulation of specific IgE and IgG4 antibodies. Our results indicate that distinct cytokine profiles inducing allergic and nonallergic responses can be attributed to thresholds of T cell activation generated by the specific binding properties of individual MHC-II molecules to immunogenic T cell epitopes and their presentation to TcR.

Allergens↗
PubMed1997

"Peptabody": a new type of high avidity binding protein.

A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.

Animals↗
PubMed1997

Chicken antibodies to a recombinant fragment of the equine immunoglobulin epsilon heavy-chain recognising native horse IgE.

An equine immunoglobulin E (IgE) heavy-chain cDNA fragment (CH3-CH4, nucleotides 1132 to 1592) was cloned, expressed in Escherichia coli as a fusion protein with a [His]6-tag and purified over a Ni-NTA column. The recombinant protein was used to immunise hens. Testing of the raised egg yolk immunoglobulin G (IgG) in Western-blot and ELISA revealed high titres against the recombinant equine IgE fragment (reqIgEf). The reqIgEf-specific IgG was successfully affinity-purified on an unconventional affinity matrix: the [His]6-tagged recombinant IgE fragment was bound to Ni-NTA agarose and used to adsorb specific immunoglobulins. In Western-blot of ammonium sulphate precipitated horse serum and bronchoalveolar lavage fluid, separated by SDS-PAGE under denaturing-reducing conditions, the raised antibodies reacted with a protein of approximately 80 kDa. A reaction of the reqIgEf-specific IgG was seen with a 190-200 kDa band when the same horse serum or bronchoalveolar fluid (BALF) was separated under non-reducing conditions. These reactions could be inhibited by preincubation of the immune IgG with reqIgEf, while preincubation with horse IgG did not inhibit the reaction. Antibody-affinity chromatography of horse serum with the reqIgEf-specific chicken IgG resulted in an enrichment of the 80 kDa protein in denaturing Western-blot. Determination of the amino acid composition of this protein and comparison with the equine IgE heavy- chain sequence strongly indicates that the 80 kDa band corresponds to the heavy chain of the horse IgE. The reqIgEf-specific chicken IgG was further characterised in an ELISA for the detection of allergen-specific horse IgE. It was demonstrated that heating IgE positive horse sera at 54 degrees C for 10 min drastically diminished the recognition by the reqIgEf-specific chicken IgG. The reaction is inhibitable by preincubation with reqIgEf in a concentration dependent manner. In addition, preincubation with horse IgG, a nonrelevant [His]6-tagged protein or 2% equine colostrum had no influence on the reqIgEf-specific chicken IgG binding characteristic. This antibody recognising horse IgE will be useful for further studies on the pathogenesis of equine allergic diseases.

Amino Acids↗
PubMed1997

Allergens of Aspergillus fumigatus and Candida boidinii share IgE-binding epitopes.

From an Aspergillus fumigatus complementary deoxyribonucleic acid (cDNA) library displayed on phage surface, an allergen formally termed rAsp f 3 was cloned. The open-reading frame of the cloned gene for the allergen encodes a protein of 168 amino acids with a predicted molecular mass of 18.5 kD, showing 36% identity and 58% similarity to two peroxisomal membrane proteins of Candida boidinii. Recombinant Asp f 3 was expressed as a [His]6-tagged fusion protein in Escherichia coil at yields of 30 mg/L, and was purified by Ni(2+)-chelate chromatography. In an enzyme-linked immunosorbent assay (ELISA), serum IgE antibody reactivity to rAsp f 3 could be detected in 72% of 89 individuals sensitized to A. fumigatus, demonstrating that the protein represents a major allergen of the mold. IgE specific to rAsp f 3 and the two recombinant Candida proteins was further demonstrated by IgE-immunoblot analysis. IgE binding to rAsp f 3 could be inhibited in the ELISA by adding either of the recombinant Candida peroxisomal proteins to sera containing IgE directed against Asp f 3. Taken together, these observations prove that the Asperigillus allergen and the two Candida proteins share IgE-binding epitopes.

Allergens↗
PubMed1997

Increased specificity of diagnostic tests with recombinant major bee venom allergen phospholipase A2.

BACKGROUND: In diagnosis of type I allergy recombinant allergens have potential advantages over conventional allergenic extracts, both regarding specificity and reproducibility. OBJECTIVES: We therefore decided to study honey bee venom (BV) and its major allergen phospholipase A2 (PLA) in native and recombinant form for diagnosis of bee sting allergy. METHOD: We investigated 85 patients with a history of a recent systemic allergic bee sting reaction and positive intracutaneous skin test to BV, and 21 controls with no history of allergic bee sting reaction and negative skin test to BV. Intracutaneous skin tests and determination of specific IgE by ImmunoCAP(R) to BV, native PLA (nPLA) and recombinant PLA (rPLA) were done in all patients and controls. RESULTS: In skin testing 84 (99%) of the 85 patients reacted to nPLA and 81 (95%) to rPLA, while none of the 21 controls was positive with nPLA or rPLA. Specific serum IgE to BV could be detected in 82 of the patients (96%), to nPLA in 73 (86%) and to rPLA in 66 (78%). Four (19%) of the controls had a positive CAP to BV, one (4.8%) to nPLA and none to rPLA. Analysis of discordant results in CAP showed, that most patients with specific IgE to BV, but not to nPLA and rPLA, had positive skin tests to both PLA preparations and low levels of BV specific IgE. Patients with specific IgE to nPLA but not to rPLA were usually sensitized to minor allergens of BV which contaminated the commercial nPLA. CONCLUSIONS: PLA is the major allergen in BV. While diagnostic tests with BV are more sensitive, the specificity of tests with PLA, especially rPLA is clearly increased as compared with BV.

Allergens↗