PubMed Health⌕ Search

Biomedical subjects

R Crameri

Publications and source records attributed to R Crameri.

At least 73 records · Page 4Linked to original sources
PubMed1996

Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus.

A cDNA encoding an allergenic protein was isolated from an Aspergillus fumigatus (A. fumigatus) cDNA library displayed on the surface of filamentous phage. Serum immunoglobulin E (IgE) from A. fumigatus-sensitized individuals was used to enrich phage-expressing gene products binding to IgE. One of the cDNAs encoded a 26.7-kD protein that was identified as a manganese superoxide dismutase (MnSOD) sharing 51.5% identity and 67.2% homology to the corresponding human enzyme. Both human and A. fumigatus MnSOD coding sequences were expressed in Escherichia coli as [His]6-tagged fusion proteins and purified by Ni(2+)-chelate affinity chromatography. The two recombinant MnSODs were both recognized by IgE antibodies from subjects allergic to the A. fumigatus MnSOD and elicited specific immediate type allergic skin reactions in these individuals. Moreover, both human and A. fumigatus MnSOD induced proliferation in peripheral blood mononuclear cells of A. fumigatus-allergic subjects who showed specific IgE responses and reacted in skin tests to MnSOD. These observations provide evidence for autoreactivity to the human MnSOD in allergic persons sensitized to an environmental allergen from A. fumigatus who share a high degree of sequence homology to the corresponding human enzyme.

Amino Acid Sequence↗
PubMed1996

Expression and function of the Fas receptor on human blood and tissue eosinophils.

The interaction between activated T cells and eosinophils has been proposed to play an important role in the pathogenesis of allergic diseases. T cell-derived cytokines such as interleukin-5 and granulocyte/macrophage colony-stimulating factor inhibit eosinophil apoptosis and may therefore contribute to the development of tissue and blood eosinophilia in these disorders. Withdrawal of these cytokines leads to eosinophil apoptosis in vitro. In contrast, the mechanisms which actively induce apoptosis in eosinophils are at present not completely understood. In this study, we demonstrate that freshly isolated human eosinophils express mRNA and protein for the Fas receptor. Using anti-Fas monoclonal antibody (mAb), we show that Fas activation accelerates apoptotic eosinophil death in vitro. Moreover, treatment of nasal polyps ex vivo with anti-Fas mAb decreased eosinophilic tissue inflammation. However, we observed that blood as well as tissue eosinophils derived from some eosinophilic donors do not express functional Fas receptors, although Fas protein is normally expressed in these cells. This implies that the susceptibility of the Fas receptor is a matter of regulation in eosinophils as previously observed in other systems. These data suggest that Fas ligand/Fas interactions are involved in the regulation of eosinophil apoptosis and that defects in this system could contribute to the accumulation of these cells in allergic and asthmatic diseases.

Antibodies, Monoclonal↗
PubMed1996

Automated serology with recombinant allergens. A feasibility study.

Diagnosis of allergic conditions is based on skin prick- and/or intradermal tests and on serology. Both methods make use of allergenic preparations and the results mainly depend on the quality of the substances applied and on the reactivity of the subjects. Recombinant allergens can be produced as highly pure proteins. They may contribute to a significant improvement of the diagnosis of allergic diseases provided that the allergens became available in a pure, fully antigenic form suitable for routine assessments. We have studied the diagnostic properties of recombinant Aspergillus fumigatus allergen I (rAsp f I/a), a major allergen recognised by about 60% of the A. fumigatus allergic individuals, to test the feasibility of using recombinant allergens for automated serology in the Pharmacia CAP System, a new solid-phase immunoassay. ImmunoCAPs carrying immobilised rAsp f I/a were produced and evaluated using sera from well characterised patients with allergic bronchopulmonary aspergillosis (ABPA), allergic asthmatics with or without A. fumigatus sensitisation, and healthy control persons. The clinical parameters used for the diagnosis of allergy to A. fumigatus were RAST and skin test reactivity to commercial A. fumigatus extracts. IgE antibodies recognising rAsp f I/a were determined by a direct antigen-specific ELISA and with the newly developed rAsp f I/a ImmunoCAPs. Quantitative results from the two rAsp f I/a detection systems correlated closely, indicating that this recombinant allergen can be incorporated in the Pharmacia CAP System with unaltered antigenic activity. The behaviour of a panel of recombinant A. fumigatus allergens in the Pharmacia CAP System will be evaluated in the near future to assess the potential of recombinant allergens for the automated diagnosis of allergic conditions.

Allergens↗
PubMed1996

Cloning Aspergillus fumigatus allergens by the pJuFo filamentous phage display system.

A cDNA library from Aspergillus fumigatus has been displayed on the surface of filamentous phage M13 and screened for gene products binding to human serum IgE. The physical linkage of cDNA gene products to the genetic information required for their production, achieved by exploiting the high-affinity interaction of the Jun and Fos leucine zippers, allows rapid and easier screening of large libraries in semifluid systems. The pJuFo cloning vector is designed to display proteins on the surface of phage and allows selective isolation of genes by gene product-ligand interaction. Thus the system is applicable to clone cDNA that encodes proteins for which a ligand is available. Herein we show that phage expressing IgE binding proteins from A. fumigatus can be enriched and separated from nonspecific phage by using serum IgE from A. fumigatus-allergic individuals coated to plastic dishes. Subsequently, the proteins can be produced in high amounts in Escherichia coli and purified for usage in allergy testing.

Allergens↗
PubMed1996

Skin test reactivity to recombinant Aspergillus fumigatus allergen I/a in patients with cystic fibrosis.

We evaluated the diagnostic value of recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) for skin testing in cystic fibrosis (CF) patients with allergic bronchopulmonary aspergillosis (ABPA), allergy to A. fumigatus and in CF controls not allergic to A. fumigatus. Skin prick test reactions to rAsp f I/a were 8.5-fold stronger in A.-fumigatus-allergic and 3.3-fold stronger in ABPA patients compared to the CF controls. Three out of 6 well-characterized ABPA patients had positive skin prick test reactions to rAsp f I/a; the same number reacted to crude A. fumigatus extracts. The skin prick test results to rAsp f I/a were significantly correlated with those to crude and commercial preparations. According to these results, rAsp f I/a represents a defined allergen that provides the possibility to perform skin prick tests in CF patients in a highly standardized way. In future, a larger panel of recombinant allergens might also pick up those CF patients with ABPA and allergy to A. fumigatus who did not react to this single allergen.

Adolescent↗
PubMed1996

Automated specific IgE assay with recombinant allergens: evaluation of the recombinant Aspergillus fumigatus allergen I in the Pharmacia Cap System.

BACKGROUND: We report the results of a study comparing the recombinant Aspergillus fumigatus allergen 1 (rAsp f I) to commercial A. fumigatus extracts in serological assays. Pharmacia CAP System and skin tests. OBJECTIVE: The study was designed to test the feasibility of using recombinant allergens in an automated serology system for determination of allergen-specific IgE. METHODS: Patients with allergic bronchopulmonary aspergillosis (ABPA), asthmatics with A. fumigatus allergy and control subjects, who included allergic asthmatics without allergy to A. fumigatus and healthy subjects, were investigated. All subjects were characterized with respect to their total IgE level, radio allergosorbent test to A. fumigatus and skin test reactivity to both commercial A. fumigatus extracts and recombinant rAsp f I protein. RESULTS: All patients with ABPA (n = 30) showed positive skin test reactions with commercial A. fumigatus extracts, and 24 were sensitized to rAsp f I by the same criterion. The 10 patients with asthma and A. fumigatus allergy showed positive skin reactions to at least one commercial extract, and five reacted to rAsp f I. All control subjects (n = 19) scored negatively in skin tests to A. fumigatus extracts and rAsp f I and showed no detectable rAsp f I-specific IgE. ImmunoCAP carrying immobilized rAsp f I were evaluated using sera from all individuals described and the results compared with those obtained with the rAsp f I-specific ELISA for IgE. The data obtained with the two rAsp f I-specific detection systems correlated closely (r = 0.997) and were in perfect agreement with the skin test results. CONCLUSION: The data show that rAsp f I can be used as immobilized allergen in the Pharmacia CAP System indicating the feasibility of using recombinant allergens for an automated serological diagnosis of allergic diseases. However, every recombinant allergen needs to be evaluated individually for its performance if applied to a new diagnostic technology.

Adult↗
PubMed1995

Type I skin reactivity to native and recombinant phospholipase A2 from honeybee venom is similar.

Phospholipase A2 is the major allergen in honeybee venom. Recombinant phospholipase A2 was produced in prokaryotes and tested for its biologic activity by intracutaneous skin testing with serial 10-fold dilutions in comparison with native and deglycosylated phospholipase A2 in patients allergic to bee venom. Linear regressions of the log of the wheal area versus the log of the allergen concentration were calculated for all allergens in each patient. The relative allergenic potency of the various preparations was analyzed by comparing the linear regressions. Native phospholipase A2 was about 10 times more potent than whole bee venom. None of 58 patients allergic to bee venom was missed by testing with native phospholipase A2 alone. This allergen and deglycosylated native phospholipase A2 resulted in similar skin reactions, indicating that the sugar residues were of little relevance for IgE-binding in the patients tested. Native phospholipase A2 also had relative potency similar to that of recombinant refolded phospholipase A2, whereas recombinant nonrefolded phospholipase A2 had almost no biologic activity in skin testing. These results demonstrate in vivo activity of the recombinant bee venom allergen phospholipase A2. Although correct refolding is a prerequisite for type I skin reactivity, glycosylation seems to be less important.

Adult↗
PubMed1995

Different reactivity to recombinant Aspergillus fumigatus allergen I/a in patients with atopic dermatitis or allergic asthma sensitised to Aspergillus fumigatus.

We report a clinical study comparing the skin test reactivity to recombinant Aspergillus fumigatus allergen 1 (rAsp fI/a) in patients with atopic dermatitis and A. fumigatus sensitisation (n = 15), A. fumigatus-allergic patients with asthma (n = 10) and healthy control subjects (n = 10). All patients sensitised to A. fumigatus reacted at intradermal skin tests with commercial A. fumigatus extracts in contrast to the healthy subjects. Six out of 10 patients with well-characterised A. fumigatus allergic asthma were sensitised to rAsp fI/a as shown by a positive skin test. The patients with skin test reactivity to rAsp fI/a also showed rAsp f I/a-specific serum IgE as determined by ELISA. None of the patients with atopic dermatitis, healthy control subjects and 4 out of 10 A. fumigatus-allergic asthmatics reacted in intradermal tests to rAsp fI/a. Serologic investigations revealed that these subjects did not express detectable amounts of rAsp fI/a-specific IgE in agreement with the negative skin test results. Extended serologic investigations have not shown significant differences in rAsp fI/a-specific IgA, IgG4 and IgG1 serum levels between atopic dermatitis patients and healthy control subjects. The results suggest that sensitisation to A. fumigatus in patients with atopic dermatitis is not related to the major A. fumigatus allergen I in contrast to the high incidence of sensitisation to Asp fI/a occurring in allergic asthmatics.

Allergens↗
PubMed1995

Identification of allergic bronchopulmonary aspergillosis in cystic fibrosis patients by recombinant Aspergillus fumigatus I/a-specific serology.

Before specific therapy, such as oral corticosteroids, can be commenced it is essential to distinguish full-blown allergic bronchopulmonary aspergillosis (ABPA) from allergy to A. fumigatus in patients with cystic fibrosis (CF). For this purpose we have evaluated the diagnostic value of recombinant A. fumigatus allergen I/a (rAsp f I/a)-specific serology in 55 patients with CF. Based on clinical presentation and laboratory data, 10 CF patients had ABPA, 27 had Aspergillus allergy, and 18 were not allergic to A. fumigatus (CF control group). The serologic assays revealed a 10-fold increase in rAsp fI/a-specific IgE, a 5-fold increase in rAsp fI/a-specific IgG1, and a 4-fold increase in rAsp fI/a-specific IgG4 antibodies in ABPA patients compared with the Aspergillus allergy and CF control groups. Sera from 11 CF patients were analyzed without knowledge of their clinical state or diagnosis of ABPA. All ABPA patients (4 of 11) were accurately identified. We conclude that rAsp fI/a-specific serology is a highly sensitive and specific test that can be used to identify ABPA reliably in CF patients.

Adolescent↗
PubMed1994

Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of genes by specific gene-product/ligand interaction.

Techniques for cloning cDNAs from bacteriophage libraries immobilised on solid supports are well established. However, these techniques do not allow selective enrichment of clones expressing proteins of interest. Screening of cDNA libraries would be simplified if the proteins encoded by cDNAs could be expressed on the surface of phage. Phage carrying genes encoding proteins for which a ligand is available can be selected directly by affinity interaction [Crameri, R. & Suter, M. (1993) Gene (Amst.) 137, 69-75]. The expression products from a cDNA library from Aspergillus fumigatus have been displayed on the surface of the filamentous phage M13 and screened for gene products binding to human serum IgE. The physical linkage of cDNA-encoded proteins to the genetic information required for their production, achieved by exploiting the high-affinity interaction of the Jun and Fos leucine zippers, allows screening of up to 1 x 10(10) independent clones in 50-microliters aliquots applied to a well of a microtiter plate coated with the ligand. Phage displaying IgE-binding proteins were selectively enriched 10(5)-10(6)-fold over non-specific phage after six rounds of growth and selection. The apparent molecular mass of the proteins selected from the cDNA library was in the range 20-40 kDa. Restriction enzyme analysis and preliminary sequence determination of 12 selected inserts revealed different sequences. The ability of the proteins to bind to human serum IgE was corroborated by enzyme-linked immunosorbent assay and by Western-blot analysis. The developed cloning strategy allows isolation of cDNAs encoding proteins for which a ligand is available and circumvents immobilisation of the libraries on solid-phase supports which hamper selective enrichment of clones expressing the desired protein.

Aspergillus fumigatus↗
PubMed1994

Diagnostic value of recombinant Aspergillus fumigatus allergen I/a for skin testing and serology.

BACKGROUND: We report a clinical study comparing the recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) to two commercial A. fumigatus extracts in skin prick tests, intradermal tests, and serologic assays. METHODS: Patients with allergic bronchopulmonary aspergillosis and A. fumigatus-allergic patients with asthma, and control subjects, including allergic patients with asthma without allergy to A. fumigatus and healthy subjects, were investigated. RESULTS: All patients with allergic bronchopulmonary aspergillosis (n = 15) reacted to skin prick tests with the commercial extracts, and eight were sensitized to rAsp f I/a. Of 10 patients with well-characterized A. fumigatus-allergic asthma nine showed positive skin prick test results to at least one of the commercial extracts, and five reacted to rAsp f I/a. There was a strong correlation between skin test reactivity to rAsp f I/a and rAsp f I/a-specific serum IgE as determined by an antigen-specific ELISA. The healthy control subjects (n = 7) and allergic patients with asthma without A. fumigatus allergy (n = 6) did not react in skin prick and intradermal tests to rAsp f I/a, nor did they have detectable amounts of rAsp f I/a-specific IgE. In addition, patients with allergic bronchopulmonary aspergillosis showed significant elevated levels of rAsp f I/a-specific IgG4 and IgG1 but no significant differences in rAsp f I/a-specific serum IgA levels when compared with the healthy control subjects. CONCLUSIONS: The data show that rAsp f I/a is a major allergen with biologic relevance in some A. fumigatus-allergic individuals as evaluated by skin prick tests, intradermal tests, or serologic methods. Furthermore, no discrepancies were observed between skin test results and rAsp f I/a-specific IgE. Hence the correlation between rAsp f I/a skin test results and serologic data indicates the potential of recombinant allergens for clinical applications and diagnosis of allergies.

Adult↗
PubMed1994

Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus.

A 32-kDa nonglycosylated alkaline protease (EC 3.4.1.14) with elastolytic activity, secreted by the opportunistic pathogen Aspergillus fumigatus ATCC 42202, is suggested to be a virulence factor of this fungus. The enzyme is a serine protease of the subtilisin family, and its cDNA nucleotide sequence has recently been reported. We have cloned the cDNA encoding the mature protease into a high-level Escherichia coli expression plasmid and produced the recombinant protease as a fusion protein with a six-adjacent-histidine affinity tag at the carboxy terminus. Subsequently, the recombinant protease was purified to homogeneity, with affinity chromatography yielding 30 to 40 mg of recombinant protease per liter of E. coli culture. Refolded recombinant protease, in comparison with native protease, demonstrated weak enzymatic activity but similar immunochemical characteristics as analyzed by antigen-specific enzyme-linked immunosorbent assay (ELISA), competition ELISA, and immunoblotting assays. To assess the allergenic potential of the protease, sera from patients with allergic bronchopulmonary aspergillosis and sera from healthy control individuals were analyzed by ELISA and immunoblotting techniques. Sera from patients with allergic bronchopulmonary aspergillosis did not have protease-specific immunoglobulin E (IgE) antibodies and, remarkably, did not show significantly elevated protease-specific IgG antibody levels compared with those in sera from healthy control individuals. This suggests that the alkaline protease from A. fumigatus does not elicit IgE antibodies and has weak immunogenicity, a property which may explain fungus persistence in allergic individuals.

Amino Acid Sequence↗
PubMed1993

Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production.

A cloning and expression system allowing display of functional cDNAs or other gene products on the surface of filamentous phage has been developed, exploiting the high-affinity interaction of the Jun and Fos leucine zippers. Gene jun was expressed from a lacZ promoter as a fusion protein with the viral coat protein, pIII, thereby being structurally incorporated into phage particles during infection with a helper phage. Using a second lacZ promoter of the phagemid, gene fos was co-expressed as an N-terminal fusion peptide to cDNA library gene products, so that the resulting Fos-fusion proteins could become associated with the Jun-decorated phage particles. To avoid interphage exchange of fos-cDNA fusion products, cysteines were engineered at the N- and C-termini of each of the leucine zippers, providing a covalent link of the cDNA gene product to the genetic instructions required for its production. Dissociation between phage and cDNA gene products was readily achieved using reducing agents. Phages displaying gene products covalently anchored on their surface via the modified leucine zippers can be selectively enriched 10(4)-10(6)-fold over nonspecific phages using antibodies. Thus, this cloning system allows rapid isolation of rare mRNA products from complex cDNA libraries by enrichment with appropriate ligands. This approach should allow the expression and cloning of dimeric proteins by cDNA shuffling.

Amino Acid Sequence↗
PubMed1993

Recombinant Aspergillus fumigatus allergen I/a (rAsp f I/a) in the diagnosis of Aspergillus related diseases.

A. fumigatus allergen I/a (rAsp f I/a) was produced in Escherichia coli, affinity purified by Ni(2+)-chelate chromatography and refolded. rAsp f I/a binds IgE of A. fumigatus-allergic individuals as shown by ELISA, dot blots and Western blots. In addition, rAsp I/a elicited positive immediate type I skin reactions in individuals allergic to A. fumigatus but not in control individuals. Hence, rAsp f I/a can be used as a standardized antigen/allergen for an improved serological and clinical diagnosis of A. fumigatus-associated diseases.

Allergens↗