PubMed Health⌕ Search

Biomedical subjects

R Crameri

Publications and source records attributed to R Crameri.

At least 19 recordsLinked to original sources
PubMed2006

Screening the allergenic repertoires of wheat and maize with sera from double-blind, placebo-controlled food challenge positive patients.

BACKGROUND: Food allergy to wheat and maize is an increasing factor of deterioration of life quality, especially childhood and can, in rare cases, even induce anaphylaxis. Although omega-5 gliadin from wheat and maize lipid transfer protein have been characterized as major cereal allergens on the molecular level, the list of food allergens is far to be complete. METHODS: To identify the IgE-binding repertoires of wheat and maize we screened respective cDNA libraries displayed on phage surface with sera from patients with a confirmed food allergy. The study included six patients with a positive double-blind, placebo-controlled food challenge (DBPCFC) to wheat, nine patients with a positive DBPCFC to maize, and six patients with anaphylactic reactions after ingestion of wheat. RESULTS: The enriched sequences encoding IgE-binding proteins showed heterogeneous repertoires for both, wheat and maize. The selected wheat repertoire yielded 12, the maize repertoire 11 open reading frames. Among these we identified allergens belonging to already characterized allergens families, such as gliadin, profilin and beta-expansin. Besides, we found novel proteins with high cross-reactive potential, such as thioredoxins, as well as sequences that had so far not been related to cereal allergy at all. The IgE-binding capacity of some selected proteins was evaluated in vitro and cross-reactivity was demonstrated by competition ELISA. CONCLUSION: With regard to the heterogeneity of the characterized sequences as well as to the biochemical nature of the new allergens detected we conclude that wheat and maize-related food allergy is more complex than so far anticipated.

Adolescent↗
PubMed2006

Factors responsible for differences between asymptomatic subjects and patients presenting an IgE sensitization to allergens. A GA2LEN project.

The synthesis of allergen-specific IgE is required for the development of allergic diseases including allergic rhinitis and allergic asthma (patients), but many individuals with allergen-specific IgE do not develop symptoms (asymptomatic subjects). Differences may exist between asymptomatic subjects and patients. Whether the presence of allergen-specific IgE translates into clinical allergy most likely depends on a complex interplay of multiple factors. These include a family history of atopy, the levels of total serum IgE and, allergen-specific IgE or IgG, epitope-specificity of IgE and their degree of polyclonality (mono- vs polysensitized), as yet unidentified serum factors, the balance of T regulatory cells (Treg) and Th1/Th2 cells, the polymorphisms of the high affinity receptor for IgE (FcepsilonRI) and other factors regulating the activation of FcepsilonRI-bearing cells. Asymptomatic subjects may be more often monosensitized than patients who may be more often polysensitized. There are many unanswered important questions that need to be addressed in order to better understand how IgE sensitization translates into clinical allergy. The assessment of differences between the asymptomatic and symptomatic groups of subjects represent one of the scientific programs of Global Allergy and Asthma European Network funded by the European Union and the hypotheses underlying these differences are presented in this paper.

Allergens↗
PubMed2005

Molecular and cellular targets of anti-IgE antibodies.

Immunoglobulin E (IgE) was the last of the immunoglobulins discovered. It is present in very low amounts (nano- to micro-gram per ml range) in the serum of normal healthy individuals and normal laboratory mouse strains and has a very short half-life. This contrasts with the other immunoglobulin classes, which are present in much higher concentrations (micro- to milligram per ml range) and form a substantial component of serum proteins. Immunoglobulins play a role in homeostatic mechanisms and they represent the humoral arm of defence against pathogenic organisms. Since IgE antibodies play a key role in allergic disorders, a number of approaches to inhibit IgE antibody production are currently being explored. In the recent past the use of nonanaphylactic, humanized anti-IgE antibodies became a new therapeutic strategy for allergic diseases. The therapeutic rational beyond the idea derives from the ability of the anti-IgE antibodies to bind to the same domains on the IgE molecule that interact with the high-affinity IgE receptor, thereby interfering with the binding of IgE to this receptor without cross-linking the IgE on the receptor (nonanaphylactic anti-IgE antibodies). Treatment with anti-IgE antibodies leads primarily to a decrease in serum IgE levels. As a consequence thereof, the number of high-affinity IgE receptors on mast cells and basophils decreases, leading to a lower excitability of the effector cells reducing the release of inflammatory mediator such as histamine, prostaglandins and leukotrienes. Experimental studies in mice indicate that injection of some monoclonal anti-IgE antibodies also inhibited IgE production in vivo. The biological mechanism behind this reduction remains speculative. A possible explanation may be that these antibodies can also interact with membrane bound IgE on B cells, which could interfere the IgE production.

Antibodies, Anti-Idiotypic↗
PubMed2005

The potential of proteomics and peptidomics for allergy and asthma research.

Progress in the field of proteomics, the branch of biology that studies the full set of proteins derived from a given genome, is moving fast. Two-dimensional gel electrophoresis (2DG) separation of complex protein mixtures and the subsequent analysis of isolated protein spots by mass spectrometry allow fast and accurate identification of proteins. The comparison of spots from different samples separated on customized 2D gels allows the detection of punctual differences in their mobility and facilitates tracing back differences in protein expression, presence of isoforms, splice variants and posttranslational modifications by mass spectrometry. In spite of significant analytical challenges owing to the high complexity of the proteome and the challenge deriving from the necessity to process huge amounts of raw data generated by mass spectrometric profiling, proteomics has evolved to an indispensable tool in life sciences. A restricted window of the proteome that consists of peptides and small proteins not easily manageable by conventional gel electrophoresis prompted the development of separation methods based on liquid chromatography. This new research field termed peptidomics already contributed, together with proteomics to enlarge our knowledge about biological processes and supported by sophisticated bioinformatics tools, to the discovery of new diagnostic and therapeutic targets. The technological capabilities of biophysical separation, mass spectrometry and bioinformatics form the basis of discovery programs that aim at mining the proteome starting from microgram amounts of protein extracts derived from body fluids and tissues. Proteomics and peptidomics have a great potential to speed up allergy and asthma research, where disease- and tissue-specific samples are easy to obtain.

Asthma↗
PubMed2005

Metabolic activity and collagen turnover in human tendon in response to physical activity.

Connective tissue of the human tendon plays an important role in force transmission. The extracellular matrix turnover of tendon is influenced by physical activity. Blood flow, oxygen demand, and the level of collagen synthesis and matrix metalloproteinases increase with mechanical loading. Gene transcription and especially post-translational modifications of proteins of the extracellular matrix are enhanced following exercise. Conversely, inactivity markedly decreases collagen turnover. Training leads to a chronically increased collagen turnover, and dependent on the type of collagen also to some degree of net collagen synthesis. These changes modify the biomechanical properties of the tissue (for example, viscoelastic characteristics) as well as the structural properties of the in collagen (for example, cross-sectional area). Mechanical loading of human tendon does result in a marked interstitial increase in growth factors that are known potentially to stimulate synthesis of collagen and other extracellular matrix proteins. Taken together, human tendon tissue mounts a vigorous acute and chronic response to mechanical loading in terms of metabolic-circulatory changes as well as of extracellular matrix formation. These changes may contribute to training-induced adaptation of biomechanical properties consisting of altered resistance to loading and enhanced tolerance to strenuous exercise. Understanding of such changes is a pre-requisite in the development of measures aimed at prevention of overuse tendon injuries occurring during sport, work or leisure-related activities.

Adaptation, Physiological↗
PubMed2003

Nuclear transport factor 2 represents a novel cross-reactive fungal allergen.

BACKGROUND: Ubiquitously occuring moulds are important allergenic sources known to elicit IgE-mediated allergic diseases and to share cross-reactive allergens. Limited information is available about the molecular structures involved in cross-reactivity. We aimed to clone and characterize cross-reactive mould allergens. METHODS: Phage surface-displayed Alternaria alternata and Cladosporium herbarum cDNA libraries were screened using sera from Aspergillus fumigatus-sensitized patients. Inserts encoding putative allergens were sequenced, and recombinant proteins used to demonstrate cross-reactivity by inhibition experiments and skin test. Three-dimensional homology models of cloned putative nuclear transport factor 2 (NTF2) were constructed based on known NTF2 structure to corroborate the functional and structural properties of the novel allergens. RESULTS: After six rounds of affinity selection, the libraries were enriched for clones displaying allergens. Sequencing of inserts showed that some clones derived from Alternaria alternata and Cladosporium herbarum contain open reading frames predicting proteins of 124 and 125 amino acids corresponding to NTF2. The recombinant proteins were able to bind and cross-inhibit IgE binding and to elicit type I skin reactions in mould-sensitized individuals, demonstrating the allergenicity of the proteins. CONCLUSIONS: NTF2 represents a novel cross-reactive fungal allergen as demonstrated by sequence homology, three-dimensional modelling, inhibition experiments and skin test reactivity.

Allergens↗
PubMed2003

Immunoglobulin E-binding and skin test reactivity to hydrophobin HCh-1 from Cladosporium herbarum, the first allergenic cell wall component of fungi.

BACKGROUND: For many years, fungal spores have been recognized as potential causes of respiratory allergies. All fungal allergens cloned so far represent either secreted or cytoplasmatic proteins, but nothing is known about the involvement of fungal surface proteins in allergic diseases. METHODS: A phage surface displayed cDNA-library from the mould Cladosporium herbarum was constructed and phage displaying IgE-binding proteins were selectively enriched with immobilized serum IgE from C. herbarum-sensitized individuals. Inserts encoding putative allergens were sequenced, subcloned and used to produce recombinant proteins. Allergenicity of the proteins was evaluated by IgE binding in Western blots, enzyme-linked immunosorbent assay (ELISA) and skin prick test in a total of 84 patients sensitized to either C. herbarum or Aspergillus fumigatus and three healthy controls. RESULTS: After four rounds of affinity selection, the cDNA-library was enriched for clones displaying IgE-binding molecules. Sequencing of inserts showed that one clone contained an open reading frame predicting a protein of 105 amino acids and a calculated molecular weight of 10.5 kDa showing the classical signature of members of the hydrophobin family. The recombinant protein, termed HCh-1, was able to bind IgE from six patients sensitized to fungi in vitro. Two of those patients were also included in a skin prick test survey and showed strong type I skin reactions to HCh-1, demonstrating the allergenic nature of C. herbarum hydrophobin and indicating a prevalence of sensitization in the range of 8-9%. In contrast, the hydrophobin HYP1 from Aspergillus fumigatus was not recognized by the sera of the same patients and controls investigated with HCh-1. CONCLUSION: C. herbarum hydrophobin represents the first component of the cell wall of fungi demonstrated to act as a rare but clinically relevant allergen in vitro and in vivo.

Allergens↗
PubMed2003

In vitro and in vivo allergenicity of recombinant Bet v 1 compared to the reactivity of natural birch pollen extract.

BACKGROUND: Diagnostic procedures using natural extracts show only limited quantitative correlation between in vivo and in vitro results. Highly pure recombinant allergens might show more predictive findings. OBJECTIVE: The aim of this study was to compare natural birch pollen extract (BPE) and recombinant Betula verrucosa (rBet v 1) for their diagnostic value comparing skin prick tests (SPTS) and nasal provocation tests (NPTS) with specific IgE in the serum. METHODS: Thirty-four patients allergic to birch pollen and five healthy controls were investigated. SPT and NPT were performed with BPE and rBet v 1 at different concentrations. Specific serum IgE was measured by the Pharmacia CAP system. RESULTS: Commercial BPE and rBet v 1 (10 micro g/mL) were able to elicit similar allergenic reactions in vivo and IgE binding in vitro. SPT reflects immediate-type allergy as determined by NPT to a higher degree than specific IgE, for both reagents. To cause allergic reactions in NPT, higher amounts of rBet v 1 were needed than for skin tests and the sensitivity was lower than with BPE. CONCLUSION: rBet v 1 alone is sufficient for a reliable diagnosis of birch pollen allergy in most patients and induces comparable skin test reactivity as BPE, but less allergic reactions in nasal provocations.

Adult↗
PubMed2002

Allergy and immunity to fungal infections and colonization.

Innate and cell-mediated immunity are considered as the principal defence lines against fungal infections in humans. Most opportunistic mycoses occur in individuals with defective innate and/or adaptive cellular immunity. The morbidity and mortality rates associated with infections caused by fungal pathogens are high, and prevention, diagnosis and treatment of these infections remain quite difficult. A variety of pathological conditions, including impaired immune function, are believed to cause host susceptibility to fungal infections as well as to determine the severity and characteristics of the associated pathology. Nonspecific cellular immunity, mediated by macrophages, neutrophils and natural killer cells, provides efficient protection against fungal infections in healthy individuals. A major reason for the increase in systemic mycoses is undoubtedly related to an increased number of patients with congenital or acquired immunodeficiencies. However, there is increasing clinical and experimental evidence indicating that antigen-specific cellular immunity may also play a critical role in host protection against fungi. A better understanding of reciprocal regulation between innate, humoral and adaptive immune responses in the development of an optimal antifungal immunity and, in particular, the improved definition of fungal antigens, may lead to a clarification of the mechanisms involved in host immunity to fungal infections. Molecular cloning and characterization of fungal antigens reveals the involvement of related cross-reactive molecular structures produced by different fungi as pathological molecules involved in development of allergic reactions.

Antibody Formation↗
PubMed2001

Patient-tailored cloning of allergens by phage display: peanut (Arachis hypogaea) profilin, a food allergen derived from a rare mRNA.

A peanut cDNA phage surface display library was constructed and screened for the presence of IgE-binding proteins. We used a serum from a peanut-sensitized individual with a low specific IgE level to peanut extract and suffering from mild symptoms after peanut ingestion. A total of 10(11) cDNA clones were screened by affinity selection towards serum IgE immobilized to solid-phase supports. After five rounds of selective enrichment, sequence determination of 25 inserts derived from different clones revealed presence of a single cDNA species. The cDNA-encoded gene product, formally termed Ara h 5, shows up to 80% amino acid sequence identity to the well-known plant allergen profilin, a 14 kD protein present only in low amount in peanut extracts. Immunoblot analysis of fifty sera from individuals sensitized to peanut showed that 16% had mounted a detectable IgE response to the newly identified peanut profilin. High-level expression as non-fusion protein in BL21 (DE3) was carried under control of the inducible T7 promoter. Peanut profilin was purified by affinity chromatography on poly-(L-proline)-Sepharose and yielded 30 mg l(-1) culture of highly pure recombinant allergen. In spite of the high level of up to 80% amino acid identity to other plant profilins, inhibition experiments with recombinant profilins of peanut, cherry, pear, celery and birch revealed marked differences regarding their IgE-binding capacity.

Allergens↗
PubMed2001

Somatic diversity of the immunoglobulin repertoire is controlled in an isotype-specific manner.

We have studied two aspects of the IgE immune response. First, we have compared the kinetics of the IgE response to the T cell-dependent antigen ph-Ox coupled to ovalbumin with that of the IgG1 response and we have assessed the quality of the IgE response. Second, we have studied the generation of somatic diversity, understood as the combined effect of somatic mutation and the selection of D(iversity) and J(oining) elements, in germinal center B cells at the molecular level, using the germ-line sequence of the prototype anti-ph-Ox heavy chain variable element V(H)Ox1 as reference. We evaluated sequences derived from mu-, gamma 1- and epsilon-variable elements and showed that somatic diversification was different for all isotypes studied. We further compared the IgE responses of wild-type mice with those of mice expressing a truncated cytoplasmic IgE tail (IgE(KVK Delta tail)). IgE(KVK Delta tail) mice showed a more diverse sequence pattern. We corroborated previous results suggesting that short CDR3 regions are indicative for high-affinity antibodies by measuring relative affinities of phage-expressed Fab fragments with prototype long and short CDR3 regions. Therefore, the composition of the antigen-receptor is responsible for the selection process and the expansion of antigen-specific cells, leading to an isotype-specific antibody repertoire.

Amino Acid Sequence↗
PubMed2001

Purified recombinant A. fumigatus allergens induce different responses in mice.

Aspergillus fumigatus an opportunistic fungus is associated with a number of diseases in humans. Allergy resulting from exposure to the A. fumigatus allergens has been recognized frequently. The damage caused by the disease is very striking in patients with atopy and those with cystic fibrosis. Avoidance to exposure is not feasible because A. fumigatus spores are ubiquitously distributed in the environment. Hence, immunotherapeutic regimens in severe forms of A. fumigatus allergy may have a high potential. However, before such forms of therapy can be envisaged, it is essential to understand the immunopathogenesis. In the present study, we investigated the role of purified A. fumigatus allergens in the development of allergic asthma in mice. We have used four major recombinant A. fumigatus allergens in the murine model. Mice exposed to Asp f 1, f 3, and f 4 showed inflammatory changes in the lungs and airway hyperreactivity. The immune responses, including elevated serum IgE, enhanced eosinophils, recruitment in the peripheral blood and lungs, and expression of regulatory cytokines, are characteristic of a Th2 response. Asp f 6 demonstrated only a reduced response in these animals. The results suggest that the pathology induced by crude A. fumigatus extract results from the cumulative effects of the allergens and the individual responses varied considerably with different purified antigens.

Airway Resistance↗
PubMed2001

High throughput screening: a rapid way to recombinant allergens.

Complex allergenic sources such as moulds, foods and mites contain large panels of IgE-binding molecules which need to be cloned, produced and characterized in order to mimic the entire allergenicity of whole extracts reconstituted by mixing single standardized recombinant allergens. Phage display of cDNA libraries allows selective enrichment of allergen-expressing clones using IgE from allergic patients. For the characterization of all different clones present in enriched cDNA libraries in a fast and cost-effective way, however, high-throughput screening technology is required. We have used a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries in order to characterize whole allergenic repertoires from complex allergenic sources. The strategy, based on a combination of phage display and high-density arrays, allowed fast discovery of panels of related structures from different allergenic sources. They cover secreted, cytoplasmic and structural proteins with or without enzymatic activity and offer a rational explanation for the IgE-mediated cross-reactivity frequently encountered in clinical practice.

Allergens↗
PubMed2001

Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology.

The dust mite Lepidoglyphus destructor is a common species in Europe and a major cause of dust mite allergy in rural surroundings, but it also contributes to dust mite allergy in urban areas. One major allergen, Lep d 2, has been expressed as a recombinant protein and evaluated both in vivo and in vitro and shown to detect 60% or more of L. destructor-sensitized subjects. Additional recombinant allergens are needed to obtain a reliable diagnostic tool for L. destructor allergy. The aim of this study was to clone and express new allergens from L. destructor and determine their recognition frequency among sensitized individuals. A phage display cDNA expression library was constructed and screened with sera from L. destructor-sensitized individuals. The cDNAs encoding the allergens were cloned into the pET17b vector and subsequently expressed in Escherichia coli as C-terminal His6-tagged proteins. Immunoblotting of the recombinant proteins was performed using sera from 45 subjects allergic to L. destructor. Three new allergens from L. destructor, Ld 5 (originating from a partial Lep d 5 clone), Lep d 7 and Lep d 13, were identified and recognized by 4/45 (9%), 28/45 (62%) and 6/45 (13%) sera from L. destructor-sensitized subjects, respectively.

Allergens↗