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Reto Crameri

Publications and source records attributed to Reto Crameri.

At least 37 records · Page 2Linked to original sources
PubMed2005

The future of post-genomic biology at the proteomic level: an outlook.

Drug discovery and early-stage drugs and biomarkers development is a continuous adaptation and maturation process. The cycle of changes based on new findings is coupled with shifts in research priorities and make this part of pharmaceutical research a challenging endeavour. Over the last years, the emphasis on genomics has shifted to proteomics, the science of understanding how proteins translate gene information into function, and metabonomics, the science of small metabolites that are further apart from genomic projects. Proteomics describes the analysis of the protein complement of a biological sample with respect to temporal and spatial resolution. This technology is based on separation of complex protein mixtures by 2D gel-electrophoresis, in gel digest and mass spectrometric analysis of the protein fragments. Proteomics has been recently flanked by peptidomics, a new research direction aimed at the comprehensive analysis of small (1-20 kDa) polypeptides, thus covering the gap between proteomics and metabonomics. The refinement of peptidomics is based on an essential paradigm related to modularity and diversity. Peptides are a paramount example of how one single gene can release multiple functionalities. We can expect fast progress in understanding protein and peptide networks from a systems biology approach ending in the discovery of new peptide targets. However, the way from a complex sample to potential diagnostic and therapeutic targets will depend on technological developments and from the ability to discriminate true disease-related signals from false positive and negative signals, and the way from target discovery to target validation will not be short.

Aged↗
PubMed2005

Peptide sequence prediction supported by correlation-associated networks in human cerebrospinal fluid.

During the course of biosynthesis, processing and degradation of a peptide, many structurally related intermediate peptide products are generated. Human body fluids and tissues contain several thousand peptides that can be profiled by reversed-phase chromatography and subsequent MALDI-ToF-mass spectrometry. Correlation-Associated Peptide Networks (CAN) efficiently detect structural and biological relations of peptides, based on statistical analysis of peptide concentrations. We combined CAN with recognition of probable cleavage sites for peptidases and proteases in cerebrospinal fluid, resulting in a model able to predict the sequence of unknown peptides with high accuracy. On the basis of this approach, identification of peptide coordinates can be prioritized, and a rapid overview of the peptide content of a novel sample source can be obtained.

Animals↗
PubMed2005

Datamining methodology for LC-MALDI-MS based peptide profiling.

This report will provide a brief overview of the application of data mining in proteomic peptide profiling used for medical biomarker research. Mass spectrometry based profiling of peptides and proteins is frequently used to distinguish disease from non-disease groups and to monitor and predict drug effects. It has the promising potential to enter clinical laboratories as a general purpose diagnostic tool. Data mining methodologies support biomedical science to manage the vast data sets obtained from these instrumentations. Here we will review the typical workflow of peptide profiling, together with typical data mining methodology. Mass spectrometric experiments in peptidomics raise numerous questions in the fields of signal processing, statistics, experimental design and discriminant analysis.

Animals↗
PubMed2004

Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells.

The mechanisms by which immune responses to nonpathogenic environmental antigens lead to either allergy or nonharmful immunity are unknown. Single allergen-specific T cells constitute a very small fraction of the whole CD4+ T cell repertoire and can be isolated from the peripheral blood of humans according to their cytokine profile. Freshly purified interferon-gamma-, interleukin (IL)-4-, and IL-10-producing allergen-specific CD4+ T cells display characteristics of T helper cell (Th)1-, Th2-, and T regulatory (Tr)1-like cells, respectively. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals; in contrast, there is a high frequency of allergen-specific IL-4-secreting T cells in allergic individuals. Tr1 cells use multiple suppressive mechanisms, IL-10 and TGF-beta as secreted cytokines, and cytotoxic T lymphocyte antigen 4 and programmed death 1 as surface molecules. Healthy and allergic individuals exhibit all three allergen-specific subsets in different proportions, indicating that a change in the dominant subset may lead to allergy development or recovery. Accordingly, blocking the suppressor activity of Tr1 cells or increasing Th2 cell frequency enhances allergen-specific Th2 cell activation ex vivo. These results indicate that the balance between allergen-specific Tr1 cells and Th2 cells may be decisive in the development of allergy.

Adult↗
PubMed2004

Serological cloning of cancer/testis antigens expressed in prostate cancer using cDNA phage surface display.

Serological cloning of tumor-associated antigens (TAAs) using patient autoantibodies and tumor cDNA expression libraries (SEREX) has identified a wide array of tumor proteins eliciting B-cell responses in patients. However, alternative cloning strategies with the possibility of high throughput analysis of patient sera and tumor libraries may be of interest. We explored the pJuFo phage surface display system, allowing display of recombinant tumor proteins on the surface of M13 filamentous phage, for cloning of TAAs in prostate cancer (PC). Control experiments established that after a few rounds of selection on immobilized specific IgG, a high degree of enrichment of seroreactive clones was achieved. With an increasing number of selection rounds, a higher yield of positive clones was offset by an apparent loss of diversity in the repertoire of selected clones. Using autologous patient serum IgG in a combined biopanning and immunoscreening approach, we identified 13 different TAAs. Three of these (NY-ESO-1, Lage-1, and Xage-1) were known members of the cancer/testis family of TAAs, and one other protein had previously been isolated by SEREX in cancer types other than PC. Specific IgG responses against NY-ESO-1 were found in sera from 4/20 patients with hormone refractory PC, against Lage-1 in 3/20, and Xage-1 in 1/20. No reactivity against the remaining proteins was detected in other PC patients, and none of the TAAs reacted with serum from healthy subjects. The results demonstrate that phage surface display combined with postselection immunoscreening is suitable for cloning a diverse repertoire of TAAs from tumor tissue cDNA libraries. Furthermore, candidate TAAs for vaccine development of PC were identified.

Antigens, Neoplasm↗
PubMed2004

Cloning allergens via phage display.

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

Allergens↗
PubMed2004

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

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

Adult↗
PubMed2003

Correlating IgE reactivity with three-dimensional structure.

This Commentary discusses the work of Neudecker et al. in this issue of the Biochemical Journal in which site-directed mutagenesis and NMR spectroscopy have been used to analyse in detail the IgE-binding capacity of two cross-reactive allergens: Apg1.0101 from celery ( Apium graveolens ) and Pru av 1 from cherry ( Prunus avium ), which are both members of the pathogenesis-related allergen family. The study, showing that the IgE-binding epitopes are highly patient specific, will have a profound impact on our understanding of conformational IgE-binding epitopes, raising serious questions about the therapeutic usefulness of conventional site-directed-mutagenic approaches for the production of hypo-allergenic protein variants.

Allergens↗
PubMed2003

Allergic bronchopulmonary aspergillosis in cystic fibrosis--state of the art: Cystic Fibrosis Foundation Consensus Conference.

Because of the difficulties of recognizing allergic bronchopulmonary aspergillosis (ABPA) in the context of cystic fibrosis (because of overlapping clinical, radiographic, microbiologic, and immunologic features), advances in our understanding of the pathogenesis of allergic aspergillosis, new possibilities in therapy, and the need for agreed-upon definitions, an international consensus conference was convened. Areas addressed included fungal biology, immunopathogenesis, insights from animal models, diagnostic criteria, epidemiology, the use of new immunologic and genetic techniques in diagnosis, imaging modalities, pharmacology, and treatment approaches. Evidence from the existing literature was graded, and the consensus views were synthesized into this document and recirculated for affirmation. Virulence factors in Aspergillus that could aggravate these diseases, and particularly immunogenetic factors that could predispose persons to ABPA, were identified. New information has come from transgenic animals and recombinant fungal and host molecules. Diagnostic criteria that could provide a framework for monitoring were adopted, and helpful imaging features were identified. New possibilities in therapy produced plans for managing diverse clinical presentations.

Animals↗
PubMed2003

Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadin.

Coeliac disease (CD), a gastrointestinal illness characterized by intestinal malabsorption, results from gluten intolerance accompanied with immunological responses towards gliadin, an ethanol-soluble protein fraction of wheat and other cereals. The role of gliadin in eliciting immune responses in CD is still partly unclear; however, the occurrence of anti-gliadin in the sera of patients suffering from CD correlates well with clinical symptoms. In this work we report the construction of isotype-specific, phage-displayed scFv libraries from peripheral blood lymphocytes of a patient with CD and from a healthy control individual. VH and VL chains were amplified by reverse transcription-polymerase chain reaction (RT-PCR) using a set of oligonucleotides recognizing all human variable gene families. The three scFv libraries (IgA, IgG and IgM) were selectively enriched for gliadin-binding phage. After four rounds of affinity selection, polyclonal enrichment of gliadin-binding phage was observed in all libraries from the CD patient but in none from the healthy donor. Phagemid particles generated from single clones were demonstrated to be gliadin-specific, as shown by strongly positive enzyme-linked immunosorbent assay (ELISA) and BiaCore signals. The VH and VL chains from samples of these monoclonal isotype-specific phage were sequenced to identify the most common variable regions used by the immune system to elicit antibody responses against gliadin.

Amino Acid Sequence↗
PubMed2003

Native Art v 1 and recombinant Art v 1 are able to induce humoral and T cell-mediated in vitro and in vivo responses in mugwort allergy.

BACKGROUND: Mugwort pollen is an important allergen source in hay fever and pollen-related food allergy. Little is known about the clinical relevance of the major mugwort allergen Art v 1 and its importance in allergy. OBJECTIVE: In this study we aimed to investigate the allergenicity of mugwort extract compared with the allergenicity of native (n)Art v 1 and recombinant (r)Art v 1, one major allergen of mugwort, in vivo and in vitro. METHODS: Thirty-two patients allergic to mugwort and 10 control subjects were investigated by means of skin prick and nasal provocation testing with different concentrations of mugwort extract, nArt v 1, and rArt v 1. nArt v 1 was purified from aqueous mugwort extract, and rArt v 1 was cloned, expressed in Escherichia coli, and then purified. The in vitro allergenicity was measured by means of ImmunoCAP, ELISA, ELISA-inhibition experiments, and T-cell proliferation assays. RESULTS: nArt v 1 and rArt v 1 were able to elicit positive in vivo and in vitro reactions. The IgE-binding capacity, as determined by means of ELISA, was slightly higher for nArt v 1 than for rArt v 1, and both allergens were able to induce T-cell proliferation in sensitized patients. However, rArt v 1 elicited a reduced response in skin and nasal provocation tests compared with nArt v 1. Compared with mugwort extract, both nArt v 1 and rArt v 1 showed lower sensitivity in patients with mugwort allergy in vivo. CONCLUSIONS: Art v 1, either in its native or recombinant form, is able to induce allergic reactions in patients with mugwort allergy. rArt v 1 induced comparable humoral and cell-mediated responses in vitro but showed reduced in vivo allergenicity compared with biochemically purified nArt v 1.

Adult↗
PubMed2003

Conidial hydrophobins of Aspergillus fumigatus.

The surface of Aspergillus fumigatus conidia, the first structure recognized by the host immune system, is covered by rodlets. We report that this outer cell wall layer contains two hydrophobins, RodAp and RodBp, which are found as highly insoluble complexes. The RODA gene was previously characterized, and DeltarodA conidia do not display a rodlet layer (N. Thau, M. Monod, B. Crestani, C. Rolland, G. Tronchin, J. P. Latgé, and S. Paris, Infect. Immun. 62:4380-4388, 1994). The RODB gene was cloned and disrupted. RodBp was highly homologous to RodAp and different from DewAp of A. nidulans. DeltarodB conidia had a rodlet layer similar to that of the wild-type conidia. Therefore, unlike RodAp, RodBp is not required for rodlet formation. The surface of DeltarodA conidia is granular; in contrast, an amorphous layer is present at the surface of the conidia of the DeltarodA DeltarodB double mutant. These data show that RodBp plays a role in the structure of the conidial cell wall. Moreover, rodletless mutants are more sensitive to killing by alveolar macrophages, suggesting that RodAp or the rodlet structure is involved in the resistance to host cells.

Amino Acid Sequence↗
PubMed2003

Association between the MHC gene region and variation of serum IgE levels against specific mould allergens in the horse.

To investigate whether the equine major histocompatibility complex (MHC) gene region influences the production of mould-specific immunoglobulin E antibodies (IgE), alleles of the equine leukocyte antigen (ELA-A) locus and three microsatellite markers (UM-011, HTG-05 and HMS-42) located on the same chromosome as the equine MHC were determined in 448 Lipizzan horses. Statistical analyses based on composite models, showed significant associations of the ELA-A and UM-011 loci with IgE titres against the recombinant Aspergillus fumigatus 7 antigen (rAsp f 7). UM-011 was also significantly associated with IgE titres against the recombinant Aspergillus fumigatus 8 antigen (rAsp f 8). In addition to the loci mentioned above, the MHC class II DQA and DRA loci were determined in 76 Lipizzans from one stud. For IgE levels against rAsp f 7, the composite model showed the strongest association for DQA ([Formula: see text]P) while for rAsp f 8 specific IgE levels, similarly to the results found with all 448 horses, the strongest association was found with UM-011 (P=0.01), which is closely linked with the MHC class II DRB locus. These results suggest that the equine MHC gene region and possibly MHC class II loci, influence the specific IgE response in the horse. However, although the strongest associations were found with DQA and UM-011, this study did not distinguish if the observed effects were due to the MHC itself or to other tightly linked genes.

Allergens↗
PubMed2003

IL-10 controls Aspergillus fumigatus- and Pseudomonas aeruginosa-specific T-cell response in cystic fibrosis.

Up to 90% of patients with cystic fibrosis (CF) are chronically colonized with Pseudomonas aeruginosa, and 10% to 50% of CF patients are colonized with Aspergillus fumigatus. Despite an extensive inflammatory reaction, patients cannot eliminate the microorganisms. The present study demonstrates that an IL-10 mediated T-cell tolerance to major infectious agents A. fumigatus and P. aeruginosa plays an important role in the control of T-cell-mediated inflammatory responses in CF. Peripheral blood mononuclear cells of CF patients secreted significantly higher amounts of IL-10. T-cell response against recombinant A. fumigatus antigens rAsp f 3, rAsp f 4, rAsp f 6, and heat-inactivated P. aeruginosa was controlled by IL-10. Proliferation and interferon-gamma production was significantly increased when endogenous IL-10 was blocked in aspergillus and pseudomonas antigen-stimulated cells of CF patients. The role of IL-10 was further documented by increased spontaneous proliferation of peripheral blood mononuclear cells of CF patients after preincubation with antisense oligonucleotides blocking the synthesis of IL-10 receptor-associated kinases janus tyrosine kinase 1 and tyrosine kinase 2. Together, these data demonstrate an important role of IL-10-mediated peripheral T-cell tolerance to P. aeruginosa and A. fumigatus in the control of the intensity of the inflammatory T-cell response in CF.

Adolescent↗
PubMed2003

Rapid identification of allergen-encoding cDNA clones by phage display and high-density arrays.

We describe a high-throughput, quantitative technology for fast identification of all different clones present in selectively enriched phage surface-displayed cDNA libraries. The strategy is based on a combination of phage display and high-density arrays. To demonstrate the utility of the method cDNAs of Aspergillus fumigatus cloned into phagemid pJuFo were expressed on the tip of filamentous M13 phage and affinity-selected on solid phase-immobilized serum IgE from allergic patients. Enriched phagemid libraries were amplified in bacteria, plated and arrayed into 384-well microtitre plates by robotic colony picking. cDNA inserts were amplified by high-throughput PCR and gridded onto high-density filter membranes. Filters were iteratively probed with randomly-sequenced inserts until all clones were identified. Eighty-one different sequences encoding IgE-binding proteins likely to cover a large part of the allergen repertoire of the mould were found. This approach represents a widely applicable method for rapid high-throughput identification of all individual cDNAs present in selectively enriched libraries.

Allergens↗
PubMed2002

Intracutaneous tests with recombinant allergens in cystic fibrosis patients with allergic bronchopulmonary aspergillosis and Aspergillus allergy.

Allergic bronchopulmonary aspergillosis (ABPA), an intensive inflammatory reaction to Aspergillus fumigatus, can cause irreversible lung damage in patients with cystic fibrosis (CF). The aim of this study was to assess if intracutaneous testing with recombinant A. fumigatus allergens (rAsp f ) allowed a reliable diagnosis of ABPA. Fifty patients with CF were tested, 12 suffering from ABPA, 21 with allergy to A. fumigatus, and 17 CF control patients not sensitized to A. fumigatus. All patients with ABPA reacted to at least one of the two intracellular A. fumigatus allergens rAsp f 4, a 30-kD protein of unknown biologic function, and rAsp f 6, a 23-kD manganese superoxide dismutase, at a concentration of 10(-2) microg/ml. The intracutaneous tests were negative or only marginally positive in the patients with allergy to A. fumigatus and completely negative in the CF control patients. The differential responses to the recombinant A. fumigatus allergens were in perfect agreement with our previous serologic results, so that rAsp f 4 and rAsp f 6 can be considered specific markers for ABPA. Early diagnosis of the disease might help to prevent irreversible lung damage and minimize possible steroid-mediated side effects as a consequence of an optimized control of the disease.

Adolescent↗