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Biomedical subjects

F Ferreira

Publications and source records attributed to F Ferreira.

At least 73 records · Page 4Linked to original sources
PubMed1997

Genomic characterization of members of the Bet v 1 family: genes coding for allergens and pathogenesis-related proteins share intron positions.

Bet v 1, the major birch pollen allergen, is a member of a multigene family; a number of isoforms and homologous proteins from closely related species (alder, hazel and hornbeam) has been isolated and their cDNAs cloned and characterized. Genomic clones coding for Bet v 1 and homologues from apple and hazel were isolated and sequenced. Some of these clones contained intervening sequences. The exon-intron formation is highly conserved throughout this family of pathogenesis-related proteins in dicot plants and is also found in Aopr1 (Asparagus officinalis), a monocol species. Phylogenetic analysis suggested a possible common origin of the intron position in these homologous proteins at codon 62 in various families of flowering plants, including Fagaceae, Rosaceae and Apiaceae. This conserved 'proto-splice site' may point to a structure/function relationship. A conserved sequence motif (P-loop) was also found in all members of this protein family. Moreover, there is a certain degree of sequence similarity among the proteins derived from various species throughout the dicots and the only monocot examined. This fact is reflected by cross-reactivity from monoclonal and polyclonal antibodies raised against Bet v 1.

Allergens↗
PubMed1997

Structural studies of the extracellular polysaccharide from Butyrivibrio fibrisolvens strain CF3.

The structure of the Butyrivibrio fibrisolvens strain CF3 capsular polysaccharide has been investigated mainly by sugar and methylation analyses, Smith degradation, NMR spectroscopy, and mass spectrometry. The results indicate that the polysaccharide is composed of pentasaccharide repeating units having the following structure: -->4)-beta-L-Altp-(1-->4)-beta-D-Glcp-(1-->3)-4-O-[(R)-1-carboxyet hyl]-beta- D-Glcp-(1-->4)-6-O-[(R)-1-carboxyethyl]-alpha-D-Galp-(1--> 2 increases 1 beta-D-Glcp.

Bacteroidaceae↗
PubMed1997

Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen.

A cDNA coding for a newly identified Bermuda grass pollen allergen, Cyn d 7, with significant sequence similarity to Ca2+-binding proteins, was isolated from a cDNA expression library using serum IgE from an allergic individual. The deduced amino acid sequence of Cyn d 7 contained two typical Ca2+-binding sites (EF hand domains). Depletion of Ca2+ with EGTA led to a loss of IgE-binding capacity of rCyn d 7. A synthetic peptide based on domain II showed high IgE reactivity. Cyn d 7 therefore represents a grass pollen allergen that belongs to a novel class of Ca2+-binding proteins.

Allergens↗
PubMed1997

High-level expression and purification of the major birch pollen allergen, Bet v 1.

Bet v 1, the single major allergen from birch pollen, shares IgE epitopes with all major tree pollen allergens from closely related species such as alder, hazel, hornbeam, beech, and European chestnut. Because of high sequence homologies among these allergens and the well-studied cross-reactivities on B cell epitopes, Bet v 1 is a representative model protein which can be used for in vitro studies. The cDNA coding for Bet v 1, the single major allergen from birch pollen, was cloned into the T7-based Escherichia coli expression system pMW 175/BL21(DE3) and synthesized as a nonfusion protein. In contrast to other E. coli systems (e.g., pKK233-2/JM105), this system produces high levels of readily extractable proteins corresponding to 5-10% of E. coli total protein, the percentage varying with culture conditions. The overall yield was 8-10 mg of purified recombinant protein per liter of culture medium. The recombinant allergen was purified by several steps, including ion-exchange and hydrophobic interaction chromatography. The purified recombinant allergen showed identical immunological properties with the respective natural counterpart. The use of recombinant allergens of high purity is expected to result in more accurate diagnostic procedures, but possibly also in a superior immunotherapy of Type I allergic diseases when compared with methods using crude allergen extracts containing various amounts of allergen concentrations.

Allergens↗
PubMed1997

A collaborative approach to the diagnosis of a lethal short limb skeletal dysplasia.

We report a male infant with a lethal short limb Skeletal Dysplasia, born in a District Hospital in the South of Portugal. Local paediatricians investigated clinical and radiographical data. At the Perinatal Pathology Unit of the Egas Moniz hospital the diagnosis of Atelosteogenesis type II was proposed by the Clinical Geneticist and was supported by histopathological findings. Only a collaborative approach turned possible the diagnosis of this unusual entity. Atelosteogenesis type II and Diastrophic Dysplasia are closely related diseases, with similarities in phenotypic and histopathological presentation. Recently, these similarities were extended to molecular levels. The DNA analysis, in progress, will be able to establish a final diagnosis for this affected family.

Abnormalities, Multiple↗
PubMed1997

Allergic contact dermatitis caused by Lithraea molleoides and Lithraea brasiliensis: identification and characterization of the responsible allergens.

BACKGROUND: Allergic contact dermatitis caused by species of Lithraea genus (Anacardiaceae) is frequent in South America. Nevertheless, it has been scarcely reported in the literature, hitherto the responsible allergens have not been studied in some species. OBJECTIVE: The purpose of this study was to identify and characterize the allergenic compounds of Lithraea molleoides and brasiliensis, and to investigate the existence of cross-reactions with Toxicodendron allergens. METHODS: Twenty-seven South American subjects (17 with previous Lithraea dermatitis and 10 controls without any plant dermatitis) and four North American subjects who are highly sensitive to poison oak were tested with both purified Lithraea molleoides and brasiliensis extracts and poison oak urushiol. Lithraea extracts were analyzed by gas liquid chromatography (GLC) and gas chromatography-mass spectrometry (GC-MS). RESULTS: All 17 Lithraea-sensitive subjects reacted to poison oak urushiol and 13 of them also reacted to Lithraea molleoides and/or brasiliensis extracts. All 4 poison oak sensitive subjects reacted to poison oak urushiol and to Lithraea extracts. In both groups of sensitive subjects, the responses to poison oak urushiol were stronger and occurred at lower concentration than those to Lithraea extracts. The allergenic fraction in both Lithraea species consisted of: 3-pentadecylcatechol, 3-pentadecenylcatechol, 3-heptadecenylcatechol and 3-hepta-dec-dienilcatechol. CONCLUSION: We concluded that Lithraea molleoides and brasiliensis allergens are closely related to urushiol, although their eliciting potential seems to be lower in comparison with poison oak urushiol, even for Lithraea-sensitive subjects.

Adolescent↗
PubMed1996

Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy.

We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE-binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen-allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell-recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects.

Allergens↗
PubMed1996

Biological and immunological importance of Bet v 1 isoforms.

In 2D-PAGE analysis of Bet v 1, the major birch pollen allergen, up to 12 isoforms can be demonstrated that differ in their isoelectric points from about pH 4.9 to pH 5.9. The molecular weights of these isoforms seem to be rather similar, but minor variations can also be seen. Preliminary experiments with birch leaves seem to indicate that in aging leaves some isoforms can be found that do not occur in pollen. In birch cells cultured in vitro, Bet v 1 isoforms can be induced by bacterial infection that do not occur in pollen (Swoboda et al. (1995), Pant, Cell and Environment 18, 865-874). In a recent paper (Swoboda et al (1995)., J. Biol. Chem. 270, 2607-2613) we show that in natural Bet v 1 from pollen the isoforms are due to different protein sequences. The derived protein sequences of 10 different isoforms (corresponding to 13 different cDNAs) were determined and confirmed by plasma desorption mass spectrometry of purified natural Bet v 1 after trypsin and endoproteinase Glu-C digestion. These experiments also showed that pollen Bet v 1 isoforms were reactive to patients' sera to different degrees and that common post-synthetic modifications (besides N-terminal methionine cleavage) did not occur on Bet v 1. Recombinant isoforms were produced in E. coli, purified and tested with selected patients allergic to birch pollen (Ferreira et al., J. Exp. Med., in the press). The pattern of IgE binding to Bet v 1 isoforms widely differs. Also, T-cell clones from individual patients in some cases are specific to peptides occurring only in certain isoforms. It was of particular interest that three of the naturally occurring pollen Bet v 1 isoforms do not or hardly bind IgE of untreated patients allergic to Bet v 1. However, a comparison of IgE reactivity in patients before and after conventional immunotherapy with natural pollen extract clearly showed that this form of immunotherapy induced IgE to the isoforms that had been unreactive in untreated patients. One of these, Bet v 1d, showed a particularly strong potency towards T-cell stimulation. The isoform(s) that do not bind IgE in untreated patients but still show T-cell reactivity could be potentially utilized for a new form of immunotherapy that avoids the risk of anaphylaxis.

Allergens↗
PubMed1996

Exploring the use of prosody during language comprehension using the auditory moving window technique.

Researchers in psycholinguistics have speculated about the possible role of prosody in resolving syntactic ambiguities. We argue in this paper that the issue is complicated by the following considerations: first, prosody may be even more effective at conveying semantic information than syntactic structure, yet the question how prosody signals meaning is essentially unstudied. Second, the one-to-many relation between syntactic and prosodic structure leads to a great deal of variability across speakers and contexts in the way a given sentence will be produced. The parser must somehow deal with this variability. Third, resolution of architectural debates in the parsing literature requires the use of sensitive, online techniques for measuring processing load during comprehension. In the auditory domain, no optimal technique is presently available. We discuss the strengths and weaknesses of a technique we introduced in previous work, which is an analogue of the visual moving window. We present the results of an experiment demonstrating that the technique preserves some aspects of the prosody of a spoken sentence but disrupts others, and we discuss ways of dealing with this problem. We conclude that the technique is useful for studying language processing, including the use of prosody during parsing. However, we also argue that researchers should study not just the role of prosody in parsing, but also its role in establishing sentence meaning.

Cognition↗
PubMed1996

Steroid saponins from Solanum laxum.

Two new steroid saponins, named laxumins A and B, were isolated from the ethanolic extract of the aerial parts of Solanum laxum. These compounds were characterized, using mainly NMR spectroscopy, mass spectrometry and chemical methods, as (23S,25S)-spirost-5-en-3 beta, 15 alpha, 23-triol 3-O-{beta-D-glucopyranosyl-(1-->2)- beta-D-glucopyranosyl-(1-->4)-[alpha-L-rhamnopyranosyl-(1-->2)]-beta-D- galactopyranoside} and 3-O-{beta-D-glucopyranosyl-(1--> 4)-alpha-L-rhamnopyranosyl-(1-->2)]-beta-D-galactopyranoside}, respectively.

Carbohydrate Conformation↗
PubMed1996

Skin testing with recombinant allergens rBet v 1 and birch profilin, rBet v 2: diagnostic value for birch pollen and associated allergies.

OBJECTIVE: This study assesses the value of two recombinant birch allergens for diagnosis of patients sensitized to birch pollen with or without associated food allergy. METHODS: Fifty-one patients with positive skin test responses to Betulaceae and seven nonallergic control subjects were investigated; specific IgE antibodies were evaluated by specific immunoassay and blot immunodetection. RESULTS: Among 51 patients, 47 reacted to rBet v 1 and 10 to rBet v 2. Seven patients reacted to both recombinant allergens. In skin prick tests we found a correlation between the wheal produced by the commercial birch extract and the wheal produced by rBet v 1. Among 47 patients with positive test responses to rBet v 1, 83% had IgE binding to the Bet v 1 protein as determined by immunoblotting. Among 10 patients sensitized to rBet v 2, six had IgE binding to Bet v 2. Eleven patients with negative results, as determined by immunoblotting, had low levels of birch IgE in the sera (less than 10 kU/L) and low concentrations of IgE to rBet v 1 or rBet v 2 in ELISA. The nonallergic control subjects (n = 7) did not react to rBet v 1 or rBet v 2 in skin prick tests, nor did they have detectable amounts of specific IgE to rBet v 1 or rBet v 2. Histamine release tests confirmed sensitization to Bet v 1 in two patients with discordant results; for Bet v 2, one patient had positive results only at a high concentration, and one had results that remained negative. Thirty-four patients had birch pollinosis, and all reacted to rBet v 1. Patients who were monosensitized to birch never reacted to rBet v 2. Sensitization to rBet v 2 was only found in patients who reacted to other pollens (mainly grass). Twenty-nine patients demonstrated allergy to apples, cherries, or hazelnuts; and all reacted to rBet v 1. Among 11 patients with allergy to Umbelliferae, only three reacted to rBet v 2. CONCLUSIONS: Use of the two recombinant allergens (rBet v 1 and rBet v 2) always permits the diagnosis of birch sensitization. Sensitization to rBet v 1 is specific for birch and Rosaceae allergies, whereas sensitization to birch profilin, Bet v 2, is encountered in multisensitized subjects and is not always related to Umbelliferae allergy.

Adult↗
PubMed1996

Induction of IgE antibodies in mice and rhesus monkeys with recombinant birch pollen allergens: different allergenicity of Bet v 1 and Bet v 2.

BACKGROUND: Serologic measurements with recombinant birch pollen allergens, rBet v 1 and rBet v 2 (birch profilin), have shown that more than 95% of patients allergic to tree pollen mount high levels of IgE against rBet v 1, whereas only approximately 10% of the patients display rather low levels of IgE against rBet v 2. OBJECTIVE: In this study an attempt was made to determine whether the different allergenicity of the major birch pollen allergen, rBet v 1, and a minor birch pollen allergen, rBet v 2, might be related to a different immunogenicity of the proteins as evaluated in experimental animal systems (mice and rhesus monkeys). METHODS: Purified recombinant allergens were injected into mice and rhesus monkeys with aluminum hydroxide as adjuvant for elicitation of specific IgE responses. Antibody responses to the allergens were detected by immunoblotting, and time courses of immune responses were measured by ELISA. RESULTS: In both animal models more than the 10-fold dose of rBet v 2 was required to induce IgE antibodies, and even then, the amount of specific IgE antibodies elicited with rBet v 1 was substantially higher than that induced by rBet v 2. It was noted that rBet v 2 formed stable polymers through disulfide bonds. CONCLUSION: In two different animal models (mice and rhesus monkeys) the major birch pollen allergen, rBet v 1, induced substantially higher levels of IgE than rBet v 2. A reduced allergenicity of Bet v 2 caused by polymer formation would be in agreement with previous studies indicating reduced allergenicity of proteins on chemical polymerization.

Adjuvants, Immunologic↗
PubMed1996

Serological and skin-test diagnosis of birch pollen allergy with recombinant Bet v I, the major birch pollen allergen.

BACKGROUND: Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer from allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis, leading to death. OBJECTIVE: The aim of this study was to evaluate recombinant Bet v I, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy. METHODS: A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradermal testing, comparing commercial birch pollen extracts with recombinant Bet v I. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Recombinant Bet v I allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA, Western blot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v I. CONCLUSION: Our results document that recombinant Bet v I produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients.

Adult↗
PubMed1996

High-level expression of tree pollen isoallergens in Escherichia coli.

cDNAs coding for the major allergen of alder (Alnus glutinosa) pollen Aln g 1, for nine isoforms of Bet v 1, the major birch (Betula verrucosa) pollen allergen, and for four isoforms of Cor a 1, the major allergen of hazel (Corylus avellana) pollen, were inserted into the plasmid pMW175 or pMW 172 and expressed in Escherichia coli as recombinant non-fusion proteins. These constructs produced between 20 and 160 mg protein/l. The recombinant tree pollen isoallergens were tested in immunoblots for their antibody binding properties. For this purpose, we used two monoclonal antibodies (BIP 1 and BIP 4) raised against natural Bet v 1, a polyclonal rabbit anti-recombinant Bet v 1a, as well as serum IgE from allergic patients. Our results show that this expression system is suitable for the production of milligram amounts of tree pollen isoallergens which can be used for the characterization of allergenic epitopes recognized by T and B cells.

Allergens↗
PubMed1996

[Serologic and skin test diagnosis of birch pollen allergy with recombinant Bet v 1, the chief allergen of birch].

BACKGROUND: Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffers from allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis, leading to death. OBJECTIVE: The aim of this study was to evaluate recombinant Bet v 1, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy. METHODS: A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradermal testing, comparing commercial birch pollen extracts with recombinant Bet v 1. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Recombinant Bet v 1 allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA, Westernblot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v 1. CONCLUSION: Our results document that recombinant Bet v 1 produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients.

Adult↗