Quantitative analysis of immunoglobulin E reactivity profiles in patients allergic or sensitized to natural rubber latex (Hevea brasiliensis).

BACKGROUND: Characterized native and recombinant Hevea brasiliensis (rHev b) natural rubber latex (NRL) allergens are available to assess patient allergen sensitization profiles. OBJECTIVE: Quantification of individual IgE responses to the spectrum of documented NRL allergens and evaluation of cross-reactive carbohydrate determinants (CCDs) for more definitive diagnosis. METHODS: Sera of 104 healthcare workers (HCW; 51 German, 21 Portuguese, 32 American), 31 spina bifida patients (SB; 11 German, 20 Portuguese) and 10 Portuguese with multiple surgeries (MS) were analysed for allergen-specific IgE antibody (sIgE) to NRL, single Hev b allergens and CCDs with ImmunoCAP technology. RESULTS: In all patient groups rHev b 5-sIgE concentrations were the most pronounced. Hev b 2, 5, 6.01 and 13 were identified as the major allergens in HCW and combined with Hev b 1 and Hev b 3 in SB. In MS Hev b 1 displayed an intermediate relevance. Different sIgE antibody levels to native Hevea brasiliensis (nHev b) 2 and rHev b 6.01 allowed discrimination of SB with clinical relevant latex allergy vs. those with latex sensitization. Sensitization profiles of German, Portuguese and American patients were equivalent. rHev b 5, 6.01 and nHev b 13 combined detected 100% of the latex-allergic HCW and 80.1% of the SB. Only 8.3% of the sera showed sIgE response to CCDs. CONCLUSIONS: Hev b 1, 2, 5, 6.01 and 13 were identified as the major Hev b allergens and they should be present in standardized latex extracts and in vitro allergosorbents. CCDs are only of minor relevance in patients with clinical relevant latex allergy. Component-resolved diagnostic analyses for latex allergy set the stage for an allergen-directed immunotherapy strategy.

Percutaneous reactivity to natural rubber latex proteins persists in health-care workers following avoidance of natural rubber latex.

BACKGROUND: Long-term avoidance of natural rubber latex [Hevea brasiliensis (Hev b)] is currently recommended for health-care workers (HCWs) with established natural rubber latex (NRL) allergy. Percutaneous sensitivity to eight Hev b NRL allergens was evaluated in HCWs in 2000. To date, no studies have evaluated the longitudinal effects of NRL avoidance on percutaneous sensitivity to NRL allergens. OBJECTIVE: The aims of this study were to evaluate changes in percutaneous reactivity to non-ammoniated latex (NAL) and NRL allergens in HCWs 5 years after a recommendation to avoid NRL and to evaluate factors that predict the persistence of in vivo sensitivity to NAL and NRL allergens. METHODS: Skin prick testing was performed with NAL, seven NRL allergens (Hev b 1, 2, 3, 4, 6.01, 7.01, and 13), and recombinant Hev b 5 (rHev b 5) in 34 HCWs who were initially evaluated in 2000 for occupationally related NRL allergy. Serial 10-fold dilutions of NAL and NRL allergens were employed in skin testing. Sera from the HCWs were assayed for latex and enhanced latex (rHev b 5-enriched allergosorbent)-specific IgE antibodies using the ImmunoCAP assay. RESULTS: The prevalence of work-related symptoms significantly decreased between 2000 and 2005 with avoidance of NRL (P<0.05). A >/=100-fold reduction in percutaneous sensitivity to Hev b 2 and Hev b 7 was less likely in those with prior history of systemic reactions to NRL (P=0.0053), reported history of reaction to cross-reactive foods (P=0.014), continued local reactions to NRL gloves (P<0.0001), or high NRL glove exposure since the initial study (P=0.0075). The diagnostic sensitivity and specificity of the latex-specific IgE serology was 54% and 87.5%, respectively, in comparison with NAL skin tests. The addition of rHev b 5 to the ImmunoCAP (enhanced latex) allergosorbent altered the diagnostic sensitivity and specificity of the ImmunoCAP to 77% and 75%, respectively. CONCLUSION: While symptoms may resolve quickly with NRL avoidance therapy, detectable IgE indicating continued sensitization remains beyond 5 years, and thus continued avoidance of NRL should be recommended.

Generation of allergen-enriched protein fractions of Hevea brasiliensis latex for in vitro and in vivo diagnosis.

BACKGROUND: The latex of Hevea brasiliensis trees contains a complex proteome that includes a range of allergenic proteins. Current latex extracts that are used for the diagnosis of latex allergy still lack important allergens. We aimed to devise a production process for an improved reagent that would ideally contain the complete latex allergome. METHODS: Latex C-serum was fractionated by ammonium sulfate precipitation, and B- and C-serum proteins were then separated by anion exchange chromatography. Proteins eluting within defined salt concentration ranges were pooled into six final fractions. Fractions were evaluated by two-dimensional electrophoresis and subsequent IgE immunoblot for their spectrum of allergens. The presence of the most important latex allergens in the fractions was checked by Western blot analyses. Each fraction was further evaluated by skin prick test (SPT). RESULTS: Reproducibility of the preparation method was demonstrated with two batches of latex. Comparison of latex B- and C-serum to the six fractions showed a remarkable increase in the number of detectable allergens in the fractions. The presence of the latex allergens Hev b 1-8 and Hev b 13 in the fractions was demonstrated. In SPTs, the fractions produced wheal-and-flare reactions comparable to commercial latex extracts. CONCLUSIONS: This method provides reproducible latex protein fractions of high allergen content for the diagnosis of latex allergy.

Allergen concentration in natural rubber latex.

BACKGROUND: Hevea brasiliensis latex serum is commonly used as the in vivo and in vitro reference antigen for latex allergy diagnosis as it contains the full complement of latex allergens. OBJECTIVE: This study quantifies the concentrations of the significant allergens in latex serum and examines its suitability as an antigen source in latex allergy diagnosis and immunotherapy. METHODS: The serum phase was extracted from centrifuged latex that was repeatedly freeze-thawed or glycerinated. Quantitation of latex allergens was performed by two-site immunoenzymetric assays. The abundance of RNA transcripts of the latex allergens was estimated from the number of their clones in an Expressed Sequence Tags library. RESULTS: The latex allergens, Hev b 1, 2, 3, 4, 5, 6, 7 and 13, were detected in freeze-thawed and glycerinated latex serum at levels ranging from 75 (Hev b 6) to 0.06 nmol/mg total proteins (Hev b 4). Hev b 6 content in the latex was up to a thousand times higher than the other seven latex allergens, depending on source and/or preparation procedure. Allergen concentration was reflected in the abundance of mRNA transcripts. When used as the antigen, latex serum may bias the outcome of latex allergy diagnostic tests towards sensitization to Hev b 6. Tests that make use of latex serum may fail to detect latex-specific IgE reactivity in subjects who are sensitized only to allergens that are present at low concentrations. CONCLUSION: Latex allergy diagnostics and immunotherapy that use whole latex serum as the antigen source may not be optimal because of the marked imbalance of its constituent allergens.

Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4.

BACKGROUND: Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53-55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53-55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information. OBJECTIVE: We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding. METHODS: The 5'/3' rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients. RESULTS: The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue. CONCLUSION: The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA.

The polycotyledon mutant of tomato shows enhanced polar auxin transport.

The polycotyledon mutant of tomato (Lycopersicon esculentum L. cv Ailsa Craig) showed altered development during embryogenesis and during vegetative and reproductive phases. The phenotype was pleiotropic and included the formation of extra cotyledons, changes in leaf shape, increased number of flowers (indeterminacy) with abnormal floral organs, the formation of epiphyllous structures, and altered gravitropism. The earliest defects were observed at the transition from the globular to the heart stage of embryogenesis with the formation of multiple cotyledons. Epidermal cells in the mutant embryo were smaller and less expanded compared with wild type. Examination of polar auxin transport (PAT) showed a striking enhancement in the case of the mutant. Increase in PAT did not appear to be caused by a decrease in flavonoids because the mutant had normal flavonoid levels. Application of 2,3,5-triiodobenzoic acid, an inhibitor of polar transport of auxin, rescued postgermination phenotypes of young seedlings. Our analysis reveals a level of control that negatively regulates PAT in tomato and its contribution to plant development and organogenesis.

Appraisal of latex glove proteins in the induction of sensitivity to multiple latex allergens.

Six Hevea brasiliensis latex protein allergens, Hevb 1, Hev b 2, Hev b 3, Hev b 4, and two variants of Hev b 7 (7b and 7c), were purified from Hevea latex, while a seventh protein, Hev b 5, was prepared in recombinant form. The presence of these proteins in glove extracts was indicated by their respective antibodies in the serum of rabbits immunized against the extracts. The relative propensities of IgE binding to the individual latex allergens were compared using sera from latex-allergic patients. IgE recognition of Hev b 4, Hev b 7b, Hev b 5 and Hev b 2 was most frequently encountered, with 75, 61, 31 and 28%, respectively, of the patient sera reacting. Sensitivity to multiple latex proteins was common, and out of the 31 seropositive patients, 23 (74%/ ) had IgE against at least two latex allergens, while 12 (39%) had IgE specific for at least three allergens. Statistical analysis of the data suggested that many patients might have acquired sensitivity to Hev b 2, Hev b 4 and Hev b 7b from a common source. (e.g., from latex products). On the other hand, sensitivity to Hev b 5 and to Hev b 7c were interrelated. It is plausible that sensitivity to these two proteins might have been acquired from sources other than latex products (e.g., from certain foods).

Molecular and immunologic characterization of new isoforms of the Hevea brasiliensis latex allergen hev b 7: evidence of no cross-reactivity between hev b 7 isoforms and potato patatin and proteins from avocado and banana.

BACKGROUND: Hev b 7 is a Hevea brasiliensis latex allergen with sequence identities of 39% to 42% to patatins recently identified as potato allergens. The complementary DNAs encoding 2 different Hev b 7 isoforms were previously reported. OBJECTIVE: The aim of this study was to determine the sequence variation of Hev b 7 and to compare the IgE reactivity of individual isoforms in vitro and in vivo. A further objective was to evaluate possible cross-reactivities between Hev b 7 and patatins and proteins from banana and avocado. METHODS: An H brasiliensis lambda ZAP complementary DNA (cDNA) library was screened with use of a Hev b 7 cDNA probe. Four Hev b 7 isoforms were produced in recombinant form and their IgE-binding capacities were compared. IgE immunoblot inhibitions and ELISA inhibition assays were used to investigate the possible cross-reactivity between Hev b 7 and recombinant potato patatin and proteins from avocado and banana. RESULTS: Two new isoforms, S2 and D2, were identified by sequencing 32 cDNA clones with full-length coding regions. All 4 recombinant isoforms displayed esterase activity and identical IgE-binding capacities. The new isoforms S2 and D2 were evaluated in skin prick tests and provoked responses equivalent to natural Hev b 7. No cross-reactivity was observed between Hev b 7 isoforms and potato patatin and proteins from avocado and banana. CONCLUSIONS: All 4 recombinant Hev b 7 isoforms have equivalent IgE-binding capacity and therefore represent suitable reagents for the development of in vitro and in vivo diagnostic tests. Hev b 7, patatins, and their homologs appear not to contribute to cross-reactivity in the latex-fruit syndrome.

Avidin is a promising tag for fusion proteins produced in baculovirus-infected insect cells.

The baculovirus expression vector system (BEVS) has become one of the most versatile and powerful eukaryotic systems for recombinant protein expression. We have constructed a novel baculovirus transfer vector (pbacAVs+C) which allows for the efficient production, detection, and single-step purification of the desired molecule as a secretion-compatible avidin fusion protein in insect cells. It also enables fast construction of the baculoviruses by site-specific transposition in Escherichia coli. To demonstrate the power of this vector, we report here on the production of immunologically intact hevein, a major cysteine-rich latex allergen, as avidin fusion protein. Our results indicate that avidin is a stable and versatile tag in the BEVS. It retains its extraordinarily high biotin-binding activity and also enables independent folding of the fusion partner. The versatility with which avidin fusion proteins can be detected, purified, and immobilized is the basis for the use of our system as a useful alternative in eukaryotic fusion protein production.

Purified and recombinant latex proteins stimulate peripheral blood lymphocytes of latex allergic patients.

BACKGROUND: Natural rubber latex proteins have been implicated in severe allergy in individuals exposed to latex products, particularly health care workers. Until recently, only crude antigens were available to study the immune response in these patients. In recent years a number of relevant allergens have been purified, but few have been used in lymphocyte studies. Hence, to better understand the immunological mechanisms involved in latex allergy, we investigated the response of peripheral blood mononuclear cells (PBMCs) to various purified natural rubber latex allergens. METHODS: Using conventional protein purification methods and gene cloning, we have obtained 6 natural rubber latex proteins. We studied allergen-specific IgE levels and PBMC responses to these allergens along with 3 crude latex antigen preparations. RESULTS: Of the 28 latex-allergic health care workers studied, 16 reacted to one or more of the allergens studied, but PBMCs from controls failed to respond to these antigens. Serum IgE to the antigens was detected in 11-90% of the patients. CONCLUSION: Fifty-seven percent of the latex-allergic patients demonstrated PBMC responses to at least one of the latex allergens tested, but there was no direct correlation between serum IgE levels and PBMC responses. However, since none of the control subjects showed any PBMC stimulation, this may prove useful in determining sensitization to latex. Among the allergens studied, the predominant mononuclear cell responses were directed against Hev b 2, while serum IgE against rHev b 6 was demonstrable in the greatest number of patients. The crude latex allergens were toxic to PBMCs and hence, the purified allergens may be of greater value in demonstrating sensitization of patients to latex allergens.

Characterisation of the S-adenosylmethionine decarboxylase (SAMDC) gene of potato.

S-adenosylmethionine decarboxylase (SAMDC) is involved in the biosynthesis of the polyamines, spermidine and spermine. Recently, we reported the isolation of a putative cDNA clone of the SAMDC clone of potato (Plant Mol Biol 20; 641-651). In order to confirm that the potato genes does encode SAMDC, a complementation experiment with a yeast strain that possesses a null mutation in the SAMDC gene was performed. The yeast strain contains a deletion-insertion mutation in the SAMDC gene and has an absolute requirement for the addition of exogenous spermidine for growth. When the full-length potato cDNA was expressed in the mutant yeast strain there was no longer a requirement for exogenous spermidine. Immunoblotting experiments suggest that the potato SAMDC gene product has an apparent molecular mass of 39 kDa. Expression of the SAMDC gene was high in the young and actively dividing tissues and low in the mature and non-dividing tissues of both vegetative and reproductive organs. Additionally, isolation and characterisation of the corresponding genomic clone is reported. The gene has one intron in its 5'-untranslated sequence but otherwise the transcribed portion is identical to the cDNA clone.

Expression and sequence analysis of cDNAs induced during the early stages of tuberisation in different organs of the potato plant (Solanum tuberosum L.).

cDNA clones of two genes (TUB8 and TUB13) which show a 25-30-fold increase in transcript in the stolon tip during the early stages of tuberisation, have been isolated by differential screening. These genes are also expressed in leaves, stems and roots and the expression pattern in these organs changes on tuberisation. Southern analysis shows homologous sequences in the non-tuberising wild type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUB13 cDNA, and a human S-adenosylmethionine decarboxylase gene. The predicted TUB8 peptide sequence shows several repeats of alanine, glutamate and proline which suggests a structural role for the encoded protein.

Differential Expression and Sequence Analysis of Ribosomal Protein Genes Induced in Stolon Tips of Potato (Solanum tuberosum L.) during the Early Stages of Tuberization.

cDNA clones of two genes, TUBS19 and TUBL7, which show a 15- to 20-fold increase in transcript level in the stolon tip during the early stages of tuberization, have been isolated by differential screening. These genes are also expressed in leaves, stems, and roots, and the expression pattern in these organs changes on tuberization. Southern analysis shows that there are similar sequences in the genome of nontuberizing wild-type potato species Solanum brevidens and in Lycopersicon esculentum (tomato). Sequence analysis reveals a high degree of similarity between the TUBS19 cDNA and the eukaryotic S19 ribosomal protein gene. TUBL7 cDNA shows similarity to another eukaryotic ribosomal protein gene, L7.