Evidence of leptospirosis in the kidneys and serum of feral swine (Sus scrofa) in the United States.

Leptospirosis is the most widespread zoonosis in humans worldwide. In the United States, widespread detection of antibodies to leptospirosis have been identified in feral swine (Sus scrofa) with the highest detection of serovars, Bratislava, Icterohaemorrhagiae, and Pomona. Over the past few years, feral swine populations have expanded their geographical range and distribution in the United States with reports in at least 39 of 50 states. Since feral swine serve as reservoirs for serovars that can infect humans, it is important to understand the risk of transmission. In order to learn more about the probability that feral swine shed infectious leptospires, we collected kidneys and paired serum when possible from 677 feral swine in 124 counties of 29 states. These counties had previously been identified as antibody positive for Leptospira interrogans serovars Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae or Pomona. Although exposure to these same six serovars of leptospirosis continued to be high (53% overall) in the counties we sampled, we detected leptospiral DNA in only 3·4% of feral swine kidneys tested. Based on these results, it appears that although feral swine can serve as a source of infection to humans, especially in those who are more likely to encounter them directly such as wildlife biologists, veterinarians, and hunters, the risk may be relatively low. However, further studies to examine the relationship between leptospiral shedding in the urine and kidneys in addition to culturing the organism are recommended in order to better understand the risk associated with feral swine.

Multi-institutional randomized control study of haemolysis in stored red cell units prepared manually or by an automated system.

BACKGROUND: The haemolysis level at the end of storage is a performance parameter for RBC preparations. In the evaluation of new devices or new processes for processing blood, it is relevant to evaluate whether the haemolysis is linked to (1) specific characteristics of the blood donor, or (2) the nature of the blood-processing methodologies. MATERIALS AND METHODS: As part of the validation of a new automated whole blood processing system compared to the current manual methods, randomized, paired crossover studies were conducted evaluating measures of blood component quality, including RBC haemolysis over 42 days of storage. RESULTS: The association between haemolysis and the individual subject was evaluated by modelling haemolysis with independent predictors of treatment (control and test processing) and leucocyte reduction as fixed factors with donor and laboratory as random effects in a mixed-effects ANOVA model. It was found that the day 42 haemolysis values were strongly dependent on the donor subject, with an intraclass correlation coefficient of 0.81. CONCLUSIONS: The data reported in this study suggest a link between the specific whole blood donor and the haemolysis levels observed in red-blood-cell units stored refrigerated for 42 days. Additional research to identify possible donor characteristics associated with haemolysis during storage is warranted.

In vivo and in vitro characteristics of double units of RBCs collected by apheresis with a single in-line WBC-reduction filter.

BACKGROUND: A novel apheresis procedure for a blood separator (MCS+, Haemonetics) enables the collection of 2 WBC-reduced RBC units in a single donation by using one disposable set with one in-line WBC-reduction filter (RC2H, Pall Corp.). The objective of this study was to evaluate the filtration performance in connection with different prefiltration RBC storage conditions and with the in vitro and in vivo storage quality of the filtered units. STUDY DESIGN AND METHODS: Sixty-six 2-unit RBC collection and gravity-filtration procedures were completed at three sites, resulting in 132 RBC units. Filtration of the double RBC units was performed at room temperature (RT) within 8 hours of collection (n = 36) and under refrigeration (1-6 degrees C) for up to 24 hours (n = 10) and 72 hours (n = 20) before filtration. RBC quality was compared to that of nonfiltered apheresis RBC units (n = 10). RESULTS: Median filtration time was 6.5 and 14 minutes for units stored at RT and under refrigeration, respectively. All 132 RBC units had residual WBC counts <0.4 x 10(6). The refrigerated units showed a greater mean log reduction in WBCs: 5.06 +/- 0.16 (24 hour) and 4.74 +/- 0.48 (72 hour), respectively, than did RT units: 4.47 +/- 0.28 (p<0.05). RBC loss was less than 12 percent in all cases (mean, 7.8 +/- 1.8%). Minimal differences in volume were observed between the paired RBC units. In vitro RBC storage characteristics of the filtered units were as expected and similar to those of the nonfiltered units. For RBC units held at RT (n = 24), the mean in vivo 24-hour recovery was 81.8 +/- 8.4 percent (double-label). CONCLUSION: Satisfactory filter performance in terms of WBC removal and RBC loss was observed with all 66 procedures, irrespective of storage conditions before filtration.

A hypoallergenic derivative of the major allergen of the dust mite Lepidoglyphus destructor, Lep d 2.6Cys, induces less IgE reactivity and cellular response in the skin than recombinant Lep d 2.

BACKGROUND: The major allergen of the dust mite Lepidoglyphus destructor, Lep d 2, has been produced as a recombinant allergen (rLep d 2) with IgE reactivity both in vivo and in vitro. A modified form of rLep d 2 (rLep d 2.6Cys) obtained by site-directed mutagenesis has been shown to have a reduced IgE reactivity in vitro. In this study we have compared the ability of rLep d 2 and rLep d 2.6Cys to elicit positive skin prick tests and cellular responses among L. destructor-sensitized subjects. METHODS: Seventeen subjects were skin prick-tested with rLep d 2, rLep d 2.6Cys, histamine and negative controls and 17-20 h later skin biopsy specimens were taken from the skin prick-tested sites. The biopsy specimens were stained immunohistochemically for EG2+, CD3+, CD1a+, mast cell tryptase+, and IgE+ cells. Dermal cell infiltrates were judged in hematoxylin and eosin staining. Total IgE and allergen-specific IgE were determined by CAP-RAST. RESULTS: Compared to rLep d 2, rLep d 2.6Cys induced significantly smaller and fewer skin prick test reactions (p < 0.001) and dermal cell infiltrates (p < 0.05). Further, rLep d 2.6Cys induced fewer EG2+ cells (p < 0.001) but more tryptase+ cells (p < 0.05) than rLep d 2. A positive RAST to rLep d 2 was obtained for 88.2% of the subjects, while only 35.2% displayed a positive RAST to rLep d 2.6Cys. CONCLUSION: This study demonstrates that rLep d 2.6Cys is less able to evoke IgE-mediated reactions and cellular responses, as measured both in skin and in serum, than rLep d 2. In the future this hypoallergenic derivative may be a promising candidate molecule for immunotherapy of L. destructor-allergic patients.

The effect of leukocyte-reduction method on the amount of human cytomegalovirus in blood products: a comparison of apheresis and filtration methods.

This study examined the effectiveness of 3 leukocyte-reduction (LR) methods in depleting the residual level of cytomegalovirus (CMV) in blood products measured by quantitative polymerase chain reaction (QA-PCR). At 2 locations over 3 allergy seasons, apheresis platelets and whole blood were collected from 52 healthy CMV seropositive subjects having an elevated titer of CMV DNA (median = 2400 genome equivalents [GE]/mL) resulting in 32 evaluable LR apheresis platelets, 31 filtered platelets from whole blood, and 31 filtered red blood cells (RBCs) from whole blood. Leukoreduction by apheresis and filtration resulted in substantial reduction of detectable CMV DNA levels with 99.9% of the LR products expected to have less than 500 GE/mL of CMV DNA. No difference was found between methods (P =.52). CMV genomic leukocyte subset localization was determined by QA-PCR of fluorescence-activated cell sorter (FACS)-sorted peripheral blood from 20 seropositive subjects (n = 10 > 100 GE/mL, n = 10 QA-PCR negative). CMV was detected in monocyte (13 of 20) and granulocyte (3 of 20) fractions. Presence of competent virus in QA-PCR positive (> 100 GE/mL) peripheral blood samples was verified with 4 of 19 subjects positive in shell vial assay, and 8 of 18 positive for CMV gene products (messenger RNA). We observed a seasonal DNAemia variation in seropositive subjects. CMV seropositive subjects (n = 45) entered into longitudinal monitoring in March/April 1999 were QA-PCR negative at baseline. Subjects converted to a positive QA-PCR coincident with increased seasonal allergen levels (Norfolk 15 of 18 evaluable in 43.4 +/- 9.48 days; Denver, 16 of 23 evaluable in 96 +/- 26.3 days). These data demonstrate effective reduction of CMV load by LR during periods of DNAemia in CMV seropositive subjects. (Blood. 2001;97:3640-3647)

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.

Relationships of cervical, thoracic, and lumbar bone mineral density by quantitative CT.

PURPOSE: The purpose of this work was to compare, using quantitative CT (QCT), vertebral bone mineral density (BMD) in the cervical, thoracic, and lumbar spine in healthy volunteers. METHOD: QCT of the vertebral bodies of C2, C5, T12, and L4 was performed on 50 healthy volunteers (25 women, 25 men; mean age 31.7 years). Trabecular BMD analysis was performed at each level. RESULTS: Mean BMDs (mg/cm3 calcium hydroxyapatite) for women and men were highest at C5 (BMD women/men 341.6/300.6 mg/cm3) and C2 (297.2/269.6 mg/cm3) and lowest at T12 (193.1/184.9 mg/cm3) and L4 (186.2/180.1 mg/cm3). The BMD of C2 was statistically significantly different from that of C5, T12, and L4 (p < 0.0001) for both genders. Also, the BMD of C5 differed significantly from that of T12 and L4 (p < 0.0001). The BMD of C5 showed significant gender differences (p = 0.002). Correlation coefficient showed a strong correlation between the BMD of T12 and L4 for both genders (women, r = 0.67; men, r = 0.90). CONCLUSION: Trabecular BMD of C2 and C5 measured by QCT is significantly higher than trabecular BMD of T12 and L4 in nonosteoporotic volunteers of both genders.

Molecular population genetics of male accessory gland proteins in Drosophila.

Drosophila seminal proteins have an unusually high rate of molecular sequence evolution, suggesting either a high rate of neutral substitution or rapid adaptive evolution. To further quantify patterns of polymorphism and divergence in genes encoding seminal proteins, also called accessory gland proteins (Acp's), we conducted a sequencing survey of 10 Acp genes in samples of Drosophila melanogaster and D. simulans (Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB). Mean heterozygosity at replacement sites in D. simulans was 0.0074 for Acp genes and 0.0013 for a set of 19 non-Acp genes, and mean melanogaster-simulans divergence at replacement sites was 0.0497 for Acp genes and 0.0107 at non-Acp genes. The elevated divergence of Acp genes is thus accompanied by elevated within-species polymorphism. In addition to the already-reported departures of Acp26A, Acp29AB, and Acp70A from neutrality, our data reject neutrality at Acp29AB and Acp36DE in the direction of excess replacements in interspecific comparisons.

Evaluation of an automated system for the collection of packed RBCs, platelets, and plasma.

BACKGROUND: This study evaluated the quality of WBC-reduced platelets, RBCs, and plasma collected on a new system (Trima, Gambro BCT) designed to automate the collection of all blood components. The study also evaluated donor safety and suitability of these components for transfusion. STUDY DESIGN AND METHODS: In Phase I, the quality of the components collected on the new system was evaluated by standard in vitro and in vivo testing methods. Results were compared to those from control components collected by currently approved standard methods. In Phase II, additional collections were performed to evaluate the acceptability of the new system and the safety of platelets collected. RESULTS: In vivo 24-hour RBC recovery was 76.8 +/- 3.1 percent for the test RBC units and 77.1 +/- 4.4 percent recovery for whole-blood (control) RBCs. The differences between test and control platelet results in the in vivo and in vitro assays were not clinically significant. Plasma clotting factors and fibrinogen levels met international standards. The system was well accepted by donors, and no major adverse donor reactions were reported for the 68 procedures performed. No problems were reported with transfusing the blood components collected. CONCLUSION: Blood components collected with the Trima are equivalent to currently available components, and they meet the applicable regulatory standards. This system provides consistent, standardized components with predictable yields. It provides the option of fully automating the collection of all blood components.

Genetics of alpha-amanitin resistance in a natural population of Drosophila melanogaster.

The genetic basis of variation in resistance to natural toxins is of interest for both ecological and evolutionary genetics. The wide variety of larval resources used by Drosophila, both within and between species, makes flies an excellent system for studying causes and consequences of selection resulting from exposure to natural toxins associated with different resources. In this study we carry out a genetic analysis of alpha-amanitin resistance in a population sample of Drosophila melanogaster. Data from mapping crosses of chromosome III support a role for a naturally occurring polymorphism in a multidrug resistance gene (Mdr65A) in alpha-amanitin resistance. However, there are no amino acid differences between resistant and sensitive chromosomes at Mdr65A. Therefore, if Mdr65A mutants contribute to the difference between alpha-amanitin-resistant and alpha-amanitin-sensitive third chromosome lines, the underlying cause is a gene regulatory mutation.

Cloning, characterization and expression of complete coding sequences of three IgE binding Malassezia furfur allergens, Mal f 7, Mal f 8 and Mal f 9.

Malassezia furfur, formerly known as Pityrosporum orbiculare or P. ovale, is a yeast that colonizes human skin. Normally, this yeast is nonpathogenic but under the influence of predisposing factors it may induce IgE reactivity in patients with atopic dermatitis. Approximately 40-65% of atopic dermatitis patients have IgE antibodies and/or skin reactivity against M. furfur and a higher T-cell response against this yeast is found in atopic dermatitis patients than in healthy individuals. By making a cDNA library displayed on a phage surface, we previously cloned five different IgE-binding proteins, Mal f 5, Mal f 6, MF 7, MF 8 and MF 9, from this yeast. The cDNAs encoding these allergens were sequenced and expressed in Escherichia coli. The sequences of MF 7, MF 8 and MF 9 were not full length (missing their 5'-ends) giving only partial gene products. To obtain complete cDNA sequences, we performed RACE-PCR to amplify the 5'-ends of each cDNA. These PCR products were sequenced and analyzed. The coding sequences of Mal f 7, Mal f 8 and Mal f 9 encode proteins with ORFs of 141 (16.2 kDa), 179 (19.2 kDa) and 126 (14.0 kDa) amino-acid residues, respectively. None of the putative proteins showed significant sequence homology with other known proteins in the searched database. The proteins encoded by the complete cDNA sequences were expressed in E. coli as recombinant proteins. Immunoblotting and radioallergosorbant test data showed that all of the expressed recombinant proteins have the ability to bind serum IgE from atopic dermatitis patients and furthermore, the M. furfur extract could specifically inhibit this IgE binding.

Reduced X-linked nucleotide polymorphism in Drosophila simulans.

Population genetic theory predicts that selectively driven changes of allele frequency for both beneficial and deleterious mutants reduce polymorphism at tightly linked sites. All else being equal, these reductions in polymorphism are expected to be greater when recombination rates are lower. Therefore, the empirical observation of a positive correlation between recombination rates and amounts of DNA polymorphism across the Drosophila melanogaster genome can be explained by two very different types of natural selection. Here, we evaluate alternative models of effects of selection on linked sites by comparison of X-linked and autosomal variation. We present polymorphism data from 40 genes distributed across chromosome arms X and 3R of Drosophila simulans, a sibling species of D. melanogaster. We find significantly less silent polymorphism in D. simulans on the X chromosome than on 3R, but no difference between arms for silent divergence between species. This pattern is incompatible with predictions from theoretical studies on the effect of negative selection on linked sites. We propose that some form of positive selection having greater effects on sex chromosomes than on autosomes is the better explanation for the D. simulans data.

Adaptive evolution of relish, a Drosophila NF-kappaB/IkappaB protein.

NF-kappaB and IkappaB proteins have central roles in regulation of inflammation and innate immunity in mammals. Homologues of these proteins also play an important role in regulation of the Drosophila immune response. Here we present a molecular population genetic analysis of Relish, a Drosophila NF-kappaB/IkappaB protein, in Drosophila simulans and D. melanogaster. We find strong evidence for adaptive protein evolution in D. simulans, but not in D. melanogaster. The adaptive evolution appears to be restricted to the IkappaB domain. A possible explanation for these results is that Relish is a site of evolutionary conflict between flies and their microbial pathogens.

Different conformations of nascent polypeptides during translocation across the ER membrane.

BACKGROUND: In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated alpha-helices) may be possible within the ribosome. RESULTS: We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 271: 6241-6244). Using this approach, we now show that model segments composed of residues with strong helix-forming properties in water (Ala, Leu) have a more compact conformation in the ribosome-translocon channel than model segments composed of residues with weak helix-forming potential (Val, Pro). CONCLUSIONS: The main conclusions from the work reported here are (i) that the propensity to form an extended or more compact (possibly alpha-helical) conformation in the ribosome-translocon channel does not depend on whether or not the model segment has stop-transfer function, but rather seems to reflect the helical propensities of the amino acids as measured in an aqueous environment, and (ii) that stop-transfer sequences may adopt a helical structure and integrate into the ER membrane at different times relative to the time of glycan addition to nearby upstream glycosylation acceptor sites.

Quality and clinical response to transfusion of prestorage white cell-reduced apheresis platelets prepared by use of an in-line white cell-reduction system.

BACKGROUND: This study evaluated the quality and clinical effectiveness of white cell (WBC)-reduced apheresis platelets collected by the use of a new technology, fluidized particle-bed separation. STUDY DESIGN AND METHODS: In phase 1, six suitable donors underwent two separate plateletpheresis procedures on one occasion, each separated by less than 10 minutes. In random order, a control unit was collected with the COBE Spectra and a test unit with the Spectra Leukocyte-Reduction System (LRS). The quality of apheresis platelet components was assessed by an in vitro test panel, and residual WBCs were counted by Nageotte chamber and flow cytometric methods. For the in vivo studies, the test and control units were randomly labeled with either 51Cr or 111In at the end of storage and transfused simultaneously to the donor. Samples were taken for calculation of platelet survival and recovery. In phase II, 109 thrombocytopenic patients were given platelets collected by use of the Spectra LRS. RESULTS: Test platelets had significantly fewer residual WBCs (median 7.6 x 10(4)) than control platelets (median 3.9 x 10(5)), with equivalent in vitro function values. Test and control platelets had similar recovery and survival. Transfused platelets collected by use of the LRS achieved a mean 1-hour corrected-count increment of 19.3. CONCLUSION: The LRS collects platelet components with significantly lower WBC contamination without adverse effects on the function or in vivo survival of the platelets.

Evaluation of specific IgE to the recombinant group 2 mite allergens Lep d 2 and Tyr p 2 in the Pharmacia CAP system.

BACKGROUND: Several recombinant allergens have been shown to be potentially useful for diagnosis of IgE-mediated allergy, but only a few recombinant allergens are at present commercially available in serological assays for detection of specific IgE. The aim of this study was to evaluate the IgE binding to the recombinant major dust mite allergens rLep d 2 and rTyr p 2 and compare it with the IgE binding to the commercial mite extracts Lepidoglyphus destructor and Tyrophagus putrescentiae in the Pharmacia RAST CAP System. METHODS: The recombinant allergens rLep d 2 and rTyr p 2 were immobilised on ImmunoCAPs, and sera from 461 Swedish farmers who are frequently exposed to mites were analysed for specific IgE antibodies. Immunoblotting was performed to evaluate discrepancies between the results obtained with the recombinant and the commercial CAP assays. RESULTS: The IgE values of each recombinant assay significantly correlated with the IgE values of the corresponding commercial CAP assay. The sensitivity of the rLep d 2 assay was 73.3% and that of the rTyr p 2 assay, 60.5% of that provided by the commercial L. destructor and T. putrescentiae assays. Two subjects out of 416, who tested negative in the commercial L. destructor assay, were positive to rLep d 2. The corresponding figures for rTyr p 2 and the T. putrescentiae extract were 5/418. The possibility that these subjects were sensitised to L. destructor and T. putrescentiae could not be excluded. CONCLUSIONS: The data suggest that it may be possible to use rLep d 2 and rTyr p 2 on ImmunoCAPs to detect and quantify IgE antibodies to these, the major allergens of L. destructor and T. putrescentiae. It appears likely that the addition of just a few more recombinant L. destructor and T. putrescentiae allergens in the CAP assay will be sufficient for in vitro diagnosis of IgE mediated allergy to L. destructor and T. putrescentiae.

Selective cloning of allergens from the skin colonizing yeast Malassezia furfur by phage surface display technology.

The yeast Malassezia furfur, also known as Pityrosporum orbiculare (ovale), is part of the normal microflora of the human skin but has also been associated with different skin diseases including atopic dermatitis. More than 50% of atopic dermatitis patients have positive skin test and specific IgE to M. furfur extracts; however, the pathophysiologic role of these IgE-mediated reactions in the development of the disease remains unknown. The yeast is able to produce a wide panel of IgE-binding proteins, variably recognized by sera of individual patients. In order to assess the contribution of individual components to the disease, highly pure allergen preparations are required. We have cloned M. furfur allergens from a cDNA library displayed on the phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. Phage displaying IgE-binding proteins were selectively enriched from the library using IgE from a M. furfur-sensitized atopic dermatitis patient as a ligand. We were able to identify five different inserts coding for IgE-binding polypeptides. Three of the sequenced cDNA encode incomplete gene products with molecular masses of 21.3 kDa (MF 7), 14.4 kDa (MF 8), and 9.7 kDa (MF 9), respectively, having no sequence similarity to known proteins. The other two cDNA encode allergens of 18.2 kDa (Mal f 5) and 17.2 kDa (Mal f 6). Mal f 5 shows significant homology to M. furfur allergens Mal f 2, Mal f 3 and an Aspergillus fumigatus allergen Asp f 3. Mal f 6 has significant homology with cyclophilin. All of the recombinant polypeptides were capable of binding serum IgE from atopic dermatitis patients in immunoblotting experiments. The availability of pure recombinant M. furfur allergens will allow the careful investigation of the role of IgE-binding proteins in atopic dermatitis.

Identification and characterisation of two allergens from the dust mite Acarus siro, homologous with fatty acid-binding proteins.

BACKGROUND: Dust mites are a major cause of allergic disease worldwide. The dust mite Acarus siro is an inducer of occupational allergy among farmers, but sensitisation has also been found in non-farming populations. METHODS: A degenerate primer was designed to the N-terminal amino acid sequence of a 15-kD IgE-binding protein in A. siro extract. The cDNA sequence was obtained by using reverse transcriptase polymerase chain reaction, standard cloning and sequencing techniques. The protein was expressed in Escherichia coli with a 6-histidine tag at its C-terminus. Immunoblotting of the recombinant protein and whole extract was performed using patient sera. RESULTS AND CONCLUSION: 15 and 17-kD allergens were identified in a fraction of A. siro extract. The cDNA of the 15-kD allergen was isolated, cloned and sequenced and the allergen was expressed as a recombinant protein. The calculated molecular weight of the cDNA-encoded protein is 14.2 kD. The predicted amino acid sequence has one potential N-glycosylation site at position 4-6 and a cytosolic fatty acid-binding protein signature at position 5-22. The protein has 64% sequence identity with Blo t 13, an allergen from the dust mite Blomia tropicalis, as well as homology with several other fatty acid-binding proteins (FABPs) from different organisms. The allergen was named Aca s 13 and was recognised strongly by 3 of 13 (23%) of the subjects investigated. The amino acid sequence of the 17-kD protein was partly determined and it also showed high sequence homology with Blo t 13 and FABPs.

Immunologic characterization of natural and recombinant Mal f 1 yeast allergen.

BACKGROUND: Individuals with atopic dermatitis (AD) often have IgE antibodies against protein components of Malassezia furfur. The cDNA encoding one of these proteins (Mal f 1) has recently been cloned and sequenced. OBJECTIVE: We sought to express recombinant Mal f 1 (rMal f 1) allergen in large quantities by using different expression systems. The primary aim was to characterize the IgE-binding properties of rMal f 1 in comparison with its natural counterpart in M furfur extract. METHODS: We have expressed and purified Mal f 1 from prokaryotic (Escherichia coli) and eukaryotic cells (baculovirus-infected insect cells). The rMal f 1 produced in both systems has been tested for the ability to be recognized by IgE from patients with specific serum IgE to M furfur by using immunoblotting and the Pharmacia CAP System RAST FEIA. RESULTS: Sixty-one percent of sera from 95 patients showed positive RAST responses to the rMal f 1 produced in the baculovirus expression system and 43% to the E coli -produced rMal f 1. Both the E coli - and baculovirus-produced proteins can specifically inhibit IgE binding to a 36-kd protein band (Mal f 1) in immunoblotting, indicating that the recombinant proteins contain the majority, if not all, the IgE-binding epitopes of Mal f 1. Recombinant Mal f 1 is able to release histamine from basophils of an atopic individual. CONCLUSION: We have expressed and purified rMal f 1, which can bind IgE in a way resembling natural Mal f 1. The ability to produce recombinant allergens with similar properties to their native counterparts has many potential uses, such as accurately diagnosing causes of IgE-mediated allergy.

IgE binding capacity of synthetic and recombinant peptides of the major storage mite (Lepidoglyphus destructor) allergen, Lep d 2.

BACKGROUND: Lepidoglyphus destructor is an important non-pyroglyphid mite species in Europe and a dominant allergen in farming environments. The major allergen of L. destructor, Lep d 2, is a protein of 13.2 kD that is recognised by about 90% of sera RAST positive to this mite species. METHODS: The cDNA of two isoallergens of the Lep d 2 has previously been sequenced and the protein expressed in different protein expression systems. In order to map the B-cell epitopes, the full length protein and the truncated forms of the protein have been expressed in Escherichia coli as glutathione-S-transferase (GST) fusion proteins. Recombinant Lep d 2 fragments and synthetic overlapping 15 mer peptides spanning Lep d 2 were probed with sera from patients allergic to storage mite. RESULTS: The full-length (125 amino acids) GST fusion protein reacted strongly with patient IgE in Western blots and dot blots. Synthetic peptides failed to react with IgE antibodies from mite-allergic patients and the truncated fusion proteins displayed weak IgE-binding capacity. CONCLUSION: We conclude that there are no dominant linear IgE-binding epitopes in Lep d 2. Recombinant or synthetic Lep d 2 fragments may, however, be further evaluated as hypoallergenic candidate molecules for specific immunotherapy.