Cloning and characterization of a major allergen of the house dust mite, Dermatophagoides pteronyssinus, homologous with glutathione S-transferase

A major allergen of the house dust mite, Dermatophagoides pteronyssinus, has been identified and characterized from a lambda gt11 cDNA library of the mite. IgE antibodies from the sera of allergic patients that recognise the cloned polypeptide bind to an approximately 26 kDa polypeptide on a Western blot of reduced mite polypeptides. Nucleotides sequencing of the clone revealed a 219 amino acid open reading frame encoding a protein with a derived molecular mass of 25,589 Da and a pI of 6.3. Comparison of the deduced amino acid sequence with amino acid sequence databanks revealed a strong homology with glutathione S-transferases. The nucleotide sequence of the clone displayed a strong homology with the active glutathione binding site of glutathione transferases and contained all but one of the 19 positionally conserved amino acid residues found in glutathione transferases. The cloned polypeptide was expressed in Escherichia coli and affinity-purified on glutathione agarose.     

Glutathione S-transferase a major allergen of the house dust mite, Dermatophagoides pteronyssinus

Cutaneous electrogastrography (EGG) allows the measurement of gastric electrical activity. An association of EGG with gastrointestinal motility disorders has been shown. Abnormalities of electrical rate or rhythm are accepted as the most important parameters in EGG. However, the reliability of the magnitude of electrical amplitude in the assessment of motility is discussed controversially. Therefore in a prospective study we investigated the relation between amplitude and antral contractions by means of ultrasonography. 8 healthy volunteers (4 men, 4 women, 24-31 years) ingested 400 ml carbonated mineral water after an overnight fast at two separate study days. Over a period of 10 min preprandial and 10 min postprandial small and intense antral contractions were measured employing sagittal antral planimetry. Simultaneous amplitudes were determined during contractions and at 1 min intervals (average amplitude) by cutaneous electrogastrography. Data were analyzed by Wilcoxon's rank sum test and Spearman rank correlation test. The coefficient of variation of the postprandial/preprandial amplitude ratio was nearly two times greater between subjects than between recordings in the same subject, which reflects a moderate intraindividual reproducibility. We found a significant increase in the average amplitude postprandially (p < 0.05). Although postprandial contractions (n = 243) predominated preprandial contractions (n = 127) significantly (p = 0.02), no significant correlation between the number of contractions and the average amplitude existed (R = 0.1; p = 0.7). Moreover the average amplitude did not differ from amplitudes during intense and small contractions significantly (p = 0.7; p = 0.1). The magnitude of the amplitude measured by EGG does not correlate with the mechanical gastral activity significantly.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence and determinants of house dust mite allergen in East German homes

BACKGROUND: In 1990/91, allergic sensitization to house dust mites (HDM) and other allergens was more prevalent in children from West Germany than from East Germany. OBJECTIVE: To test the hypothesis that low indoor exposure to HDM allergen in East Germany has contributed to this difference. METHODS: HDM allergen concentrations were determined in 634 East German dwellings shortly after the German reunification. RESULTS: HDM group I allergen (Der p 1 + Der f 1) levels in mattresses (median 2.16, geometric mean 2.07, maximum 278.9 microg/g dust) and carpets (median 0.41, geometric mean 0.48, maximum 96.3 microg/g dust) were within the range of levels determined in West Germany in other studies. One particular East German type of dwelling (light concrete buildings) was associated with lower mite allergen exposure, but only a minority of the population lived there. Coal heating, installed in the majority of dwellings before 1989, was associated with higher allergen exposure. Higher relative humidity (RH) was a main risk factor for higher Der p 1 exposure (odds ratio [OR] for exposure to > 0.05 microg/g dust on carpets: 1.4 [95% confidence interval (CI) 1.2-1.8] for + 10% RH) but not for higher Der f 1 exposure. Higher temperature was associated with a lower risk for elevated Der p 1 levels (> 0.05 microg/g dust on carpets): OR 0.6 (95% CI 0.5-0.8) for + 2 degrees C. CONCLUSION: Mite allergen exposure is not lower in East Germany than in West Germany. The data does not support the hypothesis, that low HDM allergen exposure in East Germany is a cause for the lower prevalence of HDM sensitization in East German children.     

Tertiary structure of the major house dust mite allergen Der p 2: sequential and structural homologies

Sensitization to indoor allergens, especially those of the house dust mite, is strongly correlated with the development of asthma. We report the tertiary structure of the major house dust mite allergen, Der p 2, determined by NMR methods. The structure of Der p 2 is a beta-barrel and is composed of two three-stranded antiparallel beta-pleated sheets. This arrangement of beta-strands is similar to the immunoglobulin fold with respect to the orientation of the two sheets and the interactions of the strands. However, the three-dimensional structure of Der p 2 aligns equivalently with a number of proteins from different families within the immunoglobulin superfamily. The structural homology with the highest significance score from analysis by DALI is to Der f 2. Although Der p 2 and Der f 2 are 87% identical in amino acid sequence, they align in three dimensions rather poorly (4.85 A RMSD; Z-score, 8.58). This unexpected finding is likely due to the different solution conditions used during structure determination by NMR for both proteins. While the structural comparisons did not elucidate a clear homologue for the function of Der p 2 in mites, we report that Der p 2 is sequentially homologous to esr16. This is a protein from moths that is expressed coincident with molting. Thus, this homology has important ramifications for the study of mite allergy. The structure of Der p 2 provides a useful tool in the design of recombinant immunotherapeutics for the group 2 allergens.     

Cloning and characterisation of a novel P-glycoprotein homologue from barley

P-glycoproteins are members of a large superfamily of transport proteins (the 'traffic ATPases') that utilize ATP to translocate a wide range of substrates across biological membranes. Using a PCR-based approach, and degenerate oligonucleotides corresponding to conserved motifs, two 300-bp cDNA fragments (pBMDR1 and pBMDR2) with a significant sequence similarity to mammalian P-glycoproteins were amplified from barley (Hordeum vulgare) root poly A+ RNA and used as probes to screen a barley root cDNA library. A single full-length clone pHVMDR2 coding for a polypeptide of 1232 residues (c. 134 kDa) was isolated. Comparison of this barley sequence with Arabidopsis ATPGP1 and human MDR1 and MDR3 P-glycoprotein sequences showed that the barley cDNA has 44%, 37% and 38% amino acid (aa) identity, respectively, with these sequences, and conserved structural features. RNase protection analysis showed that HVMDR2 mRNA is expressed at low levels in both barley roots and leaves. Southern blot analyses indicated that there is a small multigene family related to P-glycoproteins in barley. Possible functions for these barley P-glycoproteins are discussed.     

Cloning and sequencing of the Dermatophagoides pteronyssinus group III allergen, Der p III

House dust mites are widely recognized as major factors involved in the triggering of allergic diseases such as asthma. It is now apparent that the group III allergens of the Dermatophagoides mite species may play a significant role in a number of house dust mite allergic cases. Natural Der p III was isolated by gel filtration of salt precipitated Dermatophagoides pteronyssinus extract and as reported previously ran as a doublet of Mr 28 and 30 K on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Natural Der fIII was isolated by affinity purification with the 5A12 monoclonal antibody. Amino acid sequence data was generated for both these proteins which was used to construct DNA probes to screen a Dermatophagoides pteronyssinus cDNA library by hybridization and resulted in the isolation of a recombinant Der p III cDNA clone, P3WS1. The 1059 bp cDNA fragment included a 786 bp open reading frame which encodes a pre-pro region of 29 amino acids and a mature protein of 232 amino acids with a calculated Mr 24,985. A search of the BLAST protein database has confirmed that the Der pIII P3WS1 clone is approximately 50% homologous with other trypsin proteins. We have confirmed with both our natural protein sequence and the P3WS1 amino acid sequence data that the group III allergens are trypsin-like proteins.     

Cloning and immunological characterization of the allergen Hel a 2 (profilin) from sunflower pollen

Sunflower (Helianthus annuus) sensitization is not always related with occupational allergy. We have isolated the allergen profilin (Hel a 2) from this Compositae plant, cloned and sequenced five cDNAs encoding for full-length or partial Hel a 2. Natural sunflower profilin reacted with specific IgE in the 121 sera tested, at a frequency of 30.5%. Expression of the cDNA encoding Hel a 2 in Escherichia coli and a simple purification procedure by poly-L-proline chromatography allowed immunological characterization of the recombinant allergen. Binding of monoclonal antibodies against sunflower profilin revealed that some epitopes responsible for antigen-specific IgG production were not present in the recombinant allergen. High cross-reactivity has been found between recombinant Hel a 2 and profilins from other Compositae plants and also from botanically distant plants.     

Cloning and characterisation of a thermostable alpha-DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. TY

The mortality patterns of men and women of working age, in terms of the major causes of death, have changed over the past three decades. This study assesses the extent to which mortality among persons of working age represents an economic loss to society. This economic loss is measured by the per capita loss of productive working life, defined as the number of years, on the average, a person can expect to be an active member of the labor force. Causes of death affecting primarily older Americans (heart disease, cancer, stroke) had a relatively small and declining impact on the working lives of men and women. Major causes of death affecting the young (motor vehicle accidents, homicide, AIDS), although accounting for fewer deaths, were responsible for many more years of lost productivity. Gender and socioeconomic differentials in mortality suggest that different strategies are necessary for future reductions in lost work-years.     

Prediction of murine MHC class I epitopes in a major house dust mite allergen and induction of T1-type CD8+ T cell responses

The group I (Der p 1) allergen of Dermatophagoides pteronyssinus (house dust mite, HDM) contains several T helper (Th) epitopes recognized by C57BL/6 mice, with the peptide (111-139) containing a dominant MHC class II-restricted epitope (113-127). Since CD8+ T cells are thought to play a role in the regulation of allergic disease, we examined the Der p 1 sequence for potential MHC class I-binding motifs and observed that residues 111-119 (FGISNYCQI) contain motifs for H-2Db and Kb. Furthermore, immunization of C57BL/6 mice with unadjuvanted Ty virus-like particles (VLP) carrying Der p 1 (111-139), a method known to induce murine cytotoxic T lymphocyte (CTL) responses, primed Der p 1 (111-119)-specific Db-restricted CTL which produce high levels of IFN-gamma and low levels of IL-5 and IL-6 in vitro (T1-type CTL). VLP carrying the minimal epitope (FGISNYCQI) also induced a CTL response following immunization without adjuvant by various routes. Der p 1 (111-139)-VLP adjuvanted with alum did not prime CTL in C57BL/6 mice but were found to prime Th1-type CD4+ T cells that recognize the overlapping peptide (113-127) and native protein. The ability to successfully predict allergen-specific CD8+ T cell epitopes and prime CD8+ and/or CD4+ T cell responses provides an opportunity to dissect the relative roles of these T cells in the regulation of allergic responses and may offer therapeutic potential for reprogramming Th2-type allergic responses.     

Production of a mouse/human chimeric IgE monoclonal antibody to the house dust mite allergen Der p 2 and its use for the absolute quantification of allergen-specific IgE

1. We evaluated the antianginal effects of YM430 in several experimental models in vitro and in vivo. 2. In isolated dog coronary artery, YM430 (10(-8)-10(-6) M) inhibited 3,4-diaminopyridine-induced rhythmic contractions with an IC50 value of 59.2 nM. 3. In anesthetized rats, YM430 (10-100 mg/kg PO) inhibited arginine vasopressin-induced ST-segment depression with an IC50 value of 36.6 mg/kg PO. 4. In anesthetized dogs, YM430 (0.3 mg/kg IV) significantly inhibited ST-segment elevation induced by coronary artery occlusion. 5. These findings suggest that YM430 may be of value in the treatment of various types of angina pectoris such as variant and stable angina.     

The mite allergen and allergoid stimulation of histamine secretion by mast cells

Rat peritoneal mast cells were incubated with serum from highly mite-sensitive patients. It was demonstrated that exposure of passive sensitized mast cells to allergen from mites Dermatophagoides farinae induced the release of histamine. Exposure of mast cells to 10 micrograms/ml and 50 micrograms/ml mite allergen resulted in an increase of histamine secretion to 48% of the basal level. The allergoid (formaldehyde-modified mite allergen) had poor histamine-releasing activity compared to allergen. The allergoid (50 micrograms/ml) induced a 2.5-fold decrease in histamine release. The allergen at the same concentrations and the same release as allergen in dose 0.1 microgram/ml.     

Cloning, expression and characterization of the major latex allergen prohevein

BACKGROUND: About 70-80% of latex allergic health care workers are sensitized to prohevein (Hev b 6.01), a 20 kDa cysteine-rich chitin-binding protein of Hevea latex. OBJECTIVE: This study reports on the bacterial cloning, expression and immunochemical characterization of rHev b 6.01. METHODS: Prohevein was expressed in the periplasmatic space of Escherichia coli as maltose binding protein (MBP) fusion protein and purified to homogeneity after factor Xa cleavage. The IgE binding capacity of both rHev b 6.01 and prohevein isolated from fresh Hevea latex was compared by immunoblotting experiments using sera of latex-allergic patients. The diagnostic value of rHev b 6.01 was analysed by enzyme allergosorbent test (EAST). RESULTS: Two different cDNA clones of rHev b 6.01 were established. The deduced amino acid sequence of both clones revealed two and three amino acid differences in the C-terminal domain of prohevein compared with the original database entry. Purified rHev b 6.01 bound with high affinity to chitin as its natural counterpart isolated from natural latex. In IgE-immunblotting using sera of affected subjects binding intensity to both proteins was comparable indicating a very high antigenic similarity. The diagnostic value of MBP-prohevein was tested in EAST using sera of 33 latex-allergic subjects. The in vitro test showed high sensitivity and specificity and proved the diagnostic value of uncleaved MBP-prohevein. CONCLUSIONS: The production of recombinant latex key allergens with defined quality like prohevein is a straightforward strategy for the development of standardized in vitro test systems.     

Effect of a bed covering system in children with asthma and house dust mite hypersensitivity

Allergen avoidance is regarded as an important approach to management of atopic asthma. The effect of Intervent bed covering systems on house dust mite (HDM) allergen exposure, asthma symptoms and markers of inflammation was investigated in 31 HDM sensitive asthmatic children. Dust concentrations of Dermatophagoides pteronyssinus allergen 1 (Der p 1) were monitored before and after covering the mattress, duvet and pillow with active and placebo covers for 3 months, in a single-blind, cross-over trial. Twice daily peak expiratory flow rate (PEFR), daily symptom scores and treatment schedule were recorded. Bronchial hyperresponsiveness was monitored by histamine challenge (provocative concentration of histamine causing a 20% fall in forced expiratory volume in one second (PC20)), and inflammation by measuring eosinophil cationic protein (ECP), eosinophil protein X (EPX), eosinophil peroxidase (EPO), and soluble interleukin-2 receptor (sIL-2R) in serum. There was a significant reduction in Der p 1 when the mattress, duvet and pillow were covered with the active bedding. There was no significant improvement in symptoms of asthma, PEFR, bronchodilator usage of PC20. Also, ECP, EPX, sIL-2R concentrations did not change for either treatment. EPO concentrations were significantly lower in the active compared to the placebo period. The active bed covers reduced retrievable Dermatophagoides pteronyssinus allergen 1 (Der p 1) from the bedding, with short term clinical benefit.     

Analysis of the IgE-epitope of Der f 2, a major mite allergen, by in vitro mutagenesis

Der f 2 is a major mite allergen composed of 129 amino acid residues. To determine the major epitopes on Der f 2 recognized by human IgE antibodies, artificial mutations were introduced to Der f 2 protein. The IgE-binding activity of Der f 2 was significantly decreased by deletion of 10 amino acids at the N-terminus or nine amino acids at the C-terminus. Site-directed mutagenesis with a single amino acid replacement by Ala or Leu in both N- and C-terminal regions as well as a central portion was performed to generate 42 single-site mutations. Amino acid replacement around a disulfide bond of Cys8-Cys119 caused a marked decrease in IgE-binding activity. Furthermore, a distinct decrease in IgE-binding was also caused by Ala-substitution close to a disulfide bond of Cys73-Cys78 and by mutations of a few charged residues. From these results, it was concluded that the two disulfide-forming regions of Der f 2 and several charged residues are important for forming major epitope structures recognized by human IgE antibodies.     

Epithelial repair in asthma. Do the benefits of house dust mite avoidance result from proteinase avoidance?

OBJECTIVE: To describe the timing of recovery of lung function after severe acute hypoxemic respiratory failure (AHRF) in children. DESIGN: A serial observational follow-up study of clinical and lung function measurements up to 53 months after acute illness. SETTING: University pediatric intensive care unit in a national children's hospital. PATIENTS: Five critically ill children aged 5-14 years. INTERVENTIONS: None RESULTS: Clinical recovery: each patient required a 3-5 month convalescence before being able to attend full-time school because of lethargy and dyspnea. All patients developed wheeze 3-12 months after illness and four received long-term bronchodilator therapy. Lung function recovery: for both the forced vital capacity (FVC) and forced vital capacity in the first second (FEV1) four patients had abnormally low values, regaining only 60-70% of predicted values for their height and sex, and all of this improvement had occurred by 6-12 months after illness. Beyond this interval, patients remained on their same FVC and FEV1 centile. FEV1/FVC ratios were consistently within the normal range, indicating a predominantly restrictive defect. Changes in peak expiratory flow exhibited a time course of improvement similar to the other lung function tests. CONCLUSION: In children, pulmonary recovery after severe AHRF may occur for 6-12 months. A 1-year follow-up could offer a rational single point for assessment of outcome and long-term counselling of child and parents.     

Inhibition of human T-cell responses to house dust mite allergens by a T-cell receptor peptide

Recent analysis of the usage of T-cell receptor (TcR) beta chain variable region (V beta) gene elements by house dust mite (HDM)-reactive T cells from an atopic donor suggested that TcR-V beta 3 gene products may form a major component of the human T-cell repertoire reactive to this common allergen. In this study a peptide analog of the TcR-V beta 3 complementarity determining region 2 (CDR2) is shown to inhibit the polyclonal human T-cell response to HDM; this effect is specific because inhibition is dependent on the presence of V beta 3 + T cells. This experimental approach has been used to determine whether the pattern seen in T-cell clones derived from one atopic donor reflects TcR-V beta usage in the polyclonal response to allergen in the general population. Inhibition of more than 50% of the polyclonal response to allergen by V beta 3-CDR2 peptide was observed in 16 of 21 donors tested, suggesting that TcR-V beta 3 gene usage may form a major component of the human HDM repertoire and as such offer a suitable target for T cell-directed specific immunotherapy in HDM-allergic individuals. Depletion of CD8+ T cells abolishes peptide-mediated inhibition of CD4+ T-cell proliferation to HDM, suggesting that induction of a CD8+ regulatory T-cell subset by the CDR2 peptide may modulate HDM-specific allergic T-cell responses.     

Cloning and functional characterisation of two novel nicotinic acetylcholine receptor alpha subunits from the insect pest Myzus persicae

Nicotinic acetylcholine receptors play a major role in excitatory neurotransmission in insect CNSs and constitute an important target for insecticides. Here, we report the isolation and functional characterisation of two cDNAs encoding nicotinic acetylcholine receptor alpha subunits from a major insect pest, the peach-potato aphid Myzus persicae. These two subunits, termed Mp alpha1 and Mp alpha2, are respective structural homologues of the Drosophila D alpha2/Schistocerca gregaria alphaL1 alpha-subunit pair and the Drosophila ALS alpha subunit. Xenopus oocyte expression confirmed that each Myzus subunit can form functional acetylcholine- or nicotine-gated channels. However, some electrophysiological and pharmacological properties of the Myzus subunits were distinct from those encoded by the corresponding Drosophila subunits. Coexpression of the Myzus subunits with the chick beta2 subunit revealed other differences from the Drosophila system, as only very limited potentiation of agonist-induced currents was observed with Mp alpha2 and none with Mp alpha1. Available data therefore indicate that structurally homologous insect nicotinic acetylcholine receptor alpha subunits from different species can exhibit distinctive physiological and pharmacological characteristics.