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Biomedical subjects

W R Thomas

Publications and source records attributed to W R Thomas.

At least 19 recordsLinked to original sources
PubMed1992

Molecular polymorphisms of house dust mite allergens.

Previous studies from this laboratory have described the primary amino acid sequences of the group I and group II allergens from Dermatophagoides pteronyssinus and D. farinae. This report concentrates on polymorphisms of allergens within a species. Firstly, four cDNA clones of Der fII produced by polymerase chain reaction have been sequenced and are compared to the sequences published previously by ourselves and others. Although the sequences come from different sources, Australia and Japan, the overriding conclusion is one of similarity, with only two possible non-conservative changes in the six sequences. The nucleotides were also very conserved including the 3' untranslated regions, although some non-coding differences could be found which may provide a genetic marker. Experiments are reported to help define the group III D. pteronyssinus allergens. Previous studies have characterised the group III of D. farinae as a Mr 29-kDa molecule which can be defined by monoclonal antibodies. A Mr 17-kDa molecule of D. pteronyssinus has been reported with an almost identical N-terminal sequence. Here it is described that Der fIII isolated from different preparations of spent mite media by affinity chromatography have predominantly Mr 32-, 28- and 21-kDa forms which vary in degree from batch to batch. 83% of adults and 38% of children react with the preparation by radioimmune dot-blot. The difference between the children and adults is statistically significant and reactivity can be to at least the 32- and 28-kDa form. Antisera produced in mice against the Der fIII react to D. pteronyssinus mite extract by Western blotting primarily to a 32-kDa moiety, but also 28- and 21-kDa forms in some extracts.

Allergens↗
PubMed1992

T cell responses to the purified major allergens from the house dust mite Dermatophagoides pteronyssinus.

Proliferation assays were used to determine peripheral blood T cell responses to affinity-purified Der p I, Der p II, and Der f II allergens. Patients studied were sensitive to the house dust mite (HDM) either alone or in combination with other allergens. Control subjects were both nonatopic and atopic non-HDM sensitive. In general, only HDM-sensitive patients responded to the mite allergens. Mean stimulation indices (SI) were 10.2 +/- 2.8 (SEM) for Der p I and 10.0 +/- 2.2 for Der p II in HDM-sensitive individuals, and 2.0 +/- 0.2 and 2.8 +/- 0.6 for non-HDM sensitive control subjects (p less than 0.001). Patients sensitive to HDM and other allergens had higher responses than subjects sensitive to HDM alone, and the degree of proliferation to Der p I and Der p II was well correlated in individual patients (r = 0.71; p less than 0.001). Total IgE levels were elevated in the allergic patients, and values were highly correlated with the SI for both Der p I and Der p II (p less than 0.001). Responses to Der p II and Der f II were equivalent in 82% of individuals; however, in 18%, there was a marked discordance with strong responses to Der p II only. SIs were compared between different clinical groups of patients, and patients with asthma had significantly higher values to both Der p I and Der p II than patients with rhinitis alone (17.0 +/- 5.4 versus 5.7 +/- 1.0 for Der p I, p less than 0.05; 14.3 +/- 6.8 versus 5.0 +/- 1.0 for Der p II, p less than 0.05).

Adult↗
PubMed1992

IgE binding structures of the major house dust mite allergen Der p I.

The group I allergens Der p I and Der f I are potent allergens of mites from the genus Dermatophagoides. IgE radioimmune dot blots and immunoabsorption with recombinant peptides have been used to define areas of antigenicity. Four linear binding regions comprising residues 15-33, 60-80, 81-94 and 101-111 were found in the N terminal domain and one, 155-187, in the C-terminal domain, but direct evidence for their discontinuous nature is shown. Firstly, the binding activity of residues 60-80 required either C- or N-terminal flanking sequences to express reactivity and secondly a discontinuous determinant was directly demonstrated by the two non-overlapping peptides 53-99 and 101-154 which significantly cross absorbed specificities to one another. This also indicates considerable homogeneity in the antibodies recognising these peptides. The IgE binding peptides could be located to equivalent residues on the X-ray crystallographic structure of the homologous proteins actinidin and papain. The residues 81-94 and 101-111 which gave strong reactivity were located on a flexible loop connecting the domains and represent areas in which synthetic peptides could be expected to retain activity.

Allergens↗
PubMed1992

High-frequency binding of IgE to the Der p allergen expressed in yeast.

The production of allergens from cDNA clones will provide a clonally pure source of material for experimental and perhaps clinical studies. Attempts to produce the major mite allergen, Der p I, in a highly antigenic form in bacteria have, to date, had limited success. In this study, a high level of production of Der p I from a Cup1 gene cassette from pYELC5-13T in Saccharomyces cerevisiae is described. Although the protein was insoluble, it could be readily solubilized in a urea solution and remained in solution when it was returned to more physiologic buffers. An amount equivalent to about 1 mg/L of yeast culture could then be isolated by affinity chromatography with an immobilized monoclonal antibody. This product reacted strongly with IgE in 9/11 sera from mite-allergic patients compared to the 50% reactivity achieved for Der p I previously produced as a fusion by bacteria. Similarly, the intensity of binding and ability to absorb out Der p I specificities were much greater for the yeast, pYELC5-13T, product. Studies with monoclonal antibodies also demonstrated the yeast, Der p I, had a high degree of antigenicity, although clear differences with the native allergen were demonstrated. The high frequency of reactivity with IgE of the pYELC5-13T formally demonstrates that a single gene product of Der p I is a major allergen and demonstrates that even for Der p I, which is synthesized from a proenzyme, considerable antigenicity can be obtained by expressing the mature protein.

Allergens↗
PubMed1992

Genetic 'risk' for atopy is associated with delayed postnatal maturation of T-cell competence.

Recent in vitro studies suggest that IgE production in adults is co-ordinately regulated by negative signals from gamma IFN-producing CD4+ T-helper-1 (TH-1) and positive signals from IL-4 producing (TH-2) T-cells. Additionally, seroepidemiological evidence has pinpointed infancy as the period of maximum lifetime risk for T-cell sensitization to ubiquitous environmental antigens. The present study sought to elucidate the relationship between these observations, by examination of CD4+ T-cell function in normal children and those genetically at 'high risk' for atopy, spanning the age range (up to 4 years) in which IgE responses to environmental allergens is typically manifest. Immunocompetent T-cell precursor frequencies (determined by cloning at limiting dilution) were markedly reduced in 'high risk' children relative to normals (0.53 +/- 0.29 vs 0.26 +/- 0.19; P = 0.0025). Consistent with reports from other laboratories employing bulk T-cell culture techniques, the gamma IFN producing capacity of CD4+ T-cell clones from both groups of children were markedly reduced relative to adults, and was lowest in the high risk group (P < 0.02). IL-4 production by CD4+ T-cell clones from the normal children was within the adult range, but again was significantly lower in the high risk group (P < 0.00005). This indicates that initial immune responses to environmental allergens in early childhood occur against a background of maturational 'deficiency' in CD4+ T-cell function, and suggests the possibility that variations in the rate of postnatal maturation of T-cell competence may be a contributing factor in the development of differing patterns of immunological responsiveness to environmental allergens.

Adult↗
PubMed1991

IgE and IgG binding of peptides expressed from fragments of cDNA encoding the major house dust mite allergen Der p I.

Large peptides expressed from cDNA fragments of a clone encoding the mite allergen Der p I were able to bind IgE and IgG in sera from allergic individuals. The binding was found for peptides from sequences throughout the molecule, with at least five regions, comprising residues 1-56, 53-99, 98-140, 166-194, and 188-222. The only limitation was that more than 30 amino acid residues were required for consistent binding. Each of seven sera examined showed a different profile of antibody binding to the peptides. For the most part the pattern of IgE and IgG binding to the peptides for each serum was similar, demonstrating a concordant repertoire. In 5/7 sera, however, IgG bound to some peptides which had little or no IgE binding activity, thus showing more diverse specificities. It is suggested that some divergence of repertoire can develop during the maturation of the B cell response.

Allergens↗
PubMed1991

Sequence analysis of cDNA coding for a major house dust mite allergen, Der f I.

A cDNA clone coding for Der f I, a major allergen from the house dust mite Dermatophagoides farinae has been isolated and sequenced. It codes for a putative 18-residue signal peptide, an 80-residue proenzyme region, and a 223-residue mature protein with a derived molecular weight of 25,191. The deduced amino-acid sequence shows significant homology to other cysteine proteases in the proregion as well as in the mature protein. Sequence alignment of the mature Der f I protein with the homologous allergen Der p I from the related mite D. pteronyssinus revealed a high degree of homology (81%) between the two proteins, as predicted by previous sequencing at the protein level. In particular, the residues comprising the active site of these enzymes and the cysteine residues were conserved. A potential N-glycosylation site was present at an equivalent position in both mite allergens. It is anticipated that the availability of recombinant Der f I will facilitate epitope mapping studies and studies of T-cell function in mite allergy by providing high levels of pure allergen.

Allergens↗
PubMed1991

cDNA encoding the major mite allergen Der f II.

cDNA encoding the major house dust mite allergen Der f II from Dermatophagoides farinae was amplified using the polymerase chain reaction and cloned into E. coli. It encoded a 129-residue protein with a calculated molecular weight of 14,021 D and had the expected high homology (88%) with Der p II including the absence of N-glycosylation sites and conserved cysteine residues. These results are consistent with the high degree of antibody crossreactivity and may help identify the differences in T-cell epitopes revealed for these molecules so far.

Allergens↗
PubMed1991

IgE binding studies with large peptides expressed from Der p II cDNA constructs.

The major mite allergen Der p II shows marked resistance to denaturation and is expressed from cDNA in bacteria with almost all of its IgE binding activity. Despite these properties, the IgE binding activity appears to be dependent on maintaining the complete primary structure. Random fragment libraries of cDNA, able to code for up to 93 of the 129 amino acid residue protein, did not express IgE binding peptides. Large overlapping peptides 1-69, 69-129 and 42-117 expressed as the fusions from the glutathione transferase of pGEX vectors only had binding activity with IgE in 15 out of 57 sera, and this was typically weak. Sera from children with atopic dermatitis bound IgE in seven out of eight cases but this was also weak compared with their strong reactivity to intact recombinant Der p II. The inability of such large peptides to form IgE binding structures suggests that the antigenic determinants of Der p II are highly conformational and restricted.

Allergens↗
PubMed1991

Regulation of T-cell sensitization at epithelial surfaces in the respiratory tract: suppression of IgE responses to inhaled antigens by CD3+ Tcr alpha-/beta- lymphocytes (putative gamma/delta T cells).

Repeated exposure of Brown Norway rats to an aerosol of ovalbumin (OVA) induced a state of antigen-specific immunological tolerance, particularly in the IgE isotype. Tolerance was transferable to naive syngeneic animals by inoculation of splenic T cells from tolerant rats. Sequential depletion of tolerant spleen cells by sorting techniques prior to adoptive transfer, employing T-cell subset-specific monoclonal antibodies, indicated that the cells mediating tolerance were CD3+, CD4-, CD5+ and CD8+, but lacked alpha or beta chains in the T-cell receptor (TcR), suggesting that they may be part of the gamma/delta T-cell lineage. Consistent with this suggestion, the sorted population demonstrated considerable enrichment for TcR gamma chain-specific mRNA. As few as 2 x 10(3) cells are sufficient to adoptively transfer tolerance in 200-g adult rats in this model.

Administration, Inhalation↗
PubMed1990

Expression in Escherichia coli of a high-molecular-weight protective surface antigen found in nontypeable and type b Haemophilus influenzae.

An Escherichia coli clone producing a high-molecular-weight surface antigen of Haemophilus influenzae type b (Hib) was isolated from a library of Hib DNA fragments cloned as lysogens in a lambda replacement vector. The antigen is found in sarcosyl-insoluble outer membrane protein preparations and was produced by all 36 H. influenzae isolates tested. Absorption studies indicated that the antigen is a surface determinant on all isolates tested. Antibodies to the antigen (D15) were found in eight of nine convalescent-phase sera from children with invasive Hib infection. Affinity-purified antibodies prepared against the cloned antigen gave protection against the development of bacteremia in a rat pup model.

Animals↗
PubMed1990

Isolation of cDNA coding for the major mite allergen Der p II by IgE plaque immunoassay.

A lambda gt11 library made with cDNA from the house dust mite Dermatophagoides pteronyssinus was screened with human allergic serum by IgE plaque radioimmunoassay. This resulted in the isolation of clones coding for the major allergen Der p II. The cDNA coded for a 129-residue protein of 14,131 daltons with no N-glycosylation sites. No sequence homology with other proteins was evident. The Der p II expressed in Escherichia coli reacted with IgE in 14 of 17 sera from mite-allergic patients giving clonal evidence for its designation as a major allergen. This, along with previous work, has resulted in the cloning of the two major mite allergens.

Allergens↗
PubMed1990

Expression of Dermatophagoides pteronyssinus allergen, Der p II, in Escherichia coli and the binding studies with human IgE.

Lambda gt11 clones expressing the major house dust mite allergen, Der p II, have been reported to react with IgE in the serum of a high proportion of allergic patients. The clones described, however, only produced small quantities of protein which was not fused to the beta-galactosidase of the vector. A construct of the Der p II is described which produces a fusion of Der p II, minus its leader sequence, with the glutathione-S transferase in the pGEX vector. This could be readily isolated and was shown to react with IgE in 22 of 24 patient sera showing reactivity to native Der p II, the sera not reacting having low reactivity to native protein. Absorption analysis showed that the recombinant material removed most of the IgE reactivity of patients to native Der p II. The construct described, therefore, should be valuable for quantitative studies of a pure mite allergen.

Allergens↗
PubMed1990

Antigenic analysis of group I house dust mite allergens using random fragments of Der p I expressed by recombinant DNA libraries.

Antigenic regions of a major house dust mite allergen, Der p I, were identified by a recombinant DNA strategy employing the technique of random fragmentation. Fragments of cDNA coding for Der p I were produced by sonication and used to construct lambda gt 11 expression libraries. Analyses of recombinant fragments reactive with a rabbit anti-Der p I antiserum showed that the B cell determinants expressed in Escherichia coli were limited, with the majority (86%) of antigenic clones isolated mapping to the region comprising amino acid sequence position 60-80. To define antigenic regions of Der p I more precisely, selected overlapping fragments were subcloned into the expression vector pGEX-1. Dot blot immunoassay and immunoabsorption studies using individual fusion proteins revealed five regions - 34-47, 60-72, 82-99, 112-140, and 166-194 - to contain B cell determinants responsible for the antigenicity of recombinant Der p I. Absorption of the antiserum with Dermatophagoides pteronyssinus extract removed reactivity to all fragments, whereas absorption with an extract from the related mite Dermatophagoides farinae removed reactivity to peptides containing residues 34-47, 60-72, and 166-194, but not 82-99 and 112-140. Similarly, rabbit anti-D.farinae reacted strongly with peptides containing residues 34-47, 60-72, and 166-194, but not residues 82-99 and 112-140 which again showed antigenic differences in these residues between the group I allergens.

Allergens↗
PubMed1989

In vivo arming of cutaneous mast cell receptors by IgE released from macrophages.

Intracutaneous injection of purified peritoneal macrophages harvested from ovalbumin (OVA)-hypersensitive high-IgE-responder BN rats into naive animals sensitised the injection sites for subsequent OVA-specific passive cutaneous anaphylaxis (PCA) reactions. The underlying mechanism(s) were investigated using a macrophage cell line (WEHI 265.1), which exhibited comparable sensitising activity in rat or mouse skin, after initial pulsing in vitro with antiserum rich in OVA-specific IgE. Transfer of OVA-hypersensitivity by the cell line (1) was IgE-dependent and did not occur when the cells were pre-exposed to antiserum containing OVA-specific IgG alone, (2) was blockable by saturation of cell surface receptors in the recipient with myeloma IgE (but not myeloma IgG), and (3) did not occur in mast cell-deficient mice carrying the W/Wv mutation, in contrast to their normal heterozygous littermates which developed marked OVA-hypersensitivity at the injection site. These results are consistent with arming of IgE-receptors on cutaneous mast cells by IgE antibody released from macrophages, and hint at a possible role for phagocytes in amplifying IgE-mediated reactions in tissues.

Animals↗