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W R Thomas

Publications and source records attributed to W R Thomas.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular characterization of a complex chromosomal translocation breakpoint t(10;14) including the HOX11 oncogene locus.

Based on cytogenetic studies, non-random chromosomal translocations which involve the HOX11 gene at locus 10q24 and the TCR genes at loci 7q35 or 14q11 have been reported to occur in 5% of T-ALL. HOX11, a member of the homeobox family of genes, has been shown to play a role in T-ALL. The activation of the HOX11 gene by translocations to the TCR locus results in the inappropriate expression of a 2.3 kb transcript. In this paper we describe a t(10;14)(q24;q11) breakpoint from a T-ALL patient specimen. The breakpoint appears to be mediated by errors in the TCR/V(D)J recombination system, but is more complex than commonly described reciprocal translocations between the HOX11 and TCR genes, since it involves an inversion event of the TCRdelta genes. In addition, the breakpoint was characterised to a previously unsequenced area of the 10q24 locus, 3.4 kb upstream of the HOX11 gene. This breakpoint is more centromeric than the breakpoint cluster region previously shown to be involved in the majority of reported t(10;14)(q24;q11) translocations. Hence, our investigations of the translocation breakpoint in this patient identify another breakpoint region in the 10q24 locus and may define a novel recombination 'hot spot'. Surprisingly, our studies provide a mechanism for a previously unexplained complex translocation described by another group which involves the same region of the HOX11 promoter.

Base Sequence↗
PubMed1999

HLA-DPB1 alleles in house dust mite allergic patients.

Associations were sought between the presence of allergic sensitivity to Der p 1, a major allergen from the house dust mite, and the HLA-DPB1 genotype. Whilst allergic patients did not differ from controls in DPB1 allelic distribution, there was a correlation of DPB1*11011 with strongly reactive T-cell proliferative responses to Der p 1 and high titre specific IgE to Dermatophagoides pteronyssinus.

Alleles↗
PubMed1999

Molecular characterization of the group 4 house dust mite allergen from Dermatophagoides pteronyssinus and its amylase homologue from Euroglyphus maynei.

BACKGROUND: Of the ten recognised groups of Dermatophagoides pteronyssinus allergens, the group 4 is the only group that has not been characterised at the molecular level. METHODS: Primers were designed to PCR amplify Der p 4 (D. pteronyssinus) and Eur m 4 (Euroglyphus maynei) cDNA. These fragments were used to screen the corresponding cDNA libraries and the cDNA clones obtained were subsequently sequenced. The coding regions of Der p 4 and Eur m 4 were cloned into the pET expression vector and recombinant histidine-tagged proteins expressed in Escherichia coli. RESULTS: cDNA clones which included the mature protein coding sequence for Der p 4 and Eur m 4 were sequenced. The Der p 4 and Eur m 4 genes were found to code for 496 amino acid mature proteins with residues important for the function of alpha-amylase highly conserved. Der p 4 and Eur m 4 were calculated to be 90% identical and a BLAST search of the GenBank database found these sequences to be approximately 50% identical to insect and mammalian alpha-amylases. The calculated molecular weights of Der p 4 and Eur m 4 were approximately 57,000, although recombinant Der p 4 and Eur m 4 migrate on SDS-PAGE at about 60,000. Der p 4 recombinant protein was found to bind specific IgE in 3 of the 10 house dust mite allergic patients tested. CONCLUSIONS: This paper describes the first cDNA sequence of Der p 4 and Eur m 4 confirming that this allergen is house dust mite alpha-amylase.

Allergens↗
PubMed1999

Murine allergic respiratory responses to the major house dust mite allergen Der p 1.

BACKGROUND: Although many studies have examined chronic asthma, limited data exist on acute immunopathogenic events induced by allergens. The aim of the study was to investigate the acute cellular, serologic and histopathologic events in airway inflammation produced by intranasal challenge of mice sensitised to the major house dust mite allergen Der p 1. METHODS: C57BL/6 mice were immunised subcutaneously with Der p 1 in alum. Mice were bled and challenged intranasally with Der p 1 on day 14 and killed on day 17. Lungs were fixed in situ, processed and stained with haematoxylin and eosin. The degree of inflammation and eosinophil infiltration was quantified by image analysis. Specific IgE was determined by passive cutaneous anaphylaxis. Cells from spleen and draining lymph nodes were cultured for 24 h with Der p 1, and IL-3/GM-CSF released into supernatants was measured by bioassay. RESULTS: Intranasal challenge of sensitised mice induced eosinophilic influx into the large and small airways and the alveolar regions of the lung, mucus plugging and in severe cases numerous Charcot-Leyden crystals. The quantitation of the inflammation induced by different sensitisation and challenge doses showed that optimal inflammation could be produced using only 1 microg of allergen for both sensitisation and challenge. The degree of inflammation was not related to the titre of IgE antibody and was indeed produced in its absence. T cell reactivity of spleen cells to the allergen was decreased suggesting cell migration or inactivation. CONCLUSIONS: Mice sensitised and challenged intranasally with as little as 1 microg of Der p 1 produced an extensive pulmonary eosinophilic inflammation which shared many of the features of the inflammation found in asthma. The small amount of allergens required and the use of intranasal challenge should provide a useful model.

Allergens↗
PubMed1999

High-molecular-weight allergens of the house dust mite: an apolipophorin-like cDNA has sequence identity with the major M-177 allergen and the IgE-binding peptide fragments Mag1 and Mag3.

BACKGROUND: A 349-residue recombinant polypeptide of Dermatophagoides farinae, Mag 3, has been shown to represent part of a larger 177-kD (M-177) allergen with very high IgE-binding activity. METHODS: Cloning and sequencing of cDNA from the house dust mites Dermatophagoides pteronyssinus and Euroglyphus maynei was used to characterise the polypeptide containing the Mag 3 sequence. RESULTS: cDNA clones containing the complete sequence of the E. maynei homologue of the M-177 allergen were isolated and analysed. The translation contained not only an amino acid sequence with 90% identity to the 349-residue Mag-3 fragment but also a further sequence with 90% identity to another IgE-binding recombinant D. farinae polypeptide designated Mag 1. The complete sequence encoded a mature polypeptide of 1,650 residues and a molecular mass of 189.5 kD. cDNA clones from D. pteronyssinus also encoded sequences equivalent to the Mag 1 and 3 polypeptides. The M-177 sequence showed strong similarity to the lipid transport apolipophorins found in insect lipophorins. CONCLUSIONS: cDNA sequence data show that the D. pteronyssinus and E. maynei homologues of the M-177 high-molecular-weight D. farinae allergen contain sequences equivalent to both the Mag 1 and Mag 3 recombinant IgE-binding fragments. The N-terminal sequence of the full-length 1,650 amino-acid allergen showed strong similarity to the insect apolipophorins which are poorly soluble in aqueous extracts and exist in the lipid transport particles in haemolymph. It is proposed that presentation in lipid particles could be a factor which enhances the immunogenicity of this group of allergens.

Allergens↗
PubMed1998

Expression of the house dust mite allergen Der p 2 in the baker's yeast Saccharomyces cerevisiae.

BACKGROUND AND RESULTS: The major house dust mite allergen Der p 2 was expressed as a recombinant mature protein in the baker's yeast Saccharomyces cerevisiae. The yeast produces the protein fused to the invertase signal peptide, leading to the secretion of Der p 2 as a soluble protein into the culture medium. The signal peptide is hereby cleaved off, resulting in a mature allergen. In this system Der p 2 was produced in 7.6 (+/-2.9) mg/L growth culture. Purification of the recombinant allergen was achieved by a single gel filtration step, resulting in a purity > or = 95%. The yeast-derived Der p 2 was almost indistinguishable from natural Der p 2 with respect to IgE-reactivity and binding to the majority of Der p 2 specific MoAbs -- as was shown in RAST analysis (n = 168) and a sandwich ELISA and RIA analysis, respectively. Recombinant and natural Der p 2 also showed similar biological activity in histamine release assays (n = 4). CONCLUSION: An expression system for Der p 2 was developed that enables the production of a soluble allergen in the culture supernatant with immunological characteristics similar to the natural allergen. In addition, yeast offers the advantage of the absence of endotoxin in comparison to E. coli. This might facilitate acceptance of recombinant allergens for in vivo applications as immunotherapy or skin-prick testing.

Allergens↗
PubMed1998

Reactivity to recombinant house-dust-mite allergens in asthma and rhinitis in a tropical environment.

BACKGROUND: House-dust mites contain components that are allergenic in mite-sensitive patients, and a number of these have been produced in recombinant form. METHODS: In the present study, we evaluated by skin prick testing the positivity to native Der p 2 and recombinant Der p 2, Der p 5, and Der p 7 allergens of Dermatophagoides pteronyssinus in patients with rhinitis, asthma, or a combination of these diseases, who were positive to whole-mite extract. RESULTS: In all patients, the positivity to both native and recombinant Der p 2 was high. In patients with either rhinitis or asthma, the reactivity to Der p 5 and 7 was significantly lower than to Der p 2. However, in patients with combined disease, the positivity to the minor allergens was almost as high as that to Der p 2. CONCLUSIONS: These results raise the question of whether patients with combined allergic rhinitis and asthma, when compared to those with either of these diseases alone, are predisposed to react to a wider range of mite allergens, or, inversely, whether patients who respond to the minor allergens are more susceptible to suffering the combined disease.

Administration, Inhalation↗
PubMed1998

A 20-kilodalton N-terminal fragment of the D15 protein contains a protective epitope(s) against Haemophilus influenzae type a and type b.

A conserved 80-kDa minor outer membrane protein, D15, of Haemophilus influenzae has been shown to be a protective antigen in laboratory animals against H. influenzae type a (Hia) or type b (Hib) infection. To localize the protective B-cell epitope(s) within the D15 protein and to further explore the possibility of using synthetic peptides as vaccine antigens, a 20-kDa N-terminal fragment of D15 protein (truncated D15 [tD15]) was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. The tD15 moiety was cleaved from glutathione S-transferase by using thrombin and purified to homogeneity. The purified soluble tD15 appeared to contain immunodominant protective epitope(s) against Hia and Hib, since rabbit antisera directed against tD15 were capable of protecting infant rats from Hia or Hib bacteremia. The ease of purification of soluble tD15, therefore, makes it a better candidate antigen than the full-length recombinant D15 which is produced as inclusion bodies in E. coli. Furthermore, both the purified tD15 fragment and a mixture of tD15-derived peptides spanning amino acid residues 93 to 209 of the mature D15 protein were capable of inhibiting the protection against Hib conferred on infant rats by rabbit anti-tD15 antiserum, indicating that the protective epitopes of D15 may not be conformational. However, the administration of pooled rabbit immune sera raised against the same panel of peptides failed to protect infant rats from Hib infection.

Amino Acid Sequence↗
PubMed1998

House dust mite allergen characterisation: implications for T-cell responses and immunotherapy.

T cells are central regulators and mediators of allergic sensitisation and disease. The house dust mite allergens are a biochemically diverse group of proteins which are present in extracts and the environment in a wide range of concentrations. Here the importance of ascertaining the contribution of the different allergens to Th2 lymphokine production and the development of allergy is discussed as well as the effect of allelic polymorphisms and inter-species sequence diversity on T-cell activation. The imbalances in allergen concentrations in commercial extracts, the sequence variations from allergens in the environment and the poor discrimination of allergy due to different mite species points to areas of improvement and the development of novel immunotherapeutic strategies and reagents.

Alleles↗
PubMed1997

Characterization of the specificity and duration of T cell tolerance to intranasally administered peptides in mice: a role for intramolecular epitope suppression.

Mucosal administration of antigens in experimental animals leads to the induction of peripheral T cell tolerance. We have previously reported that in H-2b mice, intranasal (i.n.) or oral administration of a peptide containing the immunodominant T cell epitope will down-regulate the function of CD4+ T cells reactive with Der P 1, a major target antigen in both B and T cell responses to house dust mite. In the present study we have investigated the tolerogenicity of peptides containing both dominant and subdominant determinants when given i.n. to nalve mice. Induction of tolerance by the nasally administered immunodominant peptide leads to a diminution in all T cell-derived cytokines and modulation of delayed-type hypersensitivity responses, but IgE production did not seem to be affected, furthermore the induction of T cell tolerance was stable, lasting beyond 6 months. We have also examined the specificity of intramolecular epitope suppression which is a feature of mucosal tolerance induced by nasally administered peptides and demonstrate that regulatory CD4+ T cells may exert their suppressive effect by linked recognition of epitopes on the same or neighbouring antigen-presenting cells.

Administration, Intranasal↗
PubMed1997

Biological activity of recombinant Der p 2, Der p 5 and Der p 7 allergens of the house-dust mite Dermatophagoides pteronyssinus.

Der p 2, Der p 5 and Der p 7 are three allergens of the house-dust mite Dermatophagoides pteronyssinus that have been cloned and expressed in Escherichia coli as fusion proteins with glutathione-S-transferase (GST). We showed that these recombinant allergens produced immediate hypersensitivity skin-test reactions in 70, 60 and 52% respectively of a group of mite-sensitive allergic patients who were strongly positive to whole mite extract (WME). Comparable positivities were found for serum levels of specific IgE antibody against these allergens, as measured by the radioallergosorbant test (RAST). Overall, for the group of allergic patients that we evaluated, the serum IgE antibody concentrations against Der p 2, 5 and 7 were calculated to represent about one third, one quarter and one fifth respectively of the levels measured against the WME. However, for some patients the activity determined against the separate allergens was far higher than that detected against the WME, thus indicating that the concentration of these can be limiting in the WME. We found no significant correlations between the RAST levels against Derp 2 and either Derp 5 or 7, and RAST-inhibition tests indicated a lack of cross-reactivity between Der p 2 and the other two allergens. In contrast, the RAST results revealed the existence of a significant immunological relationship between Der p 5 and 7. Although a certain degree of reactivity against the GST fusion partner was found in the allergic patients studied, this was not a significant influence in determining the positivity against the recombinant allergens. These results confirm the in vivo biological activity of recombinant Der p 2, 5 and 7, and indicate that whilst Der p 2 is undoubtedly a major mite allergen, both Der p 5 and 7 make important contributions toward the overall allergenic activity of house-dust mites.

Adult↗
PubMed1997

House dust mite immunotherapy results in a decrease in Der p 2-specific IFN-gamma and IL-4 expression by circulating T lymphocytes.

BACKGROUND: Allergen-specific immunotherapy (IT) can be an important adjunctive therapy in the treatment of allergic disorders. Although it has now been used for over 80 yr, the precise mechanism of action remains unclear. Recently a number of studies have shown that cytokine production may be modified by IT, but different protocols have been used and different results obtained. OBJECTIVES: The aims of the present study were: (1) to document the allergen-specific expression of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) by peripheral blood cells in both untreated house dust mite (HDM) allergic patients (non-IT) and following at least 10 months of HDM-specific IT (post-IT); and (2) to determine whether alterations in these critical regulatory cytokines correlated with the clinical outcome of IT. METHODS: IT was undertaken with nine fortnightly subcutaneous injections of increasing amounts of a Dermatophagoides pteronyssinus (Dpt) extract, reaching a final dose of 10,000 PNU. This was followed by 6- to 8-monthly maintenance injections of 5000 PNU. For cytokine measurement, mononuclear cells were separated from peripheral blood and stimulated with the major Dpt allergen, Der p 2, for 18 h, after which mRNA was isolated and IL-4 and IFN-gamma cDNA were amplified by polymerase chain reaction (PCR). The presence of the particular cytokine was determined by visualization following electrophoresis on an agarose gel. The study was observational in nature being open and without a placebo group. RESULTS: Fifteen Dpt-sensitive patients who had not received HDM IT (non-IT), and 16 who had, were studied. In the non-IT group, 80% expressed IL-4 and 75% expressed IFN-gamma. In those post-IT, only 12.5% expressed IL-4 and 19% IFN-gamma. The two patients still expressing IL-4 post-IT had had very little clinical response. Six patients were studied both pre- and post-IT. Prior to IT, three were positive for both cytokines, two positive for IL-4 alone and one for IFN-gamma. Post-IT, all six were negative for IL-4 and five were negative for IFN-gamma. CONCLUSION: Allergen-specific IT results in a reduction in expression of the critical cytokines IL-4 and IFN-gamma in circulating lymphocytes. It is possible that this is a contributary mechanism in the beneficial effect of IT.

Adolescent↗
PubMed1997

Characterization of the allergen Der f 7 from house dust mite extracts by species-specific and crossreactive monoclonal antibodies.

BACKGROUND: The group 7 mite allergens react with IgE in 50% of sera from allergic patients. OBJECTIVE: To determine the molecular and antigenic characteristics and heterogeneity of Der f 7 in mite extracts. METHODS: Monoclonal antibodies (MoAbs) produced from mice immunized with recombinant Der f 7 were examined for crossreactivity to Der p 7 and then used for immunoblotting of 1 and 2-D gel electrophoresis. Deglycosylation was studied with N-glycosidase-F and N-terminal sequencing by Edman degradation. The epitopes of the monoclonal antibodies were compared by cross-inhibitory immunoassays. RESULTS: Immunoblotting of D. farinae extracts with all the anti Der f 7 MoAbs showed major reactivities at 31, 30 and 25 kDa. The strongest immunostaining was at 25 kDa which contrasted with Der p 7 where the 31 and 30 kDa bands were strongest. The relative strength of staining however varied between extracts. The 31 and 30 kDa components were glycosylation products of the 25 kDa form which had the N-terminal sequence predicted from cDNA analysis. Two MoAbs stained an 18 kDa band consistent with a degradation product. The 2-D gels showed that different components with pIs from 5.6-6.4. Both species-specific and Der p 7 crossreactive MoAbs were produced and a two-site ELISA assay for detecting group 7 allergen was developed with MoAbs recognizing different epitopes. CONCLUSIONS: Der f 7 has been defined by its natural N-terminal sequence and MoAbs. It apparently exists as different glycosylation and degradation products in mite extracts, the relative abundance of which differs with different preparations. A two-site ELISA to measure the allergen was developed.

Allergens↗
PubMed1997

T-cell sensitization to epitopes from the house dust mites Dermatophagoides pteronyssinus and Euroglyphus maynei.

BACKGROUND: Dermatophagoides pteronyssinus and Euroglyphus maynei frequently occur in house dust but little is known about primary sensitization to the less abundant E. maynei. OBJECTIVE: To determine the occurrence of primary sensitization to E. maynei by T-cell responses and the crossreactivity to D. pteronyssinus. METHODS: The proliferative response of peripheral blood cells to overlapping peptides from Der p 1 and Eur m 1 were measured as well as to peptides from Der f 1, an allergen not found in the study environment. RESULTS: The most frequent and strongest responses were to Der p 1 peptides especially in the region 105-133. However, 3/17 responders to mite peptides were stimulated predominantly by Eur m 1 peptides and a further two had their highest response to an Eur m 1 peptide. There was very little crossreactivity between Der p 1 and Eur m 1 peptides and very little response to peptides from Der f 1. CONCLUSION: E. maynei group 1 allergens are a significant source of primary T-cell sensitization and have little T-cell crossreactivity with D. pteronyssinus or D. farinae.

Adult↗