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Biomedical subjects

W R Thomas

Publications and source records attributed to W R Thomas.

At least 55 records · Page 3Linked to original sources
PubMed1996

Immunologic responses following respiratory sensitization to house dust mite allergens in mice.

Allergens from the house dust mite Dermatophagoides pteronyssinus are a major cause of human respiratory diseases, including asthma. In order to help in understanding the early events in allergen sensitization, a murine model of allergic respiratory disease was developed. Mice were immunized by intranasal inoculation of Der p 1 or Der p 2 on days 0, 3, 7, 10, 14, 17, 21 and 29. T cell reactivity was determined using in vitro assays of allergen-specific cytokine production by cells from the spleen, the draining superficial cervical lymph nodes (SCLN) and the non-draining brachial and inguinal nodes. The cytokines assayed in supernatants were IL-4, as a measure of Th2 activation, IL-2 as a measure of Th 1 activation, and IL-3/GM-CSF as an overall marker of T cell stimulation. There was evidence of local and systemic T cell activation by day 7, with the release of IL-2 and of IL-3/GM-CSF by SCLN and spleen cells, respectively. IL-4 production by SCLN and spleen cells was not evident until day 21. T cell sensitization in non-draining node groups was not detected. Intradermal skin tests were performed at the above specified times and positive wheal responses indicated that specific IgE was present from day 3. The above data suggest that respiratory immunization to allergen is rapid and is associated with early systemic sensitization. In this model both Th1 and Th2 responses were evident, with the Th1 response occurring early and the Th2 following after repeated immunizations.

Allergens↗
PubMed1996

Regulation of house dust mite responses by intranasally administered peptide: transient activation of CD4+ T cells precedes the development of tolerance in vivo.

We have previously demonstrated that intranasal (i.n.) administration of an immunodominant peptide (p1-111-139) derived from the house dust mite (HDM) allergen Der p 1 inhibits antigen-specific CD4+ T cell responses in H-2b mice. Here we report that i.n. peptide induced a rapid but transient activation of MHC class II restricted CD4+ T cells that peaked 4 days after peptide treatment and was of similar magnitude to that induced by parenteral immunization with antigen in adjuvant. During the early phase of the response lymph node and splenic T cells secreted a range of lymphokines when re-stimulated in vitro with p1 111-139; however, by day 14 IL-2 and IFN-gamma secretion by T cells were down-regulated. Mice deficient in CD8+ T cells became tolerant by i.n. treatment with peptide, suggesting that CD8+ T cells are not involved in down-regulating the CD4+ T cell response. Rechallenging mice with a single dose of p1 111-139 21 days after the initial treatment elicited a further transient T cell response, which was subsequently down-regulated over time. Although the i.n. peptide induced a strong transient CD4+ T cell response, only low levels of peptide-specific antibodies were detected either after the initial or subsequent i.n. exposures to p1 111-139. Our findings address the mechanisms underlying peripheral T cell tolerance following i.n. administration of a high dose of immunogenic peptide and have implications for understanding the consequences of peptide immunothearapy.

Animals↗
PubMed1996

Comparative analysis of the genes encoding group 3 allergens from Dermatophagoides pteronyssinus and Dermatophagoides farinae.

Group 3 allergens of the genus Dermatophagoides represent one of the major groups of house dust mite allergens. The cDNA sequence data for Der p 3, in combination with both N-terminal amino acid sequences and substrate affinity data, have confirmed that the group 3 allergens are trypsin-like proteases. Using the information from the Der p 3 P3WS1 cDNA clone, genes encoding both Der f3 and Der p 3 have now been amplified by the polymerase chain reaction and analysed. Two Der f3 clones and three Der p 3 genomic clones were sequenced. Each of the clones contained a single small intron and encoded a mature protein of 233 amino acids. The nucleotide sequence was identical for both Der f3 clones. There was 81% identity between the Der f3 sequence and the original Der p 3 P3WS1 clone. The calculated molecular weight of Der f3 was 25.27 kDa compared to 24.98 kDa for Der p 3. All the amino acid residues required for the catalytic activity and the substrate specificity were conserved between the two homologues. The coding sequences of two of the three Der p 3 genomic clones were identical to the original Der p 3 P3WS1 clone with the third having nucleotide changes resulting in four non-conservative amino acid substitutions in the mature protein. These substitutions resulted in a molecule with a slightly larger molecular weight and a more acidic pI value than the original Der p 3 clone. This third Der p 3 genomic clone is, therefore, an isoform of the Der p 3 P3WS1 clone and is classified as an isovariant of the allergen. The nucleotide sequence data presented are the first reported for Der f 3. The Der f 3 gene, like the Der p 3 gene, encoded a trypsin-like protease, but with a slightly larger molecular weight.

Allergens↗
PubMed1996

IgE and monoclonal antibody binding by the mite allergen Der p 7.

BACKGROUND: The recently characterized group 7 house dust mite allergens give positive skin-test reactions in 53% of allergic patients. OBJECTIVE: This study was performed to compare the IgE binding activity of natural and recombinant Der p 7, to measure the binding in allergic sera in comparison to major allergen Der p 2 and characterize the response by competitive inhibition with monoclonal antibodies. METHODS: IgE anti Der p 2 and Der p 7 antibodies against the recombinant allergens and monoclonal binding activities were measured by a solid phase radioimmune assay. RESULTS: A competitive binding assay showed that rDer p 7 inhibited 91% of IgE-binding to natural Der p 7 in 2 sera and 73% in a further two. The IgE binding of rDer p 2 and Der p 7 from 41 sera was then compared. Of the sera 88% and 46% respectively showed positive binding. All of the 19 sera which bound Der p 7 also bound Der p 2 but 11 (58%) had bound IgE to Der p 7 as high or higher than the binding to Der p 2. These sera were mostly high responders to both allergens. A panel of six monoclonal antibodies produced against either rDer p 7 or rDer f7 was used for epitope analysis. All of these reacted with each allergen by enzyme linked immunosorbent assay (ELISA) and immunoblotting. Two patterns of cross inhibition of monoclonal antibody binding were observed and of five monoclonal antibodies tested, four could inhibit the binding of IgE (WH9, WH22, WP8 and HD19) while one (WH14) could not. CONCLUSIONS: Although Der p 7 only reacts with 50% of allergic sera it often has a high IgE binding activity and may be more important than the major Der p 2 allergen in a high percentage of subjects. The combined competitive inhibition experiments show the IgE response is directed at several specificities.

Antibodies, Monoclonal↗
PubMed1996

Sequence polymorphisms of the Der p 3 house dust mite allergen.

BACKGROUND: The trypsin-like protein Der p 3 is a major allergen of Dermatophagoides pteronyssinus. Like other vertebrate and invertebrate trypsin-like molecules, isoelectric-focusing studies with the natural Der p 3 protein have indicated that several isoforms exist. OBJECTIVE: To determine the extent of the sequence variation of the Der p 3 allergen and distinguish at the molecular level, whether the sequence isoforms represent allelic variants or multiple genes of the allergen. METHODS: Five cDNA clones of Der p 3 have been isolated from a lambda gt10 D. pteronyssinus library, using a radiolabelled polymerase chain reaction (PCR) Der p 3 P3WS1 probe and sequenced. Southern blot and inverse PCR analysis of Eco R1 digested genomic DNA was performed. RESULTS: Southern blot analysis of Eco R1 digested genomic DNA showed that the DNA encoding Der p 3 was located on a single 3.5 kb fragment and inverse polymerase chain reaction analysis (PCR) of this DNA showed that there was only a single Der p 3 gene on this 3.5 kb fragment. The nucleotide sequence of one of the clones was identical to the original Der p 3 P3WS1 clone and two clones differed only in their 3' untranslated sequences. The other two contained nucleotide changes which lead to several substitutions at the amino acid level, both conservative and non conservative. Clone 3 had 98.7% identity with Der p 3 P3WS1. One clone for which the full sequence was not available (clone 4) had only 84.4% identity with the original clone and is therefore consistent with an isoallergen. CONCLUSIONS: These data along with our previous genomic sequence shows that for the most part, the Der p 3 allergen has only minor sequence variations (variants) although the isoallergen indicated by clone 4 needs further investigation. It is now evident that Der p 3 is encoded by a single gene and that most cDNA clones constructed from commercial mites show only minor sequence variation similar to that observed for the group 1 and group 2 house dust mite allergens.

Allergens↗
PubMed1996

Analysis of sequence polymorphism of a major mite allergen, Der p 2.

BACKGROUND: The major house dust mite allergen Der p 2 has been regarded as an important allergen involved in the immunopathogenesis of allergic asthma and eczema. OBJECTIVES: To determine the degree of sequence polymorphism exists in Der p 2 at both the genomic DNA and cDNA levels. METHODS: Isolation of cDNA clones was performed by screening the cDNA libraries with Der p 2 cDNA probe and the genomic sequences for Der p 2 were obtained by PCR amplification from environmental dust mites with Der p 2-specific primers. DNA sequencing was carried out by the dideoxynucleotide chain termination method. The study of Der p 2 isoforms was performed by 2-D gel immunoblot analysis using mouse anti-Der p 2 serum. RESULTS: In this study, we have characterized the sequences of three different gene alleles coding for the major house dust mite allergen Der p 2 at the cDNA level. The translated polypeptides from these clones differed from each other by 3-4 amino-acid residues. These polymorphic residues determined were also found in Der f 2 and they were located in regions containing T-epitopes. In addition, the genomic DNA sequences of Der p 2 which were obtained by PCR amplification using the environmental mites isolated from Taiwan and Australia have helped to confirm the authenticity of the polymorphisms detected in the cDNA clones generated from CSL cultured mites. Furthermore, 2D- immunoblot analysis indicated that there were at least 10 different isoforms (p 1 values range from greater than 7.0-5.9) of Der p 2 proteins produced by CSL cultured mites. CONCLUSION: The results showed that there was a small but significant degree of sequence polymorphisms exists in Der p 2 gene alleles. Interestingly, the polymorphic residues were found in regions containing previously determined T-epitopes. The polymorphism data reported here will be important for the understanding of the immune responses of mite allergens as well as for the development of the peptide-based immunotherapeutic reagents for mite allergy.

Allergens↗
PubMed1996

Cloning and sequencing of the group 6 allergen of Dermatophagoides pteronyssinus.

BACKGROUND: The allergen Der p 6 from Dermatophagoides pteronyssinus has been described by substrate affinity as mite chymotrypsin. OBJECTIVE: The aim of this paper was to describe a cDNA clone encoding the allergen. METHODS: cDNA was cloned from a lambda library using oligonucleotides published for Der p 6. RESULTS: The clone P6.1.1 had the N-terminal amino acid residues as reported for Der p 6, and at position 189 the Ser was conserved in chymotrypsin for substrate specificity as well as the catalytic triad of His57, Asp102 and Ser195 for serine proteases. The estimated M(r) was 24.9K and it was 37% identical to the trypsin allergen Der p 3. CONCLUSION: cDNA encoding Der p 6 has been described.

Allergens↗
PubMed1995

The sequencing of the 80-kDa D15 protective surface antigen of Haemophilus influenzae.

The 80-kDa D15 antigen (D-15-Ag) has previously been shown to be a target for protective immunity and conserved amongst typeable and nontypeable Haemophilus influenzae. Here, the gene encoding D-15-Ag is shown to encode a 797-aa polypeptide which, after cleavage of the predicted signal peptide, would have a molecular mass of 85,632 Da.

Amino Acid Sequence↗
PubMed1995

Molecular cloning and immunological characterization of the house dust mite allergen Der f 7.

BACKGROUND: The allergen Der p 7 from Dermatophagoides pteronyssinus has been defined by molecular cloning and shown to be an important specificity in 50% of mite-allergic patients. OBJECTIVE: This study compares the cDNA sequence and serological reactivity of Der f 7 from D. farinae with Der p 7. METHOD: cDNA encoding Der f 7 was amplified by polymerase chain reaction, sequenced and expressed as a fusion with glutathione-S-transferase for IgE and monoclonal antibody binding studies. RESULTS: Der f 7 cDNA encoded a 213 polypeptide containing a predicted 17 amino acid leader sequence, no cysteines and a single N-glycosylation site similar to Der p 7. The predicted 196 residue mature polypeptide had 86% identity to Der p 7 and a calculated molecular weight of 22,348Da. No homologues were found in searches of the data banks. The Der f 7 fusion protein showed a single band of 46 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reacted with IgE antibodies in 19/41 (46%) of sera from asthmatic children. The degree of binding was usually 30% of that to Der p 7 consistent with the exposure of the patients to D. pteronyssinus. Monoclonal antibodies (WH9 and WH22) against Der p 7 reacted with Der f 7 but inhibition studies showed a 10-fold difference in reactivity. CONCLUSION: Der f 7 has a predicted 213 residue polypeptide with 86% homology and serological crossreactivity to Der p 7.

Allergens↗
PubMed1995

Characterization of the house dust mite allergen Der p 7 by monoclonal antibodies.

The house dust mite allergen Der p 7, which was defined by cDNA cloning, has been shown to react with about 50% of allergic sera and corresponds to or is antigenically related to at least three different sized components in mite extracts. To characterize these entities, monoclonal antibodies (MoAbs) were generated by immunizing BALB/c mice affinity-purified Der p 7-GST (glutathione S-transferase) fusion protein. MoAbs WH9 and WH22 showed positive reactivity to recombinant Der p 7 negative reactivity to GST and the Der p 5-GST fusion protein in ELISA and immunoblotting. The specificity of both MoAbs was confirmed by inhibition of the ELISA activity by recombinant Der p 7 but not by the recombinant Der p 5. Immunoblot analysis demonstrated that both MoAbs showed reactivities to components with molecular weights (mol. wt.) of 31, 30 and 26 kDa reactive to both MoAbs. At least six major forms with different pI or size were indicated by 2-D gel analysis. In addition to characterization of Der p 7, both MoAbs may also be considered for use in the standardization of Der p 7 in mite extracts.

Allergens↗
PubMed1995

Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.

BACKGROUND: It is widely held that in vitro T cell responses to allergens are more prominent in atopic than in normal individuals, though this conclusion is based upon culture techniques which fail to detect proliferative responses in a significant minority of atopics and many normals. OBJECTIVES: Study allergen-specific proliferative responses of T cells cultured in serum-free medium (SFM). Examine associations between atopic status, age and T cell reactivity. METHODS: Initially, peripheral blood mononuclear cells were stimulated with allergens or antigens in SFM, and compared with cells cultured in RPMI + 10% fetal calf serum or human AB serum. Subsequently, T cell reactivity was studied in 34 adults (20-49 years), 27 children (2-13 years), and 19 infants (< or = 10 weeks) using SFM alone. RESULTS: Compared with serum-supplemented medium, SFM enhanced net T cell proliferation, both in bulk culture and when cloning at limiting dilution. In many subjects, SFM unmasked T cell reactivity to allergens which was not otherwise evident, and lowered the threshold allergen levels required for in vitro T cell triggering. For most allergens, T cell proliferative responses did not differ between adults who had specific IgE, and those who did not. The most vigorous responses observed were to ubiquitous inhalant allergens, which stimulated T cells from close to 100% of adults and children, and over 60% of infants. In contrast, responses to the 'vaccine' antigen tetanus toxoid were completely absent in the latter age group, but present in the majority of adults and children. CONCLUSIONS: These findings suggest that the extent of active T cell recognition of environmental allergens has been hitherto underestimated, and further that these responses may frequently be initiated in very early life. Additionally, these findings reinforce the notion that qualitative (as opposed to quantitative) variations in specific T cell reactivity ultimately determine allergen responder phenotype.

Adult↗
PubMed1995

T-cell "priming" against environmental allergens in human neonates: sequential deletion of food antigen reactivity during infancy with concomitant expansion of responses to ubiquitous inhalant allergens.

The study below comprises prospective analysis of patterns of allergen-specific T-cell reactivity in a cohort of 23 children bled at regular intervals from 6-10 weeks to 2 years of age, together with cross sectional studies on panels of cord and adult blood samples. The results indicate reciprocal patterns of responses to dietary and inhalant allergens, the former being frequent in infancy but rare in adults, whereas the latter are preserved and expand between infancy and adulthood. These findings are consistent with a recently proposed model for the development of immunity to environmental allergens which involves allergen-driven T-cell "selection" during early life leading to deletion of food allergen-specific T-cells via the induction of specific anergy, with concomitant selection and ultimately expansion of mutually exclusive TH-1-like or TH-2-like reactivity to inhalant allergens via Immune Deviation mechanisms.

Administration, Inhalation↗
PubMed1995

An immunogenetic analysis of the T-cell recognition of the major house dust mite allergen Der p 2: identification of high- and low-responder HLA-DQ alleles and localization of T-cell epitopes.

Cellular reactivity to Der p 2, a major allergen of the house dust mite (HDM) Dermatophagoides pteronyssinus, was studied in a group of 41 symptomatic HDM sensitive patients, using fresh peripheral blood mononuclear cells (PBMC) and assays of proliferation. Sixty per cent of the patients responded to Der p2, with reactivities being greater in patients with asthma as one of their clinical manifestations and also in those who had skin-test reactivity to a number of allergens. HLA-DR and -DQ serotyping was undertaken in 39 of the patients and the magnitude of T-cell proliferative responses to Der p 2 were found to be positively associated with DQ7 and negatively associated with DQ2. T-cell determinants within the Der p 2 molecule were identified by assays using a series of overlapping peptides (15- to 19-mers) spanning the entire protein. Fifty-nine per cent of the 41 HDM-sensitive patients responded to one or more of the peptides. All of the peptides were antigenic for at least one of the individuals, indicating the heterogeneity of the human repertoire reactive with Der p 2. There was a substantial variability in the number and location of epitopes recognized by T cells from the different allergic patients, the mean number per patient being 2.3 +/- 1.3 (SD). The most frequently recognized peptide was that spanning residues 111-129, being stimulatory in 66.7%, the other peptides were each recognized by between 8 to 25% of individuals. There was no correlation between the epitope recognized and the presence of particular HLA-DQ antigens.

Adolescent↗