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Biomedical subjects

W R Thomas

Publications and source records attributed to W R Thomas.

At least 73 records · Page 4Linked to original sources
PubMed1995

T-cell responses to orally administered antigens. Study of the kinetics of lymphokine production after single and multiple feeding.

Mice fed a protein antigen develop a phenomenon called oral tolerance which is defined classically by the inability to respond to a parenteral challenge with the same antigen. In a recent report we showed that antigen-reactive T cells are not depleted following the development of oral tolerance to the soluble antigen ovalbumin (OVA). Instead mice remain highly sensitized so OVA-reactive T cells can be detected in the mesenteric lymph nodes (MLN), Peyer's patches and spleen. In the present study we show that OVA-specific T cells become sensitized in the MLN within 24 hr of feeding and that lymphokine responses peak 48-96 hr after feeding. T cells produced large amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-gamma) but no interleukin-2 (IL-2) following activation in vitro. Responsiveness as measured by GM-CSF declined by days 8-11 while the ability to stimulate IFN-gamma secretion was more persistent. It was found in experiments with repeated feeding, 1 week apart, that the T-cell responsiveness was restimulated after each feed and that the magnitude and duration of the IFN-gamma or GM-CSF responses were almost identical to primary, even after 10 feeds.

Animals↗
PubMed1994

Characterization of Der p V allergen, cDNA analysis, and IgE-mediated reactivity to the recombinant protein.

A lambda gt11 library for cDNA from Dermatophagoides pteronyssinus was screened by plaque immunoassay, and a number of related clones that produced IgE-binding proteins were isolated. Their sequences were homologous to that of a cDNA described previously, which contained a reading frame encoding a 17 kd polypeptide and which as determined by serologic analysis corresponded to a protein with relative molecular mass of 14 kd in mite extracts. The cDNA from the lambda gt11 clones was truncated so it was used to obtain longer clones from a lambda gt10 library. Analysis of the sequence of these clones showed that the allergen now designated Der p V is produced from a 132-residue polypeptide, which has a putative 19-residue leader peptide and a 113-residue mature protein. This would have a molecular weight of 14 kd, corresponding to that found in mite extracts. IgE binding studies with the lambda gt11 clone and a fusion of the mature sequence in a pGEX construct showed that it reacted with 50% of allergic sera. Further studies with skin tests indicated that it caused reactions in about 60% of patients with asthma and 29% of those with allergic rhinitis alone.

Allergens↗
PubMed1994

An immunogenetic analysis of T-cell reactive regions on the major allergen from the house dust mite, Der p I, with recombinant truncated fragments.

Proliferation assays were used to localize the T-cell reactive sites on the major allergen from the house dust mite Dermatophagoides pteronyssinus, Der p I. Seven overlapping recombinant fragments of the Der p I molecule, synthesized with the pGEX expression vector system, were used to stimulate peripheral blood lymphocytes from 35 HDM-sensitive individuals. The fusion fragments were from 39 to 114 amino acids in length and spanned the entire Der p I molecule. Significant proliferative responses to one or more of the fragments were evident in 18 of the allergic individuals, and each of the fragments led to T-cell stimulation in at least one of the subjects. Although T-cell reactive regions were located throughout the molecule, in 12 of the 18 responsive individuals, major immunogenic sites were contained within the 56 amino acids of the N-terminus, and in 11 of these individuals T-cell reactive regions were only present between amino acid positions 1 and 94. In two individuals reactive sites could be mapped in the C-terminal half of the molecule, and in five subjects, epitopes were present in both N- and C-terminal regions. HLA class I and class II DR and DQ specificities were determined serologically in 16 of the 18 individuals, and no strong pattern of association between HLA type and the T-cell immunogenic region could be detected.

Animals↗
PubMed1994

Differences in epitopes recognized by T cells during oral tolerance and priming.

Feeding protein antigens to mice normally leads to the development of oral tolerance but under some circumstances, feeding can lead to immunity, for example, following pretreatment of mice with cyclophosphamide (CY). In both cases, however, it is possible to detect sensitized T cells in the spleen and mesenteric lymph nodes (MLN) by in vitro lymphokine release for granulocyte-macrophage-CSF (GM-CSF) and IFN-gamma. This study examines the recognition of the immunodominant T cell epitope on ovalbumin (OVA) following intragastric priming and tolerance. T cells from CY/OVA treated mice and cells from mice injected subcutaneously with OVA in CFA responded well to both OVA and the H2d restricted peptide epitope pOVA323-339 releasing GM-CSF. On the other hand MLN or spleen T cells from tolerized mice which responded to the protein in vitro did not recognize the immunodominant determinant. The cells responding from tolerized mice were restricted by the class II MHC so these results show there can be differential recognition of T cell epitopes between oral priming and tolerance.

Administration, Oral↗
PubMed1994

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.

Allergens↗
PubMed1994

Immune reactivity to Der p I and Der p II in house dust mite sensitive patients attending paediatric and adult allergy clinics.

A comparison was made of immune responses to house dust mite allergens in symptomatic patients from paediatric and adult allergy clinics. IgE-specific immune reactivity to Dermatophagoides pteronyssinus was examined by Western blotting and, for Der p I and Der p II, by radioimmune dot-blot using purified allergens. Nineteen mite-sensitive children (mean age 9 years) and 26 adults (mean age 31 years) were compared. Positive IgE responses by dot-blot were found to Der p I and Der p II in 79% of children, whereas reactivity was only present in 23% and 19% respectively of adults, and densitometry indicated a weaker response. In children, Western blotting indicated that the majority of the serologic reactivity was directed to Der p II (17/19, 89%) or a 100 kDa fraction whereas in adults, reactivities were generally directed to other fractions with only 15/26 (58%) recognizing Der p II. Consistent with results of others, a fraction corresponding to Der p I was poorly detected on Western analysis despite positive dot-blots. To determine whether the relative lack of IgE serological immune reactivity in adults was associated with a similar lack of cellular recognition, T-cell proliferation studies were performed, also using purified allergens. Interestingly, these revealed that cellular responsiveness, without serological reactivity, was present in 29% of subjects to Der p I and 50% to Der p II. Proliferative responses were evident in all individuals with specific IgE.

Adolescent↗
PubMed1994

In vivo biological activity of recombinant Der p II allergen of house-dust mite.

The cDNA coding for one of the major allergens of Dermatophagoides pteronyssinus, Der p II, has been cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The recombinant material (r Der p II) was tested in the skin of house-dust allergic patients, and was found to be almost as active as the purified native allergen (n Der p II) in stimulating immediate hypersensitivity reactions. The positivity to r Der p II in these patients, who were selected for their skin-test reactivity to house-dust extract (HDE) and whole mite extract (WME), was over 60%, and 80% of their sera contained detectable IgE antibody against the recombinant allergen. The level of specific IgE antibody against r Der p II was a mean of 40% of that against WME in the sera of patients who were skin test positive to both allergen preparations. In contrast to the house-dust allergic patients, none of a group of allergic patients whose clinical history did not implicate house dust, and who were skin test negative to HDE or WME, were positive to r Der p II. Of interest was the observation that only 37% of a group of subjects who were skin test positive to HDE and WME, but who reported no clinical history of allergic disease upon natural exposure to house dust, reacted to r Der p II. These results demonstrate the biological activity of r Der p II, and indicate, in persons skin test positive to house-dust mite extract, an association between reactivity to Der p II and the manifestation of clinical symptoms.

Adolescent↗
PubMed1994

Inhibition of T-cell responses by feeding peptides containing major and cryptic epitopes: studies with the Der p I allergen.

H-2b mice respond to the 222 residue allergen Der p I by producing T cells sensitized to the dominant epitopes encompassed in peptides 21-49, 78-100, 110-131 and 197-212. Immunization with the synthetic peptides 120-143 and 144-169, however, revealed cryptic epitopes which could sensitize T cells for responses to the respective peptides and, providing splenic adherent cells were added to lymph node cultures, to the whole allergen. It is shown that feeding recombinant fusion peptides can markedly inhibit the ability of the whole antigen to immunize mice, as measured by the in vitro interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 release on stimulation with protein or peptides, although inhibition measured by IL-2 release was more marked. The inhibition extended to epitopes other than those in the fusion peptides used for feeding. Thus feeding peptide 101-154 inhibited responses to 110-131 and 78-100. Fusion peptides 1-14 and 188-222 did not inhibit responses, although 188-222 did contain an epitope. Inhibition was also obtained when mice were fed a fusion containing the cryptic epitope 144-169. The ability of peptides containing the cryptic epitopes to inhibit responses has significant implications for peptide-based immunotherapy.

Administration, Oral↗
PubMed1993

Inhibition of T cell and antibody responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive and sensitized mice.

Antigen-specific CD4+ T cells play an important role in the allergic immune response to house dust mite (HDM) allergens in humans. The group 1 allergen of Dermatophagoides spp. is a major target antigen in both B and T cell recognition of HDM. In vitro studies have shown that the presentation of peptides to human T cells under appropriate conditions may lead to a state of specific nonresponsiveness. Therefore, to determine if peptides are able to modulate the function of allergen-reactive T cells in vivo, we have used a murine model of T cell recognition of the HDM allergen Der p 1. The results demonstrate that inhalation of low concentrations of peptide containing the major T cell epitope of Der p 1 (residues 111-139), induces tolerance in naive C57BL/6J mice such that they become profoundly unresponsive to an immunogenic challenge with the intact allergen. When restimulated in vitro with antigen, lymph node T cells isolated from tolerant mice secrete very low levels of interleukin 2, proliferative poorly, and are unable to provide cognate help to stimulate specific antibody production. Furthermore, intranasal peptide therapy was able to inhibit an ongoing immune response to the allergen in mice and this has potential implications in the development of allergen-based immunotherapy.

Administration, Inhalation↗
PubMed1993

Expression of the leukocyte common antigen CD45 by endothelium.

The CD45 family of Ag expressed by leukocytes play a key role in lymphocyte activation. In this study, we identify both a restricted pattern of expression of CD45 Ag by human endothelial cells and differences in m.w. of endothelial CD45 compared with lymphocyte CD45. Initially, immunoperoxidase staining of endothelial cells in culture revealed the presence of the CD45RO isoform provided the endothelial cells had been stimulated by IL-1 for several days. No other isoform of CD45 was detected by immunoperoxidase staining of endothelium. Leukocyte common antibodies, reputed to detect all isoforms of CD45, did not detect endothelial CD45RO. A polymerase chain reaction method was then used to demonstrate that the message for the CD45RO isoform was constitutively present, with increased levels after IL-1 stimulation. Western blot confirmed the presence of the RO isoform. No other CD45 isoform was detected, either by PCR amplification for message or by Western blot. There were clear differences between lymphocyte and endothelial CD45. The RO isoform expressed by endothelium has an estimated M(r) of 235,000 in contrast to lymphocyte RO that, on our gels, has an estimated M(r) of 190,000. Given the large surface area of endothelium (approximately 1 km2 in the human), the close apposition of lymphocytes and endothelium in immunologic tissues such as lymph nodes, and the pivotal role CD45 plays in activation, expression of CD45RO by endothelium may have important implications in lymphocyte-endothelial interactions. This is most likely to occur in endothelium after a few days of IL-1 stimulation, e.g., at sites of chronic inflammation, or in the draining lymph node of an inflammatory focus.

Actins↗
PubMed1993

Analysis of human T cell responses to the group II allergen of Dermatophagoides species: localization of major antigenic sites.

BACKGROUND: IgE antibodies reactive with the group II allergens of Dermatophagoides species (house dust mite [HDM]) are a major component of the allergic immune response in HDM-allergic atopic individuals. METHODS: Here we demonstrate, with the use of overlapping synthetic peptides of the group II allergen of Dermatophagoides pteronyssinus (Der p II), that polyclonal T cells isolated from the majority of atopic HDM-allergic individuals and healthy nonatopic control subjects respond to Der p II and that T-cell epitopes are present in all regions of the protein. RESULTS: From comparison of peptide-specific T-cell proliferation in both groups of individuals, it appears that together peptides 61-86 and 78-104 are the most frequently recognized (16 of 18 individuals). We also observed that nine of the 18 individuals responded to T-cell epitopes in the region 11-50, and with Der p II-reactive T-cell clones, three distinct T-cell epitopes were mapped within the sequence 11-35. Also, with the use of T-cell clones, two additional epitopes were identified at residues 81-96 and 91-101. CONCLUSIONS: These results suggest that T-cell epitopes located in these regions (11-50 and 61-104) are immunodominant. The value of this information in the potential application of Der p II peptides to desensitize HDM allergic responses is discussed.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of a house dust mite allergen with common antibody binding specificities with multiple components in mite extracts.

Plaque radio-immuno assay has been used to isolate an IgE-binding clone from a lambda gt11 library of Dermatophagoides pteronyssinus cDNA. The clone HD6 contained DNA encoding a 215 residue protein which contained a predicted 17 amino acid residue leader sequence, no cysteines and a single N-glycosylation site. The 198 residue mature protein would have a predicted MW of 22,177 D. No homologues were found in searches of the data banks. Sera from 14/38 allergic children reacted strongly with the polypeptide produced by the clone (37%). Skin tests showed reactivity in 16/30 (53%) allergic patients and 0/10 of controls. Affinity purification of rabbit antibodies with the clone showed that antibodies to the polypeptide had specificities to multiple products in mite extracts corresponding to components of Mr 29, 27 and 24 K by Western blotting. Absorption studies of IgE in allergic serum indicated further entities at 13 and 11.5 kD. It is proposed to name this allergen Der p VII.

Allergens↗
PubMed1993

House dust mite allergy: from T-cell epitopes to immunotherapy.

CD4+ T-lymphocytes induce and regulate allergic inflammatory responses to common environmental aeroallergens derived from Dermatophagoides spp. (house dust mite, HDM), which cause clinical symptoms in approximately 10% of the population. Definition of the molecular structure of HDM proteins combined with the ability to isolate monoclonal populations of human CD4+ T-cells representative of the 'interleukin-4 (IL-4) dominant' functional phenotype, which support immunoglobulin E (IgE) synthesis, has allowed T-cell recognition of HDM to be examined in detail. The results of these investigations demonstrated extensive heterogeneity in both the antigen and HLA class II restriction specificity of the HDM reactive T-cell repertoire. Furthermore, long-lived clones of T-cells with oligoclonality in T-cell antigen receptor (TcR) usage, driven by chronic stimulation with HDM, have been identified in human peripheral blood. The presentation of specific peptides and superantigens under conditions that induce T-cell non-responsiveness has provided an in vitro model for analysing the mechanisms of CD4+ T-cell targeted immunotherapy. It appears that the mechanisms underlying T-cell anergy are accompanied by a transient downregulation of TcR and CD28 and mediated by a shift in the cytokine profile from that of the 'IL-4 dominant' to the 'interferon-gamma (IFN-gamma) dominant' functional phenotype of CD4+ T-cells. In parallel, using a murine model, it has been demonstrated that administration of an immunodominant peptide via the mucosal surfaces of the respiratory and alimentary tracts may tolerize an established response to intact HDM proteins. The potential application of these models in the development of novel approaches to immunotherapy is discussed.

Allergens↗
PubMed1993

Sequence polymorphisms of cDNA clones encoding the mite allergen Der p I.

Sequence information has been obtained from 5 cDNA clones encoding the major house dust mite allergen Der p I, including one which codes for the full length preproenzyme form of the molecule. All translated sequences were unique with 1-3 differences between each clone. 4/5 of the substitutions found were to residues found in Der f I and two positions had a substitution in more than one clone. The polymorphisms included residues already described to be in T cell epitopes, so as well as being necessary to consider them in the construction of synthetic allergens, the substitutions will be important for the interpretation of experimental and genetic studies.

Allergens↗
PubMed1993

Characterization of T-cell responses to the house dust mite allergen Der p II in mice. Evidence for major and cryptic epitopes.

Major histocompatibility complex (MHC) congenic strains can be defined as high and low responders to the major house dust mite allergen Der p II on the basis of the ability to sensitize T cells for in vitro lymphokine release. Mice of the H-2b haplotype were high responders, H-2k were intermediate and H-2d low responders. Like responses to other proteins, only a limited number of epitopes could be located by the response of T cells from mice immunized with allergen to a series of overlapping peptides. The epitopes for H-2b mice were 11-35, 78-104 and 105-129, 36-50 and 78-104 for H-2k mice and 36-60 for H-2d. Immunization with the peptides however revealed that spleen-adherent cells were required for lymph node cells to recall responses to the whole protein and in addition that mice could be sensitized by cryptic epitopes defined by peptides 22-50 and 1-20 for H-2b mice. Peptides containing these cryptic epitopes did not normally induce responses in mice primed with the allergen, but when they were used for immunizing they could prime mice for responses to the peptide and the whole allergen. The results both help to define a model for studying the presentation of allergens and have significant implications for peptide-based immunotherapy.

Allergens↗
PubMed1993

Presentation of peptides and proteins by intestinal epithelial cells.

Murine intestinal epithelial cells (IEC) constitutively express major histocompatibility complex (MHC) class II molecules, which enable them to present foreign antigens to T cells in vitro. In this study we have compared the ability of freshly isolated IEC and spleen adherent cells to present both protein and peptide antigens to T cells in vitro. Consistently IEC were unable to present protein but could readily present immunogenic peptides in an MHC-dependent fashion to stimulate lymphokine release from T cells. Consistent with their inability to process protein antigens, IEC were also unable to present a synthetic peptide which requires intracellular processing, whereas spleen antigen-presenting cells (APC) could readily present the same peptide. These findings indicate that IEC may lack the necessary intracellular machinery to process exogenous antigens but they raise the possibility that IEC may play an important immunological role in vivo by presenting processed peptides to T cells.

Animals↗