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W R Thomas

Publications and source records attributed to W R Thomas.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1988

Sequence analysis of cDNA coding for a major house dust mite allergen, Der p 1. Homology with cysteine proteases.

A cDNA clone coding for Der p 1, a major allergen from the house dust mite Dermatophagoides pteronyssinus, has been sequenced. It codes for a 222 residue mature protein with a derived molecular weight of 25,371 and contains 1 potential N-glycosylation site. In addition, the cDNA appears to code for a 13 residue proregion, and an incomplete signal peptide. The deduced sequence shows a high degree of homology with animal and plant cysteine proteases, particularly in the region of the contact residues making up the active site. Southern analysis of genomic DNA indicates that the allergen is coded by a noncontiguous gene. These data will now facilitate epitope mapping studies.

Allergens↗
PubMed1987

Physicochemical characterization of a major protein allergen, Der p I, from the house dust mite, Dermatophagoides pteronyssinus. Amino acid analysis and circular dichroism studies.

A major house dust mite allergen, Der p I, was isolated from spent growth medium and physicochemically characterized. These studies show that the allergen is monomeric, contains approximately 216 residues and 4 intra-chain disulphide bonds. The N-terminal amino acid is threonine. Circular dichroism studies show that the allergen contains 10% alpha-helical, 50% beta-pleated sheet and 40% random structures.

Allergens↗
PubMed1987

In vitro translation of messenger RNA from the house dust mite Dermatophagoides pteronyssinus.

RNA was extracted from the house dust mite Dermatophagoides pteronyssinus and a polyadenylated [poly(A)+] and non-polyadenylated [poly(A)-] mRNA isolated by oligothymidylic acid-cellulose chromatography. Both preparations were translated in vitro using a cell-free rabbit reticulocyte system. The relative incorporation of 35S-methionine into trichloroacetic acid-precipitable translation products obtained using poly(A)+ and poly(A)- mRNA was 21- and 1.3-fold, respectively, when compared with control translations performed without added mRNA. Sodium dodecylsulphate-polyacrylamide gel electrophoretic analysis of the translation products in combination with autoradiography showed that many proteins with apparent molecular weights in the range 14,000- greater than 67,000 daltons were synthesized. Immunoprecipitation studies performed using a sheep anti-whole mite antiserum showed that several of the synthesized proteins corresponded with mite antigens. These findings were confirmed by coprecipitation studies using crossed immunoelectrophoresis (CIE). A comparison of the CIE data with those obtained with crossed radio-immunoelectrophoresis showed that 6 of the 9 allergens previously described in our laboratory were synthesized, namely Der p I, Dpt 1, 3, 4, 8 and 17. The allergenicity of the lysates was also confirmed by immunoprecipitation studies using a specific anti-human IgE reagent.

Allergens↗
PubMed1987

Early experience with the use of anti-inhibitor coagulant complex to treat bleeding in hemophiliacs with inhibitors to factor VIII.

Early clinical experience with anti-inhibitor coagulant complex in the treatment of bleeding in patients with factor VIII inhibitors is described. Sixty patients with a total of 120 bleeding episodes were studied. Satisfactory responses were obtained in 86.6% of bleeding episodes. Efficacy was slightly better in open bleeding than in closed bleeding. Higher doses were significantly better than lower doses in treating open bleeding. The incidence of adverse reactions was low. The results reported support the safety and efficacy of anti-inhibitor coagulant complex in the treatment of bleeding in patients with factor VIII inhibitors.

Adolescent↗
PubMed1986

Molecular cloning of DNA coding for outer membrane proteins of Haemophilus influenzae type b.

DNA from Haemophilus influenzae type b was cloned in Escherichia coli with a vector lambda gt11 Amp1. Clones producing antigens reactive with hyperimmune rabbit antisera were identified by colony radioimmune assay. A second screening with hyperimmune serum adsorbed with intact H. influenzae type b bacteria was used to identify those clones producing surface-exposed outer membrane proteins. The proteins expressed in E. coli were coupled to Sepharose and used to affinity purify antibodies which were tested for reactivity with outer membrane vesicles. It was found by Western blotting that the clones were producing antigens corresponding to Mr 49,000, 39,500, and 35,000 major outer membrane protein or antigens of H. influenzae type b. Additional clones could be detected by human serum, but their reactivity was not removed when serum was adsorbed with intact bacteria. One of these studied in more detail was found to produce an antigen present in H. influenzae type b lysate but not in outer membrane vesicle preparations.

Antigens, Bacterial↗
PubMed1986

P cell stimulating factor release: a useful assay of T cell activation in vitro.

The coordinate production of multiple lymphokines by activated T cells allows the investigator a choice of assays for antigen recognition in vitro. In this paper, we examine the production of P cell stimulating factor (PSF, a lymphokine identical to interleukin 3) by L3T4+ Lyt-2-T cell clones. PSF production is antigen-specific, Ia-restricted and inhibited by a monoclonal antibody to L3T4, PSF, interferon-gamma (IFN-gamma) and interleukin 2 (IL 2) are all rapidly secreted after antigen or mitogen stimulation. The measurement of PSF avoids problems inherent in the IL 2 and IFN-gamma assays, namely absorption of IL 2 by the producer T cells and failure of the interferon assay to distinguish between interferons of the alpha, beta and gamma classes.

Animals↗
PubMed1985

Characterization of hemopoietic growth factors from T cells and the myelomonocytic leukemia WEHI-3B.

P-cell-stimulating factor (PSF) (also termed interleukin 3) produced by the T-cell clone A3-37.4, the T-cell hybridoma 123, the T-lymphoma EL4, spleen cells, and the myelomonocytic cell line WEHI-3B had a similar apparent mol. wt. and in each case eluted from a Waters C18 silica column at a concentration of acetonitrile of 38%. Both the PSF from the T-cell clones and from WEHI-3B stimulated the in vitro growth of cloned T-dependent mast cells and of colonies from normal bone marrow cells. The T-cell sources--but not WEHI-3B--also produced an additional, distinct hemopoietic growth factor that stimulated the growth of colonies of neutrophils and macrophages but did not support the growth of P cells. This factor was termed T-cell granulocyte-macrophage colony-stimulating factor (T-cell GM-CSF). T-cell GM-CSF eluted from a C18 silica column at an acetonitrile concentration of 41%, differing in this respect from both PSF, which eluted at 38% acetonitrile, and the GM-CSF produced by endotoxin-stimulated mouse lungs.

Animals↗
PubMed1984

In vivo transfer of persisting (P) cells; further evidence for their identity with T-dependent mast cells.

Previously we described the persistent in vitro growth of lines of cells (persisting [P] cells) that shared many cytochemical, biochemical, and functional characteristics with mast cells and depended for their survival and growth on a specific T cell-derived factor, P cell-stimulating factor (PSF). Here we present further evidence for their identity with the T-dependent or atypical subset of mast cells and show that they retain characteristics of T-dependent mast cells when transferred in vivo. One week after the injection of P cells into the dermis of mutant Wf/Wf mice, which have a genetically determined deficiency in mast cells, large numbers of mast cells were present at the injection site, although by 2 wk or later these had disappeared. These mast cells resembled T-dependent mast cells rather than connective tissue mast cells in terms of their size and staining characteristics. Further evidence that these mast cells belonged to the T-dependent subset was that they retained their sensitivity to PSF. Thus, if P cells were injected into the dermis of Wf/Wf mice that bore in one groin a subcutaneous tumor (WEHI-3B) that produced PSF, increased numbers of mast cells were still evident at the injection site 4 wk later; this was not the case in mice bearing a non-PSF-producing variant of the same tumor. Experiments with cloned P cells generated from mice bearing the beige (bgJ/bgJ) mutation and with the giant granules of cells of this genotype used as a marker showed conclusively that the mast cells at the injection sites were derived from the injected P cells. P cells sensitized in vitro with monoclonal antigen-specific IgE or IgG1 antibodies and then injected intracutaneously into W/Wv mice transferred local cutaneous anaphylactic responses. P cells sensitized with IgG1 transferred local cutaneous anaphylactic responses to rats. These results support the view that P cell lines are cognate with the atypical or T-dependent subset of mast cells and that these cells retain their functional capabilities when injected in vivo.

Anaphylaxis↗
PubMed1983

Delayed hypersensitivity induced by anti-T-cell-line antisera is enhanced by pertussigen and not restricted by histocompatibility genes.

Anti-T-cell-line antisera were raised by repeatedly injecting mice with syngeneic, antigen-specific, delayed hypersensitivity (DH) inducing cells grown as continuous T-cell lines in vitro. Many of these antisera could induce antigen-specific DH responses which, in some cases, were rather slight. For example, the DH reaction to azobenzenearsonate induced in A/J mice by an A/J antiserum produced against the syngeneic azobenzenearsonate-specific cell line AA3 was only a weak response. Administration of pertussigen, a protein purified from Bordetella pertussis, markedly enhanced this response and prolonged its duration. Histologically the reaction showed the classical mononuclear infiltration of DH. It could be transferred to naive mice by lymph node cells that were anti-Thy-1.2 sensitive, and the transfer was restricted by genes of the major histocompatibility complex (MHC). The induction of DH by antiserum, however, was not influenced by MHC or non-MHC gene products.

Animals↗
PubMed1983

Hapten-specific help mediated by an anti-ABA T cell line.

Long-term cultures of functional T cells were grown from mice injected with azobenzene arsonate (ABA) coupled spleen cells or mice painted with the diazonium salt of p-phenylarsonate. All cell lines produced delayed hypersensitivity to ABA. When tested for helper function in an antibody-forming cell assay only one line, AA3, helped plaque-forming cell formation. It was able to help anti-FLU response of FLU-HGG primed B cells to ABA-KLH-FLU. The help required linkage of the ABA and FLU determinants because cultures stimulated with a mixture of ABA-KLH and TNP-KLH-FLU did not show increased PFC. These experiments demonstrate hapten-specific help in a system which eliminates involvement of passively absorbed antibody in hapten recognition.

Animals↗
PubMed1983

Induction of suppressor T cells by monoclonal anti-idiotope antibody in strains of mice not expressing the idiotope in hyperimmune serum.

A monoclonal antibody, reactive with an idiotope present on some but not all A/J anti-azobenzenearsonate antibodies bearing the cross-reactive idiotype, was used to induce suppressor T cells. These cells suppressed the effector phase of the delayed hypersensitivity response to ABA but not to oxazolone. They could be enriched on dishes coated with ABA but not with trinitrophenylated human gamma-globulin, and the enrichment could be blocked by the anti-idiotope. Suppressor activity was also enhanced by first incubating T cells with the anti-idiotope and then collecting immunoglobulin-coated cells on dishes coated with anti-mouse immunoglobulin. The suppressor cells could be induced in A/J and CBA mice, even though hyperimmune CBA mice did not produce antibodies bearing this idiotope.

Animals↗
PubMed1983

Delayed hypersensitivity in mast-cell-deficient mice.

The ability of mast cell-deficient Wf/Wf and W/Wv mice to produced delayed hypersensitivity responses was examined. The W/Wv mice did not have detectable mast cells and could not produce IgE-mediated passive cutaneous anaphylaxis. Mice of both genotypes produced large delayed hypersensitivity responses to the contact sensitizers oxazolone and picryl chloride. The responses were indistinguishable from responses of control mice when challenged with optimal or suboptimal doses of antigen. Delayed hypersensitivity could be transferred into Wf/Wf mice by an antigen-specific T cell line, and the proliferative responses in the lymph nodes of these mice after, painting with sensitizer, were normal.

Animals↗
PubMed1982

A search for messenger RNA molecules bearing immunoglobulin VH nucleotide sequences in T cells.

Expression of VH-coded mRNA molecules in T cells, antigen-specific T cell lines, or T cell hybridomas was not detected using four different VH DNA probes under conditions that permitted cross-hybridization between distantly related VH genes. In contrast, VH gene expression was readily detected in two B cell lymphomas and in splenic B cells. Less than one molecule per cell of RNA, exactly complementary to the DNA probes used, would have been detected in these T cell populations. The results thus seriously question the proposition that T cells use the B cell VH repertoire to code for antigen receptors.

Animals↗
PubMed1982

Hapten-specific T cell lines mediating delayed hypersensitivity to contact-sensitizing agents.

Continuous cultures of T cells from the lymph nodes of mice sensitized to the contact sensitizers 4-ethoxymethylene-2-phenyl oxazolone or picryl chloride have been established. For continuous proliferation, the lines required specific antigen, syngeneic antigen-presenting cells, and growth factors from the supernatant of concanavalin-A-stimulated lymphoid cultures. Cells from the lines showed antigen specificity and major histocompatibility complex restriction in proliferation assays and in delayed hypersensitivity. They could mediate delayed hypersensitivity to the sensitizer presented as a reactive hapten or coupled to keyhold limpet hemocyanin.

Animals↗
PubMed1982

Antigen-specific production of immune interferon by T Cells lines.

Continuous cultures of T cells reactive to the hapten 4-ethoxymethylene-2-phenyloxazolone were tested for interferon production after antigenic stimulation in vitro. Induction of interferon was antigen-specific and also restricted by the I region of the major histocompatibility complex. Kinetics of antigen induced interferon production were different from those reported for mitogen induced synthesis.

Animals↗