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Biomedical subjects

D G Marsh

Publications and source records attributed to D G Marsh.

At least 37 records · Page 2Linked to original sources
PubMed1996

Association of responsiveness to the major pollen allergen of Parietaria officinalis with HLA-DRB1* alleles: a multicenter study.

Parietaria, a plant belonging to the family of Urticaceae, is a major source of allergenic pollen in Europe. In the context of a multinational study, we investigated whether in allergic subjects antibody response towards Par o 1, the major allergen from P. officinalis, was associated with defined HLA-DRB1* alleles. The study population consisted of 234 allergic patients: 65 from Bulgaria, 30 from Israel, 99 from Italy, and 40 from Spain. In the Italian study group, the prevalence of ST positivity to Parietaria was 77%. In Parietaria ST-positive subjects, the prevalences of IgG and IgE serum Ab towards Par o 1 were 91% and 75%, respectively. HLA-DRB1*1101 and/or 1104 were significantly positively associated with the presence of IgG Ab and with high levels of IgE Ab towards this allergen (p = 0.0007 and p = 0.012, respectively). In the Spanish study group, the positive association of DR1100 with responsiveness to Par o 1 was confirmed (p = 0.02, RR = 4, and p = 0.002, RR = 7, for IgG and IgE Ab, respectively). None of the Bulgarian patients had IgE Ab to Par o 1, whereas IgG Ab response was observed in 7 out of 65 subjects and was positively associated with DRB1*1101 and/or 1104 (p = 0.025). In the Israeli study group, responsiveness to Par o 1 was not associated with specific HLA-DRB1* alleles. In conclusion, this study shows that in allergic patients from three European populations antibody response to the major allergen from the pollen of Parietaria is associated with HLA-DRB1*1101 and/or 1104. Our data suggest that this association is stronger in subjects monosensitized to Parietaria.

Adolescent↗
PubMed1996

Nucleotide sequence analysis of a complementary DNA coding for a Blomia tropicalis allergen.

Blomia tropicalis is a mite of allergenic importance in tropical and subtropical areas. A clone (Bt11a) from a B. tropicalis complementary DNA library was expressed in lambda phage and analyzed by plaque radioimmunoassay. The recombinant allergen produced by this clone was bound by IgE in 16 of 32 sera from individuals with asthma with a positive RAST response and none of 3 control sera from healthy individuals with negative RAST response to B. tropicalis. The cDNA insert was amplified by polymerase chain reaction with use of universal primers. A 582-base-pair (bp) fragment was cloned into a pCR II vector. The complete sequence of both strands was determined by using T7, SP6, and internal primers. The sequence shows a 432 bp reading frame with a 34 bp 5' untranslated region and a 116 bp 3' untranslated region with a poly A tail. Analysis of the sequence suggests that it encodes a putative signal peptide of 20 residues and a 124-residue mature protein allergen of 14,206 Da. The nucleotide and the inferred amino acid sequences did not show homology to any known sequence. No potential N-linked glycosylation site was found. The recombinant protein appears to represent a major allergen of the mite B. tropicalis.

Adult↗
PubMed1996

The conserved lymphokine element-0 in the IL5 promoter binds to a high mobility group-1 protein.

The conserved lymphokine elements-0 (CLE0) in the IL5 promoter is essential for the expression of IL-5. Here, we report the cloning and expression of a cDNA encoding a novel CLE0-binding protein, CLEBP-1 from a mouse Th2 clone, D10.G4.1. Interestingly, it was found that the CLEBP1 cDNA sequence was almost identical to the sequences of known high mobility group-1 (HMG1) cDNAs. When expressed as a recombinant fusion protein in Escherichia coli, CLEBP-1 was shown to bind to the IL5-CLE0 element in electrophoretic mobility-shift assays (EMSA) and southwestern blot analysis. The CLEBP-1 fusion protein cross-reacts with and-HMG-1/2 in Western blot analysis. It also binds to the CLE0 elements of IL4, GMCSF and GCSF genes. CLEBP-1 and closely related HMG-1 and HMG-2 proteins may play key roles in facilitating the expression of the lymphokine genes that contain CLE0 elements.

Animals↗
PubMed1995

IL-13 expression at the sites of allergen challenge in patients with asthma.

Atopic asthma is characterized by inflammatory responses of the airway and is associated with up-regulation of Th2 cytokines, notably IL-4 and IL-5. A recently described human cytokine, IL-13, is a potent in vitro modulator of various cell types, including monocytes, B cells, and endothelial cells. Similar to IL-4, it is also involved in the induction of IgE synthesis. However, the in vivo expression and function of IL-13 and its relation to disease remain to be defined. Using a segmental allergen challenge model, we have examined the in vivo expression of IL-13 in the bronchoalveolar lavage (BAL) cells of atopic patients. We found a significant enhancement of both IL-13 transcripts and secreted proteins in the allergen-challenged BAL compared with the saline-challenged control sites of asthmatic and rhinitic patients. In contrast, the expression of IL-13 transcripts was not detected in the BAL of two normal subjects challenged with the same dose of ragweed allergen. The cellular source of IL-13 mRNA was identified in the mononuclear cell fraction of the allergen-challenged BAL. The allergen-induced quantitative differences in the level of transcripts were confirmed by competitive PCR assays. These results suggest that the significant increase in IL-13 in the allergen-challenged BAL is primarily from the mononuclear cells and is involved in the regulation of allergen-induced late phase inflammatory responses.

Adult↗
PubMed1995

A dominant T cell receptor beta-chain in response to a short ragweed allergen, Amb a 5.

HLA-DR alpha, beta 1 *1501 (and the closely related DR alpha, beta 1 *1502) heterodimers are the primary class II MHC restriction elements controlling T cell responses to a ragweed pollen allergen, Amb a 5 (M(r) = 5000). Using a novel, quantitative, competitive PCR (QC-PCR) assay and a TCR beta-gene internal standard (IS), we have examined the TCR beta-gene use in Amb a 5-specific T cells. Multiple TCR V beta (and V alpha) genes were found in polyclonal T cell lines from two unrelated subjects, and 30% of the cells were stained positive for V beta 5.2/5.3 as judged by a flow cytometry analysis. An Amb a 5-specific Th2 clone (AP1.2) was found to express V beta 5.2 (and V alpha 8) and to have a unique V-D-J junctional region sequence. To determine the relative frequency of clone AP1.2 beta-gene expression in the polyclonal T cell lines and in the PBMC using a QC-PCR assay, we first created a TCR IS by duplicating the J beta 1.5 gene segment of the AP1.2 beta-gene. A QC-PCR assay was performed using the modified AP1.2 beta-gene as a competitor and a V beta 5.2 or a D beta-specific primer, each paired with a C beta primer. Results showed the presence of 30% of AP1.2 beta transcripts in the polyclonal T cell lines, but no detectable amplified products were found in the total RNAs (200 ng) of autologous PBMC. These findings were confirmed by sequencing independent clones of PCR-amplified V beta 5.2-C beta fragments (from two unrelated subjects) from each cell line, which suggested that the frequency of the clone AP1.2 beta sequence is quite low in the PBMC but is dominant in polyclonal Amb a 5-specific cell lines.

Allergens↗
PubMed1994

Linkage analysis of IL4 and other chromosome 5q31.1 markers and total serum immunoglobulin E concentrations.

Sib-pair analysis of 170 individuals from 11 Amish families revealed evidence for linkage of five markers in chromosome 5q31.1 with a gene controlling total serum immunoglobulin E (IgE) concentration. No linkage was found between these markers and specific IgE antibody concentrations. Analysis of total IgE within a subset of 128 IgE antibody-negative sib pairs confirmed evidence for linkage to 5q31.1, especially to the interleukin-4 gene (IL4). A combination of segregation and maximum likelihood analyses provided further evidence for this linkage. These analyses suggest that IL4 or a nearby gene in 5q31.1 regulates IgE production in a nonantigen-specific (noncognate) fashion.

Adolescent↗
PubMed1994

Immunologic and molecular characterization of Amb p V allergens from Ambrosia psilostachya (western Ragweed) pollen.

We have purified and characterized the Amb p V allergen (A1 variant) from western ragweed (Ambrosia psilostachya) pollen. This allergen was found to be highly cross-reactive with the Amb a VA1 allergen from short ragweed (A. artemisiifolia) pollen in a competitive double-Ab radioimmunoassay (DARIA) and the two allergens showed concordant allergenic potency in histamine-release experiments. We cloned and sequenced several Amb p V genes from western ragweed pollen and flowers by direct PCR of genomic DNA. The amino acid sequences deduced from the nucleotide sequences indicated the presence of multiple forms of Amb p V that could be broadly classified into two groups: Amb p VA and Amb p VB variants. The sequences of the Amb p VA variants are highly homologous to Amb a V (about 90% identity) and very similar to the protein sequence that we obtained. The Amb p VB variants share approximately 65% amino acid homology with Amb a V and have five to seven cysteine residues as compared with the eight found in Amb a V and Amb t V. Two cysteine residues that form disulfide bonds in other Amb Vs (positions 19 and 43 in Amb a V) are replaced by serine and alanine in the Amb p VB1 and Amb p VB2 variants. We have generated model structures of Amb p VA1, VA2, VA3, and VB1 variants from the nuclear magnetic resonance-derived structure of Amb a VA1 by homology modeling. Comparison of antigenic epitopes predicted for the structures of Amb p V variants and Amb a VA1 explains the observed cross-reactivity of the two ragweed proteins and suggests the epitopes likely to be involved in Ab recognition.

Allergens↗
PubMed1994

Immunogenetic aspects of IgE-mediated responses.

The reported significant HLA association is consistent with the codominant expression of MHC-linked Ir genes. Similar studies are needed in the case of the other HLA-immune response associations. It seems unlikely that any unique HLA-D genetic sequence will be found only among subjects responding to a particular Ag. It is likely, however, that we will find that a particular sequence is a necessary, but not sufficient, requirement for responsiveness to a particular antigenic epitope. Previous family studies, in which failed to observed parent-to-child transmission of specific immune responsiveness to a particular allergen, suggest that further genetic and/or environmental factors are required for the expression of specific immune responsiveness. These factors include variations in the degree of antigenic exposure. Of particular importance is the need for Ag-specific TcR genes to be expressed in the mature T-cell repertoire. In study of the specific immune response, there have been several studies of the protein and DNA sequences of allergenic proteins; therefore, in regard to understanding the genetics of specific immune responsiveness to allergens and its relationship to atopic diseases, rapid advances can be anticipated over the next several years.

Allergens↗
PubMed1994

Association of class II sequences encoding DR1 and DQ5 specificities with hypersensitivity to chironomid allergen Chi t I.

A panel of 188 unrelated Caucasian subjects who were exposed to the larvae of Chironomus thummi (Diptera, nonbiting midges) was HLA-typed by polymerase chain reaction amplification of the second exons of the DRB, DQA1, and DQB1 genes followed by dot-blot hybridization with sequence-specific oligonucleotide probes. Type I sensitization to the allergen Chi t I and a large number of other inhalant allergens was determined by RAST and skin testing. Sixty-one individuals were found to be sensitized to Chi t I, of whom 24 were sensitive to this allergen and to no other allergens tested. Statistical analyses showed that only in the latter group were the HLA-D genes DRB1*0101, DQA1*0101, and DQB1*0501 associated with IgE-responsiveness to Chi t I. These results suggest that HLA associations with responsiveness to certain allergens may be more striking in monosensitized subjects.

Adolescent↗
PubMed1993

Cloning and expression of immunologically active recombinant Amb a V allergen of short ragweed (Ambrosia artemisiifolia) pollen.

We have cloned and sequenced Amb a V, an Ambrosia artemisiifolia (short ragweed) pollen allergen that has proved to be particularly useful in the genetic analysis of human immune responsiveness. The amino acid sequence deduced from the cloned cDNA sequence corresponds to the published sequence of the protein, except that the cDNA sequence encodes an extra 10 amino acids at the C-terminus. The expressed 55-residue protein is then presumably cleaved enzymatically at the C-terminal lysine found in the 45-residue protein isolated from the pollen. The cloning and sequencing of Amb a V genomic DNA confirmed the cDNA sequence and showed that the Amb a V gene has no introns. Recombinant Amb a V allergen, expressed in Escherichia coli, bound to IgG and IgE antibodies in all Amb a V-allergic individuals tested and inhibition studies demonstrated that the recombinant protein contains a subset of the antigenic epitopes found on native Amb a V. In addition, recombinant Amb a V released histamine efficiently from basophils from Amb a V-allergic patients. The recombinant Amb a V allergen and mutants of Amb a V should, therefore, be useful in studies of allergen epitopes in humans, as well as providing a diagnostic tool.

Allergens↗
PubMed1993

Detection of specific IgE-secreting cells with an enzyme-linked immunospot assay.

BACKGROUND: An in vitro assay system for monitoring the induction of human allergen-specific IgE synthesis with an enzyme-linked immunospot assay is described. METHODS: IgE-secreting cells, responding specifically to short ragweed pollen allergens. Amb a V and Amb a VI, were directly enumerated after the coculture of high-density, resting B cells with either autologous T cells or allergen-specific T-cell clones from the peripheral blood of two ragweed-allergic individuals. Both T-cell clones were type 2-like T helper cells (TH2) as determined by reverse transcription polymerase chain reaction. The IgE-producing cells were detected on a nitrocellulose-based paper disc coated with antigen after treatment with biotinylated, anti-human IgE and avidin-peroxidase conjugate. RESULTS: We demonstrated the induction of Amb a V- or Amb a VI-specific IgE synthesis from either peripheral blood B cells or purified resting B cells. Specific IgE-secreting B cells could be detected only when T cells were present in the culture, providing that they were not separated by a membrane. The addition of interleukin-4 had an enhancing effect on the overall numbers of IgE-secreting cells and allergen-specific T cells and a synergistic effect in the coculture of B cells and an autoreactive T-cell line. CONCLUSIONS: These results suggest that cognate interaction T and B cells is required for the de novo induction of IgE responses. This in vitro system could thus provide a useful model for analysis of the molecular and cellular mechanisms that regulate the allergen-specific IgE responses in human beings.

B-Lymphocytes↗
PubMed1993

Relationship among asthma, serum IgE levels and skin test sensitivity to inhaled allergens.

In two groups of subjects, we have shown that increased high total serum IgE levels (with and without age and sex correction) are significantly associated with the asthmatic phenotype, as diagnosed by a physician. This association was confirmed by discriminant analyses; as compared to variables such as specific sensitivity to allergens and smoking history, age- and sex-corrected log[total serum IgE] was the best predictor of the status of physician-diagnosed asthma in these subjects. Additionally, we observed significantly increased skin test sensitivity to house dust allergens in asthmatics. These findings support previous studies showing that high total serum IgE levels and IgE-mediated sensitivity to house dust allergens are associated with the presence of asthma.

Adult↗