Search PubMed⌕ Search

Biomedical subjects

D G Marsh

Publications and source records attributed to D G Marsh.

At least 55 records · Page 3Linked to original sources
PubMed1993

Analysis of cytokine transcripts in the bronchoalveolar lavage cells of patients with asthma.

A panel of steady-state cytokine mRNAs was analyzed in the bronchoalveolar lavage (BAL) cells from asthmatic subjects or patients challenged with ragweed allergen. This was achieved by combining both qualitative and quantitative assays using the reverse transcription-polymerase chain reaction (RT-PCR). Analysis of BAL cells from six mild allergic asthmatic and five nonasthmatic, nonallergic subjects showed no qualitative differences in the profile of cytokine mRNAs (including interleukin [IL]-1 beta, IL-2, IL-5, IL-6, IL-8, and granulocyte/macrophage colony-stimulating factor), except for tumor necrosis factor-alpha, which was detected in three out of six asthmatic BAL samples but in none of the controls. A key cytokine, IL-5, has been implicated in the pathogenesis of allergic inflammation through the recruitment of eosinophils. We found a significant enhancement of steady-state IL-5 transcripts in the BAL cells from allergen-challenged as compared with the saline-challenged control sites of four asthmatic patients; furthermore, the cellular source for IL-5 mRNA was identified in the mononuclear cell fraction, but not in the purified eosinophils, of the allergen-challenged BALs. These results suggest that the significant increase of IL-5 transcripts is primarily from the infiltrating mononuclear cells. Our study also demonstrates the power of qualitative and quantitative PCR analysis in determining the molecular basis of allergic inflammatory diseases.

Adult↗
PubMed1993

The effect of interferon-gamma, interleukin-4 and immunoglobulin receptor cross-linking of monocytes on allergen-specific T-cell response.

Modulation of the proliferative responses of an allergen-specific human Th2 cell line by cytokine-treated monocytes was examined. The response of this cell line to the specific allergen, Amb a V (from short ragweed pollen), increased following the addition of interleukin-1 beta (IL-1 beta). However, in the presence of exogenous interferon-gamma (IFN-gamma), there was greater than 40% reduction in the responsiveness of these T cells. The addition of IL-1 beta did not reverse the inhibitory effect of IFN-gamma. To determine the primary target cell type for IFN-gamma, autologous monocytes were pretreated with IL-4, IFN-gamma, or medium alone, and used as antigen-presenting cells (APC). We showed that the responses of T cells to Amb a V were significantly down-regulated in the presence of autologous monocytes pretreated with IFN-gamma, but not for monocytes pretreated with IL-4. Similar inhibitory effect of IFN-gamma was confirmed using a human T-cell line specific for a ragweed allergen, Amb a I, and a human T-cell clone raised against ragweed extract. Cross-linking of CD23 (Fc epsilon RII) on monocytes pretreated with IFN-gamma increased this inhibitory effect in an additive fashion, but, in the absence of IFN-gamma treatment, such cross-linking had no effect. These inhibitory effects were not due to alterations in the surface expression of HLA-DR on the monocytes, and the addition of exogenous IL-1 beta was unable to reverse these effects. In similar experiments, cross-linking of CD64 (Fc gamma R) on monocytes showed no significant effects. In conclusion, IFN-gamma is important in regulating the function of monocytes involved in Th2 cell responses to allergens. IL-4 treatment, as well as cross-linking of FcR of monocytes, have no direct effect on such response.

Allergens↗
PubMed1992

Expression and analysis of recombinant Amb a V and Amb t V allergens. Comparison with native proteins by immunological assays and NMR spectroscopy.

The Amb V allergens are small, highly disulfide-bonded ragweed pollen allergens that serve as useful models for understanding the molecular basis of the human immune response. We have produced recombinant Amb a V and Amb t V (from short and giant ragweed pollens, respectively) in Escherichia coli and have compared their structural and functional characteristics to those of the native proteins. Recombinant Amb t V was indistinguishable from native Amb t V as determined by NMR spectroscopy and antibody-binding studies. Whereas inhibition analysis showed that recombinant Amb a V possessed only approximately 50% of the antibody-binding activity of native Amb a V, the two proteins were similarly effective in stimulating Amb a V-specific T-cells. Our results demonstrate that even highly homologous proteins exhibit different abilities to fold into their native three-dimensional conformations and establish the potential and limits of expressing the recombinant Amb V allergens intracellularly in E. coli.

Allergens↗
PubMed1992

Proton resonance assignments and three-dimensional solution structure of the ragweed allergen Amb a V by nuclear magnetic resonance spectroscopy.

Essentially complete assignment of the proton resonances in the allergenic protein Amb a V has been made by analysis of two-dimensional NMR experiments. Conformational constraints were obtained in three forms: interproton distances derived from NOE cross-peak intensities of NOESY spectra, torsion angle constraints derived from J-coupling constants of COSY and PE-COSY spectra, and hydrogen bond constraints derived from hydrogen-exchange experiments. Conformations of Amb a V with low constraint violations were generated using dynamic simulated annealing in the program XPLOR. The refined structures are comprised of a C-terminal alpha-helix, a small segment of antiparallel beta-sheet, and several loops. A hydrophobic core exists at the interface of the alpha-helix and beta-sheet. The derived structure accounts for the several anomalous proton chemical shifts that are observed. The structure determined here for Amb a V is topologically similar to the structure determined previously for the homologous allergenic protein Amb t V [Metzler, W. J., Valentine, K., Roebber, M., Friedrichs, M. S., Marsh, D., & Mueller, L. (1992) Biochemistry 31, 5117-5127]; however, significant differences exist in the packing of side chains in the hydrophobic core of the molecules. Comparison of the detailed structural features of these two proteins will allow us to suggest surface substructures for the Amb V allergens that are likely to participate in B cell epitopes.

Allergens↗
PubMed1992

Determination of the three-dimensional solution structure of ragweed allergen Amb t V by nuclear magnetic resonance spectroscopy.

Analysis of two-dimensional NMR experiments has afforded essentially complete assignment of all proton resonances in the allergenic protein Amb t V. Conformational constraints were obtained from the NMR data in three forms: interproton distances derived from NOE cross-peak intensities of NOESY spectra, torsion angle constraints derived from J-coupling constants of COSY and PE-COSY spectra, and hydrogen bond constraints derived from hydrogen-exchange experiments. Conformations of Amb t V with low constraint violations were generated using dynamic simulated annealing in the program XPLOR. The refined structures are comprised of a C-terminal alpha-helix, a short stretch of triple-stranded antiparallel beta-sheet, and several loops. In addition, the cystine partners of the four disulfide linkages (for which there are no biochemical data) have been assigned. The refined structures of Amb t V will allow us to suggest surface substructures for the Amb V allergens that are likely to participate in B cell epitopes and will assist us in defining the Ia/T cell epitopes that interact with the MHC class II (or Ia) molecule and the T cell receptor leading to the induction of the immune response to Amb t V.

Allergens↗
PubMed1992

Outcome of urgent percutaneous transluminal coronary angioplasty in acute myocardial infarction: comparison of single-vessel versus multivessel coronary artery disease.

Despite recent clinical trials of percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction, specific groups of patients that may benefit from adjunctive or alternative therapy have yet to be adequately characterized. The in-hospital outcome of 151 consecutive patients treated for acute myocardial infarction with urgent PTCA of the infarct-related artery was studied to identify a subgroup of patients at high risk. Patients were divided into two groups based on the angiographic presence of either single-vessel (n = 86) or multivessel (n = 65) coronary artery disease. Despite PTCA of only the infarct-related artery and similar baseline clinical characteristics such as age, peak serum creatine kinase concentration, left ventricular ejection fraction, and time from the onset of chest pain to arrival at the hospital, the group with multivessel disease had a lower rate of successful angioplasty (75% vs 92%, p < 0.005), with higher incidences of persistent total occlusion of the infarct-related artery (14% vs 3%, p < 0.02) and procedural complications during PTCA (28% vs 13%, p < or = 0.02), and were more likely to have multiple complications (12% vs 1%, p < 0.004). In addition, the group with multivessel disease had a higher rate of urgent (< or = 24 hours) coronary artery bypass graft surgery (13% vs 2%, p < 0.05) and a trend toward a higher in-hospital mortality rate (6% vs 1%, p < or = 0.17). By stepwise logistic regression, only the presence of single-vessel versus multivessel disease was predictive of PTCA success (p < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

A novel HLA-haplotype containing a DRB5 gene not associated with DRB1*15 or DRB1*16 alleles.

In Caucasians, HLA-DR2 haplotypes usually express two DRB genes encoding the DR specificity. The DRB5 genes are in strong linkage disequilibrium with the respective DRB1*15 or DRB1*16 alleles. So far, no other DRB1 alleles have been found in association with DRB5 genes. Here, we report evidence for a probably recombinant DRB5 haplotype with a DRB1 allele not specific for DR2. From our results the haplotype DQB1*0501, DRB1*0101 and DRB5*0101 seems most likely.

Adult↗
PubMed1991

Cloning the cDNA encoding the AmbtV allergen from giant ragweed (Ambrosia trifida) pollen.

Ragweed (Ambrosia) pollens contain a number of proteins that cause allergic disease in ragweed-sensitive people. The cloning of the AmbtV cDNA is important, since the 4.4-kDa AmbtV, one of the allergens in giant ragweed (Ambrosia trifida) pollen, serves as a simple model system to study the basic structural requirements for immune recognition of foreign protein allergens. We report the cloning of the AmbtV cDNA by means of the polymerase chain reaction (PCR) using degenerate primers. We generated three sets of overlapping cDNA clones by a combination of PCR and anchored-PCR, and determined the complete nucleotide (nt) sequence. From the nt sequence, the amino acid (aa) sequence of the protein was confirmed and the leader sequence was deduced. This general approach can be used to clone allergen and other cDNAs from complex biological sources provided partial aa sequence information is available. It may be the best available approach in cases where the isolation of clones from a cDNA library is difficult, which proved to be the case for AmbtV.

Allergens↗
PubMed1991

Class II major histocompatibility complex restriction of human T cell responses to short ragweed allergen, Amb a V.

Although T cells are known to play a crucial role in the induction of IgE synthesis, the class II major histocompatibility complex (MHC) restriction of aeroallergen-induced T cell responses in humans is incompletely defined. We have previously shown that, in allergic Caucasoid individuals, HLA-DR2 and Dw2 (DR2.2) is strongly associated with specific IgE and IgG antibody responses to highly purified Ambrosia (ragweed) allergen, Amb a V, from the artemisiifolia (short) species. For example, 95% of IgE antibody responders to Amb a V were typed as DR2.2. In a novel study of the genetic control of T cell responses to the Amb a V allergen, we have investigated the MHC class II restriction specificity of three CD4, Amb a V-specific T cell clones derived from a DR2.2+ atopic patient, and a polyclonal Amb a V-reactive T cell line from another DR2.2+ patient. We observed proliferative responses of all three clones to Amb a V only when either HLA-DR2.2 or DR2, Dw12 (DR2.12; found on Mongoloid populations) was present on the antigen-presenting cells, regardless of the HLA-DQ phenotype of the cells. Moreover, the responses of T cell line and clones were abolished by anti-DR but not by anti-DQ nor by anti-DP monoclonal antibodies, and, significantly, anti-DR alpha/beta I2 (anti-DR alpha /beta Iw15/w16; anti-"DR2b") monoclonal antibody blocked, in a dose-dependent manner, the cloned T cell responses to Amb a V. These findings demonstrate that DR alpha/beta I2.2 (DR alpha/beta I1501) and DR alpha/beta I2.12 (DR alpha/beta I1502) are functional in the restriction of the T cell recognition of Amb a V. These findings also illustrate the power of the allergy model for definitive investigation of the molecular basis of the human immune response.

Allergens↗
PubMed1991

Genetics of total serum IgE levels: a regressive model approach to segregation analysis.

The genetics of basal total serum IgE levels was investigated in 278 individuals from 42 randomly ascertained nuclear families. The data were analyzed using the regressive model approach to segregation analysis with age, sex, and a measure of skin test responsiveness as covariates in the Class D models. The best fitting model was that of recessive inheritance of high IgE levels with a gene frequency of 0.99 for the "high" allele. Only 3 families showed evidence for segregation of the rare "low" allele, and, if extended further, these families could be useful for molecular genetic linkage studies. These results suggest that there may be a rare allele for very low total serum IgE levels that can be detected even after a measurement of allergic responsiveness (skin test results) is considered as a covariate. Therefore, this major gene for IgE levels appears independent of any similar locus controlling atopy.

Adolescent↗
PubMed1991

Sequencing of HLA-D in responders and nonresponders to short ragweed allergen, Amb a V.

We investigated the molecular basis for the striking association between HLA-DR2,Dw2 and human immune responsiveness to the Ambrosia artemisiifolia (short ragweed) pollen allergen Amb a V by sequencing the second exons of the DRB and DQB1 genes of 17 selected ragweed-allergic Caucasoid subjects. We also studied the DQA1 allelic polymorphic regions (APRs) in these patients by dot-blotting using sequence-specific oligonucleotides (SSOs). The deduced amino acid sequences of the respective class II beta and alpha polypeptides were compared, with particular emphasis on residues in the APRs that are implicated in antigen binding. No evidence for "new" HLA-DRB or DQB sequences unique to Amb a V responders were found on sequencing seven Dw2+ subjects. This suggests that the presence of a particular Dw2-associated class II molecule usually provides a necessary, but not always sufficient condition for responsiveness to Amb a V. The HLA phenotypes of three subjects suggest that they possess novel recombinant haplotypes containing either DRB1*1501 and DRB5*0101 (DR2.2-associated) or DQB1*0602 (DQ1.2-associated) sequences. In these subjects, responsiveness to Amb a V was associated with the DR2.2 but not the DQ1.2 sequences, suggesting that DR alpha beta I or DR alpha beta V class II molecules are involved in antigen presentation. We investigated whether there may be shared HLA-D-encoded responder sequences present in all responders, including some exceptional DR2- Amb a V responders. The 13 subjects producing antibody (Ab) responses to Amb a V [either from natural exposure and/or after ragweed immunotherapy (Rx)] possessed DRB1*1501, 1601, 1602, 0103, 0402, 0404, 0801, or 1101 sequences, which share the majority of their aa residues in the APRs 2-4. Some of these shared residues might be important for the binding of a common Amb a V agretope prior to presentation of the class II Amb a V complex to the T-cell receptor (Tcr). An alternative postulate is that the recognition of two different Amb a V agretopes may be determined by the beta 1 polypeptides of molecules having the DR2 and DQw3 specificities.

Alleles↗
PubMed1991

HLA-D gene studies in relation to immune responsiveness to a grass allergen Lol p III.

The grass pollen allergen Lol p III (Mr 11,000) is a well-characterized antigen that has been found useful in immunogenetic studies of human immune responsiveness. Since immune responsiveness to this allergen is associated with HLA-DR3, we investigated whether there was any sequence in the HLA-D region that would render a person "susceptible" [antibody (Ab)-positive] to the allergen. By sequence-specific oligonucleotide (SSO) slot-blot and sequence analyses of polymerase chain reaction (PCR)-amplified genomic DNA from Lol p III responder and nonresponder subjects, Ab responsiveness was found to be strongly associated with the sequence Glu-Tyr-Ser-Thr-Ser (EYSTS), present in the first polymorphic regions of DR beta I polypeptide chains of DR3, DR11 (split of DR5), and DRw6. Of the 41 grass-allergic subjects investigated, 19 had the EYSTS sequence, of whom 18 (95%) were Lol p III immunoglobulin G (IgG) Ab responders; among the 22 EYSTS- subjects, ten were Lol p III responders (P = 0.001, relative risk = 21.6). No such association was found with any polymorphic sequences in other DR beta chains, or in DQ alpha I and DQ beta I chains. These findings suggest that the EYSTS sequence is important in the presentation of an epitope of Lol p III; other sequence(s) may be involved in the presentation of other epitope(s). To our knowledge, this is the first demonstration of a strong association between a specific HLA sequence and immune responsiveness to a well-defined antigen. The paper shows that presence of the EYSTS sequence classifies subjects as Lol p III responders in 18/19 cases.

Allergens↗
PubMed1991

Human T-cell responses to ragweed allergens: Amb V homologues.

Specific IgE and IgG responses to highly purified Ambrosia (ragweed) allergens, Amb a V, Amb t V and Amb p V from the artemisiifolia (short), trifida (giant) and psilostachya (western) species are strongly associated with HLA-DR2 and Dw2 (DR2.2) in allergic Caucasoid individuals. To investigate the molecular basis of these HLA associations, we examined the human T-cell responses to these Amb V homologues using three Amb a V-specific, DR alpha beta I 2.2-restricted T-cell clones from an atopic patient. We first examined the cross-reactivity of Amb a V-specific T-cell clones upon challenge with the Amb a V homologues, Amb t V and Amb p V, in the presence of autologous antigen-presenting cells (APC). Neither Amb t V nor Amb p V was able to stimulate the T-cell clones directly. However, both Amb t V and Amb p V specifically blocked, in a dose-dependent fashion, the ability of APC to present Amb a V to all three T-cell clones. Taken together, these results suggest that Amb t V and Amb p V possess distinct T-cell epitopes, but that all Amb V homologues share similar or identical regions (agretopes) interacting with the DR alpha beta I 2.2 (DR alpha beta I 1501) heterodimer. The agretope was potentially localized to a 14-residue C-terminal Amb a V peptide (with Ala-Cys substitutions), which was able to block presentation of native Amb a V by the APC to the T-cell clones.

Amino Acid Sequence↗
PubMed1991

Molecular and cellular studies of human immune responsiveness to the short ragweed allergen, Amb a V.

Specific immune responsiveness to the Amb a V allergen (from Ambrosia artemisiifolia, short ragweed pollen) is significantly associated with the Class II specificities, human leucocyte antigen (HLA)-DR2 and Dw2 determined by serological and MLR typing ("DR2.2"). Similarly, responsiveness to homologous Amb t V and Amb p V allergens is associated with DR2.2. We examined the deoxyribonucleic acid (DNA) sequences of HLA-DRB1, DRB5, DQB1 and DQA1 genes associated with Amb a V responsiveness using a combination of polymerase chain reaction (PCR), dot-blot and DNA sequencing methodologies. Our focus was on the highly polymorphic regions within the second-exon gene segments that are believed to encode antigen (Ag)-binding portions of the respective Class II molecules. Analysis of three patients having unusual sequence combinations of HLA-D gene sequences implicate an HLA-DR molecule (either DR alpha beta I 2.2 or DR alpha beta V 2.2), rather than a DQ Class II molecule, as the major Amb a V immune response (Ir) gene product. Our studies suggest that this DR2.2 molecule is usually a necessary, and almost always a sufficient, requirement for high immunoglobulin E and G (IgE) and (IgG) antibody responsiveness in ragweed-allergic individuals. From an atopic DR2.2+ subject, we isolated three Amb a V-specific T-cell clones. Analysis revealed these clones to be DR-restricted, supporting the conclusion that the Amb a V-Ir gene is a DR and not a DQ molecule. The DR beta I polypeptide of DR2.2 and 2.12 was implicated in Ag presentation, since monoclonal antibody (MoAb) Hu30 (antibody specific for DR beta I) blocked T-cell proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗