Search PubMed⌕ Search

Biomedical subjects

F Shakib

Publications and source records attributed to F Shakib.

At least 37 records · Page 2Linked to original sources
PubMed1998

Primary sequence and molecular model of the variable region of a mouse monoclonal anti-Der p 1 antibody showing a similar epitope specificity as human IgE.

BACKGROUND: Der p 1, a major mite allergen, elicits IgE antibody responses in 80% of patients suffering from dust mite allergy. Given the potent IgE eliciting properties of Der p 1, there is considerable interest in studying the molecular architecture of the variable (Fv) region of IgE antibodies specific for this allergen. OBJECTIVES: IgE is present in human serum at extremely low concentrations, and as such it is practically impossible to purify sufficient quantities for structural studies. We have therefore sought to sequence and model a representative murine monoclonal (MoAb) anti-Der p 1 antibody, as a surrogate human IgE. METHODS: The cDNA coding for the Fv region of an anti-Der p 1 MoAb (2C7), that mimics the binding of human IgE to Der p 1, was amplified by PCR, cloned and sequenced. The predicted amino acid sequences were then compared with a directory of human germline V-gene segments. Modelling of the Fv region of MoAb 2C7 was carried out using the extensive database of existing immunoglobulin structures in the Brookhaven PDB. RESULTS: The MoAb 2C7 heavy chain showed greater than 70% homology with three members of the VH3 family, DP-35, DP-53 and DP-54. Similarly, the light chain showed greater than 70% homology with 11 VK sequences, including the VKII sequences DPK18, DPK19 and DPK28. A molecular model of the Fv region of MoAb 2C7 was generated and can be accessed from the EMBL databank. CONCLUSIONS: Antibodies similar to MoAb 2C7 could be generated as part of the human repertoire. The availability of 3-dimensional model of MoAb 2C7, as a surrogate human IgE antibody, combined with further data on its epitope specificity, will facilitate studies into IgE antibody responses to Der p 1.

Amino Acid Sequence↗
PubMed1998

A sensitive fluorescent assay for measuring the cysteine protease activity of Der p 1, a major allergen from the dust mite Dermatophagoides pteronyssinus.

The potent allergenicity of Der p 1, a major allergen of the house dust mite Dermatophagoides pteronyssinus, is thought to be related to its cysteine protease activity. Therefore, there is considerable interest in developing a sensitive assay for measuring Der p 1 activity to screen for specific inhibitors. This study demonstrates for the first time that the activity of Der p 1 can be measured conveniently in a continuous rate assay with the fluorogenic substrate Boc-Gln-Ala-Arg-AMC (K(m) = 280 microM and kcat/K(m) = 4.6 x 10(3)/M/s).

Allergens↗
PubMed1997

Cleavage of the low-affinity receptor for human IgE (CD23) by a mite cysteine protease: nature of the cleaved fragment in relation to the structure and function of CD23.

Der p I, a cysteine protease representing a major allergen of the house dust mite Dermatophagoides pteronyssinus, has recently been shown to cleave CD23 from the surface of cultured human B cells (RPMI 8866 B cell line). We have now undertaken a detailed investigation of CD23 cleavage by Der p I. We demonstrate that Der p I cleaves CD23 at two sites (Ser155-Ser156 and Glu298-Ser299) to produce a 17-kDa fragment containing the lectin domain and only part of the C-terminal tail. No such effect was demonstrable with mouse CD23, a finding which was anticipated based on its lack of the cleavage sites identified on human CD23. Based on the cleavage pattern and the model of CD23, we propose a sequence of events leading to the liberation of the 17-kDa soluble CD23 fragment. The biological significance of such cleavage is underlined by the demonstration that Der p I-treated B lymphocytes lose their ability to bind IgE, and that the 17-kDa fragment (amino acids 156-298) contains the minimum structural requirement (amino acids 156-288) for binding to both IgE and CD21.

Animals↗
PubMed1996

Peripheral blood based T cell-containing and T cell-depleted culture systems for human IgE synthesis: the role of T cells.

BACKGROUND: Comparable T cell-containing and T cell-depleted culture systems for human IgE synthesis are currently not available. OBJECTIVE: This has prompted us to develop peripheral blood mononuclear cell (PBMC) based culture systems for human IgE synthesis in the presence and absence of T cells. METHODS: In this paper we describe simplified conditions for in vitro synthesis of high levels of IgE by human peripheral blood B cells, both in T cell-containing cultures and in anti-CD40 stimulated T cell-depleted cultures. RESULTS: T cell-depleted cultures released approximately 20 times more IgE [range 410-2220 ng/mliter (mean 1270 ng/mliter); based on six experiments using cells from three donors] than did T cell-containing cultures [range 23-105 ng/mliter (mean 58 ng/mliter); based on 15 experiments using cells from three donors]. Reconstitution experiments were performed to investigate the role of T cells on IgE synthesis. Adding T cells back to the anti-CD40 stimulated T cell-depleted cultures resulted in a dose-dependent inhibition of IgE production. In the absence of anti-CD40 low numbers of T cells stimulated, while high numbers suppressed, IgE production: the optimal ratio of T cells to non-T cells for maximal IgE production was found to be 1:1. At this ratio, irradiated (non-replicating) T cells supported a much greater IgE synthesis than did non-irradiated T cells. CONCLUSION: The development of these systems provides directly comparable T cell-containing and T cell-depleted cultures for human IgE synthesis from peripheral blood, allowing further study of the role of T cells in IgE regulation. These systems will also be of use for determining whether potential modulators of IgE synthesis act on the T cells or on other cell types.

Cells, Cultured↗
PubMed1995

Der p I, a major allergen of the house dust mite, proteolytically cleaves the low-affinity receptor for human IgE (CD23).

The nature of the proteases that cleave CD23 in vivo is of considerable interest, but remains unknown. Here, we demonstrate that Der p I, a major allergen of the house dust mite Dermatophagoides pteronyssinus, cleaves CD23 from the surface of cultured human B cells (RPMI 8866 B cell line). The cleavage of the receptor from the B cell surface was associated with a parallel increase in soluble CD23 (sCD23) in the culture supernatant. Furthermore, the proteolytic effect of Der p I was specific for CD23, since none of the other B cell markers tested (CD20, HLA-DR, CD71 and CD49d) were affected. Labeled antibody experiments and protease inhibition assays clearly demonstrate that Der p I is a cysteine protease that directly cleaves a 25-kDa fragment of CD23. These data suggest that the cysteine protease Der p I, in addition to being highly immunogenic, may up-regulate IgE synthesis by virtue of its ability to cleave CD23.

Allergens↗
PubMed1995

Do autoantibodies to IgE play a role in IgE-mediated events?

Elevated blood levels of IgG anti-IgE are detectable in individuals exhibiting enhanced IgE production, namely those with allergic conditions and helminthic parasite infections. The fact that there are epitope-specific subpopulations of autoanti-IgE suggests that this autoantibody could potentially have multiple effects in immunological events involving IgE.

Animals↗
PubMed1995

Potentiation of in vitro synthesis of human IgE by cyclosporin A (CsA).

In this study, we investigated the modulatory effects of CsA on in vitro synthesis of IgE, IgG1 and IgG4 by human peripheral blood mononuclear cells (PBMC). In contrast to its known immunosuppressive effect, we have demonstrated that a low dose of CsA (10(-7) M, 120 ng/ml) potentiated IgE production by up to 40-fold (i.e. from 33 +/- 4.5 to 1346 +/- 290 ng/ml). This potentiation was specific for IgE since no such effect was demonstrable with IgG1 and IgG4. Potentiation of IgE synthesis by CsA in the PBMC cultures was partly due to CsA acting on T cells, as demonstrated by the addition of CsA-treated T cells to T cell-depleted cultures. However, potentiation was also demonstrable in a T cell-depleted, anti-CD40-stimulated culture (four-fold increase from 400 +/- 48 to 1606 +/- 127 ng/ml). Our data therefore suggest that there are at least two mechanisms for CsA-induced potentiation of IgE synthesis, one T cell-dependent and the other T cell-independent. The clinical implications of these findings are discussed with regard to the use of CsA in the treatment of Th2-mediated diseases.

Adjuvants, Immunologic↗
PubMed1995

The prevalence of IgG1 and IgG4 autoantibodies to IgE in patients with allergic and non-allergic rhinitis.

In this study we have compared the levels of IgG1 and IgG4 autoantibodies to IgE in the sera of patients with allergic rhinitis and non-allergic rhinitis. A group of patients undergoing cosmetic nasal surgery, but who did not have rhinitis or any history of atopy, acted as control. The frequency of positive titres of IgG1 and/or IgG4 anti-IgE was 70% (14/20) in patients with allergic rhinitis, 50% (10/20) in patients with non-allergic rhinitis and 20% (4/20) in the control group. The mean titres of IgG1 anti-IgE and IgG4 anti-IgE were in the order allergic rhinitis > non-allergic rhinitis > controls. In the allergic rhinitis group levels of IgG1 anti-IgE (P = 0.0055) and IgG4 anti-IgE (P = 0.0028) were significantly higher than those found in the control group. The non-allergic rhinitis group also showed significantly higher levels of IgG1 anti-IgE (P = 0.0182) and IgG4 and anti-IgE (P = 0.0359) than the control group. The existence of IgG autoantibodies to IgE in both allergic and non-allergic rhinitis suggests a possible role for these antibodies in the disease process, particularly in patients whose symptoms are not due to an allergic trigger.

Adolescent↗
PubMed1995

Circulating human IgG autoanti-IgE antibodies in asthma patients block the binding of IgE to its high affinity receptor.

Aims-To investigate the ability of circulating human IgG autoanti-IgE antibodies from asthma patients to block the binding of IgE to the alpha chain of the high affinity receptor (FcepsilonRI).Methods-This involved the use of a well validated flow cytometric method to detect inhibition of FITC labelled IgE binding to a fibroblast cell line (CHK1E1) transfected with the alpha chain of FcepsilonRI.Results-IgG autoanti-IgE-containing sera blocked the binding of IgE-FITC to the CHK1E1 cells. No such inhibition was demonstrable with rheumatoid sera containing autoanti-IgG (that is, rheumatoid factor) but lacking autoanti-IgE. Percentage inhibition (up to 50%) of IgE binding to the CHK1E1 cells was directly related to the titre of IgG1, but not IgG4, autoanti-IgE in the sera tested (correlation coefficient 0.66, probability 0.003).Conclusions-The capacity of anti-IgE to block the binding of IgE to FcepsilonRI has important clinical implications, particularly in terms of downregulation of allergic reactions.

Journal Article↗
PubMed1995

Circulating IgG autoanti-IgE antibodies in atopic patients block the binding of IgE to its low affinity receptor (CD23).

Aims-To investigate the ability of circulating IgG autoanti-IgE antibodies from atopic rhinitis patients to block the binding of IgE to its low affinity receptor (FcepsilonRII), also termed CD23.Methods-This involved the use of a well validated flow cytometric method to detect inhibition of FITC labelled IgE binding to human B cells expressing CD23 (RPMI 8866 cell line).Results-Taking inhibition values greater than 20% as being significant, 15 out of 20 IgG anti-IgE containing sera inhibited the binding of IgE-FITC to the RPMI 8866 cells. The inhibitory effect was recoverable in the IgG fraction of serum, but was not related to the titre of either IgG1 anti-IgE or IgG4 anti-IgE, thus suggesting that it might be related to epitope specificity. No such inhibition was demonstrable with rheumatoid sera containing autoanti-IgG (that is, rheumatoid factor), but lacking autoanti-IgE.Conclusions-The capacity of anti-IgE to block the binding of IgE to CD23 has important implications, particularly in terms of upregulation of IgE synthesis and the consequent perpetuation of the inflammatory response.

Journal Article↗
PubMed1994

In vitro basophil histamine-releasing activity of circulating IgG1 and IgG4 autoanti-IgE antibodies from asthma patients and the demonstration that anti-IgE modulates allergen-induced basophil activation.

In this study we have examined the relationship between the in vitro basophil histamine-releasing activity of human IgG anti-IgE, isolated as euglobulin fractions from sera of asthmatic patients, and its IgG1/IgG4 subclass distribution. In particular, we have investigated whether IgG anti-IgE modulates allergen-induced basophil activation. The study has revealed that only a small proportion of IgG anti-IgE samples triggered histamine release from basophils of an asthmatic individual (4/21; 19%), a hay fever sufferer (4/10; 40%) and a healthy person (7/21; 33%). The basophil histamine-releasing activity of IgG anti-IgE did not seem to be determined by the IgG1/IgG4 subclass composition of the IgG anti-IgE preparation used. Furthermore, we have demonstrated that autoanti-IgE antibodies modulate allergen-induced basophil histamine release. The three modulatory effects exerted by IgG anti-IgE antibodies on allergen-triggered basophil activation (i.e. additive, synergistic and blocking) were not dependent on the subclass nature of IgG anti-IgE or the use of histamine-releasing anti-IgE preparations. Our data suggest that IgG anti-IgE antibodies in asthma patients may consist of two functionally distinct subpopulations: those which up-regulate (pro-allergic) and those which down-regulate (anti-allergic) the allergic release of mediators from mast cells and basophils.

Allergens↗
PubMed1994

Circulating levels of IgG1 and IgG4 anti-IgE antibodies and asthma severity.

In this paper, we have determined the levels of IgG1 and IgG4 anti-IgE in the sera of 66 asthma patients suffering from mild (n = 24), moderate (n = 23), or severe (n = 19) symptoms, and 20 nonatopic, healthy subjects. The study has revealed that although asthma patients have significantly elevated levels of IgG1 and IgG4 anti-IgE antibodies, the concentration of these autoantibodies is not related to the severity of asthma. This conclusion may be related to the known heterogeneity of autoanti-IgE antibodies in terms of their ability to trigger basophil histamine release.

Adolescent↗
PubMed1993

The detection of autoantibodies to IgE in plasma of individuals infected with hookworm (Necator americanus) and the demonstration of a predominant IgG1 anti-IgE autoantibody response.

In this study we have demonstrated significantly elevated levels of circulating IgG autoanti-IgE antibody in hookworm infected individuals from Kebasob village on Karkar Island, Papua New Guinea. Although anti-IgE activity was demonstrable in IgG1, IgG3 and IgG4, IgG1 was by far the most important subclass of IgG anti-IgE in terms of frequency of detection (34/39; 87.2%) and magnitude of increase (P = 0.0000); with IgG3 (16/39; 41.0%) and IgG4 (15/39; 38.5%) antibodies being considerably less prevalent. Plasma levels of IgG1 anti-IgE (P = 0.0019) and IgG3 anti-IgE (P = 0.0034) showed significant correlations with total IgE concentrations, but not with IgE specific to excretory-secretory worm products; thus suggesting that anti-IgE synthesis is more related to polyclonal hyper IgE production than to antigen-specific IgE stimulation. No correlation was seen between IgG subclass anti-IgE levels and faecal egg counts or worm burden. Given that our data failed to show a negative or a positive correlation between anti-IgE and the degree of infection with hookworm, it is tempting to speculate that the main role of autoanti-IgE is to provide the host with protection against immune complex- and IgE-mediated hypersensitivity reactions to parasitic antigens.

Adolescent↗
PubMed1992

The recognition of a recombinant human Fc epsilon fragment by the subclasses of IgG autoanti-IgE: disproportional subclasses distribution of complexed autoantibody.

Circulating IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma where it is suggested to be potentially involved in the removal of IgE-allergen complexes. Since such a putative role will largely be determined by the subclass profile of complexed (i.e. IgE-bound) IgG anti-IgE, a study was undertaken to determine the subclass distribution of complexed IgG anti-IgE antibody in the sera of asthmatic patients. The study exploits the heat-labile property of IgE by heating (30 min at 56 degrees C) serum to liberate bound anti-IgE, pre- and post-heated sera are then assayed for IgG subclass anti-recombinant human Fc epsilon (rFc epsilon) activities by ELISA and any heat-induced increase in antibody activity is taken as a measure of complexed anti-IgE. This has revealed a disproportionately high amount of IgG4 in complexed (but not free) IgG anti-IgE. The propensity of IgG4 to form complexes with IgE has important biological consequences, particularly with regard to the activation of C1q and Fc gamma R by other subclasses.

Adult↗
PubMed1991

Elucidation of the epitope locations of human autoanti-IgE: recognition of two epitopes located within the C epsilon 2 and the C epsilon 4 domains.

IgG autoanti-IgE is detectable in a large proportion of individuals with allergic asthma, where it is suggested to be potentially involved in modulating IgE-mediated hypersensitivity. Using a series of overlapping recombinant human epsilon-chain peptides, we have shown that circulating IgG anti-IgE antibodies recognise at least 2 epitopes located within the C epsilon 2 and the C epsilon 4 domains, respectively. The C epsilon 2 recognition site is located within the C-terminal portion of the C epsilon 2 domain (i.e. aa301-339) which is thought to contribute residues to the Fc epsilon RI-binding site on IgE. The recognition by autoanti-IgE of an effector function site of such pivotal importance in IgE-mediated hypersensitivity suggests that it plays a possible modulatory role during mast cell and basophil activation.

Adult↗