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Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.

Harrer A, Lang R, Grims R, Braitsch M, Hawranek T, Aberer W, Vogel L, Schmid W, Ferreira F, Himly M - PLoS ONE (2010)

Bottom Line: In none of the patients' samples evidence for drug/metabolite-specific IgE in serum or bound to allergic effector cells was found.In contrast, a small group of patients (8/59, 14%) displayed drug/metabolite-specific IgG.We found no evidence for an IgE-mediated effector mechanism based on haptenation of protein carriers in DF-hypersensitive patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.

ABSTRACT

Background: Hypersensitivity reactions against nonsteroidal antiinflammatory drugs (NSAIDs) like diclofenac (DF) can manifest as Type I-like allergic reactions including systemic anaphylaxis. However, except for isolated case studies experimental evidence for an IgE-mediated pathomechanism of DF hypersensitivity is lacking. In this study we aimed to investigate the possible involvement of drug- and/or metabolite-specific antibodies in selective DF hypersensitivity.

Methodology/principal findings: DF, an organochemically synthesized linkage variant, and five major Phase I metabolites were covalently coupled to carrier proteins. Drug conjugates were analyzed for coupling degree and capacity to crosslink receptor-bound IgE antibodies from drug-sensitized mice. With these conjugates, the presence of hapten-specific IgE antibodies was investigated in patients' samples by ELISA, mediator release assay, and basophil activation test. Production of sulfidoleukotrienes by drug conjugates was determined in PBMCs from DF-hypersensitive patients. All conjugates were shown to carry more than two haptens per carrier molecule. Immunization of mice with drug conjugates induced drug-specific IgE antibodies capable of triggering mediator release. Therefore, the conjugates are suitable tools for detection of drug-specific antibodies and for determination of their anaphylactic activity. Fifty-nine patients were enrolled and categorized as hypersensitive either selectively to DF or to multiple NSAIDs. In none of the patients' samples evidence for drug/metabolite-specific IgE in serum or bound to allergic effector cells was found. In contrast, a small group of patients (8/59, 14%) displayed drug/metabolite-specific IgG.

Conclusions/significance: We found no evidence for an IgE-mediated effector mechanism based on haptenation of protein carriers in DF-hypersensitive patients. Furthermore, a potential involvement of the most relevant metabolites in DF hypersensitivity reactions could be excluded.

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Related in: MedlinePlus

Characterization of hapten-HSA conjugates.(A) Coupling degree of eluting HPSEC fractions derived from hapten-HSA conjugates was determined from (B) the ratio of refractive index (black) to UV signal (gray) resulting in (C) several haptens per carrier molecules for all conjugates compared to respective mock controls; determined coupling degree for total, monomeric, and aggregated HPSEC fractions are illustrated in black, light, and dark gray, respectively. (D) Determination of optimal concentration of DF-HSA conjugate for FcεRI-crosslinking/mediator release; determined values for total, monomeric, and aggregated HPSEC fractions are illustrated in black, light, and dark gray, respectively, mock controls in white. (E) All hapten-HSA conjugates (dark gray) showed mediator release (n = 1–4) from rat basophil leukemia cells when incubated with sera from immunized mice; mock controls in light gray. (F) Investigation of cytotoxicity of hapten-HSA conjugates coincubated at 1 (dark gray) and 20 µg/ml (light gray) with anti-IgE by basophil activation test (100% without conjugate).
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pone-0013707-g002: Characterization of hapten-HSA conjugates.(A) Coupling degree of eluting HPSEC fractions derived from hapten-HSA conjugates was determined from (B) the ratio of refractive index (black) to UV signal (gray) resulting in (C) several haptens per carrier molecules for all conjugates compared to respective mock controls; determined coupling degree for total, monomeric, and aggregated HPSEC fractions are illustrated in black, light, and dark gray, respectively. (D) Determination of optimal concentration of DF-HSA conjugate for FcεRI-crosslinking/mediator release; determined values for total, monomeric, and aggregated HPSEC fractions are illustrated in black, light, and dark gray, respectively, mock controls in white. (E) All hapten-HSA conjugates (dark gray) showed mediator release (n = 1–4) from rat basophil leukemia cells when incubated with sera from immunized mice; mock controls in light gray. (F) Investigation of cytotoxicity of hapten-HSA conjugates coincubated at 1 (dark gray) and 20 µg/ml (light gray) with anti-IgE by basophil activation test (100% without conjugate).

Mentions: Human serum albumin was chosen for chemical conjugation of DF/DF5der/DF-metabolites (Figure 1) as it is the most abundant plasma protein and a physiological carrier of slightly acidic xenobiotics like DF [23]. Mock-HSA was used to control for a formation of neoepitopes, which might result from inter- and intramolecular crosslinkings. Antigenic bivalency as minimum theoretical requirement for a receptor cross-linking activity was given as coupling degrees were shown to exceed 2 DF/HSA in all conjugates as determined from the ratio of UV to RI signals from HPSEC (Figure 2A–C).


Diclofenac hypersensitivity: antibody responses to the parent drug and relevant metabolites.

Harrer A, Lang R, Grims R, Braitsch M, Hawranek T, Aberer W, Vogel L, Schmid W, Ferreira F, Himly M - PLoS ONE (2010)

Characterization of hapten-HSA conjugates.(A) Coupling degree of eluting HPSEC fractions derived from hapten-HSA conjugates was determined from (B) the ratio of refractive index (black) to UV signal (gray) resulting in (C) several haptens per carrier molecules for all conjugates compared to respective mock controls; determined coupling degree for total, monomeric, and aggregated HPSEC fractions are illustrated in black, light, and dark gray, respectively. (D) Determination of optimal concentration of DF-HSA conjugate for FcεRI-crosslinking/mediator release; determined values for total, monomeric, and aggregated HPSEC fractions are illustrated in black, light, and dark gray, respectively, mock controls in white. (E) All hapten-HSA conjugates (dark gray) showed mediator release (n = 1–4) from rat basophil leukemia cells when incubated with sera from immunized mice; mock controls in light gray. (F) Investigation of cytotoxicity of hapten-HSA conjugates coincubated at 1 (dark gray) and 20 µg/ml (light gray) with anti-IgE by basophil activation test (100% without conjugate).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965666&req=5

pone-0013707-g002: Characterization of hapten-HSA conjugates.(A) Coupling degree of eluting HPSEC fractions derived from hapten-HSA conjugates was determined from (B) the ratio of refractive index (black) to UV signal (gray) resulting in (C) several haptens per carrier molecules for all conjugates compared to respective mock controls; determined coupling degree for total, monomeric, and aggregated HPSEC fractions are illustrated in black, light, and dark gray, respectively. (D) Determination of optimal concentration of DF-HSA conjugate for FcεRI-crosslinking/mediator release; determined values for total, monomeric, and aggregated HPSEC fractions are illustrated in black, light, and dark gray, respectively, mock controls in white. (E) All hapten-HSA conjugates (dark gray) showed mediator release (n = 1–4) from rat basophil leukemia cells when incubated with sera from immunized mice; mock controls in light gray. (F) Investigation of cytotoxicity of hapten-HSA conjugates coincubated at 1 (dark gray) and 20 µg/ml (light gray) with anti-IgE by basophil activation test (100% without conjugate).
Mentions: Human serum albumin was chosen for chemical conjugation of DF/DF5der/DF-metabolites (Figure 1) as it is the most abundant plasma protein and a physiological carrier of slightly acidic xenobiotics like DF [23]. Mock-HSA was used to control for a formation of neoepitopes, which might result from inter- and intramolecular crosslinkings. Antigenic bivalency as minimum theoretical requirement for a receptor cross-linking activity was given as coupling degrees were shown to exceed 2 DF/HSA in all conjugates as determined from the ratio of UV to RI signals from HPSEC (Figure 2A–C).

Bottom Line: In none of the patients' samples evidence for drug/metabolite-specific IgE in serum or bound to allergic effector cells was found.In contrast, a small group of patients (8/59, 14%) displayed drug/metabolite-specific IgG.We found no evidence for an IgE-mediated effector mechanism based on haptenation of protein carriers in DF-hypersensitive patients.

View Article: PubMed Central - PubMed

Affiliation: Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.

ABSTRACT

Background: Hypersensitivity reactions against nonsteroidal antiinflammatory drugs (NSAIDs) like diclofenac (DF) can manifest as Type I-like allergic reactions including systemic anaphylaxis. However, except for isolated case studies experimental evidence for an IgE-mediated pathomechanism of DF hypersensitivity is lacking. In this study we aimed to investigate the possible involvement of drug- and/or metabolite-specific antibodies in selective DF hypersensitivity.

Methodology/principal findings: DF, an organochemically synthesized linkage variant, and five major Phase I metabolites were covalently coupled to carrier proteins. Drug conjugates were analyzed for coupling degree and capacity to crosslink receptor-bound IgE antibodies from drug-sensitized mice. With these conjugates, the presence of hapten-specific IgE antibodies was investigated in patients' samples by ELISA, mediator release assay, and basophil activation test. Production of sulfidoleukotrienes by drug conjugates was determined in PBMCs from DF-hypersensitive patients. All conjugates were shown to carry more than two haptens per carrier molecule. Immunization of mice with drug conjugates induced drug-specific IgE antibodies capable of triggering mediator release. Therefore, the conjugates are suitable tools for detection of drug-specific antibodies and for determination of their anaphylactic activity. Fifty-nine patients were enrolled and categorized as hypersensitive either selectively to DF or to multiple NSAIDs. In none of the patients' samples evidence for drug/metabolite-specific IgE in serum or bound to allergic effector cells was found. In contrast, a small group of patients (8/59, 14%) displayed drug/metabolite-specific IgG.

Conclusions/significance: We found no evidence for an IgE-mediated effector mechanism based on haptenation of protein carriers in DF-hypersensitive patients. Furthermore, a potential involvement of the most relevant metabolites in DF hypersensitivity reactions could be excluded.

Show MeSH
Related in: MedlinePlus