Definition of Haptens Derived from Sulfamethoxazole: In Vitro and in Vivo.

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.

Application of in Vitro T Cell Assay Using Human Leukocyte Antigen-Typed Healthy Donors for the Assessment of Drug Immunogenicity.

It is unclear whether priming of naïve T cells to drugs is detectable in healthy human donors expressing different human leukocyte antigen (HLA) alleles. Thus, we examined T cell priming with drugs associated with HLA risk alleles and control compounds in 14 HLA-typed donors. Nitroso sulfamethoxazole and piperacillin activated T cells from all donors, whereas responses to carbamazepine and oxypurinol were only seen in donors expressing HLA-B*15:02 and HLA-B*58:01, respectively. Weak flucloxacillin-specific T cell responses were detected in donors expressing HLA-B*57:01 and HLA-B*58:01. These data show that the priming of T cells with certain drugs is skewed toward donors expressing specific HLA alleles.

The Effect of Inhibitory Signals on the Priming of Drug Hapten-Specific T Cells That Express Distinct Vß Receptors.

Drug hypersensitivity involves the activation of T cells in an HLA allele-restricted manner. Because the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T cell response. Thus, we have used a T cell-priming assay and nitroso sulfamethoxazole (SMX-NO) as a model Ag to investigate the activation of specific TCR Vß subtypes, the impact of programmed death -1 (PD-1), CTL-associated protein 4 (CTLA4), and T cell Ig and mucin domain protein-3 (TIM-3) coinhibitory signaling on activation of naive and memory T cells, and the ability of regulatory T cells (Tregs) to prevent responses. An expansion of the TCR repertoire was observed for nine Vß subtypes, whereas spectratyping revealed that SMX-NO-specific T cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vß-restricted Ag-specific T cell responses is governed by regulatory signals. Blockade of PD-L1/CTLA4 signaling dampened activation of SMX-NO-specific naive and memory T cells, whereas blockade of TIM-3 produced no effect. Programmed death-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T cell activation, and expression of each receptor was enhanced on dividing T cells. Because these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO-induced T cell activation was investigated. Tregs significantly dampened the priming of T cells. In conclusion, our findings demonstrate that distinct TCR Vß subtypes, dysregulation of coinhibitory signaling pathways, and dysfunctional Tregs may influence predisposition to hypersensitivity.

Structural considerations on the selectivity of an immunoassay for sulfamethoxazole.

Sulfamethoxazole (SMX), a sulfonamide, is a widely used bacteriostatic antibiotic and therefore a promising marker for the entry of anthropogenic pollution in the environment. SMX is frequently found in wastewater and surface water. This study presents the production of high affinity and selective polyclonal antibodies for SMX and the development and evaluation of a direct competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of SMX in environmental water samples. The crystal structures of the cross-reacting compounds sulfamethizole, N(4)-acetyl-SMX and succinimidyl-SMX were determined by x-ray diffraction aiming to explain their high cross-reactivity. These crystal structures are described for the first time. The quantification range of the ELISA is 0.82-63µg/L. To verify our results, the SMX concentration in 20 environmental samples, including wastewater and surface water, was determined by ELISA and tandem mass spectrometry (MS/MS). A good agreement of the measured SMX concentrations was found with average recoveries of 97-113% for the results of ELISA compared to LC-MS/MS.

Negative regulation by PD-L1 during drug-specific priming of IL-22-secreting T cells and the influence of PD-1 on effector T cell function.

Activation of PD-1 on T cells is thought to inhibit Ag-specific T cell priming and regulate T cell differentiation. Thus, we sought to measure the drug-specific activation of naive T cells after perturbation of PD-L1/2/PD-1 binding and investigate whether PD-1 signaling influences the differentiation of T cells. Priming of naive CD4(+) and CD8(+) T cells against drug Ags was found to be more effective when PD-L1 signaling was blocked. Upon restimulation, T cells proliferated more vigorously and secreted increased levels of IFN-γ, IL-13, and IL-22 but not IL-17. Naive T cells expressed low levels of PD-1; however, a transient increase in PD-1 expression was observed during drug-specific T cell priming. Next, drug-specific responses from in vitro primed T cell clones and clones from hypersensitive patients were measured and correlated with PD-1 expression. All clones were found to secrete IFN-γ, IL-5, and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL-22-secreting clones expressed the skin-homing receptors CCR4, CCR10, and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the Ag-specific proliferative response or the secretion of cytokines/cytolytic molecules. This study shows that PD-L1/PD-1 binding negatively regulates the priming of drug-specific T cells. ELISPOT analysis uncovered an Ag-specific FasL/IL-22-secreting T cell subset with skin-homing properties.

Development of a screening assay to evaluate the potential of drugs to cause immune-mediated hypersensitivity reactions.

Evidence suggests that bio-activation of drugs to generate chemically reactive metabolites (RM) that act as haptens to form immunogenic protein conjugates may be an important cause of immune-mediated drug hypersensitivity reactions (IDHR). Although many drugs that form RMs raise concerns about producing IDHR, standard non-clinical testing methods are rarely able to identify compounds with the potential to produce IDHR in humans. The objective of this study was to develop a predictive assay for IDHR that involves: (1) the use of an in vitro drug-metabolizing system to generate the RM that is captured by GSH, (2) conjugating the RM-GSH conjugate to mouse serum albumin (MSA) by using a chemical cross-linker, (3) immunization of mice with RM-GSH-MSA adducts, and (4) ex vivo challenge with RM-GSH-MSA adduct and measurement of lymphocyte proliferation to determine if the RM is immunogenic. The predictivity of the assay was evaluated by using drugs that produce RM and have been strongly, weakly, or not associated with IDHRs in the clinic. While this method requires additional validation with more drugs, the results demonstrate the feasibility of identifying drugs strongly associated with IDHR and the utility of the assay for rank ordering drugs with respect to their potential to cause IDHR.

Enhanced antigenicity leads to altered immunogenicity in sulfamethoxazole-hypersensitive patients with cystic fibrosis.

BACKGROUND: Exposure of patients with cystic fibrosis to sulfonamides is associated with a high incidence of hypersensitivity reactions. OBJECTIVE: To compare mechanisms of antigen presentation and characterize the phenotype and function of T cells from sulfamethoxazole-hypersensitive patients with and without cystic fibrosis. METHODS: T cells were cloned from 6 patients and characterized in terms of phenotype and function. Antigen specificity and mechanisms of antigen presentation to specific clones were then explored. Antigen-presenting cell metabolism of sulfamethoxazole was quantified by ELISA. The involvement of metabolism in antigen presentation was evaluated by using enzyme inhibitors. RESULTS: Enzyme inhibitable sulfamethoxazole-derived protein adducts were detected in antigen-presenting cells from patients with and without cystic fibrosis. A significantly higher quantity of adducts were detected with cells from patients with cystic fibrosis. Over 500 CD4(+) or CD8(+) T-cell clones were generated and shown to proliferate and kill target cells. Three patterns of MHC-restricted reactivity (sulfamethoxazole-responsive, sulfamethoxazole metabolite-responsive, and cross-reactive) were observed with clones from patients without cystic fibrosis. From patients with cystic fibrosis, sulfamethoxazole metabolite-responsive and cross-reactive, but not sulfamethoxazole-responsive, clones were observed. The response of the cross-reactive clones to sulfamethoxazole was dependent on adduct formation and was blocked by glutathione and enzyme inhibitors. Antigen-stimulated clones from patients with cystic fibrosis secreted higher levels of IFN-γ, IL-6, and IL-10, but lower levels of IL-17. CONCLUSION: Sulfamethoxazole metabolism and protein adduct formation is critical for the stimulation of T cells from patients with cystic fibrosis. T cells from patients with cystic fibrosis secrete high levels of IFN-γ, IL-6, and IL-10.

Haemolytic anaemia and renal failure associated with antibodies to trimethoprim and sulfamethoxazole.

AIM: The aim of this study was to support a clinical diagnosis of drug-induced immune haemolytic anaemia (DIIHA). BACKGROUND: DIIHA is rare and has only been described twice with the antibiotic combination of trimethoprim (TMP) and sulfamethoxazole (SMX). METHODS/MATERIALS: Serologic tests for drug antibodies were performed using methods previously published by our laboratory. RESULTS: A 44-year-old woman experienced body aches, chills, chest pressure, nausea and a rash while receiving TMP-SMX; a week later her haemoglobin was low and she was in renal failure. At the hospital, the direct antiglobulin test (DAT) was positive (C3 only) and the serum reacted with all red blood cells (RBCs) by the gel method only (TMP-SMX is present in the RBC diluent used for the gel method). At the Red Cross immunohaematology laboratory, the patient's serum was reactive in the presence of TMP-SMX (haemolysis and positive antiglobulin test), pure TMP (positive antiglobulin test using anti-IgG only) and pure SMX (haemolysis and positive antiglobulin test using both anti-IgG and anti-C3). The patient was treated with transfusions and haemodialysis and was discharged after a week in stable condition. CONCLUSION: We describe a patient who appeared to have haemolytic anaemia and renal failure associated with antibodies to both TMP and SMX.

Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism.

Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein-generating antigenic determinants for T cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant Ags to functional immune responses is lacking. The aim of this study was to develop an integrated approach using animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship among adduct formation, cell death, costimulatory signaling, and stimulation of a T cell response. Formation of SMX-derived adducts in APCs was dose and time dependent, detectable at nontoxic concentrations, and dependent on drug-metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell costimulatory molecule expression, and cytokine secretion. APCs cultured with SMX for 16 h, the time needed for drug metabolism, stimulated T cells from sensitized mice and lymphocytes and T cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T cells were stimulated with adducts derived from SMX metabolism in APCs, not the parent drug. This study shows that APCs metabolize SMX; subsequent protein binding generates a functional T cell Ag. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

Drug-dependent clearance of human platelets in the NOD/scid mouse by antibodies from patients with drug-induced immune thrombocytopenia.

Drug-induced immune thrombocytopenia (DITP) is a relatively common and sometimes life-threatening condition caused by antibodies that bind avidly to platelets only when drug is present. How drug-dependent antibodies (DDAbs) are induced and how drugs promote their interaction with platelets are poorly understood, and methods for detecting DDAbs are suboptimal. A small animal model of DITP could provide a new tool for addressing these and other questions concerning pathogenesis and diagnosis. We examined whether the nonobese diabetic/severe combined immunodeficient (NOD/scid) mouse, which lacks xenoantibodies and therefore allows infused human platelets to circulate, can be used to study drug-dependent clearance of platelets by DDAbs in vivo. In this report, we show that the NOD/scid model is suitable for this purpose and describe studies to optimize its sensitivity for drug-dependent human antibody detection. We further show that the mouse can produce metabolites of acetaminophen and naproxen for which certain drug-dependent antibodies are specific in quantities sufficient to enable these antibodies to cause platelet destruction. The findings indicate that the NOD/scid mouse can provide a unique tool for studying DITP pathogenesis and may be particularly valuable for identifying metabolite-specific antibodies capable of causing immune thrombocytopenia or hemolytic anemia.

Drug metabolite-specific lymphocyte responses in sulfamethoxazole allergic patients with cystic fibrosis.

Sulfamethoxazole (SMX) is an important antibiotic in the management of patients with cystic fibrosis, but allergic reactions may develop thus restricting therapy. The aim of this study was to utilize drug (metabolite) antigens to diagnose SMX-mediated allergic reactions in patients with cystic fibrosis. Lymphocytes from 2/12 allergic patients were stimulated to proliferate strongly with the SMX metabolite nitroso SMX (SMX-NO). In contrast, responses to SMX were weak. The introduction of an antigen-driven T-cell enrichment step prior to the analysis of proliferation increased the sensitivity of the assay. SMX-NO responses were detected with lymphocytes from all patients with cutaneous signs.

Cross-reactivity in drug hypersensitivity reactions to sulfasalazine and sulfamethoxazole.

BACKGROUND: Sulfonamides are generally classified into 2 groups: antibiotics and non-antibiotics. Recent studies showed that patients allergic to sulfonamide antibiotics do not have a specific risk for an allergy to sulfonamide non-antibiotic. However, the anti-inflammatory drug sulfasalazine represents an important exception. Used in rheumatic diseases, it is classified as a non-antibiotic sulfonamide, but is structurally related to antibiotic sulfonamides. Therefore, we aimed to analyze in vitro the cross-reactivity between the antimicrobial sulfamethoxazole and the anti-inflammatory drug sulfasalazine. METHODS: PBMC from 2 patients with severe hypersensitivity syndrome to sulfasalazine, 3 patients with sulfamethoxazole allergy and 5 healthy donors were isolated and incubated with medium only (negative control), 2 concentrations (10, 100 µg/ml) of sulfapyridine, 2 concentrations (100, 200 µg/ml) of sulfamethoxazole, and tetanus toxoid (10 µg/ml) as a positive control. After 6 days of culture, (3)H-thymidine was added and cell proliferation was measured. RESULTS: In all patients tested, the lymphocyte transformation tests were positive for both sulfapyridine and sulfamethoxazole, suggesting a strong cross-reactivity to these drugs. None of the healthy donors reacted to any of the drugs tested. We refrained from provoking our patients with either sulfasalazine or sulfamethoxazole, as they had a clear, typical history, severe symptoms and were positive on in vitro tests to both compounds. CONCLUSIONS: We demonstrate that in the case of sulfamethoxazole and sulfasalazine, cross-reactivity is dependent on chemical features rather than the indication of the drugs. Therefore, patients with hypersensitivity to sulfasalazine or sulfamethoxazole should be specifically advised to avoid both drugs.

Stimulation of human T cells with sulfonamides and sulfonamide metabolites.

BACKGROUND: Exposure to sulfonamides is associated with a high incidence of hypersensitivity reactions. Antigen-specific T cells are involved in the pathogenesis; however, the nature of the antigen interacting with specific T-cell receptors is not fully defined. OBJECTIVE: We sought to explore the frequency of sulfamethoxazole (SMX)- and SMX metabolite-specific T cells in hypersensitive patients, delineate the specificity of clones, define mechanisms of presentation, and explore additional reactivity with structurally related sulfonamide metabolites. METHODS: SMX- and SMX metabolite-specific T-cell clones were generated from 3 patients. Antigen specificity, mechanisms of antigen presentation, and cross-reactivity of specific clones were then explored. Low-lying energy conformations of drugs (metabolites) were modeled, and the energies available for protein binding was estimated. RESULTS: Lymphocytes proliferated with parent drugs (SMX, sulfadiazine, and sulfapyridine) and both hydroxylamine and nitroso metabolites. Three patterns of drug (metabolite) stimulation were seen: 44% were SMX metabolite specific, 43% were stimulated with SMX metabolites and SMX, and 14% were stimulated with SMX alone. Most metabolite-responsive T cells were stimulated with nitroso SMX-modified protein through a hapten mechanism involving processing. In contrast to SMX-responsive clones, which were highly specific, greater than 50% of nitroso SMX-specific clones were stimulated with nitroso metabolites of sulfapyridine and sulfadiazine but not nitrosobenzene. Pharmacophore modeling showed that the summation of available binding energies for protein interactions and the preferred spatial arrangement of atoms in each molecule determine a drug's potential to stimulate specific T cells. CONCLUSIONS: Nitroso sulfonamide metabolites form potent antigenic determinants for T cells from hypersensitive patients. T-cell responses against drugs (metabolites) bound directly to MHC or MHC/peptide complexes can occur through cross-reactivity with the haptenic immunogen.

Metabolic and chemical origins of cross-reactive immunological reactions to arylamine benzenesulfonamides: T-cell responses to hydroxylamine and nitroso derivatives.

Exposure to sulfamethoxazole (SMX) is associated with T-cell-mediated hypersensitivity reactions in human patients. T-cells can be stimulated by the putative metabolite nitroso SMX, which binds irreversibly to protein. The hydroxylamine and nitroso derivatives of three arylamine benzenesulfonamides, namely, sulfamethozaxole, sulfadiazine, and sulfapyridine, were synthesized, and their T-cell stimulatory capacity in the mouse was explored. Nitroso derivatives were synthesized by a three-step procedure involving the formation of nitro and hydroxylamine sulfonamide intermediates. For immune activation, female Balb-c strain mice were administered nitroso sulfonamides four times weekly for 2 weeks. After 14 days, isolated splenocytes were incubated with the parent compounds, hydroxylamine metabolites, and nitroso derivatives to measure antigen-specific proliferation. To explore the requirement of irreversible protein binding for spleen cell activation, splenocytes were incubated with nitroso derivatives in the presence or absence of glutathione. Splenocytes from nitroso sulfonamide-sensitized mice proliferated and secreted interleukin (IL)-2, IL-4, IL-5, and granulocyte monocyte colony-stimulating factor following stimulation with nitroso derivatives but not the parent compounds. Splenocytes from sensitized mice were also stimulated to proliferate with hydroxylamine and nitroso derivatives of the structurally related sulfonamides. The addition of glutathione inhibited the nitroso-specific T-cell response. Hydroxylamine metabolites were unstable in aqueous solution: Spontaneous transformation yielded appreciable amounts of nitroso and azoxy compounds as well as the parent compounds within 0.1 h. T-cell cross-reactivity with nitroso sulfonamides provides a mechanistic explanation as to why structurally related arylamine benzenesulfonamides are contraindicated in hypersensitive patients.

"Danger" conditions increase sulfamethoxazole-protein adduct formation in human antigen-presenting cells.

Antigen-presenting cells (APC) are thought to play an important role in the pathogenesis of drug-induced immune reactions. Various pathological factors can activate APC and therefore influence the immune equilibrium. It is interesting that several diseases have been associated with an increased rate of drug allergy. The aim of this project was to evaluate the impact of such "danger signals" on sulfamethoxazole (SMX) metabolism in human APC (peripheral blood mononuclear cells, Epstein-Barr virus-modified B lymphocytes, monocyte-derived dendritic cells, and two cell lines). APC were incubated with SMX (100 microM-2 mM; 5 min-24 h), in the presence of pathological factors: bacterial endotoxins (lipopolysaccharide and staphylococcal enterotoxin B), flu viral proteins, cytokines [interleukin (IL)-1beta, IL-6, IL-10; tumor necrosis factor-alpha; interferon-gamma; and transforming growth factor-beta], inflammatory molecules (prostaglandin E2, human serum complement, and activated protein C), oxidants (buthionine sulfoximine and H(2)O(2)), and hyperthermia (37.5-39.5 degrees C). Adduct formation was evaluated by enzyme-linked immunosorbent assay and confocal microscopy. SMX-protein adduct formation was time- and concentration-dependent for each cell type tested, in both physiological and danger conditions. A danger environment significantly increased the formation of SMX-protein adducts and significantly shortened the delay for their detection. An additive effect was observed with a combination of danger signals. Dimedone (chemical selectively binding cysteine sulfenic acid) and antioxidants decreased both baseline and danger-enhanced SMX-adduct formation. Various enzyme inhibitors were associated with a significant decrease in SMX-adduct levels, with a pattern varying depending on the cell type and the culture conditions. These results illustrate that danger signals enhance the formation of intracellular SMX-protein adducts in human APC. These findings might be relevant to the increased frequency of drug allergy in certain disease states.

Sulfamethoxazole and its metabolite nitroso sulfamethoxazole stimulate dendritic cell costimulatory signaling.

Different signals in addition to the antigenic signal are required to initiate an immunological reaction. In the context of sulfamethoxazole allergy, the Ag is thought to be derived from its toxic nitroso metabolite, but little is known about the costimulatory signals, including those associated with dendritic cell maturation. In this study, we demonstrate increased CD40 expression, but not CD80, CD83, or CD86, with dendritic cell surfaces exposed to sulfamethoxazole (250-500 microM) and the protein-reactive metabolite nitroso sulfamethoxazole (1-10 microM). Increased CD40 expression was not associated with apoptosis or necrosis, or glutathione depletion. Covalently modified intracellular proteins were detected when sulfamethoxazole was incubated with dendritic cells. Importantly, the enzyme inhibitor 1-aminobenzotriazole prevented the increase in CD40 expression with sulfamethoxazole, but not with nitroso sulfamethoxazole or LPS. The enzymes CYP2C9, CYP2C8, and myeloperoxidase catalyzed the conversion of sulfamethoxazole to sulfamethoxazole hydroxylamine. Myeloperoxidase was expressed at high levels in dendritic cells. Nitroso sulfamethoxazole immunogenicity was inhibited in mice with a blocking anti-CD40L Ab. In addition, when a primary nitroso sulfamethoxazole-specific T cell response using drug-naive human cells was generated, the magnitude of the response was enhanced when cultures were exposed to a stimulatory anti-CD40 Ab. Finally, increased CD40 expression was 5-fold higher on nitroso sulfamethoxazole-treated dendritic cells from an HIV-positive allergic patient compared with volunteers. These data provide evidence of a link between localized metabolism, dendritic cell activation, and drug immunogenicity.

Predicting drug hypersensitivity by in vitro tests.

Recently it was found that drugs causing drug hypersensitivities do not only rely on the formation of hapten-carrier conjugates but can stimulate T cells directly via their T cell receptors for antigen. This new mechanism was termed pharmacological interaction of drugs with immune receptors (p-i concept). It is frequent in systemic drug hypersensitivity reactions and has major implications for predicting them. First experiments to identify such drugs able to interact with T cells are presented.

Association of drug-serum protein adducts and anti-drug antibodies in dogs with sulphonamide hypersensitivity: a naturally occurring model of idiosyncratic drug toxicity.

BACKGROUND: Sulphonamide antimicrobials, such as sulphamethoxazole (SMX), provide effective infection prophylaxis in immunocompromised patients, but can lead to drug hypersensitivity (HS) reactions. These reactions also occur in dogs, with a similar time course and clinical presentation as seen in humans. OBJECTIVES: Drug-serum adducts and anti-drug antibodies have been identified in sulphonamide HS humans. The aim of this study was to determine whether similar markers were present in dogs with sulphonamide HS. METHODS: Thirty-four privately owned sulphonamide HS dogs, 10 sulphonamide-'tolerant' dogs, 18 sulphonamide-naïve dogs, and four dogs experimentally dosed with SMX and the oxidative metabolite SMX-nitroso, were tested for drug-serum adducts by immunoblotting, and anti-drug antibodies by ELISA. RESULTS: Sulphonamide-serum adducts were found in 10/20 HS dogs tested (50%), but in no tolerant dogs. Anti-sulphonamide IgG antibodies were detected in 17/34 HS dogs (50%), but in only one tolerant dog; antibody absorbance values were significantly higher in HS dogs. There was a significant association between the presence of sulphonamide-serum adducts and anti-sulphonamide antibodies (P = 0.009). Anti-drug antibodies were also found in dogs experimentally dosed with SMX-nitroso followed by SMX, but not in a dog dosed with drug vehicle, followed by SMX. CONCLUSION: Similar humoral markers are present in dogs and humans with sulphonamide HS, supporting the use of dogs as a naturally occurring model for this syndrome in humans. These data suggest the potential use of drug-serum adducts and anti-drug antibodies as markers for sulphonamide HS. Preliminary data indicate that anti-sulphonamide antibodies may be triggered by the SMX-nitroso metabolite, not by the parent drug, in dogs.