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.

Drug-specific in vitro release of IL-2, IL-5, IL-13 and IFN-gamma in patients with delayed-type drug hypersensitivity.

BACKGROUND: The most prevalent drug hypersensitivity reactions are T-cell mediated. The only established in vitro test for detecting T-cell sensitization to drugs is the lymphocyte transformation test, which is of limited practicability. To find an alternative in vitro method to detect drug-sensitized T cells, we screened the in vitro secretion of 17 cytokines/chemokines by peripheral blood mononuclear cells (PBMC) of patients with well-documented drug allergies, in order to identify the most promising cytokines/chemokines for detection of T-cell sensitization to drugs. METHODS: Peripheral blood mononuclear cell of 10 patients, five allergic to beta-lactams and five to sulfanilamides, and of five healthy controls were incubated for 3 days with the drug antigen. Cytokine concentrations were measured in the supernatants using commercially available 17-plex bead-based immunoassay kits. RESULTS: Among the 17 cytokines/chemokines analysed, interleukin-2 (IL-2), IL-5, IL-13 and interferon-gamma (IFN-gamma) secretion in response to the drugs were significantly increased in patients when compared with healthy controls. No difference in cytokine secretion patterns between sulfonamide- and beta-lactam-reactive PBMC could be observed. The secretion of other cytokines/chemokines showed a high variability among patients. CONCLUSION: The measurement of IL-2, IL-5, IL-13 or IFN-gamma or a combination thereof might be a useful in vitro tool for detection of T-cell sensitization to drugs. Secretion of these cytokines seems independent of the type of drug antigen and the phenotype of the drug reaction. A study including a higher number of patients and controls will be needed to determine the exact sensitivity and specificity of this test.

Antigenicity of sulfanilamide and its metabolites using fluorescent-labelled compounds.

In order to clarify the onset mechanisms of drug-induced allergies, three fluorescent-labelled compounds were synthesized by subjecting sulfanilamide (SA), a base compound for sulfonamides, and its active metabolites, i.e. sulfanilamide hydroxylamine and sulfanilamide nitroso, to dansylation using dansylchloride. In other words, 5-dimethylamino-N-(4-aminobenzyl)-naphthalenesulfonamide (DNS-4ABA), 5-dimethylamino-N-(4-hydroxylaminobenzyl)-1-naphthalenesulfonamide (DNS-4HABA) and 5-dimethylamino-N-(4-nitrosobenzyl)-1-naphthalenesulfonamide (DNS-4NSBA) were synthesized as model haptens. When analysed by HPLC, a conjugate of DNS-4HABA and glutathione (GSH) with nucleophilic amino acids had two peaks (P-1 and P-2). FAB-MS and 1H-NMR revealed that the DNS-4HABA-GSH conjugate consisted of sulphinamide and semimercaptal. The reactivity of GSH to DNS-4ABA, DNS-4HABA and DNS-4NSBA was quantified by HPLC using an oxidization system (horseradish peroxidase/H2O2). The results show that production of DNS-4NSBA-GSH-conjugate was four to eight times higher than that of DNS-4HABA-GSH conjugate, but that DNS-4ABA did not bind with GSH. Skin reactions were assessed using guinea pigs, and strong delayed erythema was seen with DNS-4NSBA, which bound most strongly with GSH, whereas weak delayed erythema was seen with DNS-4ABA, which did not bind with GSH. This suggests a correlation between GSH conjugate production and skin reactions. DNS-4HABA enzymatically bound with proteins in rat and guinea pig liver cytosol and microsomal fractions. The proteins that bound to DNS-4HABA were purified by HPLC and then subjected to N-terminal amino acid analysis. Ubiquitin (10 kDa) and fatty acid binding protein (30 kDa) were detected in the rat liver cytosol fraction; retinol-dehydrogenase (35 kDa) in the rat microsomal fraction; and glutathione-S-transferase B (mmu) (25 kDa) in the guinea pig liver cytosol fraction. When DNS-4HABA or DNS-4NSBA binds to proteins that play important roles in the body, unexpected adverse reactions may occur. Furthermore, by utilizing our technique using model compounds, it may be possible to identify the carrier proteins of various compounds, including pharmaceutical agents.

The spectrum of allergic (cross-)sensitivity in clinical patch testing with 'para amino' compounds.

BACKGROUND: Allergic contact sensitization to 'para amino' compounds is frequent and the spectrum of cross-reactivity between members of this chemical group is variable. METHODS: A retrospective analysis of clinical patch test data obtained with a special test series in the centres of the Information Network of Departments of Dermatology (IVDK) between 1995 and 1999. RESULTS: In the 638 patients tested with the above test panel positive reactions were observed most often to p-aminoazobenzene (16.2%), p-phenylenediamine (14.1%), p-toluylenediamine (10.0%), followed by 4,4'-diaminodiphenylmethane (8.5%), Disperse Orange 3 (8.4%) and p-aminophenol (3.1%). Among the 544 patients tested with p-phenylenediamine and all seven additional 'para amino' compounds, concordance between reactions varied greatly. The stronger the positive test reaction to p-phenylenediamine, p-toluylenediamine or p-aminoazobenzene, the more frequently additional positive reactions to the other compounds were observed. CONCLUSIONS: A screening employing several 'para amino' compounds is necessary to describe the individual spectrum of allergic contact sensitization, as there is no reliable marker substance. Further research should aim at (i) establishing the mechanism of cross-reactivity to 'para amino' compounds and (ii) identifying exposures in the environment.

Determination of sulfonamides in swine meat by immunoaffinity chromatography.

A procedure was developed for the preparation of anti-sulfonamide (SA) group-specific antibodies and immunosorbents. Sulfonamide haptens and conjugates were synthesized by building spacer arms on an N1 group of 4-aminobenzensulfonamide. The anti-SA group-specific antibodies and immunosorbents were prepared successfully. After extraction with methanol-water (8 + 2), sulfamonomethoxine, sulfadimethoxine, and sulfaquinoxaline were cleaned up on immunoaffinity columns and determined by reversed-phase liquid chromatography with UV detection at 270 nm. The recoveries from fortified swine meat (10-100 microg/kg) ranged from 70.8 to 94.1%, with coefficients of variation of 3.4-12.9%. Limits of detection were 1-2 microg/kg.

Spontaneous formation of immunogenic conjugates by a biotransformation product of sulfanilamide.

3-hydroxy-sulfanilamide (3-HS) has been previously shown to be formed in significant amounts in rabbits and humans treated with sulfanilamide. In the present paper we demonstrate that 3-HS reacts readily with proteins at alkaline pH and forms conjugates capable to induce hapten specific antibodies in rabbits. According to our previous observations on ortho-aminophenol (OAP), the haptenic properties of 3-HS are apparently due to the presence in its molecule of an ortho-aminophenolic structure, rendering it capable to form a highly reactive quinoneimine derivative. In spite of a similar mechanism of linking and of the presence of an ortho-aminophenolic structure in the two haptens, 3-HS and OAP immunological systems do not cross react with each other. The data presented here support the hypothesis that 3-HS may represent the hapten or one of the haptens involved in sulfonamide allergy.

[Effect of comprehensive therapy using antibiotics and sulfanilamide and stimulant preparations on natural body resistance in acute dysentery]. / Vliianie kompleksnoi terapii s primenenien antibiotikov, sul'fanilamidnykh i stimuliruiushchikh preparatov na estestvennuiu rezistentnost' organizma bol'nykh ostroi dizenteriei.

A total of 489 patients at the age of 18 to 74 with acute dysentery of medium severity without concomitant diseases were observed. The patients were subjected to combined therapy with the use of antibiotics, sulfanilamides and stimulants. The state of the patients' natural resistance was determined by the dynamics and levels of the bactericidal activity of lysozyme, blood serum complement as well as lysozyme and beta-lysine coprofiltrates. It was found that the use of tetracycline, furazolidone combinations with sulfanilamides and leukopoiesis-stimulating drugs had a favourable effect on the dynamics of the host natural resistance reduction. The reduction of the natural resistance in the patients treated with levomycetin in combination with sulfanilamides and stimulants was much slower than the clinical recovery.

Characterisation of the reaginic response to sulphonamides in mice.

Reaginic responses were induced in immunised mice with either 4-sulphanilamidobenzoic acid (4-SABA) or sulphamethoxazole (SMX) coupled to chicken gamma-globulin (CGG). The former was coupled through the carboxylic group of benzoic acid and the latter through the diazo derivative of the sulphanilamide group. The specificity of the reaginic responses obtained was assessed in each case by inhibition of passive cutaneous anaphylaxis in the rat. Immunisation with 4-SABA-CGG resulted in antibodies which recognised the sulphanilamide group as immunodominant and, therefore, they cross-reacted strongly with other sulphonamides. In contrast, SMX-CGG-induced IgE antibodies directed primarily to the methoxazole end of the molecule and cross-reactivity with other sulphonamides could not be demonstrated. This model may be useful for a more comprehensive study of allergic reactions induced by sulphonamides.

[Resistance to hypoglycemic sulfanilamides and biguanidines and its immune genesis]. / Ustoichivost' k gipoglikemiziruiushchim sul'fanilamidam i biguanidam i ee immunnyi genez.

In blood of patients with diabetes mellitus receiving bucarban or adebit antibodies to these drugs were discovered. Patients medicated with bucarban demonstrated an elevated content of antibodies to insulin. The appearance of specific antibodies occurred in rabbits and rats following introduction of bucarban, glucophage or adebit per se or when mixed with adjuvants. An increased production of antibodies to endogenous insulin was observed in all animals reveiving bucarban. Introduction to rats of inactivated (at 56 degrees C for 30 minutes) serum of patients with diabetes mellitus that contained antibodies to bucarban or adebit as well as immune rabbit sera greatly mitigated the hypoglycemic effect of the latter. Introduction to rats of the blood serum taken in patients reveiving bucarban allso attenuated the effect of exogenous insulin. These findings suggest that, possessing neutralizing properties, these antibodies to bucarban, adebit and glucophage cause habituation to these substances. Accumulation in the blood of autoantibodies to insulin following introduction of bacarban reduces the sensitivity to the exogenous hormone and manifests the existence of an immune mechanism securing the preservation of chemical homeostasis in the organism.

Positive skin reaction induced by 4,4'-azoxybenzenedisulfonamide in relationship to the sulfanilamide allergy.

All guinea pigs sensitized to sulfanilamide or 4-hydroxylaminobenzenesulfonamide in the absence of ultraviolet irradiation exhibited positive skin reactions to 4,4'-azoxybenzenedisulfonamide, 4-nitrosobenzenesulfonamide, and 4-hydroxylaminobenzenesulfonamide.