Nitroso Sulfamethoxazole Research Articles

A blinded in vitro analysis of the intrinsic immunogenicity of hepatotoxic drugs: implications for preclinical risk assessment.

In vitro preclinical drug-induced liver injury (DILI) risk assessment relies largely on the use of hepatocytes to measure drug-specific changes in cell function or viability. Unfortunately, this does not provide indications toward the immunogenicity of drugs and/or the likelihood of idiosyncratic reactions in the clinic. This is because the molecular initiating event in immune DILI is an interaction of the drug-derived antigen with MHC proteins and the T-cell receptor. This study utilized immune cells from drug-naïve donors, recently established immune cell coculture systems and blinded compounds with and without DILI liabilities to determine whether these new methods offer an improvement over established assessment methods for the prediction of immune-mediated DILI. Ten blinded test compounds (6 with known DILI liabilities; 4 with lower DILI liabilities) and 5 training compounds, with known T-cell-mediated immune reactions in patients, were investigated. Naïve T-cells were activated with 4/5 of the training compounds (nitroso sulfamethoxazole, vancomycin, Bandrowski's base, and carbamazepine) and clones derived from the priming assays were activated with drug in a dose-dependent manner. The test compounds with DILI liabilities did not stimulate T-cell proliferative responses during dendritic cell-T-cell coculture; however, CD4+ clones displaying reactivity were detected toward 2 compounds (ciprofloxacin and erythromycin) with known liabilities. Drug-responsive T-cells were not detected with the compounds with lower DILI liabilities. This study provides compelling evidence that assessment of intrinsic drug immunogenicity, although complex, can provide valuable information regarding immune liabilities of some compounds prior to clinical studies or when immune reactions are observed in patients.

Checkpoint Inhibition Reduces the Threshold for Drug-Specific T-Cell Priming and Increases the Incidence of Sulfasalazine Hypersensitivity.

An emerging clinical issue associated with immune-oncology agents is the collateral effects on the tolerability of concomitant medications. One report of this phenomenon was the increased incidence of hypersensitivity reactions observed in patients receiving concurrent immune checkpoint inhibitors (ICIs) and sulfasalazine (SLZ). Thus, the aim of this study was to characterize the T cells involved in the pathogenesis of such reactions, and recapitulate the effects of inhibitory checkpoint blockade on de-novo priming responses to compounds within in vitro platforms. A regulatory competent human dendritic cell/T-cell coculture assay was used to model the effects of ICIs on de novo nitroso sulfamethoxazole- and sulfapyridine (SP) (the sulfonamide component of SLZ) hydroxylamine-specific priming responses. The role of T cells in the pathogenesis of the observed reactions was explored in 3 patients through phenotypic characterization of SP/sulfapyridine hydroxylamine (SPHA)-responsive T-cell clones (TCC), and assessment of cross-reactivity and pathways of T-cell activation. Augmentation of the frequency of responding drug-specific T cells and intensity of the T-cell response was observed with PD-1/PD-L1 blockade. Monoclonal populations of SP- and SPHA-responsive T cells were isolated from all 3 patients. A core secretory effector molecule profile (IFN-γ, IL-13, granzyme B, and perforin) was identified for SP and SPHA-responsive TCC, which proceeded through Pi and hapten mechanisms, respectively. Data presented herein provides evidence that drug-responsive T cells are effectors of hypersensitivity reactions observed in oncology patients administered ICIs and SLZ. Perturbation of drug-specific T-cell priming is a plausible explanation for clinical observations of how an increased incidence of these adverse events is occurring.

Characterization of T-Cell Responses to SMX and SMX-NO in Co-Trimoxazole Hypersensitivity Patients Expressing HLA-B*13:01.

HLA-B*13:01-positive patients in Thailand can develop frequent co-trimoxazole hypersensitivity reactions. This study aimed to characterize drug-specific T cells from three co-trimoxazole hypersensitive patients presenting with either Stevens-Johnson syndrome or drug reaction with eosinophilia and systemic symptoms. Two of the patients carried the HLA allele of interest, namely HLA-B*13:01. Sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones were generated from T cell lines of co-trimoxazole hypersensitive HLA-B*13:01-positive patients. Clones were characterized for antigen specificity and cross-reactivity with structurally related compounds by measuring proliferation and cytokine release. Surface marker expression was characterized via flow cytometry. Mechanistic studies were conducted to assess pathways of T cell activation in response to antigen stimulation. Peripheral blood mononuclear cells from all patients were stimulated to proliferate and secrete IFN-γ with nitroso sulfamethoxazole. All sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones expressed the CD4+ phenotype and strongly secreted IL-13 as well as IFN-γ, granzyme B and IL-22. No secretion of IL-17 was observed. A number of nitroso sulfamethoxazole-specific clones cross-reacted with nitroso dapsone but not sulfamethoxazole whereas sulfamethoxazole specific clones cross-reacted with nitroso sulfamethoxazole only. The nitroso sulfamethoxazole specific clones were activated in both antigen processing-dependent and -independent manner, while sulfamethoxazole activated T cell responses via direct HLA binding. Furthermore, activation of nitroso sulfamethoxazole-specific, but not sulfamethoxazole-specific, clones was blocked with glutathione. Sulfamethoxazole and nitroso sulfamethoxazole specific T cell clones from hypersensitive patients were CD4+ which suggests that HLA-B*13:01 is not directly involved in the iatrogenic disease observed in co-trimoxazole hypersensitivity patients.

Performance evaluation of waste iron shavings (Fe0) for catalytic ozonation in removal of sulfamethoxazole from municipal wastewater treatment plant effluent in a batch mode pilot plant

Feasibility of iron shavings (Fe0) as a low-cost catalyst was evaluated in catalytic ozonation for degradation of sulfamethoxazole (SMX). The characterization of the catalyst studied by a variety of techniques like scanning electron microscopy with energy dispersive spectroscopy (SEM-EDS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR). Experimental results indicated a first order kinetics for Fe-based catalytic ozonation (O3/Fe0) and sole-ozonation (O3) in the removal of SMX with slightly greater rate constant for O3. In the both processes, the removal efficiency of SMX was more than 99% at the first 5 min. Average COD removal efficiency in the O3/Fe0 process was about 1.36 times greater than O3. The main intermediates detected by LC-MS/MS were 3-Amino-5-methylisoxazole, benzoquinone, sulfanilic acid, hydroxyl-SMX and nitroso SMX. The mineralization of SMX significantly increased concentration of sulfate and nitrate. Fe0 enhanced ozone utilization and transfer rate. Fe0 is not requiring any preparation before use and could be a promising catalyst for practical advanced wastewater treatment.

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.

Mechanisms leading to T-cell activation in drug hypersensitivity.

Delayed-type or nonimmediate drug hypersensitivity reactions often involve the activation of drug-specific T cells. As such, the molecular initiating event is an interaction between HLA proteins, HLA-binding peptides and the drug. For many years, the formation of covalently modified drug protein adducts was assumed to be a prerequisite for T-cell activation. The purpose of this article is to review recent studies using human PBMC, T-cell lines and clones, which show that drugs are in fact loaded onto HLA molecules in different forms to activate T cells. We now know that protein-reactive drugs such as β-lactam antibiotics activate T cells via direct noncovalent interactions with HLA or HLA-binding peptides, direct covalent modification of HLA-binding peptides and covalent binding of non-HLA associated proteins. Adducts formed inside and outside of the cells undergo protein processing to generate HLA-binding peptides that are assumed to contain the drug modification. Studies using synthetic stable (e.g. oxypurinol) and reactive (e.g. nitroso sulfamethoxazole) metabolites show that metabolites activate T cells via the same pathways. A variety of drugs with different structural features have also been shown to activate T cells though a direct HLA-binding interaction. Of note, abacavir behaves in an unexpected way, binding deep in the peptide binding cleft of one HLA, selectively activating CD8 T cells. In-vitro studies have revealed that a number of drug HLA-binding interactions lead to the activation of T cells. These can be categorized according to two hypotheses, namely hapten and pharmacological interactions. As we move forward with the development of diagnostic and predictive T-cell assays, it is critical to reach a consensus that direct drug HLA binding and the formation of drug protein adducts are important events for T-cell activation.

Correction: The Effect of Inhibitory Signals on the Priming of Drug Hapten-Specific T Cells That Express Distinct Vβ 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 Vβ 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 Vβ 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 Vβ-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 Vβ subtypes, dysregulation of coinhibitory signaling pathways, and dysfunctional Tregs may influence predisposition to hypersensitivity.

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 Vβ 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 Vβ 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 Vβ 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 Vβ-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 Vβ subtypes, dysregulation of coinhibitory signaling pathways, and dysfunctional Tregs may influence predisposition to hypersensitivity.

Identification of drug- and drug-metabolite immune responses originating from both naive and memory T cells

Identification of drug- and drug-metabolite immune responses originating from both naive and memory T cells

Detection of Primary T Cell Responses to Drugs and Chemicals in HLA-Typed Volunteers: Implications for the Prediction of Drug Immunogenicity.

A number of serious adverse drug reactions are caused by T cells. An association with HLA alleles has been identified with certain reactions, which makes it difficult to develop standardized preclinical tests to predict chemical liability. We have recently developed a T cell priming assay using the drug metabolite nitroso sulfamethoxazole (SMX-NO). We now report on reproducibility of the assay, establishment of a biobank of PBMC from 1000 HLA-typed volunteers, and generation of antigen-specific responses to a panel of compounds. Forty T cell priming assays were performed with SMX-NO; 5 gave weak responses (1.5-1.9) and 34 showed good (SI 2.0-3.9) or strong responses (SI > 4.0) using readouts for proliferation and cytokine release. Thus, SMX-NO can be used as a model reagent for in vitro T cell activation. Good to strong responses were also generated to haptenic compounds (amoxicillin, piperacillin and Bandrowski's base) that are not associated with an HLA risk allele. Furthermore, responses were detected to carbamazepine (in HLA-B*15:02 donors), flucloxacillin (in 1 HLA-B*57:01 donor) and oxypurinol (in HLA-B*58:01 donors), which are associated with HLA-class I-restricted forms of hypersensitivity. In contrast, naïve T cell priming to ximelagatran, lumiracoxib, and lapatinib (HLA-class II-restricted forms of hypersensitivity) yielded negative results. Abacavir, which activates memory T cells in patients, did not activate naïve T cells from HLA-B*57:01 donors. This work shows that the priming assay can be used to assess primary T cell responses to drugs and to study mechanisms T cell priming for drugs that display HLA class I restriction. Additional studies are required to investigate HLA-class II-restricted reactions.

HLA-DQ allele-restricted activation of nitroso sulfamethoxazole-specific CD4-positive T lymphocytes from patients with cystic fibrosis.

For certain HLA allele-associated drug hypersensitivity reactions, the parent drug has been shown to associate directly with the risk allele. In other forms of hypersensitivity, HLA risk alleles have not been identified and T cells are activated in an allele unrestricted manner. Chemically reactive drug metabolites bind to multiple proteins; thus, it is assumed that the derived peptide antigens interact with a number of HLA molecules to activate T cells; however, HLA restriction of the drug metabolite-specific T-cell response has not been studied. To utilize T cells from sulfamethoxazole (SMX) hypersensitive patients with cystic fibrosis to examine the HLA molecules that interact with nitroso SMX (SMX-NO)-derived antigens. T-cell clones were generated from 4 hypersensitive patients. Drug-specific proliferative responses and cytokine secretion were measured. Anti-human class I and class II antibodies were used to analyse HLA restriction. Antigen-presenting cells expressing different HLA molecules were used to determine the alleles involved in the presentation of SMX-NO-derived antigens to T cells. A total of 976 clones were tested for SMX-NO reactivity. Thirty-nine CD4+ clones were activated with SMX-NO and found to proliferate and secrete cytokines. The SMX-NO-specific response was blocked with an antibody against HLA-DQ. SMX-NO-specific responses were detected with antigen-presenting cells expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. HLA-DQ plays an important role in the activation of SMX-NO-specific CD4+ T cells. Detection of HLA-DQ allele-restricted responses suggests that T cells are activated by a limited repertoire of SMX-NO-modified peptides.

Characterization of Peroxidases Expressed in Human Antigen Presenting Cells and Analysis of the Covalent Binding of Nitroso Sulfamethoxazole to Myeloperoxidase.

Drug hypersensitivity remains a major concern, as it causes high morbidity and mortality. Understanding the mechanistic basis of drug hypersensitivity is complicated by the multiple risk factors implicated. This study utilized sulfamethoxazole (SMX) as a model drug to (1) relate SMX metabolism in antigen presenting cells (APCs) to the activation of T-cells and (2) characterize covalent adducts of SMX and myeloperoxidase, which might represent antigenic determinants for T-cells. The SMX metabolite nitroso-SMX (SMX-NO) was found to bind irreversibly to APCs. Time- and concentration-dependent drug-protein adducts were also detected when APCs were cultured with SMX. Metabolic activation of SMX was significantly reduced by the oxygenase/peroxidase inhibitor methimazole. Similarly, SMX-NO-specific T-cells were activated by APCs pulsed with SMX, and the response was inhibited by pretreatment with methimazole or glutaraldehyde, which blocks antigen processing. Western blotting, real-time polymerase chain reaction (RT-PCR), and mass spectrometry analyses suggested the presence of low concentrations of myeloperoxidase in APCs. RT-PCR revealed mRNA expression for flavin-containing monooxygenases (FMO1-5), thyroid peroxidase, and lactoperoxidase, but the corresponding proteins were not detected. Mass spectrometric characterization of SMX-NO-modified myeloperoxidase revealed the formation of N-hydroxysulfinamide adducts on Cys309 and Cys398. These data show that SMX's metabolism in APCs generates antigenic determinants for T-cells. Peptides derived from SMX-NO-modified myeloperoxidase may represent one form of functional antigen.

Skewing of the TCR V repertoire in SMX‐NO specific T cell responses

Background A role for drug-specific T cells has been demonstrated in many instances of drug hypersensitivity. In the case of -lactam antibiotics, such as flucloxacillin, we have shown that drug-protein conjugates are present in the plasma of all patients treated with this antibiotic, however only a small subset of patients develop hypersensitivity. Similarly, when a strong HLA association to drug hypersensitivity has been demonstrated such as for abacavir, not all patients with the risk allele will develop a hypersensitivity reaction. The mechanism underlying this variation in susceptibility is not fully understood. In carbamazepineinduced Stevens Johnson syndrome it is known that only patients with both the HLA risk allele and T cells with a specific TCR V will develop a reaction. This shows that an individual’s T cell repertoire may confer some susceptibility to drug hypersensitivity. Method In initial experiments, SMX-NO-specific CD4+ clones were generated from hypersensitive patients and analysed for TCR V expression. Nitroso sulfamethoxazole (SMXNO)-specific T cells were generated from 6 individuals by priming naive T-cells using drug-treated autologous dendritic cells. The responding cells were isolated using positive CD45RO magnetic bead selection. Naive and SMX-NO specific T cells were analysed by serology for 24 TCR Vs and by RT-PCR spectra-typing of the CDR3 region.

Negative regulation by Programmed Death Ligand‐1 during drug‐specific priming of T‐cells and the influence of Programmed Death‐1 on effector T‐cell function

PD-1 (Programmed Death-1) has been classified as a marker of T-cell exhaustion, however, several recent studies suggest that most PD-1 high T-cells are highly proliferative effector memory cells that maintain effector function during chronic infection. Furthermore, activation of PD-1 on T-cells is thought to inhibit antigen-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 signalling influences the differentiation of T-cells. Naive T-cells were cocultured with monocyte-derived dendritic cells in the presence of a drug (flucloxacillin, nitroso sulfamethoxazole) for a period of 8 days (±PD-L1/2 block), to expand the number of drug-responsive T-cells. The T-cells were then incubated with fresh dendritic cells and the drug. The antigen responsiveness was analyzed using readouts for proliferation, cytokine secretion, and cell phenotype. Cell phenotype was characterised by flow cytometry. T-cell clones were generated following priming and from drug hypersensitive patients to characterize the cytokine signature(s) of antigen specific T-cells and to study whether PD-1 expression/signalling governs the differentiation of T-cells into effector/helper subsets. Priming of naive CD4+ and CD8+ T-cells against drug antigens 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, CFSE analysis revealed a transient increase during priming. Drug-specific T-cell clones generated through priming and from hypersensitive patients 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 antigen-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 antigen-specific FasL/IL-22 secreting T-cell subset with skin homing properties.

HLA‐DQ restricted activation of nitroso‐sulfamethoxazole‐specific CD4+ T‐lymphocytes

Background Sulfamethoxazole hypersensitivity has been used as a paradigm to study the role of drug metabolism in the activation of T-cells. CD4+ T-cells isolated from blood/ skin of 100% of hypersensitive patients are activated with the protein-reactive metabolite nitroso sulfamethoxazole (SMX.NO). Since SMX.NO binds to multiple cellular proteins it is assumed that peptides derived from the modified protein interact with a number of diverse HLA molecules to activate T-cells; however, the HLA molecules that interact with SMX-NO modified peptides has not been studied. Method The aims of this study were to examine the HLA molecules that present SMX.NO (derived peptides) to T-cells and determine the extent of alloreactivity. T-cell clones were generated from 4 hypersensitive patients with cystic fibrosis. Drug-specific proliferative responses and cytokine secretion were measured using [3H]-thymidine incorporation and ELISpot, respectively. Anti-human class I and class II (DR, DP, and DQ) antibodies were used to determine HLA restriction. Antigen presenting cells expressing different HLAs were used to define the alleles involved in the presentation of SMX.NO-derived antigens to T-cells. Results

Human Leukocyte Antigen (HLA)-B*57:01-Restricted Activation of Drug-Specific T cells Provides the Immunological Basis for Flucloxacillin-Induced Liver Injury

The role of the adaptive immune system in adverse drug reactions that target the liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of human leukocyte antigen (HLA)-B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, we characterize flucloxacillin-responsive CD4+ and CD8+ T cells from patients with liver injury and show that naive CD45RA+CD8+ T cells from volunteers expressing HLA-B*57:01 are activated with flucloxacillin when dendritic cells present the drug antigen. T-cell clones expressing CCR4 and CCR9 migrated toward CCL17 and CCL 25, and secreted interferon-gamma (IFN-γ), T helper (Th)2 cytokines, perforin, granzyme B, and FasL following drug stimulation. Flucloxacillin bound covalently to selective lysine residues on albumin in a time-dependent manner and the level of binding correlated directly with the stimulation of clones. Activation of CD8+ clones with flucloxacillin was processing-dependent and restricted by HLA-B*57:01 and the closely related HLA-B*58:01. Clones displayed additional reactivity against β-lactam antibiotics including oxacillin, cloxacillin, and dicloxacillin, but not abacavir or nitroso sulfamethoxazole. This work defines the immune basis for flucloxacillin-induced liver injury and links the genetic association to the iatrogenic disease.

The Development of In Vitro Culture Methods to Characterize Primary T-Cell Responses to Drugs

Adverse drug reactions represent a major stumbling block to drug development and those with an immune etiology are the most difficult to predict. We have developed an in vitro T-cell priming culture method using peripheral blood from healthy volunteers to assess the allergenic potential of drugs. The drug metabolite nitroso sulfamethoxazole (SMX-NO) was used as a model drug allergen to establish optimum assay conditions. Naive T cells were cocultured with monocyte-derived dendritic cells at a ratio of 25:1 in the presence of the drug for a period of 8 days, to expand the number of drug-responsive T cells. The T cells were then incubated with fresh dendritic cells, and drug and their antigen responsiveness analyzed using readouts for proliferation, cytokine secretion, and cell phenotype. All five volunteers showed dose-dependent proliferation as measured by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester content and by (3)H-thymidine uptake. CD4 T cells that had divided in the presence of SMX-NO had changed from a naive phenotype (CD45RA+) to a memory phenotype (CD45RO+). These memory T cells expressed the chemokine receptors CCR2, CCR4, and CXCR3 suggesting a mixture of T(H)1 and T(H)2 cells in the responding population, with a propensity for homing to the skin. Drug stimulation was also associated with the secretion of a mixture of T(H)1 cytokines (interferon γ) and T(H)2 cytokines (interleukin [IL]-5 and IL-13) as detected by ELISpot. We are currently developing this approach to investigate the allergenic potential of other drugs, including those where an association between specific human leucocyte antigen alleles and susceptibility to an immunological reaction has been established.

Multiple Adduction Reactions of Nitroso Sulfamethoxazole with Cysteinyl Residues of Peptides and Proteins: Implications for Hapten Formation

Sulfamethoxazole (SMX) induces immunoallergic reactions that are thought to be a result of intracellular protein haptenation by its nitroso metabolite (SMX-NO mass, 267 amu). SMX-NO reacts with protein thiols in vitro, but the conjugates have not been defined chemically. The reactions of SMX-NO with glutathione (GSH), a synthetic peptide (DS3), and two model proteins, human GSH S-transferase pi (GSTP) and serum albumin (HSA), were investigated by mass spectrometry. SMX-NO formed a semimercaptal (N-hydroxysulfenamide) conjugate with GSH that rearranged rapidly (1-5 min) to a sulfinamide. Reaction of SMX-NO with DS3 also yielded a sulfinamide adduct (mass increment, 267 amu) on the cysteine residue. GSTP was exclusively modified at the reactive Cys47 by SMX-NO and exhibited mass increments of 267, 283, and 299 amu, indicative of sulfinamide, N-hydroxysulfinamide, and N-hydroxysulfonamide adducts, respectively. HSA was modified at Cys34, forming only the N-hydroxysulfinamide adduct. HSA modification by SMX-NO under these conditions was confirmed with ELISA and immunoblotting with an antisulfonamide antibody. It is proposed that cysteine-linked N-hydroxysulfinamide and N-hydroxysulfonamide adducts of SMX are formed via the reaction of SMX-NO with cysteinyl sulfoxy acids. Evidence for a multistep assembly of model sulfonamide epitopes on GSH and polypeptides via hydrolyzable intermediates is also presented. In summary, novel, complex, and metastable haptenic structures have been identified on proteins exposed in vitro to the nitroso metabolite of SMX.

Activation of T-Cells from Allergic Patients and Volunteers by p-Phenylenediamine and Bandrowski's Base

Allergic contact dermatitis is commonly associated with exposure to p-phenylenediamine. The aim of this study was to determine whether p-phenylenediamine (PPD) and/or Bandrowski's base (BB) stimulate T cells from allergic patients and volunteers, and to explore the relationship between T-cell immunogenicity and allergy. Lymphocytes from allergic patients proliferated with PPD and BB (n=8). Lymphocytes from 14/16 non-allergic individuals also proliferated following stimulation, but only with BB; cord blood lymphocytes failed to respond (n=6). Glutathione, which prevented BB formation, but not binding of PPD to cells and serum, did not prevent p-phenylenediamine-specific stimulation of patient lymphocytes. T-cell clones generated from allergic patients were stimulated separately with PPD and BB, while clones from volunteers proliferated with BB alone. Patient and volunteer clones secreted IL-4, IL-5, IL-13, TNF-alpha, MIP-1alpha, MIP-1beta, and RANTES. These data show that activation of T lymphocytes from allergic individuals alone with PPD represents an important discrimination between allergic and non-allergic groups. BB-specific T cells are found in both allergic patients and volunteers, but not in cord blood. Their presence seems to reflect an acquired immune response, which is not translated into an allergic reaction.