Sidonie N. Lavergne

Mass Spectrometric Characterization of Circulating and Functional Antigens Derived from Piperacillin in Patients with Cystic Fibrosis

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys541 observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys190, Lys195, Lys432, and Lys541) were detected in patients’ plasma. Piperacillin-specific T lymphocyte responses (proliferation, cytokines, and granzyme B release) were detected ex vivo with cells from hypersensitive patients, and analysis of incubation medium showed that modification of the same lysine residues in albumin occurred in situ. The antigenicity of piperacillin-modified albumin was confirmed by stimulation of T cells with characterized synthetic conjugates. Analysis of minimally modified T cell-stimulatory albumin conjugates revealed peptide sequences incorporating Lys190, Lys432, and Lys541 as principal functional epitopes for T cells. This study has characterized the multiple haptenic structures on albumin in patients and showed that they constitute functional antigenic determinants for T cells.

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.

“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 μM–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)-1β, IL-6, IL-10; tumor necrosis factor-α; interferon-γ; and transforming growth factor-β], inflammatory molecules (prostaglandin E2, human serum complement, and activated protein C), oxidants (buthionine sulfoximine and H2O2), and hyperthermia (37.5–39.5°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.

The roles of drug metabolism in the pathogenesis of T-cell-mediated drug hypersensitivity.

Purpose of review The purpose of this review is to highlight recent studies on drug metabolism in T-cell-mediated reactions. Although the hapten theory and the danger hypothesis imply an important role of reactive metabolites in the pathogenesis of drug hypersensitivity, the more recent pi concept gives the central role to the parent drug. It is therefore important to have a broad vision on data supporting each theory to understand the potential role(s) of drug metabolism in T-cell-mediated hypersensitivity. Recent findings Reactive metabolites have been identified for most drugs associated with hypersensitivity. Recent studies have further characterized drug metabolism outside the liver, in key tissues such as the skin and immune cells. Localized drug metabolism is associated with oxidative stress, adduct formation and toxicity creating danger signals for antigen presenting cells, influencing whether a drug antigen will induce tolerance or immunity. Summary The involvement of metabolic drug activation in the initiation and propagation of hypersensitivity is intriguing since metabolites are generated in different quantities throughout the body, can be directly or indirectly toxic to cells, might stimulate innate immunity, and can bind to protein to generate neoantigens for cellular and humoral responses.

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 hypersensitivity reactions targeting the skin in dogs and cats.

Adverse drug reactions (ADRs) can be dose dependent or idiosyncratic. Most idiosyncratic reactions are believed to be immune-mediated; such drug hypersensitivities and allergies are unpredictable. Cutaneous reactions are the most common presentation of drug allergies. In veterinary medicine it can be difficult to assess the true prevalence of adverse drug reactions, although reports available suggest that they occur quite commonly. There are multiple theories that attempt to explain how drug allergies occur, because the pathogenesis is not yet well understood. These include the (pro)-hapten hypothesis, the Danger Theory, the pi concept, and the viral reactivation theory. Cutaneous drug allergies in veterinary medicine can have a variety of clinical manifestations, ranging from pruritus to often fatal toxic epidermal necrolysis. Diagnosis can be challenging, as the reactions are highly pleomorphic and may be mistaken for other dermatologic diseases. One must rely heavily on history and physical examination to rule out other possibilities. Dechallenge of the drug, histopathology, and other diagnostic tests can help to confirm the diagnosis. New diagnostic tools are beginning to be used, such as antibody or cellular testing, and may be used more in the future. There is much yet to learn about drug allergies, which makes future research vitally important. Treatment of drug allergies involves supportive care, and additional treatments, such as immunosuppressive medications, depend on the manifestation of the disease. Of utmost importance is to avoid the use of the incriminating drug in future treatment of the patient, as subsequent reactions can be worse, and ultimately can prove fatal.

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.

Characterization of drug-specific lymphocyte responses in a patient with drug-induced liver injury

To the Editor: Drug-induced liver injury takes many forms and can mimic naturally occurring liver disease. In 1997,Maria andVictorino described lymphocyte responses to drugs in more than 50% of patients with drug-induced liver injury. More recently, histologic examination of inflamed liver tissue from a patient exposed to sulfasalazine revealed an infiltration of granzyme B–secreting T lymphocytes. Importantly, the phenotype and function of T cells frompatientswith drug-induced liver injury have not been studied. In the present investigationwe used lymphocytes from a patient with trimethoprim-induced liver injury as part of a generalized hypersensitivity reaction (drug rash with eosinophilia and systemic symptoms) to define the frequency of circulating drugspecific T cells and to characterize the cellular response in terms of phenotype and function. The patient was admitted to the hospital in the fourth week after onset of trimethoprim treatment for acnewith fever, desquamating rash, and pruritus. Initial results on admission were as follows: white cell count of 24.93 10/mL with eosinophilia of 13% and increased liver function test results (see Table E1 in this article’s Online Repository at www. jacionline.org). She was later transferred to a specialized liver unit at Queen Elizabeth Hospital in Birmingham, where her liver test results continued to deteriorate. Blood samples were taken for the immunologic investigations on referral and 3, 12, and 24 months after recovery. Details of the methods used are available in the Methods section of this article’s Online repository at www.jacionline.org. Ex vivo lymphocytes isolated from the patient, but not shortand long-term drug-exposed control subjects, were shown to readily proliferate and secrete cytokines in vitro after challenge with trimethoprim (Fig 1, A) to confirm that the reaction had an immunologic cause. The frequency of trimethoprim-specific T cells measured by generating libraries of amplified CD4 and CD8 T cells from peripheral blood were estimated to be 75/10 cells (95% CI, 51-110/10 cells) and 31/10 cells (95% CI, 17-56/10 cells), respectively (Fig 1, B). Cells were expanded and proliferation and IFN-g/IL-13 levels were measured to confirm that the amplified cultures contained T cells responsive against trimethoprim (Fig 1, C and D). Similar experiments were conducted with lymphocytes from trimethoprim-exposed control subjects and patients with trimethoprim-mediated skin reactions (with no liver injury, n 5 7), but it was not possible to estimate a drug-specific precursor frequency. Beeler et al recently demonstrated that the frequency of drug-specific T cells in patients with cutaneous reactions was 100 to 4000/10 cells. Although themethodological approach in the 2 studies differs, the data suggest that reaction severity, organ selectivity, or both are not directly related to the frequency of antigen-specific T cells. Forty-five CD4 and CD8 clones expressing different Vb receptors were generated to define the cellular pathophysiology of the reaction to trimethoprim. Proliferative responses to a number of clones were detected with trimethoprim at concentrations as low as 1 mg/mL, indicating that these clones might be preferentially activated in vivo. Cytotoxicity measured with a classical Cr release assay and by monitoring transient increases in CD107a (lysosomal-associated membrane protein 1) expression was detected with both CD4 and CD8 T cells after trimethoprim exposure (see Fig E1 in this article’s Online Repository at www.jacionline.org). Thus, as described previously with T cells isolated from epicutaneous test reactions, both populations of T cells might play a role in the development of clinical signs. Trimethoprim-specific T-cell clones secreted a restricted cytokine profile (Fig 2, A, and see Table E2 in this article’s Online Repository at www.jacionline.org); IL-13, IFN-g, and TNF-a were detected after trimethoprim stimulation. Clones seemed to corelease IFN-g and TNF-a (R 5 0.73), and an association between the levels of these cytokines and cytolytic activity was observed. Cytotoxicity was seen with CD4 and CD8 clones secreting IFN-g and TNF-a at concentrations of greater than 500 pg/mL. Picard et al recently characterized activated IFNg/TNF-a CD8 T lymphocytes, which were largely directed against herpes viruses in hypersensitive patients with systemic symptoms. Furthermore, the authors suggested that reactivation of virus-specific T cells contributes to the development of clinical signs of hypersensitivity. Although screening for the underlying infection or infections was beyond the scope of the current study, our data describing drug-responsive cytotoxic CD4 T cells clearly highlight the need for further mechanistic studies to unequivocally define the disease’s pathogenesis. A particular area of interest was why the T-cell response observed in this patient targeted the liver. For lymphocytes to exert a cytotoxic response in a particular organ, they must be recruited from the circulation. The expression of specific chemokines produced in response to inflammatory stress and their subsequent interaction with chemokine receptors on the surface of lymphocytes is known to promote T-cell migration through the endothelium and into tissues. T cells express distinct chemokine receptors that direct cellular migration. T cells infiltrating skin express high levels of cutaneous lymphocyte antigen and the chemokine receptors CCR4 and CCR10, and indeed, drugspecific T cells from patients with cutaneous drug-induced hypersensitivity reactions express high levels of these receptors. Although the expression of chemokine receptors on liver-homing T cells is less well defined, CXCR3, CCR5, CCR9, and CXCR6 have been found on T cells isolated from patients with various forms of liver disease. In the current study CD11aCD27 (memory and terminally differentiated) trimethoprim-specific CD4 and CD8 clones expressed high levels of CXCR3 and CCR9 but only low levels of skin-homing receptors, which might explain the relatively mild cutaneous symptoms (Fig 2, B, and see Table E3 in this article’s Online Repository at www.jacionline. org). CXCR3-expressing T cells are a common feature of inflammatory conditions in the liver, whereas CCR9 is expressed on T cells that predominantly migrate to the small intestine and enter the liver after activation because of hepatic expression of the CCR9 chemokine CCL25 (thymus-expressed chemokine). In line with this observation, it is possible that an undiagnosed disease or infection of the gut might have played a role in priming dendritic cells to imprint trimethoprim-specific T cells with a profile of homing receptors that directed the tissue injury. CXCR6, which interacts with the chemokine expressed on liver endothelium CXCL16 (Bonzo), was also detected on the majority of

Contact dermatitis: A comparative and translational review of the literature

BACKGROUND Contact dermatitis (CD) is an inflammatory skin condition induced by direct contact with a specific chemical. Irritant CD (ICD) is a nonspecific inflammatory cutaneous reaction to an irritating agent. Allergic CD (ACD) is an immune-mediated antigen-specific skin reaction to an allergenic chemical. OBJECTIVES AND METHODS The biomedical literature (human, basic science, veterinary) was reviewed to evaluate the current state of knowledge regarding CD. RESULTS The incidence of human CD remains unclear, but represents up to 90-95% of all occupational skin diseases. The prevalence of CD has not been established in veterinary medicine. The pathogenesis of CD is not fully understood, but involves a complex cascade of events between resident skin cells, relocated immune cells, pro-inflammatory cytokines and chemokines. The main difference between ICD and ACD is that ACD is an antigen-specific reaction to an allergenic irritating agent whereas ICD is not antigen-specific. To date, there is no fully validated diagnostic test available for CD. Thus, its clinical diagnosis relies on the patients history, clinical examination, dermatological tests and, in some cases, research-based assays. The most important factor in CD management is the identification and avoidance of the culprit irritant or allergen. In addition, various topical and systemic therapies can be considered. CONCLUSION AND CLINICAL RELEVANCE CD is a relatively common occupational skin disease in human beings, but the prevalence in veterinary medicine is undefined. It can lead to debilitating clinical signs. Further research in human medicine and even more so in veterinary patients, will be required in order to allow for an evidence-based approach in its diagnosis and management.

Trimethoprim Stimulates T-Cells through Metabolism-Dependent and -Independent Pathways

Pathways of drug-specific T-cell stimulation have not been fully defined. The aim of this study was to use T-cell clones from a patient hypersensitive to the drug trimethoprim to characterize the involvement of drug metabolism and processing in antigen presentation and cross-reactivity patterns. The MHC-restricted CD4+ and CD8+ T-cell response was dependent on the presence of antigen-presenting cells, and both processing-dependent and -independent pathways of antigen presentation were detected. Stimulation of certain clones was blocked through inhibition of drug-metabolizing enzyme activity. Trimethoprim clones were additionally stimulated with diaveridine and pyrimethamine but not other closely related structures.