Werner J. Pichler

HLA-A*3101 and Carbamazepine-Induced Hypersensitivity Reactions in Europeans

BACKGROUND Carbamazepine causes various forms of hypersensitivity reactions, ranging from maculopapular exanthema to severe blistering reactions. The HLA-B*1502 allele has been shown to be strongly correlated with carbamazepine-induced Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS-TEN) in the Han Chinese and other Asian populations but not in European populations. METHODS We performed a genomewide association study of samples obtained from 22 subjects with carbamazepine-induced hypersensitivity syndrome, 43 subjects with carbamazepine-induced maculopapular exanthema, and 3987 control subjects, all of European descent. We tested for an association between disease and HLA alleles through proxy single-nucleotide polymorphisms and imputation, confirming associations by high-resolution sequence-based HLA typing. We replicated the associations in samples from 145 subjects with carbamazepine-induced hypersensitivity reactions. RESULTS The HLA-A*3101 allele, which has a prevalence of 2 to 5% in Northern European populations, was significantly associated with the hypersensitivity syndrome (P=3.5×10(-8)). An independent genomewide association study of samples from subjects with maculopapular exanthema also showed an association with the HLA-A*3101 allele (P=1.1×10(-6)). Follow-up genotyping confirmed the variant as a risk factor for the hypersensitivity syndrome (odds ratio, 12.41; 95% confidence interval [CI], 1.27 to 121.03), maculopapular exanthema (odds ratio, 8.33; 95% CI, 3.59 to 19.36), and SJS-TEN (odds ratio, 25.93; 95% CI, 4.93 to 116.18). CONCLUSIONS The presence of the HLA-A*3101 allele was associated with carbamazepine-induced hypersensitivity reactions among subjects of Northern European ancestry. The presence of the allele increased the risk from 5.0% to 26.0%, whereas its absence reduced the risk from 5.0% to 3.8%. (Funded by the U.K. Department of Health and others.).

General considerations for skin test procedures in the diagnosis of drug hypersensitivity.

K. Brockow, A. Romano, M. Blanca, J. Ring, W. Pichler, P. Demoly Klinik und Poliklinik fur Dermatologie und Allergologie, Muenchen, Germany; Department of Internal Medicine and Geriatrics, UCSC, Allergy Unit, CI Columbus, Rome and IRCS Oasi Maria SS, Troina, Italy; Research Unit for Allergic Diseases, Carlos Haya Hospital, Malaga, Spain; Clinic for Rheumatology and Clinical Immunology/Allergology, Inselspital, Bern, Switzerland; Maladies Respiratoires, Hopital Arnaud de Villeneuve, Montpellier, France

Drug provocation testing in the diagnosis of drug hypersensitivity reactions: general considerations.

A drug provocation test (DPT) is the controlled administration of a drug in order to diagnose drug hypersensitivity reactions. DPTs are performed under medical surveillance, whether this drug is an alternative compound, or structurally/pharmacologically related, or the suspected drug itself. DPT is sometimes termed controlled challenge or reexposure (1), drug challenge (2), graded (2) or incremental challenge (3), test dosing (2), W. Aberer, A. Bircher, A. Romano, M. Blanca, P. Campi, J. Fernandez, K. Brockow, W. J. Pichler, P. Demoly for ENDA, and the EAACI interest group on drug hypersensitivity Department of Environmental Dermatology, University of Graz, Graz, Austria; Department of Dermatology, Basle, Switzerland; Allergy Service, Catholic University of Rome, Italy; Allergy Service, University La Paz, Madrid, Spain; Clinic for Allergy and Immunology, Florence, Italy; Allergy Section, Dept. Clin. Med., UMH, Elche, Spain; Klinik und Poliklinik f1r Dermatologie und Allergologie, Muenchen, Germany; Clinic for Rheumatology and Clinical Immunology/Allergology, Inselspital, Bern, Switzerland; Maladies Respiratoires-INSERM U454, H7pital Arnaud de Villeneuve, Montpellier, France

Delayed Drug Hypersensitivity Reactions

Drug-induced adverse reactions are a major health problem (1-3). Most adverse effects, so-called type A reactions, are due to the pharmacologic action of a drug. Idiosyncratic and immune-mediated side effects, which are not predictable, are called type B reactions (4). Drug hypersensitivity reactions (drug allergy) account for about one seventh of adverse reactions and manifest themselves in many diseases, some of which are severe (5, 6). The most common allergic reactions occur in the skin and are observed in about 2% to 3% of hospitalized patients (7-9). To correlate the clinical symptoms with the underlying immune mechanism, drug hypersensitivity and other immune reactions are frequently classified into 4 categories described by Coombs and Gell (10). Type I reactions are due to IgE mediation and mainly cause urticaria, anaphylaxis, and asthma; type II reactions are based on immunoglobulin-mediated cytotoxic mechanisms, accounting mainly for blood cell dyscrasias; type III reactions are immune complexmediated (for example, vasculitis); and type IV reactions are mediated by T cells, causing so-called delayed hypersensitivity (Table 1). Table 1. Relationship of Clinical Symptoms to Drug Reactivity This classification system has proven to be helpful in clinical practice and can guide diagnostic decisions. However, the term delayed hypersensitivity reactions, originally coined to describe T-cell reactions to tuberculin, became an umbrella term for various T-cellmediated immune mechanisms leading to clinically distinct diseases. Indeed, T cells have been found to differ in the cytokines they produce, which result in distinct disorders. T-helper 1 T cells activate macrophages by secreting large amounts of interferon drive the production of complement-fixing antibody isotypes, and costimulate proinflammatory responses (tumor necrosis factor-, interleukin [IL]-12) and CD8+ T-cell responses. T-helper 2 T cells secrete the cytokines IL-4 and IL-5 (11), which promote B-cell production of IgE and IgG4, macrophage deactivation, and mast-cell and eosinophil responses. CD8+ T cells can produce similarly polarized patterns of cytokines. Newer immunology textbooks have recognized this heterogeneity of T-cell function and consequently subdivide delayed hypersensitivity reactions into type IVa, type IVb, and type IVc reactions, which correspond to T-helper 1, T-helper 2, and cytotoxic reactions (Table 1) (12). T cells recognize small peptide antigens but are also involved in immune reactions to small chemicals. Indeed, the original description of cellular immunity is based on immune responses to haptens (13). The role of T cells in contact dermatitis elicited by small chemicals has been extensively documented (14, 15), and animal models have been used to dissect the immunopathogenesis (16, 17). Understanding of allergies to orally or parentally administered drugs has, in contrast, only slowly evolved, since clinical manifestations are extremely heterogeneous and animal models do not exist for most side effects. Nevertheless, the observation of T-cell infiltrates in drug-related allergic reactions that affect the skin, liver, and kidney, as well as drug-specific reactions found in vitro or indicated in the results of skin tests (16-21), strongly suggested T-cellmediated pathogenesis. This review presents newer concepts of the role of T cells in drug hypersensitivity, which evolved from the study of drug-specific T cells in various drug-induced hypersensitivity diseases. On the basis of in vitro analysis of drug-specific T-cell clones, novel methods of drug presentation to T cells can be defined, extending the hapten concept (22-24). Moreover, functional analysis of T-cell clones from the peripheral blood as well as from the affected tissue, together with immunohistologic analysis, reveals that distinct types of T-cell reactions can lead to different clinical forms of drug hypersensitivity reactions (25-28). How Do T Cells Recognize Drugs? T cells recognize the antigen by their antigen receptors, which are heterodimers of 2 chains designated as either T-cell receptors (the majority of T cells) or T-cell receptors (about 5% of circulating T cells). An enormous variety (>107) of T-cell receptors can be generated with distinct specificities because of different recombinations of genes related to T-cell receptors and the addition of N-region nucleotide insertions. Each T cell displays thousands of identical T-cell receptors, which bind a bimolecular complex displayed at the surface of another cell called an antigen-presenting cell. This complex consists of a fragment of a protein antigen (peptide) bound in the groove of a major MHC molecule (Figure 1). Two classes of MHC molecules present peptides of different origin and stimulate different T cells. Peptides that are derived from proteins synthesized and degraded in the cytosol are presented by MHC class I molecules and activate CD8+ T cells. The reactive CD8+ T cells secrete cytokines and are able to kill cells displaying foreign peptides derived from cytosolic pathogens, such as viruses. In contrast, MHC class II molecules present peptides derived from proteins degraded in endocytic vesicles. These structures interact with CD4+ T cells, which activate other immune effector cells as dictated by their cytokines (for example, macrophages, B cells, and CD8+ T cells) (11, 12). CD4+ T cells can also be cytotoxic (34). Figure 1. The hapten and prohapten concept and the noncovalent drug presentation to T cells. APC NO TCR The recognition of small molecules (such as drugs) by B cells and T cells is usually explained by the hapten concept. Haptens are small molecules (mostly <1000 Da) that are chemically reactive and thus able to undergo stable, covalent binding to a larger protein or peptide (13, 29, 30-33, 35, 36). This modification of a protein or peptide makes it immunogenic (Figure 1): Cell-bound or soluble immunoglobulins can recognize it directly, while T cells recognize a haptenpeptide fragment that is generated by intracellular processing of the haptenprotein complex and is presented to T cells by MHC molecules (Figure 1). Penicillin G is a typical hapten that tends to bind covalently to lysine groups within soluble or cell-bound proteins, thereby modifying them and eliciting B-cell and T-cell reactions (36). It is also possible that the hapten may bind directly to the immunogenic peptide presented by the MHC molecule itself or alter the MHC molecule directly. In this case, no processing is required (26, 37-39) (Figure 1). Alternatively, if the drug is not chemically reactive itself, it may represent a prohapten, which becomes reactive during metabolism (26-28, 34) (Figure 1). Sulfamethoxazole has been proposed as a typical example of a prohapten,since it is not chemically reactive but gains immunogenicity by intracellular metabolism. Cytochrome P450dependent metabolism can lead to sulfamethoxazolehydroxylamine, which becomes sulfamethoxazole-nitroso after oxidation, a chemically reactive compound that is able to bind covalently to proteins and peptides (Figure 1) (31, 37-40). The finding that keratinocytes might also process sulfamethoxazole to sulfamethoxazolehydroxylamine supports this concept and may explain the manifestation of drug allergy in the skin (38). Recently, a third possibility has been considered, namely a pharmacologic interaction of drugs with immune receptors (the pi concept) (Figure 1) (41-46). Chemically inert drugs, unable to covalently bind to peptides or proteins, may still activate certain T cells that happen to bear T-cell receptors that can interact with the drug. This model has been expanded by in vitro studies using T-cell clones specific for such drugs as sulfamethoxazole, lidocaine, mepivacaine, celecoxib, lamotrigine, carbamazepine, and p-phenylenediamine (41-43, 47-49). It relies on the following findings: Glutaraldehyde-fixed antigen-presenting cells, unable to process, can still present the drug and stimulate specific T cells (41); inhibited generation of reactive metabolites actually enhances the reactivity of T cells, suggesting that the inert drug but not the reactive metabolite is recognized (50); the drug is bound in a labile way since it can be washed away from the cell surface, in contrast to covalently bound drugs, which cannot (41, 42); and a drug-reactive T-cell clone reacts to the drug within seconds, before metabolism and processing can take place (42). This stimulation by inert drugs is MHC dependent, implying that for full stimulation of the T cell, the T-cell receptor needs to interact with the drug and the MHC molecule. This new concept has a major impact on our understanding of drug hypersensitivity and its distinct clinical manifestations (Figure 1, Table 1). Haptens are primarily immunogenic because of their chemical reactivity. They modify peptides and make them more or newly immunogenic. In contrast, chemical inert drugs are immunogenic only because of their structural features, which enable them to interact with immune receptors (certain T-cell receptors and possibly MHC). These structural features have never been considered in drug development but may account for a substantial portion of unforeseen side effects (51). The clinical symptoms elicited by drugs that are immunogenic because of their chemical or structural features may well differ. A hapten-like drug (for example, amoxicillin) is able to alter many different proteins, either soluble or cell-bound, and can even modify different MHC molecules and their embedded peptides directly (Figure 1). These distinct antigenic determinants can stimulate T cells and B cells and elicit more or less all types of immune reactions. Indeed, penicillins are reported to cause different antibody-mediated diseases, such as anaphylaxis or hemolytic anemia, but also various T-cellmediated reactions, such as maculopapular exanthema, drug-induced hypersensitivity syndrome, acute generalized

Correlation of Intravascular Ultrasound Findings With Histopathological Analysis of Thrombus Aspirates in Patients With Very Late Drug-Eluting Stent Thrombosis

Background— Intravascular ultrasound of drug-eluting stent (DES) thrombosis (ST) reveals a high incidence of incomplete stent apposition (ISA) and vessel remodeling. Autopsy specimens of DES ST show delayed healing and hypersensitivity reactions. The present study sought to correlate histopathology of thrombus aspirates with intravascular ultrasound findings in patients with very late DES ST. Methods and Results— The study population consisted of 54 patients (28 patients with very late DES ST and 26 controls). Of 28 patients with very late DES ST, 10 patients (1020±283 days after implantation) with 11 ST segments (5 sirolimus-eluting stents, 5 paclitaxel-eluting stents, 1 zotarolimus-eluting stent) underwent both thrombus aspiration and intravascular ultrasound investigation. ISA was present in 73% of cases with an ISA cross-sectional area of 6.2±2.4 mm2 and evidence of vessel remodeling (index, 1.6±0.3). Histopathological analysis showed pieces of fresh thrombus with inflammatory cell infiltrates (DES, 263±149 white blood cells per high-power field) and eosinophils (DES, 20±24 eosinophils per high-power field; sirolimus-eluting stents, 34±28; paclitaxel-eluting stents, 6±6; P for sirolimus-eluting stents versus paclitaxel-eluting stents=0.09). The mean number of eosinophils per high-power field was higher in specimens from very late DES ST (20±24) than in those from spontaneous acute myocardial infarction (7±10), early bare-metal stent ST (1±1), early DES ST (1±2), and late bare-metal stent ST (2±3; P from ANOVA=0.038). Eosinophil count correlated with ISA cross-sectional area, with an average increase of 5.4 eosinophils per high-power field per 1-mm2 increase in ISA cross-sectional area. Conclusions— Very late DES thrombosis is associated with histopathological signs of inflammation and intravascular ultrasound evidence of vessel remodeling. Compared with other causes of myocardial infarction, eosinophilic infiltrates are more common in thrombi harvested from very late DES thrombosis, particularly in sirolimus-eluting stents, and correlate with the extent of stent malapposition.

The lymphocyte transformation test in the diagnosis of drug hypersensitivity

Diagnosis of drug hypersensitivity is difficult, as an enormous amount of different drugs can elicit various immune‐mediated diseases with distinct pathomechanism. The lymphocyte transformation test (LTT) measures the proliferation of T cells to a drug in vitro– from which one concludes to a previous in vivo reaction due to a sensitization. This concept of the LTT has been confirmed by the generation of drug‐specific T‐cell clones and the finding that drugs can directly interact with the T‐cell receptor, without previous metabolism or need to bind to proteins.

Diagnosis of immediate allergic reactions to beta-lactam antibiotics

Allergic reactions to betalactams are the most common cause of adverse drug reactions mediated by specific immunological mechanisms. Reactions may be induced by all betalactams currently available, ranging from benzylpenicillin (BP) to other more recently introduced betalactams, such as aztreonam or the related betalactamase-inhibitor clavulanic acid (Fig. 1) (1–5). Although the production process of betalactams has improved over the years, the number of reactions has not decreased, M. J. Torres, M. Blanca, J. Fernandez, A. Romano, A. de Weck, W. Aberer, K. Brockow, W. J. Pichler, P. Demoly for ENDA, and the EAACI interest group on drug hypersensitivity Allergy Service, Carlos Haya Hospital, Malaga, Spain; Allergy Service, University La Paz, Madrid, Spain; Allergy Section, Dept. Clin. Med., UMH, Elche, Spain; Allergy Service, Catholic University of Rome, Italy; Fondation Gerimmun, Beaumont 18, CH1700, Fribourg, Switzerland; Department of Environmental Dermatology, Graz, Austria; Klinik und Poliklinik f5r Dermatologie und Allergologie, Muenchen, Germany; Clinic for Rheumatology and Clinical Immunology/Allergy, Inselspital, Bern, Switzerland; Maladies Respiratoires-INSERM U454, Hopital Arnaud de Villeneuve, Montpellier, France

T-cell involvement in drug-induced acute generalized exanthematous pustulosis

Acute generalized exanthematous pustulosis (AGEP) is an uncommon eruption most often provoked by drugs, by acute infections with enteroviruses, or by mercury. It is characterized by acute, extensive formation of nonfollicular sterile pustules on erythematous background, fever, and peripheral blood leukocytosis. We present clinical and immunological data on four patients with this disease, which is caused by different drugs. An involvement of T cells could be implied by positive skin patch tests and lymphocyte transformation tests. Immunohistochemistry revealed a massive cell infiltrate consisting of neutrophils in pustules and T cells in the dermis and epidermis. Expression of the potent neutrophil-attracting chemokine IL-8 was elevated in keratinocytes and infiltrating mononuclear cells. Drug-specific T cells were generated from the blood and skin of three patients, and phenotypic characterization showed a heterogeneous distribution of CD4/CD8 phenotype and of T-cell receptor Vbeta-expression. Analysis of cytokine/chemokine profiles revealed that IL-8 is produced significantly more by drug-specific T cells from patients with AGEP compared with drug-specific T cells from patients that had non-AGEP exanthemas. In conclusion, our data demonstrate the involvement of drug-specific T cells in the pathomechanism of this rather rare and peculiar form of drug allergy. In addition, they indicate that even in some neutrophil-rich inflammatory responses specific T cells are engaged and might orchestrate the immune reaction.

Functional expression of the eotaxin receptor CCR3 in T lymphocytes co-localizing with eosinophils.

BACKGROUND The chemokine eotaxin is produced at sites of allergic inflammation, binds selectively to the chemokine receptor CCR3 and attracts eosinophil and basophil leukocytes, which express high numbers of this receptor. Responses of T lymphocytes to eotaxin have not been reported so far. We have investigated the expression of CCR3 in T lymphocytes and analysed the properties and in vivo distribution of T lymphocytes expressing this receptor. RESULTS In search of chemokine receptors with selective expression in T lymphocytes, we have isolated multiple complementary DNAs (cDNAs) encoding CCR3 from a human CD4+ T-cell cDNA library. T-lymphocyte clones with selectivities for protein and non-protein antigens were analysed for expression of CCR3 and production of Th1- and Th2-type cytokines. Of 13 clones with surface CCR3, nine secreted enhanced levels of interleukin-4 and/or interleukin-5, indicating that CCR3 predominates in Th2-type lymphocytes. CCR3+ T lymphocytes readily migrated in response to eotaxin, and showed the characteristic changes in cytosolic free calcium. Immunostaining of contact dermatitis, nasal polyp and ulcerative colitis tissue showed that CCR3+ T lymphocytes are recruited together with eosinophils and, as assessed by flow cytometry, a large proportion of CD3+ cells extracted from the inflamed skin tissue were CCR3+. By contrast, CCR3+ T lymphocytes were absent from tissues that lack eosinophils, as demonstrated for normal skin and rheumatoid arthritis synovium. CONCLUSIONS We show that T lymphocytes co-localizing with eosinophils at sites of allergic inflammation express CCR3, suggesting that eotaxin/CCR3 represents a novel mechanism of T-lymphocyte recruitment. These cells are essential in allergic inflammation, as mice lacking mature T lymphocytes were insensitive to allergen challenge. Surface CCR3 may mark a subset of T lymphocytes that induce eosinophil mobilization and activation through local production of Th2-type cytokines.

Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alphabeta T cell clones.

T cells can recognize small molecular compounds like drugs. It is thought that covalent binding to MHC bound peptides is required for such a hapten stimulation. Sulfamethoxazole, like most drugs, is not chemically reactive per se, but is thought to gain the ability to covalently bind to proteins after intracellular drug metabolism. The purpose of this study was to investigate how sulfamethoxazole is presented in an immunogenic form to sulfamethoxazole-specific T cell clones. The stimulation of four CD4(+) and two CD8(+) sulfamethoxazole-specific T cell clones by different antigen-presenting cells (APC) was measured both by proliferation and cytolytic assays. The MHC restriction was evaluated, first, by inhibition using anti-class I and anti-class II mAb, and second, by the degree of sulfamethoxazole-induced stimulation by partially matched APC. Fixation of APC was performed with glutaraldehyde 0.05%. The clones were specific for sulfamethoxazole without cross-reaction to other sulfonamides. The continuous presence of sulfamethoxazole was required during the assay period since pulsing of the APC was not sufficient to induce proliferation or cytotoxicity. Stimulation of clones required the addition of MHC compatible APC. The APC could be fixed without impairing their ability to present sulfamethoxazole. Sulfamethoxazole can be presented in an unstable, but MHC-restricted fashion, which is independent of processing. These features are best explained by a direct, noncovalent binding of sulfamethoxazole to the MHC-peptide complex.