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.

Assessment of Antipiperacillin IgG Binding to Structurally Related Drug Protein Adducts.

The risk of developing hypersensitivity to alternative antibiotics is a concern for penicillin hypersensitive patients and healthcare providers. Herein we use piperacillin hypersensitivity as a model to explore the reactivity of drug-specific IgG against alternative ß-lactam protein adducts. Mass spectrometry was used to show the drugs (amoxicillin, flucloxacillin, benzyl penicillin, aztreonam, and piperacillin) bind to similar lysine residues on the protein carrier bovine serum albumin. However, the hapten-specific IgG antibodies found in piperacillin hypersensitive patient plasma did not bind to other ß-lactam protein conjugates. These data outline the fine specificity of piperacillin-specific IgG antibodies that circulate in patients with hypersensitivity.

Definition of the Nature and Hapten Threshold of the ß-Lactam Antigen Required for T Cell Activation In Vitro and in Patients.

Covalent modification of protein by drugs may disrupt self-tolerance, leading to lymphocyte activation. Until now, determination of the threshold required for this process has not been possible. Therefore, we performed quantitative mass spectrometric analyses to define the epitopes formed in tolerant and hypersensitive patients taking the ß-lactam antibiotic piperacillin and the threshold required for T cell activation. A hydrolyzed piperacillin hapten was detected on four lysine residues of human serum albumin (HSA) isolated from tolerant patients. The level of modified Lys541 ranged from 2.6 to 4.8%. Analysis of plasma from hypersensitive patients revealed the same pattern and levels of modification 1-10 d after the commencement of therapy. Piperacillin-responsive skin-homing CD4+ clones expressing an array of Vß receptors were activated in a dose-, time-, and processing-dependent manner; analysis of incubation medium revealed that 2.6% of Lys541 in HSA was modified when T cells were activated. Piperacillin-HSA conjugates that had levels and epitopes identical to those detected in patients were shown to selectively stimulate additional CD4+ clones, which expressed a more restricted Vß repertoire. To conclude, the levels of piperacillin-HSA modification that activated T cells are equivalent to the ones formed in hypersensitive and tolerant patients, which indicates that threshold levels of drug Ag are formed in all patients. Thus, the propensity to develop hypersensitivity is dependent on other factors, such as the presence of T cells within an individual's repertoire that can be activated with the ß-lactam hapten and/or an imbalance in immune regulation.

Detection of drug-responsive B lymphocytes and antidrug IgG in patients with ß-lactam hypersensitivity.

BACKGROUND: Delayed-type ß-lactam hypersensitivity develops in subset of patients. The cellular immunological processes that underlie the drug-specific response have been described; however, little is known about involvement of the humoral immune system. Thus, the aim of this study was to utilize piperacillin hypersensitivity as an exemplar to (i) develop cell culture methods for the detection of drug-specific B-cell responses, (ii) characterize drug-specific IgG subtypes and (iii) assess reactivity of IgG antibodies against proteins modified to different levels with piperacillin haptens. METHODS: IgG secretion and CD19+ CD27+ expression on B cells were measured using ELISPOT and flow cytometry, respectively. A piperacillin-BSA adduct was used as an antigen in ELISA antibody binding studies. Adducts generated using different ratios of drug to protein were used to determine the degree of conjugation required to detect IgG binding. RESULTS: B cells from hypersensitive patients, but not controls, were stimulated to secrete IgG and increase CD27 expression when cultured with soluble piperacillin. A piperacillin-BSA adduct with cyclized and hydrolysed forms of the hapten bound to eight lysine residues was used to detect hapten-specific IgG 1-4 subclasses in patient plasma. Hapten inhibition and the use of structurally unrelated hapten-BSA adducts confirmed antigen specificity. Antibody binding was detected with antigens generated at piperacillin/BSA ratios of 10:1 and above, which corresponded to a minimum epitope density of 1 for antibody binding. CONCLUSION: These data show that antigen-specific B lymphocytes and T lymphocytes are activated in piperacillin-hypersensitive patients. Further work is needed to define the role different IgG subtypes play in regulating the iatrogenic disease.

The incidence and features of systemic reactions to skin prick tests.

BACKGROUND: Skin prick testing (SPT) has been regarded as a safe procedure with few systemic reactions. OBJECTIVE: To evaluate the rate of systemic reactions and their associations after SPT in the largest population to date. METHODS: In this study reactions were recorded prospectively in a specialist UK allergy clinic for 6 years (2007-2013). An estimated 31,000 patients underwent SPT. RESULTS: Twenty-four patients (age range 7 months to 56 years, mean 23.5 years, 17 female patients, 12 with asthma) had systemic reactions. The rate of systemic reactions to SPT was 0.077%. The likely allergens causing the reaction were foods (18; peanut, 7; walnut, 1; Brazil nut, 2; pistachio, 1; lupin, 1; cow's milk, 2; shrimp, 1; spinach, 1; legume, 1; soy, 1), aeroallergens (4; rabbit, 1; rat, 1; ragwort, 1; grass pollen, 1), wasp venom (1), and Tazocin (1). The causative SPT wheal was larger than 8 mm in 75%. The reaction to Tazocin was severe, with anaphylaxis occurring minutes after SPT. Reactions were treated immediately in the clinic and did not require further medical care. CONCLUSION: In this largest single-center study, the rate of systemic reactions after SPT was 77 per 100,000 patients. It is the first study to identify foods as a common and important cause (75%), with nuts posing the highest risk. This study reports the first systemic reaction to venom SPT and the first anaphylactic reaction after drug SPT. There was an association with a history of severe reactions and large skin test reaction. There are risks, albeit small, when undertaking SPT.

ß-Lactam antibiotics form distinct haptenic structures on albumin and activate drug-specific T-lymphocyte responses in multiallergic patients with cystic fibrosis.

ß-Lactam antibiotics provide the cornerstone of treatment for respiratory exacerbations in patients with cystic fibrosis. Unfortunately, approximately 20% of patients develop multiple nonimmediate allergic reactions that restrict therapeutic options. The purpose of this study was to explore the chemical and immunological basis of multiple ß-lactam allergy through the analysis of human serum albumin (HSA) covalent binding profiles and T-cell responses against 3 commonly prescribed drugs; piperacillin, meropenem, and aztreonam. The chemical structures of the drug haptens were defined by mass spectrometry. Peripheral blood mononuclear cells (PBMC) were isolated from 4 patients with multiple allergic reactions and cultured with piperacillin, meropenem, and aztreonam. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ /IL-13 ELIspot. T-cell clones were generated from drug-stimulated T-cell lines and characterized in terms of phenotype, function, and cross-reactivity. Piperacillin, meropenem, and aztreonam formed complex and structurally distinct haptenic structures with lysine residues on HSA. Each drug modified Lys190 and at least 6 additional lysine residues in a time- and concentration-dependent manner. PBMC proliferative responses and cytokine release were detected with cells from the allergic patients, but not tolerant controls, following exposure to the drugs. 122 CD4+, CD8+, or CD4+CD8+ T-cell clones isolated from the allergic patients were found to proliferate and release cytokines following stimulation with piperacillin, meropenem, or aztreonam. Cross-reactivity with the different drugs was not observed. In conclusion, our data show that piperacillin-, meropenem-, and aztreonam-specific T-cell responses are readily detectable in allergic patients with cystic fibrosis, which indicates that multiple ß-lactam allergies are instigated through priming of naïve T-cells against the different drug antigens. Characterization of complex haptenic structures on distinct HSA lysine residues provides a chemical basis for the drug-specific T-cell response.

Characterization of the antigen specificity of T-cell clones from piperacillin-hypersensitive patients with cystic fibrosis.

ß-Lactam antibiotics provide the cornerstone of treatment and reduce the rate of decline in lung function in patients with cystic fibrosis, but their use is limited by a high frequency of delayed-type allergic reactions. The objective of this study was to use cloned T-cells expressing a single T-cell receptor from five piperacillin-hypersensitive patients to characterize both the cellular pathophysiology of the reaction and antigen specificity to define the mechanism of activation of T-cells by piperacillin. More than 400 piperacillin-responsive CD4+, CD4+CD8+, or CD8+ T-cell clones were generated from lymphocyte transformation test and ELIspot-positive patients. The T-cell response (proliferation, T helper 2 cytokine secretion, and cytotoxicity) to piperacillin was concentration-dependent and highly specific. Enzyme-linked immunosorbent assay, gel electrophoresis, and mass spectrometry revealed that piperacillin bound exclusively to albumin in T-cell culture. Irreversible piperacillin binding at Lys 190, 195, 199, 432, and 541 on albumin and the stimulation of T-cells depended on incubation time. A synthetic piperacillin albumin conjugate stimulated T-cell receptors via a major histocompatibility complex- and processing-dependent pathway. Flucloxacillin competes for the same Lys residues on albumin as piperacillin, but the resulting conjugate does not stimulate T-cells, indicating that binding of the ß-lactam hapten in peptide conjugates confers structural specificity on the activation of the T-cell receptors expressed on drug-specific clones. Collectively, these data describe the cellular processes that underlie the structural specificity of piperacillin antigen binding in hypersensitive patients with cystic fibrosis.

A case of piperacillin-induced occupational anaphylaxis: detection of serum IgE to piperacillin-HSA conjugate.

This is the first reported detection of serum IgE antibody to piperacillin-human serum albumin (HSA) conjugate in a patient presenting with anaphylaxis that developed after occupational exposure. A 24-yr-old nurse, who had worked at a University Hospital for 2 yr, experienced chest tightness, dizziness, generalized urticaria, abdominal pain, and diarrhea 10 min after administering a piperacillin injection. She had previously suffered from atopic dermatitis. A skin prick test for common inhalant allergens was entirely negative; in contrast, her serum total IgE was elevated (283 IU/mL). A high level of piperacillin-specific serum IgE was detected by ELISA using piperacillin-HSA conjugate. Significant inhibition upon addition of both free piperacillin and piperacillin-HSA conjugate was detected by inhibition ELISA. These data suggest that piperacillin exposure in the workplace can induce occupational anaphylaxis and urticaria mediated by an interaction of IgE with the hapten of piperacillin.

Piperacillin and vancomycin induced severe thrombocytopenia in a hospitalized patient.

In hospitalized patients with complex medical problems on numerous drugs, thrombocytopenia may have a multiple confounding etiology. Keeping this in mind, it is of utmost importance to monitor the platelet count regularly during hospitalization and on subsequent follow-up visits, even after the most probable etiology has been identified/most likely causative drug has been withdrawn. Isolated thrombocytopenia with no evidence of microangiopathic hemolysis on the peripheral blood smear in an acutely ill hospitalized patient implicated sepsis, disseminated intravascular coagulation and drugs as the most probable causes. Our patient represents an uncommon case of antibiotic-induced severe immune thrombocytopenia, as he developed both vancomycin-dependent and piperacillin-dependent antibodies, while being treated for cellulitis (vancomycin-specific antibodies of the IgG isotype, and both IgG and IgM antibodies specific for piperacillin were identified in laboratory testing). Vancomycin was stopped before the reports were available. Following this, the patient's platelet count showed a transient upward trend, but then the thrombocytopenia worsened drastically reaching a nadir of 10,000/µL. The platelet count returned to normal only after piperacillin/tazobactam was stopped after a week, thus establishing it as the cause of the more severe thrombocytopenia, which occurred later on; this was subsequently confirmed by the laboratory results. Vancomycin is an established cause of drug-induced immune thrombocytopenias, especially in acutely ill, hospitalized or elderly patients, whereas incidents of piperacillin/tazobactam-induced immune thrombocytopenia are uncommon. In case clinical suspicion is high, workup should include immunoprecipitation and flow cytometry studies to confirm antiplatelet antibodies.

A Case of Piperacillin-induced Occupational Anaphylaxis: Detection of Serum IgE to Piperacillin-HSA Conjugate

This is the first reported detection of serum IgE antibody to piperacillin-human serum albumin (HSA) conjugate in a patient presenting with anaphylaxis that developed after occupational exposure. A 24-yr-old nurse, who had worked at a University Hospital for 2 yr, experienced chest tightness, dizziness, generalized urticaria, abdominal pain, and diarrhea 10 min after administering a piperacillin injection. She had previously suffered from atopic dermatitis. A skin prick test for common inhalant allergens was entirely negative; in contrast, her serum total IgE was elevated (283 IU/mL). A high level of piperacillin-specific serum IgE was detected by ELISA using piperacillin-HSA conjugate. Significant inhibition upon addition of both free piperacillin and piperacillin-HSA conjugate was detected by inhibition ELISA. These data suggest that piperacillin exposure in the workplace can induce occupational anaphylaxis and urticaria mediated by an interaction of IgE with the hapten of piperacillin.

Serological studies of piperacillin antibodies.

BACKGROUND: Penicillin-induced immune hemolytic anemia (IHA) is associated with immunoglobulin G antipenicillin detected by testing penicillin-coated red blood cells (RBCs). Antibodies to piperacillin, a semisynthetic penicillin, would be expected to react similarly; however, antipiperacillin can be detected by testing in the presence of the drug. Piperacillin is commonly used in combination with tazobactam, which causes nonimmunologic protein adsorption onto RBCs. In six cases of piperacillin-induced IHA, reactivity with piperacillin-coated RBCs was not similar to reactivity of antipenicillin with penicillin-coated RBCs. STUDY DESIGN AND METHODS: Antipiperacillin was tested against piperacillin-coated RBCs prepared using different pH buffers. Plasma from blood donors and sera/plasma from patients were tested with piperacillin-coated, penicillin-coated, and uncoated RBCs. Hapten inhibition studies were performed using different concentrations of piperacillin. Donors' plasma were tested in the presence of piperacillin; sera from patients with IHA were tested in the presence of tazobactam. RESULTS: Piperacillin required high pH for binding to RBCs. Agglutination of piperacillin-coated RBCs was observed in 91 percent of donors' and 49 percent of patients' plasma and was inhibited by piperacillin. In contrast to patients with IHA due to piperacillin, donors' plasma tested in the presence of piperacillin did not react. Tazobactam antibodies were not detected. CONCLUSION: A high percentage of donors' and patients' plasma contain an antibody to piperacillin or a chemically related structure detected by testing with piperacillin-coated RBCs. A diagnosis of piperacillin-induced IHA should not be made solely on the reactivity of a patient's plasma/serum with piperacillin- or piperacillin/tazobactam-coated RBCs; testing in the presence of piperacillin is more reliable.

Cephalosporin induced toxic epidermal necrolysis and subsequent penicillin drug exanthem.

BACKGROUND: Drug hypersensitivity is classically divided into IgE mediated and non-IgE mediated disease. We report a rare case of consequent IgE mediated and non-IgE mediated reactions within the beta lactam class of antibiotics. CASE SUMMARY: An 84-year-old man developed toxic epidermal necrolysis (TEN) due to ceftriaxone, a third generation cephalosporin, involving 72% of the body surface area. The patient recovered but within weeks subsequently developed an acute IgE mediated allergic reaction to piperacillin/tazobactam, an extended spectrum penicillin. Further IgE RAST revealed positive results to penicillin major determinant. DISCUSSION: This case demonstrates the complexity of drug hypersensitivity reactions. While it is accepted that IgE mediated penicillin allergy is a predisposition to cephalosporin allergy, this case displays an unusual correlation between drug hypersensitivity and drug class. There have been few studies that evaluate the cross reactivity with penicillin or other beta-lactams in subjects with primary hypersensitivity to cephalosporins. This clinical scenario emphasizes the need of more studies on cephalosporin allergy in particular as shown by this case of sequential non-IgE mediated cephalosporin induced TEN reaction pursuant by an IgE mediated penicillin allergy.