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.

The presence of Ag-specific T cells in blood and target organs of drug-hypersensitive patients provides a robust case for their involvement in the pathogenesis of a reaction (16). It is thought that drugs activate T cells by covalent modification of protein generating novel antigenic determinants (2, 3, 79). However, the paucity of studies that define the chemistry of drug–protein binding in patients has severely restricted mechanistic studies that relate the chemistry of Ag formation to immune function. Indeed, the simple concept of the hapten hypothesis of drug hypersensitivity has been brought into question by studies that have demonstrated that drugs may activate T cells through noncovalent interactions (4, 5, 1016).

Hypersensitivity reactions to β-lactam antibiotics remain an important clinical problem. For Ag formation, the β-lactam ring is targeted by nucleophilic lysine residues, leading to ring opening and binding of the penicilloyl group (1719). We have developed novel mass spectrometric techniques to define unequivocally the chemistry of drug–protein conjugation in patients under physiological conditions (2023). In this study, we report on the methods we have developed to detect and fully characterize circulating Ags derived from piperacillin and its metabolite in patients undergoing therapy. Using the same mass spectrometry (MS) methods, it was possible to characterize the nature of the drug-derived epitopes on a protein that can function as an Ag and a potential immunogen to stimulate T cells from patients with clinically characterized drug hypersensitivity. For this purpose, we have studied piperacillin hypersensitivity reactions in patients with cystic fibrosis. In these patients, i.v. antibiotics provide the cornerstone of treatment for recurrent respiratory infections and help reduce the rate of decline in lung function and overall health. The overall prevalence of clinically relevant β-lactam reactions in patients with cystic fibrosis is 26–50% (2426). We found that the frequency of drug-specific T cells in such patients was >75%. It was therefore possible to investigate the chemistry of functional Ags formed from piperacillin and albumin not only in patients’ blood, but also in ex vivo incubations with patients’ T cells to relate the chemistry of protein modification to drug antigenicity and immunogenicity.

A sterile i.v. preparation of Tazocin (Wyeth Pharmaceuticals) was purchased for skin testing. Histamine and saline controls, together with lancets for skin prick testing, were purchased from ALK-Abelló (Hørsholm, Denmark). The following products were purchased from Sigma-Aldrich (Gillingham, U.K.): HBSS, penicillin-streptomycin, l-glutamine, HEPES, RPMI 1640, human AB serum, and piperacillin. Invitrogen (Paisley, U.K.) provided FBS. Radiolabeled thymidine was obtained from Moravek International.

The time- and concentration-dependent modification of human serum albumin was investigated in vitro. Human serum albumin (66 mg/ml, 1 mM) in phosphate buffer (KH2PO4, 13.08 mM; K2HPO4, 62.27 mM [pH 7.4]) was incubated at 37°C with piperacillin at molar ratios of piperacillin to human serum albumin of 0.01:1, 0.1:1, 1:1, 10:1, and 50:1 for 24 h and at 50:1 for 1, 24, 48, 72, and 96 h. The protein was precipitated by the addition of nine volumes of ice-cold methanol followed by centrifugation at 14,000 × g and 4°C for 15 min. The precipitation was repeated to ensure the removal of non-covalently bound drug. The concentration of human serum albumin was determined by Bradford assay (27), and aliquots were prepared in serum-free RPMI 1640 for application in T cell assays in 50 mM ammonium bicarbonate for mass spectrometric analysis and in Laemmli sample buffer for Western blotting. Prior to MS, all samples were incubated with DTT (10 mM) at room temperature for 15 min and with iodoacetamide (55 mM) for a further 15 min at room temperature before again being subjected to methanol precipitation. They were reconstituted in ammonium bicarbonate buffer (50 mM), digested with trypsin overnight at 37°C, and then desalted using C18 Zip-Tips (Millipore, Watford, U.K.).

Human serum albumin was recovered from 100-μl aliquots of clarified culture supernatants using the same protocol as above. For higher sensitivity detection of adducts in human plasma, samples from piperacillin-exposed patients were processed individually for three-dimensional liquid chromatography (LC) tandem MS analysis. Human serum albumin was first isolated from plasma by affinity chromatography using a POROS anti-human serum albumin column (ABSciex, Foster City, CA) (21). Aliquots of 400 μg affinity-isolated human serum albumin were precipitated and digested as described above, and the digests were fractionated on a Polysulfoethyl A strong cation-exchange column (200 × 4.6 mm, 5 μm, 300 Å; Poly LC, Columbia, MD). Fractions of 2 ml were collected and dried by centrifugation under vacuum (SpeedVac; Eppendorf). All samples were analyzed by reversed-phase LC-MS.

Samples were reconstituted in 2% acetonitrile (ACN)/0.1% formic acid (v/v), and aliquots of 2.4–5 pmol were delivered into a QTRAP 5500 hybrid quadrupole-linear ion trap mass spectrometer (ABSciex) by automated in-line LC (U3000 HPLC System, 5 mm C18 nano-precolumn and 75 μm × 15 cm C18 PepMap column; Dionex) via a 10-μm inner diameter PicoTip (New Objective). A gradient from 2% ACN/0.1% formic acid (v/v) to 50% ACN/0.1% formic acid (v/v) in 70 min was applied at a flow rate of 280 nl/min. The ionspray potential was set to 2200–3500 V, the nebulizer gas to 18, and the interface heater to 150°C. Multiple reaction monitoring (MRM) transitions specific for drug-modified peptides were selected as follows: the mass/charge ratio (m/z) values were calculated for all possible peptides with a missed cleavage at a lysine residue; to these were added the mass of the appropriate hapten (cyclized, 517 atomic mass units [amu]; hydrolyzed, 535 amu; desethyl cyclized, 489 amu; and desethyl hydrolyzed, 507 amu); the parent ion masses were then paired with a fragment mass of 160 ([M+H]+ of cleaved thiazolidine ring present in all of the haptens) and/or a fragment mass of 106 ([M+H]+ of cleaved benzylamine group of hydrolyzed haptens). MRM transitions were acquired at unit resolution in both the Q1 and Q3 quadrupoles to maximize specificity, they were optimized for collision energy and collision cell exit potential, and the dwell time was 20 ms. MRM survey scans were used to trigger enhanced product ion MS/MS scans of drug-modified peptides, with Q1 set to unit resolution, dynamic fill selected, and dynamic exclusion for 20 s. Total ion counts were determined from a second aliquot of each sample analyzed by conventional LC tandem MS and were used to normalize sample loading on column. MRM peak areas were determined by MultiQuant 1.2 software (ABSciex). Epitope profiles were constructed by comparing the relative intensity of MRM peaks for each of the modified lysine residues within a sample and normalization of those signals across samples.

Aliquots of 5 μg protein were separated by electrophoresis on a 10% SDS-polyacrylamide gel and electroblotted onto nitrocellulose membrane. Nonspecific binding was blocked using Tris/saline/Tween buffer (TST; NaCl, 150 mM; Tris-HCl, 10 mM; Tween 20, 0.05% [pH 8]) containing 10% nonfat dry milk for 16 h at 4°C. The blot was incubated with primary anti-penicillin Ab (mouse anti-penicillin mAb; AbD Serotec) diluted 1:20,000 in 5% milk/TST for 1 h, followed by incubation with HRP-conjugated anti-mouse IgG Ab (Abcam) diluted 1:10,000 in 5% milk/TST for a further 1 h. Signal was detected by ECL (Western Lightning; PerkinElmer, Boston, MA) using autoradiography film and a GS800 calibrated scanning densitometer (Bio-Rad, Hemel Hempstead, U.K.).

The medical records of 350 patients with cystic fibrosis attending the Regional Adult Cystic Fibrosis Unit in Leeds, U.K., were reviewed. Eighty-eight patients (25%) had a previous nonimmediate reaction to piperacillin. Reactions were defined as an adverse reaction occurring after at least 48 h of i.v. piperacillin. This study enrolled 28 hypersensitive patients; the reactions mainly consisted of maculopapular exanthema, fevers, urticarial eruptions, and flulike symptoms. In every case, treatment had to be discontinued, and patients received antihistamines and/or oral steroids when clinically indicated. Detailed information on each of the hypersensitive patients is presented in Table II.

Table II.
Clinical characteristics of the hypersensitive patients
Patient No.Age (y)/GenderDrugReactionTime to Reaction (d)Time Since Reaction (y)No. of Courses Prior to ReactionLTT
18/M Tazocin MPE/fevers +++ 
29/F Piperacillin MPE 12 11 ++++ 
29/F Tazocin Fever/eosinophilia 0.5 14 ++ 
26/M Tazocin MPE ++++ 
29/M Tazocin MPE ++ 
24/F Tazocin Delayed angioedema − 
17/M Piperacillin Flulike illness ++++ 
24/M Piperacillin MPE >5a NA +++ 
23/M Tazocin MPE 11 +++ 
10 30/F Tazocin Flulike illness ++ 
11 45/F Tazocin Fevers 11 14 − 
12 19/M Tazocin MPE/fever 0.5 ++++ 
13 22/F Tazocin MPE ++ 
14 21/F Tazocin Arthralgia ++++ 
15 17/M Tazocin MPE 11 − 
16 24/F Tazocin Arthralgia/MPE 
17 22/M Tazocin Pruritis 11 
18 28/F Tazocin Fever/arthralgia +++ 
19 31/F Tazocin Flulike illness 12 
20 34/M Piperacillin MPE 10 11 − 
21 32/M Piperacillin MPE 10 
22 23/F Tazocin Fevers/unwell 12 
23 29/M Tazocin Tight chest − 
24 18/M Piperacillin Urticarial rash >5a NA − 
25 40/M Piperacillin Fevers 10 12 11 − 
26b 29/F Piperacillin MPE − 
27b 34/F Tazocin MPE 17 − 
28b 35/F Piperacillin MPE 10 17 − 
Patient No.Age (y)/GenderDrugReactionTime to Reaction (d)Time Since Reaction (y)No. of Courses Prior to ReactionLTT
18/M Tazocin MPE/fevers +++ 
29/F Piperacillin MPE 12 11 ++++ 
29/F Tazocin Fever/eosinophilia 0.5 14 ++ 
26/M Tazocin MPE ++++ 
29/M Tazocin MPE ++ 
24/F Tazocin Delayed angioedema − 
17/M Piperacillin Flulike illness ++++ 
24/M Piperacillin MPE >5a NA +++ 
23/M Tazocin MPE 11 +++ 
10 30/F Tazocin Flulike illness ++ 
11 45/F Tazocin Fevers 11 14 − 
12 19/M Tazocin MPE/fever 0.5 ++++ 
13 22/F Tazocin MPE ++ 
14 21/F Tazocin Arthralgia ++++ 
15 17/M Tazocin MPE 11 − 
16 24/F Tazocin Arthralgia/MPE 
17 22/M Tazocin Pruritis 11 
18 28/F Tazocin Fever/arthralgia +++ 
19 31/F Tazocin Flulike illness 12 
20 34/M Piperacillin MPE 10 11 − 
21 32/M Piperacillin MPE 10 
22 23/F Tazocin Fevers/unwell 12 
23 29/M Tazocin Tight chest − 
24 18/M Piperacillin Urticarial rash >5a NA − 
25 40/M Piperacillin Fevers 10 12 11 − 
26b 29/F Piperacillin MPE − 
27b 34/F Tazocin MPE 17 − 
28b 35/F Piperacillin MPE 10 17 − 
a

Indicates that the exact time period since reaction is not known due to missing medical records or occurrence at another cystic fibrosis center.

b

Indicates patient on long-term immunosuppressive therapy. Patients 26 and 27 were receiving hydroxychloroquine for cystic fibrosis-related arthritis. Patient 28 received long-term low-dose steroids as prophylaxis against hypersensitive bronchopulmonary aspergillosis.

+, SI 2–5; ++, SI 5–10; +++, SI 10–20; ++++, >20; −, no response; F, female; ID, intradermal; LTT, lymphocyte transformation test; M, male; MPE, maculopapular exanthema; NT, not tested.

Nine patients (five males and four females) identified as tolerant had received piperacillin without any adverse event. Blood samples were also collected from 11 healthy naive volunteers who had never received piperacillin. There were no significant differences when the tolerant and hypersensitive groups were compared for age, lung function, and sputum classification. Dose and treatment duration were identical in both groups. Skin and biological tests were performed when patients were clinically well and had not received i.v. antibiotics for at least 6 wk. Further blood samples were taken from the tolerant group during a course of piperacillin to characterize albumin conjugates in vivo. Written informed consent was obtained from all patients, and the study was approved by the Leeds East Ethics Committee.

Freshly isolated PBMCs from heparinized venous blood were dispensed into a 96-well U-bottom culture plate (0.15 × 106 cells/well in 200 μl cell-culture medium [RPMI 1640 supplemented with penicillin (100 μg/ml), streptomycin (100 μg/ml), HEPES (25 mM), l-glutamine (2 mM), 10% pooled human AB serum, and transferrin (12.5 mg)]). Piperacillin was first tested from 7.5 μM to 4 mM. Tetanus toxoid (0.5 μg/ml) was used as a positive control. Cell cultures were incubated in a CO2-ventilated (5%) incubator at 37°C for 6 d. On the fifth day, 0.5 μCi [3H]thymidine was added to each well. Cells were finally harvested onto filter membranes, and the amount of incorporated radioactivity was measured (counts per minute) using a β-counter (MicroBeta Trilux; PerkinElmer). Thereafter, the results were expressed as stimulation index (SI; calculated as average counts per minute in drug replicates/average counts per minute in medium replicates). An SI >2 was considered positive.

T cell lines were generated by culturing purified CD3+ T cells (4 × 106; 1 ml) with piperacillin (2 mM) and autologous irradiated PBMCs (1 × 106). IL-2 was added on day 3 to sustain the drug-specific proliferative response. Lines were restimulated with piperacillin and autologous irradiated PBMCs weekly for 4 wk prior to analysis of drug-specific proliferation and IFN-γ secretion by ELISPOT (see below). Ag-specific T cell clones were generated by serial dilution using established methodology (15, 16). To test the specificity of the clones, T cells (0.5 × 105) were incubated with autologous EBV-transformed B cells (0.1 × 105) and piperacillin (2 mM). After 48 h, [3H]thymidine (0.5 μCi) was added, and 16 h later, proliferation was measured by scintillation counting.

PBMCs (1.5 × 106; 200 μl) were pulsed with piperacillin (0.25–2 mM) for 4 d. After 1 h, 1, 2, 3, and 4 d, supernatant was collected for mass spectrometric analysis of piperacillin-albumin binding. At each time point, cells were washed to remove unbound drug, suspended in drug-free medium, and dispensed into a second culture plate. On the fifth day, 0.5 μCi [3H]thymidine was added to each well for the analysis of lymphocyte proliferation.

The proliferative response of PBMCs and T cell clones to piperacillin (0.25–2 mM) and piperacillin-modified albumin (0.25–4 mg/ml) was also evaluated. The protocols used were essentially the same as described above for the parent drug, with the exception that unconjugated albumin subjected to the same extraction protocol as piperacillin-conjugated albumin was used as a control.

ELISPOT (IFN-γ, IL-13, and granzyme B) was used to monitor secretory profiles from piperacillin-hypersensitive patients. PBMCs (0.5 × 106; 0.5 ml) were incubated with or without piperacillin (0.5–2 mM) for 48 h in Ab-coated plates prior to development.

Supernatants (25 μl) were also collected prior to the addition of [3H]thymidine for the analysis of cytokine/chemokine secretion using a Millipore multiplex assay kit (Millipore). Cytokine and chemokine concentrations (IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-17, TNF-α, IFN-γ, eotaxin, MIP-1α, and MIP-1β) were measured using a Bio-Plex Suspension Array System (model Luminex 100) and its Bio-Plex Manager 3.0 Software (Bio-Rad).

Skin prick tests were performed using a previously published protocol (28) against Tazocin (piperacillin-tazobactam), histamine (10 mg/ml positive control), and 0.9% saline (negative control) to exclude a diagnosis of immediate hypersensitivity. Intravenous piperacillin preparations were used at final concentrations of 2 mg/ml and 20 mg/ml in 0.9% saline. The reagents were prepared under sterile conditions and tested on the volar forearm. Readings were performed at 20 min. A wheal ≥3 mm in diameter than the negative control was considered positive. Intradermal injections were also performed with delayed readings at 48 and 72 h for the diagnosis of nonimmediate reactions. An infiltrated erythema >5 mm in diameter was considered a positive reaction.

Results were analyzed using a Mann–Whitney U test or a Wilcoxon test for paired data sets when comparing lymphocyte proliferation and cytokine concentrations. A Fisher exact test was used to compare frequencies among and between groups, and a Spearman analysis allowed nonparametric correlation analysis. A p value <0.05 was considered statistically significant.

Mass spectrometric analysis of piperacillin-albumin conjugates formed in phosphate buffer revealed a hapten of the predicted mass of 517 amu, which was formed from direct adduction of piperacillin (cyclized hapten [1], Fig. 1), but also a second hapten of mass 535 amu hypothesized to be formed through hydrolysis of the 2,3-dioxopiperazine ring (hydrolyzed hapten [2], Fig. 1). As shown in the tandem MS spectra of the peptide 182LDELRDEGK*ASSAK195 modified with the cyclized hapten (Fig. 2A), the presence of the most characteristic fragmentation ions of m/z at 160 and 143 indicated the incorporation of piperacillin in this peptide. In addition, the presence of an abundant ion of m/z at 868, corresponding to the doubly charged peptide mass plus 216 (a portion associated with penicilloyl adducts of the piperacillin structure), provided further evidence that the hapten of mass of 517 amu was formed by the addition to the β-lactam ring rather than the dioxopiperazine ring. In the mass spectrum of the hydrolyzed hapten, the most characteristic fragment ions were detected at m/z of 106 and 160 (Fig. 2A). Similarly, the ion at m/z of 867.8 confirmed that the nucleophilic addition took place at the β-lactam ring, whereas the hydrolysis occurred at the 2,3-dioxopiperazine ring. In addition, an equilibration between cyclized and hydrolyzed haptens was observed (results not shown), further confirming that the hydrolysis occurred at the 2,3-dioxopiperazine ring, as the hydrolysis of β-lactam ring is irreversible.

FIGURE 1.

Chemical structures of cyclized and hydrolyzed piperacillin and desethyl piperacillin haptens showing the dominant MS-induced fragmentation sites.

FIGURE 1.

Chemical structures of cyclized and hydrolyzed piperacillin and desethyl piperacillin haptens showing the dominant MS-induced fragmentation sites.

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FIGURE 2.

Mass spectrometric characterization of piperacillin haptens formed on albumin in vitro and isolated from plasma of patients undergoing piperacillin treatment. Representative MS/MS spectrum of the albumin peptide 182LDELRDEGKASSAK195 modified at Lys190 with the cyclized and hydrolyzed piperacillin haptens (A) and the hydrolyzed desethyl piperacillin hapten (B). Characteristic fragment ions derived from partial cleavage of the hapten are circled. C, Epitope profile showing the lysine residues of albumin modified in vivo with the cyclized and hydrolyzed piperacillin and desethyl piperacillin haptens. M, unmodified peptide mass; M*, modified peptide mass.

FIGURE 2.

Mass spectrometric characterization of piperacillin haptens formed on albumin in vitro and isolated from plasma of patients undergoing piperacillin treatment. Representative MS/MS spectrum of the albumin peptide 182LDELRDEGKASSAK195 modified at Lys190 with the cyclized and hydrolyzed piperacillin haptens (A) and the hydrolyzed desethyl piperacillin hapten (B). Characteristic fragment ions derived from partial cleavage of the hapten are circled. C, Epitope profile showing the lysine residues of albumin modified in vivo with the cyclized and hydrolyzed piperacillin and desethyl piperacillin haptens. M, unmodified peptide mass; M*, modified peptide mass.

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The masses of the modified peptides in combination with masses of the signature fragment ions enabled high-sensitivity detection of modified peptides in human serum albumin exposed to piperacillin in vitro. The cyclized and hydrolyzed forms of the piperacillin hapten were detected after 24 h at a drug/protein ratio of 50:1 at 8 out of 59 and 13 out of 59 lysine residues in human serum albumin, respectively. Modification at Lys190, Lys432, and Lys541 resulted in the strongest MRM signals, and, notwithstanding the differences in ionization efficiency, the hydrolyzed hapten was more prevalent than the cyclized (Fig. 3B, Table I shows the amino acid sequence of the modified peptides). The time and concentration dependency of the modification revealed by Western blotting was confirmed by MRM-MS, whereas the exquisite sensitivity of the mass spectrometric approach revealed modification on Lys541 at a molar ratio of drug to protein of 0.01:1 (Fig. 3).

FIGURE 3.

Time- and concentration-dependent binding of piperacillin to albumin in vitro. A, Western blotting with an anti-drug Ab and mass spectrometric analysis of the time- and concentration-dependent binding of piperacillin to albumin. B, Epitope profile showing the lysine residues of albumin modified in vitro with the cyclized and hydrolyzed piperacillin haptens.

FIGURE 3.

Time- and concentration-dependent binding of piperacillin to albumin in vitro. A, Western blotting with an anti-drug Ab and mass spectrometric analysis of the time- and concentration-dependent binding of piperacillin to albumin. B, Epitope profile showing the lysine residues of albumin modified in vitro with the cyclized and hydrolyzed piperacillin haptens.

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Table I.
Human serum albumin-derived tryptic peptides containing piperacillin-modified lysine residues
LysinePeptideCyclized HaptenHydrolyzed Hapten
DAHK*SEVAHR ✓ 
12 FK*DLGEENFK ✓ ✓ 
137 K*YLYEIAR ✓ 
162 YK*AAFTECCQAADK ✓ 
190 LDELRDEGK*ASSAK ✓ ✓ 
195 ASSAK*QR ✓ 
199 LK*CASLQK ✓ ✓ 
212 AFK*AWAVAR ✓ ✓ 
351 LAK*TYETTLEK ✓ ✓ 
432 NLGK*VGSK ✓ ✓ 
525 K*QTALVELVK ✓ ✓ 
541 ATK*EQLK ✓ ✓ 
545 EQLK*AVMDDFAAFVEK ✓ 
LysinePeptideCyclized HaptenHydrolyzed Hapten
DAHK*SEVAHR ✓ 
12 FK*DLGEENFK ✓ ✓ 
137 K*YLYEIAR ✓ 
162 YK*AAFTECCQAADK ✓ 
190 LDELRDEGK*ASSAK ✓ ✓ 
195 ASSAK*QR ✓ 
199 LK*CASLQK ✓ ✓ 
212 AFK*AWAVAR ✓ ✓ 
351 LAK*TYETTLEK ✓ ✓ 
432 NLGK*VGSK ✓ ✓ 
525 K*QTALVELVK ✓ ✓ 
541 ATK*EQLK ✓ ✓ 
545 EQLK*AVMDDFAAFVEK ✓ 

Piperacillin/albumin ratio 50:1; incubation time 24 h.

*

, Site of piperacillin modification.

Albumin was isolated from four piperacillin-exposed patients with cystic fibrosis to characterize Ag formation in vivo. To enhance the sensitivity of detection of the modified peptides, a three-dimensional LC approach was adopted that enabled the detection of the cyclized [1] and hydrolyzed [2] forms of the piperacillin hapten at Lys190. A further mass addition of 507 amu, which was associated with fragment ions of 106 and 160 amu, was detected at Lys190 (Fig. 2B), Lys195, Lys432, and Lys541 (Fig. 2C), and we propose that this is the hapten formed from the desethyl metabolite of piperacillin (29) with hydrolysis of the dioxopiperazine ring. Fig. 1 shows the structure of desethyl piperacillin and the cyclized [3] and hydrolyzed [4] forms of the piperacillin metabolite-derived hapten. No desethyl cyclized hapten was detected. The possibility that the desethyl structure was formed by in-source fragmentation was ruled out, as the retention times of the peptide containing hydrolyzed and desethyl hydrolyzed Lys190 differed by 1 to 2 min during both cation exchange and reversed-phase chromatography (data not shown).

PBMCs from 19 out of the 28 piperacillin-hypersensitive patients (68% sensitivity) were found to proliferate in the presence of piperacillin (Fig. 4A, Table II). The sensitivity of the assay was increased to 76% when excluding the three patients receiving oral steroids at the time of blood sampling (patients 26–28; Table II). The number of positive patients did not differ when those with and without cutaneous manifestation were compared. The lymphocyte transformation test was repeated on 10 hypersensitive patients with at least a 1-mo interval between assays. Although the strength of the proliferative response varied slightly, the number of lymphocyte transformation test-positive patients remained the same (results not shown). PBMCs from patients hypersensitive to piperacillin alone were not stimulated with other structurally related β-lactam antibiotics.

FIGURE 4.

Piperacillin-specific stimulation of PBMCs and T cell lines from hypersensitive patients. A, PBMCs from 19 hypersensitive patients were specifically stimulated with piperacillin (SI >2). B, Positive intradermal skin test from one of four patients presenting with cutaneous signs and a strong in vitro proliferative response against piperacillin. C, A biopsy of the maculopapular reaction site. Immune stain confirmed the lymphocytic infiltrate as being almost entirely T cell in character, with a CD3+CD45RO+ phenotype (original magnification ×100). Both CD4+ and CD8+ subsets were present. D, Piperacillin-specific IFN-γ, IL-13, and granzyme B ELISPOT. The figure shows PBMCs from four hypersensitive patients stimulated with piperacillin for 48 h. E, Multiplex analysis of cytokines/chemokines secreted from hypersensitive patient PBMCs (n = 5) incubated with stimulatory concentrations of piperacillin. F, Concentration-dependent proliferation and IFN-γ secretion by a piperacillin-responsive T cell line. T cell lines were generated by repetitive stimulation of blood lymphocytes with piperacillin and irradiated autologous PBMCs in IL-2–containing medium. ●, patient 22; ○, patient 10; ■, patient 2; ▲, patient 4; ♦, patient 5.

FIGURE 4.

Piperacillin-specific stimulation of PBMCs and T cell lines from hypersensitive patients. A, PBMCs from 19 hypersensitive patients were specifically stimulated with piperacillin (SI >2). B, Positive intradermal skin test from one of four patients presenting with cutaneous signs and a strong in vitro proliferative response against piperacillin. C, A biopsy of the maculopapular reaction site. Immune stain confirmed the lymphocytic infiltrate as being almost entirely T cell in character, with a CD3+CD45RO+ phenotype (original magnification ×100). Both CD4+ and CD8+ subsets were present. D, Piperacillin-specific IFN-γ, IL-13, and granzyme B ELISPOT. The figure shows PBMCs from four hypersensitive patients stimulated with piperacillin for 48 h. E, Multiplex analysis of cytokines/chemokines secreted from hypersensitive patient PBMCs (n = 5) incubated with stimulatory concentrations of piperacillin. F, Concentration-dependent proliferation and IFN-γ secretion by a piperacillin-responsive T cell line. T cell lines were generated by repetitive stimulation of blood lymphocytes with piperacillin and irradiated autologous PBMCs in IL-2–containing medium. ●, patient 22; ○, patient 10; ■, patient 2; ▲, patient 4; ♦, patient 5.

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PBMCs from tolerant patients with cystic fibrosis and drug-naive volunteers were not stimulated to proliferate with piperacillin.

Skin prick tests, which are traditionally used in patients with immediate hypersensitivity (28), did not generate positive readings with piperacillin in any of the patients tested. Four out of the 28 piperacillin-hypersensitive patients had positive intradermal readings to piperacillin. Positive readings were only detected in patients with cutaneous signs and a positive lymphocyte transformation test (Fig. 4B). Patients 2 and 9 developed marked erythema and induration 24 h following injection. A biopsy of the skin reaction from patient 2 revealed epidermal basal layer vacuolation, necrotic keratinocytes, and a dermal perivascular infiltration of T lymphocytes (Fig. 4C). Patients 4 and 21 showed significant induration at 48 h.

The drug-specific proliferative response was associated with the secretion of IFN-γ, IL-13, and the granulation molecule granzyme B, a key mediator of cell killing (PBMCs, Fig. 4D; T cell line, Fig. 4F). Multiplex analysis of cell-culture supernatant was performed to obtain a more global analysis of cytokines secreted from hypersensitive and tolerant patients’ PBMCs. In addition to the cytokines IFN-γ and IL-13, significantly higher levels of IL-1β (p = 0.031), IL-6 (p = 0.016), TNF-α (p = 0.012), and MIP-1α (p = 0.031) were found in cell cultures containing piperacillin-treated PBMCs from hypersensitive patients when compared with the tolerant and naive controls (Fig. 4E).

The response of PBMCs from hypersensitive patients was concentration dependent and detectable over a wide range of piperacillin concentrations (Fig. 5A). Therefore, the lymphocyte transformation test was used to explore the relationship between drug albumin binding and drug immunogenicity. Cyclized [1] and hydrolyzed [2] forms of the piperacillin hapten were detectable on albumin, and the levels of albumin binding increased progressively over 96 h (Fig. 5B). Binding was concentration dependent and observed at piperacillin concentrations that stimulate the proliferation of PBMCs from hypersensitive patients. Both piperacillin haptens (hydrolyzed and cyclized forms) were found to bind preferentially to Lys190, followed by Lys432 and Lys541 on albumin. Drug modifications were also detected at an additional five lysine residues (Lys195, Lys199, Lys212, Lys351, and Lys525) (Fig. 5C).

FIGURE 5.

Concentration-dependent stimulation of patient PBMCs with piperacillin and characterization of the major Ag formed in cell culture. A, Concentration-dependent piperacillin-specific proliferation of PBMCs from hypersensitive patients. Proliferative responses were analyzed by incorporation of [3H]thymidine in the final 16 h of the experiment. B, Analysis of total levels of albumin binding with piperacillin concentrations associated with a significant lymphocyte-proliferative response after 1–120 h. C, Epitope profile showing the lysine residues of albumin modified with the cyclized and hydrolyzed piperacillin haptens in culture medium.

FIGURE 5.

Concentration-dependent stimulation of patient PBMCs with piperacillin and characterization of the major Ag formed in cell culture. A, Concentration-dependent piperacillin-specific proliferation of PBMCs from hypersensitive patients. Proliferative responses were analyzed by incorporation of [3H]thymidine in the final 16 h of the experiment. B, Analysis of total levels of albumin binding with piperacillin concentrations associated with a significant lymphocyte-proliferative response after 1–120 h. C, Epitope profile showing the lysine residues of albumin modified with the cyclized and hydrolyzed piperacillin haptens in culture medium.

Close modal

To determine which piperacillin-modified lysine residues in albumin are the key epitopes involved in the stimulation of a lymphocyte proliferative response, PBMCs from hypersensitive patients were cultured with piperacillin (0.25–2 mM). After 1–96 h, PBMCs were washed repeatedly to remove soluble drug, suspended in culture medium, and dispensed into fresh culture plates for the remainder of the assay. Modified Lys190 and Lys432 were detectable at 2 mM after a 1-h incubation; however, the level of modification was extremely low, coinciding with a negative lymphocyte proliferative response (Fig. 6). An increase in the level of modification at Lys190 and Lys432 and an increase in the number of sites modified (Lys199 and Lys541) at 24 h was associated with a weak proliferative response of patient PBMCs (Fig. 6). At intermediate time points (48–72 h), there was a further increase in the number of sites modified and a concentration-dependent increase in the level of modification at each site. At 72 h, piperacillin modifications were detectable at six lysine residues (Lys190, Lys199, Lys212, Lys351, Lys432, and Lys541). This correlated with the concentration-dependent proliferation of T cells from hypersensitive patients, reaching a maximum response at 2 mM. Finally, at 96 h, when the highest levels of albumin binding were detected, a maximal proliferative response was seen with each stimulatory concentration of piperacillin (0.25–2 mM). Hydrolyzed [1] and/or cyclized [2] forms of the piperacillin hapten were detected on eight lysine residues (Fig. 6C), including Lys190, Lys195, Lys432, and Lys541, which were modified with piperacillin in patients.

FIGURE 6.

Identification of the key piperacillin-modified lysine residues in albumin involved in a lymphocyte-proliferative response. A, Kinetic profile of the hydrolyzed and cyclized haptens of piperacillin bound to each modified lysine residue on albumin in cell culture. Profiles derive from MRM-MS analysis of the modified tryptic peptides. B, Proliferative response of PBMCs from hypersensitive patients pulsed with piperacillin for 1–96 h. Proliferative responses were analyzed by incorporation of [3H]thymidine in the final 16 h of the experiment. C, Model of albumin showing piperacillin binding sites at positions Lys190, Lys195, Lys199, Lys212, Lys351, Lys432, Lys525, and Lys541.

FIGURE 6.

Identification of the key piperacillin-modified lysine residues in albumin involved in a lymphocyte-proliferative response. A, Kinetic profile of the hydrolyzed and cyclized haptens of piperacillin bound to each modified lysine residue on albumin in cell culture. Profiles derive from MRM-MS analysis of the modified tryptic peptides. B, Proliferative response of PBMCs from hypersensitive patients pulsed with piperacillin for 1–96 h. Proliferative responses were analyzed by incorporation of [3H]thymidine in the final 16 h of the experiment. C, Model of albumin showing piperacillin binding sites at positions Lys190, Lys195, Lys199, Lys212, Lys351, Lys432, Lys525, and Lys541.

Close modal

To generate a synthetic piperacillin-albumin conjugate for use as an Ag in in vitro assays, human serum albumin was modified with piperacillin at a molar ratio of drug to protein of 50:1 for 24 h in phosphate buffer. The epitope profile is shown in Fig. 7A; this largely mirrored the profile of albumin binding detected in culture supernatant in that: 1) hydrolyzed and cyclized forms of the piperacillin hapten were detected on the eight lysine residues modified in culture; and 2) the three sites most readily detected by MRM-MS were Lys190, Lys432, and Lys541.

FIGURE 7.

Stimulation of patient PBMCs and T cell clones with a synthetic piperacillin-albumin conjugate. A, Epitope profile of the piperacillin-albumin conjugate derived from MRM-MS analysis of modified tryptic peptides. Specific proliferation of PBMCs (B) and T cell clones (C) from hypersensitive patients with the synthetic albumin conjugate. Unconjugated albumin subjected to the same extraction protocol as the piperacillin-albumin conjugate was used as a control in each experiment. Proliferation in cultures containing unconjugated albumin were consistently <2000 cpm, and no significant difference was observed when unconjugated albumin and medium controls were compared.

FIGURE 7.

Stimulation of patient PBMCs and T cell clones with a synthetic piperacillin-albumin conjugate. A, Epitope profile of the piperacillin-albumin conjugate derived from MRM-MS analysis of modified tryptic peptides. Specific proliferation of PBMCs (B) and T cell clones (C) from hypersensitive patients with the synthetic albumin conjugate. Unconjugated albumin subjected to the same extraction protocol as the piperacillin-albumin conjugate was used as a control in each experiment. Proliferation in cultures containing unconjugated albumin were consistently <2000 cpm, and no significant difference was observed when unconjugated albumin and medium controls were compared.

Close modal

The synthetic piperacillin-albumin conjugate stimulated the proliferation of PBMCs and CD4+ T cell clones from hypersensitive patients (Fig. 7B).

Although little is known about the mechanisms that lead to drug hypersensitivity reactions, several hypotheses to explain drug immunogenicity have been postulated, and one of the most popular is the hapten hypothesis. This is based on the concept that drugs form haptenic structures, compounds with a propensity to bind covalently to biological macromolecules, that modify endogenous protein to stimulate an immune response (3032). Processed peptides derived from the modified protein are presumed to interact with MHC molecules prior to stimulating T cells through the TCR. Many drugs associated with a high prevalence of hypersensitivity reactions in humans form protein adducts; however, the absence of sufficiently sensitive bioanalytical methods to characterize functional drug–protein conjugates has effectively prohibited any attempt to study the relationship between Ag formation and immunogenicity.

We have recently developed and employed MS methods to qualify the site(s) of drug–protein conjugation (2023). Drugs and drug metabolites bind in a dose-dependent manner to proteins such as albumin and can display different preferences for the sites of modification. In the current study, we sought to exploit these methods to investigate piperacillin hypersensitivity in patients with cystic fibrosis. Specific objectives of the project were to characterize haptenic structures on albumin and the relationship between drug modification of protein and drug-specific lymphocyte responses, thereby determining the fundamental relationship between the chemistry of Ag formation and drug hypersensitivity.

Mass spectrometric analysis, after protein digestion, revealed that piperacillin forms multiple haptenic structures on human serum albumin in vitro. Consistent with the known chemistry of β-lactam antibiotics, we have shown that piperacillin can form adducts with lysine residues by direct opening of the β-lactam ring. However, the chemistry of Ag formation is complex. There is also a hapten formed in which the 2,3-dioxopiperazine ring has undergone hydrolysis. Thus, two distinct haptens can be formed. In principle, adducts resulting from opening of the 2,3-dioxopiperazine ring or cross-linking adducts could also be generated. However, these adducts were not detected, indicating that the β-lactam ring is more susceptible to nucleophiles than the 2,3-dioxopiperazine ring. No evidence of modification at other amino acid residues could be found. This might be because adducts resulting from piperacillin binding to other nucleophilic amino acids such as serine, histidine, and cysteine are not formed. However, such adducts might be too labile to be detected under current MS conditions. The extent of piperacillin albumin binding was dependent on incubation time at each drug concentration studied. This is important, as cystic fibrosis patients are treated by rapid infusion with high doses of drug (4 g over 20 min, three times a day), leading to a plasma maximum concentration of 0.4–0.65 mM and plasma clearance of the drug after 4.5 h (33). The concentration of human serum albumin in plasma is ∼35 g/l (0.53 mM) (34), and the molar ratio of drug to protein at maximum concentration is 0.75–1.22:1. These data indicate that conjugates would be formed in vivo, and, because the t1/2 of human serum albumin is ∼19 d (35), the modified protein is likely to accumulate over the course of the therapeutic intervention, which is usually 14 d in duration.

To characterize haptens formed in vivo, albumin was isolated from patients receiving piperacillin and subjected to affinity, cation exchange, and reverse-phase chromatography after trypsin digestion and prior to MS. In addition to the hydrolyzed and cyclized haptens of piperacillin, a further haptenic structure was detected that was derived from the desethyl metabolite of piperacillin with a hydrolyzed piperazine ring. This reveals that not all of the products of piperacillin metabolism in the liver are excreted in the bile or urine, but a significant amount is released back into the circulation.

A restricted profile of piperacillin albumin binding was detected in vitro and in vivo. At the lowest drug concentration investigated (drug/protein ratio of 0.01:1), modification of a single lysine (Lys541) could be detected (Fig. 3B). Modified Lys541 was also detected on albumin isolated from patients, alongside modification of Lys190, Lys195, and Lys432. The selective modification of lysine residues on albumin was not simply related to the pKa of individual lysine residues and therefore the reactivity of the side-chain amino group. Instead, the majority of the sites of modification were associated with Sudlow sites I and II, hydrophobic pockets in human serum albumin involved in the noncovalent interaction with endogenous ligands and drugs (21, 36, 37). This indicates that the orientation of the noncovalent interaction of drug with protein and the subsequent stabilization by hydrogen bonding influences which lysine residues are in close proximity with the drug and are therefore preferentially covalently modified.

Circulating piperacillin-specific lymphocytes were detected in the majority of piperacillin-hypersensitive patients with cystic fibrosis. The piperacillin-specific proliferative response was reproducible on repeated testing and dose dependent, with the highest response detected with therapeutic piperacillin concentrations that are estimated to be in the range of 0.5–1 mM. Piperacillin-specific lymphocytes were detected in hypersensitive patients with and without cutaneous symptoms, suggesting that drug-specific lymphocytes are involved in other clinical features of the disease pathogenesis (arthralgia, fevers, or flulike symptoms). The cytokine profile associated with a particular reaction determines the nature of the induced immune response (1, 3842). Ag stimulation of PBMCs from piperacillin-hypersensitive patients was associated with the secretion of a mixed panel of cytokines, with high levels of IFN-γ, IL-13, TNF-α, IL-1β, IL-6, and MIP-1α/β detected. IL-5 secretion, a common feature of drug reactions in non-cystic fibrosis patients (43, 44), was not seen in piperacillin-hypersensitive patients, and this may relate to the absence of eosinophilia as a clinical feature. The detection of IL-1β, TNF-α, and IL-6 in culture supernatant containing piperacillin-stimulated PBMCs from hypersensitive patients and tolerant controls (albeit to a lesser extent) are suggestive of a dendritic cell response against piperacillin, as has recently been described with amoxicillin (45, 46). In support of this argument, piperacillin-specific T cell clones do not secrete IL-1β (Sabah El-Ghaiesh, B. Kevin Park, and Dean J. Naisbitt, unpublished observations). Whether this cytokine profile is a function of the chemistry of the drug or the disease status of the patient is not known and is an area of ongoing work. A positive granzyme B ELISPOT in patients with a positive proliferative response shows that piperacillin stimulates cytotoxic T cells and that they may play a role in the hypersensitivity reaction.

In view of the fact that the lymphocyte transformation test can be used to confirm the immunological etiology of piperacillin hypersensitivity reactions, MS methods were used to explore the chemical basis of drug antigenicity in PBMC cultures and to define that albumin conjugates are antigenic per se. Piperacillin-albumin binding at individual lysine residues was profiled with respect to incubation time, dose, and stimulation of patient PBMCs. Hydrolyzed and/or cyclized forms of the piperacillin hapten were detected at each lymphocyte-stimulating concentration of piperacillin (Fig. 6). Modified Lys190, Lys199, Lys432, and Lys541 were detectable at 2 mM piperacillin after a 24-h incubation. The level of modification at each site was low, but coincided with a weak proliferative response. Piperacillin haptens were detectable on six lysine residues after 48–72 h, and a concentration-dependent increase in the level of modification at each site was observed. This correlated with the concentration-dependent stimulation of patient PBMCs. Analysis of minimally modified but lymphocyte-stimulatory albumin conjugates formed in culture indicate that peptide sequences around positions Lys190, Lys432, and Lys541 may be the principal functional epitopes generated in the lymphocyte transformation test. To confirm that piperacillin-albumin conjugates are indeed antigenic, a conjugate with hydrolyzed and cyclized forms of the piperacillin hapten detectable on each lysine residue modified in culture was generated under physiological conditions and shown to stimulate PBMCs and T cell clones to proliferate. These data are consistent with previous studies showing that synthetic penicillin–albumin constructs generated under forced chemical conditions can stimulate T cells (2) but crucially relate to Ags that are formed under physiological conditions. We are therefore in a position to prepare fully characterized peptide conjugates of piperacillin of physiological relevance and determine their fit into relevant MHC molecules (8).

To conclude, using mass spectrometric methods, we have defined piperacillin modifications on albumin, with respect to hapten formation and peptide epitope profile, that are able to stimulate T cells ex vivo and shown that such modifications can be detected on circulating albumin in patients receiving the drug. Drug–peptide conjugates derived from modified albumin clearly represent functional Ags for T cells and may indeed function as immunogens in patients with cystic fibrosis. However, it also possible that alternative proteins, which generate similar drug-modified peptide epitopes, may constitute the functional immunogen in the patients. A prospective clinical study of piperacillin hypersensitivity is required to explore immunological consequences of Ag formation before, during, and after the development of hypersensitivity using the techniques developed in this investigational study.

We thank the nurses of the Adult Cystic Fibrosis Unit who helped to collect samples as well as all of the volunteers and patients with cystic fibrosis who participated in the project. We also thank Prof. Mark Taylor and postdoctoral fellow Dr. Kelly Johnston (School of Tropical Medicine, University of Liverpool) for the contribution to the cytokine analysis.

This work was supported by a grant from the Wellcome Trust (078598/Z/05/Z) as part of the Centre for Drug Safety Science supported by the Medical Research Council (G0700654). X.M. is supported by the National Institute for Health Research Biomedical Research Centre in Microbial Diseases. M.P. is a National Institute for Health Research senior investigator. S.E. and M.M. are Ph.D. students funded by the Egyptian and Saudi Arabian governments, respectively.

Abbreviations used in this article:

ACN

acetonitrile

amu

atomic mass units

LC

liquid chromatography

MRM

multiple reaction monitoring

MS

mass spectrometry

m/z

mass/charge ratio

SI

stimulation index

TST

Tris/saline/Tween.

1
Beeler
A.
,
Engler
O.
,
Gerber
B. O.
,
Pichler
W. J.
.
2006
.
Long-lasting reactivity and high frequency of drug-specific T cells after severe systemic drug hypersensitivity reactions.
J. Allergy Clin. Immunol.
117
:
455
462
.
2
Brander
C.
,
Mauri-Hellweg
D.
,
Bettens
F.
,
Rolli
H.
,
Goldman
M.
,
Pichler
W. J.
.
1995
.
Heterogeneous T cell responses to beta-lactam-modified self-structures are observed in penicillin-allergic individuals.
J. Immunol.
155
:
2670
2678
.
3
Castrejon
J. L.
,
Berry
N.
,
El-Ghaiesh
S.
,
Gerber
B.
,
Pichler
W. J.
,
Park
B. K.
,
Naisbitt
D. J.
.
2010
.
Stimulation of human T cells with sulfonamides and sulfonamide metabolites.
J. Allergy Clin. Immunol.
125
:
411
418
.e4.
4
Nassif
A.
,
Bensussan
A.
,
Boumsell
L.
,
Deniaud
A.
,
Moslehi
H.
,
Wolkenstein
P.
,
Bagot
M.
,
Roujeau
J. C.
.
2004
.
Toxic epidermal necrolysis: effector cells are drug-specific cytotoxic T cells.
J. Allergy Clin. Immunol.
114
:
1209
1215
.
5
Schnyder
B.
,
Burkhart
C.
,
Schnyder-Frutig
K.
,
von Greyerz
S.
,
Naisbitt
D. J.
,
Pirmohamed
M.
,
Park
B. K.
,
Pichler
W. J.
.
2000
.
Recognition of sulfamethoxazole and its reactive metabolites by drug-specific CD4+ T cells from allergic individuals.
J. Immunol.
164
:
6647
6654
.
6
Wu
Y.
,
Farrell
J.
,
Pirmohamed
M.
,
Park
B. K.
,
Naisbitt
D. J.
.
2007
.
Generation and characterization of antigen-specific CD4+, CD8+, and CD4+CD8+ T-cell clones from patients with carbamazepine hypersensitivity.
J. Allergy Clin. Immunol.
119
:
973
981
.
7
Elsheikh
A.
,
Lavergne
S. N.
,
Castrejon
J. L.
,
Farrell
J.
,
Wang
H.
,
Sathish
J.
,
Pichler
W. J.
,
Park
B. K.
,
Naisbitt
D. J.
.
2010
.
Drug antigenicity, immunogenicity, and costimulatory signaling: evidence for formation of a functional antigen through immune cell metabolism.
J. Immunol.
185
:
6448
6460
.
8
Padovan
E.
,
Bauer
T.
,
Tongio
M. M.
,
Kalbacher
H.
,
Weltzien
H. U.
.
1997
.
Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy.
Eur. J. Immunol.
27
:
1303
1307
.
9
Padovan
E.
,
Mauri-Hellweg
D.
,
Pichler
W. J.
,
Weltzien
H. U.
.
1996
.
T cell recognition of penicillin G: structural features determining antigenic specificity.
Eur. J. Immunol.
26
:
42
48
.
10
Burkhart
C.
,
von Greyerz
S.
,
Depta
J. P.
,
Naisbitt
D. J.
,
Britschgi
M.
,
Park
K. B.
,
Pichler
W. J.
.
2001
.
Influence of reduced glutathione on the proliferative response of sulfamethoxazole-specific and sulfamethoxazole-metabolite-specific human CD4+ T-cells.
Br. J. Pharmacol.
132
:
623
630
.
11
Depta
J. P.
,
Altznauer
F.
,
Gamerdinger
K.
,
Burkhart
C.
,
Weltzien
H. U.
,
Pichler
W. J.
.
2004
.
Drug interaction with T-cell receptors: T-cell receptor density determines degree of cross-reactivity.
J. Allergy Clin. Immunol.
113
:
519
527
.
12
Farrell
J.
,
Naisbitt
D. J.
,
Drummond
N. S.
,
Depta
J. P.
,
Vilar
F. J.
,
Pirmohamed
M.
,
Park
B. K.
.
2003
.
Characterization of sulfamethoxazole and sulfamethoxazole metabolite-specific T-cell responses in animals and humans.
J. Pharmacol. Exp. Ther.
306
:
229
237
.
13
Hashizume
H.
,
Takigawa
M.
,
Tokura
Y.
.
2002
.
Characterization of drug-specific T cells in phenobarbital-induced eruption.
J. Immunol.
168
:
5359
5368
.
14
Keller
M.
,
Lerch
M.
,
Britschgi
M.
,
Tâche
V.
,
Gerber
B. O.
,
Lüthi
M.
,
Lochmatter
P.
,
Kanny
G.
,
Bircher
A. J.
,
Christiansen
C.
,
Pichler
W. J.
.
2010
.
Processing-dependent and -independent pathways for recognition of iodinated contrast media by specific human T cells.
Clin. Exp. Allergy
40
:
257
268
.
15
Naisbitt
D. J.
,
Farrell
J.
,
Wong
G.
,
Depta
J. P.
,
Dodd
C. C.
,
Hopkins
J. E.
,
Gibney
C. A.
,
Chadwick
D. W.
,
Pichler
W. J.
,
Pirmohamed
M.
,
Park
B. K.
.
2003
.
Characterization of drug-specific T cells in lamotrigine hypersensitivity.
J. Allergy Clin. Immunol.
111
:
1393
1403
.
16
Schnyder
B.
,
Mauri-Hellweg
D.
,
Zanni
M.
,
Bettens
F.
,
Pichler
W. J.
.
1997
.
Direct, MHC-dependent presentation of the drug sulfamethoxazole to human alphabeta T cell clones.
J. Clin. Invest.
100
:
136
141
.
17
Batchelor
F. R.
,
Dewdney
J. M.
,
Gazzard
D.
.
1965
.
Penicillin allergy: the formation of the penicilloyl determinant.
Nature
206
:
362
364
.
18
Levine
B. B.
1960
.
Studies on the mechanism of the formation of the penicillin antigen. I. Delayed allergic cross-reactions among penicillin G and its degradation products.
J. Exp. Med.
112
:
1131
1156
.
19
Levine
B. B.
,
Ovary
Z.
.
1961
.
Studies on the mechanism of the formation of the penicillin antigen. III. The N-(D-alpha-benzylpenicilloyl) group as an antigenic determinant responsible for hypersensitivity to penicillin G.
J. Exp. Med.
114
:
875
904
.
20
Callan
H. E.
,
Jenkins
R. E.
,
Maggs
J. L.
,
Lavergne
S. N.
,
Clarke
S. E.
,
Naisbitt
D. J.
,
Park
B. K.
.
2009
.
Multiple adduction reactions of nitroso sulfamethoxazole with cysteinyl residues of peptides and proteins: implications for hapten formation.
Chem. Res. Toxicol.
22
:
937
948
.
21
Jenkins
R. E.
,
Meng
X.
,
Elliott
V. L.
,
Kitteringham
N. R.
,
Pirmohamed
M.
,
Park
B. K.
.
2009
.
Characterisation of flucloxacillin and 5-hydroxymethyl flucloxacillin haptenated HSA in vitro and in vivo.
Proteomics Clin. Appl.
3
:
720
729
.
22
Jenkinson
C.
,
Jenkins
R. E.
,
Aleksic
M.
,
Pirmohamed
M.
,
Naisbitt
D. J.
,
Park
B. K.
.
2010
.
Characterization of p-phenylenediamine-albumin binding sites and T-cell responses to hapten-modified protein.
J. Invest. Dermatol.
130
:
732
742
.
23
Jenkinson
C.
,
Jenkins
R. E.
,
Maggs
J. L.
,
Kitteringham
N. R.
,
Aleksic
M.
,
Park
B. K.
,
Naisbitt
D. J.
.
2009
.
A mechanistic investigation into the irreversible protein binding and antigenicity of p-phenylenediamine.
Chem. Res. Toxicol.
22
:
1172
1180
.
24
Burrows
J. A.
,
Nissen
L. M.
,
Kirkpatrick
C. M.
,
Bell
S. C.
.
2007
.
Beta-lactam allergy in adults with cystic fibrosis.
J. Cyst. Fibros.
6
:
297
303
.
25
Koch
C.
,
Hjelt
K.
,
Pedersen
S. S.
,
Jensen
E. T.
,
Jensen
T.
,
Lanng
S.
,
Valerius
N. H.
,
Pedersen
M.
,
Høiby
N.
.
1991
.
Retrospective clinical study of hypersensitivity reactions to aztreonam and six other beta-lactam antibiotics in cystic fibrosis patients receiving multiple treatment courses.
Rev. Infect. Dis.
13
(
Suppl 7
):
S608
S611
.
26
Pleasants
R. A.
,
Walker
T. R.
,
Samuelson
W. M.
.
1994
.
Allergic reactions to parenteral beta-lactam antibiotics in patients with cystic fibrosis.
Chest
106
:
1124
1128
.
27
Bradford
M. M.
1976
.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72
:
248
254
.
28
Torres
M. J.
,
Blanca
M.
,
Fernandez
J.
,
Romano
A.
,
Weck
A.
,
Aberer
W.
,
Brockow
K.
,
Pichler
W. J.
,
Demoly
P.
ENDA
; 
EAACI Interest Group on Drug Hypersensitivity
.
2003
.
Diagnosis of immediate allergic reactions to beta-lactam antibiotics.
Allergy
58
:
961
972
.
29
Ghibellini
G.
,
Bridges
A. S.
,
Generaux
C. N.
,
Brouwer
K. L.
.
2007
.
In vitro and in vivo determination of piperacillin metabolism in humans.
Drug Metab. Dispos.
35
:
345
349
.
30
Landsteiner
K.
,
Jacobs
J.
.
1935
.
Studies on the sensitization of animals with simple chemical compounds.
J. Exp. Med.
61
:
643
656
.
31
Lavergne
S. N.
,
Park
B. K.
,
Naisbitt
D. J.
.
2008
.
The roles of drug metabolism in the pathogenesis of T-cell-mediated drug hypersensitivity.
Curr. Opin. Allergy Clin. Immunol.
8
:
299
307
.
32
Park
B. K.
,
Pirmohamed
M.
,
Kitteringham
N. R.
.
1998
.
Role of drug disposition in drug hypersensitivity: a chemical, molecular, and clinical perspective.
Chem. Res. Toxicol.
11
:
969
988
.
33
Hayashi
Y.
,
Roberts
J. A.
,
Paterson
D. L.
,
Lipman
J.
.
2010
.
Pharmacokinetic evaluation of piperacillin-tazobactam.
Expert Opin. Drug Metab. Toxicol.
6
:
1017
1031
.
34
Anderson
N. L.
,
Anderson
N. G.
.
2002
.
The human plasma proteome: history, character, and diagnostic prospects.
Mol. Cell. Proteomics
1
:
845
867
.
35
Müller
N.
,
Schneider
B.
,
Pfizenmaier
K.
,
Wajant
H.
.
2010
.
Superior serum half life of albumin tagged TNF ligands.
Biochem. Biophys. Res. Commun.
396
:
793
799
.
36
Fasano
M.
,
Curry
S.
,
Terreno
E.
,
Galliano
M.
,
Fanali
G.
,
Narciso
P.
,
Notari
S.
,
Ascenzi
P.
.
2005
.
The extraordinary ligand binding properties of human serum albumin.
IUBMB Life
57
:
787
796
.
37
Sudlow
G.
,
Birkett
D. J.
,
Wade
D. N.
.
1975
.
Spectroscopic techniques in the study of protein binding. A fluorescence technique for the evaluation of the albumin binding and displacement of warfarin and warfarin-alcohol.
Clin. Exp. Pharmacol. Physiol.
2
:
129
140
.
38
Hertl
M.
,
Bohlen
H.
,
Jugert
F.
,
Boecker
C.
,
Knaup
R.
,
Merk
H. F.
.
1993
a
.
Predominance of epidermal CD8+ T lymphocytes in bullous cutaneous reactions caused by beta-lactam antibiotics.
J. Invest. Dermatol.
101
:
794
799
.
39
Hertl
M.
,
Geisel
J.
,
Boecker
C.
,
Merk
H. F.
.
1993
b
.
Selective generation of CD8+ T-cell clones from the peripheral blood of patients with cutaneous reactions to beta-lactam antibiotics.
Br. J. Dermatol.
128
:
619
626
.
40
Lochmatter
P.
,
Beeler
A.
,
Kawabata
T. T.
,
Gerber
B. O.
,
Pichler
W. J.
.
2009
.
Drug-specific in vitro release of IL-2, IL-5, IL-13 and IFN-gamma in patients with delayed-type drug hypersensitivity.
Allergy
64
:
1269
1278
.
41
Rozieres
A.
,
Hennino
A.
,
Rodet
K.
,
Gutowski
M. C.
,
Gunera-Saad
N.
,
Berard
F.
,
Cozon
G.
,
Bienvenu
J.
,
Nicolas
J. F.
.
2009
.
Detection and quantification of drug-specific T cells in penicillin allergy.
Allergy
64
:
534
542
.
42
Yawalkar
N.
,
Hari
Y.
,
Frutig
K.
,
Egli
F.
,
Wendland
T.
,
Braathen
L. R.
,
Pichler
W. J.
.
2000
.
T cells isolated from positive epicutaneous test reactions to amoxicillin and ceftriaxone are drug specific and cytotoxic.
J. Invest. Dermatol.
115
:
647
652
.
43
Pichler
W. J.
,
Zanni
M.
,
von Greyerz
S.
,
Schnyder
B.
,
Mauri-Hellweg
D.
,
Wendland
T.
.
1997
.
High IL-5 production by human drug-specific T cell clones.
Int. Arch. Allergy Immunol.
113
:
177
180
.
44
Yawalkar
N.
,
Shrikhande
M.
,
Hari
Y.
,
Nievergelt
H.
,
Braathen
L. R.
,
Pichler
W. J.
.
2000
b
.
Evidence for a role for IL-5 and eotaxin in activating and recruiting eosinophils in drug-induced cutaneous eruptions.
J. Allergy Clin. Immunol.
106
:
1171
1176
.
45
Lima
C. M.
,
Schroeder
J. T.
,
Galvão
C. E.
,
Castro
F. M.
,
Kalil
J.
,
Adkinson
N. F.
 Jr.
2010
.
Functional changes of dendritic cells in hypersensivity reactions to amoxicillin.
Braz. J. Med. Biol. Res.
43
:
964
968
.
46
Rodriguez-Pena
R.
,
Lopez
S.
,
Mayorga
C.
,
Antunez
C.
,
Fernandez
T. D.
,
Torres
M. J.
,
Blanca
M.
.
2006
.
Potential involvement of dendritic cells in delayed-type hypersensitivity reactions to beta-lactams.
J. Allergy Clin. Immunol.
118
:
949
956
.

The authors have no financial conflicts of interest.