Shared Clavulanate and Tazobactam Antigenic Determinants Activate T-Cells from Hypersensitive Patients.

ß-Lactamase inhibitors such as clavulanic acid and tazobactam were developed to overcome ß-lactam antibiotic resistance. Hypersensitivity reactions to these drugs have not been studied in detail, and the antigenic determinants that activate T-cells have not been defined. The objectives of this study were to (i) characterize clavulanate- and tazobactam-responsive T-cells from hypersensitive patients, (ii) explore clavulanate and tazobactam T-cell crossreactivity, and (iii) define the antigenic determinants that contribute to T-cell reactivity. Antigen specificity, pathways of T-cell activation, and crossreactivity with clavulanate- and tazobactam-specific T-cell clones were assessed by proliferation and cytokine release assays. Antigenic determinants were analyzed by mass spectrometry-based proteomics methods. Clavulanate- and tazobactam-responsive CD4+ T-cell clones were stimulated to proliferate and secrete IFN-γ in an MHC class II-restricted and dose-dependent manner. T-cell activation with clavulanate- and tazobactam was dependent on antigen presenting cells because their fixation prevented the T-cell response. Strong crossreactivity was observed between clavulanate- and tazobactam-T-cells; however, neither drug activated ß-lactam antibiotic-responsive T-cells. Mass spectrometric analysis revealed that both compounds form multiple antigenic determinants with lysine residues on proteins, including an overlapping aldehyde and hydrated aldehyde adduct with mass additions of 70 and 88 Da, respectively. Collectively, these data show that although clavulanate and tazobactam are structurally distinct, the antigenic determinants formed by both drugs overlap, which explains the observed T-cell cross-reactivity.

Towards a more precise diagnosis of hypersensitivity to beta-lactams - an EAACI position paper.

A recent survey of the European Academy of Allergy and Clinical Immunology (EAACI) Drug Allergy Interest Group (DAIG) on how European allergy specialists deal with beta-lactam (BL) hypersensitivity demonstrated a significant heterogeneity in current practice, suggesting the need to review and update existing EAACI guidelines in order to make the diagnostic procedures as safe and accurate, but also as cost-effective, as possible. For this purpose, a bibliographic search on large studies regarding BL hypersensitivity diagnosis was performed by an EAACI task force, which reviewed and evaluated the literature data using the GRADE system for quality of evidence and strength of recommendation. The updated guidelines provide a risk stratification in BL hypersensitivity according to index reaction(s), as well as an algorithmic approach, based on cross-reactivity studies, in patients with a suspicion of BL hypersensitivity and an immediate need for antibiotic therapy, when referral to an allergist is not feasible. Furthermore, the update addresses availability and concentrations of skin test (ST) reagents, ST and drug provocation test (DPT) protocols, and diagnostic algorithms and administration of alternative BL in allergic subjects. Specifically, distinct diagnostic algorithms are suggested depending on risk stratification of the patient into high and low risk based on the morphology and chronology of the reaction, immediate (ie, occurring within 1-6 hours after the last administered dose) or nonimmediate (ie, occurring more than 1 hour after the initial drug administration), and the reaction severity. Regarding the allergy workup, the main novelty of this document is the fact that in some low-risk nonimmediate reactions ST are not mandatory, especially in children. For DPT, further studies are necessary to provide data supporting the standardization of protocols, especially of those regarding nonimmediate reactions, for which there is currently no consensus.

Cell Membrane Transporters Facilitate the Accumulation of Hepatocellular Flucloxacillin Protein Adducts: Implication in Flucloxacillin-Induced Liver Injury.

Flucloxacillin is a ß-lactam antibiotic associated with a high incidence of drug-induced liver reactions. Although expression of HLA-B*57:01 increases susceptibility, little is known about the pathological mechanisms involved in the induction of the clinical phenotype. Irreversible protein modification is suspected to drive the reaction through the presentation of flucloxacillin-modified peptides by the risk allele. In this study, the binding of flucloxacillin to proteins of liver-like cells was characterized. Flucloxacillin was shown to bind to proteins localized in bile canaliculi regions, coinciding with the site of clinical disease. The localization of flucloxacillin was mediated primarily by the membrane transporter multidrug resistance-associated protein 2. Modification of multiple proteins by flucloxacillin in bile canaliculi regions may provide a potential local source of neo-antigens for HLA presentation in the liver.

A EAACI drug allergy interest group survey on how European allergy specialists deal with ß-lactam allergy.

An accurate diagnosis of ß-lactam (BL) allergy can reduce patient morbidity and mortality. Our aim was to investigate the availability of BL reagents, their use and test procedures in different parts of Europe, as well as any differences in the diagnostic workups for evaluating subjects with BL hypersensitivity. A survey was emailed to all members of the EAACI Drug Allergy Interest Group (DAIG) between February and April 2016, and the questionnaire was meant to study the management of suspected BL hypersensitivity. The questionnaire was emailed to 82 DAIG centres and answered by 57. Amoxicillin alone or combined to clavulanic acid were the most commonly involved BL except in the Danish centre, where penicillin V was the most frequently suspected BL. All centres performed an allergy workup in subjects with histories of hypersensitivity to BL: 53 centres (93%) followed DAIG guidelines, two national guidelines and two local guidelines. However, there were deviations from DAIG recommendations concerning allergy tests, especially drug provocation tests. A significant heterogeneity exists in current practice not only among countries, but also among centres within the same country. This suggests the need to re-evaluate, update and standardize protocols on the management of patients with suspected BL allergy.

Definition of Haptens Derived from Sulfamethoxazole: In Vitro and in Vivo.

Hypersensitivity reactions occur frequently in patients upon treatment with sulfamethoxazole (SMX). These adverse effects have been attributed to nitroso sulfamethoxazole (SMX-NO), the reactive product formed from auto-oxidation of the metabolite SMX hydroxylamine. The ability of SMX-NO to prime naïve T-cells in vitro and also activate T-cells derived from hypersensitive patients has illustrated that T-cell activation may occur through the binding of SMX-NO to proteins or through the direct modification of MHC-bound peptides. SMX-NO has been shown to modify cysteine residues in glutathione, designer peptides, and proteins in vitro; however, the presence of these adducts have not yet been characterized in vivo. In this study a parallel in vitro and in vivo analysis of SMX-NO adducts was conducted using mass spectrometry. In addition to the known cysteine adducts, multiple SMX-NO-derived haptenic structures were found on lysine and tyrosine residues of human serum albumin (HSA) in vitro. On lysine residues two haptenic structures were identified including an arylazoalkane adduct and a Schiff base adduct. Interestingly, these adducts are labile to heat and susceptible to hydrolysis as shown by the presence of allysine. Furthermore, SMX-modified HSA adducts were detected in patients on long-term SMX therapy illustrated by the presence of an arylazoalkane adduct derived from a proposed carboxylic acid metabolite of SMX-NO. The presence of these adducts could provide an explanation for the immunogenicity of SMX and the strong responses to SMX-NO observed in T-cell culture assays. Also, the degradation of these adducts to allysine could lead to a stress-related innate immune response required for T-cell activation.

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.

ß-Lactam hypersensitivity involves expansion of circulating and skin-resident TH22 cells.

BACKGROUND: ß-Lactam hypersensitivity has been classified according to the phenotype and function of drug-specific T cells. However, new T-cell subsets have not been considered. OBJECTIVE: The objective of this study was to use piperacillin as a model of ß-lactam hypersensitivity to study the nature of the drug-specific T-cell response induced in the blood and skin of hypersensitive patients and healthy volunteers. METHODS: Drug-specific T cells were cloned from blood and inflamed skin, and cellular phenotype and function were explored. Naive T cells from healthy volunteers were primed to piperacillin, cloned, and subjected to the similar analyses. RESULTS: PBMC and T-cell clones (n = 570, 84% CD4+) from blood of piperacillin-hypersensitive patients proliferated and secreted TH1/TH2 cytokines alongside IL-22 after drug stimulation. IL-17A secretion was not detected. Drug-specific clones from inflamed skin (n = 96, 83% CD4+) secreted a similar profile of cytokines but displayed greater cytolytic activity, secreting perforin, granzyme B, and Fas ligand when activated. Blood- and skin-derived clones expressed high levels of skin-homing chemokine receptors and migrated in the presence of the ligands CCL17 and CCL27. Piperacillin-primed naive T cells from healthy volunteers also secreted IFN-γ, IL-13, IL-22, and cytolytic molecules. Aryl hydrocarbon receptor blockade prevented differentiation of the naive T cells into antigen-specific IL-22-secreting cells. CONCLUSION: Together, our results reveal that circulating and skin-resident, antigen-specific, IL-22-secreting T cells are detectable in patients with ß-lactam hypersensitivity. Furthermore, differentiation of naive T cells into antigen-specific TH22 cells is dependent on aryl hydrocarbon receptor signaling.

Up-Regulation of T-Cell Activation MicroRNAs in Drug-Specific CD4+ T-Cells from Hypersensitive Patients.

Dysregulation in the expression of microRNAs (miRNAs), single-stranded RNAs which regulate gene expression, has been associated with diseases such as Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), although their cellular origin has not been explored. Thus, the focus of this work was to study expression patterns of reported miRNAs involved in T-cell activation following drug-specific stimulation in peripheral blood mononuclear cells (PBMCs) and drug-specific CD4+ T-cell clones (TCC) from patients with different cutaneous manifestations of delayed-type drug hypersensitivity reactions. CD4+ T-cells from hypersensitive patients were stimulated to proliferate, secreted cytokines (IFN-γ and IL-22), cytolytic molecules (Granzyme B) and up-regulate miRNAs 24 to 48 h after drug exposure. Carbamazepine-specific CD4+ T-cells that proliferated to the greatest extent and secreted the highest levels of IFN-γ showed an up-regulation of miR-18a and miR-155. In contrast, piperacillin-specific CD4+ T-cells displaying high expression of miR-9 and miR-21 showed an association with the extent of proliferation, but not IFN-γ secretion. MiR-155 up-regulation was detected in PBMCs from all hypersensitive patients 24 h after drug treatment, while miR-18a and miR-21 expression was up-regulated after 48 h. These findings demonstrate that miRNAs are expressed during drug-specific CD4+ T-cell activation and shows a new regulation path for drug hypersensitivity reactions.

Invasive Pulmonary Fungal Infections in Cystic Fibrosis.

Invasive pulmonary mycosis is after allergic bronchopulmonary aspergillosis (ABPA) a frequent and severe complication of CF lung disease. Among CF caregivers, there is an insecurity when and how to treat infections of the lung parenchyma caused by different fungi in patients with CF. This case series provides a multicenter experience on diagnostic, manifestation, and treatment of non-ABPA cases of pulmonary. Non-ABPA cases of pulmonary mycoses in patients with CF have been collected from the CF Centers in Berlin, Essen, Worms, Frankfurt (Germany), Leeds (UK), and Barcelona (Spain). Non-ABPA was defined as total serum IgE level <500 kU/L. Scedosporium and Lomentospora species seem to be more virulent in patients with CF and have been successfully treated with triple antifungal drug regimens in several cases. Rare fungi including yeasts can have pathogenic potential in CF. In this series, antibiotic treatment failure was the main indicator for the initiation of antifungal treatment. For an early and effective treatment of pulmonary mycoses in CF, the identification of biomarkers and of risk factors beyond antibiotic treatment failure is crucial and urgently needed. Furthermore, treatment efficacy studies are necessary for the different causative agents of these infections.

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.

Amoxicillin and Clavulanate Form Chemically and Immunologically Distinct Multiple Haptenic Structures in Patients.

Amoxicillin-clavulanate (AC) is one of the most common causes of drug induced liver injury (DILI). The association between AC-DILI and HLA alleles and the detection of drug-specific T cells in patients with AC-DILI indicate that the adaptive immune system is involved in the disease pathogenesis. In this study, mass spectrometric methods were employed to characterize the antigen formed by AC in exposed patients and the antigenic determinants that stimulate T cells. Amoxicillin formed penicilloyl adducts with lysine residues on human serum albumin (HSA) in vitro, with K190 and K199 being the most reactive sites. Amoxicillin-modified K190 and K199 have also been detected in all patients, and more extensive modification was observed in patients exposed to higher doses of amoxicillin. In contrast, the binding of clavulanic acid to HSA was more complicated. Multiple adducts were identified at high concentrations in vitro, including those formed by direct binding of clavulanic acid to lysine residues, novel pyrazine adducts derived from binding to the degradation products of clavulanic acid, and a cross-linking adduct. Stable adducts derived from formylacetic acid were detected in all patients exposed to the drug. Importantly, analysis of hapten-protein adducts formed in the cell culture medium revealed that the highly drug-specific T-cell responses were likely driven by the markedly different haptenic structures formed by these two drugs. In this study, the unique haptenic structures on albumin in patients formed by amoxicillin and clavulanic acid have been characterized and shown to function as chemically distinct antigens which can stimulate separate, specific T-cell clones.

Detection of Drug-Responsive T-Lymphocytes in a Case of Fatal Antituberculosis Drug-Related Liver Injury.

Antituberculosis (TB) drug exposure is associated with a mild elevation of liver enzymes that occasionally develops into severe liver injury. Herein, we identify ethambutol- and rifampicin-specific CD4+ and CD8+ T-cell clones in a patient with fatal anti TB-related liver injury. The clones were activated to proliferate and secrete IFN-γ, Il-13, and granzyme B following drug treatment. Drug-responsive T-cells may contribute to the pathogenesis of antituberculosis-related liver failure.

Negative regulation by PD-L1 during drug-specific priming of IL-22-secreting T cells and the influence of PD-1 on effector T cell function.

Activation of PD-1 on T cells is thought to inhibit Ag-specific T cell priming and regulate T cell differentiation. Thus, we sought to measure the drug-specific activation of naive T cells after perturbation of PD-L1/2/PD-1 binding and investigate whether PD-1 signaling influences the differentiation of T cells. Priming of naive CD4(+) and CD8(+) T cells against drug Ags was found to be more effective when PD-L1 signaling was blocked. Upon restimulation, T cells proliferated more vigorously and secreted increased levels of IFN-γ, IL-13, and IL-22 but not IL-17. Naive T cells expressed low levels of PD-1; however, a transient increase in PD-1 expression was observed during drug-specific T cell priming. Next, drug-specific responses from in vitro primed T cell clones and clones from hypersensitive patients were measured and correlated with PD-1 expression. All clones were found to secrete IFN-γ, IL-5, and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL-22-secreting clones expressed the skin-homing receptors CCR4, CCR10, and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the Ag-specific proliferative response or the secretion of cytokines/cytolytic molecules. This study shows that PD-L1/PD-1 binding negatively regulates the priming of drug-specific T cells. ELISPOT analysis uncovered an Ag-specific FasL/IL-22-secreting T cell subset with skin-homing properties.

Auto-oxidation of Isoniazid Leads to Isonicotinic-Lysine Adducts on Human Serum Albumin.

Isoniazid (INH), a widely used antituberculosis drug, has been associated with serious drug-induced liver injury (DILI). INH-modified proteins have been proposed to play important roles in INH DILI; however, it remains to be determined whether INH or reactive metabolites bind irreversibly to proteins. In this study, mass spectrometry was used to define protein modifications by INH in vitro and in patients taking INH therapy. When INH was incubated with N-acetyl lysine (NAL), the same isonicotinic-NAL (IN-NAL) adducts were detected irrespective of the presence or absence of any oxidative enzymes, indicating auto-oxidation may have been involved. In addition, we found that INH could also bind to human serum albumin (HSA) via an auto-oxidation pathway, forming isonicotinic amide adducts with lysine residues in HSA. Similar adducts were detected in plasma samples isolated from patients taking INH therapy. Our results show that INH forms protein adducts in the absence of metabolism.

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

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

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.

Mass spectrometric characterization of circulating and functional antigens derived from piperacillin in patients with cystic fibrosis.

A mechanistic understanding of the relationship between the chemistry of drug Ag formation and immune function is lacking. Thus, mass spectrometric methods were employed to detect and fully characterize circulating Ags derived from piperacillin in patients undergoing therapy and the nature of the drug-derived epitopes on protein that can function as an Ag to stimulate T cells. Albumin modification with piperacillin in vitro resulted in the formation of two distinct haptens, one formed directly from piperacillin and a second in which the dioxopiperazine ring had undergone hydrolysis. Modification was time and concentration dependent, with selective modification of Lys(541) observed at low concentrations, whereas at higher concentrations, up to 13 out of 59 lysine residues were modified, four of which (Lys(190), Lys(195), Lys(432), and Lys(541)) 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 Lys(190), Lys(432), and Lys(541) 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.

Nebuliser systems for drug delivery in cystic fibrosis.

BACKGROUND: Nebuliser systems are used to deliver medications to control the symptoms and the progression of lung disease in people with cystic fibrosis. Many types of nebuliser systems are available for use with various medications; however, there has been no previous systematic review which has evaluated these systems. OBJECTIVES: To evaluate effectiveness, safety, burden of treatment and adherence to nebulised therapy using different nebuliser systems for people with cystic fibrosis. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register comprising references identified from comprehensive electronic database searches, handsearching of relevant journals and abstract books of conference proceedings. We searched the reference lists of each study for additional publications and approached the manufacturers of both nebuliser systems and nebulised medications for published and unpublished data. Date of the most recent search: 15 Oct 2012. SELECTION CRITERIA: Randomised controlled trials or quasi-randomised controlled trials comparing nebuliser systems including conventional nebulisers, vibrating mesh technology systems, adaptive aerosol delivery systems and ultrasonic nebuliser systems. DATA COLLECTION AND ANALYSIS: Two authors independently assessed studies for inclusion. They also independently extracted data and assessed the risk of bias. A third author assessed studies where agreement could not be reached. MAIN RESULTS: The search identified 40 studies with 20 of these (1936 participants) included in the review. These studies compared the delivery of tobramycin, colistin, dornase alfa, hypertonic sodium chloride and other solutions through the different nebuliser systems. This review demonstrates variability in the delivery of medication depending on the nebuliser system used. Conventional nebuliser systems providing higher flows, higher respirable fractions and smaller particles decrease treatment time, increase deposition and may be preferred by people with CF, as compared to conventional nebuliser systems providing lower flows, lower respirable fractions and larger particles. Nebulisers using adaptive aerosol delivery or vibrating mesh technology reduce treatment time to a far greater extent. Deposition (as a percentage of priming dose) is greater than conventional with adaptive aerosol delivery. Vibrating mesh technology systems may give greater deposition than conventional when measuring sputum levels, but lower deposition when measuring serum levels or using gamma scintigraphy. The available data indicate that these newer systems are safe when used with an appropriate priming dose, which may be different to the priming dose used for conventional systems. There is an indication that adherence is maintained or improved with systems which use these newer technologies, but also that some nebuliser systems using vibrating mesh technology may be subject to increased failures. AUTHORS' CONCLUSIONS: Clinicians should be aware of the variability in the performance of different nebuliser systems. Technologies such as adaptive aerosol delivery and vibrating mesh technology have advantages over conventional systems in terms of treatment time, deposition as a percentage of priming dose, patient preference and adherence. There is a need for long-term randomised controlled trials of these technologies to determine patient-focused outcomes (such as quality of life and burden of care), safe and effective dosing levels of medications and clinical outcomes (such as hospitalisations and need for antibiotics) and an economic evaluation of their use.

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.