Involvement of autologous myeloid dendritic cells in the evaluation of immediate hypersensitivity reactions to betalactams.

BACKGROUND: Amoxicillin (AX) and clavulanic acid (CLV) are the betalactam antibiotics (BLs) most used to treat bacterial infections, although they can trigger immediate hypersensitivity reactions (IDHRs). The maturation analysis of monocyte-derived dendritic cells (moDCs) and their capacity to induce proliferative response of lymphocytes are useful to test the sensitisation to a drug, although without optimal sensitivity. Nevertheless, this can be improved using directly isolated DCs such as myeloid DCs (mDCs). METHODS: mDCs and moDCs were obtained from 28 allergic patients (AP), 14 to AX, 14 to CLV and from 10 healthy controls (HC). The expression of CCR7, CD40, CD80, CD83, and CD86 was analysed after stimulation with both BLs. We measured the capacity of these pre-primed DCs to induce drug-specific activation of different lymphocyte subpopulations, CD3+, CD4+, CD8+, CD4+Th1, and CD4+Th2, by flow cytometry. RESULTS: Higher expression of CCR7, CD40, CD80, CD83, and CD86 was observed on mDCs compared to moDCs from AP after stimulating with the culprit BL. Similarly, mDCs induced higher proliferative response, mainly of CD4+Th2 cells, compared to moDCs, reaching up to 67% of positive results with AX, whereas of only 25% with CLV. CONCLUSIONS: mDCs from selective AP efficiently recognise the culprit drug which trigger the IDHR. mDCs also trigger proliferation of lymphocytes, mainly those with a Th2 cytokine pattern, although these responses depend on the nature of the drug, mimicking the patient's reaction.

Diagnosis of immediate reactions to amoxicillin: Comparison of basophil activation markers CD63 and CD203c in a prospective study.

BACKGROUND: Amoxicillin (AX) combined or not with clavulanic acid (CLV) is frequently involved in IgE-mediated reactions. Drug provocation test (DPT) is considered as the gold standard for diagnosis, although contraindicated in high-risk patients. Basophil activation test (BAT) can help diagnose immediate reactions to beta-lactams, although controversy exists regarding the best activation marker. We have performed a real-life study in a prospective cohort to analyze the real value of BAT as diagnostic tool and the best activation marker, CD63 and CD203c, for the evaluation of immediate reactions to these drugs. METHODS: We prospectively evaluated patients with a clinical suspicion of immediate reactions after AX or AX-CLV administration during a 6-year period. The allergological work-up was done following the EAACI recommendations. BAT was performed in all patients using CD63 and CD203c as activation markers. RESULTS: In AX-allergic patients, both activation markers, CD63 and CD203c, showed similar SE values (48.6% and 46.7%, respectively); however, specificity was of 81.1% and 94.6%, respectively, with CD203c showing good positive predictive value and like-hood ratio. In CLV-allergic patients, CD203c showed higher SE (50%) than CD63 (42.9%), maintaining the same value of SP (80%). Combining the results of both markers can slightly increase the sensitivity (51.4% for AX and 54.8% for CLV), although decreasing the specificity (79.7% and 73%, respectively). Interestingly, all patients with an anaphylactic shock showed a positive BAT to CLV using CD203c. CONCLUSIONS: BAT using CD203c showed a good confirmatory power, especially for AX allergy. Placing BAT as a first step in the diagnostic procedure can help reduce the need of performing a complete allergological work-up in 46.6% of patients, diminishing the risk of reinducing allergic reactions.

Detection of Serum-Specific IgE by Fluoro-Enzyme Immunoassay for Diagnosing Type I Hypersensitivity Reactions to Penicillins.

Diagnosis of type I hypersensitivity reactions (IgE-mediated reactions) to penicillins is based on clinical history, skin tests (STs), and drug provocation tests (DPTs). Among in vitro complementary tests, the fluoro-enzyme immunoassay (FEIA) ImmunoCAP® (Thermo-Fisher, Waltham, MA, USA) is the most widely used commercial method for detecting drug-specific IgE (sIgE). In this study, we aimed to analyze the utility of ImmunoCAP® for detecting sIgE to penicillin G (PG) and amoxicillin (AX) in patients with confirmed penicillin allergy. The study includes 139 and 250 patients evaluated in Spain and Italy, respectively. All had experienced type I hypersensitivity reactions to penicillins confirmed by positive STs. Additionally, selective or cross-reactive reactions were confirmed by DPTs in a subgroup of patients for further analysis. Positive ImmunoCAP® results were 39.6% for PG and/or AX in Spanish subjects and 52.4% in Italian subjects. When only PG or AX sIgE where analyzed, the percentages were 15.1% and 30.4%, respectively, in Spanish patients; and 38.9% and 46% in Italian ones. The analysis of positive STs showed a statistically significant higher percentage of positive STs to PG determinants in Italian patients. False-positive results to PG (16%) were detected in selective AX patients with confirmed PG tolerance. Low and variable sensitivity values observed in a well-defined population with confirmed allergy diagnosis, as well as false-positive results to PG, suggest that ImmunoCAP® is a diagnostic tool with relevant limitations in the evaluation of subjects with type I hypersensitivity reactions to penicillins.

Basophil Activation Test for Allergy Diagnosis.

The basophil activation test (BAT) is a complementary in vitro diagnostic test that can be used in addition to clinical history, skin test (ST), and specific IgE (sIgE) determination in the evaluation of IgE-mediated allergic reactions to food, insect venom, drugs, as well as some forms of chronic urticaria. However, the role of this technique in the diagnostic algorithms is highly variable and not well determined. BAT is based on the determination of basophil response to allergen/drug cross-linking IgE activation through the measurement of activation markers (such as CD63, CD203c) by flow cytometry. This test can be a useful and complementary tool to avoid controlled challenge tests to confirm allergy diagnosis, especially in subjects experiencing severe life-threatening reactions. In general, the performance of BAT should be considered if i) the allergen/drug produces false positive results in ST; ii) there is no allergen/drug source to use for ST or sIgE determination; iii) there is discordance between patient history and ST or sIgE determination; iv) symptoms suggest that ST may result in systemic response; v) before considering a CCT to confirm the culprit allergen/drug. The main limitations of the test are related to non-optimal sensitivity, especially in drug allergy, the need to perform the test no longer than 24 h after sample extraction, and the lack of standardization between laboratories in terms of procedures, concentrations, and cell markers.

Amoxicillin Inactivation by Thiol-Catalyzed Cyclization Reduces Protein Haptenation and Antibacterial Potency.

Serum and cellular proteins are targets for the formation of adducts with the ß-lactam antibiotic amoxicillin. This process could be important for the development of adverse, and in particular, allergic reactions to this antibiotic. In studies exploring protein haptenation by amoxicillin, we observed that reducing agents influenced the extent of amoxicillin-protein adducts formation. Consequently, we show that several thiol-containing compounds, including dithiothreitol, N-acetyl-L-cysteine, and glutathione, perform a nucleophilic attack on the amoxicillin molecule that is followed by an internal rearrangement leading to amoxicillin diketopiperazine, a known amoxicillin metabolite with residual activity. Increased diketopiperazine conversion is also observed with human serum albumin but not with L-cysteine, which mainly forms the amoxicilloyl amide. The effect of thiols is catalytic and can render complete amoxicillin conversion. Interestingly, this process is dependent on the presence of an amino group in the antibiotic lateral chain, as in amoxicillin and ampicillin. Furthermore, it does not occur for other ß-lactam antibiotics, including cefaclor or benzylpenicillin. Biological consequences of thiol-mediated amoxicillin transformation are exemplified by a reduced bacteriostatic action and a lower capacity of thiol-treated amoxicillin to form protein adducts. Finally, modulation of the intracellular redox status through inhibition of glutathione synthesis influenced the extent of amoxicillin adduct formation with cellular proteins. These results open novel perspectives for the understanding of amoxicillin metabolism and actions, including the formation of adducts involved in allergic reactions.

Early Biomarkers for Severe Drug Hypersensitivity Reactions.

Drug hypersensitivity reactions (DHRs) are typically classified into immediate and delayed reactions based on the time interval between drug exposure and onset of symptoms. Clinical manifestations range from mild to severe and life-threatening reactions. The most severe clinical entities are anaphylaxis and anaphylactic shock for immediate reactions, and severe cutaneous adverse reactions such as Steven Johnson Syndrome and Toxic Epidermal Necrolysis for delayed reactions. The diagnosis is complex and challenging, as drug provocation tests and even skin tests can be very risky procedures, which makes them not recommended. Therefore, it is necessary to search for useful early biomarkers to manage the diagnosis of these reactions. These biomarkers could be useful to determine the clinical entity, but not to identify the culprit drug. Some of the currently available biomarkers are few genetic associations of drug allergy with polymorphisms of human leukocyte antigen (HLA), the detection of inflammatory and lipid mediators in serum, or the detection of cytokines, chemokines, and cytotoxic markers in skin biopsies. In this literature review, it has been summarize the immunological mechanisms involved in severe reactions, both immediate and delayed, and different early biomarkers: those currently used for the diagnosis of these reactions as well as possible early biomarkers that could be useful with further studies to standardize their clinical use.

The Basophil Activation Test Can Be of Value for Diagnosing Immediate Allergic Reactions to Omeprazole.

BACKGROUND: Proton pump inhibitors (PPIs) are commonly used to treat gastrointestinal diseases. Incidence of hypersensitivity reactions to PPIs has risen, likely because of increased consumption. Their diagnosis is difficult, with skin tests (STs) presenting low sensitivity, making it necessary to perform drug provocation tests (DPTs). The value of in vitro tests for the diagnosis of immediate reaction to PPI is unclear. OBJECTIVE: To analyze the diagnostic value of the basophil activation test (BAT) in a group of patients diagnosed with immediate allergy to omeprazole. METHODS: The study included 42 patients with confirmed immediate allergic reactions to omeprazole confirmed by positive ST results or DPT results and 22 age- and sex-matched subjects tolerant to PPIs. BAT was performed with omeprazole, pantoprazole, and lansoprazole using CD63 and CD203c as activation markers. RESULTS: ST sensitivity was 66.7% with a specificity of 100%. BAT using CD63 with a stimulation index of more than 2 as positive revealed a sensitivity of 73.8%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 66.7%. BAT was positive in 57.1% of patients with negative ST result, and thus by combining ST and BAT we can correctly diagnose 85.7% of patients with immediate allergy to omeprazole. CONCLUSION: BAT represents a complementary tool for inclusion in the allergological workup for patients allergic to omeprazole. When combined with ST, it can be of value to guide the clinician as to whether to perform a DPT.

Patients Taking Amoxicillin-Clavulanic Can Become Simultaneously Sensitized to Both Drugs.

BACKGROUND: Patients can react to amoxicillin (AX) and clavulanic acid (CLV) taken in combination because of selective reactions to either drug. However, scant information exists concerning patients who react simultaneously to both compounds. OBJECTIVE: To analyze the mechanisms involved in 4 patients who developed allergic reactions to AX-CLV administration (3 with immediate IgE-mediated reaction and 1 with nonimmediate T-cell-mediated reaction) and who responded specifically to both AX and CLV. METHODS: Skin tests with benzylpenicillin (BP), AX, and CLV were done and, if necessary, drug provocation tests with BP/penicillin V, AX, and AX-CLV were carried out. In immediate reactors, serum specific IgE to benzylpenicilloyl and amoxicilloyl was determined by using the CAP-FEIA system (Pharmacia Diagnostics, Uppsala, Sweden), and basophil activation test to BP, AX, CLV, and AX-CLV was done. In nonimmediate reactors, immunohistochemistry of skin biopsy and analysis of dendritic cell maturation and T-cell-specific response to BP, AX, CLV, and AX-CLV at both acute and resolution phases of the reaction were conducted. RESULTS: All patients with immediate reactions (N = 3) had good tolerance to BP and penicillin V. Two cases also had specific IgE to AX and all had a basophil activation test positive to AX, CLV, and AX-CLV. The patient with a nonimmediate reaction exhibited dendritic cell and T-lymphocyte responses specific to both AX and CLV. Finally, the analysis of the cells infiltrating the skin and peripheral blood during the acute phase indicated a TH1 pattern response. CONCLUSIONS: Our study provides evidence that reactions to both AX and CLV can appear in the same patient.

Understanding the mechanisms in accelerated drug reactions.

PURPOSE OF REVIEW: The purpose is to understand the underlying mechanisms of accelerated allergic reactions to drugs, defined here as reactions occurring between 1 and 24 h after drug intake. RECENT FINDINGS: Recent publications have shown that accelerated reactions are T cell-mediated, although an IgE mechanism cannot be ruled out in some cases. SUMMARY: Classification of allergic reactions to drugs is complex. Based on the time interval between drug administration and appearance of the clinical reaction, as well as the type of clinical symptoms, they can be classified as: immediate, typically appearing from less than 1 to 6 h after the last drug administration and nonimmediate, occurring at any time from 1 h after drug administration. Therefore, overlap exists in what the Levine classification defined as accelerated reactions, where clinical symptoms are mainly urticaria and less often exanthema and serum sickness-like reactions. The immunological mechanisms involved suggest that they are T cell-mediated reactions with a Th1 pattern, comprising increased production of IFNγ, TNFα, the chemokine CXCL9 and its corresponding receptor CXCR3. In most cases an IgE-mediated response is ruled out because of negative immediate skin test results, no detection of serum-specific IgE antibodies or tryptase, and no skin-secreted tryptase. However, an IgE-mediated response can be demonstrated in exceptional situations. Finally, serum sickness-like reactions have been reported as an immune complex-mediated accelerated reaction. However, the exact mechanism has not been confirmed.

Mass spectrometric strategies for the identification and characterization of human serum albumin covalently adducted by amoxicillin: ex vivo studies.

This study addresses the detection and characterization of the modification of human serum albumin (HSA) by amoxicillin (AX) in ex vivo samples from healthy subjects under oral amoxicillin administration (acute intake of 1 g every 8 h for 48 h). To reach this goal, we used an analytical strategy based on targeted and untargeted mass spectrometric approaches. Plasma samples withdrawn before AX oral intake represented the negative control samples to test the method selectivity, whereas HSA incubated in vitro with AX was the positive control. Different MS strategies were developed, particularly (1) multiple reaction monitoring (MRM) and precursor ion scan (PIS) using a HPLC system coupled to a triple quadrupole MS analyzer and (2) a dedicated data-dependent scan and a customized targeted MS/MS analysis carried out using a nano-LC system coupled to a high-resolution MS system (LTQ Orbitrap XL). Lys 190 was identified as the only modification site of HSA in the ex vivo samples. The AX adduct was identified and fully characterized by complementary targeted approaches based on triple quadrupole (MRM mode) and orbitrap (SIC mode) mass analyzers. The SIC mode also permitted the relative amount of AX-adducted HSA to be measured, ranging from 1 to 2% (6-12 µM) at 24 and 48 h after the oral intake. No adduct in any ex vivo sample was identified by the untargeted methods (PIS and data-dependent scan mode analysis). The results on one hand indicate that MS, in particular high-resolution MS, analysis represents a suitable analytical tool for the identification/characterization of covalently modified proteins/peptides; on the other hand, they give deeper insight into AX-induced protein haptenation, which is required to better understand the mechanisms involved in AX-elicited allergic reactions.

Study of protein haptenation by amoxicillin through the use of a biotinylated antibiotic.

Allergic reactions towards ß-lactam antibiotics pose an important clinical problem. The ability of small molecules, such as a ß-lactams, to bind covalently to proteins, in a process known as haptenation, is considered necessary for induction of a specific immunological response. Identification of the proteins modified by ß-lactams and elucidation of the relevance of this process in allergic reactions requires sensitive tools. Here we describe the preparation and characterization of a biotinylated amoxicillin analog (AX-B) as a tool for the study of protein haptenation by amoxicillin (AX). AX-B, obtained by the inclusion of a biotin moiety at the lateral chain of AX, showed a chemical reactivity identical to AX. Covalent modification of proteins by AX-B was reduced by excess AX and vice versa, suggesting competition for binding to the same targets. From an immunological point of view, AX and AX-B behaved similarly in RAST inhibition studies with sera of patients with non-selective allergy towards ß-lactams, whereas, as expected, competition by AX-B was poorer with sera of AX-selective patients, which recognize AX lateral chain. Use of AX-B followed by biotin detection allowed the observation of human serum albumin (HSA) modification by concentrations 100-fold lower that when using AX followed by immunological detection. Incubation of human serum with AX-B led to the haptenation of all of the previously identified major AX targets. In addition, some new targets could be detected. Interestingly, AX-B allowed the detection of intracellular protein adducts, which showed a cell type-specific pattern. This opens the possibility of following the formation and fate of AX-B adducts in cells. Thus, AX-B may constitute a valuable tool for the identification of AX targets with high sensitivity as well as for the elucidation of the mechanisms involved in allergy towards ß-lactams.