Cytokine expression profile of sensitized human T lymphocytes following in vitro stimulation with amoxicillin.

Since the withdrawal of penicillin determinants from the market, in addition to the hazard of re-exposing the patient to the drug, skin testing for the diagnosis of penicillin allergy has become less accurate and less standardized. The assay currently used, the lymphocyte transformation test (LTT), lacks sufficient sensitivity, and requires the use of radioactive material. The objective of this study was to establish an accessible and reliable method for the safe diagnosis of penicillin allergy. Peripheral blood mononuclear cells (PBMC) were isolated from 18 patients who were allergic to penicillin and 12 control subjects using the Ficoll-Hypaque method. The isolated, sensitized cells were stimulated in vitro with amoxicillin (1 mg/mL). Stimulation with phytohemagglutinin A (PHA) was used as the positive control. Transcriptional expression of specific cytokines (IL-2, -4, -5 and -13, TGF-beta, TNF-alpha and IFN-gamma) was assessed by RT-PCR. IFN-gamma expression was also evaluated by ELISPOT. Secreted levels of IL-2, -5 and IFN-gamma were measured by ELISA. All of these assays were performed two or five days, post-stimulation. This study of the in vitro diagnosis of penicillin allergy by the measurement of cytokine concentration in the supernatants of sensitized lymphocytes cultures involved the largest number of patients to-date. The Delta values (difference in cytokine concentration in the supernatants before and after stimulation) were compared between cases and controls using different statistical tests (Student's t test and the Mann-Whitney rank test). Of the various tests performed in this study, measurement of secreted cytokines using ELISA was the most sensitive and specific (80% and 100% respectively). In vitro stimulation of human lymphocytes sensitized to amoxicillin is a safe and useful test for the diagnosis of penicillin allergy if the ELISA is used to measure cytokine expression. The advantages are that it can be performed by many laboratories since kits to determine cytokines are widely available, and it can be done without the need for particularly specialized equipment.