glycerol was used as a negative (non-stimulated) control. On the
basis of preliminary time-course studies, 24 h of stimulation cor-
responded to the best time point for the responses of cytokines to
bacteria stimulated-PBMCs. After 24 h of stimulation at 37 C in an
atmosphere of air with 5% CO2, the culture supernatants were
collected, clarified by centrifugation and stored at À20 C until
cytokine analysis. Neither medium acidification nor bacterial pro-
liferation was observed. Cytokines were measured by ELISA using
BD Pharmingen antibody pairs (BD Biosciences, San Jose, Ca, USA)
for IL-10 and IL-12p70, according to the manufacturer's recom-
mendations. Data were expressed as means of all donors ± SEM, in
either pg.LÀ1 for cytokines or relative values for the IL10/IL12 ratio.