Diminished cellular immunity and cytokine secretion could also affect the level of protection from infections leading to frequent AOMs. vaccination. We used fluorescently conjugated vaccine antigens to label antigen receptors on the surface of memory B cells and examined the frequency of antigen-specific CD19+ CD27+ memory B cells in the peripheral blood. sOP children showed a significantly lower percentage of antigen-specific CD19+ CD27+ memory B cells than NOP children. We also found a linear correlation between the frequencies of memory B cells and circulating IgG titres for DT, TT and PT proteins. To our knowledge, this is the first study to show significant differences in memory B cell responses to DTaP vaccine antigens and their correlation with the circulating antibodies in young children with recurrent AOM. Keywords: acute otitis media, diphtheria toxoid, memory B cells, pertussis toxoid, tetanus Tanshinone IIA sulfonic sodium toxoid Introduction Acute otitis media (AOM) is one of the most common infectious diseases affecting 6C30-month-old children, and can lead to hearing impairment and delayed speech development 1,2. Approximately 80% of children experience at least one episode of AOM and approximately Nrp2 30% experience three Tanshinone IIA sulfonic sodium to four episodes diagnosed clinically within a 6C12-month time-span during their first 3 years of life, termed otitis-prone (OP). We have recently defined a subset of OP children, representing 5% of the total population of children, who meet the above definition and whose diagnosis was based on tympanocentesis confirmation of otopathogens in the middle ear fluid; we termed these children stringently defined otitis-prone (sOP) 3C5. These sOP children have recurrent ear infections associated with elevated bacterial burden, and higher proinflammatory cytokine levels within the middle ear space despite tympanocentesis and individualized antibiotic treatment 6,7. Predominant bacterial otopathogens for otitis media include (((with AOM 8C10. Circulating antibodies in the serum that transudates to mucosal surfaces and/or mucosal immunoglobulin (IgA) antibodies play a role in blocking adherence of these pathogens to mucosal epithelial cells and/or interfere with microbial invasion of the bloodstream 11,12. Diminished cellular immunity and cytokine secretion could also affect the level of protection from infections leading to frequent AOMs. We previously showed that sOP children generate low humoral and cellular immune responses to otopathogens following nasal colonization and AOM caused by and resembling a neonatal immune profile 6. The Center for Disease Control (CDC) immunization schedule for children aged 0C18 years recommends primary doses of DTaP vaccine at ages 2, 4 and 6 months, followed by a booster at 15C18 months, and a fifth dose at age 4C6 years. Despite these multiple vaccine doses, pertussis remains poorly controlled, resulting in morbidity and mortality in vaccinated and non-vaccinated children. Recent reports of Tanshinone IIA sulfonic sodium pertussis outbreaks show that this disease remains dangerous in the United States and other countries 13,14. In our recent studies, sOP children failed to generate protective antibody responses to many common vaccine antigens, including DTaP components 6,10. In this study, to delineate a more precise immunological mechanism Tanshinone IIA sulfonic sodium for the lower antibody levels in sOP children, for the first time to our knowledge we describe an evaluation of the memory B cell (CD19+ CD27+) responses to DTaP vaccine antigens in age-matched sOP and non-otitis-prone (NOP) children and correlated the observations with serum IgG levels. Material and methods Subjects Subjects in this study are from our 7-year, prospective, longitudinal study funded by the National Institutes of Health, National Institute on Deafness and Other Communication Disorders (NIDCD Tanshinone IIA sulfonic sodium R0108671) to study immunological dysfunction in OP children. For the studies reported here, all 35 children were aged 9C18 months (mean age 105 months) from a middle-class, suburban sociodemographic population in Rochester, New York, who had received three doses of DTaP vaccine (Sanofi Pasteur, Swiftwater, PA, USA) at 2, 4 and 6 months of age. A written informed consent was obtained from parents of the children in accordance with.