Safety and Effect of L. reuteri on Biomarkers of Inflammation in Healthy Infants

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DESCRIPTION (provided by applicant): Colic is a severe problem characterized by fussing, crying and screaming for more than 3 hours daily and affecting ~15% of otherwise normal infants in the first 3 months of life. We recently published our trial defining key clinical and microbiological characteristics of infants with colic. Compared to normal infants, those with colic demonstrated a four-fold increase in crying time, a doubling in the level of fecal calprotectin (a measure of gut inflammation), and changes in the fecal microbiota characterized by reduced bacterial diversity and an increase in the prevalence of Klebsiella. These results suggested a novel hypothesis: that the infants are crying because their bowels are inflamed, possibly as a result from an abnormal microbiota. Our investigation will focus on a population of healthy infants with colic to show safety of a probiotic that has been shown to reduce crying time in infants with colic. Our basic science laboratory trials showed that feeding of L. reuteri reduced gut inflammation and reduced mortality by > 50% in a neonatal rat model of necrotizing enterocolitis. We are currently close to completion of a double-blind placebo-controlled Phase 1 safety trial of L. reuteri in adult volunteers (U01AT003519). In this study, we are measuring how the probiotic influences the effector cells that modulate inflammation. We are quantifying circulating T cells, B cells, mononuclear cells, T cell subsets (by flow cytometry). We are measuring cytokine production by peripheral blood mononuclear cells, fecal calprotectin, and fecal microbiota. This trial includes measurement of regulatory T-cells (Tregs), the cells proposed to mediate the immunomodulatory effects of probiotics. Thus, our Primary Aim is to demonstrate (in a two-year trial) if Lactobacillus reuteri is safe in healthy newborns with colic (n=45), at 3 different doses: 5 x 106, 5 x 107, and 5 x 108 c.f.u. given by mouth once daily for si weeks. Our Secondary Aim is to compare outcomes to determine the optimal dose for a future randomized controlled trial (RCT). Dose-effect relationships will be determined for the following measures: (a) crying + fussing time; (b) fecal calprotectin; (c) serum cytokines; (d) toll-like receptor expression on peripheral blood mononuclear cells; (e) circulating regulatory T-cells; and (f) fecal levels of colic-related organisms (Klebsiella and E. coli, determined by qPCR) and diversity of fecal microbiota (assessed by 454 pyrosequencing).