Lactobacillus rhamnosus HN001 decreases the severity of necrotizing enterocolitis in neonatal mice and preterm piglets: evidence in mice for a role of TLR9.

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and develops partly from an exaggerated intestinal epithelial immune response to indigenous microbes. There has been interest in administering probiotic bacteria to reduce NEC severity, yet concerns exist regarding infection risk. Mechanisms of probiotic activity in NEC are unknown although activation of the microbial DNA receptor Toll-like receptor-9 (TLR9) has been postulated. We now hypothesize that the Gram-positive bacterium Lactobacillus rhamnosus HN001 can attenuate NEC in small and large animal models, that its microbial DNA is sufficient for its protective effects, and that protection requires activation of the Toll-like receptor 9 (TLR9). We now show that oral administration of live or UV-inactivated Lactobacillus rhamnosus HN001 attenuates NEC severity in newborn mice and premature piglets, as manifest by reduced histology score, attenuation of mucosal cytokine response, and improved gross morphology. TLR9 was required for Lactobacillus rhamnosus-mediated protection against NEC in mice, as the selective decrease of TLR9 from the intestinal epithelium reversed its protective effects. Strikingly, DNA of Lactobacillus rhamnosus HN001 reduced the extent of proinflammatory signaling in cultured enterocytes and in samples of resected human ileum ex vivo, suggesting the therapeutic potential of this probiotic in clinical NEC. Taken together, these findings illustrate that Lactobacillus rhamnosus HN001 is an effective probiotic for NEC via activation of the innate immune receptor TLR9 and that Lactobacillus rhamnosus DNA is sufficient for its protective effects, potentially reducing concerns regarding the infectious risk of this novel therapeutic approach.

Novel nutritional immune formula maintains host defense mechanisms.

Military combat and training stress induce immune changes that increase the risk of infection and ultimately influence soldiers' performance and readiness. Strenuous military training/assessment provides a uniform stress and the opportunity to evaluate nutritional strategies to minimize stress-induced immune changes that predispose soldiers to infection. Immunological changes and effects of a novel nutritional immune formula (NNIF) were examined prospectively in a double-blind, controlled study of 200 soldiers attending Special Forces Assessment and Selection School. Immune function was measured by skin delayed-type hypersensitivity, lymphocyte phenotyping, mitogenic proliferative responses, and granulocyte function. Approximately 50% of soldiers completed the study (control, n = 57; NNIF, n = 50). Several stress-induced lymphocyte changes were observed (decreased mitogen-induced proliferation, T and total lymphocytes, and interferon-gamma-producing lymphocytes and increased percentage of neutrophils). NNIF modified several changes, including delayed-type hypersensitivity responses (NNIF, 78%; control, 59%; p < 0.05), increased proportions of helper T cells, activation of B cells, enhanced neutrophil phagocytosis, and attenuation of declines in certain functional subpopulations (i.e., cytotoxic/ suppressor lymphocytes). Soldiers who consumed NNIF experienced less stress-induced immune impairment, thereby lowering the risk of infection.

Effect of dietary ribonucleotides on infant immune status. Part 2: Immune cell development.

The objective of this study was to determine whether dietary ribonucleotides alter immune cell phenotypes or function in the first year of life. Newborn term infants in a double-blind, 12-mo, multicenter trial were randomized to cow milk formula groups with (FN, n = 138) or without (F, n = 147) 72 mg/L supplemental ribonucleotides. A nonrandomized HMF cohort (n = 192) was concurrently enrolled. Eighty-eight immune blood cell types were characterized by flow cytometry. Data were analyzed by multivariate ANOVA (MANOVA), ANOVA, and repeated measures analysis (RMA), with adjustments made for multiple comparisons. Ribonucleotide feeding changed subpopulations of T and natural killer (NK) cells. FN had higher numbers and percentages of memory/effector (M/E) cytotoxic/suppressor (CD45R0(+)CD8(+), RMA) T, Fas(+) M/E (CD45R0(+)CD95(+)CD3(+), 6 mo) T, and CD56(+)CD16(-) NK cells (CD56(+)CD16(-)CD3(-)CD8(-), 12 mo), and higher percentages of M/E helper (CD45R0(+)CD4(+), RMA) T, Tc1 (IFN gamma(+)CD4(-)CD3(+), RMA), total interferon (IFN)gamma T (IFN gamma(+)CD4(+/-)CD3(+), RMA), Th2 (IL-4(+)CD4(+)CD3(+), 7 mo), and CD57(+) NK-T cells (CD57(+)CD56(-)CD3(+), 6 mo, 7 mo) compared with F. Percentages of naive helper T (CD45RA(+)CD4(+), 12 mo) and numbers and percentages of CD56(+) NK-T cells (CD56(+)CD16(-)CD3(+)CD8(-), 2 mo, 6 mo) were lower in FN than F. Percentages of M/E cytotoxic/suppressor, Th2, and CD56(+)CD16(-) NK cells in FN were significantly higher than F but were not different from HMF, whereas F was significantly lower than HMF. Ribonucleotide supplementation of infant formula supported increased T-cell maturation and affected immunoregulatory NK cell subsets. These FN-associated immune cell profiles either did not differ from those infants fed HMF or tended to be more like those fed HMF than those fed F.

Effect of dietary ribonucleotides on infant immune status. Part 1: Humoral responses.

The objective of this study was to further explore previously identified effects of supplemental ribonucleotides on infant immune status as measured by antibody responses to routine infant immunizations. Infants were randomized to a milk-based formula with (FN, n = 138) or without (F, n = 147) 72 mg ribonucleotides/L. A cohort of human milk-fed (HMF, n = 192) infants was also followed. Subjects were given Haemophilus influenzae type b (Hib), diphtheria tetanus acellular pertussis, and oral poliovirus vaccinations at 2, 4, and 6 mo of age, and specific antibody responses were assessed at 2, 6, 7, and 12 mo. Growth and safety data were also monitored. Using a two-group repeated measures analysis (RMA), FN-fed infants had significantly higher poliovirus type 1 neutralizing antibody (PV-VN1) responses than F-fed infants (p = 0.045). Using three-group RMA, PV-VN1 responses in HMF infants were not different from FN-fed infants, while HMF-fed infant PV-VN1 responses were significantly higher than F-fed infants at 6 (p = 0.0004) and 12 mo (p = 0.0001). FN-fed infants had responses to Hib Farr, diphtheria, tetanus toxoid, oral poliovirus-specific IgA, and PV-VN3 not significantly different from those of F and HMF infants. Growth, gastrointestinal tolerance, and adverse events were equivalent among the three groups. The FN-associated increase in PV-VN1 response and nonstatistically significant trends toward increased Hib and diphtheria antibody responses were consistent with observations from earlier studies, indicating immune benefits of nucleotide supplementation of infant formula.