The human milk oligosaccharides 2'-fucosyllactose and 6'-sialyllactose protect against the development of necrotizing enterocolitis by inhibiting toll-like receptor 4 signaling.

BACKGROUND: Necrotizing enterocolitis (NEC) develops through exaggerated toll-like receptor 4 (TLR4) signaling in the intestinal epithelium. Breast milk is rich in non-digestible oligosaccharides and prevents NEC through unclear mechanisms. We now hypothesize that the human milk oligosaccharides 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) can reduce NEC through inhibition of TLR4 signaling. METHODS: NEC was induced in newborn mice and premature piglets and infant formula was supplemented with 2'-FL, 6'-SL, or lactose. Intestinal tissue was obtained at surgical resection. HMO inhibition of TLR4 was assessed in IEC-6 enterocytes, mice, and human tissue explants and via in silico modeling. RESULTS: Supplementation of infant formula with either 2'-FL and/or 6'-SL, but not the parent sugar lactose, reduced NEC in mice and piglets via reduced apoptosis, inflammation, weight loss, and histological appearance. Mechanistically, both 2'-FL and 6'-SL, but not lactose, reduced TLR4-mediated nuclear factor kappa light-chain enhancer of activated B cells (NF-kB) inflammatory signaling in the mouse and human intestine. Strikingly, in silico modeling revealed 2'-FL and 6'-SL, but not lactose, to dock into the binding pocket of the TLR4-MD2 complex, explaining their ability to inhibit TLR4 signaling. CONCLUSIONS: 2'-FL and 6'-SL, but not lactose, prevent NEC in mice and piglet models and attenuate NEC inflammation in the human ileum, in part through TLR4 inhibition. IMPACT: Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants that occurs in the setting of bacterial colonization of the gut and administration of formula feeds and activation by the innate immune receptor toll-like receptor 4 (TLR4). Breast milk prevents NEC through unclear mechanisms. We now show that breast milk-enriched human milk oligosaccharides (HMOs) that are derived from lactose prevent NEC through inhibition of TLR4. The human milk oligosaccharides 2'-FL and 6'-SL, but not the backbone sugar lactose, prevent NEC in mice and piglets. 2'-FL and 6'-SL but not lactose inhibited TLR4 signaling in cultured enterocytes, in enteroids derived from mouse intestine, and in human intestinal explants obtained at the time of surgical resection for patients with NEC. In seeking the mechanisms involved, 2'-FL and 6'-SL but not lactose were found to directly bind to TLR4, explaining the inhibition and protection against NEC. These findings may impact clinical practice by suggesting that administration of HMOs could serve as a preventive strategy for premature infants at risk for NEC development.

The role of in utero endotoxin exposure in the development of inflammatory bowel disease in mice.

PROBLEM: Although early environmental influences are thought to influence the development of inflammatory bowel disease (IBD), little is known about the role of the in utero environment on subsequent IBD risk. We hypothesized that prenatal exposure to bacterial lipopolysaccharide (LPS) could modify the subsequent development of dextran sulfate sodium (DSS)-induced ulcerative colitis in adulthood by influencing the associated cellular and immune response. METHOD OF STUDY: To test this hypothesis, we exposed developing mice in utero to LPS or saline (PBS) at E17.5, and then induced colitis at 5 weeks. We then assessed colitis severity and effects on the microbiome. In order to define the developmental impact of any potential LPS effect, we also exposed 1-week-old mice to either LPS or saline before inducing colitis at 5 weeks. RESULTS: Mice that had been exposed to LPS but not saline in utero were protected from subsequent colitis development, and their intestinal barrier integrity and tight junction expression distribution were similar to that of control mice that were not exposed to DSS. By contrast, mice exposed to either LPS or saline at day 7 of life all developed severe colitis upon subsequent DSS exposure. CONCLUSION: These results identify an informative time window during fetal development during which exposure to an otherwise pro-inflammatory agent like LPS protects against an inflammatory disease in adulthood.

A Novel Role for Necroptosis in the Pathogenesis of Necrotizing Enterocolitis.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by Toll-like receptor 4 (TLR4)-dependent intestinal inflammation and enterocyte death. Given that necroptosis is a proinflammatory cell death process that is linked to bacterial signaling, we investigated its potential role in NEC, and the mechanisms involved. METHODS: Human and mouse NEC intestine were analyzed for necroptosis gene expression (ie, RIPK1, RIPK3, and MLKL), and protein activation (phosphorylated RIPK3). To evaluate a potential role for necroptosis in NEC, the effects of genetic (ie, Ripk3 knockout or Mlkl knockout) or pharmacologic (ie, Nec1s) inhibition of intestinal inflammation were assessed in a mouse NEC model, and a possible upstream role of TLR4 was assessed in Tlr4-deficient mice. The NEC-protective effects of human breast milk and its constituent milk oligosaccharides on necroptosis were assessed in a NEC-in-a-dish model, in which mouse intestinal organoids were cultured as either undifferentiated or differentiated epithelium in the presence of NEC bacteria and hypoxia. RESULTS: Necroptosis was activated in the intestines of human and mouse NEC in a TLR4-dependent manner, and was up-regulated specifically in differentiated epithelium of the immature ileum. Inhibition of necroptosis genetically and pharmacologically reduced intestinal-epithelial cell death and mucosal inflammation in experimental NEC, and ex vivo in the NEC-in-a-dish system. Strikingly, the addition of human breast milk, or the human milk oligosaccharide 2 fucosyllactose in the ex vivo system, reduced necroptosis and inflammation. CONCLUSIONS: Necroptosis is activated in the intestinal epithelium upon TLR4 signaling and is required for NEC development, and explains in part the protective effects of breast milk.

Lifestyle behavior patterns and mortality among adults in the NHANES 1988-1994 population: A latent profile analysis.

Evidence suggests interdependent associations of individual modifiable behaviors with health outcomes. However, such interrelations have not been accounted for in previous behavior-outcome associations. We conducted latent profile analysis (LPA) on self-reported levels of alcohol consumption, restaurant dining, vitamin/mineral supplement use, physical activity (PA) and smoke exposure (first- and second-hand smoke) separately for smokers (N = 4530) and non-smokers (N = 13,421) using data from the third National Health and Nutrition Examination Survey (NHANES III) to identify subgroups with similar levels within and across behaviors. Cox-proportional hazards models were used to compare mortality rates between subgroups from cancer, cardiovascular disease (CVD) and all-causes at an average of 16.4 (±6.1) years follow-up. Five behavioral typologies were identified in non-smokers ("Moderates", "Low Risk Factors", "Restaurant Diners", "Moderate Passive Smokers" and "Heavy Passive Smokers"), and four in smokers ("Moderates", "Low Risk Factors", "Heavy Smokers" and "Physically Active"). As a group, "Moderates" had levels of each behavior that were not significantly different from at least one other group. Compared to "Moderates", in non-smokers "Restaurant Diners" had lower hazard from all-cause (hazard ratio (HR):0.84, 95% CI:0.74-0.97) and CVD (HR:0.59, 0.43-0.82) mortality, while "Low Risk Factors" had higher cancer mortality (HR:1.38,1.03-1.84). In smokers, compared to "Moderates", higher hazards for mortality were found for "Heavy Smokers" (all cause: HR:1.34, 1.12-1.60; CVD: HR:1.52, 1.04-2.23; cancer: HR:1.41 1.02-1.96) and "Low Risk Factors" (all-cause: HR:1.58, 1.14-2.17). Taken together, when restaurant dining, PA and smoking exposures are grouped together, novel predictions for mortality occur, suggesting data on multiple behaviors may be informative for risk stratification.

Use of non-steroidal anti-inflammatory drugs in US adults: changes over time and by demographic.

OBJECTIVE: Aspirin and non-aspirin non-steroidal anti-inflammatory drugs (NSAIDs) are preventive against cardiovascular disease (CVD) and several cancer types, but long-term use has been associated with significant health risks, resulting in conflicting recommendations on NSAID use for prevention of CVD and cancer. Previous research indicates that aspirin use increases with age and CVD risk factors and that a large percentage of the US population regularly use analgesics, including NSAIDs, but there has not been a recent, in-depth assessment of NSAID use prevalence, changes in use over time or predictors of NSAID use in the USA. METHODS: We used the cross-sectional, National Health And Nutrition Examination Survey (NHANES) from 1988 to 1994 and three continuous cycles (1999-2004) to assess regular NSAID use prevalence, changes over time and predictors of regular NSAID use. RESULTS: Overall, regular NSAID use increased over time and varied by demographic features. Participants over 60 years of age, women, participants with high body mass index, increased waist circumference or heart disease were significantly more likely to be regular NSAID users. By contrast, non-Hispanic African American and Mexican American participants were significantly less likely to regularly use NSAIDs. CONCLUSIONS: This study uses a nationally representative data set (NHANES) to provide an exploration of regular NSAID use patterns over time, highlighting several demographic, lifestyle and clinical conditions associated with regular NSAID use. Understanding who is likely to regularly use NSAIDs enables more targeted messaging both for increasing the preventive benefits and for limiting the toxicities associated with regular use of NSAIDs.

Exploratory analysis of ERCC2 DNA methylation in survival among pediatric medulloblastoma patients.

AIM: Medulloblastoma is the most frequent malignant pediatric brain tumor. While survival rates have improved due to multimodal treatment including cisplatin-based chemotherapy, there are few prognostic factors for adverse treatment outcomes. Notably, genes involved in the nucleotide excision repair pathway, including ERCC2, have been implicated in cisplatin sensitivity in other cancers. Therefore, this study evaluated the role of ERCC2 DNA methylation profiles on pediatric medulloblastoma survival. METHODS: The study population included 71 medulloblastoma patients (age <18years at diagnosis) and recruited from Texas Children's Cancer Center between 2004 and 2009. DNA methylation profiles were generated from peripheral blood samples using the Illumina Infinium Human Methylation 450 Beadchip. Sixteen ERCC2-associated CpG sites were evaluated in this analysis. Multivariable regression models were used to determine the adjusted association between DNA methylation and survival. Cox regression and Kaplan-Meier curves were used to compare 5-year overall survival between hyper- and hypo-methylation at each CpG site. RESULTS: In total, 12.7% (n=9) of the patient population died within five years of diagnosis. In our population, methylation of the cg02257300 probe (Hazard Ratio=9.33; 95% Confidence Interval: 1.17-74.64) was associated with death (log-rank p=0.01). This association remained suggestive after correcting for multiple comparisons (FDR p<0.2). No other ERCC2-associated CpG site was associated with survival in this population of pediatric medulloblastoma patients. CONCLUSION: These findings provide the first evidence that DNA methylation within the promoter region of the ERCC2 gene may be associated with survival in pediatric medulloblastoma. If confirmed in future studies, this information may lead to improved risk stratification or promote the development of novel, targeted therapeutics.

Poor Adherence to US Dietary Guidelines for Children and Adolescents in the National Health and Nutrition Examination Survey Population.

BACKGROUND: Poor diet quality in childhood and adolescence is associated with adverse health outcomes throughout life, yet the dietary habits of American children and how they change across childhood and adolescence are unknown. OBJECTIVES: This study sought to describe diet quality among children and adolescents by assessing adherence to the 2010 Dietary Guidelines for Americans (DGA) and to determine whether any differences in adherence occurred across childhood. DESIGN, SETTING, AND PARTICIPANTS: We employed a cross-sectional design using data from the National Health and Nutrition Examination Survey (NHANES). Of 9,280 children aged 4 to 18 years who participated in NHANES from 2005 to 2010, those with insufficient data on dietary recall (n=852) or who were pregnant or lactating during the time of interview (n=38) were excluded from the final study sample (n=8,390). MAIN OUTCOME MEASURES: We measured adherence to the DGA using the Healthy Eating Index 2010 (HEI-2010) and stratified participants into three age groups (4 to 8, 9 to 13, and 14 to 18 years of age). We analyzed each of 12 HEI-2010 components and total HEI-2010 score. RESULTS: The youngest children had the highest overall diet quality due to significantly greater scores for total fruit, whole fruit, dairy, and whole grains. These children also had the highest scores for sodium, refined grains, and empty calories. Total HEI-2010 scores ranged from 43.59 to 52.11 out of 100, much lower than the minimum score of 80 that is thought to indicate a diet associated with good health. CONCLUSIONS: Overall, children and adolescents are failing to meet the DGA and may be at an increased risk of chronic diseases throughout life. By analyzing which food groups show differences between age groups, we provide data that can inform the development of dietary interventions to promote specific food groups targeting specific ages and improve diet quality among children and adolescents.