A high fiber diet or supplementation with Lactococcus lactis subspecies cremoris to pregnant mice confers protection against intestinal injury in adult offspring.

The diet during pregnancy, or antenatal diet, influences the offspring's intestinal health. We previously showed that antenatal butyrate supplementation reduces injury in adult murine offspring with dextran sulfate sodium (DSS)-induced colitis. Potential modulators of butyrate levels in the intestine include a high fiber diet or dietary supplementation with probiotics. To test this, we supplemented the diet of pregnant mice with high fiber, or with the probiotic bacteria Lactococcus lactis subspecies cremoris or Lactobacillus rhamnosus GG. We then induced chronic colitis with DSS in their adult offspring. We demonstrate that a high fiber antenatal diet, or supplementation with Lactococcus lactis subspecies cremoris during pregnancy diminished the injury from DSS-induced colitis in offspring. These data are evidence that antenatal dietary interventions impact offspring gut health and define the antenatal diet as a therapeutic modality to enhance offspring intestinal health.

Butyrate supplementation to pregnant mice elicits cytoprotection against colonic injury in the offspring.

BACKGROUND: Maternal diet during pregnancy can impact progeny health and disease by influencing the offspring's gut microbiome and immune development. Gut microbial metabolism generates butyrate, a short-chain fatty acid that benefits intestinal health. Here we assess the effects of antenatal butyrate on the offspring's gastrointestinal health. We hypothesized that antenatal butyrate supplementation will induce protection against colitis in the offspring. METHODS: C57BL/6 mice received butyrate during pregnancy and a series of experiments were performed on their offspring. RNA sequencing was performed on colonic tissue of 3-week-old offspring. Six-8-week-old offspring were subjected to dextran sulfate sodium-induced colitis. Fecal microbiome analysis was performed on the 6-8-week-old offspring. RESULTS: Antenatal butyrate supplementation dampened transcript enrichment of inflammation-associated colonic genes and prevented colonic injury in the offspring. Antenatal butyrate increased the offspring's stool microbiome diversity and expanded the prevalence of specific gut microbes. CONCLUSIONS: Antenatal butyrate supplementation resulted in downregulation of genes in the offspring's colon that function in inflammatory signaling. In addition, antenatal butyrate supplementation was associated with protection against colitis and an expanded fecal microbiome taxonomic diversity in the offspring. IMPACT: Dietary butyrate supplementation to pregnant mice led to downregulation of colonic genes involved in inflammatory signaling and cholesterol synthesis, changes in the fecal microbiome composition of the offspring, and protection against experimentally induced colitis in the offspring. These data support the mounting evidence that the maternal diet during pregnancy has enduring effects on the offspring's long-term health and disease risk. Although further investigations are needed to identify the mechanism of butyrate's effects on fetal gut development, the current study substantiates the approach of dietary intervention during pregnancy to optimize the long-term gastrointestinal health of the offspring.

Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is an inflammatory disease affecting premature infants. Intestinal microbial composition may play a key role in determining which infants are predisposed to NEC and when infants are at highest risk of developing NEC. It is unclear how to optimize antibiotic therapy in preterm infants to prevent NEC and how to optimize antibiotic regimens to treat neonates with NEC. This article discusses risk factors for NEC, how dysbiosis in preterm infants plays a role in the pathogenesis of NEC, and how probiotic and antibiotic therapy may be used to prevent and/or treat NEC and its sequelae.

To start or not: Factors to consider when implementing routine probiotic use in the NICU.

Supplementation of probiotics to very low birth weight (VLBW) infants has been extensively studied, with multiple meta-analyses reporting probiotics decrease the risk of necrotizing enterocolitis (NEC) and death. Despite availability of this evidence, the decision to initiate routine probiotic supplementation to preterm infants continues to be a complex one. There are uncertainties regarding the use of probiotics, including selecting the appropriate product, dose and target population. Additionally, availability of specific probiotic products and regulatory oversight varies by country, raising concerns regarding the safety and efficacy of specific probiotic products. In this review, we summarize the latest evidence on probiotic use in preterm infants and discuss considerations that may help guide clinicians who are considering routine probiotic supplementation.

Routine Supplementation of Lactobacillus rhamnosus GG and Risk of Necrotizing Enterocolitis in Very Low Birth Weight Infants.

OBJECTIVE: To evaluate if routine supplementation of Lactobacillus rhamnosus GG ATCC 53103 (LGG) is associated with a decreased risk of necrotizing enterocolitis in very low birth weight (VLBW) infants. STUDY DESIGN: Retrospective observational cohort study of VLBW (<1500 g) infants at a single center from 2008 to 2016. LGG supplementation with Culturelle at a dose of 2.5 to 5 × 109 CFU/day began in 2014. We used multivariable logistic regression to evaluate the association between LGG supplementation and necrotizing enterocolitis (modified Bell stage IIA or greater), after adjusting for potential confounders. We also compared changes in necrotizing enterocolitis incidence before and after implementation of LGG using a statistical process control chart. RESULTS: We evaluated 640 VLBW infants with a median gestational age of 28.7 weeks (IQR 26.3-30.6); 78 (12%) developed necrotizing enterocolitis. The median age at first dose of LGG was 6 days (IQR 3-10), and duration of supplementation was 32 days (IQR 18-45). The incidence of necrotizing enterocolitis in the epoch before LGG implementation was 10.2% compared with 16.8% after implementation. In multivariable analysis, LGG supplementation was associated with a higher risk of necrotizing enterocolitis (aOR 2.10, 95 % CI 1.25-3.54, P = .005). We found no special cause variation in necrotizing enterocolitis after implementation of LGG supplementation. There were no episodes of Lactobacillus sepsis during 5558 infant days of LGG supplementation. CONCLUSIONS: In this study, routine LGG supplementation was not associated with a decreased risk of necrotizing enterocolitis. Our findings do not support the use of the most common probiotic preparation currently supplemented to VLBW infants in the US.

Timing of developmental reduction in epithelial glutathione redox potential is associated with increased epithelial proliferation in the immature murine intestine.

BackgroundThe intracellular redox potential of the glutathione (GSH)/glutathione disulfide (GSSG) couple regulates cellular processes. In vitro studies indicate that a reduced GSH/GSSG redox potential favors proliferation, whereas a more oxidized redox potential favors differentiation. Intestinal growth depends upon an appropriate balance between the two. However, how the ontogeny of intestinal epithelial cellular (IEC) GSH/GSSG redox regulates these processes in the developing intestine has not been fully characterized in vivo.MethodsOntogeny of intestinal GSH redox potential and growth were measured in neonatal mice.ResultsWe show that IEC GSH/GSSG redox potential becomes increasingly reduced (primarily driven by increased GSH concentration) over the first 3 weeks of life. Increased intracellular GSH has been shown to drive proliferation through increased poly-ADP-ribose polymerase (PARP) activity. We show that increasing IEC poly-ADP-ribose chains can be measured over the first 3 weeks of life, indicating an increase in IEC PARP activity. These changes are accompanied by increased intestinal growth and IEC proliferation as assessed by villus height/crypt depth, intestinal length, and Ki67 staining.ConclusionUnderstanding how IEC GSH/GSSG redox potential is developmentally regulated may provide insight into how premature human intestinal redox states can be manipulated to optimize intestinal growth and adaptation.

Pathogenesis of NEC: Role of the innate and adaptive immune response.

Necrotizing enterocolitis (NEC) is a devastating disease in premature infants with high case fatality and significant morbidity among survivors. Immaturity of intestinal host defenses predisposes the premature infant gut to injury. An abnormal bacterial colonization pattern with a deficiency of commensal bacteria may lead to a further breakdown of these host defense mechanisms, predisposing the infant to NEC. Here, we review the role of the innate and adaptive immune system in the pathophysiology of NEC.

Intestinal microbiota and its relationship with necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in infants born prematurely. After birth, the neonatal gut must acquire a healthy complement of commensal bacteria. Disruption or delay of this critical process, leading to deficient or abnormal microbial colonization of the gut, has been implicated as key risk factor in the pathogenesis of NEC. Conversely, a beneficial complement of commensal intestinal microbiota may protect the immature gut from inflammation and injury. Interventions aimed at providing or restoring a healthy complement of commensal bacteria, such as probiotic therapy, are currently the most promising treatment to prevent NEC. Shifting the balance of intestinal microbiota from a pathogenic to protective complement of bacteria can protect the gut from inflammation and subsequent injury that leads to NEC. Herein, we review the relationship of intestinal microbiota and NEC in preterm infants.

The role of intestinal epithelial barrier function in the development of NEC.

The intestinal epithelial barrier plays an important role in maintaining host health. Breakdown of intestinal barrier function is known to play a role in many diseases such as infectious enteritis, idiopathic inflammatory bowel disease, and neonatal inflammatory bowel diseases. Recently, increasing research has demonstrated the importance of understanding how intestinal epithelial barrier function develops in the premature neonate in order to develop strategies to promote its maturation. Optimizing intestinal barrier function is thought to be key to preventing neonatal inflammatory bowel diseases such as necrotizing enterocolitis. In this review, we will first summarize the key components of the intestinal epithelial barrier, what is known about its development, and how this may explain NEC pathogenesis. Finally, we will review what therapeutic strategies may be used to promote optimal development of neonatal intestinal barrier function in order to reduce the incidence and severity of NEC.

Small intestinal intraepithelial TCRγδ+ T lymphocytes are present in the premature intestine but selectively reduced in surgical necrotizing enterocolitis.

BACKGROUND: Gastrointestinal barrier immaturity predisposes preterm infants to necrotizing enterocolitis (NEC). Intraepithelial lymphocytes (IEL) bearing the unconventional T cell receptor (TCR) γδ (γδ IEL) maintain intestinal integrity and prevent bacterial translocation in part through production of interleukin (IL) 17. OBJECTIVE: We sought to study the development of γδ IEL in the ileum of human infants and examine their role in NEC pathogenesis. We defined the ontogeny of γδ IEL proportions in murine and human intestine and subjected tcrδ-/- mice to experimental gut injury. In addition, we used polychromatic flow cytometry to calculate percentages of viable IEL (defined as CD3+ CD8+ CD103+ lymphocytes) and the fraction of γδ IEL in surgically resected tissue from infants with NEC and gestational age matched non-NEC surgical controls. RESULTS: In human preterm infants, the proportion of IEL was reduced by 66% in 11 NEC ileum resections compared to 30 non-NEC controls (p<0.001). While γδ IEL dominated over conventional αß IEL early in gestation in mice and in humans, γδ IEL were preferential decreased in the ileum of surgical NEC patients compared to non-NEC controls (50% reduction, p<0.05). Loss of IEL in human NEC was associated with downregulation of the Th17 transcription factor retinoic acid-related orphan nuclear hormone receptor C (RORC, p<0.001). TCRδ-deficient mice showed increased severity of experimental gut injury (p<0.05) with higher TNFα expression but downregulation of IL17A. CONCLUSION: Complimentary mouse and human data suggest a role of γδ IEL in IL17 production and intestinal barrier production early in life. Specific loss of the γδ IEL fraction may contribute to NEC pathogenesis. Nutritional or pharmacological interventions to support γδ IEL maintenance in the developing small intestine could serve as novel strategies for NEC prevention.

Human breast milk and the gastrointestinal innate immune system.

The gastrointestinal (GI) tract is a large potential portal for multiple infectious agents to enter the human body. The GI system performs multiple functions as part of the neonate's innate immune system, providing critical defense during a vulnerable period. Multiple mechanisms and actions are enhanced by the presence of human breast milk. Bioactive factors found in human milk work together to create and maintain an optimal and healthy environment, allowing the intestines to deliver ideal nutrition to the host and afford protection by a variety of mechanisms.

Gut commensal bacteria and regional Wnt gene expression in the proximal versus distal colon.

Regional expression of Wingless/Int (Wnt) genes plays a central role in regulating intestinal development and homeostasis. However, our knowledge of such regional Wnt proteins in the colon remains limited. To understand further the effect of Wnt signaling components in controlling intestinal epithelial homeostasis, we investigated whether the physiological heterogeneity of the proximal and distal colon can be explained by differential Wnt signaling. With the use of a Wnt signaling-specific PCR array, expression of 84 Wnt-mediated signal transduction genes was analyzed, and a differential signature of Wnt-related genes in the proximal versus distal murine colon was identified. Several Wnt agonists (Wnt5a, Wnt8b, and Wnt11), the Wnt receptor frizzled family receptor 3, and the Wnt inhibitory factor 1 were differentially expressed along the colon length. These Wnt signatures were associated with differential epithelial cell proliferation and migration in the proximal versus distal colon. Furthermore, reduced Wnt/ß-catenin activity and decreased Wnt5a and Wnt11 expression were observed in mice lacking commensal bacteria, an effect that was reversed by conventionalization of germ-free mice. Interestingly, myeloid differentiation primary response gene 88 knockout mice showed decreased Wnt5a levels, indicating a role for Toll-like receptor signaling in regulating Wnt5a expression. Our results suggest that the morphological and physiological heterogeneity within the colon is in part facilitated by the differential expression of Wnt signaling components and influenced by colonization with bacteria.

Commensal and probiotic bacteria may prevent NEC by maturing intestinal host defenses.

Necrotizing enterocolitis (NEC) is a devastating disease of prematurity with significant morbidity and mortality. Immaturity of intestinal host defenses predisposes the premature infant gut to injury. An abnormal bacterial colonization pattern with a deficiency of commensal bacteria may lead to a further breakdown of these host defense mechanisms, predisposing the infant to NEC. The presence of probiotic and commensal bacteria within the gut has been shown to mature the intestinal defense system through a variety of mechanisms. We have shown that commensal and probiotic bacteria can promote intestinal host defenses by reducing apoptotic signaling, blocking inflammatory signaling, and maturing barrier function in immature intestinal epithelia. Future studies aimed at elucidating the mechanisms by which probiotic and commensal bacteria exert their effects will be critical to developing effective preventive therapies for NEC.

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species.

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.

Therapeutic use of prebiotics, probiotics, and postbiotics to prevent necrotizing enterocolitis: what is the current evidence?

Necrotizing enterocolitis (NEC) is a leading cause of neonatal morbidity and mortality, and preventive therapies that are both effective and safe are urgently needed. Current evidence from therapeutic trials suggests that probiotics are effective in decreasing NEC in preterm infants, and probiotics are currently the most promising therapy for this devastating disease. However, concerns regarding safety and optimal dosing have limited the widespread adoption of routine clinical use of probiotics in preterm infants. This article summarizes the current evidence regarding the use of probiotics, prebiotics, and postbiotics in the preterm infant, including their therapeutic role in preventing NEC.

Lactobacillus rhamnosus (LGG) regulates IL-10 signaling in the developing murine colon through upregulation of the IL-10R2 receptor subunit.

The intestinal microflora is critical for normal development, with aberrant colonization increasing the risk for necrotizing enterocolitis (NEC). In contrast, probiotic bacteria have been shown to decrease its incidence. Multiple pro- and anti-inflammatory cytokines have been identified as markers of intestinal inflammation, both in human patients with NEC and in models of immature intestine. Specifically, IL-10 signaling attenuates intestinal responses to gut dysbiosis, and disruption of this pathway exacerbates inflammation in murine models of NEC. However, the effects of probiotics on IL-10 and its signaling pathway, remain poorly defined. Real-time PCR profiling revealed developmental regulation of MIP-2, TNF-α, IL-12, IL-10 and the IL-10R2 subunit of the IL-10 receptor in immature murine colon, while the expression of IL-6 and IL-18 was independent of postnatal age. Enteral administration of the probiotic Lactobacillus rhamnosus GG (LGG) down-regulated the expression of TNF-α and MIP-2 and yet failed to alter IL-10 mRNA and protein expression. LGG did however induce mRNA expression of the IL-10R2 subunit of the IL-10 receptor. IL-10 receptor activation has been associated with signal transducer and activator of transcription (STAT) 3-dependent induction of members of the suppressors of cytokine signaling (SOCS) family. In 2 week-old mice, LGG also induced STAT3 phosphorylation, increased colonic expression of SOCS-3, and attenuated colonic production of MIP-2 and TNF-α. These LGG-dependent changes in phosphoSTAT3, SOCS3, MIP-2 and TNF-α were all inhibited by antibody-mediated blockade of the IL-10 receptor. Thus LGG decreased baseline proinflammatory cytokine expression in the developing colon through upregulation of IL-10 receptor-mediated signaling, most likely due to the combined induction of phospho-STAT3 and SOCS3. Furthermore, LGG-dependent increases in IL-10R2 were associated with reductions in TNF-α, MIP-2 and disease severity in a murine model of intestinal injury in the immature colon.

Risk factors for mumps at a university with a large mumps outbreak.

OBJECTIVES: Routine measles-mumps-rubella (MMR) vaccine use has greatly decreased the incidence of mumps in the U.S. However, a resurgence of mumps occurred in 2006. We investigated the large outbreak at a university and assessed risk factors for disease. METHODS: We described the outbreak and conducted a case-control study. We interviewed case students (identified from student health service and health department records) and control students (selected from a randomly ordered administrative list) and assessed their vaccination status. We compared case students with > or = 2 MMR doses and control students with > or = 2 MMR doses in univariate and multivariate analyses. RESULTS: The mean age of the 174 case students was 20.9 years; 65% were women. Ninety-seven case students and 147 control students were enrolled in the study. Two-dose MMR coverage was 99% among case and control students with complete records. Only 33% of case students reported exposure to someone with mumps. Case students were more likely than control students to be aged 18 to 19 years (vs. aged 22 years, adjusted odds ratio [AOR] = 5.55; 95% confidence interval [CI] 2.09, 14.74), to report exposure to mumps (AOR=2.31, 95% CI 1.13, 4.73), and to have worked/volunteered on campus (AOR=2.91, 95% CI 1.33, 6.33). Also, women in dormitories had increased odds of mumps compared with men in dormitories. CONCLUSION: High two-dose MMR coverage was not sufficient to prevent the outbreak. Further study is needed to better understand the effects of dormitory residency and gender on mumps transmission. Clinicians should be vigilant for mumps in young adults presenting with parotitis regardless of immunization history.

Mumps vaccine performance among university students during a mumps outbreak.

BACKGROUND: The largest reported mumps outbreak at a US college in 19 years occurred in 2006 at a Kansas university with a 2-dose measles-mumps-rubella (MMR) vaccination policy. We assessed vaccine performance and mumps risk factors, including the possibility of waning vaccine protection. METHODS: Case students were compared with a cohort of the university's approximately 19,000 undergraduates. The secondary attack rate for clinical mumps was determined among roommates exposed to case students. Time from receipt of the second dose of MMR vaccine was compared between case students and roommates without mumps. RESULTS: Coverage with > or =2 dose of MMR vaccine was > or =95% among 140 undergraduate case students and 444 cohort students. The secondary attack rate for clinical mumps among roommates who had received 2 doses of vaccine ranged from 2.2% to 7.7%, depending on the case definition. Compared with roommates without mumps, case students were more likely (odds ratio, 2.46; 95% confidence interval, 1.25-4.82) to have received their second dose of MMR vaccine > or =10 years earlier. The odds of being a case student increased with each 1-year increase in time from receipt of the second dose of MMR vaccine (odds ratio, 1.36; 95% confidence interval, 1.10-1.68) among case students and roommates aged 18-19 years but not among those aged > or =20 years. Students aged 18-19 years had a higher risk of mumps (risk ratio, 3.14; 95% confidence interval, 1.60-6.16), compared with students aged > or =22 years; women living in dormitories had increased risk of mumps (risk ratio, 1.95; 95% confidence interval, 1.01-3.76), compared with men not living in dormitories. CONCLUSION: High 2-dose MMR coverage protected many students from developing mumps but was not sufficient to prevent the mumps outbreak. Vaccine-induced protection may wane. Similar US settings where large numbers of young adults from wild-type naive cohorts live closely together may be at particular risk for mumps outbreaks.