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Gastrointestinal (GI) symptoms often affect children with autism spectrum disorders (ASD) and GI symptoms have been associated with an abnormal fecal microbiome. There is limited evidence of Candida species being more prevalent in children with ASD. We enrolled 20 children with ASD and GI symptoms (ASD + GI), 10 children with ASD but no GI symptoms (ASD - GI), and 20 from typically developing (TD) children in this pilot study. Fecal mycobiome taxa were analyzed by Internal Transcribed Spacer sequencing. GI symptoms (GI Severity Index [GSI]), behavioral symptoms (Social Responsiveness Scale -2 [SRS-2]), inflammation and fungal immunity (fecal calprotectin and serum dectin-1 [ELISA]) were evaluated. We observed no changes in the abundance of total fungal species (alpha diversity) between groups. Samples with identifiable Candida spp. were present in 4 of 19 (21%) ASD + GI, in 5 of 9 (56%) ASD - GI, and in 4 of 16 (25%) TD children (overall P = 0.18). The presence of Candida spp. did not correlate with behavioral or GI symptoms (P = 0.38, P = 0.5, respectively). Fecal calprotectin was normal in all but one child. Finally, there was no significance in serum dectin-1 levels, suggesting no increased fungal immunity in children with ASD. Our data suggest that fungi are present at normal levels in the stool of children with ASD and are not associated with gut inflammation.

Background Probiotic Lactobacillus reuteri DSM 17938 (LR 17938) is beneficial to infants with colic. To understand its mechanism of action, we assessed ultrasonic vocalizations (USV) and brain pain/stress genes in newborn mice exposed to maternal separation stress. Methods Pups were exposed to unpredictable maternal separation (MSU or SEP) or MSU combined with unpredictable maternal stress (MSU + MSUS or S + S), from postnatal days 5 to 14. USV calls and pain/stress/neuroinflammation-related genes in the brain were analyzed. Results We defined 10 different neonatal call patterns, none of which increased after MSU. Stress reduced overall USV calls. Orally feeding LR 17938 also did not change USV calls after MSU. However, LR 17938 markedly increased vocalizations in mice allowed to stay with their dams. Even though LR 17938 did not change MSU-related calls, LR 17938 modulated brain genes related to stress and pain. Up-regulated genes following LR 17938 treatment were opioid peptides, kappa-opioid receptor 1 genes, and CD200, important in anti-inflammatory signaling. LR 17938 down-regulated CCR2 transcripts, a chemokine receptor, in the stressed neonatal brain. Conclusions USV calls in newborn mice are interpreted as "physiological calls" instead of "cries." Feeding LR 17938 after MSU did not change USV calls but modulated cerebral genes favoring pain and stress reduction and anti-inflammatory signaling. Impact We defined mouse ultrasonic vocalization (USV) call patterns in this study, which will be important in guiding future studies in other mouse strains. Newborn mice with maternal separation stress have reduced USVs, compared to newborn mice without stress, indicating USV calls may represent "physiological calling" instead of "crying." Oral feeding of probiotic Lactobacillus reuteri DSM 17938 raised the number of calls when newborn mice continued to suckle on their dams, but not when mice were under stress. The probiotic bacteria had a dampening effect on monocyte activation and on epinephrine and glutamate-related stress gene expression in the mouse brain.

Treg deficiency causes a lethal, CD4(+) T cell-driven autoimmune disease called IPEX syndrome (immunodysregulation, polyendocrinopathy, and enteropathy, with X-linked inheritance) in humans and in the scurfy (SF) mouse, a mouse model of the disease. Feeding Limosilactobacillus reuteri DSM 17938 (LR 17938, LR) to SF mice reprograms the gut microbiota, reduces disease progression, and prolongs lifespan. However, the efficacy and mechanism of LR, compared with other probiotics, in producing these effects is unknown. We compared LR with Lacticaseibacillus rhamnosus GG (LGG), an extensively investigated probiotic. LR was more effective than LGG in prolonging survival. Both probiotics restored the fecal microbial alpha diversity, but they produced distinct fecal bacterial clusters and differentially modulated microbial relative abundance (RA). LR increased the RA of phylum_Firmicutes, genus_Oscillospira whereas LR reduced phylum_Bacteroidetes, genus_Bacteroides and genus_Parabacteroides, reversing changes attributed to the SF phenotype. LGG primarily reduced the RA of genus_Bacteroides. Both LR and LGG reduced the potentially pathogenic taxon class_γ-proteobacteria. Plasma metabolomics revealed substantial differences among 696 metabolites. We observed similar changes of many clusters of metabolites in SF mice associated with treatment with either LR or LGG. However, a unique effect of LR was to increase the abundance of plasma adenosine metabolites such as inosine, which we previously showed had immune modulatory effects. In conclusion: 1) different probiotics produce distinct signatures in the fecal microbial community in mice with Treg deficiency; and 2) when comparing different probiotics, there are strain-specific microbial products with different anti-inflammatory properties, reinforcing the concept that “one size does not fit all” in the treatment of autoimmune disease. NEW & NOTEWORTHY In the treatment of Treg-deficiency-induced autoimmunity, Limosilactobacillus reuteri DSM 17938 (LR) showed greater efficacy than Lacticaseibacillus rhamnosus GG (LGG). The study demonstrated that two different probiotics produce distinct signatures in the fecal microbial community in mice with Treg deficiency, but with many similarities in global plasma metabolites in general. However, there are strain-specific microbial products with different anti-inflammatory properties, reinforcing the concept that “one size does not fit all” in the treatment of autoimmune disease.

Levels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

Few treatments for human diseases have received as much investigation in the past 20 years as probiotics. In 2017, English-language meta-analyses totaling 52 studies determined the effect of probiotics on conditions ranging from necrotizing enterocolitis and colic in infants to constipation, irritable bowel syndrome, and hepatic encephalopathy in adults. The strongest evidence in favor of probiotics lies in the prevention or treatment of 5 disorders: necrotizing enterocolitis, acute infectious diarrhea, acute respiratory tract infections, antibiotic-associated diarrhea, and infant colic. Probiotic mechanisms of action include the inhibition of bacterial adhesion; enhanced mucosal barrier function; modulation of the innate and adaptive immune systems (including induction of tolerogenic dendritic cells and regulatory T cells); secretion of bioactive metabolites; and regulation of the enteric and central nervous systems. Future research is needed to identify the optimal probiotic and dose for specific diseases, to address whether the addition of prebiotics (to form synbiotics) would enhance activity, and to determine if defined microbial communities would provide benefit exceeding that of single-species probiotics.

Lactobacillus reuteri DSM 17938 (LR 17938) has been shown to reduce the incidence and severity of necrotizing enterocolitis (NEC). It is unclear if preventing NEC by LR 17938 is mediated by Toll-like receptor 2 (TLR2), which is known to mediate proinflammatory responses to bacterial cell wall components. NEC was induced in newborn TLR2−/− or wild-type (WT) mice by the combination of gavage-feeding cow milk-based formula and exposure to hypoxia and cold stress. Treatment groups were administered formula supplemented with LR 17938 or placebo (deMan-Rogosa-Sharpe media). We observed that LR 17938 significantly reduced the incidence of NEC and reduced the percentage of activated effector CD4+T cells, while increasing Foxp3+ regulatory T cells in the intestinal mucosa of WT mice with NEC, but not in TLR2−/− mice. Dendritic cell (DC) activation by LR 17938 was mediated by TLR2. The percentage of tolerogenic DC in the intestine of WT mice was increased by LR 17938 treatment during NEC, a finding not observed in TLR2−/− mice. Furthermore, gut levels of proinflammatory cytokines IL-1β and IFN-γ were decreased after treatment with LR 17938 in WT mice but not in TLR2−/− mice. In conclusion, the combined in vivo and in vitro findings suggest that TLR2 receptors are involved in DC recognition and DC-priming of T cells to protect against NEC after oral administration of LR 17938. Our studies further clarify a major mechanism of probiotic LR 17938 action in preventing NEC by showing that neonatal immune modulation of LR 17938 is mediated by a mechanism requiring TLR2. NEW & NOTEWORTHY Lactobacillus reuteri DSM 17938 (LR 17938) has been shown to protect against necrotizing enterocolitis (NEC) in neonates and in neonatal animal models. The role of Toll-like receptor 2 (TLR2) as a sensor for gram-positive probiotics, activating downstream anti-inflammatory responses is unclear. Our current studies examined TLR2 −/− mice subjected to experimental NEC and demonstrated that the anti-inflammatory effects of LR 17938 are mediated via a mechanism requiring TLR2.

He et al. show that T reg deficiency markedly induces autoimmunity and shifts gut microbiota. Remodeling microbiota by Lactobacillus reuteri was found to inhibit autoimmunity via the metabolite inosine, which interacts with the adenosine A2A receptor. This finding establishes a link between the gut microbiota, A2A receptors, and autoimmunity induced by T reg cell deficiency., Regulatory T (T reg) cell deficiency causes lethal, CD4+ T cell–driven autoimmune diseases. Stem cell transplantation is used to treat these diseases, but this procedure is limited by the availability of a suitable donor. The intestinal microbiota drives host immune homeostasis by regulating the differentiation and expansion of T reg, Th1, and Th2 cells. It is currently unclear if T reg cell deficiency–mediated autoimmune disorders can be treated by targeting the enteric microbiota. Here, we demonstrate that Foxp3+ T reg cell deficiency results in gut microbial dysbiosis and autoimmunity over the lifespan of scurfy (SF) mouse. Remodeling microbiota with Lactobacillus reuteri prolonged survival and reduced multiorgan inflammation in SF mice. L. reuteri changed the metabolomic profile disrupted by T reg cell deficiency, and a major effect was to restore levels of the purine metabolite inosine. Feeding inosine itself prolonged life and inhibited multiorgan inflammation by reducing Th1/Th2 cells and their associated cytokines. Mechanistically, the inhibition of inosine on the differentiation of Th1 and Th2 cells in vitro depended on adenosine A2A receptors, which were also required for the efficacy of inosine and of L. reuteri in vivo. These results reveal that the microbiota–inosine–A2A receptor axis might represent a potential avenue for combatting autoimmune diseases mediated by T reg cell dysfunction.

Background Regulatory T cell (Treg) deficiency leads to IPEX syndrome, a lethal autoimmune disease, in Human and mice. Dysbiosis of the gut microbiota in Treg-deficient scurfy (SF) mice has been described, but to date, the role of the gut microbiota remains to be determined. Results To examine how antibiotic-modified microbiota can inhibit Treg deficiency-induced lethal inflammation in SF mice, Treg-deficient SF mice were treated with three different antibiotics. Different antibiotics resulted in distinct microbiota and metabolome changes and led to varied efficacy in prolonging lifespan and reducing inflammation in the liver and lung. Moreover, antibiotics altered plasma levels of several cytokines, especially IL-6. By analyzing gut microbiota and metabolome, we determined the microbial and metabolomic signatures which were associated with the antibiotics. Remarkably, antibiotic treatments restored the levels of several primary and secondary bile acids, which significantly reduced IL-6 expression in RAW macrophages in vitro. IL-6 blockade prolonged lifespan and inhibited inflammation in the liver and lung. By using IL-6 knockout mice, we further identified that IL-6 deletion provided a significant portion of the protection against inflammation induced by Treg dysfunction. Conclusion Our results show that three antibiotics differentially prolong survival and inhibit lethal inflammation in association with a microbiota—IL-6 axis. This pathway presents a potential avenue for treating Treg deficiency-mediated autoimmune disorders.

Background and objective: Breast milk has many growth-promoting and immune-active components, including transforming growth factor-β, lactoferrin, lysozyme, immunoglobulin A, and prebiotics such as the human milk oligosaccharides. Treatment with Lactobacillus reuteri DSM 17938 (LR), a probiotic with immunomodulatory functions, significantly increases regulatory T cells (Tregs) in the intestinal mucosa of newborn suckling rats. In humans, treatment with LR of infants with colic reduces crying optimally if the infants are breast-fed. Therefore, we examined the effects of human breast milk (HBM) on LR-associated immune modulation. Methods: Newborn rats were divided into 8 feeding groups, including dam-fed ± LR (106 CFU/kg bw/day, daily), formula-fed ± LR, formula with 20% (v/v) HBM-fed ± LR, and HBM-fed ± LR. Pups were fed by gavage from d1 to d3 of age. Subsequently, we measured intestinal immune cell profiles, including Tregs and tolerogenic dendritic cells (tDCs) by flow cytometry. We also measured inflammatory cytokine and chemokine levels of interleukin (IL)-1β and cytokine-induced neutrophil chemoattratant (CINC)-1 in intestinal tissue lysates by ELISA. Results and Conclusion: (1) Formula feeding increased intestinal CD3+ T cells, CD4+ helper T (TH) cells and CD11c+ DCs, pro-inflammatory effects which were reversed by HBM. (2) When comparing HBM-fed with formulafed newborns, HBM supplementation produced a lower percentage of CD4+ TH cells and a higher percentage of CD8+ (cytotoxic) T cells, while reducing protein levels of IL-1β and CINC-1 in the intestine. (3) Probiotic LR feeding maximally stimulated the percentage of intestinal Tregs and tDCs when the pups were fed HBM. In conclusion, HBM reduced formula-induced intestinal gut immune activation, and the addition of LR further promoted immune tolerance.

Twenty years ago, there was profound, international interest in developing oral human, bovine, or chicken egg-derived immunoglobulin (Ig) for the prevention and nutritional treatment of childhood malnutrition and gastrointestinal disease, including acute diarrhea and necrotizing enterocolitis. Although such Ig products were shown to be effective, with both nutritional and antidiarrheal benefits, interest waned because of their cost and because of the perceived risk of bovine serum encephalitis (BSE). BSE is no longer considered a barrier to use of oral Ig, because the WHO has declared the United States to be BSE-free since the early 2000s. Low-cost bovine-derived products with high Ig content have been developed and are regulated as medical foods. These new products, called serum bovine Igs (SBIs), facilitate the management of chronic or severe gastrointestinal disturbances in both children and adults and are regulated by the US Food and Drug Administration. Well-established applications for use of SBIs include human immunodeficiency virus (HIV)-associated enteropathy and diarrhea-predominant irritable bowel syndrome. However, SBIs and other similar products could potentially become important components of the treatment regimen for other conditions, such as inflammatory bowel disease, by aiding in disease control without immunosuppressive side effects. In addition, SBIs may be helpful in conditions associated with the depletion of circulating and luminal Igs and could potentially play an important role in critical care nutrition. The rationale for their use is to facilitate intraluminal microbial antibody coating, an essential process in immune recognition in the gut which is disturbed in these conditions, thereby leading to intestinal inflammation. Thus, oral Ig may emerge as an important "add-on" therapy for a variety of gastrointestinal and nutritional problems during the next decade.

Probiotics have been used to ameliorate gastrointestinal symptoms since ancient times. Over the past 40 years, probiotics have been shown to exert major effects on the immune system, both in vivo and in vitro. This interaction is clearly linked to gut microbes, their polysaccharide antigens, and key metabolites produced by these bacteria. At least four metabolic pathways have been implicated in mechanistic studies of probiotics, based on carefully studied animal models. Microbial-immune system crosstalk has been linked to short chain fatty acid production and signaling, tryptophan metabolism and the activation of aryl hydrocarbon receptors, nucleoside signaling in the gut, and activation of the intestinal histamine-2 receptor. Several randomized controlled trials have now shown that microbial modification by probiotics may improve gastrointestinal symptoms and multi-organ inflammation in rheumatoid arthritis, ulcerative colitis, and multiple sclerosis. Future work will need to carefully assess safety issues, selection of optimal strains and combinations, and attempts to prolong the duration of colonization of beneficial microbes.

OBJECTIVE: To dissect potential confounding effects of breast milk and formula feeding on crying + fussing, fecal calprotectin, and gut microbiota in babies with colic. We hypothesized that infant colic is associated with gut inflammation linked to intestinal dysbiosis. STUDY DESIGN: A nested case-control design of 3 of our studies was used to analyze clinical and laboratory data at presentation, comparing babies with colic with controls. All investigators other than the biostatistician were blinded during data analysis. Subjects were recruited based on their age and crying + fussy time. We screened 65 infants, 37 with colic, as defined by Barr diary (crying + fussing time >3 hours daily), who were compared with 28 noncolicky infants. RESULTS: Fecal calprotectin was elevated in babies with colic. For each mode of infant feeding (breast milk, formula, or breast + formula), infants’ fecal calprotectin was higher in babies with colic. Infants with colic had similar levels of fecal alpha diversity (richness) when compared with controls, and alpha diversity was lower in breast-fed babies. Beta diversity at the phylum level revealed significant differences in microbial population. A phylum difference resulted from reduced Actinobacteria (95% of which are Bifidobacilli) in babies with colic. Species significantly associated with colic were Acinetobacter and Lactobacillus iners. CONCLUSIONS: Colic is linked with gut inflammation (as determined by fecal calprotectin) and dysbiosis, independent of mode of feeding, with fewer Bifidobacilli. (J Pediatr 2018;203:55–61). TRIAL REGISTRATION: Clinicaltrials.gov: https://register.clinicaltrials.gov/ and https://register.clinicaltrials.gov/.

Objective To assess the safety of probiotic Lactobacillus reuteri strain Deutsche Sammlung von Mikroorganismen (DSM) 17938 with daily administration to healthy infants with colic and to determine the effect of L reuteri strain DSM 17938 on crying, fussing, inflammatory, immune, and microbiome variables. Study design We performed a controlled, double-blinded, phase 1 safety and tolerability trial in healthy breast-fed infants with colic, aged 3 weeks to 3 months, randomly assigned to L reuteri strain DSM 17938 (5 × 108 colony-forming units daily) or placebo for 42 days and followed for 134 days. Results Of 117 screened infants, 20 were randomized to L reuteri strain DSM 17938 or placebo (sunflower oil) (in a 2:1 ratio) with 80% retention. Eleven of the 20 (55%) presented with low absolute neutrophil counts ( Conclusions Daily administration of L reuteri strain DSM 17938 appears to be safe in newborn infants with colic, including those with neutropenia, which frequently coexists. A placebo response of 66% suggests that many infants with colic will have resolution within 3 weeks. Trial registration ClinicalTrials.gov : NCT01849991 .

Necrotizing enterocolitis (NEC) is an inflammatory disease with evidence of increased production of proinflammatory cytokines in the intestinal mucosa. Lactobacillus reuteri DSM 17938 (LR17938) has been shown to have anti-inflammatory activities in an experimental model of NEC. Activated effector lymphocyte recruitment to sites of inflammation requires the sequential engagement of adhesion molecules such as CD44. The phenotype of CD44+CD45RBlo separates T effector/memory (Tem) cells from naive (CD44−CD45RBhi) cells. It is unknown whether these Tem cells participate in the inflammation associated with NEC and can be altered by LR17938. NEC was induced in 8- to 10-day-old C57BL/6J mice by gavage feeding with formula and exposure to hypoxia and cold stress for 4 days. Survival curves and histological scores were analyzed. Lymphocytes isolated from mesenteric lymph nodes and ileum were labeled for CD4, CD44, CD45RB, intracellular Foxp3, and Helios and subsequently analyzed by flow cytometry. LR17938 decreased mortality and the incidence and severity of NEC. The percentage of Tem cells in the ileum and mesenteric lymph nodes was increased in NEC but decreased by LR17938. Conversely, the percentage of CD4+Foxp3+ regulatory T (Treg) cells in the intestine decreased during NEC and was restored to normal by LR17938. The majority of the Treg cells preserved by LR17938 were Helios+ subsets, possibly of thymic origin. In conclusion, LR17938 may represent a useful treatment to prevent NEC. The mechanism of protection by LR17938 involves modulation of the balance between Tem and Treg cells. These T cell subsets might be potential biomarkers and therapeutic targets during intestinal inflammation.

Key Clinical Message Muscular dystrophy has been traditionally associated with common gastrointestinal symptoms such as reflux, constipation, and dysphasia. In myotonic dystrophy, there are rare reports of chronic intestinal pseudoobstruction (CIPOS). We herein present a case of CIPOS requiring colectomy and with good results.

L-selectin (CD62L) is normally highly expressed in naive T cells. The expression levels of CD62L have been reported to be decreased on T cells during the inflammatory state. It is currently unknown whether the frequency of CD62L+ T cell subsets in the peripheral blood can be used as a marker to indicate is disease severity during inflammation. Our study evaluated whether circulating CD62L+ T cell subsets correlate with the severity of disease by testing an autoimmune condition of scurfy (sf) mouse associated with multi-organ inflammation due to regulatory T cell deficiency. We observed that scurfy mice spontaneously developed an inflammatory phenotype with a significant decrease in the percentage of CD62L-expressing CD4+ T and CD8+ T cells in the peripheral blood. The percentage of CD62L+CD4+ T and CD62L+CD8+ T cells negatively correlated with disease severity, as determined by the weight of spleen and liver, as well as the mean area of lymphocyte infiltrates in lung and liver. The percentage of CD8+ T cells also correlated directly with these markers of disease severity. To conclude, our results support the concept that circulating CD62L-expressing T cells may be used as markers of disease severity in sf mice which is equivalent to a syndrome characterized by immune dysregulation with polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX syndrome) in humans, or in other autoimmune or inflammatory conditions.

This review deals with the complications and treatment of short bowel syndrome (SBS), addressing the psychosocial, medical, and surgical complications in children receiving long-term parenteral nutrition (PN) support, as well as factors that affect the intestinal adaptation process. Whenever possible, as much of the colon as possible is retained in continuity because the colon is an avid absorber of sodium. It is also important for clinicians to be aware of the important absorption mechanisms in the different regions of the bowel. For example, resection of the terminal ileum removes vitamin B12 transporters and active sodium-coupled bile salt transporters. Treatment of patients missing the terminal ileum may require monthly vitamin B12 injections and oral bile salt binders, such as cholestyramine, when the colon is present to reduce the volume of diarrhea. Patients who do not have ileocecal valves (ICVs) are prone to small bowel bacterial overgrowth that requires treatment to facilitate the intestinal adaptation process. We discuss how the PN is decreased as enteral feedings are advanced as well as clinical monitoring and routine laboratory tests. Although much has been learned over the past 20 years about PN, major questions remain, including determining the optimal form of intravenous lipid (omega-3 preparations versus omega-6 lipids versus a combination of both) to prevent liver disease.

Recent identification of the mammalian target of rapamycin (mTOR) pathway as an amino acid-sensing mechanism that regulates protein synthesis led us to investigate its role in rotavirus diarrhea. We hypothesized that malnutrition would reduce the jejunal protein synthetic rate and mTOR signaling via its target, ribosomal p70 S6 kinase (p70S6K). Newborn piglets were artificially fed from birth and infected with porcine rotavirus on day 5 of life. Study groups included infected (fully fed and 50% protein calorie malnourished) and noninfected fully fed controls. Initially, in “worst-case scenario studies,” malnourished infected piglets were killed on days 1, 3, 5, and 11 postinoculation, and jejunal samples were compared with controls to determine the time course of injury and p70S6Kactivation. Using a 2 × 2 factorial design, we subsequently determined if infection and/or malnutrition affected mTOR activation on day 3. Western blot analysis and immunohistochemistry were used to measure total and phosphorylated p70S6K; [3H]phenylalanine incorporation was used to measure protein synthesis; and lactase specific activity and villus-crypt dimensions were used to quantify injury. At the peak of diarrhea, the in vitro jejunal protein synthetic rate increased twofold (compared with the rate in the uninfected pig jejunum), concomitant with increased jejunal p70S6Kphosphorylation (4-fold) and an increased p70S6Klevel (3-fold, P < 0.05). Malnutrition did not alter the magnitude of p70S6Kactivation. Immunolocalization revealed that infection produced a major induction of cytoplasmic p70S6Kand nuclear phospho-p70S6K, mainly in the crypt. A downregulation of semitendinosus muscle p70S6Kphosphorylation was seen at days 1–3 postinoculation. In conclusion, intestinal activation of p70S6Kwas not inhibited by malnutrition but was strongly activated during an active state of mucosal regeneration.

Three relatively new lactobacillus species that were developed from fecal samples of a 26-year-old woman (LOCK0900), a 6-year-old girl (LOCK0908), and a 5-year-old boy (LOCK0919) have been studied by Kozakova et al.1 in an attempt to define the mechanism by which the probiotics modify the allergic response. The researchers colonized germ-free (GF) mice with individual or combined strains and demonstrated a reduced allergic response to intraperitoneal sensitization with birch pollen allergen by evaluating intestinal lavage IgA, IgE, and IgG1/G2 levels. Increased Th1 versus Th2 cytokine responses in splenic and mesenteric lymph node cells were also observed. In addition, the ultrastructural appearance of the ileal tight junctions appeared to be improved with increased immunoreactive ZO-1 in the intestinal homogenate, suggesting an impact on the gut epithelial barrier.

To determine if serum levels of CIT (a nonprotein amino acid synthesized by the intestine) correlate with total parenteral nutrition (PN)-independence in children with short bowel syndrome (SBS).We prospectively obtained serum amino acid profiles over a 24-month interval from all infants with SBS 3 weeks to 4 years of age. Remaining small intestine length was recorded at surgery, and percent enteral calories tolerated (enteral calories divided by enteral plus parenteral calories x 100) was determined in 24 infants with SBS and 21 age-matched controls (blood drawn for non-gastrointestinal symptoms).Mean CIT for controls was 31 +/- 2 micromol/L. In patients with SBS (n = 24), serum CIT correlated linearly with percent enteral calories (R = 0.85; P.001) and with bowel length (R = 0.47; Por =.03). CIT level in patients with SBS weaned off PN was 30 +/- 2 micromol/L; in those subsequently weaned off PN, 20 +/- 2 micromol/L; and in those who would remain PN-dependent, 11 +/- 2 micromol/L ( Por =.01). Serum CITor =19 micromol/L had 94% sensitivity and 67% specificity for being off or coming off total PN.Serum CIT level19 micromol/L in children with SBS is associated with development of enteral tolerance and may be a useful predictive test.

Arginine, an amino acid that is nutritionally essential for the fetus and neonate, is crucial for ammonia detoxification and the synthesis of molecules with enormous importance (including creatine, nitric oxide, and polyamines). A significant nutritional problem in preterm infants is a severe deficiency of arginine (hypoargininemia), which results in hyperammonemia, as well as cardiovascular, pulmonary, neurological, and intestinal dysfunction. Arginine deficiency may contribute to the high rate of infant morbidity and mortality associated with premature births. Although hypoargininemia in preterm infants has been recognized for more than 30 years, it continues to occur in neonatal intensive care units in the United States and worldwide. On the basis of recent findings, we propose that intestinal citrulline and arginine synthesis (the major endogenous source of arginine) is limited in preterm neonates owing to the limited expression of the genes for key enzymes (e.g., pyrroline-5-carboxylate synthase, argininosuccinate synthase and lyase), thereby contributing to hypoargininemia. Because premature births in humans occur before the normal perinatal surge of cortisol (an inducer of the expression of key arginine-synthetic enzymes), its administration may be a useful tool to advance the maturation of intestinal arginine synthesis in preterm neonates. Additional benefits of cortisol treatment may include the following: 1) allowing early introduction of enteral feeding to preterm infants, which is critical for intestinal synthesis of citrulline, arginine, and polyamines as well as for intestinal motility, integrity, and growth; and 2) shortening the expensive stay of preterm infants in hospitals as a result of accelerated organ maturation and the restoration of full enteral feeding. Further studies of fetal and neonatal arginine metabolism will continue to advance our understanding of the mechanisms responsible for the survival and growth of preterm infants. This new knowledge will be beneficial for designing the next generation of enteral and parenteral amino acid solutions to optimize nutrition and health in this compromised population.

An 11-month-old female with failure to thrive was seen in an emergency room for a seizure that progressed to status epilepticus. She received several doses of diazepam and a loading dose of fosphen...

Studies have suggested a link between diet and behavior in children with autism spectrum disorders (ASDs). Parental reports of behavioral changes upon exposure to gluten and/or casein are common in clinical practice. An association between diet type, intestinal permeability (IP) ('leaky gut'), and behavior has been long proposed but not substantiated. We explored this possible association in this trial.This randomized double-blind, placebo-controlled study explored the effects of gluten and milk on IP and behavior in children with ASDs over a period of 4 weeks. IP assessed by lactulose:mannitol (L/M) sugar permeability test and behavior assessed by the Aberrant Behavior Checklist and Conners Parent Rating were measured. Gastrointestinal symptoms in both groups were also monitored.Neither the L/M ratio nor behavioral scores were different between groups exposed to gluten/dairy or placebo. The changes observed were noted to be small and not clinically significant.Our study although underpowered to show small differences does not support an association between dietary gluten/milk, IP, and behavioral changes in subjects with ASD.

Background: Epidermal growth factor (EGF) signals enterocyte proliferation via extracellular regulated kinases (ERKs). Because glutamine is required for EGF-stimulated proliferation and stimulates ERKs in intestinal cell culture, we hypothesized that glutamine and the EGF-related peptide transforming growth factor–alpha (TGF-α) would synergistically enhance repair associated with stimulation of ERKs. Methods: Thiry-Vella loops were created in juvenile pigs. One half of the loop was subjected to 2 hours of ischemia, and the other half served as control. Loops were infused daily with Ringer's solution containing 140 mmol/L glucose, 140 mmol/L glutamine, 140 mmol/L glucose plus 60 μg/L TGF-α, or 140 mmol/L glutamine plus 60 μg/L TGF-α. Results: After 2 hours of ischemia, complete villous epithelial sloughing was present. By 18 hours, villous epithelium had fully restituted, but villi remained stunted until 144 hours after injury. Glutamine + TGF-α triggered sustained increases in ERK activity compared with glucose-treated tissues (maximal at 18 hours), whereas glutamine alone or glucose + TGF-α caused only transient elevations in ERK activity. By 72 hours, villous surface area had increased to normal values with glutamine plus TGF-α treatment, whereas villi remained stunted with glucose alone, glutamine alone, or glucose plus TGF-α. Conclusions: Glutamine plus TGF-α treatment restored mucosal architecture within 72 hours of severe ischemic injury associated with sustained elevations in ERK activity. (Surgery 1999;125:186-94.)

Recovery of the ability to digest and absorb lipids is essential to the maintenance of normal nutrition in infants with bowel damage. Two intrinsic microsomal enzymes, monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT), catalyze the major pathway for intestinal triacylglycerol biosynthesis. This study describes the effects of intestinal ischemia on epithelial DGAT and MGAT activities and their recovery in response to two luminal treatments: L-glutamine (Gln), the primary intestinal fuel, and transforming growth factor-alpha (TGF-alpha), a mitogenic hormone similar to epidermal growth factor present in breast milk. Ischemic damage and recovery were analyzed in mucosa from Thiry-Vella loops in the mid-ileum of 7-wk-old pigs. Loops were subjected to 2-h occlusion of local mesenteric arteries, followed by 6 or 72 h of recovery in the presence of luminal glucose (control), Gln, or TGF-alpha. Ischemic tissue followed by 6-h recovery exhibited an approximate 50% decrease in both MGAT and DGAT activities compared with nonischemic loop tissue. At 72 h, MGAT and DGAT recovery in Gln plus TGF-alpha-treated loops was significantly greater than their corresponding 6-h peak damage levels (p < 0.05). From 6 to 72 h, MGAT increased 4-fold and DGAT increased 3.6-fold after Gln plus TGF-alpha treatment. With other treatments, MGAT and DGAT activities increased

We report on 4 children from 2 unrelated families who appear to have the lethal ARC syndrome (arthrogryposis, renal tubular dysfunction, and cholestasis) together with the additional findings of nephrogenic diabetes insipidus and cerebral anomalies, including deafness. With increased survival time in our patients, paucity of the intrahepatic bile ductules and cholestasis progressed to cirrhosis, growth was severely impaired, and severe mental retardation became apparent. No evidence was found for peroxisomal, chromosomal, or mitochondrial disorders. We propose to amend the ARC mnemonic to ARCC-NDI (A-Arthrogryposis, R-renal Fanconi, C-cerebral, C-cholestasis, NDI-nephrogenic diabetes insipidus) to name the major manifestations of this syndrome, several of which have not been appreciated.

Necrotizing enterocolitis (NEC) results from severe intestinal inflammation in premature infants. FoxP3(+) regulatory T cells (Tregs) are central to gut homeostasis. While Treg proportions are significantly reduced in the ileums of premature infants with NEC, it is unknown whether they play a critical function in preventing NEC. This study investigated Treg development in newborn rat pups and their role in experimental NEC induction. Utilizing an established rat model of experimental NEC, the ontogeny of T cells and Tregs in newborn pups was characterized by flow cytometry. To investigate the functions of Tregs, newborn pups were given Tregs harvested from adult rats prior to NEC induction to assess clinical improvement and mechanisms of immune regulation. The results revealed that there were few Treg numbers in the terminal ileums of newborn rats and 8-fold reduction after NEC. Adoptive transfer of Tregs significantly improved weight loss, survival from 53% to 88%, and NEC incidence from 87% to 35%. The Tregs modulated the immune response as manifested in reduced CD80 expression on antigen presenting cells and decreased T cell activation within the mesenteric lymph nodes. These findings suggest that while Tregs are present in the intestines, their numbers might be insufficient to dampen the excessive inflammatory state in NEC. Adoptive transfer of Tregs attenuates the severity of NEC by limiting the immune response. Strategies to enhance Tregs have a therapeutic potential in controlling the development of NEC.

To investigate the effect of protein-energy malnutrition on intestinal barrier function during rotavirus enteritis in a piglet model.Newborn piglets were allotted at day 4 of age to the following treatments: (1) full-strength formula (FSF)/noninfected; (2) FSF/rotavirus infected; (3) half-strength formula (HSF)/noninfected; or (4) HSF/rotavirus infected. After one day of adjustment to the feeding rates, pigs were infected with rotavirus and acute effects on growth and diarrhea were monitored for 3 d and jejunal samples were collected for Ussing-chamber analyses.Piglets that were malnourished or infected had lower body weights on days 2 and 3 post-infection (P0.05). Three days post-infection, marked diarrhea and weight loss were accompanied by sharp reductions in villus height (59%) and lactase activity (91%) and increased crypt depth (21%) in infected compared with non-infected pigs (P0.05). Malnutrition also increased crypt depth (21%) compared to full-fed piglets. Villus:crypt ratio was reduced (67%) with viral infection. There was a trend for reduction in transepithelial electrical resistance with rotavirus infection and malnutrition (P = 0.1). (3)H-mannitol flux was significantly increased (50%; P0.001) in rotavirus-infected piglets compared to non-infected piglets, but there was no effect of nutritional status. Furthermore, rotavirus infection reduced localization of the tight junction protein, occludin, in the cell membrane and increased localization in the cytosol.Overall, malnutrition had no additive effects to rotavirus infection on intestinal barrier function at day 3 post-infection in a neonatal piglet model.

To investigate recruitment, retention, and estimates for effects of formula supplementation with Lactobacillus rhamnosus GG (LGG) on inflammatory biomarkers and fecal microbial community in infants with colic.A prospective, double-blind, placebo-controlled trial was conducted in otherwise healthy infants with colic. We screened 74 infants and randomized and analyzed results in 20 infants [9 receiving LGG (LGG+) and 11 not receiving LGG (LGG-)]. LGG was incorporated in the formula (Nutramigen(®)) (minimum of 3 × 10(7) CFU/d) in the LGG+ group. Fecal microbiota and inflammatory biomarkers, including fecal calprotectin (FC), plasma cytokines, circulating regulatory T cells (Tregs), and crying + fussing time were analyzed to determine optimal time points and effect sizes for a larger trial.Recruitment in this population was slow, with about 66% of eligible infants willing to enroll; subject retention was better (75%). These rates were influenced by parents' reluctance to volunteer their infant for a clinical trial and by their tendency to change formulas. The maximal difference of crying + fussing time was observed at day 14, comparing the 2 groups, with a mean difference of -91 (95%CI: -76, 259) min (P = NS). FC showed no significant difference, but the optimal time to determine a potential effect was at day 90 [with a mean difference of 121 (95%CI: -48, 291) μg/g stool], observing a lower level of FC in the LGG+ group. The fecal microbial communities were chaotic, as determined by Shannon's diversity index and not apparently influenced by the probiotic. No significant change was observed in plasma inflammatory cytokines or Tregs, comparing LGG+ to LGG- groups.Designing future colic trials involving a probiotic-supplemented formula for infants in the United States will require consideration for difficult enrollment. Infants with colic have major variations in feal microbiota and calprotectin, both of which improve with time, with optimal time points for measurement at days 14 and 90 after treatment.

Arginine (Arg) is deficient in the serum of the preterm neonate and is lower in those developing intestinal ischemia. We investigated whether Arg or its precursor, citrulline (Cit), protects intestinal tight junctions (TJs) from hypoxia (HX) and determined whether inducible nitric oxide (NO) plays a role.Neonatal piglet jejunal IPEC-J2 cell monolayers were treated with Arg or Cit, reversible and irreversible NO synthetase (NOS) inhibitors, and were exposed to HX. TJs were assessed by serial measurements of transepithelial electrical resistance (TEER), flux of inulin-fluorescein isothiocyanate, and immunofluorescent staining of TJ proteins.We found that Arg and Cit were protective against HX-related damage. At the final time point (14 h), the mean TEER ratio (TEER as compared with baseline) for Arg + HX and Cit + HX was significantly higher than that for HX alone. Both Arg and Cit were associated with decreased inulin flux across hypoxic monolayers and qualitatively preserved TJ proteins. Irreversible inhibition of NOS blocked this protective effect. Lipid peroxidation assay showed that our model did not produce oxidant injury.Arg and Cit, via a mechanism dependent on NO donation, protected intestinal epithelial integrity.

Background There are few carefully-designed studies investigating the safety of individual probiotics approved under Investigational New Drug policies. Objectives The primary aim of this prospective, double-blind placebo-controlled trial was to investigate if daily treatment of adults with Lactobacillus reuteri DSM 17938 (LR) for 2 months is safe and well-tolerated. Our secondary aim was to determine if LR treatment has immune effects as determined by regulatory T cell percentages, expression of toll-like receptors (TLR)-2 and −4 on circulating peripheral blood mononuclear cells (PMBCs), cytokine expression by stimulated PBMC, and intestinal inflammation as measured by fecal calprotectin. Methods Forty healthy adults were randomized to a daily dose of 5×108 CFUs of LR (n = 30) or placebo (n = 10) for 2 months. Participants completed a daily diary card and had 7 clinic visits during treatment and observation. Results There were no severe adverse events (SAEs) and no significant differences in adverse events (AEs). There were no differences in PBMC subclasses, TLRs, or cytokine expression after treatment. The probiotic-treated group had a significantly higher fecal calprotectin level than the placebo group after 2 months of treatment: 50 µg/g (IQR 24–127 µg/g) vs. 17 µg/g (IQR 11–26 µg/g), p = 0.03, although values remained in the normal clinical range (0–162.9 µg/g). LR vials retained >108 CFUs viable organisms/ml. Conclusions LR is safe and well tolerated in adults, without significant changes in immunologic markers. There was a small but significant increase in fecal calprotectin, perhaps indicating some element of immune recognition at the intestinal level. Trial Registration Clinical Trials.gov NCT00922727

Rotavirus enteritis is the leading cause of diarrhea in infants worldwide. A research priority of the World Health Organization is to develop oral rehydration solutions containing amino acids or other additives that will stimulate intestinal absorption more efficiently than the current glucose-based oral rehydration solutions. Glutamine is the principal metabolic fuel of the small bowel and a putative stimulator of mucosal repair. This report describes the transport response to mucosal L-glutamine following intestinal injury caused by porcine rotavirus. Peak symptoms and mucosal damage were observed 2-7 days after oral rotavirus inoculation. In vitro transport studies of the maximally injured region, the midjejunum (80% reduction in lactase), surprisingly, showed transport responses to L-glutamine (30 mmol/L) and L-alanine (30 mmol/L) that were similar qualitatively and quantitatively to those observed in control tissue. Subsequent application of mucosal D-glucose (30 mmol/L) caused additional stimulation of electrogenic Na+ transport, but the response to glucose was blunted (P less than 0.05) in the infected tissues. Glutamine and alanine enhanced Na+ absorption to a similar degree (2-2.5 muEq.cm-2.h-1), but glutamine stimulated equal amounts of electrogenic and electroneutral NaCl absorption, whereas alanine had no significant effect on net Cl- flux. Glutamine is a potentially useful substrate for investigation in oral rehydration solutions for infant diarrhea.

Microvillus inclusion disease is an inherited intestinal brush border membrane defect that causes severe fluid and electrolyte malabsorption. In an infant with microvillus inclusion disease (confirmed by electron microscopic evaluation of rectal, jejunal, and gallbladder mucosae), basal stool output was massive (> 125 mL · kg −1 · day −1 ) and was not altered by treatment with clonidine or octreotide. A proximal jejunostomy with mucous fistula was placed, allowing separation of proximal from distal tract outputs (60 mL · kg −1 · day −1 and 100 mL · kg −1 · day −1 , respectively). A 10-cm jejunal segment was excised during surgery and mounted in Ussing chambers for determination of transepithelial Na + and Cl − fluxes. Compared with intestine of normal infants, this infant's epithelium showed transmural conductance and unidirectional ion fluxes that were only 30% of normal. With respect to both Na + and Cl − , the excised jejunum was in a net secretory state. Theophylline (5 mmol/L) increased net Cl − secretion slightly. In response to mucosal d-glucose (30 mmol/L), jejunal mucosal-to-serosal Na + flux doubled. In the infant, glucose-electrolyte solution administered intrajejunally did not significantly change stool output, suggesting that all of the solution (40 mL/kg) was absorbed. Subtotal enterocolectomy, in theory, could have decreased purging by 66% in this infant with microvillus inclusion disease, but diarrhea would still have been significant.

l-Arginine (Arg) is synthesised from glutamine, glutamate, and proline via the intestinal-renal axis in humans and most other mammals (including pigs, sheep and rats). Arg degradation occurs via multiple pathways that are initiated by arginase, nitric-oxide synthase, Arg:glycine amidinotransferase, and Arg decarboxylase. These pathways produce nitric oxide, polyamines, proline, glutamate, creatine, and agmatine with each having enormous biological importance. Arg is also required for the detoxification of ammonia, which is an extremely toxic substance for the central nervous system. There is compelling evidence that Arg regulates interorgan metabolism of energy substrates and the function of multiple organs. The results of both experimental and clinical studies indicate that Arg is a nutritionally essential amino acid (AA) for spermatogenesis, embryonic survival, fetal and neonatal growth, as well as maintenance of vascular tone and hemodynamics. Moreover, a growing body of evidence clearly indicates that dietary supplementation or intravenous administration of Arg is beneficial in improving reproductive, cardiovascular, pulmonary, renal, gastrointestinal, liver and immune functions, as well as facilitating wound healing, enhancing insulin sensitivity, and maintaining tissue integrity. Additionally, Arg or l-citrulline may provide novel and effective therapies for obesity, diabetes, and the metabolic syndrome. The effect of Arg in treating many developmental and health problems is unique among AAs, and offers great promise for improved health and wellbeing of humans and animals.

We previously showed that phosphorylation of p70 S6 kinase (p70(S6k)) in the intestine is increased during viral enteritis. In this study, we hypothesized that during rotavirus infection, oral Arg, which stimulates p70(S6k) activation, will further stimulate intestinal protein synthesis and mucosal recovery, whereas the p70(S6k) inhibitor rapamycin (Rapa) will inhibit mucosal recovery. Newborn piglets were fed a standard milk replacer diet supplemented with Arg (0.4 g x kg(-1) x d(-1), twice daily by gavage), Rapa (2 mg x m(-2) x d(-1)), Arg + Rapa, or saline (controls). They were infected on d 6 of life with porcine rotavirus. Three days postinoculation, we measured the piglets' body weight, fecal rotavirus excretion, villus-crypt morphology, epithelial electrical resistance in Ussing chambers, and p70(S6k) activation by Western blotting and immunohistochemistry. We previously showed a 2-fold increase in jejunal protein synthesis during rotavirus diarrhea. In this experiment, Arg stimulated jejunal protein synthesis 1.3-fold above standard medium, and the Arg stimulation was partially inhibited by Rapa. Small bowel stimulation of p70(S6k) phosphorylation and p70(S6k) levels were inhibited >80% by Rapa. Immunohistochemistry revealed a major increase of p70(S6k) and ribosomal protein S6 phosphorylation in the crypt and lower villus of the infected piglets. However, in Arg-treated piglets, p70(S6k) activation occurred over the entire villus. Jejunal villi of the Rapa-treated group showed inactivation of p70(S6k) and a decrease in mucosal resistance (reflecting increased permeability), the latter of which was reversed by Arg. We conclude that, early in rotavirus enteritis, Arg has no impact on diarrhea but augments intestinal protein synthesis in part by p70(S6k) stimulation, while improving intestinal permeability via a mammalian target of rapamycin/p70(S6k)-independent mechanism.

To evaluate and compare the biochemical and histologic effect of parenteral fish oil lipid emulsion that is rich in omega-3 polyunsaturated fatty acids (O3FAs), Omegaven (Fresenius Kabi AG, Bad Homburg, Germany) with standard omega-6 polyunsaturated fatty acid (O6FA) parenteral nutrition.Comparison of hepatic explant pathology and biochemical outcome on pediatric patients with intestinal failure treated with either parental O3FA or O6FA who had received a liver-inclusive intestine transplant.Seven liver-inclusive intestinal transplants were performed in 7 patients who received O3FA for a mean of 62% ± 13% of total patient life-span (16.1 ± 7.0 months) before transplant. Median total bilirubin fell from 6.9 mg/dL at the start of treatment to 0.7 mg/dL at the time transplant (P.02), which was a significant decrease compared with the similarly matched O6FA cohort (P = .012). All 7 of the 03FA-treated patients received a liver-inclusive intestinal transplant had advanced fibrosis (stage 3 or 4) noted on explant pathologic examination, despite a resolution of cholestasis at the time of transplant. Histologic inflammatory scores were lower (P = .056) in the 03FA group with similar degrees of advanced fibrosis as in the O6FA group.In a matched comparison of patients undergoing intestinal transplantation with a history of extended O3FA lipid emulsion therapy that successfully reversed hyperbilirubinemia, significant hepatic fibrosis was present in the explanted livers despite a reduction in inflammation. This result confirms concern that the use of O3FA may have a limited role in altering the development of hepatic fibrosis from parenteral nutrition.

ResearchAssessment of mucosal immunopathology and molecular and cellular biology of persistent diarrhea in representative populations in developing countries.Studies of small bowel microbiology in PD, especially in at-risk populations e.g., malnourished children and HIV endemic areas.Evaluation of t