Fungi: Friend or Foe? A Mycobiome Evaluation in Children With Autism and Gastrointestinal Symptoms.

ABSTRACT: 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.

Infant Colic Represents Gut Inflammation and Dysbiosis.

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. TRIAL REGISTRATION: Clinicaltrials.gov: NCT01279265 and NCT01849991.

Lactobacillus reuteri for Infants with Colic: A Double-Blind, Placebo-Controlled, Randomized Clinical Trial.

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 (<1500/mm3), which resolved in all subjects by day 176. L reuteri strain DSM 17938 produced no severe adverse events and did not significantly change crying time, plasma bicarbonate, or inflammatory biomarkers. Fecal calprotectin decreased rapidly in both groups. In the infants with dominant fecal gram negatives (Klebsiella, Proteus, and Veillonella), resolution of colic was associated with marked decreases in these organisms. 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.

Oral administration of surfactant protein-a reduces pathology in an experimental model of necrotizing enterocolitis.

OBJECTIVES: Necrotizing enterocolitis (NEC) frequently results in significant morbidity and mortality in premature infants. Others reported that mice deficient in pulmonary surfactant protein-A (SP-A) born and raised in a nonhygienic environment succumb to significant gastrointestinal tract pathology, and enteral administration of purified SP-A significantly reduced mortality. We hypothesized that oral administration of purified SP-A can ameliorate pathology in an experimental model of neonatal NEC. METHODS: Experimental NEC was induced in newborn Sprague-Dawley rat pups by daily formula gavage and intermittent exposure to hypoxia. Purified human SP-A (5 µg/day) was administered by oral gavage. After 4 days, surviving pups were sacrificed, and intestinal pathology was assessed by histological examination of distal terminal ileal sections. Intestinal levels of inflammatory cytokines (IL-1ß, IFN-γ, and TNF-α) were assessed by enzyme-linked immunosorbent assay and levels of Toll-like receptor 4 (TLR4) by Western analysis. RESULTS: Sixty-one percent of the gavaged rat pups that survived to day 4 met the criteria for experimental NEC after hypoxia, whereas treatment with SP-A significantly reduced mortality and assessment of NEC. Intestinal levels of proinflammatory cytokines were significantly increased in pups exposed to hypoxia. Administration of SP-A to pups exposed to hypoxia significantly reduced IL-1ß and TNF-α levels, but had little effect on elevated levels of IFN-γ. SP-A treatment of hypoxia-exposed pups significantly reduced expression of intestinal TLR4, key in NEC pathogenesis. CONCLUSIONS: In a rat model of experimental neonatal NEC, oral administration of SP-A reduces intestinal levels of proinflammatory cytokines and TLR4 protein and ameliorates adverse outcomes associated with gastrointestinal pathologies.

Are 'leaky gut' and behavior associated with gluten and dairy containing diet in children with autism spectrum disorders?

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.

Lactobacillus reuteri DSM 17938 differentially modulates effector memory T cells and Foxp3+ regulatory T cells in a mouse model of necrotizing enterocolitis.

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(+)CD45RB(lo) separates T effector/memory (Tem) cells from naive (CD44(-)CD45RB(hi)) 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.

Lactobacillus reuteri strains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF-κB signaling in the intestine.

Necrotizing enterocolitis (NEC) is the leading gastrointestinal cause of mortality and morbidity in the premature infant. Premature infants have a delay in intestinal colonization by commensal bacteria and colonization with potentially pathogenic organisms. Lactobacillus reuteri is a probiotic that inhibits enteric infections, modulates the immune system, and may be beneficial to prevent NEC. In previous studies, L. reuteri strains DSM 17938 and ATCC PTA 4659 differentially modulated inflammation in vitro; however, the strains had equivalent anti-inflammatory responses in LPS feeding-induced ileitis in neonatal rats in vivo. The impact of these two strains in the prevention of NEC has not been previously investigated. NEC was induced in newborn rats by orogastric formula feeding and exposure to hypoxia. L. reuteri was added to the formula to prevent NEC. NEC score, Toll-like receptor (TLR)-signaling genes, phospho-IκB activity, and cytokine levels in the intestine were examined. Both strains significantly increased survival rate and decreased the incidence and severity of NEC, with optimal effects from DSM 17938. In response to probiotic, mRNA expression of IL-6, TNF-α, TLR4, and NF-κB was significantly downregulated, while mRNA levels of anti-inflammatory cytokine IL-10 were significantly upregulated. In parallel, L. reuteri treatment led to decrease intestinal protein levels of TLR4 and cytokine levels of TNF-α and IL-1ß in newborn rats with NEC. Both strains significantly inhibited not only intestinal LPS-induced phospho-IκB activity in an ex vivo study but also decreased the levels of phospho-IκB in the intestines of NEC rat model. Cow milk formula feeding produced a similar but milder proinflammatory profile in the intestine that was also ameliorated by 17938. Our studies demonstrate that each of the two L. reuteri strains has potential therapeutic value in our NEC model and in enteritis associated with cow milk feeding. These results support the concept that L. reuteri may represent a valuable treatment to prevent NEC.

Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum.

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.

Human-derived probiotic Lactobacillus reuteri strains differentially reduce intestinal inflammation.

Lactobacillus reuteri (L. reuteri) is a probiotic that inhibits the severity of enteric infections and modulates the immune system. Human-derived L. reuteri strains DSM17938, ATCC PTA4659, ATCC PTA 5289, and ATCC PTA 6475 have demonstrated strain-specific immunomodulation in cultured monocytoid cells, but information about how these strains affect inflammation in intestinal epithelium is limited. We determined the effects of the four different L. reuteri strains on lipopolysaccharide (LPS)-induced inflammation in small intestinal epithelial cells and in the ileum of newborn rats. IPEC-J2 cells (derived from the jejunal epithelium of a neonatal piglet) and IEC-6 cells (derived from the rat crypt) were treated with L. reuteri. Newborn rat pups were gavaged cow milk formula supplemented with L. reuteri strains in the presence or absence of LPS. Protein and mRNA levels of cytokines and histological changes were measured. We demonstrate that even though one L. reuteri strain (DSM 17938) did not inhibit LPS-induced IL-8 production in cultured intestinal cells, all strains significantly reduced intestinal mucosal levels of KC/GRO (∼IL-8) and IFN-γ when newborn rat pups were fed formula containing LPS ± L. reuteri. Intestinal histological damage produced by LPS plus cow milk formula was also significantly reduced by all four strains. Cow milk formula feeding (without LPS) produced mild gut inflammation, evidenced by elevated mucosal IFN-γ and IL-13 levels, a process that could be suppressed by strain 17938. Other cytokines and chemokines were variably affected by the different strains, and there was no toxic effect of L. reuteri on intestinal cells or mucosa. In conclusion, L. reuteri strains differentially modulate LPS-induced inflammation. Probiotic interactions with both epithelial and nonepithelial cells in vivo must be instrumental in modulating intrinsic anti-inflammatory effects in the intestine. We suggest that the terms anti- and proinflammatory be used only to describe the effects of a probiotic in the living host.

Changes in intestinal Toll-like receptors and cytokines precede histological injury in a rat model of necrotizing enterocolitis.

It is unclear whether the broad inflammatory response shown in neonatal necrotizing enterocolitis (NEC) is the cause or the effect of tissue injury. Toll-like receptors (TLRs) on intestinal dendritic, mononuclear, and epithelial cells recognize bacterial ligands and damaged tissues, thus activating the inflammatory response. The present study aimed to determine whether active TLR signaling would precede histological injury in NEC. Newborn rat pups were divided into four groups: dam fed, dam fed-hypoxic, formula fed, and formula fed-hypoxic (NEC). The ileal tissues were evaluated for NEC scores at 24, 48, 72, and 120 h. Quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry were used to measure and localize intestinal TLRs. Cytokines were assessed by a multispot cytokine array. Among the four groups, ileal injury was seen only after 72 h of formula feeding and hypoxia. We found selective induction of mRNA levels in NEC compared with dam-fed controls for TLR2 > TLR4 > TLR1 = TLR3, TLR7, and TLR9 > TLR6 (P < 0.01); TLR5 was downregulated (P < 0.01). All TLR changes started at 48 h, before any histological evidence of NEC. Both Th1-type cytokines (IFN-gamma, IL-1beta, TNF-alpha, and KC/GRO) and Th2-type cytokines (IL-4, IL-5 and IL-13) were significantly increased in NEC but also in nondamaged formula-fed rat ileum. In conclusion, the intestinal expression of TLRs and cytokines precedes histological injury in the experimental NEC.

Altered fecal microflora and increased fecal calprotectin in infants with colic.

OBJECTIVE: We explored whether gut inflammation, colonic fermentation, and/or an altered colonic flora could provide a pathophysiological mechanism for colic. STUDY DESIGN: The study population consisted of 36 term infants ranging in age from 14 to 81 days. We measured fecal calprotectin (a marker of neutrophil infiltration) by ELISA; stool microorganisms by denaturing gradient gel electrophoresis, cloning, and sequencing; and breath hydrogen levels using gas chromatography. RESULTS: During 24 hours, infants with colic (n = 19) cried and fussed for a mean of 314 +/- 36 (SEM) minutes, compared with control infants (n = 17, 103 +/- 17 minutes). Fecal calprotectin levels were 2-fold higher in infants with colic than in control infants (413 +/- 71 vs 197 +/- 46 microg/g, P = .042). Stools of infants with colic had fewer identifiable bands on denaturing gradient gel electrophoresis. Klebsiella species were detected in more colic patients than in control patients (8 vs 1, P = .02), whereas Enterobacter/Pantoea species were detected only in the control patients. These differences could not be attributed to differences in formula versus breast milk feeding, consumption of elemental formula, or exposure to antibiotics. CONCLUSIONS: Infants with colic, a condition previously believed to be nonorganic in nature, have evidence of intestinal neutrophilic infiltration and a less diverse fecal microflora.

Infantile Colic: New Insights into an Old Problem.

Infant colic is a characteristic group of behaviors seen in young infants. The most prominent feature is prolonged crying. Additional characteristics, including clenching of the fists and flexion of the hips, have led to the suggestion that these behaviors are related to abdominal discomfort. In this article, we show emerging evidence to support the concept that infant colic could represent gut inflammation and microbial dysbiosis that impacts brain function and even brain development.

Hypoallergenic formula with Lactobacillus rhamnosus GG for babies with colic: A pilot study of recruitment, retention, and fecal biomarkers.

AIM: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Lactobacillus reuteri DSM 17938 changes the frequency of Foxp3+ regulatory T cells in the intestine and mesenteric lymph node in experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is an inflammatory disease of the intestine in premature infants. Lactobacillus reuteri DSM 17938 improves survival and reduces the incidence and severity of NEC in a rodent model. Foxp3(+) regulatory T cells (Tregs) maintain intestinal homeostasis by controlling inflammation and inducing tolerance. To determine whether there are insufficient numbers of Tregs to control inflammation in NEC and to determine if LR17938 increases the frequency of Tregs, we studied selected groups of newborn Sprague-Dawley rats according to feeding plan: dam±LR17938, formula±LR17938, and NEC±LR17938. NEC was induced by gavage feeding with special formula and exposure to hypoxic conditions. Lymphocytes isolated from ileum, mesenteric lymph nodes (MLN), spleen and thymus were labeled for T cell surface markers (CD3, CD4, CD8) and intracellular Foxp3; and labeled cells were analyzed by flow cytometry. The percentage of CD3(+) T cells and Foxp3(+) Tregs in the ileum significantly decreased in pups with NEC, compared to normal controls. Feeding LR17938 to neonatal rats with NEC increased the % of Foxp3(+) T cells in the ileum while decreasing the percentage of cells in the MLN. Administration of LR17938 to dam-fed rats significantly increased Foxp3(+)Tregs in the ileum as early as day of life (DOL)1 but did not produce an increase in Tregs in formula-fed rats on DOL1. These results suggest that factors in breast milk may enhance the early immunomodulatory effects of LR17938. An anti-inflammatory effect of LR17938 in NEC was associated with the modulation of immune responses and induction and what appears to be migration of Foxp3(+) Tregs to the diseased gut. Probiotic-facilitated development of Tregs might play an important role in the prevention of NEC.

FoxP3⁺ regulatory T cells attenuate experimental necrotizing enterocolitis.

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.

Safety and tolerability of Lactobacillus reuteri DSM 17938 and effects on biomarkers in healthy adults: results from a randomized masked trial.

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 × 10(8) 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 >10(8) 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.