Paediatric Inflammatory Bowel Disease and its Relationship with the Microbiome.

Paediatric inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the digestive tract, comprising of Crohn's disease (CD), ulcerative colitis (UC), and, where classification is undetermined, inflammatory bowel disease unclassified (IBDU). Paediatric IBD incidence is increasing globally, with prevalence highest in the developed world. Though no specific causative agent has been identified for paediatric IBD, it is believed that a number of factors may contribute to the development of the disease, including genetics and the environment. Another potential component in the development of IBD is the microbiota in the digestive tract, particularly the gut. While the exact role that the microbiome plays in IBD is unclear, many studies acknowledge the complex relationship between the gut bacteria and pathogenesis of IBD. In this review, we look at the increasing number of studies investigating the role the microbiome and other biomes play in paediatric patients with IBD, particularly changes associated with IBD, varying disease states, and therapeutics. The paediatric IBD microbiome is significantly different to that of healthy children, with decreased diversity and differences in bacterial composition (such as a decrease in Firmicutes). Changes in the microbiome relating to various treatments of IBD and disease severity have also been observed in multiple studies. Changes in diversity and composition may also extend to other biomes in paediatric IBD, such as the virome and the mycobiome. Research into biome differences in IBD paediatric patients may help progress our understanding of the aetiology of the disease.

A Double-Blind, Placebo-Controlled Trial to Assess Safety and Tolerability of (Thetanix) Bacteroides thetaiotaomicron in Adolescent Crohn's Disease.

INTRODUCTION: Thetanix (gastroresistant capsules containing lyophilized Bacteroides thetaiotaomicron) is a live biotherapeutic, under development for Crohn's disease, that antagonizes transcription factor nuclear factor kappa B, reducing proinflammatory cytokines, particularly tumor necrosis factor alpha. We aimed to assess safety and tolerability in adolescents with Crohn's disease in remission. METHODS: Subjects who were 16-18 years with Crohn's in remission (weighted pediatric Crohn's disease activity index <12.5) were recruited. Each active dose comprised ∼108.2±1.4 colony forming units of B. thetaiotaomicron (randomized 4:1 active:placebo). Part A was single dose. Part B involved 7.5 days twice daily dosing. Serial stools were analyzed for calprotectin, 16S rRNA sequencing, and B. thetaiotaomicron real-time polymerase chain reaction. Bloods were taken serially. Subjects reported adverse events and recorded temperature twice daily. RESULTS: Fifteen subjects were treated-8 in part A (75% men, median 17.1 years) and 10 in part B, including 3 from part A (80% men, median 17.1 years); all 18 completed. Seventy percent took concurrent immunosuppression. Reported compliance was >99% in part B. Two subjects reported adverse events deemed related-one in part A with eructation, flatulence, and reflux; one in part B with dizziness, abdominal pain, and headache. No serious adverse events were reported. There was no significant change in median calprotectin across part B (87.8 [4.4-447] to 50.5 [5.3-572], P = 0.44 by the Fisher exact test in the active group). No significant differences were found in microbiota profiles, but diversity seemed to increase in treated subjects. DISCUSSION: Thetanix, after single and multiple doses, was well tolerated. Although the numbers in this study were small, the safety profile seems good. Future studies should explore efficacy.

EPCAM mutation update: Variants associated with congenital tufting enteropathy and Lynch syndrome.

The epithelial cell adhesion molecule gene (EPCAM, previously known as TACSTD1 or TROP1) encodes a membrane-bound protein that is localized to the basolateral membrane of epithelial cells and is overexpressed in some tumors. Biallelic mutations in EPCAM cause congenital tufting enteropathy (CTE), which is a rare chronic diarrheal disorder presenting in infancy. Monoallelic deletions of the 3' end of EPCAM that silence the downstream gene, MSH2, cause a form of Lynch syndrome, which is a cancer predisposition syndrome associated with loss of DNA mismatch repair. Here, we report 13 novel EPCAM mutations from 17 CTE patients from two separate centers, review EPCAM mutations associated with CTE and Lynch syndrome, and structurally model pathogenic missense mutations. Statistical analyses indicate that the c.499dupC (previously reported as c.498insC) frameshift mutation was associated with more severe treatment regimens and greater mortality in CTE, whereas the c.556-14A>G and c.491+1G>A splice site mutations were not correlated with treatments or outcomes significantly different than random simulation. These findings suggest that genotype-phenotype correlations may be useful in contributing to management decisions of CTE patients. Depending on the type and nature of EPCAM mutation, one of two unrelated diseases may occur, CTE or Lynch syndrome.

Fecal microbiota variation across the lifespan of the healthy laboratory rat.

Laboratory rats are commonly used in life science research as a model for human biology and disease, but the composition and development of their gut microbiota during life is poorly understood. We determined the fecal microbiota composition of healthy Sprague Dawley laboratory rats from 3 weeks to 2 y of age, kept under controlled environmental and dietary conditions. Additionally, we determined fecal short-chain fatty acid profiles, and we compared the rat fecal microbiota with that of mice and humans. Gut microbiota and to a lesser extent SCFAs profiles separated rats into 3 different clusters according to age: before weaning, first year of life (12- to 26-week-old animals) and second year of life (52- to 104-week-old). A core of 46 bacterial species was present in all rats but its members' relative abundance progressively decreased with age. This was accompanied by an increase of microbiota α-diversity, likely due to the acquisition of environmental microorganisms during the lifespan. Contrastingly, the functional profile of the microbiota across animal species became more similar upon aging. Lastly, the microbiota of rats and mice were most similar to each other but at the same time the microbiota profile of rats was more similar to that of humans than was the microbiota profile of mice. These data offer an explanation as to why germ-free rats are more efficient recipients and retainers of human microbiota than mice. Furthermore, experimental design should take into account dynamic changes in the microbiota of model animals considering that their changing gut microbiota interacts with their physiology.

Oral Iron Treatment Response and Predictors in Anaemic Adolescents and Adults with IBD: A Prospective Controlled Open-Label Trial.

BACKGROUND: Because of previous concerns about the efficacy and safety of oral iron for treating iron deficiency anaemia in inflammatory bowel disease [IBD], particularly in young people, we compared the effects of ferrous sulphate on haemoglobin response, disease activity and psychometric scores in adolescents and adults with IBD. We also assessed the relation of baseline serum hepcidin to haemoglobin response. METHODS: We undertook a prospective, open-label, 6-week non-inferiority trial of the effects of ferrous sulphate 200 mg twice daily on haemoglobin, iron status, hepcidin, disease activity (Harvey-Bradshaw Index, Simple Colitis Clinical Activity Index, C-reactive protein [CRP]), faecal calprotectin and psychometric scores in 45 adolescents [age 13-18 years] and 43 adults [>18 years]. RESULTS: On intention-to-treat analysis, ferrous sulphate produced similar rises in haemoglobin in adolescents {before treatment 10.3 g/dl [0.18] (mean [SEM]), after 11.7 [0.23]: p < 0.0001} and adults (10.9 g/dl [0.14], 11.9 [0.19]: p < 0.0001); transferrin saturation, ferritin [in adolescents] and hepcidin [in adults] also increased significantly. On per-protocol univariate analysis, the haemoglobin response was inversely related to baseline haemoglobin, CRP and hepcidin. Oral iron did not alter disease activity; it improved Short IBDQ and Perceived Stress Questionnaire scores in adults. CONCLUSION: Oral ferrous sulphate was no less effective or well-tolerated in adolescents than adults, and did not increase disease activity in this short-term study. The inverse relation between baseline CRP and hepcidin levels and the haemoglobin response suggests that CRP or hepcidin measurements could influence decisions on whether iron should be given orally or intravenously. [ClinTrials.gov registration number NCT01991314].

Assessing the feasibility of injectable growth-promoting therapy in Crohn's disease.

BACKGROUND: Despite optimal therapy, many children with Crohn's disease (CD) experience growth retardation. The objectives of the study are to assess the feasibility of a randomised control trial (RCT) of injectable forms of growth-promoting therapy and to survey the attitudes of children with CD and their parents to it. METHODS: A feasibility study was carried out to determine study arms, sample size and numbers of eligible patients. A face-to-face questionnaire surveyed willingness to consent to future participation in the RCT. Eligibility to the survey was any child under 18 (with their parent/guardian) with CD whose height standard deviation score (HtSDS) was ≤+1. Of 118 questionnaires, 94 (80%) were returned (48 by children and 46 by parents). RESULTS: The median age of the patients in the survey was 14.3 years (range 7.0 to 17.7), and 35 (73%) were male. Their median HtSDS was -1.2 (-3.01, 0.23), and it was lower than the median mid-parental HtSDS of -0.6 (-3.1, 1.4). We analysed the willingness of the children whose HtSDS <-1 to take part in the proposed RCT, being those most likely to require treatment. Overall, 18 (47%) children and 17 (46%) parents were willing. This increased to 61% of children who were slightly concerned about their height and 100% (4/4) of those very concerned. A common reason for not taking part in the RCT was fear of injections (44%); 111 children are required for randomisation into three study arms from nine centres. CONCLUSIONS: Almost half of children and parents surveyed would take part in an RCT of growth-promoting therapy. Allaying fears about injections may result in higher recruitment rates.

Growth problems in children with IBD.

Crohn's disease in childhood causes linear growth retardation, which has a substantial effect on management of this disease. By contrast, growth is rarely a problem in children presenting with ulcerative colitis. Depending on how growth failure is defined, approximately one-third of children with Crohn's disease have growth retardation at diagnosis. Although corticosteroids can suppress growth, decreased height at diagnosis demonstrates that this finding is a consequence of the disease and not merely an adverse effect of treatment. Both inflammation and undernutrition contribute to decreased height velocity. Increased cytokine production acts both on the hepatic expression of insulin-like growth factor 1 (IGF-1) and at chondrocytes of the growth plates of long bones. Growth hormone insensitivity caused by deranged immune function is a major mechanism in growth retardation. Resolution of inflammation is the cornerstone of treatment, but current studies on growth hormone and IGF-1 might yield therapies for those children whose inflammation is refractory to treatment.

Iron status and Helicobacter pylori infection in symptomatic children: an international multi-centered study.

OBJECTIVE: Iron deficiency (ID) and iron deficiency anaemia (IDA) are global major public health problems, particularly in developing countries. Whilst an association between H. pylori infection and ID/IDA has been proposed in the literature, currently there is no consensus. We studied the effects of H. pylori infection on ID/IDA in a cohort of children undergoing upper gastrointestinal endoscopy for upper abdominal pain in two developing and one developed country. METHODS: In total 311 children (mean age 10.7±3.2 years) from Latin America--Belo Horizonte/Brazil (n = 125), Santiago/Chile (n = 105)--and London/UK (n = 81), were studied. Gastric and duodenal biopsies were obtained for evaluation of histology and H. pylori status and blood samples for parameters of ID/IDA. RESULTS: The prevalence of H. pylori infection was 27.7% being significantly higher (p<0.001) in Latin America (35%) than in UK (7%). Multiple linear regression models revealed H. pylori infection as a significant predictor of low ferritin and haemoglobin concentrations in children from Latin-America. A negative correlation was observed between MCV (r = -0.26; p = 0.01) and MCH (r = -0.27; p = 0.01) values and the degree of antral chronic inflammation, and between MCH and the degree of corpus chronic (r = -0.29, p = 0.008) and active (r = -0.27, p = 0.002) inflammation. CONCLUSIONS: This study demonstrates that H. pylori infection in children influences the serum ferritin and haemoglobin concentrations, markers of early depletion of iron stores and anaemia respectively.

IL-1ß stimulation of CCD-18co myofibroblasts enhances repair of epithelial monolayers through Wnt-5a.

Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/ß-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1ß-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1ß-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1ß-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1ß stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1ß-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1ß-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound repair.

Prevalence and management of anemia in children, adolescents, and adults with inflammatory bowel disease.

BACKGROUND: Children and adolescents with inflammatory bowel disease (IBD) are more likely to have Crohn's disease (CD) than ulcerative colitis (UC) and their disease tends to be more extensive and severe than in adults. We hypothesized that the prevalence of anemia would therefore be greater in children and adolescents than in adults attending IBD outpatient clinics. METHODS: Using the WHO age-adjusted definitions of anemia we assessed the prevalence, severity, type, and response to treatment of anemia in patients attending pediatric, adolescent, and adult IBD clinics at our hospital. RESULTS: The prevalence of anemia was 70% (41/59) in children, 42% (24/54) in adolescents, and 40% (49/124) in adults (P < 0.01). Overall, children (88% [36/41]) and adolescents (83% [20/24]) were more often iron-deficient than adults (55% [27/49]) (P < 0.01). Multivariate logistic regression showed that both active disease (odds ratio [OR], 4.7 95% confidence interval [CI], 2.5, 8.8) and attending the pediatric clinic (OR 3.7; 95% CI, 1.6, 8.4) but not the adolescent clinic predicted iron deficiency anemia. Fewer iron-deficient children (13% [5/36]) than adolescents (30% [6/20]) or adults (48% [13/27]) had been given oral iron (P < 0.05); none had received intravenous iron compared with 30% (6/20) adolescents and 41% (11/27) adults (P < 0.0001). CONCLUSIONS: Anemia is even more common in children than in older IBD patients. Oral iron was given to half of adolescents and adults but, despite similar tolerance and efficacy, only a quarter of children with iron-deficient anemia. Reasons for the apparent underutilization of iron therapy include a perceived lack of benefit and concerns about side effects, including worsening of IBD activity.

The immunomodulatory properties of viable Lactobacillus salivarius ssp. salivarius CECT5713 are not restricted to the large intestine.

PURPOSE: The aim of this study was to better characterise the biological effects of Lactobacillus salivarius ssp. salivarius CECT5713, a probiotic with immunomodulatory properties. METHODS: Live or dead probiotic was assayed in the TNBS model of rat colitis to determine whether viability was a requisite to exert the beneficial effects. In vitro studies were also performed in Caco-2 cells to evaluate its effects on epithelial cell recovery and IL-8 production. Finally, the probiotic was assayed in the LPS model of septic shock in mice to establish its effects when there is an altered systemic immune response. RESULTS: The viability of the probiotic was required for its anti-inflammatory activity. The probiotic inhibited IL-8 production in stimulated Caco-2 cells and facilitated the recovery of damaged intestinal epithelium. In LPS-treated mice, the probiotic inhibited the production of TNFα in plasma and lungs and increased the hepatic glutathione content. These effects were associated with an improvement in the altered production of the T-cell cytokines in splenocytes, by reducing IL-2 and IL-5 and by increasing IL-10. Finally, it reduced the increased plasma IgG production in LPS-treated mice. CONCLUSION: The anti-inflammatory effects of viable L. salivarius ssp. salivarius CECT5713 are not restricted to the gastrointestinal tract.

Intestinal antimicrobial gene expression: impact of micronutrients in malnourished adults during a randomized trial.

BACKGROUND: Because both micronutrients and antimicrobial peptides protect against diarrhea, we looked for an effect on intestinal antimicrobial peptide gene expression during a randomized controlled trial of multiple micronutrient (MM) supplementation. METHODS: Consenting adults (n=287) in Lusaka, Zambia, were randomized to receive a daily MM supplement or placebo and were followed up for 3.3 years, with a crossover after 2 years. Intestinal biopsy samples were obtained at annual intervals, and messenger RNA of the intestinal antimicrobial peptides human alpha defensin (HD) 5, HD6, human beta-defensin (hBD) 1, hBD2, and LL-37 were quantified by real-time reverse-transcriptase polymerase chain reaction. Samples were also obtained during diarrhea episodes and after convalescence. RESULTS: There was no effect overall of treatment allocation. However, in malnourished adults (body mass index < or =18.5), HD5 mRNA was increased by 0.8 log transcripts/microg total RNA in MM recipients, compared with HD5 mRNA in placebo recipients (P=.007). During diarrhea, HD5 expression was reduced by 0.8 log transcripts in placebo recipients (P=.02) but was not reduced in MM recipients, nor was it reduced after the crossover. Correlations between HD5 and nutritional status were found that were sex-specific but not explained by serum leptin or adiponectin concentrations. CONCLUSIONS: Micronutrient supplementation was associated with up-regulation of HD5 only in malnourished adults. Interactions between antimicrobial gene expression and nutritional status may help to explain the increased risk of infection in individuals with malnutrition.

Gastric and intestinal barrier impairment in tropical enteropathy and HIV: limited impact of micronutrient supplementation during a randomised controlled trial.

BACKGROUND: Although micronutrient supplementation can reduce morbidity and mortality due to diarrhoea, nutritional influences on intestinal host defence are poorly understood. We tested the hypothesis that micronutrient supplementation can enhance barrier function of the gut. METHODS: We carried out two sub-studies nested within a randomised, double-blind placebo-controlled trial of daily micronutrient supplementation in an urban community in Lusaka, Zambia. In the first sub-study, gastric pH was measured in 203 participants. In the second sub-study, mucosal permeability, lipopolysaccharide (LPS) and anti-LPS antibodies, and serum soluble tumour necrosis factor receptor p55 (sTNFR55) concentrations were measured in 87 participants. Up to three stool samples were also analysed microbiologically for detection of asymptomatic intestinal infection. Gastric histology was subsequently analysed in a third subset (n = 37) to assist in interpretation of the pH data. Informed consent was obtained from all participants after a three-stage information and consent process. RESULTS: Hypochlorhydria (fasting gastric pH > 4.0) was present in 75 (37%) of participants. In multivariate analysis, HIV infection (OR 4.1; 95%CI 2.2-7.8; P < 0.001) was associated with hypochlorhydria, but taking anti-retroviral treatment (OR 0.16; 0.04-0.67; P = 0.01) and allocation to micronutrient supplementation (OR 0.53; 0.28-0.99; P < 0.05) were protective. Hypochlorhydria was associated with increased risk of salmonellosis. Mild (grade 1) gastric atrophy was found in 5 participants, irrespective of Helicobacter pylori or HIV status. Intestinal permeability, LPS concentrations in serum, anti-LPS IgG, and sTNFR55 concentrations did not differ significantly between micronutrient and placebo groups. Anti-LPS IgM was reduced in the micronutrient recipients (P <0.05). CONCLUSIONS: We found evidence of a specific effect of HIV on gastric pH which was readily reversed by anti-retroviral therapy and not mediated by gastric atrophy. Micronutrients had a modest impact on gastric pH and one marker of bacterial translocation. TRIAL REGISTRATION: Current Controlled Trials ISRCTN31173864.

Curcumin as a therapeutic agent: the evidence from in vitro, animal and human studies.

Curcumin is the active ingredient of turmeric. It is widely used as a kitchen spice and food colorant throughout India, Asia and the Western world. Curcumin is a major constituent of curry powder, to which it imparts its characteristic yellow colour. For over 4000 years, curcumin has been used in traditional Asian and African medicine to treat a wide variety of ailments. There is a strong current public interest in naturally occurring plant-based remedies and dietary factors related to health and disease. Curcumin is non-toxic to human subjects at high doses. It is a complex molecule with multiple biological targets and different cellular effects. Recently, its molecular mechanisms of action have been extensively investigated. It has anti-inflammatory, antioxidant and anti-cancer properties. Under some circumstances its effects can be contradictory, with uncertain implications for human treatment. While more studies are warranted to further understand these contradictions, curcumin holds promise as a disease-modifying and chemopreventive agent. We review the evidence for the therapeutic potential of curcumin from in vitro studies, animal models and human clinical trials.

Curcumin suppresses p38 mitogen-activated protein kinase activation, reduces IL-1beta and matrix metalloproteinase-3 and enhances IL-10 in the mucosa of children and adults with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a major source of morbidity in children and adults. Its incidence is rising, particularly in young people. IBD carries a lifelong risk of cancer, which is proportional to disease duration. Drug and surgical treatments rarely offer cure and often carry a high side effect burden. Dietary therapy is highly effective in Crohn's disease. For these reasons, there is much interest in developing novel dietary treatments in IBD. Curcumin, a component of the spice turmeric, and an anti-inflammatory and anti-cancer agent, shows preclinical and clinical potential in IBD. Its mechanisms of action are unknown. Our aim was to assess the effect of curcumin on key disease mediators p38 mitogen-activated protein kinase (MAPK), IL-1beta, IL-10 and matrix metalloproteinase-3 (MMP-3) in the gut of children and adults with IBD. Colonic mucosal biopsies and colonic myofibroblasts (CMF) from children and adults with active IBD were cultured ex vivo with curcumin. p38 MAPK, NF-kappaB and MMP-3 were measured by immunoblotting. IL-1beta and IL-10 were measured by ELISA. We show reduced p38 MAPK activation in curcumin-treated mucosal biopsies, enhanced IL-10 and reduced IL-1beta. We demonstrate dose-dependent suppression of MMP-3 in CMF with curcumin. We conclude that curcumin, a naturally occurring food substance with no known human toxicity, holds promise as a novel therapy in IBD.

Micronutrient supplementation has limited effects on intestinal infectious disease and mortality in a Zambian population of mixed HIV status: a cluster randomized trial.

BACKGROUND: Diarrheal disease remains a major contributor to morbidity and mortality in Africa, but host defense against intestinal infection is poorly understood and may depend on nutritional status. OBJECTIVE: To test the hypothesis that defense against intestinal infection depends on micronutrient status, we undertook a randomized controlled trial of multiple micronutrient supplementation in a population where there is borderline micronutrient deficiency. DESIGN: All consenting adults (> or =18 y) living in a carefully defined sector of Misisi, Lusaka, Zambia, were included in a cluster-randomized (by household), double-blind, placebo-controlled trial with a midpoint crossover. There were no exclusion criteria. Participants were given a daily tablet containing 15 micronutrients at just above the recommended nutrient intake or placebo. The primary endpoint was the incidence of diarrhea; secondary endpoints were severe episodes of diarrhea, respiratory infection, nutritional status, CD4 count, and mortality. RESULTS: Five hundred participants were recruited and followed up for 3.3 y (10,846 person-months). The primary endpoint, incidence of diarrhea (1.4 episodes/y per person), did not differ with treatment allocation. However, severe episodes of diarrhea were reduced in the supplementation group (odds ratio: 0.50; 95% CI: 0.26, 0.92; P = 0.017). Mortality was reduced in HIV-positive participants from 12 with placebo to 4 with supplementation (P = 0.029 by log-rank test), but this was not due to changes in CD4 count or nutritional status. CONCLUSION: Micronutrient supplementation with this formulation resulted in only modest reductions in severe diarrhea and reduced mortality in HIV-positive participants. The trial was registered as ISRCTN31173864.

Staphylococcal enterotoxins G and I, a cause of severe but reversible neonatal enteropathy.

BACKGROUND & AIMS: Staphylococcus aureus is recognized to produce toxins A-E and toxic shock syndrome toxin-1 associated with food poisoning and toxic shock syndrome. Enterotoxins G and I co-exist in the same S aureus strains (staphylococcal enterotoxin G and staphylococcal enterotoxin I) and are implicated in scarlet fever and toxic shock. We report these enterotoxins as causative agents of 2 cases of neonatal intractable diarrhea with enteropathy. METHODS: We used a note review for this study. Stool culture, multiplex polymerase chain reaction for enterotoxin, duodenal biopsy specimens for H&E, periodic acid-Schiff staining, and electron microscopy were used. RESULTS: Infant 1 had diarrhea from age 2 weeks and was referred at age 5 weeks with weight less than the 0.4th percentile. Infant 2 was referred at age 7 weeks with 4 weeks' of diarrhea, weight less than the 0.4th percentile. Both infants were severely malnourished. Elemental feeds were not tolerated and total parenteral nutrition was required. S aureus producing staphylococcal enterotoxin G and staphylococcal enterotoxin I was isolated in stools from both infants. Clinical improvement occurred after intravenous flucloxacillin and parenteral nutrition. Histology showed subtotal villous atrophy (H&E) with abnormal brush border (periodic acid-Schiff). Electron microscopy showed severe microvilli destruction, dilated mitochondria, and lysosomes containing cellular debris. Repeat histology was normal in infant 2, age 3 months, off parenteral nutrition, showed return to normal. Currently, both infants are 2 years of age and are thriving on a normal diet. CONCLUSIONS: Staphylococcal enterotoxin G- and I-induced enteropathy is a life-threatening condition, causing reversible disruption of enterocyte ultrastructure that responds well to supportive treatment with flucloxacillin and parenteral nutrition This condition should be a differential diagnosis of neonatal early onset diarrhea.

Dietary modulation of GALT.

Changes in diet greatly affect the mucosal immune system, particularly in diseases such as Crohn's disease and necrotizing enterocolitis. This article examines the hypothesis that alterations in the luminal environment of the intestine regulate the expression of genes in the enterocyte responsible for signaling to immune cells. Genes expressed by the epithelium orchestrate leukocytes in the lamina propria. For example, chemokine expression in the mouse intestinal epithelium, through transgenic means, induced the recruitment of neutrophils and lymphocytes into intestinal tissues. Diet alters the expression of the genes responsible for signaling by a variety of pathways. The introduction of a normal diet to a weanling mouse up-regulates MHC class II expression through a particular isoform of the class II transactivator, a protein that acts in the nucleus. SCFA concentrations in the intestinal lumen vary markedly with diet. SCFAs increase IL-8 and insulin-like growth factor binding protein-2 expression by inhibiting histone deacetylase activity in the enterocyte. Down-regulation of gene expression by butyrate can act through acetylation of the inhibitory transcription factor Sp3. The review therefore describes a number of molecular pathways, explaining how changes in diet may alter leukocyte recruitment by regulating enterocyte gene expression. Myofibroblasts enhance enterocyte chemotactic activity by cleaving inactive precursors; and myofibroblast genes also are regulated by SCFA. It is likely that other similar regulatory mechanisms remain to be discovered.

Butyrate regulates the expression of pathogen-triggered IL-8 in intestinal epithelia.

Inflammatory bowel disease (IBD) is characterized by an exaggerated immune response that involves pro-inflammatory cytokines including IL-8. Production of these pro-inflammatory cytokines is triggered by pathogen-associated molecular patterns (PAMP). Butyrate, a product of bacterial fermentation of carbohydrates, has been reported to modulate inflammation in IBD, possibly by regulating production of pro-inflammatory cytokines. However, this effect of butyrate is controversial. In this study, we used Pam3CSK4 (Pam3CysSerLys4), the acylated NH2-terminus of the bacterial lipoprotein (a PAMP), to mimic in vivo infection of pathogens. Butyrate transiently down-regulated expression of IL-8 stimulated by Pam3CSK4. Treatment of cells with butyrate before Pam3CSK4, however, enhanced production of IL-8. Furthermore, butyrate induced expression of A20, a negative regulator of the nuclear factor-kappaB pathway. Over-expression of A20 inhibited Pam3CSK4-triggered IL-8 expression. Our data suggest that the inflammatory modulation of butyrate in IBD is mediated by A20 and a short pulse rather than continuous administration of butyrate may provide a protective effect on IBD.

Genetically modified enterotoxigenic Escherichia coli vaccines induce mucosal immune responses without inflammation.

OBJECTIVE: Enterotoxigenic Escherichia coli (ETEC) is a major cause of acute diarrhoea in children in the developing world, in travellers and in the military. Safe, effective vaccines could reduce morbidity and mortality. As immunity to ETEC is strain specific, the ability to create vaccines in vitro which express multiple antigens would be desirable. It was hypothesised that three genetically attenuated ETEC strains, one with a genetic addition, would be immunogenic and safe, and they were evaluated in the first licensed UK release of genetically modified oral vaccines. METHODS: Phase 1 studies of safety and immunogenicity were carried out at a Teaching Hospital in London. Varying oral doses of any of three oral vaccines, or placebo, were administered to volunteers of both sexes (n = 98). Peripheral blood responses were measured as serum antibodies (IgG or IgA) by ELISA, antibody-secreting cell (ASC) responses by enzyme-linked immunospot (ELISPOT), and antibody in lymphocyte supernatant (ALS) by ELISA. Mucosal antibody secretion was measured by ELISA for specific IgG and IgA in whole gut lavage fluids (WGLFs). RESULTS: Significant mucosal IgA responses were obtained to colonisation factors CFA/I, CS1, CS2 and CS3, both when naturally expressed and when genetically inserted. Dose-response relationships were most clearly evident in the mucosal IgA in WGLF. Vaccines were well tolerated and did not elicit interleukin (IL) 8 or IL6 secretion in WGLF. CONCLUSIONS: Genetically modified ETEC vaccines are safe and induce significant mucosal IgA responses to important colonisation factors. Mucosal IgA responses were clearly seen in WGLF, which is useful for evaluating oral vaccines.