Tolerance development in cow's milk-allergic children receiving amino acid-based formula with synbiotics: 36-Months follow-up of a randomized controlled trial (PRESTO Study).

The objective of the present study is to assess the rates of acquired tolerance to cow's milk (CM) after 36 months in subjects who consumed amino acid-based formula with synbiotics (AAF-S) or amino acid-based formula without synbiotics (AAF) during a 1-year intervention period in early life as part of the PRESTO study (Netherlands Trial Register number NTR3725). Differences in CM tolerance development between groups were analysed using a logistic regression model. Results show that the proportion of subjects (mean [±SD] age, 3.8 ± 0.27 years) who developed CM tolerance after 36 months was similar in the group receiving AAF-S (47/60 [78%]) and in the group receiving AAF (49/66 [74%]) (p = 0.253), that is, figures comparable to natural outgrowth of CM allergy. Our data suggest that the consumption of AAF and absence of exposure to CM peptides do not slow down CM tolerance acquisition.

Dietary composition of adult eosinophilic esophagitis patients is related to disease severity.

BACKGROUND: In addition to the elimination diet, dietary composition may influence disease severity in patients with eosinophilic esophagitis (EoE) through modulation of the immune response. AIM: To explore the immunomodulatory role of nutrition before and during elimination diet in adult EoE patients. METHODS: Nutritional intake was assessed in 39 Dutch adult EoE patients participating in the Supplemental Elemental Trial (Dutch trial registry NL6014, NTR6778) using 3-day food diaries. In this randomized controlled trial, diagnosed patients received either a four-food elimination diet alone (FFED) or FFED with addition of an amino acid-based formula for 6 weeks. Multiple linear regression analyses were performed to assess associations between the intake of nutrients and food groups per 1000 kCal and peak eosinophil count/high power field (PEC), both at baseline and after 6 weeks. RESULTS: At baseline, we found a statistically significant negative (thus favorable) relationship between the intake of protein, total fat, phosphorus, zinc, vitamin B12, folate, and milk products and PEC (p < .05), while calcium (p = .058) and full-fat cheese/curd (p = .056) were borderline (favorably) significant. In contrast, total carbohydrates, prepacked fruit juice, and white bread were significantly positively (unfavorable) related to PEC (p < .05), while ultra-processed meals (p = .059) were borderline (unfavorably) significant. After dietary intervention, coffee/tea were significantly negatively (favorably) related to PEC, hummus/legumes were significantly positively (unfavorably) related with PEC, while peanuts were borderline significantly positively related (p = .058). CONCLUSION: Dietary composition may be related to inflammation in adult EoE patients. High-quality and anti-inflammatory diets may be a promising adjuvant therapy in the dietary management of EoE.

Effect of amino acid-based formula added to four-food elimination in adult eosinophilic esophagitis patients: A randomized clinical trial.

BACKGROUND: Elimination of key foods restricts dietary options in eosinophilic esophagitis (EoE) patients. Addition of amino acid-based formula (AAF) to an elimination diet might facilitate adherence and, therefore, enhance efficacy of dietary management. AIM: To evaluate whether addition of AAF to a four-food elimination diet (FFED) is more effective than FFED alone in decreasing eosinophilia, endoscopic signs, and clinical outcomes. METHODS: This randomized controlled trial enrolled 41 adult patients with active EoE (≥15 eosinophils (eos) per high power field (hpf)) at baseline biopsy. Subjects were randomized (1:1 ratio) to groups given a FFED or FFED with addition of AAF providing 30% of their daily energy needs (FFED + AAF). Histological disease activity, endoscopic signs, symptoms, and disease-related quality of life (EoEQoL) were measured at baseline and after 6 weeks of intervention. RESULTS: Patients (60% male, age 34.5 (interquartile range (IQR) 29-42.8 years)) were randomized to FFED (n = 20) or FFED + AAF (n = 21); 40 participants completed the diet. Complete histological remission (<15 eos/hpf) was achieved in 48% of FFED + AAF subjects (n = 21) vs. 25% of FFED subjects (n = 20), respectively (p = 0.204). Peak eosinophil counts (PEC) decreased significantly in both groups between baseline and week 6, but the change in PEC between groups was not different (p = 0.130). A significant but similar endoscopic and symptomatic reduction was observed in both groups (all; p<0.05). Total EoEQoL scores significantly improved in the FFED + AAF group between baseline and week 6 (p = 0.007), and not in the FFED group. CONCLUSION: The addition of AAF to a FFED did not lead to a larger decrease in PEC between baseline and 6 weeks, but may result in a significant improvement of QoL in adult EoE patients NL6014 (NTR6778).

Tolerance development in cow's milk-allergic infants receiving amino acid-based formula: A randomized controlled trial.

BACKGROUND: Tolerance development is an important clinical outcome for infants with cow's milk allergy. OBJECTIVE: This multicenter, prospective, randomized, double-blind, controlled clinical study (NTR3725) evaluated tolerance development to cow's milk (CM) and safety of an amino acid-based formula (AAF) including synbiotics (AAF-S) comprising prebiotic oligosaccharides (oligofructose, inulin) and probiotic Bifidobacterium breve M-16V in infants with confirmed IgE-mediated CM allergy. METHODS: Subjects aged ≤13 months with IgE-mediated CM allergy were randomized to receive AAF-S (n = 80) or AAF (n = 89) for 12 months. Stratification was based on CM skin prick test wheal size and study site. After 12 and 24 months, CM tolerance was evaluated by double-blind, placebo-controlled food challenge. A logistic regression model used the all-subjects randomized data set. RESULTS: At baseline, mean ± SD age was 9.36 ± 2.53 months. At 12 and 24 months, respectively, 49% and 62% of subjects were CM tolerant (AAF-S 45% and 64%; AAF 52% and 59%), and not differ significantly between groups. During the 12-month intervention, the number of subjects reporting at least 1 adverse event did not significantly differ between groups; however, fewer subjects required hospitalization due to serious adverse events categorized as infections in the AAF-S versus AAF group (9% vs 20%; P = .036). CONCLUSIONS: After 12 and 24 months, CM tolerance was not different between groups and was in line with natural outgrowth. Results suggest that during the intervention, fewer subjects receiving AAF-S required hospitalization due to infections.

Low-calcium diet in mice leads to reduced gut colonization by Enterococcus faecium.

The aim of this study was to determine whether dietary intervention influenced luminal Ca2+ levels and Enterococcus faecium gut colonization in mice. For this purpose, mice fed semi-synthetic food AIN93 were compared to mice fed AIN93-low calcium (LC). Administration of AIN93-LC resulted in lower luminal Ca2+ levels independent of the presence of E. faecium. Furthermore, E. faecium gut colonization was reduced in mice fed AIN93-LC based on culture, and which was in concordance with a reduction of Enterococcaceae in microbiota analysis. In conclusion, diet intervention might be a strategy for controlling gut colonization of E. faecium, an important opportunistic nosocomial pathogen.

A specific synbiotic-containing amino acid-based formula restores gut microbiota in non-IgE mediated cow's milk allergic infants: a randomized controlled trial.

BACKGROUND: Altered gut microbiota is implicated in cow's milk allergy (CMA) and differs markedly from healthy, breastfed infants. Infants who suffer from severe CMA often rely on cow's milk protein avoidance and, when breastfeeding is not possible, on specialised infant formulas such as amino-acid based formulas (AAF). Herein, we report the effects of an AAF including specific synbiotics on oral and gastrointestinal microbiota of infants with non-IgE mediated CMA with reference to healthy, breastfed infants. METHODS: In this prospective, randomized, double-blind controlled study, infants with suspected non-IgE mediated CMA received test or control formula. Test formula was AAF with synbiotics (prebiotic fructo-oligosaccharides and probiotic Bifidobacterium breve M-16V). Control formula was AAF without synbiotics. Healthy, breastfed infants were used as a separate reference group (HBR). Bacterial compositions of faecal and salivary samples were analysed by 16S rRNA-gene sequencing. Faecal analysis was complemented with the analysis of pH, short-chain fatty acids (SCFAs) and lactic acids. RESULTS: The trial included 35 test subjects, 36 controls, and 51 HBR. The 16S rRNA-gene sequencing revealed moderate effects of test formula on oral microbiota. In contrast, the gut microbiota was substantially affected across time comparing test with control. In both groups bacterial diversity increased over time but was characterised by a more gradual increment in test compared to control. Compositionally this reflected an enhancement of Bifidobacterium spp. and Veillonella sp. in the test group. In contrast, the control-fed infants showed increased abundance of adult-like species, mainly within the Lachnospiraceae family, as well as within the Ruminococcus and Alistipes genus. The effects on Bifidobacterium spp. and Lachnospiraceae spp. were previously confirmed through enumeration by fluorescent in situ hybridization and were shown for test to approximate the proportions observed in the HBR. Additionally, microbial activity was affected as evidenced by an increase of l-lactate, a decrease of valerate, and reduced concentrations of branched-chain SCFAs in test versus control. CONCLUSIONS: The AAF including specific synbiotics effectively modulates the gut microbiota and its metabolic activity in non-IgE mediated CMA infants bringing it close to a healthy breastfed profile.Trial registration Registered on 1 May 2013 with Netherlands Trial Register Number NTR3979.

A specific synbiotic-containing amino acid-based formula in dietary management of cow's milk allergy: a randomized controlled trial.

BACKGROUND: Here we report follow-up data from a double-blind, randomized, controlled multicenter trial, which investigated fecal microbiota changes with a new amino acid-based formula (AAF) including synbiotics in infants with non-immunoglobulin E (IgE)-mediated cow's milk allergy (CMA). METHODS: Subjects were randomized to receive test product (AAF including fructo-oligosaccharides and Bifidobacterium breve M-16V) or control product (AAF) for 8 weeks, after which infants could continue study product until 26 weeks. Fecal percentages of bifidobacteria and Eubacterium rectale/Clostridium coccoides group (ER/CC) were assessed at 0, 8, 12, and 26 weeks. Additional endpoints included stool markers of gut immune status, clinical symptoms, and safety assessments including adverse events and medication use. RESULTS: The trial included 35 test subjects, 36 controls, and 51 in the healthy reference group. Study product was continued by 86% and 92% of test and control subjects between week 8-12, and by 71% and 80%, respectively until week 26. At week 26 median percentages of bifidobacteria were significantly higher in test than control [47.0% vs. 11.8% (p < 0.001)], whereas percentages of ER/CC were significantly lower [(13.7% vs. 23.6% (p = 0.003)]. Safety parameters were similar between groups. Interestingly use of dermatological medication and reported ear infections were lower in test versus control, p = 0.019 and 0.011, respectively. Baseline clinical symptoms and stool markers were mild (but persistent) and low, respectively. Symptoms reduced towards lowest score in both groups. CONCLUSION: Beneficial effects of this AAF including specific synbiotics on microbiota composition were observed over 26 weeks, and shown suitable for dietary management of infants with non-IgE-mediated CMA.Trial Registration NTR3979.

A synbiotic-containing amino-acid-based formula improves gut microbiota in non-IgE-mediated allergic infants.

BackgroundPrebiotics and probiotics (synbiotics) can modify gut microbiota and have potential in allergy management when combined with amino-acid-based formula (AAF) for infants with cow's milk allergy (CMA).MethodsThis multicenter, double-blind, randomized controlled trial investigated the effects of an AAF-including synbiotic blend on percentages of bifidobacteria and Eubacterium rectale/Clostridium coccoides group (ER/CC) in feces from infants with suspected non-IgE-mediated CMA. Feces from age-matched healthy breastfed infants were used as reference (healthy breastfed reference (HBR)) for primary outcomes. The CMA subjects were randomized and received test or control formula for 8 weeks. Test formula was a hypoallergenic, nutritionally complete AAF including a prebiotic blend of fructo-oligosaccharides and the probiotic strain Bifidobacterium breve M-16V. Control formula was AAF without synbiotics.ResultsA total of 35 (test) and 36 (control) subjects were randomized; HBR included 51 infants. At week 8, the median percentage of bifidobacteria was higher in the test group than in the control group (35.4% vs. 9.7%, respectively; P<0.001), whereas ER/CC was lower (9.5% vs. 24.2%, respectively; P<0.001). HBR levels of bifidobacteria and ER/CC were 55% and 6.5%, respectively.ConclusionAAF including specific synbiotics, which results in levels of bifidobacteria and ER/CC approximating levels in the HBR group, improves the fecal microbiota of infants with suspected non-IgE-mediated CMA.

Esophageal and Small Intestinal Mucosal Integrity in Eosinophilic Esophagitis and Response to an Elemental Diet.

OBJECTIVES: The esophageal mucosal integrity is impaired in eosinophilic esophagitis (EoE) and it has been suggested that the duodenal permeability is increased. The absence of food allergens may restore the integrity. The aims of this study were to assess duodenal permeability in EoE and to evaluate the effect of an elemental diet on the esophageal and duodenal integrity. METHODS: In this prospective study 17 adult EoE patients and 8 healthy controls (HC) were included. Esophageal biopsy specimens were sampled before and after 4 weeks of elemental diet to measure eosinophil counts and gene expression of tight junction and barrier integrity proteins. Esophageal and duodenal impedance were measured by electrical tissue impedance spectroscopy and Ussing chambers were used to measure transepithelial resistance (TER) and transepithelial molecule flux. Small intestinal permeability was measured using a test, measuring lactulose/mannitol (L/M) ratios. RESULTS: In EoE patients, the esophageal but not the duodenal integrity was impaired, compared with HC. We observed no significant difference between L/M ratios of HC and EoE patients. After diet, eosinophil counts decreased significantly, which was paralleled by normalization of esophageal impedance and transepithelial molecule flux. The esophageal TER improved significantly, but did not reach values seen in HC. Esophageal expression of genes encoding for barrier integrity proteins filaggrin and desmoglein-1 was impaired at baseline and restored after diet. CONCLUSIONS: An elemental diet restores esophageal integrity, suggesting that it is at least partly secondary to allergen exposure. Duodenal integrity seems not to be affected in EoE, and possibly plays a minor role in its pathophysiology.

Synbiotics-supplemented amino acid-based formula supports adequate growth in cow's milk allergic infants.

BACKGROUND: Children with cow's milk allergy (CMA) are at risk for inadequate nutritional intake and growth. Dietary management of CMA, therefore, requires diets that are not only hypoallergenic but also support adequate growth in this population. This study assessed growth of CMA infants when using a new amino acid-based formula (AAF) with prebiotics and probiotics (synbiotics) and evaluated its safety in the intended population. METHODS: In a prospective, randomized, double-blind controlled study, full-term infants with diagnosed CMA received either an AAF (control; n = 56) or AAF with synbiotics (oligofructose, long-chain inulin, acidic oligosaccharides, Bifidobacterium breve M-16V) (test; n = 54) for 16 wk. Primary outcome was growth, measured as weight, length and head circumference. Secondary outcomes included allergic symptoms and stool characteristics. RESULTS: Average age (±SD) of infants at inclusion was 4.5 ± 2.4 months. Both formulas equally supported growth according to WHO 2006 growth charts and resulted in similar increases of weight, length and head circumference. At week 16, differences (90% CI) in Z-scores (test-control) were as follows: weight 0.147 (-0.10; 0.39, p = 0.32), length -0.299 (-0.69; 0.09, p = 0.21) and head circumference 0.152 (-0.15; 0.45, p = 0.40). Weight-for-age and length-for-age Z-scores were not significantly different between the test and control groups. Both formulas were well tolerated and reduced allergic symptoms; the number of adverse events was not different between the groups. CONCLUSIONS: This is the first study that shows that an AAF with a specific synbiotic blend, suitable for CMA infants, supports normal growth and growth similar to the AAF without synbiotics. This clinical trial is registered as NCT00664768.

Intestinally secreted C-type lectin Reg3b attenuates salmonellosis but not listeriosis in mice.

The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal infection with pathogens increase the expression of Reg3g and Reg3b in the murine ileum. Reg3g is directly bactericidal for gram-positive bacteria, but the exact role of Reg3b in bacterial infections is unknown. To investigate the possible protective role of Reg3b in intestinal infection, Reg3b knockout (Reg3b(-/-)) mice and wild-type (WT) mice were orally infected with gram-negative Salmonella enteritidis or gram-positive Listeria monocytogenes. At day 2 after oral Listeria infection and at day 4 after oral Salmonella infection, mice were sacrificed to collect intestinal and other tissues for pathogen quantification. Protein expression of Reg3b and Reg3g was determined in intestinal mucosal scrapings of infected and noninfected mice. In addition, ex vivo binding of ileal mucosal Reg3b to Listeria and Salmonella was investigated. Whereas recovery of Salmonella or Listeria from feces of Reg3b(-/-) mice did not differ from that from feces of WT mice, significantly higher numbers of viable Salmonella, but not Listeria, bacteria were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the Reg3b(-/-) mice than from those of WT mice. Mucosal Reg3b binds to both bacterial pathogens and may interfere with their mode of action. Reg3b plays a protective role against intestinal translocation of the gram-negative bacterium S. enteritidis in mice but not against the gram-positive bacterium L. monocytogenes.

Damage to the intestinal epithelial barrier by antibiotic pretreatment of salmonella-infected rats is lessened by dietary calcium or tannic acid.

Perturbation of the intestinal microbiota by antibiotics predisposes the host to food-borne pathogens like Salmonella. The effects of antibiotic treatment on intestinal permeability during infection and the efficacy of dietary components to improve resistance to infection have not been studied. Therefore, we investigated the effect of clindamycin on intestinal barrier function in Salmonella-infected rats. We also studied the ability of dietary calcium and tannic acid to protect against infection and concomitant diarrhea and we assessed intestinal barrier function. Rats were fed a purified control diet including the permeability marker chromium EDTA (CrEDTA) (2 g/kg) or the same diet supplemented with calcium (4.8 g/kg) or tannic acid (3.75 g/kg). After adaptation, rats were orally treated with clindamycin for 4 d followed by oral infection with Salmonella enteritidis. Two additional control groups were not treated with antibiotics and received either saline or Salmonella. Urine and feces were collected to quantify intestinal permeability, diarrhea, cytotoxicity of fecal water, and Salmonella excretion. In addition, Salmonella translocation was determined. Diarrhea, CrEDTA excretion, and cytotoxicity of fecal water were higher in the clindamycin-treated infected rats than in the non-clindamycin-treated infected control group. Intestinal barrier function was less in the Salmonella-infected rats pretreated with antibiotics compared with the non-clindamycin- treated rats. Both calcium and tannic acid reduced infection-associated diarrhea and inhibited the adverse intestinal permeability changes but did not decrease Salmonella colonization and translocation. Our results indicate that calcium protects against intestinal changes due to Salmonella infection by reducing luminal cytotoxicity, whereas tannic acid offers protection by improving the mucosal resistance.

Intestinal barrier function in response to abundant or depleted mucosal glutathione in Salmonella-infected rats.

BACKGROUND: Glutathione, the main antioxidant of intestinal epithelial cells, is suggested to play an important role in gut barrier function and prevention of inflammation-related oxidative damage as induced by acute bacterial infection. Most studies on intestinal glutathione focus on oxidative stress reduction without considering functional disease outcome. Our aim was to determine whether depletion or maintenance of intestinal glutathione changes susceptibility of rats to Salmonella infection and associated inflammation.Rats were fed a control diet or the same diet supplemented with buthionine sulfoximine (BSO; glutathione depletion) or cystine (glutathione maintenance). Inert chromium ethylenediamine-tetraacetic acid (CrEDTA) was added to the diets to quantify intestinal permeability. At day 4 after oral gavage with Salmonella enteritidis (or saline for non-infected controls), Salmonella translocation was determined by culturing extra-intestinal organs. Liver and ileal mucosa were collected for analyses of glutathione, inflammation markers and oxidative damage. Faeces was collected to quantify diarrhoea. RESULTS: Glutathione depletion aggravated ileal inflammation after infection as indicated by increased levels of mucosal myeloperoxidase and interleukin-1beta. Remarkably, intestinal permeability and Salmonella translocation were not increased. Cystine supplementation maintained glutathione in the intestinal mucosa but inflammation and oxidative damage were not diminished. Nevertheless, cystine reduced intestinal permeability and Salmonella translocation. CONCLUSION: Despite increased infection-induced mucosal inflammation upon glutathione depletion, this tripeptide does not play a role in intestinal permeability, bacterial translocation and diarrhoea. On the other hand, cystine enhances gut barrier function by a mechanism unlikely to be related to glutathione.

Ileal mucosal and fecal pancreatitis associated protein levels reflect severity of salmonella infection in rats.

BACKGROUND: Microbial infections induce ileal pancreatitis-associated protein/regenerating gene III (PAP/RegIII) mRNA expression. Despite increasing interest, little is known about the PAP/RegIII protein. Therefore, ileal mucosal PAP/RegIII protein expression, localization, and fecal excretion were studied in rats upon Salmonella infection. RESULTS: Salmonella infection increased ileal mucosal PAP/RegIII protein levels in enterocytes located at the crypt-villus junction. Increased colonization and translocation of Salmonella was associated with higher ileal mucosal PAP/RegIII levels and secretion of this protein in feces. CONCLUSIONS: PAP/RegIII protein is increased in enterocytes of the ileal mucosa during Salmonella infection and is associated with infection severity. PAP/RegIII is excreted in feces and might be used as a new and non-invasive infection marker.