A randomized controlled trial of different young child formulas on upper respiratory and gastrointestinal tract infections in Chinese toddlers.

BACKGROUND: Bioactive proteins and human milk oligosaccharides (HMOs), important ingredients in breast milk, that protect against infections are lacking in young child formula (YCF). This study investigated the effects of new YCFs on respiratory and gastrointestinal infections in toddlers. METHODS: Four hundred and sixty one healthy Chinese children aged 1-2.5 years were recruited in this randomized, controlled, double-blind, parallel-group clinical trial of different YCFs. They were randomly assigned to either standard milk formula (YCF-Ref) or one of three new YCFs containing bioactive proteins and/or the HMO 2'-fucosyllactose (2'-FL) and/or milk fat for six months. Primary outcomes were incidence of upper respiratory tract infection (URTI) and duration of gastrointestinal tract infections (GITI). RESULTS: There were no significant between-group differences in primary outcomes. For secondary outcomes, subjects receiving 2'-FL-supplemented YCF had longer URTI. Subjects receiving YCF supplemented with milk fat and intact bioactive proteins, and 2'-FL at levels found in breast milk, had more GITI episodes and shorter time to first GITI but similar effects on URTI duration than YCF-Ref recipients. No effects on URTI and GITI were observed in toddlers receiving YCF with bioactive proteins at lower levels than breast milk. Occurrence of adverse events and anthropometry were similar in all groups. CONCLUSIONS: All three YCFs supplemented with different combinations of intact bioactive proteins, 2'-FL, and milk fat are safe in toddlers. No difference is detected among YCFs on URTI incidence and GITI duration. Further studies are needed to verify these findings especially in infants who may benefit most from the immune-boosting effects of bioactive proteins and HMOs.

Functional role and mechanisms of sialyllactose and other sialylated milk oligosaccharides.

Human milk is a rich source of oligosaccharides. Acidic oligosaccharides, such as sialyllactose (SL), contain sialic acid (SA) residues. In human milk, approximately 73% of SA is bound to oligosaccharides, whereas only 3% is present in free form. Oligosaccharides are highly resistant to hydrolysis in the gastrointestinal tract. Only a small portion of the available oligosaccharides in breast milk is absorbed in the neonatal small intestine. SL and sialylated oligosaccharides are thought to have significant health benefits for the neonate, because of their roles in supporting resistance to pathogens, gut maturation, immune function, and cognitive development. The need for SA to allow proper development during the neonatal period is thought to exceed the endogenous synthesis. Therefore, these structures are important nutrients for the neonate. Based on the potential benefits, SL and sialylated oligosaccharides may be interesting components for application in infant nutrition. Once the hurdle of limited availability of these oligosaccharides has been overcome, their functionality can be explored in more detail, and supplementation of infant formula may become feasible.

Probiotics Lactobacillus reuteri DSM 17938 and Lactobacillus casei CRL 431 modestly increase growth, but not iron and zinc status, among Indonesian children aged 1-6 years.

Probiotics and milk calcium may increase resistance to intestinal infection, but their effect on growth and iron and zinc status of Indonesian children is uncertain. We investigated the hypotheses that cow milk with added probiotics would improve growth and iron and zinc status of Indonesian children, whereas milk calcium alone would improve growth but reduce iron and zinc status. A 6-mo randomized trial was conducted in low-socioeconomic urban communities of Jakarta. Healthy children (n = 494) were randomly assigned to receive low-lactose milk with a low calcium content of ∼50 mg/d (LC; n = 124), a regular calcium content of ∼440 mg/d (RC group; n = 126), regular calcium with 5 × 10(8) CFU/d Lactobacillus casei CRL 431 (casei; n = 120), or regular calcium with 5 × 10(8) CFU/d Lactobacillus reuteri DSM 17938 (reuteri; n = 124). Growth, anemia, and iron and zinc status were assessed before and after the intervention. Compared with the RC group, the reuteri group had significantly greater weight gain [0.22 (95% CI: 0.02, 0.42) kg], weight-for-age Z-score (WAZ) changes [0.09 (95% CI: 0.01, 0.17)], and monthly weight [0.03 (95% CI: 0.002, 0.05) kg/mo] and height [0.03 (95% CI: 0.01, 0.05) cm/mo] velocities. Casei significantly increased monthly weight velocity [0.03 (95% CI: 0.001, 0.05) kg/mo], but not height. However, the changes in underweight, stunting, anemia prevalence, and iron and zinc status were similar between groups. In conclusion, L. reuteri DSM 17938 modestly improved growth by increasing weight gain, WAZ changes, and weight and height velocity, whereas L. casei CRL 431 modestly improved weight velocity. Independent from probiotics supplementation, regular milk calcium did not affect growth or iron and zinc status.

The protective effect of supplemental calcium on colonic permeability depends on a calcium phosphate-induced increase in luminal buffering capacity.

An increased intestinal permeability is associated with several diseases. Previously, we have shown that dietary Ca decreases colonic permeability in rats. This might be explained by a calcium-phosphate-induced increase in luminal buffering capacity, which protects against an acidic pH due to microbial fermentation. Therefore, we investigated whether dietary phosphate is a co-player in the effect of Ca on permeability. Rats were fed a humanised low-Ca diet, or a similar diet supplemented with Ca and containing either high, medium or low phosphate concentrations. Chromium-EDTA was added as an inert dietary intestinal permeability marker. After dietary adaptation, short-chain fructo-oligosaccharides (scFOS) were added to all diets to stimulate fermentation, acidify the colonic contents and induce an increase in permeability. Dietary Ca prevented the scFOS-induced increase in intestinal permeability in rats fed medium- and high-phosphate diets but not in those fed the low-phosphate diet. This was associated with higher faecal water cytotoxicity and higher caecal lactate levels in the latter group. Moreover, food intake and body weight during scFOS supplementation were adversely affected by the low-phosphate diet. Importantly, luminal buffering capacity was higher in rats fed the medium- and high-phosphate diets compared with those fed the low-phosphate diet. The protective effect of dietary Ca on intestinal permeability is impaired if dietary phosphate is low. This is associated with a calcium phosphate-induced increase in luminal buffering capacity. Dragging phosphate into the colon and thereby increasing the colonic phosphate concentration is at least part of the mechanism behind the protective effect of Ca on intestinal permeability.

Supplemental antioxidants do not ameliorate colitis development in HLA-B27 transgenic rats despite extremely low glutathione levels in colonic mucosa.

BACKGROUND: Oxidative stress is presumed to play an important role in inflammatory bowel disease (IBD). Accordingly, antioxidant supplementation might be protective. Dietary calcium inhibited colitis development in HLA-B27 transgenic rats, an animal model mimicking IBD. As antioxidants might act at mucosa level and calcium predominantly in the gut lumen, we hypothesize that the combination has additive protective effects on colitis development. METHODS: HLA-B27 rats were fed a control diet or the same diet supplemented with the antioxidants glutathione, vitamin C, and vitamin E, or supplemented with both antioxidants and calcium. Oxidative stress in colonic mucosa, colonic inflammation, intestinal permeability, and diarrhea were quantified. RESULTS: Intestinal permeability, diarrhea, myeloperoxidase, and interleukin-1ß levels were significantly lower in rats fed both antioxidants and calcium compared to rats supplemented with antioxidants only. No beneficial effects were observed in rats fed the diet supplemented with antioxidants only. Strikingly, despite extremely low colonic mucosal glutathione levels in HLA-B27 rats, there was no oxidative stress-related damage. Subsequent analyses showed no defect in expression of glutathione synthesis genes. Additional experiments, comparing young and older HLA-B27 rats, showed that glutathione levels and also reactive oxygen species production decreased with progression of intestinal inflammation. CONCLUSIONS: Antioxidant supplementation was ineffective in HLA-B27 rats despite low mucosal glutathione levels, because colitis development did not coincide with oxidative stress in this model. This indicates that the neutrophilic respiratory burst, and thus innate immune defense, is compromised in HLA-B27 rats. As supplementation with both calcium and antioxidants attenuated colitis development, we speculate that this protective effect is attributed to calcium only.

Efficacy of various dietary calcium salts to improve intestinal resistance to Salmonella infection in rats.

Previous animal and human studies have shown protective effects of Ca on the resistance to enteropathogenic infections. Most interventions were performed with calcium phosphate and little is known about the protective effect of other dietary sources of Ca. Therefore, we investigated the efficacy of several Ca salts to enhance intestinal resistance to Salmonella enteritidis infection. Rats (n 7-8 per group) were fed a high-fat, Western human-style, purified diet with a low Ca content (20 mmol calcium phosphate/kg; negative control group) or the same diet supplemented with either (extra) calcium phosphate, milk Ca, calcium chloride or calcium carbonate (total of 100 mmol Ca supplement/kg). Diets contained Cr-EDTA for assessment of incremental changes in intestinal permeability. After an adaptation period of 2 weeks, animals were orally infected with S. enteritidis to mimic a human-relevant foodborne infection. Ca supplement-induced changes on faecal lactobacilli and enterobacteria were studied before infection. Changes in intestinal permeability were determined by measuring urinary Cr with time. Persistence of Salmonella was determined by studying faecal excretion of this pathogen in time. Overall, all Ca salts increased resistance towards Salmonella. After infection, body weight gain and food intake were higher in the calcium phosphate group. Calcium phosphate and milk Ca decreased faecal enterobacteria before infection. All Ca salts decreased infection-induced intestinal permeability and persistence of Salmonella. Calcium phosphate, milk Ca, calcium carbonate and calcium chloride are able to enhance the intestinal resistance to Salmonella in rats.

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.

Dietary calcium decreases but short-chain fructo-oligosaccharides increase colonic permeability in rats.

An increased intestinal permeability is associated with several diseases. Nutrition can influence gut permeability. Previously, we showed that dietary Ca decreases whereas dietary short-chain fructo-oligosaccharides (scFOS) increase intestinal permeability in rats. However, it is unknown how and where in the gastrointestinal tract Ca and scFOS exert their effects. Rats were fed a Western low-Ca control diet, or a similar diet supplemented with either Ca or scFOS. Lactulose plus mannitol and Cr-EDTA were added to the diets to quantify small and total gastrointestinal permeability, respectively. Additionally, colonic tissue was mounted in Ussing chambers and exposed to faecal water of these rats. Dietary Ca immediately decreased urinary Cr-EDTA excretion by 24 % in Ca-fed rats compared with control rats. Dietary scFOS increased total Cr-EDTA permeability gradually with time, likely reflecting relatively slow gut microbiota adaptations, which finally resulted in a 30 % increase. The lactulose:mannitol ratio was 15 % higher for Ca-fed rats and 16 % lower for scFOS-fed rats compared with control rats. However, no dietary effect was present on individual urinary lactulose and mannitol excretion. The faecal waters did not influence colonic permeability in Ussing chambers. In conclusion, despite effects on the lactulose:mannitol ratio, individual lactulose values did not alter, indicating that diet did not influence small-intestinal permeability. Therefore, both nutrients affect permeability only in the colon: Ca decreases, while scFOS increase colonic permeability. As faecal water did not influence permeability in Ussing chambers, probably modulation of mucins and/or microbiota is important for the in vivo effects of dietary Ca and scFOS.

Supplemental calcium attenuates the colitis-related increase in diarrhea, intestinal permeability, and extracellular matrix breakdown in HLA-B27 transgenic rats.

We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function may alleviate inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in an experimental rat model of IBD. HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium concentration (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 wk. Inert chromium EDTA (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal wet weight was determined to quantify diarrhea. Colonic inflammation was determined histologically and by measuring mucosal interleukin (IL)-1beta. In addition, colonic mucosal gene expression of individual rats was analyzed using whole-genome microarrays. The calcium diet significantly inhibited the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis compared with the control transgenic rats. Mucosal IL-1beta levels were lower in calcium-fed rats and histological colitis scores tended to be lower (P = 0.08). Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens, and fibronectin), which was confirmed by quantitative real-time PCR and gelatin zymography. In conclusion, dietary calcium ameliorates several important aspects of colitis severity in HLA-B27 transgenic rats. Reduction of mucosal irritation by luminal components might be part of the mechanism. These results show promise for supplemental calcium as effective adjunct therapy for IBD.

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.

Dietary fructooligosaccharides affect intestinal barrier function in healthy men.

In contrast to most expectations, we showed previously that dietary fructooligosaccharides (FOS) stimulate intestinal colonization and translocation of invasive Salmonella enteritidis in rats. Even before infection, FOS increased the cytotoxicity of fecal water, mucin excretion, and intestinal permeability. In the present study, we tested whether FOS has these effects in humans. A double-blind, placebo-controlled, crossover study of 2 x 2 wk, with a washout period of 2 wk, was performed with 34 healthy men. Each day, subjects consumed lemonade containing either 20 g FOS or placebo and the intestinal permeability marker chromium EDTA (CrEDTA). On the last 2 d of each supplement period, subjects scored their gastrointestinal complaints on a visual analog scale and collected feces and urine for 24 h. Fecal lactic acid was measured using a colorimetric enzymatic kit. The cytotoxicity of fecal water was determined with an in vitro bioassay, fecal mucins were quantified fluorimetrically, and intestinal permeability was determined by measuring urinary CrEDTA excretion. In agreement with our animal studies, FOS fermentation increased fecal wet weight, bifidobacteria, lactobacilli, and lactic acid. Consumption of FOS increased flatulence and intestinal bloating. In addition, FOS consumption doubled fecal mucin excretion, indicating mucosal irritation. However, FOS did not affect the cytotoxicity of fecal water and intestinal permeability. The FOS-induced increase in mucin excretion in our human study suggests mucosal irritation in humans, but the overall effects are more moderate than those in rats.

Dietary fructooligosaccharides increase intestinal permeability in rats.

We showed previously that fructooligosaccharides (FOS) decrease the resistance to salmonella infection in rats. However, the mechanism responsible for this effect is unclear. Therefore, we examined whether dietary FOS affects intestinal permeability before and after infection with Salmonella enterica serovar Enteritidis. Male Wistar rats were fed restricted quantities of a purified diet that mimicked the composition of a Western human diet. The diet was supplemented with 60 g/kg cellulose (control) or 60 g/kg FOS and with 4 mmol/kg of the intestinal permeability marker chromium EDTA (CrEDTA) (n = 8 or 10). After an adaptation period of 2 wk, rats were orally infected with 10(8) colony-forming units (cfu) of S. enteritidis. Mucin concentrations in intestinal contents and mucosa were measured fluorimetrically, as markers of mucosal irritation. Intestinal permeability was determined by measuring urinary CrEDTA excretion. Translocation of salmonella was quantified by analysis of urinary nitric oxide metabolites with time. Before infection, FOS increased mucosal lactobacilli and enterobacteria in cecum and colon, but not in the ileum. However, FOS increased cytotoxicity of fecal water and intestinal permeability. Moreover, FOS increased fecal mucin excretion and mucin concentrations in cecal and colonic contents, and in cecal mucosa before infection. After infection, mucin excretion and intestinal permeability in the FOS groups increased even further in contrast to the control group. In addition, FOS increased translocation of salmonella to extraintestinal sites. Thus, FOS impairs the intestinal barrier in rats, as indicated by higher intestinal permeability. Whether these results can be extrapolated to humans requires further investigation.

Diarrhea caused by enterotoxigenic Escherichia coli infection of humans is inhibited by dietary calcium.

BACKGROUND & AIMS: In several rat infection experiments, we have shown that dietary calcium inhibits intestinal colonization and translocation of invasive salmonella. The aim of the present study was to find out whether calcium is also protective against enterotoxigenic Escherichia coli (ETEC) infection. This was first tested in our rat model and subsequently verified in a human infection study. METHODS: Rats were fed a purified diet with either a low or a high amount of calcium phosphate and orally infected with ETEC. In addition, a parallel, double-blind, placebo-controlled intervention study of 3 weeks was performed with 32 healthy men. Subjects largely maintained their habitual diet and consumed either regular milk products (calcium supply, 1100 mg/day) or placebo milk products (calcium supply, 60 mg/day). On day 10, subjects ingested a live but attenuated ETEC strain (strain E1392/75-2A), able to induce mild although short-lived symptoms. Primary outcomes studied were infection-induced diarrhea (total fecal output and relative fecal dry weight) and fecal mucin excretion. RESULTS: In humans, ETEC induced diarrhea in both groups, in that total fecal output doubled and mean relative fecal dry weight dropped from 25% to 20%. Additionally, fecal mucin excretion was increased in both groups. All these fecal parameters were completely normalized in the calcium group on the second infection day, in contrast to the placebo group, which recovered on the third infection day. Likewise, supplemental calcium inhibited ETEC colonization and diarrhea in rats. CONCLUSIONS: Calcium in milk products improves human resistance to ETEC infection as it inhibits infectious diarrhea.