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.

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.

Association of food-hygiene practices and diarrhea prevalence among Indonesian young children from low socioeconomic urban areas.

BACKGROUND: Information on the part that poor food-hygiene practices play a role in the development of diarrhea in low socioeconomic urban communities is lacking. This study was therefore aimed at assessing the contribution of food-hygiene practice to the prevalence of diarrhea among Indonesian children. METHODS: A cross-sectional study was conducted among 274 randomly selected children aged 12-59 months in selected low socioeconomic urban areas of East Jakarta. The prevalence of diarrhea was assessed from 7-day records on frequency and consistency of the child's defecation pattern. Food-hygiene practices including mother's and child's hand washing, food preparation, cleanliness of utensils, water source and safe drinking water, habits of buying cooked food, child's bottle feeding hygiene, and housing and environmental condition were collected through home visit interviews and observations by fieldworkers. Thirty-six practices were scored and classified into poor (median and below) and better (above median) food-hygiene practices. Nutritional status of children, defined anthropometrically, was measured through height and weight. RESULTS: Among the individual food-hygiene practices, children living in a house with less dirty sewage had a significantly lower diarrhea prevalence compared to those who did not [adjusted odds ratio (OR) 0.16, 95% confidence interval (CI) = 0.03-0.73]. The overall food-hygiene practice score was not significantly associated with diarrhea in the total group, but it was in children aged < 2 years (adjusted OR 4.55, 95% CI = 1.08-19.1). CONCLUSIONS: Overall poor mother's food-hygiene practices did not contribute to the occurrence of diarrhea in Indonesian children. However, among children < 2 years from low socioeconomic urban areas they were associated with more diarrhea.

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.

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.

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.

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.

A framework to identify physiological responses in microarray-based gene expression studies: selection and interpretation of biologically relevant genes.

In whole genome microarray studies major gene expression changes are easily identified, but it is a challenge to capture small, but biologically important, changes. Pathway-based programs can capture small effects but may have the disadvantage of being restricted to functionally annotated genes. A structured approach toward the identification of major and small changes for interpretation of biological effects is needed. We present a structured approach, a framework, that addresses different considerations in 1) the identification of informative genes in microarray data sets and 2) the interpretation of their biological relevance. The steps of this framework include gene ranking, gene selection, gene grouping, and biological interpretation. Random forests (RF), which takes gene-gene interactions into account, is examined to rank and select genes. For human, mouse, and rat whole genome arrays, less than half of the probes on the array are annotated. Consequently, pathway analysis tools ignore half of the information present in the microarray data set. The framework described takes all genes into account. RF is a useful tool to rank genes by taking interactions into account. Applying a permutation approach, we were able to define an objective threshold for gene selection. RF combined with self-organizing maps identified genes with coordinated but small gene expression responses that were not fully annotated but corresponded to the same biological process. The presented approach provides a flexible framework for biological interpretation of microarray data sets. It includes all genes in the data set, takes gene-gene interactions into account, and provides an objective threshold for gene selection.

Impaired barrier function by dietary fructo-oligosaccharides (FOS) in rats is accompanied by increased colonic mitochondrial gene expression.

BACKGROUND: Dietary non-digestible carbohydrates stimulate the gut microflora and are therefore presumed to improve host resistance to intestinal infections. However, several strictly controlled rat infection studies showed that non-digestible fructo-oligosaccharides (FOS) increase, rather than decrease, translocation of Salmonella towards extra-intestinal sites. In addition, it was shown that FOS increases intestinal permeability already before infection. The mechanism responsible for this adverse effect of FOS is unclear. Possible explanations are altered mucosal integrity due to changes in tight junctions or changes in expression of defense molecules such as antimicrobials and mucins. To examine the mechanisms underlying weakening of the intestinal barrier by FOS, a controlled dietary intervention study was performed. Two groups of 12 rats were adapted to a diet with or without FOS. mRNA was collected from colonic mucosa and changes in gene expression were assessed for each individual rat using Agilent rat whole genome microarrays. RESULTS: Among the 997 FOS induced genes we observed less mucosal integrity related genes than expected with the clear permeability changes. FOS did not induce changes in tight junction genes and only 8 genes related to mucosal defense were induced by FOS. These small effects are unlikely the cause for the clear increase in intestinal permeability that is observed. FOS significantly increased expression of 177 mitochondria-related genes. More specifically, induced expression of genes involved in all five OXPHOS complexes and the TCA cycle was observed. These results indicate that dietary FOS influences intestinal mucosal energy metabolism. Furthermore, increased expression of 113 genes related to protein turnover, including proteasome genes, ribosomal genes and protein maturation related genes, was seen. FOS upregulated expression of the peptide hormone proglucagon gene, in agreement with previous studies, as well as three other peptide hormone genes; peptide YY, pancreatic polypeptide and cholecystokinin. CONCLUSION: We conclude that altered energy metabolism may underly colonic barrier function disruption due to FOS feeding in rats.

Gene expression response of the rat small intestine following oral Salmonella infection.

Data on the molecular response of the intestine to the food-borne pathogen Salmonella are derived from in vitro studies, whereas in vivo data are lacking. We performed an oral S. enteritidis infection study in Wistar rats to obtain insight in the in vivo response in time. Expression profiles of ileal mucosa (IM) and Peyer's patches (PP) were generated using DNA microarrays at days 1, 3, and 6 postinfection. An overview of Salmonella-regulated processes was obtained and confirmed by quantitative real-time PCR on pooled and individual samples. Salmonella-induced gene expression responses in vivo are fewer and smaller than observed in vitro, and the response develops over a longer period of time. Few effects are seen at day 1 and mainly occur in IM, suggesting the mucosa as the primary site of invasion. Later, a bigger response is observed, especially in PP. Decreased expression of anti-microbial peptides genes (in IM at day 1) suggests inhibition of this process by Salmonella. Newly identified target processes are carbohydrate transport (increased expression in IM at day 1) and phase I and phase II detoxification (decreased expression at days 3 and 6). Increase of cytokine and chemokine expression occurs at later time points, both in PP and IM. Pancreatitis-associated protein, lipocalin 2, and calprotectin, potential inflammatory marker proteins, showed induced expression from day 3 onward. We conclude that the in vivo gene expression response of the ileum to Salmonella differs to a large extent from the response seen in vitro.

Salmonella induces prominent gene expression in the rat colon.

BACKGROUND: Salmonella enteritidis is suggested to translocate in the small intestine. In vivo it induces gene expression changes in the ileal mucosa and Peyer's patches. Stimulation of Salmonella translocation by dietary prebiotics fermented in colon suggests involvement of the colon as well. However, effects of Salmonella on colonic gene expression in vivo are largely unknown. We aimed to characterize time dependent Salmonella-induced changes of colonic mucosal gene expression in rats using whole genome microarrays. For this, rats were orally infected with Salmonella enteritidis to mimic a foodborne infection and colonic gene expression was determined at days 1, 3 and 6 post-infection (n = 8 rats per time-point). As fructo-oligosaccharides (FOS) affect colonic physiology, we analyzed colonic mucosal gene expression of FOS-fed versus cellulose-fed rats infected with Salmonella in a separate experiment. Colonic mucosal samples were isolated at day 2 post-infection. RESULTS: Salmonella affected transport (e.g. Chloride channel calcium activated 6, H+/K+ transporting Atp-ase), antimicrobial defense (e.g. Lipopolysaccharide binding protein, Defensin 5 and phospholipase A2), inflammation (e.g. calprotectin), oxidative stress related genes (e.g. Dual oxidase 2 and Glutathione peroxidase 2) and Proteolysis (e.g. Ubiquitin D and Proteosome subunit beta type 9). Furthermore, Salmonella translocation increased serum IFN gamma and many interferon-related genes in colonic mucosa. The gene most strongly induced by Salmonella infection was Pancreatitis Associated Protein (Pap), showing >100-fold induction at day 6 after oral infection. Results were confirmed by Q-PCR in individual rats. Stimulation of Salmonella translocation by dietary FOS was accompanied by enhancement of the Salmonella-induced mucosal processes, not by induction of other processes. CONCLUSION: We conclude that the colon is a target tissue for Salmonella, considering the abundant changes in mucosal gene expression.

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.

Pancreatitis-associated protein has no additional value as a marker of disease activity in a real-life cohort of IBD patients.

BACKGROUND AND AIM: Monitoring of mucosal inflammation in inflammatory bowel disease (IBD) is of major importance. New noninvasive markers for intestinal inflammation are needed. Previous studies have reported that pancreatitis-associated protein (PAP) correlates with clinical activity in IBD subgroups. Our aim was to investigate the correlation of serum and fecal PAP with clinical and biochemical parameters of disease activity in a real-life IBD cohort. PATIENTS AND METHODS: Two hundred and five consecutive IBD patients were enrolled. Clinical disease activity was scored by the Harvey-Bradshaw Index or the Simple Clinical Colitis Activity Index; also, C-reactive protein (CRP), erythrocyte sedimentation rate, and fecal calprotectin were determined. As surrogate for endoscopy, a combination score of clinical indices with CRP or calprotectin was used to define active disease. Fecal and serum PAP were measured by ELISA. RESULTS: The median serum and fecal PAP did not differ in Crohn's disease (CD) or ulcerative colitis (UC) patients with active compared with inactive disease according to clinical activity indices. Defining active disease by a combination score of Harvey-Bradshaw Index of more than 4 and CRP of more than 5 mg/l or calprotectin more than 250 µg/g, serum PAP (P=0.01), but not fecal PAP (P=0.32), was significantly higher in active than inactive CD patients. Area under the curve of the corresponding receiver operating curve (ROC) was 0.64. No differences were found in serum or fecal PAP levels using the combination score for active disease in UC. CONCLUSION: Serum but not fecal PAP was higher in active compared with nonactive CD and may reflect mucosal inflammation in CD, but not in UC. However, the accuracy of serum PAP for the diagnosis of active disease was poor, and therefore, serum PAP does not seem to have additional value compared with the current noninvasive markers.

Randomized trial of probiotics and calcium on diarrhea and respiratory tract infections in Indonesian children.

OBJECTIVE: To investigate the effects of calcium and probiotics on the incidence and duration of acute diarrhea and acute respiratory tract infections (ARTIs) in low-socioeconomic communities of Jakarta, Indonesia. METHODS: We conducted a 6-month, double-blind, placebo-controlled study in 494 healthy children aged 1 to 6 years who received low-lactose milk with low calcium content (LC; ∼50 mg/day; n = 124), regular calcium content (RC; ∼440 mg/day; n = 126), RC with 5.10(8) colony-forming units per day of Lactobacillus casei CRL431 (casei; n = 120), or RC with 5.10(8) colony-forming units per day of Lactobacillus reuteri DSM17938 (reuteri; n = 124). Number and duration of diarrhea and ARTIs episodes were primary and secondary outcomes, respectively. RESULTS: Incidence of World Health Organization-defined diarrhea (≥3 loose/liquid stools in 24 hours) was not significantly different between RC and LC (relative risk [RR]: 0.99 [95% confidence interval (CI): 0.62-1.58]), between casei and RC (RR: 1.21 [95% CI: 0.76-1.92]), or between reuteri and RC (RR: 0.76 [95% CI: 0.46-1.25]) groups. Incidence of all reported diarrhea (≥2 loose/liquid stools in 24 hours) was significantly lower in the reuteri versus RC group (RR: 0.68 [95% CI: 0.46-0.99]). Irrespective of the definition used, reuteri significantly reduced diarrhea incidence in children with lower nutritional status (below-median height-and-weight-for-age z score). None of the interventions affected ARTIs. CONCLUSIONS: RC milk, alone or with L casei, did not reduce diarrhea or ARTIs in Indonesian children. L reuteri may prevent diarrhea, especially in children with lower nutritional status.

Dietary heme adversely affects experimental colitis in rats, despite heat-shock protein induction.

OBJECTIVE: Research on dietary modulation of inflammatory bowel disease is in its infancy. Dietary heme, mimicking red meat, is cytotoxic to colonic epithelium and thus may aggravate colitis. Alternatively, heme-induced colonic stress might also result in potential protective heat-shock proteins (HSPs). Therefore, we investigated the effect of dietary heme on trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. METHODS: Rats were fed a high-fat control diet or a similar diet supplemented with heme. After dietary adaptation, rats were rectally infused with TNBS for colitis induction or saline for sham treatment. Colitis severity was evaluated and several markers were quantified in colonic mucosa isolated 1 wk after colitis induction. Furthermore, cytotoxicity of fecal water and serum α-1-acid glycoprotein were measured. RESULTS: Dietary heme increased cytotoxicity of the fecal water. Heme-fed sham-treated rats had higher colonic HSP-25 and heme-oxygenase-1 mRNA levels, which was confirmed by immunohistochemistry. HSP induction by heme was associated with decreased protein levels of myeloperoxidase and interleukin-1ß after subsequent TNBS infusion. However, no dietary effects were observed on histologic colitis score. Furthermore, body weight gain, colon length, and food intake were lower and α-1-acid glycoprotein concentrations were higher in heme-fed colitic rats. In addition, somatostatin, involved in mucosal repair, was not changed with TNBS infusion in heme-fed rats. CONCLUSION: Dietary heme adversely affects colitis, despite HSP induction. We speculate that the irritating influence of dietary heme, being continuously present in the colon, impairs recovery after colitis induction. A diet high in red meat might be a risk factor for inflammatory bowel disease development.

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.