Lactobacillus acidophilus supplementation in human subjects and their resistance to enterotoxigenic Escherichia coli infection.

To assess the effect of Lactobacillus acidophilus (American Type Culture Collection (ATCC) 700396) on enterotoxigenic Escherichia coli (ETEC) infection, in the present study, a parallel, double-blind, placebo-controlled 4-week intervention was performed in healthy males. The subjects largely consumed their habitual diet, but had to abstain from consuming dairy foods generally high in Ca. The subjects were randomised into the L. acidophilus (dose 109 colony-forming units twice daily; n 20) or the placebo (n 19) group. After an adaptation period of 2 weeks, the subjects were orally infected with a live, but attenuated, ETEC vaccine, able to induce mild, short-lived symptoms. Before and after the challenge, the subjects recorded stool consistency, bowel habits, and frequency and severity of gastrointestinal complaints. The ETEC challenge led to a significant increase in faecal output on the 2nd day and a concomitant increase in Bristol stool scale scores. Likewise, abdominal pain, bloating, flatulence, fever, headache and nausea peaked 1 d after the oral challenge. The concentrations of faecal calprotectin and IgA peaked 2 d after and that of serum IgM peaked 9 and 15 d after the oral challenge. The concentrations of serum IgA and IgG were unaffected. The ETEC challenge led to a reduction in the number of Bacteroides-Prevotella, Bifidobacterium, Clostridium cluster XIVab and total faecal bacteria. Probiotic treatment was associated with a larger increase in Bristol stool scale scores and more fever, headache and nausea after the ETEC challenge compared with the placebo treatment. These differences were, however, small and with substantial variation within the groups. Oral application of an attenuated live ETEC vaccine provides a useful model for food-borne infections. Supplementation with L. acidophilus ATCC 700396, however, was ineffective in reducing ETEC infection symptoms in healthy men.

Dietary fructo-oligosaccharides and inulin decrease resistance of rats to salmonella: protective role of calcium.

BACKGROUND: We have shown recently that rapid fermentable fructo-oligosaccharides (FOS) decreased resistance of rats towards salmonella. It is not known whether inulin (which is fermented more gradually) has similar effects or whether buffering nutrients can counteract the adverse effects of rapid fermentation. AIMS: To compare the effects of dietary inulin and FOS on resistance of rats to Salmonella enterica serovar Enteritidis and to determine whether calcium phosphate counteracts the effects of fermentation. METHODS: Male Wistar rats (n = 8 per group) were fed a human "Western style diet". Diets with 60 g/kg cellulose (control), FOS, or inulin had either a low (30 mmol/kg) or high (100 mmol/kg) calcium concentration. After an adaptation period of two weeks, animals were orally infected with 2 x 10(9) colony forming units of Salmonella enterica serovar Enteritidis. Colonisation of salmonella was determined by quantification of salmonella in caecal contents. Translocation of salmonella was quantified by analysis of urinary nitric oxide metabolites in time. RESULTS: Inulin and FOS decreased intestinal pH and increased faecal lactobacilli and enterobacteria. Moreover, both prebiotics increased the cytotoxicity of faecal water and faecal mucin excretion. Both prebiotics increased colonisation of salmonella in caecal contents and enhanced translocation of salmonella. Dietary calcium phosphate counteracted most of the adverse effects of inulin and FOS. CONCLUSIONS: Both inulin and FOS impair resistance to intestinal infections in rats. This impairment is partially prevented by dietary calcium phosphate. The results of the present study await verification in other controlled animal and human studies.

Dietary fructo-oligosaccharides and lactulose inhibit intestinal colonisation but stimulate translocation of salmonella in rats.

BACKGROUND AND AIMS: It is frequently assumed that dietary non-digestible carbohydrates improve host resistance to intestinal infections by stimulating the protective gut microflora. However, compelling scientific evidence from in vivo infection studies is lacking. Therefore, we studied the effect of several non-digestible carbohydrates on the resistance of rats to Salmonella enteritidis infection. METHODS: Rats (n=8 per group) were fed "humanised" purified diets containing 4% lactulose, fructo-oligosaccharides (FOS), resistant starch, wheat fibre, or cellulose. After an adaptation period of 2 weeks the animals were orally infected with S enteritidis. Supplement induced changes in faecal biochemical and microbiological parameters were studied before infection. Colonisation of salmonella was determined by studying the faecal excretion of this pathogen and translocation by analysis of urinary nitric oxide metabolites over time and classical organ cultures. Intestinal mucosal myeloperoxidase activity was determined to quantify intestinal inflammation after infection. RESULTS: Despite stimulation of intestinal lactobacilli and bifidobacteria and inhibition of salmonella colonisation, FOS and lactulose significantly enhanced translocation of this pathogen. These supplements also increased cytotoxicity of faecal water and faecal mucin excretion, which may reflect mucosal irritation. In addition, caecal and colonic, but not ileal, mucosal myeloperoxidase activity was increased in infected rats fed FOS and lactulose. In contrast, cellulose, wheat fibre, and resistant starch did not affect the resistance to salmonella. CONCLUSIONS: In contrast to most expectations, FOS and lactulose impair the resistance of rats to intestinal salmonella infection. Obviously, stimulation of the endogenous lactobacilli and bifidobacteria is no guarantee of improved host defence against intestinal infections.

Urinary NOx:creatinine ratios during gluten challenge in children with celiac disease.

OBJECTIVES: Celiac disease is a gluten-induced small bowel enteropathy. Inflammation is known to be associated with enhanced nitric oxide (NO) production. An increase in urinary nitrate and nitrite (NOx) reflects increased NO production. The urinary NOx:creatinine ratio can be used as an indicator of the endogenous NO production. The aim of the study was to determine whether the urinary NOx:creatinine ratio of celiac disease patients increases during gluten challenge. METHODS: The authors studied 20 patients with unconfirmed celiac disease who had been following a gluten-free diet for at least 1 year. These patients underwent an 80-day gluten challenge. Urinary samples were obtained before and 10, 20, 40, and 80 days after starting the gluten challenge. The Griess reagent method was used for measuring urinary NOx. RESULTS: Gluten challenge confirmed the diagnosis of celiac disease in 15 of 20 patients. The NOx:creatinine ratios (mmol:mmol) of the biopsy-confirmed celiac disease patients were significantly higher than those of the unconfirmed celiac disease patients (0.67 vs. 0.17 on day 10; 0.78 vs. 0.15 on day 20; 0.85 vs. 0.25 on day 40; and 0.85 vs. 0.17 on day 80). CONCLUSIONS: Gluten challenge resulted in an increased urinary NOx:creatinine ratio in patients with biopsy-confirmed celiac disease. The NOx:creatinine ratio could be useful for the serial evaluation of disease activity.

Increased urinary nitric oxide oxidation products in children with active coeliac disease.

BACKGROUND: Nitric oxide (NO) production catalyzed by iNOS (inducible NO synthase) is thought to take place mainly in macrophages after activation by inflammatory mediators. NO is subsequently oxidized to nitrite and nitrate, which are excreted in urine. The concentration of inflammatory mediators in small bowel biopsy specimens from patients with coeliac disease is increased. The latter could induce increased NO production by stimulation of intestinal macrophage iNOS, resulting in high levels of urinary NO oxidation products, nitrite and nitrate (NOx). AIM: In the present study we evaluated the urinary NOx/creatinine ratios in children with active coeliac disease (n = 22), coeliac disease patients on a gluten-free diet (n = 9), healthy (n = 11) and sick control children (n = 18). METHODS: The Griess reagent method was used for measuring urinary NOx. RESULTS: Median NOx/creatinine ratios of active coeliac disease patients, coeliac disease patients on a gluten-free diet, healthy and sick control patients were 1.21, 0.19, 0.10 and 0.13 mmol/mmol, respectively. All active coeliac disease patients showed increased NOx/ creatinine ratios. Urinary NOx/creatinine ratios of the active coeliac disease patients were significantly higher than those of healthy controls (p < 0.0001), sick controls (p < 0.0001) and coeliac disease patients on a gluten-free diet (p < 0.0001). CONCLUSION: The urinary NOx/creatinine ratio is increased in patients with active coeliac disease and reverts to normal on a gluten-free diet.

Dietary calcium phosphate stimulates intestinal lactobacilli and decreases the severity of a salmonella infection in rats.

We have shown recently that dietary calcium phosphate (CaPi) has a trophic effect on the intestinal microflora and strongly protects against salmonella infection. It was speculated that precipitation by CaPi of intestinal surfactants, such as bile acids and fatty acids, reduced the cytotoxicity of intestinal contents and favored growth of the microflora. Because lactobacilli may have antagonistic activity against pathogens, the main purpose of the present study was to examine whether this CaPi-induced protection coincides with a reinforcement of the endogenous lactobacilli. In vitro, Salmonella enteritidis appeared to be insensitive to bile acids and fatty acids, whereas Lactobacillus acidophilus was killed by physiologically relevant concentrations of these surfactants. Additionally, after adaptation to a purified diet differing only in CaPi concentration (20 and 180 mmol CaHPO4. 2H2O/kg), rats (n = 8) were orally infected with S. enteritidis. Besides reducing the cytotoxicity and the concentration of bile acids and fatty acids of ileal contents and fecal water, CaPi notably changed the composition of ileal bile acids in a less cell-damaging direction. Significantly greater numbers of ileal and fecal lactobacilli were detected in noninfected, CaPi-supplemented rats. As judged by the lower urinary NOx excretion, which is a biomarker of intestinal bacterial translocation, dietary CaPi reduced the invasion of salmonella. Additionally, the colonization resistance was improved considering the reduction of excreted fecal salmonella. In accordance, fewer viable salmonella were detected in ileal contents and on the ileal mucosa in the CaPi group. In conclusion, reducing the intestinal surfactant concentration by dietary CaPi strengthens the endogenous lactobacilli and increases the resistance to salmonella.

Dietary calcium inhibits the intestinal colonization and translocation of Salmonella in rats.

BACKGROUND & AIMS: Dietary calcium decreases the cytotoxicity of intestinal contents and intestinal epitheliolysis by precipitating cytotoxic surfactants such as bile acids. A decreased luminal cytotoxicity might not only strengthen the barrier function of the gut mucosa but also reinforce the protective, endogenous microflora. We hypothesized, therefore, that dietary calcium increases the resistance to intestinal infections. METHODS: Rats on a low-, medium-, or high-calcium purified diet were orally infected with a single dose of Salmonella enteritidis. The kinetics of fecal Salmonella excretion was studied to determine the colonization resistance. Intestinal bacterial translocation was quantitated by measuring urinary oxidation products of nitric oxide (NOx) excretion and culturing bacteria from tissues. RESULTS: Compared with the low-calcium group, the medium- and high-calcium diet-fed rats had a substantially improved colonization resistance. Calcium supplementation also reduced translocation of Salmonella, considering the diminished urinary NOx excretion and viable Salmonella counts in the Ileal Peyer's patches and spleen. Dietary calcium decreased the bile acid concentration and cytotoxicity of fecal water. Several indicators of fecal bacterial mass were significantly increased by supplemental calcium. CONCLUSIONS: Dietary calcium improves the colonization resistance and reduces the severity of gut-derived systemic infections, which is probably attributable to its luminal cytoprotective effects.

Increasing the intestinal resistance of rats to the invasive pathogen Salmonella enteritidis: additive effects of dietary lactulose and calcium.

BACKGROUND AND AIMS: Lactulose fermentation by the intestinal microflora acidifies the gut contents, resulting in an increased resistance to colonisation by acid sensitive pathogens. The extent of fermentation should be controlled to prevent acid induced epithelial cell damage. Considering the buffering capacity of calcium phosphate and its intestinal cytoprotective effects, whether supplemental calcium phosphate adds to the increased resistance to intestinal infections by lactulose fermentations was studied. METHODS: In a strictly controlled experiment, rats were fed a purified low calcium control diet, a low calcium/lactulose diet, or a high calcium/lactulose diet, and subsequently infected orally with Salmonella enteritidis. RESULTS: Lactulose fermentation lowered the pH and increased the lactic acid concentration of the intestinal contents, which significantly reduced excretion of this pathogen in faeces; thus it improved the resistance to colonisation. This agreed with the high sensitivity of S enteritidis to lactic acid (main metabolite of lactulose fermentation) in vitro. Calcium phosphate decreased translocation of S enteritidis to the systemic circulation, an effect independent of lactulose. The unfavourable increased cytotoxicity of faecal water caused by lactulose fermentation was more than counteracted by supplemental calcium phosphate. Moreover, calcium phosphate stimulated lactulose fermentation, as judged by the reduced lactulose excretion in faeces and increased lactic acid, ammonia, and faecal nitrogen excretion. CONCLUSION: Extra calcium phosphate added to a lactulose diet improves the resistance to colonisation and translocation of S enteritidis. This is probably mediated by a calcium induced stimulation of lactulose fermentation by the intestinal microflora and reversion of the lactulose mediated increased luminal cytotoxicity, which reduces damage inflicted on the intestinal mucosa.

Nitric oxide-derived urinary nitrate as a marker of intestinal bacterial translocation in rats.

BACKGROUND/AIMS: Bacterial translocation across the gut wall may lead to bacteremia and sepsis. Bacteriological analyses are laborious and time consuming, which precludes a rapid diagnosis of bacterial translocation. Synthesis of nitric oxide by macrophages is a primary response to bacterial infections. Therefore, the aim of this study was to examine whether NO-derived nitrate excretion in urine can be used as a rapid and quantitative marker of intestinal bacterial translocation. METHODS: The kinetics of urinary nitrate excretion was determined in rats intraperitoneally injected with increasing doses of Salmonella enteritidis lipopolysaccharide. Subsequently, the response to bacterial translocation was studied in rats infected orally with different doses of viable, invasive S. enteritidis. RESULTS: Increasing the lipopolysaccharide dose from 0.05 to 0.50 mg/kg resulted in a transient, dose-dependent, almost 10-fold increase in urinary nitrate excretion. Administration of the NO synthase inhibitor NG-nitro-L-arginine methyl ester merely inhibited the increase in nitrate excretion after lipopolysaccharide injection. Increasing the infective dose of viable Salmonella resulted in a time- and dose-dependent exponential increase in nitrate output. Translocation was a prerequisite for provoking a nitrate response. Total urinary nitrate excretion after infection and classical infection parameters, such as weight of the mesenteric lymph nodes and population levels of Salmonella in feces, were highly correlated. CONCLUSIONS: Urinary nitrate excretion is a quantitative, noninvasive biomarker of intestinal bacterial translocation, which can be used to follow the course of a systemic infection.

Effects of cholesterol and oxysterols on gap junctional communication between human smooth muscle cells.

Intercellular communication is considered to play an essential role in maintaining and controlling cell growth, cell differentiation and homeostasis. Cell-cell communication can be regulated by factors that influence gap junctional function. In this study it was demonstrated that cholesterol and oxidized cholesterol have the potential to modulate gap junctional communication between human smooth muscle cells in an opposite way. Cholesterol supplementation to human smooth muscle cells resulted in an increase of gap junctional communication up to 130% with regard to the control values. However, autooxidized cholesterol inhibited gap junctional communication more than 40%. Testing of several pure cholesterol oxidation derivates on gap junctional communication demonstrated that all of them were capable to inhibit intercellular communication in the order 25-hydroxycholesterol greater than cholestan-3 beta,5 alpha,6 beta-triol greater than 7-ketocholesterol greater than cholesterol 5,6 alpha-epoxide. The cell-cell communication-inhibiting potency of these oxysterols is in accordance with their atherogenic potency. This implies that cholesterol oxidation products, instead of pure cholesterol, can be promoting factors in the atherogenesis by influencing gap junctional communication between arterial smooth muscle cells, the target cells of atherosclerotic lesions.