Administration of probiotics to healthy volunteers: effects on reactivity of intestinal mucosa and systemic leukocytes.

BACKGROUND: Oral administration of health-promoting bacteria is increasingly used in clinical practise. These bacteria have anti-inflammatory characteristics and modulate the immune system without major reported side effects. The mechanisms of action are not yet fully defined. Our aim was to study systemic effects of probiotics by measurements of leukocytes as well as local effects on rectal mucosal biopsies after adding a standardized inflammatory stimulus in vitro. METHODS: Fourteen healthy subjects were randomized to receive 1010 colony forming units/day orally of the probiotic strain Lactiplantibacillus plantarum 299 (Lp299), n = 7, or Bifidobacterium infantis CURE21 (CURE21), n = 7, for six weeks. Rectal biopsies were taken before and after ingestion of either probiotic strain product, for stimulation in vitro with tumour necrosis factor alpha (TNF-α) at 10 and 100 ng/ml respectively up to 8 h. Blood tests were sampled before and after treatment. Lactate dehydrogenase (LDH) confirmed viable tissue. RESULTS: Composition of the intestinal microbiota was not changed. Systemic leukocytes decreased after administration of CURE21 (P<0.05) and Lp299 (P<0.01). Levels of the pro-inflammatory cytokine IL-6 in rectal mucosa after stimulation with TNF-α were attenuated after ingestion of Lp299. No effect was seen with CURE21. CONCLUSIONS: Administration of these probiotic strains to healthy humans show both a systemic and local reduction of inflammatory response by lowering leukocyte counts, and for Lp299 IL-6 levels in rectal mucosa. Probiotics may play an important role in the reduction of inflammatory responses expected after trauma during surgery or after pelvic irradiation. Trial registration Clinical Trials, registration number NCT01534572, retrospectively registered ( http://www.clinicaltrials.gov ).

Effects of acute stress provocation on cortisol levels, zonulin and inflammatory markers in low- and high-stressed men.

The virtual version of the Trier Social Stress Test (V-TSST) is an effective and standardized tool for social stress induction. This study aimed to examine gut permeability and physiological and inflammatory markers of reactivity to acute psychosocial stress. Forty young men were classified as high-stressed (HIGHS) or low-stressed (LOWS) according to the Shirom-Melamed Burnout Questionnaire. Cardiovascular reactivity and gut dysfunction were studied along with cortisol, zonulin and cytokines. Gut permeability was shown to be affected within one hour after the psychosocial stress induction, and shown to be dependent on age. Interleukin-6 increased with time, most pronounced at the end of the one-hour recovery after V-TSST, and was positively correlated to age. HIGHS experienced more abdominal dysfunction compared to LOWS. In conclusion, this study is the first to show fluctuations in gut permeability after psychosocial stress induction. This was partly associated with changes in inflammatory markers.

Anchorless surface associated glycolytic enzymes from Lactobacillus plantarum 299v bind to epithelial cells and extracellular matrix proteins.

An important criterion for the selection of a probiotic bacterial strain is its ability to adhere to the mucosal surface. Adhesion is usually mediated by proteins or other components located on the outer cell surface of the bacterium. In the present study we characterized the adhesive properties of two classical intracellular enzymes glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and enolase (ENO) isolated from the outer cell surface of the probiotic bacterium Lactobacillus plantarum 299v. None of the genes encoded signal peptides or cell surface anchoring motifs that could explain their extracellular location on the bacterial surface. The presence of the glycolytic enzymes on the outer surface was verified by western blotting using polyclonal antibodies raised against the specific enzymes. GAPDH and ENO showed a highly specific binding to plasminogen and fibronectin whereas GAPDH but not ENO showed weak binding to mucin. Furthermore, a pH dependent and specific binding of GAPDH and ENO to intestinal epithelial Caco-2 cells at pH 5 but not at pH 7 was demonstrated. The results showed that these glycolytic enzymes could play a role in the adhesion of the probiotic bacterium L. plantarum 299v to the gastrointestinal tract of the host. Finally, a number of probiotic as well non-probiotic Lactobacillus strains were analyzed for the presence of GAPDH and ENO on the outer surface, but no correlation between the extracellular location of these enzymes and the probiotic status of the applied strains was demonstrated.

Green tea powder and Lactobacillus plantarum affect gut microbiota, lipid metabolism and inflammation in high-fat fed C57BL/6J mice.

BACKGROUND: Type 2 diabetes is associated with obesity, ectopic lipid accumulation and low-grade inflammation. A dysfunctional gut microbiota has been suggested to participate in the pathogenesis of the disease. Green tea is rich in polyphenols and has previously been shown to exert beneficial metabolic effects. Lactobacillus plantarum has the ability to metabolize phenolic acids. The health promoting effect of whole green tea powder as a prebiotic compound has not been thoroughly investigated previously. METHODS: C57BL/6J mice were fed a high-fat diet with or without a supplement of 4% green tea powder (GT), and offered drinking water supplemented with Lactobacillus plantarum DSM 15313 (Lp) or the combination of both (Lp + GT) for 22 weeks. Parameters related to obesity, glucose tolerance, lipid metabolism, hepatic steatosis and inflammation were examined. Small intestinal tissue and caecal content were collected for bacterial analysis. RESULTS: Mice in the Lp + GT group had significantly more Lactobacillus and higher diversity of bacteria in the intestine compared to both mice in the control and the GT group. Green tea strongly reduced the body fat content and hepatic triacylglycerol and cholesterol accumulation. The reduction was negatively correlated to the amount of Akkermansia and/or the total amount of bacteria in the small intestine. Markers of inflammation were reduced in the Lp + GT group compared to control. PLS analysis of correlations between the microbiota and the metabolic variables of the individual mice showed that relatively few components of the microbiota had high impact on the correlation model. CONCLUSIONS: Green tea powder in combination with a single strain of Lactobacillus plantarum was able to promote growth of Lactobacillus in the intestine and to attenuate high fat diet-induced inflammation. In addition, a component of the microbiota, Akkermansia, correlated negatively with several metabolic parameters known to be risk factors for the development of type 2 diabetes.

Blueberry husks and probiotics attenuate colorectal inflammation and oncogenesis, and liver injuries in rats exposed to cycling DSS-treatment.

Long-term colonic inflammation promotes carcinogenesis and histological abnormalities of the liver, and colorectal tumours frequently arise in a background of dysplasia, a precursor of adenomas. Altered colonic microbiota with an increased proportion of bacteria with pro-inflammatory characteristics, have been implicated in neoplastic progression. The composition of the microbiota can be modified by dietary components such as probiotics, polyphenols and dietary fibres. In the present study, the influence of probiotics in combination with blueberry husks on colorectal carcinogenesis and subsequent liver damage was evaluated.Colorectal tumours were induced in rats by cyclic treatment with dextran sulphate sodium (DSS). Blueberry husks and a mixture of three probiotic strains (Bifidobacterium infantis DSM 15159, Lactobacillus gasseri, DSM 16737 and Lactobacillus plantarum DSM 15313) supplemented a basic diet fortified with oats. The condition of the rats was monitored using a disease activity index (DAI). A qualitative and quantitative histological judgement was performed on segments of distal colon and rectum and the caudate lobe of the liver. The formation of short-chain fatty acids, bacterial translocation, the inflammatory reaction and viable count of lactobacilli and Enterobaceriaceae were addressed.Blueberry husks with or without probiotics significantly decreased DAI, and significantly reduced the number of colonic ulcers and dysplastic lesions. With a decreased proportion of blueberry husk in the diet, the probiotic supplement was needed to achieve a significant decrease in numbers of dysplastic lesions. Probiotics decreased faecal viable count of Enterobacteriaceae and increased that of lactobacilli. Blueberry husks with or without probiotics lowered the proportion of butyric acid in distal colon, and decreased the haptoglobin levels. Probiotics mitigated hepatic injuries by decreasing parenchymal infiltration and the incidence of stasis and translocation. The results demonstrate a dietary option for use of blueberry husks and probiotics to delay colonic carcinogenesis and hepatic injuries in the rat model.

Lactobacillus plantarum 299v does not reduce enteric bacteria or bacterial translocation in patients undergoing colon resection.

BACKGROUND: Probiotics may exert beneficial effects in the gastrointestinal tract. This randomized trial investigated the effect of the probiotic Lactobacillus plantarum 299v on the intestinal load of potentially pathogenic bacteria, bacterial translocation, and cell proliferation in elective colon surgery. METHODS: Seventy-five patients were randomized to pre- and postoperative oral intake of Lactobacillus plantarum 299v or placebo. Rectal swabs and mucosal biopsies were taken before the start of intake, after 1 week, at surgery, and after 6 days, weeks, and months. Viable counts were quantified for clostridia, Enterobacteriaceae, Gram-negative anaerobes, and lactobacilli. Bacterial translocation was determined by the analysis of bacterial DNA genes in mesenteric lymph nodes. Ki-67 was used as a marker of cell proliferation in normal mucosa and tumor. RESULTS: Lactobacillus plantarum 299v was given without adverse effects. Lactobacillus plantarum 299v as well as Enterobacteriaceae and Gram-negative anaerobes increased in the colon 1 week after the administration of Lactobacillus plantarum 299v. There were no significant differences between patients receiving Lactobacillus plantarum 299v and placebo in the incidence of bacterial translocation (27 vs. 13%) and postoperative complications (16 vs. 31%). CONCLUSIONS: Lactobacillus plantarum 299v was established in the intestine, but no inhibitory effect on enteric bacteria, bacterial translocation, or postoperative complications was found. The mechanism behind the protective effects of probiotics found in animal and some human studies remain elusive and require further explorations. No adverse effects were recorded after the administration of high doses of Lactobacillus plantarum 299v.

Colorectal Oncogenesis and Inflammation in a Rat Model Based on Chronic Inflammation due to Cycling DSS Treatments.

Inflammation is known to be linked with development of colorectal cancer, and the aim was to assess the malignant potential and degree of inflammation in a dextran-sulphate-sodium-(DSS-) induced cyclic colonic tumour model (CTM) in rats and to compare it with the azoxymethane-(AOM-) induced CTM model. Tumours developed in both groups, although, in the DSS group, the colonic mucosa appeared edematous and the number of haemorrhagic erosions and quantity of dysplastic lesions were higher as well as the mucosal concentration of myeloperoxidase and faecal viable count of Enterobacteriaceae. The livers were affected as evaluated by steatosis, parenchymal loss, haemorrhage, and inflammatory infiltrations, and higher proportions of acetate and lower proportions of butyrate in colonic content were found. The DSS model seems to mimic the clinical situation and may be valuable for investigation of inflammation-related dysplasia and colon cancer, as well as for altered liver function by endogenous inflammatory mediators.

Antioxidative protection of dietary bilberry, chokeberry and Lactobacillus plantarum HEAL19 in mice subjected to intestinal oxidative stress by ischemia-reperfusion.

BACKGROUND: Ischemia-reperfusion (I/R) in the intestines is an inflammatory condition which activates leukocytes and reactive oxygen species (ROS) and leads to lipid peroxidation and DNA damage. Bilberry and chokeberry fruits are rich sources of polyphenols which may act as antioxidants and prevent lipid peroxidation. Lactic acid bacteria (LAB) may improve microbial status in the intestines and increase the metabolic activity towards polyphenolic degradation. The aim of the study was to clarify antioxidative effects of bilberry and chokeberry fruits alone and with addition of a LAB-strain, Lactobacillus plantarum HEAL19, in an I/R-model in mice. METHODS: Male BALB/cJ mice were fed the experimental diets for 10 days. Diets consisted of standard chow supplemented with either bilberry (Vaccinium myrtillus) or chokeberry (Aronia × prunifolia) powder alone or in combination with the LAB-strain Lactobacillus plantarum HEAL19. I/R-injury was induced by holding superior mesenteric artery clamped for 30 minutes followed by reperfusion for 240 minutes. Thereafter, colonic and caecal tissues and contents were collected. Malondialdehyde (MDA) was used as indicator of lipid peroxidation and was measured by a calorimetric assay, lactobacilli were cultured on Rogosa agar plates and Enterobacteriaceae on VRBG agar plates, anthocyanins and phenolic acids were analysed by HPLC-DAD-ESI-MSn. RESULTS: MDA was significantly decreased in the colon of groups fed bilberry alone (p = 0.030) and in combination with L. plantarum HEAL19 (p = 0.021) compared to the IR-control but not in chokeberry-fed groups. Supplementation with bilberry or chokeberry alone reduced the total number of lactobacilli on the mucosa. Higher concentrations of anthocyanins were found in the colon than in the caecum content of mice. A more varied composition of different anthocyanins was also observed in the colon content compared to the caecum of bilberry-fed mice. Phenolic acids formed by microbial degradation of the dietary polyphenols in the gut could be detected. More phenolic metabolites were found in the intestines of bilberry-fed mice than in the chokeberry-fed ones. CONCLUSIONS: Bilberry alone and in combination with L. plantarum HEAL19 exerts a better protection against lipid peroxidation than chokeberry. These dietary supplements may be used to prevent or suppress oxidative stress.

Effect of lactobacilli on paracellular permeability in the gut.

Paracellular permeability is determined by the complex structures of junctions that are located between the epithelial cells. Already in 1996, it was shown that the human probiotic strain Lactobacillus plantarum 299v and the rat-originating strain Lactobacillus reuteri R2LC could reduce this permeability in a methotrexate-induced colitis model in the rat. Subsequently, many animal models and cell culture systems have shown indications that lactobacilli are able to counteract increased paracellular permeability evoked by cytokines, chemicals, infections, or stress. There have been few human studies focusing on the effect of lactobacilli on intestinal paracellular permeability but recently it has been shown that they could influence the tight junctions. More precisely, short-term administration of L. plantarum WCSF1 to healthy volunteers increased the relocation of occludin and ZO-1 into the tight junction area between duodenal epithelial cells.

Probiotics and blueberry attenuate the severity of dextran sulfate sodium (DSS)-induced colitis.

We studied the anti-inflammatory properties of probiotic strains and blueberry in a colitis model. The disease activity index (DAI) was significantly lower on days 9 and 10 in all groups compared to the colitis control. Myeloperoxidase (MPO) and bacterial translocation to the liver and to the mesenteric lymph nodes (MLN) decreased significantly in all groups compared to colitis control. Cecal Enterobacteriaceae count decreased significantly in blueberry with and without probiotics compared to the other groups. Lactobacillus plantarum reisolated from the cecal content in the presence of blueberry, contrary to Lactobacillus fermentum. Colonic MDA decreased significantly in all groups, except the L. fermentum group, compared to the colitis control. The cecal concentration of acetic, propionic, and butyricbutyric acid was significantly higher in the L. plantarum group, while the L. fermentum group yielded the highest concentration of lactic acid compared with all other groups. Lactobacillus plantarum DSM 15313, Lactobacillus fermentum 35D, and blueberry alone and in combination improve the DAI, reduce bacterial translocation, and reduce inflammation.

Ileal pelvic pouch microbiota from two former ulcerative colitis patients, analysed by DNA-based methods, were unstable over time and showed the presence of Clostridium perfringens.

OBJECTIVE: Ileal pouch anal anastomosis (IPAA) is the preferred method for restorative surgery in patients with ulcerative colitis who have to undergo proctocolectomy. The most common complication is pouchitis and several studies have pointed to the microbiota of the pouch as being a risk factor. The aim of this study was to follow the development of the bacterial microbiota in pouches during the first year. MATERIAL AND METHODS: Terminal restriction fragment length polymorphism (T-RFLP) combined with cloning and sequencing was used to identify the most predominant bacteria on the different sampling occasions. A total of 274 clones were grouped by T-RFLP and clones from each group were selected for sequencing and identified by comparison with known sequences. RESULTS: Differences in T-RFLP profiles and clone libraries were found between the patients, and also in changes apparent in each patient at different time-points. The main bacterial groups in the pouches resembled those of the normal colonic microbiota, with a predominance of the clostridia clusters XIVa and IV, Bacteroides and Enterobacteriaceae. Exceptions were clones with sequences resembling those of the Clostridium perfringens group, in both patients and on all sampling occasions, and the dominance of clones resembling Turicibacter in one of the patients at the time of pouch construction. CONCLUSIONS: The pouch microbiota showed similarities to the normal colon microbiota except for the presence of clones with sequences resembling those of the C. perfringens group and Turicibacter. The bacterial composition differed between the two patients and the microbiota changed with time, suggesting that the composition is not stable during the first year.

High proportions of proinflammatory bacteria on the colonic mucosa in a young patient with ulcerative colitis as revealed by cloning and sequencing of 16S rRNA genes.

The pathogenesis of ulcerative colitis (UC) remains unknown. It is thought to be due to an abnormal and uncontrolled immune response to normally occurring constituents of the intestine. Microbial agents appear to be involved in the pathogenesis and intestinal bacteria seem to be an important factor in the development and chronicity. The aim of this study was to investigate the colonic microbiota of a patient with UC. The colonic tissues were taken during surgery from a 12-year-old girl suffering from UC. The microbiota on the colonic samples was studied by cloning and sequencing of amplified 16S rRNA genes. Compared with healthy subjects, alteration of the dominant bacterial group was observed in the UC patient. We found a high incidence of Enterobacteriaceae, Bacteroides fragilis, and the single phylotype of the Faecalibacterium prausnitzii-like "Butyrate-producing bacterium" L2-6. Furthermore, there was a substantial presence of Pseudomonas aeruginosa in the present case of UC. The high proportion of adverse proinflammatory species is striking in the present case compared with more normal situations. Even if those bacteria are not the cause of the UC, they most probably enhance the symptoms of the disease.

Multiple displacement amplification of DNA from human colon and rectum biopsies: bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis.

Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens.

TNF-alpha sensitizes HT-29 colonic epithelial cells to intestinal lactobacilli.

The ability of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) to influence epithelial interleukin (IL)-8 responses to the intestinal bacterium Lactobacillus plantarum 299v was analyzed in the human HT-29 colonic epithelial cell line. In the absence of TNF-alpha, IL-8 mRNA expression was not detectable by Northern blot analysis in HT-29 cells alone or in HT-29 cells co-cultured with L. plantarum 299v. However, TNF-alpha induced IL-8 mRNA expression, and co-culture of TNF-alpha-treated HT-29 cells with L. plantarum 299v significantly increased IL-8 mRNA expression above levels induced by TNF-alpha alone in an adhesion-dependent manner. The increase in IL-8 mRNA expression was not observed in TNF-alpha-treated HT-29/L. plantarum 299v co-cultures using heat-killed lactobacilli or when L. plantarum adhesion was prevented using mannoside or a trans-well membrane. Paradoxically, IL-8 secretion was decreased in TNF-alpha-treated HT-29 cells with L. plantarum 299v relative to cells treated with TNF-alpha alone. TNF-alpha-mediated responsiveness to L. plantarum 299v was further investigated by analyzing expression of a coreceptor for bacterial cell wall products CD14. HT-29 cells expressed CD14 mRNA and cell-surface CD14; however, TNF-alpha did not alter CD14 mRNA or cell-surface expression, and blockade of CD14 with monoclonal antibody MY4 did not alter the IL-8 response to L. plantarum 299v in TNF-alpha-treated HT-29 cells. These results indicate that although TNF-alpha sensitizes HT-29 epithelial cells to intestinal lactobacilli, the bacteria exert a protective effect by downregulating IL-8 secretion.