Staining of stratum corneum with fluorescent ε-poly-L-lysine and its application to evaluation of skin conditions.

BACKGROUND: ε-Poly-L-lysine (PLL) is a cationic polymer consisting of 25 to 35 L-lysine residues that adheres to the surface of skin as well as hair. However, the properties of PLL regarding its adhesion to the skin remain to be elucidated. In this study, we examined the staining of stratum corneum (SC) with fluorescence-labeled PLL and explored its relationship with skin condition. MATERIALS AND METHODS: Alexa Fluor 488-labeled PLL (AF-PLL) was reacted with tape-stripped stratum corneum (SC), and the staining properties were monitored by fluorescence microscopy. Clinical study was performed by measuring the water content of the cheek SC and transepidermal water loss (TEWL), and the tape-stripped SC was subjected to staining with AF-PLL. RESULTS: AF-PLL staining of the SC was inhibited at acidic pH or by the addition of high concentration of salt solution, suggesting the involvement of ionic interaction between PLL and the SC, at least in part. The AF-PLL staining was inhibited by unlabeled PLL or various alkyl amines, but not by L-lysine monomer. AF-PLL staining was observed inside the corneocytes as well as surrounding cornified envelope. Clinical study revealed that AF-PLL staining intensity of the SC was negatively correlated with its water content and positively correlated with its TEWL. CONCLUSION: PLL can efficiently adhere to SC and AF-PLL staining of SC can be applied to evaluate skin conditions.

Novel double staining of the stratum corneum with fluorescent ε-poly-l-lysine and anionic dextran.

OBJECTIVE: ε-Poly-l-lysine (PLL) is a cationic polymer consisting of 25-35 l-lysine residues. Our previous study revealed that fluorescently labelled PLL can stain the stratum corneum (SC) via ionic interactions between PLL and SC constituents. In this study, to further clarify the mechanisms underlying the interaction between PLL and the SC, the staining properties of fluorescent PLL were compared with that of fluorescently labelled anionic dextran (aDex), which has approximately the same molecular weight as PLL. METHODS: SC samples were collected by non-invasive tape stripping and stained with fluorescent PLL and/or fluorescent aDex. Fluorescence images were acquired using a fluorescence microscope and then analysed. RESULTS: The SC could be stained with either fluorescent PLL or aDex, both of which were inhibited by the addition of high concentrations of salt solutions. In particular, aDex staining was inhibited at a lower salt concentration than PLL staining. Moreover, PLL staining was inhibited under acidic conditions, while aDex staining was inhibited under neutral to alkaline conditions. Double staining of SC with both fluorescent polymers produced heterogeneous staining patterns: corneocytes stained with both polymers, corneocytes stained with PLL or aDex in a mutually exclusive manner, and unstained corneocytes. Staining of SC samples from the face was more extensive than staining of SC samples from the inside of the upper arm with both polymers. In addition, pretreatment of the SC with ethanol resulted in enhanced staining with both polymers. These results suggest that double staining of SC with both polymers can provide information on the damaged SC. CONCLUSION: Staining of SC with fluorescent PLL depends on its properties of a cationic and hydrophobic polymer with appropriate molecular size, which can distinguish the damaged SC. Double staining of SC with fluorescent PLL and aDex is a novel approach to obtain information for the analysis of skin conditions.

OBJECTIF: La ε-poly-L-lysine (PLL) est un polymère cationique constitué de résidus de 25 à 35 L-lysines. Notre précédente étude a révélé que la PLL marquée par fluorescence peut colorer le stratum corneum (SC) par des interactions ioniques entre la PLL et les constituants du SC. Dans cette étude, afin de clarifier davantage les mécanismes sous-jacents à l'interaction entre la PLL et le SC, les propriétés de coloration de la PLL fluorescent ont été comparées à celles du dextran anionique (aDex) marqué par fluorescence, qui a à peu près le même poids moléculaire que la PLL. MÉTHODES: Les échantillons SC ont été prélevés par «tape stripping¼ non invasif et colorés avec de la PLL fluorescente et/ou de l'aDex fluorescent. Les images de fluorescence ont été acquises au microscope à fluorescence puis analysées. RÉSULTATS: Le SC pouvait être coloré avec de la PLL ou de l'aDex fluorescents, tous deux inhibés par l'ajout de fortes concentrations de solutions salines. En particulier, la coloration par aDex était inhibée à une concentration en sel inférieure à la coloration par PLL. En outre, la coloration de la PLL a été inhibée dans des conditions acides, tandis que la coloration de l'aDex a été inhibée dans des conditions neutres à alcalines. La double coloration de SC avec les deux polymères fluorescents a produit des modes de coloration hétérogènes: cornéocytes colorés avec les deux polymères, cornéocytes colorés avec de la PLL ou de l'aDex d'une manière mutuellement exclusive, et cornéocytes non colorés. La coloration des échantillons de SC sur le visage était plus étendue que la coloration des échantillons de SC sur la face intérieure du haut du bras avec les deux polymères. En outre, le prétraitement du SC avec de l'éthanol a entraîné une coloration améliorée avec les deux polymères. Ces résultats indiquent qu'une double coloration du CS avec les deux polymères peut fournir des informations sur le CS endommagé. CONCLUSION: La coloration du CS avec de la PLL fluorescente dépend de ses propriétés de polymère cationique et hydrophobe de taille moléculaire appropriée, ce qui permet de distinguer le CS endommagé. La double coloration de SC avec de la PLL et de l'aDex fluorescents est une nouvelle approche pour obtenir des informations pour l'analyse des affections cutanées.

Glutamate in the medium of Lactiplantibacillus plantarum FL-664 affects the production of IL-12(p40) on murine spleen cells.

Lactic acid bacteria (LAB) have been attracting attention for their effects on innate immunity, and therefore, it is required to develop an efficient culturing method while maintaining their functionality. In this study, first, we compared the growth and functionality of LAB cultured on food grade (FG) medium with those on standard LAB medium and found that LAB cultured in the FG medium were smaller in cell size with high yield and had a higher ability to induce IL-12(p40) production by murine spleen cells in vitro. Moreover, the higher the glutamate concentration in the medium, the smaller the cell size, and the higher the yield and the higher the ability to induce IL-12 production. Addition of glutamate to the culture medium changes the size of LAB and affects their ability to induce IL-12(p40) production. In conclusion, regulating the concentration of glutamate would be important in the efficient culturing of functional LAB.

Epidermal permeability barrier function and sphingolipid content in the skin of sphingomyelin synthase 2 deficient mice.

Sphingomyelin synthase (SMS) is an enzyme that generates sphingomyelin (SM) from ceramide (CER) and phosphatidylcholine. SM in the epidermis is a precursor of CER, an important lipid for epidermal permeability barrier function. However, the physiological role of SMS in skin is unclear. To uncover the function of SMS in skin, we investigated sphingolipid metabolism enzyme activity in skin, SM content in the epidermis, CER content in the stratum corneum (SC) and transepidermal water loss (TEWL) as an indicator of barrier function in SMS2-knockout (KO) mice. The activities of sphingolipid metabolism enzymes in skin homogenates were measured using a fluorescently labelled substrate. Enzymatic reaction products were detected by high-performance liquid chromatography (HPLC). Lipids in the epidermis or SC were extracted and quantified by high-performance thin layer chromatography (HPTLC). TEWL was measured using a Tewameter TM300. In SMS2-KO mice, SMS activity in skin homogenates, epidermal SM content and SC CER content were significantly decreased relative to wild-type (WT) mice. The TEWL of SMS2-KO mice was significantly increased compared to WT mice. Our data indicate that SMS2 generates SM in the epidermis and contributes to epidermal permeability barrier function and will support understanding of SM-related metabolic disorders.

Gut Microbiota Composition Before and After Use of Proton Pump Inhibitors.

BACKGROUND: Recently, problems associated with proton pump inhibitor (PPI) use have begun to surface. PPIs influence the gut microbiota; therefore, PPI use may increase the risk of enteric infections and cause bacterial translocation. In this study, we investigated fecal microbiota composition, fecal organic acid concentrations and pH, and gut bacteria in the blood of the same patients before and after PPI use. METHODS: Twenty patients with reflux esophagitis based on endoscopic examination received 8 weeks of treatment with PPIs. To analyze fecal microbiota composition and gut bacteria in blood and organic acid concentrations, 16S and 23S rRNA-targeted quantitative RT-PCR and high-performance liquid chromatography were conducted. RESULTS: Lactobacillus species were significantly increased at both 4 and 8 weeks after PPI treatment compared with bacterial counts before treatment (P = 0.011 and P = 0.002, respectively). Among Lactobacillus spp., counts of the L. gasseri subgroup, L. fermentum, the L. reuteri subgroup, and the L. ruminis subgroup were significantly increased at 4 and 8 weeks after treatment compared with counts before treatment. Streptococcus species were also significantly increased at 4 and 8 weeks after PPI treatment compared with counts before treatment (P < 0.01 and P < 0.001, respectively). There was no significant difference in the total organic acid concentrations before and after PPI treatment. Detection rates of bacteria in blood before and after PPI treatment were 22 and 28%, respectively, with no significant differences. CONCLUSIONS: Our quantitative RT-PCR results showed that gut dysbiosis was caused by PPI use, corroborating previous results obtained by metagenomic analysis.

Effects of synbiotics on ileal microbiota.

BACKGROUND & OBJECTIVES: Despite advancements in molecular-based methods, the composition of the human ileal microbiota and the effects of synbiotics/probiotics on its microbes remain poorly understood. The aim of this study was to determine the composition of the mucus microbiota in the human ileum and to assess the effects of oral administration of synbiotics on the microbiota. METHODS: As part of a clinical trial for synbiotics treatment and surgical infection, ileal mucus was sampled when resection of the ileocecal portion was required. The microbiota composition was examined using 16S rRNA-targeted real-time-quantitative polymerase chain reaction. RESULTS: A total of 33 samples from the synbiotics group and 39 from the control group were analyzed. Total numbers of bacteria in the ileum were 108.5 cells/g in the synbiotics group and 108.4 cells/g in the control group, in which obligate anaerobes were dominant over facultative anaerobes. The level of Enterobacteriaceae was significantly lower in the synbiotics group than in the control group. The administered probiotics species Lactobacillus casei strain Shirota and Bifidobacterium breve strain Yakult were detected in 42 and 76 per cent of the synbiotics group, respectively. No significant correlations were observed between tumour stage/size and the various microbes present, except for a negative correlation between tumour size and Bifidobacterium. INTERPRETATION & CONCLUSIONS: The present analysis of a substantial number of samples from surgically resected intestines showed an abundance of obligate anaerobes as a characteristic feature of the ileal mucus microbiota. Our results also indicated that the synbiotics intervention induced a prominent reduction in Enterobacteriaceae in the ileal microbiota.

Yakult Intestinal Flora-SCAN: A Novel Culture-Independent Analytical Method for Detection of Bacteria in the Bloodstream.

We have developed a highly sensitive microbial analytical system, the Yakult Intestinal Flora-SCAN (YIF-SCAN), based on reverse transcription-quantitative PCR, to quantify the intestinal microbiota. Targeting ribosomal RNA "molecules," which are present in abundant copies in the microorganisms, YIF-SCAN is at least hundreds of times more sensitive (detection limits: 102-3 cells/g feces, 1 cell/mL blood) than conventional DNA-based methods, and hence enables highly sensitive and precise detection of viable microorganisms in various types of samples, such as stools, blood etc. Specifically, this high analytical sensitivity enables more accurate and reliable detection (compared with conventional culture methods) of microorganisms migrating in the blood, bacteremia, in patients with different diseases such as pediatric patients with febrile neutropenia, neonates with clinically diagnosed sepsis, and patients with type 2 diabetes mellitus or ischemic stroke. Such detection of bacteremia during gastrointestinal surgery can also be particularly effective for preventing postoperative infectious complications. Herein, we review some of the studies corroborating the potential application and clinical significance of YIF-SCAN in diagnosing bacteremia (even at marginable levels) in patients with different diseases.

Effects of Synbiotics to Prevent Postoperative Infectious Complications in Highly Invasive Abdominal Surgery.

Postoperative infectious complication (POIC) is one of the most common complications following highly invasive abdominal surgeries, such as hepatectomy, esophagectomy, and pancreatoduodenectomy. The surgical stress temporarily deteriorates the intestinal microenvironment, and the fecal concentrations of beneficial bacteria such as Bifidobacterium and Lactobacillus decrease following highly invasive abdominal surgery. In parallel with these changes, the concentrations of fecal short-chain fatty acids (SCFAs) such as acetic acid, propionic acid, and butyric acid also decrease after surgery. In contrast, the fecal concentration of lactic acid increases under this condition because of the deterioration of the metabolism from lactic acid to SCFAs by normal intestinal microflora. Decreased fecal concentration of SCFAs may lead to an impaired intestinal barrier function under stressful condition. Translocation of bacteria from the gut to lymphatic and bloodstream leads to bacteremia and subsequent POICs. The incidence of POICs in patients with unhealthy intestinal microflora before surgery may be more because their intestine is more susceptible to bacterial translocation induced by surgical stress. Therefore, improving the intestinal microenvironment and intestinal barrier function before surgery is crucial to prevent POICs following highly invasive abdominal surgeries. In this regard, the use preoperative synbiotics therapy may be one of the effective ways because it has been shown to improve intestinal microflora, increase fecal SCFAs, prevent bacterial translocation, and reduce the incidence of POICs in several randomized controlled trial in patients undergoing highly invasive abdominal surgeries.

The High Incidence of Bacteremia in Children Undergoing Surgery Can Be Prevented by Bifidobacterium Supplementation.

Major surgical procedures can alter intestinal microbiota and disrupt the intestinal barrier function, leaving the patient at risk for infection. Probiotics are defined as live microorganisms that confer a health benefit on the host when administered in adequate amounts. Although the efficacy of administering probiotics perioperatively to adults has been reported, the clinical significance of probiotics in children undergoing surgery is still unclear. This study provides a brief overview of our randomized controlled trial of preoperative probiotic administration to children, and discusses the relationship between probiotics and their effects in the perioperative period, particularly focusing on bacteremia.

Protective Effect of a Synbiotic against Multidrug-Resistant Acinetobacter baumannii in a Murine Infection Model.

This study investigated the ability of the probiotic Bifidobacterium breve strain Yakult (BbY) to protect against infection, as well as the potentiation of BbY activity by the synbiotic combination of BbY and prebiotic galactooligosaccharides (GOS). The study employed a mouse model of lethal intestinal multidrug-resistant Acinetobacter baumannii (MDRAb) infection. The endogenous intestinal microbiota was disrupted by the administration of multiple antibiotics, causing the loss of endogenous Bifidobacterium Oral infection of these mice with MDRAb resulted in marked growth of this organism. Additional treatment of the infected mice with a sublethal dose of 5-fluorouracil (5-FU) induced systemic invasion by MDRAb and subsequent animal death. The continuous oral administration of BbY increased the survival rate and inhibited the intestinal growth and invasion by MDRAb in the infection model. Disruptions of the intestinal environment and barrier function in the infected mice were attenuated by BbY. Protection against the MDRAb infection was markedly potentiated by a synbiotic combination of BbY and GOS, although GOS by itself did not provide protection. Negative correlations were observed between intestinal MDRAb and BbY counts or acetic acid levels; positive correlations were observed between acetic acid levels and intestinal epithelium expression of tight-junction-related genes. These results demonstrated that the probiotic and synbiotic markedly potentiated protection against fatal intestinal infection caused by a multidrug-resistant bacterium. Probiotics and synbiotics are presumed to provide protection by compensation for the disrupted indigenous populations, thereby maintaining the intestinal environments and barrier functions otherwise targeted during opportunistic infection by MDRAb.

Intestinal Microbiota in Pediatric Surgical Cases Administered Bifidobacterium Breve: A Randomized Controlled Trial.

The efficacy of perioperative probiotic administration has been reported in adults. We examined the effects of orally administered Bifidobacterium breve strain Yakult (BBG-01) on outcomes in pediatric surgical cases by assessing intestinal and blood microbiota. BBG-01 was well tolerated without adverse effects, and postoperative infectious complications were significantly decreased. Fecal analysis showed increased Bifidobacterium and decreased Enterobacteriaceae, Clostridium difficile, and Pseudomonas. Concentrations of fecal acetic acid were significantly increased, maintaining fecal pH at <7.0. The incidence of detecting bacteria in blood was significantly reduced. BBG-01 improved the intestinal environment, and may be implicated in suppressing bacterial translocation.

Effect of Perioperative Synbiotic Treatment on Bacterial Translocation and Postoperative Infectious Complications after Pancreatoduodenectomy.

BACKGROUND/AIMS: This study investigated the effect of perioperative synbiotics on bacterial translocation to mesenteric lymph nodes (MLNs) and occurrence of infectious complications following pancreatoduodenectomy (PD; University Hospital Medical Information Network ID 000003705). METHODS: Patients who were scheduled to undergo PD were randomized to receive preoperative synbiotics or no synbiotics (control group). MLNs were harvested at laparotomy (MLN-1) and after the resection (MLN-2). Blood samples were collected before laparotomy (Blood-1) and on postoperative day 1 (Blood-2). Microorganisms in each sample were detected using a bacterium-specific ribosomal RNA-targeted reverse transcriptase-quantitative polymerase chain reaction. RESULTS: Forty-four patients were included. In all samples, the bacterial detection rate in the MLN-1, MLN-2, Blood-1, and Blood-2 was lower in the synbiotics group than in the control group, although it did not reach a statistically significant difference. There was a significant correlation between the positivity of bacteria in the MLN-2 and Blood-2 samples (p = 0.009). The incidence rate of overall infectious complications was not significantly different between the 2 groups. Among various perioperative factors, the incidence of pancreatic fistula was the only factor that had a significant association with the incidence of infectious complications. CONCLUSION: The preoperative use of synbiotics did not affect the incidence of infectious complications following PD.

The Effectiveness of Lactobacillus Beverages in Controlling Infections among the Residents of an Aged Care Facility: A Randomized Placebo-Controlled Double-Blind Trial.

BACKGROUNDS/AIMS: To clarify the usefulness of Lactobacillus casei strain Shirota (LcS)-fermented milk in the normalization of bowel movements and improvement of infection control for the elderly residents and staff of facilities for the elderly. METHODS: A randomized placebo-controlled double-blind test was performed among the elderly residents (average age, 85) and staff members (average age, 37) of facilities for the elderly. The participants randomly received either LcS-fermented milk or a placebo beverage once daily for 6 months. Clinical data and enteric conditions were compared between the 2 groups. RESULTS: A significantly lower incidence of fever and improved bowel movements were seen in the LcS-fermented milk group (n = 36) in comparison to the placebo group (n = 36). The numbers of Bifidobacterium and Lactobacillus were significantly higher (p < 0.01), the numbers of destructive bacteria such as Clostridium difficile were significantly lower (p < 0.05), and the fecal acetic acid concentration and total acidity were significantly higher in the LcS group. A significant difference in the intestinal microbiota, fecal acetic acid, and pH was also observed between the LcS and placebo groups among the facility's staff members. CONCLUSIONS: The long-term consumption of LcS-fermented milk may be useful for decreasing the daily risk of infection and improving the quality of life among the residents and staff of facilities for the elderly.

Efficacy of perioperative synbiotics treatment for the prevention of surgical site infection after laparoscopic colorectal surgery: a randomized controlled trial.

PURPOSE: The aim of this study was to assess the effect of perioperative oral administration of synbiotics on the surgical outcome in patients undergoing laparoscopic colorectal resection. METHODS: In this single-center randomized, controlled trial, patients scheduled to undergo elective laparoscopic colorectal surgery were eligible to participate and randomly assigned to a synbiotics group or a control group. The primary study outcome was the development of infectious complications, particularly surgical site infection (SSI), within 30 days of surgery. RESULTS: In this study, 379 patients were enrolled and randomly assigned (173 to the synbiotics group and 206 to the control group), of whom 362 patients (168 to the synbiotics group and 194 to the control group) were eligible for this study. SSI occurred in 29 (17.3%) patients in the synbiotics group and 44 (22.7%) patients in the control group (OR: 0.761, 95% CI 0.50-1.16; p = 0.20). Overall, the rate of postoperative complications, including anastomotic leakage, did not differ significantly between the two groups. Synbiotics treatment reversed the changes in fecal bacteria and organic acids after surgery and suppressed the increases in potentially pathogenic species, such as Clostridium difficile. CONCLUSION: The efficacy of perioperative administration of synbiotics was not validated as a treatment for reducing the incidence of infectious complications after laparoscopic colorectal resection. However, the microbial imbalance, in addition to the reduction in organic acids, could be improved by perioperative synbiotics treatment.

Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes.

BACKGROUND: Clostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its α-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of α-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults. METHODS: The plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221). RESULTS: The qPCR assays were highly specific and sensitive, with a minimum detection limit of 10(3) bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36% infants and 33% adults, with counts ranging widely (10(3)-10(7) bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0±1.5 log10 bacterial cells/g), as compared to that in adults (4.8±1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9±1.9 and 4.8±0.8 log10 bacterial cells/g in infants and adults, respectively. CONCLUSIONS: These data indicate that some healthy infants and young adults carry α-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of α-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.

Enhanced Immunostimulating Activity of Lactobacilli-Mimicking Materials by Controlling Size.

The design and synthesis of materials capable of activating the immune system in a safe manner is of great interest in immunology and related fields. Lactobacilli activate the innate immune system of a host when acting as probiotics. Here, we constructed lactobacilli-mimicking materials in which polysaccharide-peptidoglycan complexes (PS-PGs) derived from lactobacilli were covalently conjugated to the surfaces of polymeric microparticles with a wide variety of sizes, ranging from 200 nm to 3 µm. The artificial lactobacilli successfully stimulated macrophages without cytotoxicity. Importantly, we found that the size of artificial lactobacilli strongly influenced their immunostimulating activities, and that artificial lactobacilli of 1 µm exhibited 10-fold higher activity than natural lactobacilli. One major advantage of the artificial lactobacilli is facile control of size, which cannot be changed in natural lactobacilli. These findings provide new insights into the design of materials for immunology as well as the molecular biology of lactobacillus.

Synthesis and immunestimulating activity of lactobacilli-originated polysaccharide-polymeric microparticle conjugates.

The design and synthesis of biomaterials capable of activating the immune system are of interest in immunology-related fields because of their ability to tune up the immune defenses of the host. Lactobacilli are a major constituent of normal human indigenous flora, and some specific strains are known to activate the immune system of the host as probiotics. In this study, we first fabricated novel biohybrid materials in which lactobacilli (L. casei strain Shirota, LcS)-originated polysaccharide-peptidoglycan complexes (PS-PGs) are conjugated with polymeric microparticles (MPs). PS-PGs conjugated onto polymeric MPs surfaces bound its specific antibody, suggesting that PS-PGs kept their original molecular recognition ability. The PS-PGs-based hybrid MPs with an appropriate density of conjugated PS-PGs effectively induced high levels of IL-12 production from macrophages without cytotoxicity. These results suggest that LcS-originated PS-PGs could be available bio-originated materials for developing novel biomaterials capable of activating the immune system in a safe manner.

Intestinal Microbiota Profiles of Healthy Pre-School and School-Age Children and Effects of Probiotic Supplementation.

OBJECTIVES: This study aims to establish the baseline profile of intestinal microbiota in pre-school and school-age Japanese children and to investigate the effects of a probiotic on the microbiota. METHODS: We analyzed the intestinal microbiota and investigated the effects (before, during and after the ingestion period) on intestinal microbiota and the environment of 6 months of daily ingestion of a probiotic (Lactobacillus casei strain Shirota (LcS)-fermented milk). RESULTS: We performed an open trial in 23 children (14 boys, 9 girls; age 7.7 ± 2.4 years (mean ± SD); BMI 19.6 ± 4.6). The composition of intestinal microbiota of healthy pre-school and school-age children resembled that of adults. During probiotic supplementation, the population levels of Bifidobacterium and total Lactobacillus increased significantly, while those of Enterobacteriaceae, Staphylococcus and Clostridium perfringens decreased significantly. A significant increase in fecal concentrations of organic acids and also a decrease in fecal pH were observed during the ingestion period. However, the patterns of fecal microbiota and intestinal environment were found to revert to the baseline levels (i.e. before ingestion) within 6 months following the cessation of probiotic intake. CONCLUSION: Regular intake of an LcS-containing probiotic product may modify the gut microbiota composition and intestinal environment in pre-school and school-age children while maintaining the homeostasis of the microbiota.

The detection of intraoperative bacterial translocation in the mesenteric lymph nodes is useful in predicting patients at high risk for postoperative infectious complications after esophagectomy.

OBJECTIVE: To investigate the incidence of BT in the mesenteric lymph node and bacteremia after an esophagectomy using a bacterium-specific ribosomal RNA-targeted reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). BACKGROUND: There is little evidence regarding the occurrence of bacterial translocation (BT) and its correlation to postoperative infectious complications after an esophagectomy. METHODS: Eighteen patients with esophageal cancer were studied. Mesenteric lymph nodes were harvested from the jejunal mesentery before surgical mobilization (MLN-1) and after the restoration of bowel continuity (MLN-2). Blood and sputum were also sampled before surgery (Blood-1 and Sputum-1) and on postoperative day 1 (Blood-2 and Sputum-2). RESULTS: The detection rates of bacteria in the MLN-2 (56%) and Blood-2 (56%) were significantly higher than those in the MLN-1 (17%) and Blood-1 (22%), indicating that surgical stress induces BT. The detection rate was not different between Sputum-1 (80%) and Sputum-2 (78%). There was an 80% sequence homology between the RT-qPCR products in the MLN-2 and Blood-2, whereas the homology was only 20% between Blood-2 and Sputum-2. In the patients with positive bacteria in the MLN-2 sample, there was a greater incidence of postoperative infectious complications than in patients without bacteria in the MLN-2 sample (P = 0.04). The postoperative hospital stay was also longer (P = 0.037) for patients with positive bacteria in the MLN-2 sample. CONCLUSIONS: BT frequently occurs during esophagectomies, and postoperative bacteremia is likely to be gut-derived. Patients with positive bacteria in the MLN-2 sample should be carefully managed because these patients are more susceptible to postoperative infectious complications.

Probiotic Bifidobacterium breve induces IL-10-producing Tr1 cells in the colon.

Specific intestinal microbiota has been shown to induce Foxp3(+) regulatory T cell development. However, it remains unclear how development of another regulatory T cell subset, Tr1 cells, is regulated in the intestine. Here, we analyzed the role of two probiotic strains of intestinal bacteria, Lactobacillus casei and Bifidobacterium breve in T cell development in the intestine. B. breve, but not L. casei, induced development of IL-10-producing Tr1 cells that express cMaf, IL-21, and Ahr in the large intestine. Intestinal CD103(+) dendritic cells (DCs) mediated B. breve-induced development of IL-10-producing T cells. CD103(+) DCs from Il10(-/-), Tlr2(-/-), and Myd88(-/-) mice showed defective B. breve-induced Tr1 cell development. B. breve-treated CD103(+) DCs failed to induce IL-10 production from co-cultured Il27ra(-/-) T cells. B. breve treatment of Tlr2(-/-) mice did not increase IL-10-producing T cells in the colonic lamina propria. Thus, B. breve activates intestinal CD103(+) DCs to produce IL-10 and IL-27 via the TLR2/MyD88 pathway thereby inducing IL-10-producing Tr1 cells in the large intestine. Oral B. breve administration ameliorated colitis in immunocompromised mice given naïve CD4(+) T cells from wild-type mice, but not Il10(-/-) mice. These findings demonstrate that B. breve prevents intestinal inflammation through the induction of intestinal IL-10-producing Tr1 cells.