454 Pyrosequencing Analysis on Faecal Samples from a Randomized DBPC Trial of Colicky Infants Treated with Lactobacillus reuteri DSM 17938

Objective

To analyze the global microbial composition, using large-scale DNA sequencing of 16 S rRNA genes, in faecal samples from colicky infants given L. reuteri DSM 17938 or placebo.

Methods

Twenty-nine colicky infants (age 10–60 days) were enrolled and randomly assigned to receive either Lactobacillus reuteri (10⁸ cfu) or a placebo once daily for 21 days. Responders were defined as subjects with a decrease of 50% in daily crying time at day 21 compared with the starting point. The microbiota of faecal samples from day 1 and 21 were analyzed using 454 pyrosequencing. The primers: Bakt_341F and Bakt_805R, complemented with 454 adapters and sample specific barcodes were used for PCR amplification of the 16 S rRNA genes. The structure of the data was explored by using permutational multivariate analysis of variance and effects of different variables were visualized with ordination analysis.

Results

The infants’ faecal microbiota were composed of Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes as the four main phyla. The composition of the microbiota in infants with colic had very high inter-individual variability with Firmicutes/Bacteroidetes ratios varying from 4000 to 0.025. On an individual basis, the microbiota was, however, relatively stable over time. Treatment with L. reuteri DSM 17938 did not change the global composition of the microbiota, but when comparing responders with non-responders the group responders had an increased relative abundance of the phyla Bacteroidetes and genus Bacteroides at day 21 compared with day 0. Furthermore, the phyla composition of the infants at day 21 could be divided into three enterotype groups, dominated by Firmicutes, Bacteroidetes, and Actinobacteria, respectively.

Conclusion

L. reuteri DSM 17938 did not affect the global composition of the microbiota. However, the increase of Bacteroidetes in the responder infants indicated that a decrease in colicky symptoms was linked to changes of the microbiota.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00893711","term_id":"NCT00893711"}}NCT00893711     

Oral Microbiota Shift after 12-Week Supplementation with Lactobacillus reuteri DSM 17938 and PTA 5289; A Randomized Control Trial

Background

Lactobacillus spp. potentially contribute to health by modulating bacterial biofilm formation, but their effects on the overall oral microbiota remain unclear.

Methods and Findings

Oral microbiota was characterized via 454-pyrosequencing of the 16S rDNA hypervariable region V3-V4 after 12 weeks of daily Lactobacillus reuteri DSM 17938 and PTA 5289 consumption. Forty-four adults were assigned to a test group (n = 22) that received lactobacilli lozenges (10⁸ CFU of each strain/lozenge) or a control group that received placebo (n = 22). Presence of L. reuteri was confirmed by cultivation and species specific PCR. Tooth biofilm samples from 16 adults before, during, and after exposure were analyzed by pyrosequencing. A total of 1,310,292 sequences were quality filtered. After removing single reads, 257 species or phylotypes were identified at 98.5% identity in the Human Oral Microbiome Database. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria were the most abundant phyla. Streptococcus was the most common genus and the S. oralis/S. mitis/S. mitis bv2/S. infantis group comprised the dominant species. The number of observed species was unaffected by L. reuteri exposure. However, subjects who had consumed L. reuteri were clustered in a principal coordinates analysis relative to scattering at baseline, and multivariate modeling of pyrosequencing microbiota, and culture and PCR detected L. reuteri separated baseline from 12-week samples in test subjects. L. reuteri intake correlated with increased S. oralis/S. mitis/S. mitis bv2/S. infantis group and Campylobacter concisus, Granulicatella adiacens, Bergeyella sp. HOT322, Neisseria subflava, and SR1 [G-1] sp. HOT874 detection and reduced S. mutans, S. anginosus, N. mucosa, Fusobacterium periodicum, F. nucleatum ss vincentii, and Prevotella maculosa detection. This effect had disappeared 1 month after exposure was terminated.

Conclusions

L. reuteri consumption did not affect species richness but induced a shift in the oral microbiota composition. The biological relevance of this remains to be elucidated.

Trial Registration

ClinicalTrials.gov NCT02311218     

The Efficacy and Safety of the Probiotic Bacterium Lactobacillus reuteri DSM 17938 for Infantile Colic: A Meta-Analysis of Randomized Controlled Trials

Objective

To evaluate the efficacy and safety of Lactobacillus reuteri DSM 17938 for treating infantile colic.

Methods

A systematic literature retrieval was carried out to obtain randomized controlled trials of L. reuteri DSM 17938 for infantile colic. Trials were performed before May 2015 and retrieved from the PubMed, EMBASE, Cochrane library, CNKI, WanFang, VIP, and CBM databases. Data extraction and quality evaluation of the trials were performed independently by two investigators. A meta-analysis was performed using STATA version 12.0.

Results

Six randomized controlled trials of 423 infants with colic were included. Of these subjects, 213 were in the L. reuteri group, and 210 were in the placebo group. Lactobacillus reuteri increased colic treatment effectiveness at two weeks (RR = 2.84; 95% CI: 1.24–6.50; p = 0.014) and three weeks (relative risk [RR] = 2.33; 95% CI: 1.38–3.93; P = 0.002) but not at four weeks (RR = 1.41; 95% CI: 0.52–3.82; P = 0.498). Lactobacillus reuteri decreased crying time (min/d) at two weeks (weighted mean difference [WMD] = –42.89; 95% CI: –60.50 to –25.29; P = 0.000) and three weeks (WMD = –45.83; 95% CI: –59.45 to –32.21; P = 0.000). In addition, L. reuteri did not influence infants’ weight, length or head circumference and was not associated with serious adverse events.

Conclusions

Lactobacillus reuteri possibly increased the effectiveness of treatment for infantile colic and decreased crying time at two to three weeks without causing adverse events. However, these protective roles are usurped by gradual physiological improvements. The study is limited by the heterogeneity of the trials and should be considered with caution. Higher quality, multicenter randomized controlled trials with larger samples are needed.     

新生期大鼠补充罗伊乳杆菌DSM 17938 对肠道菌群及肠道发育的影响

目的 探讨新生期应用罗伊乳杆菌DSM 17938对大鼠肠道菌群的影响。 方法 24只自然分娩出生的新生SD大鼠随机分为对照组和益生菌组(每组12只),益生菌组大鼠在生后2 d(PND2)开始予罗伊乳杆菌DSM 17938[1×106 CFU/(g·bw),1次/d]灌胃,至出生后第6天(PND6),对照组同期予等量生理盐水灌胃。PND7及PND42时,两组分别处死6只SD大鼠,留取空肠、结肠黏膜及其肠内容物标本,采用16S rDNA V4区二代测序法检测空肠、结肠菌群构成;采用Image Pro Plus 6.0测量空肠、结肠绒毛长度及隐窝深度。 结果 益生菌组和对照组大鼠的体质量增量差异无统计学意义(t值分别为0.410、0.856,均P>0.05),PND42时两组大鼠空肠、结肠黏膜绒毛长度及隐窝深度差异均无统计学意义(t值分别为1.796、0.115、1.122和0.715,均P>0.05),两组大鼠空肠和结肠菌群的α多样性及β多样性差异在PND7及PND42时差异均无统计学意义(均P>0.05),不同组间的肠道菌群LEfSe分析显示PND7及PND42时益生菌组和对照组大鼠肠道菌群组成无差异。 结论 自然分娩出生的健康SD大鼠生命早期添加罗伊乳杆菌DSM 17938不影响生命早期、远期肠道菌群的总体组成及远期肠道上皮组织结构的发育。     

罗伊乳杆菌DSM 17938对生命早期抗生素暴露大鼠肠道菌群的影响

目的探讨预防性应用罗伊乳杆菌DSM 17938对生命早期抗生素暴露大鼠肠道菌群的影响。方法将24只新生SD大鼠随机分为抗生素暴露组(A组)和益生菌干预组(B组),每组12只。A组在出生后第2天(postnatal day 2,PND2)～PND6给予罗氏芬灌胃,2 h后给予生理盐水灌胃;B组同期给予罗氏芬灌胃,2 h后给予罗伊乳杆菌DSM 17938灌胃。PND7及PND42,2组大鼠中各处死6只,取空肠、结肠黏膜及其肠内容物进行16S rDNA V4区二代测序;PND42空肠、结肠组织进行病理检测,用Image Pro Plus 6.0测量绒毛长度和隐窝深度。结果PND42时,A组大鼠体质量明显低于B组。2组大鼠空肠、结肠未见明显炎症等改变,2组间空肠、结肠黏膜绒毛及隐窝深度差异无统计学意义。PND7时,2组大鼠空肠、结肠菌群多样性及门水平菌群组成差异无统计学意义;属水平上,A组、B组大鼠空肠菌属差异无统计学意义,而B组大鼠结肠菌群中乳杆菌属明显增加。PND42时,与A组大鼠比较,B组空肠菌群Alpha多样性指数明显降低,Beta多样性差异明显,门水平上拟杆菌门、变形菌门、柔膜菌门及广古菌门比例明显降低,厚壁菌门比例明显增加,属水平上假单胞菌属、乳球菌属、厌氧芽胞杆菌属、芽胞杆菌属、瘤胃球菌属、Dechloromonas及普氏菌属比例明显降低,乳杆菌属比例明显增加;结肠菌群Shannon指数明显增加,Beta多样性差异明显,门水平上拟杆菌门、疣微菌门比例明显增加,厚壁菌门比例明显降低,属水平上不动杆菌属、葡萄球菌属、金黄杆菌属及Facklamia比例明显降低,Akkermansia、瘤胃球菌属、普氏菌属及拟杆菌属比例明显增高。结论预防性应用罗伊乳杆菌DSM 17938可纠正生命早期抗生素暴露对大鼠发育期肠道菌群的影响。     

Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938

The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of beta-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known beta-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The beta-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.     

Survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG in the Human Gastrointestinal Tract with Daily Consumption of a Low-Fat Probiotic Spread

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Therefore, probiotic strains should be able to survive passage through the human gastrointestinal tract. Human gastrointestinal tract survival of probiotics in a low-fat spread matrix has, however, never been tested. The objective of this randomized, double-blind, placebo-controlled human intervention study was to test the human gastrointestinal tract survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. Forty-two healthy human volunteers were randomly assigned to one of three treatment groups provided with 20 g of placebo spread (n = 13), 20 g of spread with a target dose of 1 × 10⁹ CFU of L. reuteri DSM 17938 (n = 13), or 20 g of spread with a target dose of 5 × 10⁹ CFU of L. rhamnosus GG (n = 16) daily for 3 weeks. Fecal samples were obtained before and after the intervention period. A significant increase, compared to the baseline, in the recovery of viable probiotic lactobacilli in fecal samples was demonstrated after 3 weeks of daily consumption of the spread containing either L. reuteri DSM 17938 or L. rhamnosus GG by selective enumeration. In the placebo group, no increase was detected. The results of selective enumeration were supported by quantitative PCR, detecting a significant increase in DNA resulting from the probiotics after intervention. Overall, our results indicate for the first time that low-fat spread is a suitable carrier for these probiotic strains.The human intestinal microflora or microbiota constitutes a metabolically active microbial environment. This community is relatively stable in the guts of healthy individuals (20). Some of the microbial groups harbor species that are potentially harmful, whereas others, such as the bifidobacteria and lactobacilli, are regarded as beneficial (8). Specific members of the genera Lactobacillus and Bifidobacterium are being applied in functional foods as probiotics (25). Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (9). The current scientific consensus is that probiotics should be alive to exert their beneficial effect in the human gastrointestinal (GI) tract (6). Consequently, probiotics should remain alive in the product, such that the daily effective dose per serving is still present at the end of the shelf life (14). Food matrices, production processes, or product usages that involve heating can affect the viability of probiotics (24).Typically, those members selected for probiotic application are chosen for their resistance to passage through the upper GI tract and thus are able to transiently colonize the gut (25). Human GI tract survival of probiotics should lead to shedding of live cells in fecal samples. GI tract survival is, however, dependent on both the strain and the food matrix involved (27). Fecal recovery of several probiotic strains has been demonstrated in different food matrices, including fermented milk and yoghurt (10, 26, 29), fruit drinks (21), a cereal bar (22), supplements (13, 17, 27), and infant formula (29).For this study, we have selected two well-established probiotic strains to test the suitability of a low-fat spread as a probiotic carrier, namely, Lactobacillus reuteri DSM 17938 (BioGaia, Sweden) and Lactobacillus rhamnosus GG (ATCC 53103; Valio, Finland). L. reuteri DSM 17938 was derived from L. reuteri ATCC 55730 by curing of two plasmids harboring antibiotic resistance genes (23). A series of in vitro experiments confirmed the retention of the functional properties of the daughter strain, as no differences in colony morphology, fermentation patterns, production of reuterin, generation time, mucus-binding ability, or tolerance to bovine bile were found between L. reuteri ATCC 55730 and DSM 17938. The daughter strain is somewhat more resistant to low pH and grows to a higher density in vitro (23). Several studies have been published which provide data on the survival of L. reuteri ATCC 55730 in the human GI tract at doses of 4 × 10⁸ to 1 × 10¹⁰ CFU/day in freeze-dried matrices and chewable tablets (32-34). Furthermore, L. reuteri DSM 17938 was demonstrated to survive human GI tract passage in the same way as L. reuteri ATCC 55730 (23).L. rhamnosus GG has been isolated from a healthy human intestinal flora by Goldin et al. (10). L. rhamnosus GG is relatively resistant to acid and bile, adheres in vitro to epithelial cells, and can produce an antimicrobial substance (10, 15). A wide range of studies have been published which provide data on the survival of L. rhamnosus GG in the human GI tract (3, 4, 10, 18, 19, 27-30), as well as transient colonization of the intestinal microbiota in healthy adults in various formats, including freeze-dried powder, capsules, and tablets or via fermented milk drinks, yoghurt, or fruit juice. Saxelin et al. (28) evaluated the dose-response effect of orally administered L. rhamnosus GG in powder form on fecal colonization in healthy adults, which indicated that consumption of approximately 10¹⁰ to 10¹¹ CFU/day was required to reach detectable levels in fecal samples from volunteers. This was also the case when L. rhamnosus GG was administered in gelatin capsules (29). Additionally, Saxelin et al. (27) observed that milk, but possibly also other protective compounds in food, can improve the survival of L. rhamnosus GG. Fecal recovery of L. rhamnosus GG in milk-based products was shown at dose levels of around 2 × 10⁹ CFU/day.It is, however, not known whether probiotics can survive passage through the human GI tract after the consumption of a low-fat spread. The objective of this randomized, double-blind, placebo-controlled human intervention study was therefore to test the human GI tract survival of L. reuteri DSM 17938 and L. rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. The primary outcome parameter of this study was a significant change from the baseline in the number of probiotic bacteria of the respective strains in fecal samples.     

Pharmacokinetics,Safety and Tolerability of Melissa officinalis Extract which Contained Rosmarinic Acid in Healthy Individuals: A Randomized Controlled Trial

The aim of this study was to evaluate the safety, tolerability and pharmacokinetics of single dose of Melissa officinalis extract which contained rosmarinic acid, including food-effects in healthy individuals. A total of eleven healthy individuals were randomly assigned to treatment arms in the two studies [Study 1 (fasted state) and Study 2 (fed state)]. Rosmarinic acid in serum was measured by a coulometric detection method using High-Performance Liquid Chromatography electrochemical detector. The serum concentration of total rosmarinic acid peaked at 1 hour after administration of Melissa officinalis extract containing 500mg rosmarinic acid in fasted state, with a maximum serum concentration 162.20 nmol/ L. The area under the curve for intact rosmarinic acid was calculated from the serum concentration-time profile to be 832.13 nmol • hour/ L. Food intake increases area under the curve and delayed time at which the maximum serum concentration. Rosmarinic acid supplementation did not affect liver, kidney, or blood cell function parameters. No adverse event was reported by any of the participants due to the study treatment. Single dose of Melissa officinalis extract containing 500 mg rosmarinic acid appears to be safe and tolerable in healthy individuals. Food intake increased the exposure of rosmarinic acid and delayed absorption of rosmarinic acid in healthy individuals.

Trial Registration

Trial Registration: UMIN-CTR UMIN000004997     

Lactobacillus reuteri DSM 17938 Changes the Frequency of Foxp3+ Regulatory T Cells in the Intestine and Mesenteric Lymph Node in Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is an inflammatory disease of the intestine in premature infants. Lactobacillus reuteri DSM 17938 improves survival and reduces the incidence and severity of NEC in a rodent model. Foxp3⁺ regulatory T cells (Tregs) maintain intestinal homeostasis by controlling inflammation and inducing tolerance. To determine whether there are insufficient numbers of Tregs to control inflammation in NEC and to determine if LR17938 increases the frequency of Tregs, we studied selected groups of newborn Sprague-Dawley rats according to feeding plan: dam±LR17938, formula±LR17938, and NEC±LR17938. NEC was induced by gavage feeding with special formula and exposure to hypoxic conditions. Lymphocytes isolated from ileum, mesenteric lymph nodes (MLN), spleen and thymus were labeled for T cell surface markers (CD3, CD4, CD8) and intracellular Foxp3; and labeled cells were analyzed by flow cytometry. The percentage of CD3⁺ T cells and Foxp3⁺ Tregs in the ileum significantly decreased in pups with NEC, compared to normal controls. Feeding LR17938 to neonatal rats with NEC increased the % of Foxp3⁺ T cells in the ileum while decreasing the percentage of cells in the MLN. Administration of LR17938 to dam-fed rats significantly increased Foxp3⁺Tregs in the ileum as early as day of life (DOL)1 but did not produce an increase in Tregs in formula-fed rats on DOL1. These results suggest that factors in breast milk may enhance the early immunomodulatory effects of LR17938. An anti-inflammatory effect of LR17938 in NEC was associated with the modulation of immune responses and induction and what appears to be migration of Foxp3⁺ Tregs to the diseased gut. Probiotic-facilitated development of Tregs might play an important role in the prevention of NEC.     

Ultra-Brief Intervention for Problem Drinkers: Results from a Randomized Controlled Trial

Background

There are a number of evidence-based, in-person clinical inteventions for problem drinkers, but most problem drinkers will never seek such treatments. Reaching the population of non-treatment seeking problem drinkers will require a different approach. Accordingly, this randomized clinical trial evaluated an intervention that has been validated in clinical settings and then modified into an ultra-brief format suitable for use as an indicated public health intervention (i.e., targeting the population of non-treatment seeking problem drinkers).

Methodology/Principal Findings

Problem drinkers (N = 1767) completed a baseline population telephone survey and then were randomized to one of three conditions – a personalized feedback pamphlet condition, a control pamphlet condition, or a no intervention control condition. In the week after the baseline survey, households in the two pamphlet conditions were sent their respective interventions by postal mail addressed to ‘Check Your Drinking.’ Changes in drinking were assessed post intervention at three-month and six-month follow-ups. The follow-up rate was 86% at three-months and 76% at six-months. There was a small effect (p = .04) in one of three outcome variables (reduction in AUDIT-C, a composite measure of quantity and frequency of drinking) observed for the personalized feedback pamphlet compared to the no intervention control. No significant differences (p>.05) between groups were observed for the other two outcome variables – number of drinks consumed in the past seven days and highest number of drinks on one occasion.

Conclusions/Significance

Based on the results of this study, we tentatively conclude that a brief intervention, modified to an ultra-brief, public health format can have a meaningful impact.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00688584","term_id":"NCT00688584"}}NCT00688584.     

Safety,Tolerability, Pharmacodynamics and Pharmacokinetics of Umeclidinium and Vilanterol Alone and in Combination: A Randomized Crossover Trial

Umeclidinium bromide (GSK573719; UMEC), a new long-acting muscarinic receptor antagonist (LAMA), is in development with vilanterol ({"type":"entrez-nucleotide","attrs":{"text":"GW642444","term_id":"290522278","term_text":"GW642444"}}GW642444; VI), a selective long-acting β₂ agonist (LABA), as a once-daily inhaled combination therapy for the treatment of chronic obstructive pulmonary disease (COPD). A single dose healthy volunteer study was conducted to assess the safety and tolerability, pharmacodynamics (PD) and pharmacokinetics (PK) of inhaled umeclidinium (500 µg) and vilanterol (50 µg) when administered separately and in combination using a novel dry powder inhaler (NDPI). Co-administration of single inhaled doses of umeclidinium and vilanterol to healthy Japanese subjects was well tolerated and not associated with meaningful changes in systemic exposure or PD effects compared with administration of either compound individually. Pharmacokinetic assessments showed rapid absorption for both drugs (Tmax = 5 min for both umeclidinium and vilanterol) followed by rapid elimination with median tlast of 4–5 h for umeclidinium and median tlast of 1.5–2.0 h for vilanterol. Assessments of pharmacokinetic interaction were inconclusive since for umeclidinium, Cmax following combination was higher than umeclidinium alone but not AUC whereas for vilanterol, AUC following combination was higher than vilanterol alone but not Cmax. There were no obvious trends observed between individual maximum supine heart rate and umeclidinium Cmax or vilanterol Cmax when delivered as umeclidinium 500 µg and vilanterol 50 µg combination or when delivered as umeclidinium or vilanterol alone.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00976144","term_id":"NCT00976144"}}NCT00976144     

Randomized Trial of Glucosamine and Chondroitin Supplementation on Inflammation and Oxidative Stress Biomarkers and Plasma Proteomics Profiles in Healthy Humans

BackgroundGlucosamine and chondroitin are popular non-vitamin dietary supplements used for osteoarthritis. Long-term use is associated with lower incidence of colorectal and lung cancers and with lower mortality; however, the mechanism underlying these observations is unknown. In vitro and animal studies show that glucosamine and chondroitin inhibit NF-kB, a central mediator of inflammation, but no definitive trials have been done in healthy humans.MethodsWe conducted a randomized, double-blind, placebo-controlled, cross-over study to assess the effects of glucosamine hydrochloride (1500 mg/d) plus chondroitin sulfate (1200 mg/d) for 28 days compared to placebo in 18 (9 men, 9 women) healthy, overweight (body mass index 25.0–32.5 kg/m²) adults, aged 20–55 y. We examined 4 serum inflammatory biomarkers: C-reactive protein (CRP), interleukin 6, and soluble tumor necrosis factor receptors I and II; a urinary inflammation biomarker: prostaglandin E2-metabolite; and a urinary oxidative stress biomarker: F₂-isoprostane. Plasma proteomics on an antibody array was performed to explore other pathways modulated by glucosamine and chondroitin.ResultsSerum CRP concentrations were 23% lower after glucosamine and chondroitin compared to placebo (P = 0.048). There were no significant differences in other biomarkers. In the proteomics analyses, several pathways were significantly different between the interventions after Bonferroni correction, the most significant being a reduction in the “cytokine activity” pathway (P = 2.6 x 10^-16), after glucosamine and chondroitin compared to placebo.ConclusionGlucosamine and chondroitin supplementation may lower systemic inflammation and alter other pathways in healthy, overweight individuals. This study adds evidence for potential mechanisms supporting epidemiologic findings that glucosamine and chondroitin are associated with reduced risk of lung and colorectal cancer.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01682694","term_id":"NCT01682694"}}NCT01682694     

Safety and Immunogenicity of a Live Oral Recombinant Cholera Vaccine VA1.4: A Randomized,Placebo Controlled Trial in Healthy Adults in a Cholera Endemic Area in Kolkata,India

Background

A live oral cholera vaccine VA 1.4 developed from a non-toxigenic Vibrio cholerae O1 El Tor strain using ctxB gene insertion was further developed into a clinical product following cGMP and was evaluated in a double-blind randomized placebo controlled parallel group two arm trial with allocation ratio of 1∶1 for safety and immunogenicity in men and women aged 18–60 years from Kolkata, India.

Method

A lyophilized dose of 1.9×10⁹ CFU (n = 44) or a placebo (n = 43) reconstituted with a diluent was administered within 5 minutes of drinking 100 ml of a buffer solution made of sodium bicarbonate and ascorbic acid and a second dose on day 14.

Result

The vaccine did not elicit any diarrhea related adverse events. Other adverse events were rare, mild and similar in two groups. One subject in the vaccine group excreted the vaccine strain on the second day after first dose. The proportion of participants who seroconverted (i.e. had 4-folds or higher rise in reciprocal titre) in the vaccine group were 65.9% (95% CI: 50.1%–79.5%) at both 7 days (i.e. after 1^st dose) and 21 days (i.e. after 2^nd dose). None of the placebo recipients seroconverted. Anti-cholera toxin antibody was detected in very few recipients of the vaccine.

Conclusion

This study demonstrates that VA 1.4 at a single dose of 1.9×10⁹ is safe and immunogenic in adults from a cholera endemic region. No additional benefit after two doses was seen.

Trial Registration

Clinical Trials Registry-India, National Institute of Medical Statistics (Indian Council of Medical Research) CTRI/2012/04/002582     

Pharmacokinetics and Tolerability of Inhaled Umeclidinium and Vilanterol Alone and in Combination in Healthy Chinese Subjects: A Randomized,Open-Label,Crossover Trial

Inhaled umeclidinium (UMEC) and the combination of inhaled UMEC with vilanterol (UMEC/VI) are approved maintenance treatments for chronic obstructive pulmonary disease in the US and EU. This was a randomized, open-label, three-period crossover, single- and repeat-dose study to assess the pharmacokinetics (PK), safety, and tolerability of inhaled UMEC/VI 62.5/25 μg (delivering 55/22 μg) and UMEC/VI 125/25 μg (delivering 113/22 μg) compared with their monotherapy components (UMEC 62.5 μg, UMEC 125 μg and, VI 25 μg [delivering 55, 113, and 22 μg, respectively]) in healthy Chinese subjects (n=20). UMEC and VI were rapidly absorbed following single and repeat dosing (time to maximum plasma concentration [t_max]: UMEC = 5 min; VI = 5 min). The median t_last was 2–4 h for UMEC and 1–2 h for VI following single doses of UMEC/VI and UMEC monotherapy (both doses). UMEC reached steady-state prior to Day 10; steady-state for VI could not be assessed. UMEC accumulation following repeat dosing was 11–34% based on C_max and 19–59% based on area under the concentration-time curve from time zero to 2 h (AUC_(0-2)). VI accumulation following repeat dosing was 25–66% based on C_max and 17–43% based on AUC_(0-2). The evidence was not sufficient to suggest that systemic exposure was substantially different between UMEC/VI combination therapy and the constituent monotherapies following single or repeat dosing. Following both single- and repeat-dose administration, the inter-subject coefficient of variation for all UMEC PK parameter estimates ranged from 12% to 165% for all treatments, indicating a wide range of variability in inhaled PK parameters. Twelve subjects experienced ≥1 adverse event (AE). Six subjects experienced ≥1 treatment-related AE; the most commonly reported treatment-related AE was chest discomfort (n=3 [15%]). No clinically important changes in vital signs or electrocardiogram parameters were reported. These data suggest that single- and repeat-dose administration of UMEC/VI combination therapy in healthy Chinese subjects did not result in substantial differences in systemic exposure compared with UMEC and VI as monotherapies.

Trial Registration

Clinicaltrials.gov NCT01899638 NCT01899638     

Biochemical and crystallographic characterization of a glucansucrase from Lactobacillus reuteri 180

Glucansucrases are large extracellular transglycosidases secreted by lactic acid bacteria. Using sucrose as a substrate they synthesize high molecular mass α-glucans or, in the presence of suitable acceptor molecules, low molecular mass oligosaccharides. Although about 60 glucansucrases have been classified in glycoside hydrolase family GH70, no three-dimensional structure has been reported for any. With the aim of solving the first structure of a GH70 glucansucrase, purification and crystallization experiments were performed with a fully active, 117 kDa N-terminally truncated fragment of glucansucrase GTF180 from Lactobacillus reuteri 180 (residues 742–1772). Crystallization experiments yielded crystals that belong to two different triclinic crystal forms (space group P1) and one orthorhombic crystal form (space group P2₁2₁2₁). Native data sets for both triclinic and the orthorhombic crystals were collected at 1.7 and 2.0 Å resolution, respectively. Enzyme activity assays, pH and temperature optima show comparable values for both the full-length and the N-terminally truncated GTF180.     

罗伊乳杆菌对小鼠血清细胞因子含量和肠道菌群的影响

目的 探讨罗伊乳杆菌对小鼠免疫力及肠道菌群的影响。方法 将50只昆明系小鼠按体重随机分成5组：空白对照组、低剂量组、中剂量组、高剂量组和发酵上清液组。空白对照组灌胃等量生理盐水,其余各组分别灌胃罗伊乳杆菌菌液及发酵液上清21 d后,眼球取血收集血清,采用ELISA法检测IL-2、IFN-γ、IgA和IgG含量;采用16S rRNA基因高通量测序分析小鼠肠道菌群变化。结果 与对照组相比,其余各组小鼠血清IL-2、IFN-γ、IgA和IgG的水平均明显升高,粪便菌群丰度有所增高,多样性降低。与对照组相比,低剂量组和中剂量组小鼠粪便菌群结构无显著差异,而高剂量组和发酵上清液组小鼠粪便菌群结构差异显著。结论 罗伊乳杆菌可以增强小鼠免疫力,提高菌群丰度,改变小鼠肠道菌群结构。     

Safety and Immunogenicity of an AMA1 Malaria Vaccine in Malian Children: Results of a Phase 1 Randomized Controlled Trial

Background

The objective was to evaluate the safety and immunogenicity of the AMA1-based malaria vaccine FMP2.1/AS02_A in children exposed to seasonal falciparum malaria.

Methodology/Principal Findings

A Phase 1 double blind randomized controlled dose escalation trial was conducted in Bandiagara, Mali, West Africa, a rural town with intense seasonal transmission of Plasmodium falciparum malaria. The malaria vaccine FMP2.1/AS02_A is a recombinant protein (FMP2.1) based on apical membrane antigen 1 (AMA1) from the 3D7 clone of P. falciparum, formulated in the Adjuvant System AS02_A. The comparator vaccine was a cell-culture rabies virus vaccine (RabAvert®). One hundred healthy Malian children aged 1–6 years were recruited into 3 cohorts and randomized to receive either 10 µg FMP2.1 in 0.1 mL AS02_A, or 25 µg FMP2.1 in 0.25 mL AS02_A, or 50 µg FMP2.1 50 µg in 0.5 mL AS02_A, or rabies vaccine. Three doses of vaccine were given at 0, 1 and 2 months, and children were followed for 1 year. Solicited symptoms were assessed for 7 days and unsolicited symptoms for 30 days after each vaccination. Serious adverse events were assessed throughout the study. Transient local pain and swelling were common and more frequent in all malaria vaccine dosage groups than in the comparator group, but were acceptable to parents of participants. Levels of anti-AMA1 antibodies measured by ELISA increased significantly (at least 100-fold compared to baseline) in all 3 malaria vaccine groups, and remained high during the year of follow up.

Conclusion/Significance

The FMP2.1/AS02_A vaccine had a good safety profile, was well-tolerated, and induced high and sustained antibody levels in malaria-exposed children. This malaria vaccine is being evaluated in a Phase 2 efficacy trial in children at this site.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00358332","term_id":"NCT00358332"}}NCT00358332 [{"type":"clinical-trial","attrs":{"text":"NCT00358332","term_id":"NCT00358332"}}NCT00358332]     

Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Lactobacillus reuteri

Plasmid analysis, plasmid curing, cloning, and hybridization experiments were used to study four Lactobacillus reuteri strains showing high resistance to erythromycin. Plasmid curing with acriflavine resulted in a loss of erythromycin resistance in a frequency of 1-10%. For three of the strains this was accompanied by a loss of a 6.9-MDa plasmid, which was shown to be identical for the different strains and designated pLUL631. The erythromycin (erm) gene was located on a 5.5-MDa plasmid in the fourth strain. A restriction map of pLUL631 was constructed and the location of the erm gene on the plasmid was identified by cloning in Escherichia coli. By using a Streptococcus lactis-E. coli shuttle vector, the erm gene was also transformed to S. lactis and expressed. The erm gene from L. reuteri was shown to be related to the erm gene from pIP501 (Streptococcus agalactiae) by DNA-DNA hybridization.