Anti-Atherogenic Actions of the Lab4b Consortium of Probiotics In Vitro.

Probiotic bacteria have many protective effects against inflammatory disorders, though the mechanisms underlying their actions are poorly understood. The Lab4b consortium of probiotics contains four strains of lactic acid bacteria and bifidobacteria that are reflective of the gut of newborn babies and infants. The effect of Lab4b on atherosclerosis, an inflammatory disorder of the vasculature, has not yet been determined and was investigated on key processes associated with this disease in human monocytes/macrophages and vascular smooth muscle cells in vitro. The Lab4b conditioned medium (CM) attenuated chemokine-driven monocytic migration, monocyte/macrophage proliferation, uptake of modified LDL and macropinocytosis in macrophages together with the proliferation and platelet-derived growth factor-induced migration of vascular smooth muscle cells. The Lab4b CM also induced phagocytosis in macrophages and cholesterol efflux from macrophage-derived foam cells. The effect of Lab4b CM on macrophage foam cell formation was associated with a decrease in the expression of several key genes implicated in the uptake of modified LDL and induced expression of those involved in cholesterol efflux. These studies reveal, for the first time, several anti-atherogenic actions of Lab4b and strongly implicate further studies in mouse models of the disease in vivo and in clinical trials.

Assessment of Lab4P Probiotic Effects on Cognition in 3xTg-AD Alzheimer's Disease Model Mice and the SH-SY5Y Neuronal Cell Line.

Aging and metabolic syndrome are associated with neurodegenerative pathologies including Alzheimer's disease (AD) and there is growing interest in the prophylactic potential of probiotic bacteria in this area. In this study, we assessed the neuroprotective potential of the Lab4P probiotic consortium in both age and metabolically challenged 3xTg-AD mice and in human SH-SY5Y cell culture models of neurodegeneration. In mice, supplementation prevented disease-associated deteriorations in novel object recognition, hippocampal neurone spine density (particularly thin spines) and mRNA expression in hippocampal tissue implying an anti-inflammatory impact of the probiotic, more notably in the metabolically challenged setting. In differentiated human SH-SY5Y neurones challenged with ß-Amyloid, probiotic metabolites elicited a neuroprotective capability. Taken together, the results highlight Lab4P as a potential neuroprotective agent and provide compelling support for additional studies in animal models of other neurodegenerative conditions and human studies.

Gut Microbiome in BALB/c and C57BL/6J Mice Undergoing Experimental Thyroid Autoimmunity Associate with Differences in Immunological Responses and Thyroid Function.

Experimental models of hyperthyroid Graves' disease (GD) and Graves' orbitopathy (GO) are efficiently developed by genetic immunisation by electroporation with human thyrotropin hormone receptor (hTSHR) A-subunit plasmid in female BALB/c (H-2d) mice. We investigated susceptibility in C57BL/6 J (H-2b) animals to allow studies on disease mechanisms in transgenic and immune response gene knock-out mice. Higher numbers of female C57BL/6 J were positive for pathogenic thyroid stimulating antibodies, but induced hyperthyroidism remained at a low frequency compared to BALB/c animals. Assessment of hTSHR specific T cells showed reduced proliferation in C57BL/6 J animals accompanied with anti-inflammatory IL-10, with less pro-inflammatory IFN-γ compared to BALB/c. Whilst the orbital tissue from immune BALB/c mice showed inflammation and adipogenesis, in contrast C57BL/6 J animals showed normal pathology. We characterised the gut microbiota using 16 S ribosomal RNA gene sequencing to explore its possible pathogenic role in the model. Despite being housed under identical conditions, we observed significantly different organisation of the microbiota (beta-diversity) in the two strains. Taxonomic differences were also noted, with C57BL/6 J showing an enrichment of Operational Taxonomic Units (OTUs) belonging to the Paludibacter and Allobaculum, followed by Limibacter, Anaerophaga and Ureaplasma genera. A higher number of genera significantly correlating with clinical features was observed in C57BL/6 J compared to BALB/c; for example, Limibacter OTUs correlated negatively with thyroid-stimulating antibodies in C57BL/6 J mice. Thus, our data suggest gut microbiota may play a pivotal immunomodulatory role that differentiates the thyroid function and orbital pathology outcome in these two inbred strains undergoing experimental GO.

The microbiota and autoimmunity: Their role in thyroid autoimmune diseases.

Since the 1970s, the role of infectious diseases in the pathogenesis of Graves' disease (GD) has been an object of intensive research. The last decade has witnessed many studies on Yersinia enterocolitica, Helicobacter pylori and other bacterial organisms and their potential impact on GD. Retrospective, prospective and molecular binding studies have been performed with contrary outcomes. Until now it is not clear whether bacterial infections can trigger autoimmune thyroid disease. Common risk factors for GD (gender, smoking, stress, and pregnancy) reveal profound changes in the bacterial communities of the gut compared to that of healthy controls but a pathogenetic link between GD and dysbiosis has not yet been fully elucidated. Conventional bacterial culture, in vitro models, next generation and high-throughput DNA sequencing are applicable methods to assess the impact of bacteria in disease onset and development. Further studies on the involvement of bacteria in GD are needed and may contribute to the understanding of pathogenetic processes. This review will examine available evidence on the subject.

Long-term multi-species Lactobacillus and Bifidobacterium dietary supplement enhances memory and changes regional brain metabolites in middle-aged rats.

Ageing is associated with changes in the gut microbiome that may contribute to age-related changes in cognition. Previous work has shown that dietary supplements with multi-species live microorganisms can influence brain function, including induction of hippocampal synaptic plasticity and production of brain derived neurotrophic factor, in both young and aged rodents. However, the effect of such dietary supplements on memory processes has been less well documented, particularly in the context of aging. The main aim of the present study was to examine the impact of a long-term dietary supplement with a multi-species live Lactobacillus and Bifidobacteria mixture (Lactobacillus acidophilus CUL60, L. acidophilus CUL21, Bifidobacterium bifidum CUL20 and B. lactis CUL34) on tests of memory and behavioural flexibility in 15-17-month-old male rats. Following behavioural testing, the hippocampus and prefrontal cortex was extracted and analysed ex vivo using 1H nuclear magnetic resonance (1H NMR) spectroscopy to examine brain metabolites. The results showed a small beneficial effect of the dietary supplement on watermaze spatial navigation and robust improvements in long-term object recognition memory and short-term memory for object-in-place associations. Short-term object novelty and object temporal order memory was not influenced by the dietary supplement in aging rats. 1H NMR analysis revealed diet-related regional-specific changes in brain metabolites; which indicated changes in several pathways contributing to modulation of neural signaling. These data suggest that chronic dietary supplement with multi-species live microorganisms can alter brain metabolites in aging rats and have beneficial effects on memory.

Effect of a plant sterol, fish oil and B vitamin combination on cardiovascular risk factors in hypercholesterolemic children and adolescents: a pilot study.

BACKGROUND: Assessment of cardiovascular disease (CVD) risk factors can predict clinical manifestations of atherosclerosis in adulthood. In this pilot study with hypercholesterolemic children and adolescents, we investigated the effects of a combination of plant sterols, fish oil and B vitamins on the levels of four independent risk factors for CVD; LDL-cholesterol, triacylglycerols, C-reactive protein and homocysteine. METHODS: Twenty five participants (mean age 16 y, BMI 23 kg/m2) received daily for a period of 16 weeks an emulsified preparation comprising plant sterols esters (1300 mg), fish oil (providing 1000 mg eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA)) and vitamins B12 (50 µg), B6 (2.5 mg), folic acid (800 µg) and coenzyme Q10 (3 mg). Atherogenic and inflammatory risk factors, plasma lipophilic vitamins, provitamins and fatty acids were measured at baseline, week 8 and 16. RESULTS: The serum total cholesterol, LDL- cholesterol, VLDL-cholesterol, subfractions LDL-2, IDL-1, IDL-2 and plasma homocysteine levels were significantly reduced at the end of the intervention period (p<0.05). The triacylglycerols levels decreased by 17.6%, but did not reach significance. No significant changes in high sensitivity C-reactive protein, HDL-cholesterol and apolipoprotein A-1 were observed during the study period. After standardisation for LDL cholesterol, there were no significant changes in the levels of plasma γ-tocopherol, ß-carotene and retinol, except for reduction in α-tocopherol levels. The plasma levels of n-3 fatty acids increased significantly with the dietary supplementation (p<0.05). CONCLUSIONS: Daily intake of a combination of plant sterols, fish oil and B vitamins may modulate the lipid profile of hypercholesterolemic children and adolescents. TRIAL REGISTRATION: Current Controlled Trials ISRCTN89549017.

(+)-Catechin Attenuates Multiple Atherosclerosis-Associated Processes In Vitro, Modulates Disease-Associated Risk Factors in C57BL/6J Mice and Reduces Atherogenesis in LDL Receptor Deficient Mice by Inhibiting Inflammation and Increasing Markers of Plaque Stability.

SCOPE: A prospective study of 34492 participants shows an inverse association between (+)-catechin intake and coronary heart disease. The effects of (+)-catechin on atherosclerosis and associated risk factors are poorly understood and are investigated. METHODS AND RESULTS: (+)-Catechin attenuates reactive oxygen species production in human macrophages, endothelial cells and vascular smooth muscle cells, chemokine-driven monocytic migration, and proliferation of human macrophages and their expression of several pro-atherogenic genes. (+)-Catechin also improves oxidized LDL-mediated mitochondrial membrane depolarization in endothelial cells and attenuates growth factor-induced smooth muscle cell migration. In C57BL/6J mice fed high fat diet (HFD) for 3 weeks, (+)-catechin attenuates plasma levels of triacylglycerol and interleukin (IL)-1ß and IL-2, produces anti-atherogenic changes in liver gene expression, and reduces levels of white blood cells, myeloid-derived suppressor cells, Lin- Sca+ c-Kit+ cells, and common lymphoid progenitor cells within the bone marrow. In LDL receptor deficient mice fed HFD for 12 weeks, (+)-catechin attenuates atherosclerotic plaque burden and inflammation with reduced macrophage content and increased markers of plaque stability; smooth muscle cell and collagen content. CONCLUSION: This study provides novel, detailed insights into the cardio-protective actions of (+)-catechin together with underlying molecular mechanisms and supports further assessments of its beneficial effects in human trials.

Gut Microbiome Associated With Graves Disease and Graves Orbitopathy: The INDIGO Multicenter European Study.

CONTEXT: Gut bacteria can influence host immune responses but little is known about their role in tolerance-loss mechanisms in Graves disease (GD; hyperthyroidism caused by autoantibodies, TRAb, to the thyrotropin receptor, TSHR) and its progression to Graves orbitopathy (GO). OBJECTIVE: This work aimed to compare the fecal microbiota in GD patients, with GO of varying severity, and healthy controls (HCs). METHODS: Patients were recruited from 4 European countries (105 GD patients, 41 HCs) for an observational study with cross-sectional and longitudinal components. RESULTS: At recruitment, when patients were hyperthyroid and TRAb positive, Actinobacteria were significantly increased and Bacteroidetes significantly decreased in GD/GO compared with HCs. The Firmicutes to Bacteroidetes (F:B) ratio was significantly higher in GD/GO than in HCs. Differential abundance of 15 genera was observed in patients, being most skewed in mild GO. Bacteroides displayed positive and negative correlations with TSH and free thyroxine, respectively, and was also significantly associated with smoking in GO; smoking is a risk factor for GO but not GD. Longitudinal analyses revealed that the presence of certain bacteria (Clostridiales) at diagnosis correlated with the persistence of TRAb more than 200 days after commencing antithyroid drug treatment. CONCLUSION: The increased F:B ratio observed in GD/GO mirrors our finding in a murine model comparing TSHR-immunized with control mice. We defined a microbiome signature and identified changes associated with autoimmunity as distinct from those due to hyperthyroidism. Persistence of TRAb is predictive of relapse; identification of these patients at diagnosis, via their microbiome, could improve management with potential to eradicate Clostridiales.

Effect of different long-chain fatty acids on cholecystokinin release in vitro and energy intake in free-living healthy males.

Long-chain fatty acids have been shown to suppress appetite and reduce energy intake (EI) by stimulating the release of gastrointestinal hormones such as cholecystokinin (CCK). The effect of NEFA acyl chain length on these parameters is not comprehensively understood. An in vitro screen tested the capacity of individual NEFA (C12 to C22) to trigger CCK release. There was a gradient in CCK release with increasing chain length. DHA (C22) stimulated significantly (P < 0.01) more CCK release than all other NEFA tested. Subsequently, we conducted a randomised, controlled, crossover intervention study using healthy males (n 18). The effects of no treatment (NT) and oral doses of emulsified DHA-rich (DHA) and oleic acid (OA)-rich oils were compared using 24 h EI as the primary endpoint. Participants reported significantly (P = 0.039) lower total daily EI (29 % reduction) with DHA compared to NT. There were no differences between DHA compared to OA and OA compared to NT. There was no between-treatment difference in the time to, or EI of, the first post-intervention eating occasion. It is concluded that NEFA stimulate CCK release in a chain length-dependent manner up to C22. These effects may be extended to the in vivo setting, as a DHA-based emulsion significantly reduced short-term EI.

A multicentre randomised controlled trial evaluating lactobacilli and bifidobacteria in the prevention of antibiotic-associated diarrhoea in older people admitted to hospital: the PLACIDE study protocol.

BACKGROUND: Antibiotic associated diarrhoea complicates 5-39% of courses of antibiotic treatment. Major risk factors are increased age and admission to hospital. Of particular importance is C. difficile associated diarrhoea which occurs in about 4% of antibiotic courses and may result in severe illness, death and high healthcare costs. The emergence of the more virulent 027 strain of C. difficile has further heightened concerns. Probiotics may prevent antibiotic associated diarrhoea by several mechanisms including colonization resistance through maintaining a healthy gut flora. METHODS: This study aims to test the hypothesis that administration of a probiotic comprising two strains of lactobacilli and two strains of bifidobacteria alongside antibiotic treatment prevents antibiotic associated diarrhoea. We have designed a prospective, parallel group trial where people aged 65 years or more admitted to hospital and receiving one or more antibiotics are randomly allocated to receive either one capsule of the probiotic or a matching placebo daily for 21 days. The primary outcomes are the frequency of antibiotic associated and C. difficile diarrhoea during 8-12 weeks follow-up. To directly inform routine clinical practice, we will recruit a sufficient number of patients to demonstrate a 50% reduction in the frequency of C. difficile diarrhoea with a power of 80%. To maximize the generalizability of our findings and in view of the well-established safety record of probiotics, we will recruit a broad range of medical and surgical in-patients from two different health regions within the UK. DISCUSSION: Antibiotic associated diarrhoea constitutes a significant health burden. In particular, current measures to prevent and control C. difficile diarrhoea are expensive and disrupt clinical care. This trial may have considerable significance for the prevention of antibiotic associated diarrhoea in hospitals. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number Register ISRCTN70017204.

The Impact of Probiotic Supplementation on Cognitive, Pathological and Metabolic Markers in a Transgenic Mouse Model of Alzheimer's Disease.

Brain degenerative disorders such as Alzheimer's disease (AD) can be exacerbated by aberrant metabolism. Supplementation with probiotic bacteria is emerging as a promising preventative strategy for both neurodegeneration and metabolic syndrome. In this study, we assess the impact of the Lab4b probiotic consortium on (i) cognitive and pathological markers of AD progression and (ii) metabolic status in 3xTg-AD mice subjected to metabolic challenge with a high fat diet. The group receiving the probiotic performed better in the novel object recognition test and displayed higher hippocampal neuronal spine density than the control group at the end of the 12 weeks intervention period. These changes were accompanied by differences in localised (brain) and systemic anti-inflammatory responses that favoured the Probiotic group together with the prevention of diet induced weight gain and hypercholesterolaemia and the modulation of liver function. Compositional differences between the faecal microbiotas of the study groups included a lower Firmicutes:Bacteroidetes ratio and less numbers of viable yeast in the Probiotic group compared to the Control. The results illustrate the potential of the Lab4b probiotic as a neuroprotective agent and encourage further studies with human participants.

Analysis of a network-wide specialist palliative care out-of-hours advice and support line: a model for the future.

INTRODUCTION: Recently the palliative care literature has emphasized the need for 24-hour telephone advice and support for patients during the last year of life. As some health professionals may lack skills and knowledge regarding palliative care issues, an out-of-hours (OoH) advice line is considered a valuable resource. This report provides an analysis of both qualitative and quantative data concerning the Mount Vernon Cancer Network telephone advice line (TAL) for 2008-2010. METHOD: Data relating to the use of the TAL during OoH periods was collated and analysed on an annual basis. Data collected from a caller questionnaire relating to patient outcome, experience, and caller satisfaction was also evaluated. RESULTS: The number of calls to the OoH TAL increased by 13% over the 3-year period. The primary reason for the calls was symptom management, and 79% were made by patients' relatives. From the questionnaire responses, 66% of callers appeared to have a high rate of satisfaction, and 67% of patients were not admitted to hospital. CONCLUSIONS: It appears that the advice line is providing a highly valued service, with calls being managed by experienced nurses. Increasingly, local commissioners are placing greater emphasis on supporting care at home and reducing unnecessary hospital admissions, and advice lines seem to be an effective way of facilitating this.

Probiotics reduce self-reported symptoms of upper respiratory tract infection in overweight and obese adults: should we be considering probiotics during viral pandemics?

Gut microbiome manipulation to alter the gut-lung axis may potentially protect humans against respiratory infections, and clinical trials of probiotics show promise in this regard in healthy adults and children. However, comparable studies are lacking in overweight/obese people, who have increased risks in particular of viral upper respiratory tract infections (URTI). This Addendum further analyses our recent placebo-controlled trial of probiotics in overweight/obese people (focused initially on weight loss) to investigate the impact of probiotics upon the occurrence of URTI symptoms. As well as undergoing loss of weight and improvement in certain metabolic parameters, study participants taking probiotics experienced a 27% reduction in URTI symptoms versus control, with those ≥45 years or BMI ≥30 kg/m2 experiencing greater reductions. This symptom reduction is apparent within 2 weeks of probiotic use. Gut microbiome diversity remained stable throughout the study in probiotic-treated participants. Our data provide support for further trials to assess the potential role of probiotics in preventing viral URTI (and possibly also COVID-19), particularly in overweight/obese people.

The Lab4P Consortium of Probiotics Attenuates Atherosclerosis in LDL Receptor Deficient Mice Fed a High Fat Diet and Causes Plaque Stabilization by Inhibiting Inflammation and Several Pro-Atherogenic Processes.

SCOPE: Previous studies show that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (Lab4P) reduces diet-induced weight gain and plasma cholesterol levels in C57BL/6J mice fed a high fat diet (HFD). The effect of Lab4P on atherosclerosis is not known and is therefore investigated. METHODS AND RESULTS: Atherosclerosis-associated parameters are analyzed in LDL receptor deficient mice fed HFD for 12 weeks alone or supplemented with Lab4P. Lab4P increases plasma HDL and triglyceride levels and decreases LDL/VLDL levels. Lab4P also reduces plaque burden and content of lipids and macrophages, indicative of dampened inflammation, and increases smooth muscle cell content, a marker of plaque stabilization. Atherosclerosis arrays show that Lab4P alters the liver expression of 19 key disease-associated genes. Lab4P also decreases the frequency of macrophages and T-cells in the bone marrow. In vitro assays using conditioned media from probiotic bacteria demonstrates attenuation of several atherosclerosis-associated processes in vitro such as chemokine-driven monocytic migration, proliferation of monocytes and macrophages, foam cell formation and associated changes in expression of key genes, and proliferation and migration of vascular smooth muscle cells. CONCLUSION: This study provides new insights into the anti-atherogenic actions of Lab4P together with the underlying mechanisms and supports further assessments in human trials.

The Impact of Lab4 Probiotic Supplementation in a 90-Day Study in Wistar Rats.

The anti-inflammatory and cholesterol lowering capabilities of probiotic bacteria highlight them as potential prophylactics against chronic inflammatory diseases, particularly cardiovascular disease. Previous studies in silico, in vitro, and in vivo suggest that the Lab4 probiotic consortium may harbour such capabilities and in the current study, we assessed plasma levels of cytokines/chemokines, short chain fatty acids and lipids and faecal levels of bile acids in a subpopulation of healthy Wistar rats included in 90-day repeat dose oral toxicity study. In the rats receiving Lab4, circulating levels of pro-inflammatory interleukin-6, tumour necrosis factor-α and keratinocyte chemoattractant/growth regulated oncogene were significantly lower compared to the control group demonstrating a systemic anti-inflammatory effect. These changes occurred alongside significant reductions in plasma low density lipoprotein cholesterol and increases in faecal bile acid excretion implying the ability to lower circulating cholesterol via the deconjugation of intestinal bile acids. Correlative analysis identified significant associations between plasma tumour necrosis factor-α and the plasma total cholesterol:high density lipoprotein cholesterol ratio and faecal levels of bifidobacteria in the Lab4 rats. Together, these data highlight Lab4 supplementation as a holistic approach to CVD prevention and encourages further studies in humans.

Protective effects of a unique combination of nutritionally active ingredients on risk factors and gene expression associated with atherosclerosis in C57BL/6J mice fed a high fat diet.

Atherosclerosis, an inflammatory disorder of the vasculature and the underlying cause of cardiovascular disease, is responsible for one in three global deaths. Consumption of active food ingredients such as omega-3 polyunsaturated fatty acids, flavanols and phytosterols has many beneficial effects on cardiovascular disease. However, their combined actions on the risk factors for atherosclerosis remains poorly understood. We have previously shown that a formulation containing each of these active components at physiologically relevant doses modulated several monocyte/macrophage processes associated with atherosclerosis in vitro, including inhibition of cytokine-induced pro-inflammatory gene expression, chemokine-driven monocyte migration, expression of M1 phenotype markers, and promotion of cholesterol efflux. The objectives of the present study were to investigate whether the protective actions of the formulation extended in vivo and to delineate the potential underlying mechanisms. The formulation produced several favourable changes, including higher plasma levels of HDL and reduced levels of macrophages and myeloid-derived suppressor cells in the bone marrow. The mRNA expression of liver-X-receptor-α, peroxisome proliferator-activated receptor-γ and superoxide dismutase-1 was induced in the liver and that of interferon-γ and the chemokine (C-X-C motif) ligand 1 decreased, thereby suggesting the potential mechanisms for many beneficial effects. Other changes were also observed such as increased plasma levels of triglycerides and lipid peroxidation that may reflect potential activation of brown fat. This study provides new insights into the protective actions and the potential underlying mechanisms of the formulation in vivo, particularly in relation to risk factors together with changes in systemic inflammation and hepatic lipid alterations associated with atherosclerosis and metabolic syndrome, and supports further assessments in human trials.

Modulating gut microbiota in a mouse model of Graves' orbitopathy and its impact on induced disease.

BACKGROUND: Graves' disease (GD) is an autoimmune condition in which autoantibodies to the thyrotropin receptor (TSHR) cause hyperthyroidism. About 50% of GD patients also have Graves' orbitopathy (GO), an intractable disease in which expansion of the orbital contents causes diplopia, proptosis and even blindness. Murine models of GD/GO, developed in different centres, demonstrated significant variation in gut microbiota composition which correlated with TSHR-induced disease heterogeneity. To investigate whether correlation indicates causation, we modified the gut microbiota to determine whether it has a role in thyroid autoimmunity. Female BALB/c mice were treated with either vancomycin, probiotic bacteria, human fecal material transfer (hFMT) from patients with severe GO or ddH2O from birth to immunization with TSHR-A subunit or beta-galactosidase (ßgal; age ~ 6 weeks). Incidence and severity of GD (TSHR autoantibodies, thyroid histology, thyroxine level) and GO (orbital fat and muscle histology), lymphocyte phenotype, cytokine profile and gut microbiota were analysed at sacrifice (~ 22 weeks). RESULTS: In ddH2O-TSHR mice, 84% had pathological autoantibodies, 67% elevated thyroxine, 77% hyperplastic thyroids and 70% orbital pathology. Firmicutes were increased, and Bacteroidetes reduced relative to ddH2O-ßgal; CCL5 was increased. The random forest algorithm at the genus level predicted vancomycin treatment with 100% accuracy but 74% and 70% for hFMT and probiotic, respectively. Vancomycin significantly reduced gut microbiota richness and diversity compared with all other groups; the incidence and severity of both GD and GO also decreased; reduced orbital pathology correlated positively with Akkermansia spp. whilst IL-4 levels increased. Mice receiving hFMT initially inherited their GO donors' microbiota, and the severity of induced GD increased, as did the orbital brown adipose tissue volume in TSHR mice. Furthermore, genus Bacteroides, which is reduced in GD patients, was significantly increased by vancomycin but reduced in hFMT-treated mice. Probiotic treatment significantly increased CD25+ Treg cells in orbital draining lymph nodes but exacerbated induced autoimmune hyperthyroidism and GO. CONCLUSIONS: These results strongly support a role for the gut microbiota in TSHR-induced disease. Whilst changes to the gut microbiota have a profound effect on quantifiable GD endocrine and immune factors, the impact on GO cellular changes is more nuanced. The findings have translational potential for novel, improved treatments. Video abstract.

Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption.

BACKGROUND: The Lactic Acid Bacteria (LAB) are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD) are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two Lactobacillus strains, and also tracked the fate of these two administered strains using a RAPD technique. RESULTS: A RAPD typing scheme was developed to genetically type LAB isolates from a wide range of species, and optimised for direct application to bacterial colony growth. A high-throughput strategy for fingerprinting the cultivable diversity of human faeces was developed and used to determine: (i) the initial cultivable LAB strain diversity in the human gut, and (ii) the fate of two Lactobacillus strains (Lactobacillus salivarius NCIMB 30211 and Lactobacillus acidophilus NCIMB 30156) contained within a capsule that was administered in a small-scale human feeding study. The L. salivarius strain was not cultivated from the faeces of any of the 12 volunteers prior to capsule administration, but appeared post-feeding in four. Strains matching the L. acidophilus NCIMB 30156 feeding strain were found in the faeces of three volunteers prior to consumption; after taking the Lactobacillus capsule, 10 of the 12 volunteers were culture positive for this strain. The appearance of both Lactobacillus strains during capsule consumption was statistically significant (p < 0.05). CONCLUSION: We have shown that genetic strain typing of the cultivable human gut microbiota can be evaluated using a high throughput RAPD technique based on single bacterial colonies. Validation of this strategy paves the way for future systematic studies on the fate and efficacy of bacterial probiotics during human clinical trials.

Long term extension of a randomised controlled trial of probiotics using electronic health records.

Most randomised controlled trials (RCTs) are relatively short term and, due to costs and available resources, have limited opportunity to be re-visited or extended. There is no guarantee that effects of treatments remain unchanged beyond the study. Here, we illustrate the feasibility, benefits and cost-effectiveness of enriching standard trial design with electronic follow up. We completed a 5-year electronic follow up of a RCT investigating the impact of probiotics on asthma and eczema in children born 2005-2007, with traditional fieldwork follow up to two years. Participants and trial outcomes were identified and analysed after five years using secure, routine, anonymised, person-based electronic health service databanks. At two years, we identified 93% of participants and compared fieldwork with electronic health records, highlighting areas of agreement and disagreement. Retention of children from lower socio-economic groups was improved, reducing volunteer bias. At 5 years we identified a reduced 82% of participants. These data allowed the trial's first robust analysis of asthma endpoints. We found no indication that probiotic supplementation to pregnant mothers and infants protected against asthma or eczema at 5 years. Continued longer-term follow up is technically straightforward.