Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

The effect of intestinal microbiota and dietary supplementation of mannan oligosaccharides (MOS) on mucosal architecture and digestive physiology in broiler chicks was examined. In experiment 1, pre-sterilized eggs (Ross x Ross 308) were placed in three HEPA (high efficiency particulate air)-filtered isolator units at day 19 of incubation. Germ-free chicks in one isolator were conventionalized by exposure to cecal contents from a laying hen. Bacterial contamination occurred in one germ-free isolator such that these birds were monoassociated by a bacterium within the Acinetobacter spp. resulting in 3 categories of microbial status including germ-free (GF, n=10), conventionalized (CV, n=19) and monoassociated (Mono, n=13) birds. Dietary treatments assigned to each isolator consisted of a negative control (NC, 0 g/kg of MOS in the basal diet) and MOS (2 g/kg of MOS in the diet) resulting in a 2X3 factorial treatment arrangement. At 7 d of age, body weight was recorded and birds were killed to permit collection of visceral organs, intestinal tissues and cecal contents. Body weight, relative length of small intestinal segments and relative bursa weight were significantly increased in CV birds. These birds also had increased crypt depth and lamina propria area. Dietary MOS increased villus height and villus surface area in CV birds compared with GF and Mono birds. Transcripts for all housekeeping genes tested in ileal tissue were increased by MOS such that transcripts were normalized to unit mass of total RNA. In comparison to birds fed the NC diet, MOS significantly increased the abundance of proliferative cell nuclear antigen (PCNA), toll-like receptor (TLR)-4, avian β-defensin (GAL)-6, interleukin (IL)-8, peptide transporter 1 (PepT1) and sodium-dependent glucose transporter (SGLT)-1 transcripts in ileum per unit total RNA. However, the effect of microbial status on selected gene expression profiles was surprisingly limited. A second experiment was conducted to confirm the conventionalization protocol produced a complex microbiota similar to conventionally reared birds. Twenty day-old broiler chicks (Ross x Ross 308) were assigned to one of two wire-floored battery cages provided the NC and MOS diets ad libitum and killed at 7 d of age. Terminal restriction fragment length polymorphism (TRFLP) analysis demonstrated that microbial diversity indices (Richness, Evenness, Shannon, and Simpson) were greater in conventionalized gnotobiotic birds compared to the conventionally reared birds confirming a successful conventionalization strategy in the gnotobiotic trial. These studies demonstrate that under good hygienic conditions, CV chicks thrive similar to GF animals. Based on responses to MOS observed in GF birds, evidence indicates that MOS, independent of changes in microbial composition, directly modifies host response parameters including innate immune activation, digestive and absorptive function.

Exercise is known to induce changes in the gut, typically referred to as the ‘forgotten organ’, and changes in gut microbiota can also occur with exercise possibly imparting systemic benefits. The question remains whether or not microbiota from an exercised animal can independently affect skeletal muscle morphology. Our first objective was to examine whether an endurance exercise program could modify the microbiota in donor mice. Second, we aimed to elucidate if such an endurance-trained microbiota could be transferred to germ-free mice via fecal inoculation. Finally, we sought to determine how the morphology and functional characteristics of skeletal muscle were influenced as a result of fecal inoculation. We hypothesized that germ-free mice recipients inoculated with the microbiota from endurance trained donors would undergo morphological changes in muscle fibre type composition and physiological changes in skeletal muscle function associated with a more oxidative phenotype. Eight-week-old male C57BL/6NCrl donor mice (n = 20) were randomized into two groups: one group completed an endurance exercise training protocol on a treadmill machine 3x/week for 11 weeks (n = 10) while one group remained cage-bound (n = 10). Ten-week-old male (n = 7) and female (n = 9) germ-free mice were colonized with the cecal microbiota of the donor mice in that, equal numbers of germ-free mice were inoculated with exercised-microbiota as sedentary-microbiota. Glucose metabolism and performance measures were evaluated in the donors as well as the recipients post-inoculation. Muscle tissue was extracted for immunohistochemistry and mitochondrial assays. During the intra-peritoneal glucose tolerance test (IPGTT), significant differences in blood glucose were found at 30min between exercise-inoculated and sedentary-inoculated (23.4 ± 2.2; 29.0 ± 1.9 mmol/L, p<0.05).and change in blood glucose relative to baseline (12.04 ± 2.4; 18.3 ± 1.9 mmol/L, p<0.01). There were significant sex-based differences in the blood glucose response in inoculated animals such that there were significant differences in blood glucose between the exercise-inoculated females and sedentary-inoculated females at 15mins (28.4 ± 2.4; 30.6 ± 1.1 mmol/L, p<0.05) and 30mins (24.7 ± 3.6; 29.9 ± 2.4 mmol/L, p<0.01), however no differences between exercise-inoculated males and sedentary-inoculated males. In addition, there were significant differences in the change in blood glucose relative to baseline between the exercise-inoculated females and sedentary-inoculated females at 15mins (12.3 ± 1.9; 20.6 ± 0.8 mmol/L, p<0.01) and 30mins (10.2 ± 2.6; 19.9 ± 2.1 mmol/L, p<0.001). This novel characterization of a link between gut microbiota and skeletal muscle suggests a transmissible capacity of microbiota to impart properties of ‘healthy’ muscle into compromised populations. / Thesis / Master of Science (MSc) / The gut microbiome or microbiota describes the composition of the human gut – remarkably, over 100 trillion bacterial cells live in symbiosis with the cells of the human body. Research from the past decade has elucidated the salient nature of the human gut microbiome on immunity, metabolic homeostasis, and overall health and disease. Transformative research in the field has demonstrated the ability to transfer these bacterial colonies from one individual to another and elicit change, such as altering body mass and adiposity, respective to their donor. The interaction between gut microbiota and other organ systems i.e. brain, liver, adipose tissue has been the focus of several recent investigations, suggesting that lifestyle changes such as diet and exercise can influence communication between the gut and various other organs and contribute to changes in function. Skeletal muscle is the largest muscle in the human body accounting for 40% of total mass and although the main role of skeletal muscle is locomotion and postural stabilization, it is integral for the regulation of blood glucose as well as a reservoir for other macronutrients. Acute and chronic physical exercise cause a myriad of adaptive responses throughout the human body including in skeletal muscle and the gut. Therefore, the existence and influence of a gut-muscle link or ‘axis’ on human health cannot be ignored. What is unclear exactly, is if exercise-induced adaptations in the gut of an individual can be transferred to elicit change in the gut of a recipient and further induce adaptations at the level of the skeletal muscle.

The main goal in reversing the allergy epidemic is the development of effective prophylactic strategies. Early life events, such as exposures to microbes, have a major influence on the development of balanced immune responses. Due to their ability to interact with host immune system and to modulate host immune responses probiotics, mainly bifidobacteria and lactobacilli have been used with some success in prevention of allergic disease. In order to be referred to as probiotic, bacterial strain has to undergo rigorous testing. We have selected three new Lactobacillus (L.) strains out of 24 human isolates according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Safety of these strains was proven upon intragastric administration to mice; moreover, we have shown their ability to shift cytokine Th1 - Th2 balance towards non-allergic Th1 response in isolated splenic cells. Allergen specific prophylaxis using probiotics as vehicles for mucosal delivery of recombinant allergen is an attractive concept for development of well-tolerated and effective allergy vaccines. We have shown that neonatal mono-colonization of germ-free mice with the L. plantarum NCIMB8826 strain producing the major birch pollen allergen Bet v 1 attenuates the development of...

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Inflammatory bowel disease (IBD) is a chronic inflammatory disorder that occurs as a consequence of a genetic mutation that results in an overly aggressive immune response to normal bacteria. Metabolomics is a new born cousin to genomics and proteomics and involves a high throughput identification, characterization and quantification of small molecule metabolites generated by the organism. This study will show that metabolomics can be an effective tool in studying the differences between wild type and IL 10 KO mice as they age in axenic and conventional environments, and the onset of disease in a conventional environment. I show specific changes upon colonizing axenic mice with fecal bacteria that are similar to changes occurring over 16 weeks of conventional growth. Several bacterial metabolites have been identified that may play a role in the pathogenesis or provide clues to the interactions of the gut microbiota with the intestinal immune system. / Experimental Medicine

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Remyelination describes the regeneration of myelin sheaths, and is considered one of the most promising strategies for improving the prognosis of demyelinating diseases such as multiple sclerosis. Data from animal models and human studies have shown that remyelination can occur extensively in the central nervous system (CNS), leading to functional recovery and axonal protection. However, remyelination does not always proceed to completion, and its failure is associated with progressive neurological disability. Thus, there is clinical need for interventions that can optimise the conditions for remyelination. Recent advances in genomics and animal husbandry have kindled an interest in the microbiome as a means to influence processes throughout the body. Our commensal microbes communicate with host cells at epithelial barriers, stimulate neural and endocrine axes and directly produce a plethora of long-range signalling molecules. Critically, the development and maintenance of the immune system depend on signals from the microbiota, and we know that a well-coordinated immune response is a key determinant of the success of remyelination. This thesis explores how the microbiome can influence CNS remyelination. To do so, I have studied remyelination in three murine models of microbiome alteration. Firstly, long-term oral administration of an antibiotic cocktail was used to deplete the microbiota of adult mice. Following focal demyelination, these mice had deficits in their inflammatory response, clearance of myelin debris and differentiation of new oligodendrocytes from oligodendrocyte progenitor cells (OPCs). Faecal microbial transplant was able to rescue aspects of the inflammatory response and phagocytosis, but not OPC differentiation. Secondly, I looked at remyelination in germ-free (GF) mice following cuprizone-induced demyelina- tion. As with the antibiotics-treated mice, there were deficits in inflammation following demyelination, which tended to peak later than in control mice. Finally, I investigated the potential of a therapeutic probiotic (VSL#3) to improve remyelination in aged mice. In contrast to antibiotic treatment, probiotic administration caused a slight enhancement in the onset of inflammation following focal demyelination. However, there was no significant improvement in OPC differentiation or toluidine blue rank analysis, suggesting these changes in inflammation were not sufficient to positively modulate remyelination. The results from these three studies introduce a significant but previously unconsidered environmental influence on remyelination in the CNS. Whilst the effects are subtle relative to more direct interventions, the microbiome can be manipulated simply and non-invasively, which may provide a useful adjunct to other strategies for optimising remyelination.

<p>We are all born germ-free. Soon after birth, microbes colonize our body’s surfaces, with the intestine housing the highest density of microbes on earth. Most of us remain blissfully unaware of this co-existence because inflammatory responses to the indigenous microbes are normally not triggered. Nonetheless, intestinal microbes are true educators of our immune system, which is exemplified by the immature immune system observed in germ-free animals. Accumulating evidence suggests that microbial exposure and/or composition impacts on immune regulation. As an example, isotype switch to immunoglobulin E (IgE) is normally very tightly regulated such that in healthy individuals and mice, serum levels are maintained at very low levels. In contrast, total serum IgE levels are elevated in germ-free mice, indicating that in the absence of microbes the regulatory pathway that maintains IgE at basal levels is disrupted. We hypothesize that in the absence of stimuli from the resident intestinal bacteria the immune system does not receive adequate educational signals. We showed that in germ-free mice class switch recombination (CSR) to IgE occurred at intestinal mucosal lymphoid sites a few weeks after birth. IgE levels then remained at elevated levels throughout life, even when intestinal bacteria were introduced after weaning. In the first part of this thesis, the mechanisms involved in this hygiene-induced IgE were investigateted. In a second part, the immunoregulatory role of commensal bacteria was extended to a model of autoimmunity.</p> <p>Collectively these results demonstrate a new dimension of the impact of intestinal symbionts on the immune system: they dictate baseline immune system regulation. Elucidating the mechanisms whereby microbes induce immunoregulatory pathways may give insights into the increasing prevalence of allergic- and autoimmune diseases.</p> / Doctor of Philosophy (PhD)