Influence of the gut microbiome on appetite-regulating neuropeptides in the hypothalamus: Insight from conventional, antibiotic-treated, and germ-free mouse models of anorexia nervosa.

Recent research highlights the profound impact of the gut microbiome on neuropsychiatric disorders, shedding light on its potential role in shaping human behavior. In this study, we investigate the role of the gut microbiome in appetite regulation using activity-based anorexia (ABA) mouse model of anorexia nervosa (AN) - a severe eating disorder with significant health consequences. ABA was induced in conventional, antibiotic-treated, and germ-free mice. Our results show the clear influence of the gut microbiome on the expression of four orexigenic (neuropeptide Y, agouti-related peptide, melanin-concentrating hormone, and orexin) and four anorexigenic peptides (cocaine- and amphetamine-regulated transcript, corticotropin-releasing hormone, thyrotropin-releasing hormone, and pro-opiomelanocortin) in the hypothalamus. Additionally, we assessed alterations in gut barrier permeability. While variations were noted in germ-free mice based on feeding and activity, they were not directly attributable to the gut microbiome. This research emphasizes that the gut microbiome is a pivotal factor in AN's appetite regulation beyond just dietary habits or physical activity.

Mitigating Curtaining Artifacts During Ga FIB TEM Lamella Preparation of a 14 nm FinFET Device.

We report on the mitigation of curtaining artifacts during transmission electron microscopy (TEM) lamella preparation by means of a modified ion beam milling approach, which involves altering the incident angle of the Ga ions by rocking of the sample on a special stage. We applied this technique to TEM sample preparation of a state-of-the-art integrated circuit based on a 14-nm technology node. Site-specific lamellae with a thickness <15 nm were prepared by top-down Ga focused ion beam polishing through upper metal contacts. The lamellae were analyzed by means of high-resolution TEM, which showed a clear transistor structure and confirmed minimal curtaining artifacts. The results are compared with a standard inverted thinning preparation technique.

Crohn's disease: a role of gut microbiota and Nod2 gene polymorphisms in disease pathogenesis.

Crohn's disease is a chronic immune-mediated intestinal inflammation targeted against a yet incompletely defined subset of commensal gut microbiota and occurs on the background of a genetic predisposition under the influence of environmental factors. Genome-wide association studies have identified about 70 genetic risk loci associated with Crohn's disease. The greatest risk for Crohn's disease represent polymorphisms affecting the CARD15 gene encoding nucleotide-binding oligomerization domain 2 (NOD2) which is an intracellular sensor for muramyl dipeptide, a peptidoglycan constituent of bacterial cell wall. The accumulated evidence suggests that gut microbiota represent an essential, perhaps a central factor in the induction and maintaining of Crohn's disease where dysregulation of normal co-evolved homeostatic relationships between intestinal microbiota and host mucosal immune system leads to intestinal inflammation. Taken together, these findings identify Crohn's disease as a syndrome of overlapping phenotypes that involves variable influences of genetic and environmental factors. A deeper understanding of different genetic abnormalities underlying Crohn's disease together with the identification of beneficial and harmful components of gut microbiota and their interactions are essential conditions for the categorization of Crohn's disease patients, which enable us to design more effective, preferably causative, individually tailored therapy.

Detection of galanin receptors in the spinal cord in experimental autoimmune encephalomyelitis.

AIMS: The neuropeptide galanin is a widely distributed neurotransmitter/neuromodulator that regulates a variety of physiological processes and also participates in the regulation of stress responses. The aims of the present study were to investigate the expression of galanin receptors (GalR1, GalR2, GalR3) in the spinal cords in a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) using qPCR analysis and to determine GalR1 cellular localization (oligodendrocytes, microglia, astrocytes, ependymal cells, and endothelial cells in the capillaries) by immunohistochemistry. METHODS: Twelve samples from the EAE group and 14 samples from the control group were analyzed. Spinal cords samples were obtained at the peak of the EAE disease. RESULTS: The GalR1 mRNA level was significantly decreased in the EAE mice compared with the controls (P=0.016), whereas the mRNA levels of GalR2 and GalR3 were not significantly different for the EAE and the control mice. No significant correlations were found between the severity of the EAE disease and the mRNA levels of GalR1, GalR2 and GalR3. Immunochemical detection of the GalR1 revealed its expression in the ependymal and endothelial cells. Additionally, a weak GalR1 immunoreactivity was occasionally detected in the oligodendrocytes. CONCLUSION: This study provides additional evidence of galanin involvement in EAE pathophysiology, but this has to be further investigated.

Negative regulation of Toll-like receptor signaling plays an essential role in homeostasis of the intestine.

A healthy intestinal tract is characterized by controlled homeostasis due to the balanced interaction between commensal bacteria and the host mucosal immune system. Human and animal model studies have supported the hypothesis that breakdown of this homeostasis may underlie the pathogenesis of inflammatory bowel diseases. However, it is not well understood how intestinal microflora stimulate the intestinal mucosal immune system and how such activation is regulated. Using a spontaneous, commensal bacteria-dependent colitis model in IL-10-deficient mice, we investigated the role of TLR and their negative regulation in intestinal homeostasis. In addition to IL-10(-/-) MyD88(-/-) mice, IL-10(-/-) TLR4(-/-) mice exhibited reduced colitis compared to IL-10(-/-) mice, indicating that TLR4 signaling plays an important role in inducing colitis. Interestingly, the expression of IRAK-M, a negative regulator of TLR signaling, is dependent on intestinal commensal flora, as IRAK-M expression was reduced in mice re-derived into a germ-free environment, and introduction of commensal bacteria into germ-free mice induced IRAK-M expression. IL-10(-/-) IRAK-M(-/-) mice exhibited exacerbated colitis with increased inflammatory cytokine gene expression. Therefore, this study indicates that intestinal microflora stimulate the colitogenic immune system through TLR and negative regulation of TLR signaling is essential in maintaining intestinal homeostasis.

Lipopolysaccharide treatment suppresses spontaneously developing ankylosing enthesopathy in B10.BR male mice: the potential role of interleukin-10.

BACKGROUND: Ankylosing enthesopathy (ANKENT) is an animal model of human ankylosing spondylitis. ANKENT is an inflammatory disease affecting the ankle and tarsal joints of the hind limbs in susceptible mouse strains. In the disease, the participation of intestinal microbiota components was suggested. Therefore, we attempted to increase the incidence of ANKENT by systemic administration of lipopolysaccharide (LPS), which is a component of bacterial cellular walls and stimulates inflammatory processes. METHODS: ANKENT occurrence, serum cytokine profiles, spleen cellular composition and in vitro cytokine response to LPS were analysed in LPS-treated and control LPS-untreated B10.BR male mice. RESULTS: Contrary to expectations, LPS treatment decreased the incidence of ANKENT in LPS-treated group compared to control LPS-untreated group. Flow cytometry analysis of splenocytes showed an increased percentage of macrophages, dendritic cells and neutrophils and a decreased percentage of B cells, T cells and T helper cells in LPS-treated males following LPS administration. In addition, LPS-treated males had significantly elevated IL-6 and IL-10 serum levels. At 20-22 weeks after the final LPS application, splenocytes from LPS-treated mice were more susceptible to in vitro LPS stimulation than those of the controls and produced significantly higher levels of TNFα and IL-6. CONCLUSIONS: Repeated systemic stimulation with microbial component lipopolysaccharide in early adulthood significantly reduced the incidence of ANKENT in B10.BR mice and this finding can support the "hygiene hypothesis". In LPS-treated mice, the innate immunity parameters and the level of anti-inflammatory IL-10 cytokine were significantly increased. Nevertheless, the immunological mechanism of the LPS protective effect remains unclear.

Nod2 is required for the regulation of commensal microbiota in the intestine.

Mutations in the Nod2 gene are among the strongest genetic risk factors in the pathogenesis of ileal Crohn's disease, but the exact contributions of Nod2 to intestinal mucosal homeostasis are not understood. Here we show that Nod2 plays an essential role in controlling commensal bacterial flora in the intestine. Analysis of intestinal bacteria from the terminal ilea of Nod2-deficient mice showed that they harbor an increased load of commensal resident bacteria. Furthermore, Nod2-deficient mice had a diminished ability to prevent intestinal colonization of pathogenic bacteria. In vitro, intestinal crypts isolated from terminal ilea of Nod2-deficient mice were unable to kill bacteria effectively, suggesting an important role of Nod2 signaling in crypt function. Interestingly, the expression of Nod2 is dependent on the presence of commensal bacteria, because mice re-derived into germ-free conditions expressed significantly less Nod2 in their terminal ilea, and complementation of commensal bacteria into germ-free mice induced Nod2 expression. Therefore, Nod2 and intestinal commensal bacterial flora maintain a balance by regulating each other through a feedback mechanism. Dysfunction of Nod2 results in a break-down of this homeostasis.

Gut Microbiota Dysbiosis: Triggers, Consequences, Diagnostic and Therapeutic Options.

The global incidence of numerous immune-mediated, metabolic, neurodegenerative, and psychiatric diseases is steadily increasing [...].

Gut Microbiota and NAFLD: Pathogenetic Mechanisms, Microbiota Signatures, and Therapeutic Interventions.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Its worldwide prevalence is rapidly increasing and is currently estimated at 24%. NAFLD is highly associated with many features of the metabolic syndrome, including obesity, insulin resistance, hyperlipidaemia, and hypertension. The pathogenesis of NAFLD is complex and not fully understood, but there is increasing evidence that the gut microbiota is strongly implicated in the development of NAFLD. In this review, we discuss the major factors that induce dysbiosis of the gut microbiota and disrupt intestinal permeability, as well as possible mechanisms leading to the development of NAFLD. We also discuss the most consistent NAFLD-associated gut microbiota signatures and immunological mechanisms involved in maintaining the gut barrier and liver tolerance to gut-derived factors. Gut-derived factors, including microbial, dietary, and host-derived factors involved in NAFLD pathogenesis, are discussed in detail. Finally, we review currently available diagnostic and prognostic methods, summarise latest knowledge on promising microbiota-based biomarkers, and discuss therapeutic strategies to manipulate the microbiota, including faecal microbiota transplantation, probiotics and prebiotics, deletions of individual strains with bacteriophages, and blocking the production of harmful metabolites.

Targeting Keap1/Nrf2/ARE signaling pathway in multiple sclerosis.

Multiple sclerosis (MS) is a neurologic autoimmune disorder featured by chronic inflammation of the central nervous system, demyelination and axonal damage. Recently, the term "oxinflammation" has been proposed to depict the vicious circle of chronic inflammation and oxidative stress (OS). OS promotes demyelination and neurodegeneration directly, by oxidation of lipids, proteins, and DNA but also indirectly, by inducing a dysregulation of the immunity and favoring the state of pro-inflammatory response. Many of the actors of this delicately tuned network are controlled by Keap1/Nrf2/ARE signaling pathway, a principal regulator of antioxidant and phase II detoxification genes. This pathway also has a pivotal role in inflammation, and therefore possesses a great potential in the treatment of MS. The aim of this review is to provide the newest insights in the preclinical and clinical evidence of Nrf2 induction in the regeneration of the antioxidant response and attenuation of inflammation in MS. Preclinical studies have indicated that activators of this pathway, such as epigallocatechin gallate (EGCG), curcumin, melatonin, resveratrol, and sulforaphane might be a promising therapeutic option in amelioration of MS symptoms, nevertheless, the efficacy and safety of these compounds have to be confirmed in future clinical trials.

Severity of Experimental Autoimmune Uveitis Is Reduced by Pretreatment with Live Probiotic Escherichia coli Nissle 1917.

Non-infectious uveitis is considered an autoimmune disease responsible for a significant burden of blindness in developed countries and recent studies have linked its pathogenesis to dysregulation of the gut microbiota. We tested the immunomodulatory properties of two probiotics, Escherichia coli Nissle 1917 (EcN) and E. coli O83:K24:H31 (EcO), in a model of experimental autoimmune uveitis (EAU). To determine the importance of bacterial viability and treatment timing, mice were orally treated with live or autoclaved bacteria in both preventive and therapeutic schedules. Disease severity was assessed by ophthalmoscopy and histology, immune phenotypes in mesenteric and cervical lymph nodes were analyzed by flow cytometry and the gut immune environment was analyzed by RT-PCR and/or gut tissue culture. EcN, but not EcO, protected against EAU but only as a live organism and only when administered before or at the time of disease induction. Successful prevention of EAU was accompanied by a decrease in IRBP-specific T cell response in the lymph nodes draining the site of immunization as early as 7 days after the immunization and eye-draining cervical lymph nodes when the eye inflammation became apparent. Furthermore, EcN promoted an anti-inflammatory response in Peyer's patches, increased gut antimicrobial peptide expression and decreased production of inducible nitric oxide synthase in macrophages. In summary, we show here that EcN controls inflammation in EAU and suggest that probiotics may have a role in regulating the gut-eye axis.

Diet Rich in Simple Sugars Promotes Pro-Inflammatory Response via Gut Microbiota Alteration and TLR4 Signaling.

Diet is a strong modifier of microbiome and mucosal microenvironment in the gut. Recently, components of western-type diets have been associated with metabolic and immune diseases. Here, we studied how high-sugar diet (HSD) consumption influences gut mucosal barrier and immune response under steady state conditions and in a mouse model of acute colitis. We found that HSD significantly increased gut permeability, spleen weight, and neutrophil levels in spleens of healthy mice. Subsequent dextran sodium sulfate administration led to severe colitis. In colon, HSD significantly promoted neutrophil infiltration and increased the levels of IL-6, IL-1ß, and TNF-α. Moreover, HSD-fed mice had significantly higher abundance of pathobionts, such as Escherichia coli and Candida, in fecal samples. Although germ-free mice colonized with microbiota of conventionally reared mice that consumed different diets had equally severe colitis, mice colonized with HSD microbiota showed markedly increased infiltration of neutrophils to the gut. The induction of colitis in Toll-like receptor 4 (TLR4)-deficient HSD-fed mice led to significantly milder colitis than in wild-type mice. In conclusion, our results suggested a significant role of HSD in disruption of barrier integrity and balanced mucosal and systemic immune response. In addition, these processes seemed to be highly influenced by resident potentially pathogenic microbiota or metabolites via the TLR4 signaling pathway.

Potential for recycling of slightly radioactive metals arising from decommissioning within nuclear sector in Slovakia.

The decommissioning of nuclear installations represents a complex process resulting in the generation of large amounts of waste materials containing various concentrations of radionuclides. Selection of an appropriate strategy of management of the mentioned materials strongly influences the effectiveness of decommissioning process keeping in mind safety, financial and other relevant aspects. In line with international incentives for optimization of radioactive material management, concepts of recycling and reuse of materials are widely discussed and applications of these concepts are analysed. Recycling of some portion of these materials within nuclear sector (e.g. scrap metals or concrete rubble) seems to be highly desirable from economical point of view and may lead to conserve some disposal capacity. However, detailed safety assessment along with cost/benefit calculations and feasibility study should be developed in order to prove the safety, practicality and cost effectiveness of possible recycling scenarios. Paper discussed the potential for recycling of slightly radioactive metals arising from decommissioning of NPPs within nuclear sector in Slovakia. Various available recycling scenarios are introduced and method for overall assessment of various recycling scenarios is outlined including the preliminary assessment of safety and financial aspects.

Food Preservatives Induce Proteobacteria Dysbiosis in Human-Microbiota Associated Nod2-Deficient Mice.

The worldwide incidence of many immune-mediated and metabolic diseases, initially affecting only the wealthy Western countries, is increasing rapidly. Many of these diseases are associated with the compositional and functional alterations of gut microbiota, i.e., dysbiosis. The most consistent markers of the dysbiosis are a decrease in microbiota diversity and an expansion of Proteobacteria. The role of food preservatives as potential triggers of gut microbiota dysbiosis has been long overlooked. Using a human microbiota-associated mouse model, we demonstrate that a mixture of common antimicrobial food additives induces dysbiosis characterised by an overgrowth of Proteobacteria phylum and a decrease in the Clostridiales order. Remarkably, human gut microbiota in a Nod2-deficient genetic background is even more susceptible to the induction of Proteobacteria dysbiosis by additives than the microbiota in a wild-type background. To conclude, our data demonstrate that antimicrobial food additives trigger gut microbiota dysbiosis in both wild-type and Nod2-deficient backgrounds and at the exposure levels reached in European populations. Whether this additive-modified gut microbiota plays a significant role in the pathogenesis of immune-mediated and metabolic diseases remains to be elucidated.

The role of gut microbiota in intestinal and liver diseases.

The world-wide incidence of many immune-mediated and metabolic diseases, including those of the intestines and liver, is steadily increasing. Gut microbiota plays a central role in the pathogenesis of these diseases as it mediates environmental changes to the intestinal immune system. Various environmental factors including diet, food additives and medication also trigger the compositional and functional alterations of microbiota, that is, dysbiosis, and this dysbiosis is closely associated with many chronic inflammatory diseases. However, the causal relationship remains unclear for the majority of these diseases. In this review, we discuss essential epidemiological data, known pathogenetic factors including those of genetic and environmental nature, while mainly focusing on the role of gut microbiota in the development of selected intestinal and liver diseases. Using specific examples, we also briefly describe some of the most widely-used animal models including gnotobiotic models and their contribution to the research of pathogenetic mechanisms of the host-microbiota relationship.

Human gut microbes are susceptible to antimicrobial food additives in vitro.

The aim of this work was to test the hypothesis that antimicrobial food additives may alter the composition of human gut microbiota by selectively suppressing the growth of susceptible gut microbes. To explore the influence of antimicrobial food additives on the composition of the human gut microbiota, we examined the susceptibility of both aerobic and anaerobic gut bacteria to sodium benzoate, sodium nitrite, and potassium sorbate, and their combinations, using a broth microdilution method. The tested bacteria exhibited a wide range of susceptibilities to food additives. For example, the most susceptible strain, Bacteroides coprocola, was almost 580 times more susceptible to sodium nitrite than the most resistant strain, Enterococcus faecalis. However, most importantly, we found that gut microbes with known anti-inflammatory properties, such as Clostridium tyrobutyricum or Lactobacillus paracasei, were significantly more susceptible to additives than microbes with known proinflammatory or colitogenic properties, such as Bacteroides thetaiotaomicron or Enterococcus faecalis. Our data show that some human gut microbes are highly susceptible to antimicrobial food additives. We speculate that permanent exposure of human gut microbiota to even low levels of additives may modify the composition and function of gut microbiota and thus influence the host's immune system. Whether the effect of additive-modified gut microbiota on the human immune system could explain, at least in part, the increasing incidence of allergies and autoimmune diseases remains to be shown.

Gut microbiota and lipopolysaccharide content of the diet influence development of regulatory T cells: studies in germ-free mice.

BACKGROUND: Mammals are essentially born germ-free but the epithelial surfaces are promptly colonized by astounding numbers of bacteria soon after birth. The most extensive microbial community is harbored by the distal intestine. The gut microbiota outnumber ~10 times the total number of our somatic and germ cells. The host-microbiota relationship has evolved to become mutually beneficial. Studies in germ-free mice have shown that gut microbiota play a crucial role in the development of the immune system. The principal aim of the present study was to elucidate whether the presence of gut microbiota and the quality of a sterile diet containing various amounts of bacterial contaminants, measured by lipopolysaccharide (LPS) content, can influence maturation of the immune system in gnotobiotic mice. RESULTS: We have found that the presence of gut microbiota and to a lesser extent also the LPS-rich sterile diet drive the expansion of B and T cells in Peyer's patches and mesenteric lymph nodes. The most prominent was the expansion of CD4+ T cells including Foxp3-expressing T cells in mesenteric lymph nodes. Further, we have observed that both the presence of gut microbiota and the LPS-rich sterile diet influence in vitro cytokine profile of spleen cells. Both gut microbiota and LPS-rich diet increase the production of interleukin-12 and decrease the production of interleukin-4. In addition, the presence of gut microbiota increases the production of interleukin-10 and interferon-gamma. CONCLUSION: Our data clearly show that not only live gut microbiota but also microbial components (LPS) contained in sterile diet stimulate the development, expansion and function of the immune system. Finally, we would like to emphasize that the composition of diet should be regularly tested especially in all gnotobiotic models as the LPS content and other microbial components present in the diet may significantly alter the outcome of experiments.

Susceptibility to nasal and oral tolerance induction to the major birch pollen allergen Bet v 1 is not dependent on the presence of the microflora.

The indigenous microflora plays an integrative role in the maintenance of immunological homeostasis. Several studies reported that immunological tolerance is dependent on microbial colonization of the gut. In the present study, we investigated whether the absence of the microflora influences the sensitization process to an allergen as well as the ability to develop mucosal tolerance in a mouse model of birch pollen allergy. Germ-free or conventional BALB/c mice were intranasally or intragastrically pre-treated with the major birch pollen allergen Bet v 1 prior to sensitization with this allergen. Both germ-free and conventional mice displayed comparable Th2 biased immune responses after allergic sensitization. Oral as well as intranasal tolerization led to suppression of allergen-specific serum antibodies (IgG1, IgE, IgA) as well as cytokine production by splenocytes (IL-5, IFN-gamma) in both germ-free and conventional animals. Peyer's patches of germ-free animals were approximately 20 times smaller than in conventional animals, but the relative distribution of lymphocyte subpopulations was equal. We conclude that the absence of the microflora does not influence the ability to mount Th2 responses nor to establish tolerance towards the aeroallergen Bet v 1. Our findings may challenge the view that the commensal microflora is a key factor for breakdown of physiological tolerance and allergy development.

Segmented filamentous bacteria in a defined bacterial cocktail induce intestinal inflammation in SCID mice reconstituted with CD45RBhigh CD4+ T cells.

BACKGROUND: The aim was to analyze the influence of intestinal microbiota on the development of intestinal inflammation. We used the model of chronic inflammation that develops spontaneously in the colon of conventional severe combined immunodeficiency (SCID) mice restored with the CD45 RB(high) subset of CD4+T cells isolated from the spleen of normal BALB/c mice. METHODS: A CD4+CD45RB(high) subpopulation of T cells was purified from the spleen of conventional BALB/c mice by magnetic separation (MACS) and transferred into immunodeficient SCID mice. Germ-free (GF) SCID mice or SCID mice monoassociated with Enterococcus faecalis, SFB (segmented filamentous bacteria), Fusobacterium mortiferum, Bacteroides distasonis, and in combination Fusobacterium mortiferum + SFB or Bacteroides distasonis + SFB were used as recipients. SCID mice were colonized by a defined cocktail of specific pathogen-free (SPF) bacteria. Mice were evaluated 8-12 weeks after the cell transfer for clinical and morphological signs of inflammatory bowel disease (IBD). RESULTS: After the transfer of the CD4+CD45RB(high) T-cell subpopulation to SCID mice severe colitis was present in conventional animals and in mice colonized with a cocktail of SPF microflora plus SFB. Altered intestinal barrier in the terminal ileum of mice with severe colitis was documented by immunohistology using antibodies to ZO-1 (zona occludens). CONCLUSIONS: Only SFB bacteria together with a defined SPF mixture were effective in triggering intestinal inflammation in the model of IBD in reconstituted SCID mice, while no colitis was detected in GF mice or in mice colonized either with SPF microflora or monoassociated only with SFB or colonized by Bacteroides distasonis + SFB or Fusobacterium mortiferum + SFB.

The Microbiota Determines Susceptibility to Experimental Autoimmune Uveoretinitis.

The microbiota is a crucial modulator of the immune system. Here, we evaluated how its absence or reduction modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis (EAU). We induced EAU in germ-free (GF) or conventionally housed (CV) mice and in CV mice treated with a combination of broad-spectrum antibiotics either from the day of EAU induction or from one week prior to induction of disease. The severity of the inflammation was assessed by fundus biomicroscopy or by histology, including immunohistology. The immunophenotyping of T cells in local and distant lymph nodes was performed by flow cytometry. We found that GF mice and mice where the microbiota was reduced one week before EAU induction were protected from severe autoimmune inflammation. GF mice had lower numbers of infiltrating macrophages and significantly less T cell infiltration in the retina than CV mice with EAU. GF mice also had reduced numbers of IFN-γ and IL-17-producing T cells and increased numbers of regulatory T cells in the eye-draining lymph nodes. These data suggest that the presence of microbiota during autoantigen recognition regulates the inflammatory response by influencing the adaptive immune response.