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The applicability was evaluated of 16 different oats species and varieties of different provenance in the coeliac diet in view of the composition of the protein complex and immunological testing during two-year experiments (2001 and 2002). Determination was carried out of total nitrogen content (average of evaluated oats collection in 2001 was 2.21%, in 2002 2.78%), protein nitrogen content (average 2001 1.94%, 2002 2.28%), and crude protein (N × 6.25) content (average 2001 13.80%, 2002 17.37%). The proportions of different protein fractions play a decisive role for the aims of this study because, based on the existing knowledge, coeliacally active protein components are present particularly in the prolamin fraction. The percentage of prolamins (determined by discontinual fractionation after Osborne) in the author's evaluated collection of oats species and varieties under the conditions of Central Bohemia reached on average 17.68% of the total protein in 2001, and 15.36% in 2002. The average percentage of albumins and globulins of the total protein reached 36.97% in 2001 and 41.04% in 2002, the average percentage of glutelins of the total proteins was 37.61% in 2001 and 34.10% in 2002, and residual was on average 7.55% in 2001 and 8.70% in 2002, respectively, of the total protein. Electrophoretic analysis of reserve (gluten) proteins (SDS-PAGE ISTA) showed in the oats collection evaluated the percentage of LMW + prolamins in the range 56-77% of the total reserve proteins in 2001, and 52-73% in 2002. The results of A-PAGE electrophoretic analysis of prolamin proteins confirmed the presence of α-prolamins, that ranged in the total content of prolamins from 50 to 88% in 2001, and from 77 to 100% in 2002, while β- + γ-prolamins ranged in 2001 from 11 to 49%, and in 2002 from 0 to 22%. These values do not give serious guarantees for the possible utilisation of oats in the gluten-free diet. The results of the immunological evaluation of the amount of prolamins in oats grains using ELISA showed great differences between different varieties and the experimental years. In 2001, 7 oats samples out of 13 evaluated, and in 2002 10 samples out of 12 evaluated were below the limit for the gluten-free diet (10 mg prolamins (gliadins)/100 g of sample dry matter), but the other varieties exceeded the limit, particularly in 2001, very significantly. The results obtained in the evaluated collection of species and varieties of oats revealed a great variability in the structure of the protein complex and in the immunological testing. In addition a significant effect of the year on the results of all analyses was evident. Based on our results, the use of oats in the diet for coeliac disease can be very risky for these reasons.

Study Objectives: Microbial antigens can elicit an immune response leading to the production of autoantibodies cross-reacting with autoantigens. Still, their clinical significance in human sera in the context of brain diseases is unclear. Therefore, assessment of natural autoantibodies reacting with their neuropeptides may elucidate the autoimmune etiology of central hypersomnias. The study aims to determine whether serum autoantibody levels differ in patients with different types of central hypersomnias (narcolepsy type 1 and 2, NT1 and NT2; idiopathic hypersomnia, IH) and healthy controls and if the differences could suggest the participation of autoantibodies in disease pathogenesis., Methods: Sera from 91 patients with NT1, 27 with NT2, 46 with IH, and 50 healthy controls were examined for autoantibodies against assorted neuropeptides. Participants were screened using questionnaires related to sleep disorders, quality of life, and mental health conditions. In addition, serum biochemical parameters and biomarkers of microbial penetration through the intestinal wall were determined., Results: A higher prevalence of autoantibodies against neuropeptides was observed only for alpha-melanocytes-stimulating hormone (α-MSH) and neuropeptide glutamic acid-isoleucine (NEI), which differed slightly among diagnoses. Patients with both types of narcolepsy exhibited signs of microbial translocation through the gut barrier. According to the questionnaires, patients diagnosed with NT2 or IH had subjectively worse life quality than patients with NT1. Patients displayed significantly lower levels of bilirubin and creatinine and slightly higher alkaline phosphatase values than healthy controls., Conclusions: Overall, serum anti-neuronal antibodies prevalence is rare, suggesting that their participation in the pathophysiology of concerned sleep disorders is insignificant. Moreover, their levels vary slightly between diagnoses indicating no major diagnostic significance., Competing Interests: Declaration of competing interest All authors report there are no competing interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In recent years, there has been an increased interest in elucidating the influence of the gut microbiota on sleep physiology. The gut microbiota affects the central nervous system by modulating neuronal pathways through the neuroendocrine and immune system, the hypothalamus-pituitary-adrenal axis, and various metabolic pathways. The gut microbiota can also influence circadian rhythms. In this study, we observed the gut microbiota composition of patients suffering from narcolepsy type 1, narcolepsy type 2, and idiopathic hypersomnia. We did not observe any changes in the alpha diversity of the gut microbiota among patient groups and healthy controls. We observed changes in beta diversity in accordance with Jaccard dissimilarities between the control group and groups of patients suffering from narcolepsy type 1 and idiopathic hypersomnia. Our results indicate that both these patient groups differ from controls relative to the presence of rare bacterial taxa. However, after adjustment for various confounding factors such as BMI, age, and gender, there were no statistical differences among the groups. This indicates that the divergence in beta diversity in the narcolepsy type 1 and idiopathic hypersomnia groups did not arise due to sleep disturbances. This study implies that using metabolomics and proteomics approaches to study the role of microbiota in sleep disorders might prove beneficial., Competing Interests: Declaration of competing interest The authors report there are no competing interests to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Tumor necrosis factor-alpha (TNF- α ) agonists revolutionized therapeutic algorithms in inflammatory bowel disease (IBD) management. However, approximately every third IBD patient does not respond to this therapy in the long term, which delays efficient control of the intestinal inflammation., Methods: We analyzed the power of serum biomarkers to predict the failure of anti-TNF- α . We collected serum of 38 IBD patients at therapy prescription and 38 weeks later and analyzed them with relation to therapy response (no-, partial-, and full response). We used enzyme-linked immunosorbent assay to quantify 16 biomarkers related to gut barrier (intestinal fatty acid-binding protein, liver fatty acid-binding protein, trefoil factor 3, and interleukin (IL)-33), microbial translocation, immune system regulation (TNF- α , CD14, lipopolysaccharide-binding protein, mannan-binding lectin, IL-18, transforming growth factor- β 1 (TGF- β 1), osteoprotegerin (OPG), insulin-like growth factor 2 (IGF-2), endocrine-gland-derived vascular endothelial growth factor), and matrix metalloproteinase system (MMP-9, MMP-14, and tissue inhibitors of metalloproteinase-1)., Results: We found that future full-responders have different biomarker profiles than non-responders, while partial-responders cannot be distinguished from either group. When future non-responders were compared to responders, their baseline contained significantly more TGF- β 1, less CD14, and increased level of MMP-9, and concentration of these factors could predict non-responders with high accuracy (AUC = 0.938). Interestingly, during the 38 weeks, levels of MMP-9 decreased in all patients, irrespective of the outcome, while OPG, IGF-2, and TGF- β 1 were higher in non-responders compared to full-responders both at the beginning and the end of the treatment., Conclusions: The TGF- β 1 and CD14 can distinguish non-responders from responders. The changes in biomarker dynamics during the therapy suggest that growth factors (such as OPG, IGF-2, and TGF- β ) are not markedly influenced by the treatment and that anti-TNF- α therapy decreases MMP-9 without influencing the treatment outcome., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Stepan Coufal et al.)

Psoriasis is a chronic, immune-mediated, inflammatory disease primarily affecting the skin. It is currently coming to light that patients with psoriasis have disrupted intestinal barrier and often suffer from comorbidities associated with the gastrointestinal tract. Moreover, there is growing evidence of both cutaneous and intestinal paradoxical reactions during biologic treatment in patients with psoriasis. This review focuses on barrier defects and changes in immune responses in patients with psoriasis, which play an important role in the development of the disease but are also influenced by modern biological treatments targeting IL-17 and TNFα cytokines. Here, we highlight the relationship between the gut-skin axis, microbiota, psoriasis treatment, and the incidence of paradoxical reactions, such as inflammatory bowel disease in patients with psoriasis. A better understanding of the interconnection of these mechanisms could lead to a more personalized therapy and lower the incidence of treatment side effects, thereby improving the quality of life of the affected patients., (© 2023. The Author(s).)

Crohn's disease (CD) and ulcerative colitis (UC) are two forms of inflammatory bowel disease (IBD), where the role of gut but not skin dysbiosis is well recognized. Inhibitors of TNF have been successful in IBD treatment, but up to a quarter of patients suffer from unpredictable skin adverse events (SkAE). For this purpose, we analyzed temporal dynamics of skin microbiota and serum markers of inflammation and epithelial barrier integrity during anti-TNF therapy and SkAE manifestation in IBD patients. We observed that the skin microbiota signature of IBD patients differs markedly from healthy subjects. In particular, the skin microbiota of CD patients differs significantly from that of UC patients and healthy subjects, mainly in the retroauricular crease. In addition, we showed that anti-TNF-related SkAE are associated with specific shifts in skin microbiota profile and with a decrease in serum levels of L-FABP and I-FABP in IBD patients. For the first time, we showed that shifts in microbial composition in IBD patients are not limited to the gut and that skin microbiota and serum markers of the epithelium barrier may be suitable markers of SkAE during anti-TNF therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Reiss, Rob, Kolar, Schierova, Kreisinger, Jackova, Roubalova, Coufal, Mihula, Thon, Bajer, Novakova, Vasatko, Kostovcikova, Galanova, Lukas, Kverka, Tresnak Hercogova, Tlaskalova-Hogenova and Jiraskova Zakostelska.)

Background: Ustekinumab, is a new therapy for patients with IBD, especially for patients suffering from Crohn's disease (CD) who did not respond to anti-TNF treatment. To shed light on the longitudinal effect of ustekinumab on the immune system, we investigated the effect on skin and gut microbiota composition, specific immune response to commensals, and various serum biomarkers., Methodology/principal Findings: We recruited 11 patients with IBD who were monitored over 40 weeks of ustekinumab therapy and 39 healthy controls (HC). We found differences in the concentrations of serum levels of osteoprotegerin, TGF-β1, IL-33, and serum IgM antibodies against Lactobacillus plantarum between patients with IBD and HC. The levels of these biomarkers did not change in response to ustekinumab treatment or with disease improvement during the 40 weeks of observation. Additionally, we identified differences in stool abundance of uncultured Subdoligranulum, Faecalibacterium, and Bacteroides between patients with IBD and HC., Conclusion/significance: In this preliminary study, we provide a unique overview of the longitudinal monitoring of fecal and skin microbial profiles as well as various serum biomarkers and humoral and cellular response to gut commensals in a small cohort of patients with IBD on ustekinumab therapy., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Rob et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The composition of microbiota and the gut-brain axis is increasingly considered a factor in the development of various pathological conditions. The etiology of multiple sclerosis (MS), a chronic autoimmune disease affecting the CNS, is complex and interactions within the gut-brain axis may be relevant in the development and the course of MS. In this article, we focus on the relationship between gut microbiota and the pathophysiology of MS. We review the contribution of germ-free mouse studies to our understanding of MS pathology and its implications for treatment strategies to modulate the microbiome in MS. This summary highlights the need for a better understanding of the role of the microbiota in patients' responses to disease-modifying drugs in MS and disease activity overall.

Inflammatory bowel diseases (IBD) are chronic disorders of the gastrointestinal tract that have been linked to microbiome dysbiosis and immune system dysregulation. We investigated the longitudinal effect of anti-TNF therapy on gut microbiota composition and specific immune response to commensals in IBD patients. The study included 52 patients tracked over 38 weeks of therapy and 37 healthy controls (HC). To characterize the diversity and composition of the gut microbiota, we used amplicon sequencing of the V3V4 region of 16S rRNA for the bacterial community and of the ITS1 region for the fungal community. We measured total antibody levels as well as specific antibodies against assorted gut commensals by ELISA. We found diversity differences between HC, Crohn's disease, and ulcerative colitis patients. The bacterial community of patients with IBD was more similar to HC at the study endpoint, suggesting a beneficial shift in the microbiome in response to treatment. We identified factors such as disease severity, localization, and surgical intervention that significantly contribute to the observed changes in the gut bacteriome. Furthermore, we revealed increased IgM levels against specific gut commensals after anti-TNF treatment. In summary, this study, with its longitudinal design, brings insights into the course of anti-TNF therapy in patients with IBD and correlates the bacterial diversity with disease severity in patients with ulcerative colitis (UC).

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Anorexia nervosa (AN) is a life-threatening psychiatric disorder with not well-described pathogenesis. Besides the genetic and sociological factors, autoimmunity is also considered to take part in AN pathogenesis. We evaluated general serological factors showing the physiological state of 59 patients with AN at hospital admission and their discharge. We detected the altered levels of some general biochemical and immunological parameters. We also detected decreased levels of appetite-regulating alpha-melanocyte stimulating hormone (α-MSH) in patients at hospital admission. Moreover, elevated anti-α-MSH IgM levels and decreased anti-α-MSH IgA levels were observed in patients with AN. Therefore, we analyzed the gut microbiota composition with special focus on α-MSH antigen-mimetic containing microbes from the Enterobacteriaceae family. We correlated gut bacterial composition with anti-α-MSH Ig levels and detected decreasing IgG levels with increasing alpha diversity. The upregulation of pro-inflammatory cytokines IL-6, IL-17, and TNF-α were detected in patients with AN both prior and after hospitalization. We also evaluated the treatment outcome and improvement was observed in the majority of patients with AN. We provide new data about various serum biochemical parameters and their changes during the patients' hospitalization, with emphasis on the immune system, and its possible participation in AN pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roubalova, Prochazkova, Dvorak, Hill, Papezova, Kreisinger, Bulant, Lambertova, Holanova, Bilej and Tlaskalova-Hogenova.)

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.

The equilibrium and reciprocal actions among appetite-stimulating (orexigenic) and appetite-suppressing (anorexigenic) signals synthesized in the gut, brain, microbiome and adipose tissue (AT), seems to play a pivotal role in the regulation of food intake and feeding behavior, anxiety, and depression. A dysregulation of mechanisms controlling the energy balance may result in eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN). AN is a psychiatric disease defined by chronic self-induced extreme dietary restriction leading to an extremely low body weight and adiposity. BN is defined as out-of-control binge eating, which is compensated by self-induced vomiting, fasting, or excessive exercise. Certain gut microbiota-related compounds, like bacterial chaperone protein Escherichia coli caseinolytic protease B (ClpB) and food-derived antigens were recently described to trigger the production of autoantibodies cross-reacting with appetite-regulating hormones and neurotransmitters. Gut microbiome may be a potential manipulator for AT and energy homeostasis. Thus, the regulation of appetite, emotion, mood, and nutritional status is also under the control of neuroimmunoendocrine mechanisms by secretion of autoantibodies directed against neuropeptides, neuroactive metabolites, and peptides. In AN and BN, altered cholinergic, dopaminergic, adrenergic, and serotonergic relays may lead to abnormal AT, gut, and brain hormone secretion. The present review summarizes updated knowledge regarding the gut dysbiosis, gut-barrier permeability, short-chain fatty acids (SCFA), fecal microbial transplantation (FMT), blood-brain barrier permeability, and autoantibodies within the ghrelin and melanocortin systems in eating disorders. We expect that the new knowledge may be used for the development of a novel preventive and therapeutic approach for treatment of AN and BN., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LP declared a shared affiliation with several of the authors to the handling editor at time of review., (Copyright © 2021 Smitka, Prochazkova, Roubalova, Dvorak, Papezova, Hill, Pokorny, Kittnar, Bilej and Tlaskalova-Hogenova.)

Mucosal surfaces are colonized by highly diverse commensal microbiota. Coating with secretory IgA (SIgA) promotes the survival of commensal bacteria while it inhibits the invasion by pathogens. Bacterial coating could be mediated by antigen-specific SIgA recognition, polyreactivity, and/or by the SIgA-associated glycans. In contrast to many in vitro studies, only a few reported the effect of SIgA glycans in vivo. Here, we used a germ-free antibody-free newborn piglets model to compare the protective effect of SIgA, SIgA with enzymatically removed N-glycans, Fab, and Fc containing the secretory component (Fc-SC) during oral necrotoxigenic E. coli O55 challenge. SIgA, Fab, and Fc-SC were protective, whereas removal of N-glycans from SIgA reduced SIgA-mediated protection as demonstrated by piglets' intestinal histology, clinical status, and survival. In vitro analyses indicated that deglycosylation of SIgA did not reduce agglutination of E. coli O55. These findings highlight the role of SIgA-associated N-glycans in protection. Further structural studies of SIgA-associated glycans would lead to the identification of those involved in the species-specific inhibition of attachment to corresponding epithelial cells.

Preterm germ-free piglets were monoassociated with probiotic Bifidobacterium animalis subsp. lactis BB-12 (BB12) to verify its safety and to investigate possible protection against subsequent infection with Salmonella Typhimurium strain LT2 (LT2). Clinical signs of salmonellosis, bacterial colonization in the intestine, bacterial translocation to mesenteric lymph nodes (MLN), blood, liver, spleen, and lungs, histopathological changes in the ileum, claudin-1 and occludin mRNA expression in the ileum and colon, intestinal and plasma concentrations of IL-8, TNF-α, and IL-10 were evaluated. Both BB12 and LT2 colonized the intestine of the monoassociated piglets. BB12 did not translocate in the BB12-monoassociated piglets. BB12 was detected in some cases in the MLN of piglets, consequently infected with LT2, but reduced LT2 counts in the ileum and liver of these piglets. LT2 damaged the luminal structure of the ileum, but a previous association with BB12 mildly alleviated these changes. LT2 infection upregulated claudin-1 mRNA in the ileum and colon and downregulated occludin mRNA in the colon. Infection with LT2 increased levels of IL-8, TNF-α, and IL-10 in the intestine and plasma, and BB12 mildly downregulated them compared to LT2 alone. Despite reductions in bacterial translocation and inflammatory cytokines, clinical signs of LT2 infection were not significantly affected by the probiotic BB12. Thus, we hypothesize that multistrain bacterial colonization of preterm gnotobiotic piglets may be needed to enhance the protective effect against the infection with S. Typhimurium LT2.

Brain-gut microbiota interactions are intensively studied in connection with various neurological and psychiatric diseases. While anorexia nervosa (AN) pathophysiology is not entirely clear, it is presumably linked to microbiome dysbiosis. We aimed to elucidate the gut microbiota contribution in AN disease pathophysiology. We analyzed the composition and diversity of the gut microbiome of patients with AN (bacteriome and mycobiome) from stool samples before and after renourishment, and compared them to healthy controls. Further, levels of assorted neurotransmitters and short-chain fatty acids (SCFA) were analyzed in stool samples by MS and NMR, respectively. Biochemical, anthropometric, and psychometric profiles were assessed. The bacterial alpha-diversity parameter analyses revealed only increased Chao 1 index in patients with AN before the realimentation, reflecting their interindividual variation. Subsequently, core microbiota depletion signs were observed in patients with AN. Overrepresented OTUs (operation taxonomic units) in patients with AN taxonomically belonged to Alistipes, Clostridiales, Christensenellaceae , and Ruminococcaceae . Underrepresented OTUs in patients with AN were Faecalibacterium, Agathobacter, Bacteroides, Blautia , and Lachnospira . Patients exhibited greater interindividual variation in the gut bacteriome, as well as in metagenome content compared to controls, suggesting altered bacteriome functions. Patients had decreased levels of serotonin, GABA, dopamine, butyrate, and acetate in their stool samples compared to controls. Mycobiome analysis did not reveal significant differences in alpha diversity and fungal profile composition between patients with AN and healthy controls, nor any correlation of the fungal composition with the bacterial profile. Our results show the changed profile of the gut microbiome and its metabolites in patients with severe AN. Although therapeutic partial renourishment led to increased body mass index and improved psychometric parameters, SCFA, and neurotransmitter profiles, as well as microbial community compositions, did not change substantially during the hospitalization period, which can be potentially caused by only partial weight recovery.

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 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-1β, 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.

Aims: Epstein-Barr virus (EBV) targets predominantly B cells and these cells could acquire new phenotype characteristics. Here we analyzed whether EBV-infected and -uninfected B cells from healthy subjects differ in proportion of dominant phenotypes, maturation stage, and homing receptors expression., Methods: EBV-infected and -uninfected cells were identified by flow cytometry using fluorophore-labeled EBV RNA-specific DNA probes combined with fluorophore-labeled antibody to surface lineage markers, integrins, chemokine receptors, and immunoglobulin isotypes, including intracellular ones., Results: Our results show that the trafficking characteristics of EBER pos B cells are distinct from EBER neg B cells with most dominant differences detected for α4β1 and α4β7 and CCR5 and CCR7. EBV-positive cells are predominantly memory IgM + B cells or plasmablasts/plasma cells (PB/PC) positive for IgA or less for IgM. In comparison to uninfected B cells, less EBV-positive B cells express α4β7 and almost no cells express α4β1. EBV-positive B cells contained significantly higher proportion of CCR5 + and CCR7 + cells in comparison to EBV-negative cells. In vitro exposure of blood mononuclear cells to pro-inflammatory cytokine IL-6 reduces population of EBV-positive B cell., Conclusion: Although EBV-infected B cells represent only a minor subpopulation, their atypical functions could contribute in predisposed person to development abnormities such as some autoimmune diseases or tumors. Using multi-parameter flow cytometry we characterized differences in migration of EBV-positive and -negative B cells of various maturation stage and isotype of produced antibodies particularly different targeting to mucosal tissues of gastrointestinal and respiratory tracts.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease affecting mainly preterm newborns. It is characterized by unexpected onset and rapid progression with specific diagnostic signs as pneumatosis intestinalis or gas in the portal vein appearing later in the course of the disease. Therefore, we analyzed diagnostic and prognostic potential of the markers of early NEC pathogenesis, such as excessive inflammatory response (serum amyloid A (SAA)) and gut epithelium damage (intestinal and liver fatty acid-binding protein (I-FABP and L-FABP, respectively) and trefoil factor-3 (TFF-3)). We used ELISA to analyze these biomarkers in the urine of patients with suspected NEC, either spontaneous or surgery-related, or in infants without gut surgery (controls). Next, we compared their levels with the type of the disease (NEC or sepsis) and its severity. Already at the time of NEC suspicion, infants who developed NEC had significantly higher levels of all tested biomarkers than controls and higher levels of I-FABP and L-FABP than those who will later develop sepsis. Infants who will develop surgery-related NEC had higher levels of I-FABP and L-FABP than those who will develop sepsis already during the first 6 hours after the abdominal surgery. I-FABP was able to discriminate between infants who will develop NEC or sepsis and the SAA was able to discriminate between medical and surgical NEC. Moreover, the combination of TFF-3 with I-FABP and SAA could predict pneumatosis intestinalis , and the combination of I-FABP, L-FABP, and SAA could predict gas in the portal vein or long-term hospitalization and low SAA predicts early full enteral feeding. Thus, these biomarkers may be useful not only in the early, noninvasive diagnostics but also in the subsequent NEC management., Competing Interests: The authors declare that they have no conflicts of interests., (Copyright © 2020 Stepan Coufal et al.)

Recurrent aphthous stomatitis (RAS) is the most common disease of the oral mucosa, and it has been recently associated with bacterial and fungal dysbiosis. To study this link further, we investigated microbial shifts during RAS manifestation at an ulcer site, in its surroundings, and at an unaffected site, compared with healed mucosa in RAS patients and healthy controls. We sampled microbes from five distinct sites in the oral cavity. The one site with the most pronounced differences in microbial alpha and beta diversity between RAS patients and healthy controls was the lower labial mucosa. Detailed analysis of this particular oral site revealed strict association of the genus Selenomonas with healed mucosa of RAS patients, whereas the class Clostridia and genera Lachnoanaerobaculum , Cardiobacterium , Leptotrichia , and Fusobacterium were associated with the presence of an active ulcer. Furthermore, active ulcers were dominated by Malassezia , which were negatively correlated with Streptococcus and Haemophilus and positively correlated with Porphyromonas species. In addition, RAS patients showed increased serum levels of IgG against Mogibacterium timidum compared with healthy controls. Our study demonstrates that the composition of bacteria and fungi colonizing healthy oral mucosa is changed in active RAS ulcers, and that this alteration persists to some extent even after the ulcer is healed., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Crohn's disease (CD), ulcerative colitis (UC) and inflammatory bowel disease (IBD) associated with primary sclerosing cholangitis (PSC-IBD), share three major pathogenetic mechanisms of inflammatory bowel disease (IBD)-gut dysbiosis, gut barrier failure and immune system dysregulation. While clinical differences among them are well known, the underlying mechanisms are less explored. To gain an insight into the IBD pathogenesis and to find a specific biomarker pattern for each of them, we used protein array, ELISA and flow cytometry to analyze serum biomarkers and specific anti-microbial B and T cell responses to the gut commensals. We found that decrease in matrix metalloproteinase (MMP)-9 and increase in MMP-14 are the strongest factors discriminating IBD patients from healthy subjects and that PSC-IBD patients have higher levels of Mannan-binding lectin, tissue inhibitor of metalloproteinases 1 (TIMP-1), CD14 and osteoprotegerin than patients with UC. Moreover, we found that low transforming growth factor-β1 (TGF-β1) is associated with disease relapse and low osteoprotegerin with anti-tumor necrosis factor-alpha (TNF-α) therapy. Patients with CD have significantly decreased antibody and increased T cell response mainly to genera Eubacterium , Faecalibacterium and Bacteroides . These results stress the importance of the gut barrier function and immune response to commensal bacteria and point at the specific differences in pathogenesis of PSC-IBD, UC and CD.

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.

Diet is a major factor determining gut microbiota composition and perturbances in this complex ecosystem are associated with the inflammatory bowel disease (IBD). Here, we used gnotobiotic approach to analyze, how interaction between diet rich in proteins and gut microbiota influences the sensitivity to intestinal inflammation in murine model of ulcerative colitis. We found that diet rich in animal protein (aHPD) exacerbates acute dextran sulfate sodium (DSS)-induced colitis while diet rich in plant protein (pHPD) does not. The deleterious effect of aHPD was also apparent in chronic DSS colitis and was associated with distinct changes in gut bacteria and fungi. Therefore, we induced acute DSS-colitis in germ-free mice and transferred gut microbiota from aCD or aHPD fed mice to find that this effect requires presence of microbes and aHPD at the same time. The aHPD did not change the number of regulatory T cells or Th17 cells and still worsened the colitis in immuno-deficient RAG2 knock-out mice suggesting that this effect was not dependent on adaptive immunity. The pro-inflammatory effect of aHPD was, however, abrogated when splenic macrophages were depleted with clodronate liposomes. This treatment prevented aHPD induced increase in colonic Ly-6C high pro-inflammatory monocytes, but the ratio of resident Ly-6C -/low macrophages was not changed. These data show that the interactions between dietary protein of animal origin and gut microbiota increase sensitivity to intestinal inflammation by promoting pro-inflammatory response of monocytes.

Commensal microbiota contribute to gut homeostasis by inducing transcription of mucosal genes. Analysis of the impact of various microbiota on intestinal tissue provides an important insight into the function of this organ. We used cDNA microarrays to determine the gene expression signature of mucosa isolated from the small intestine and colon of germ-free (GF) mice and animals monoassociated with two E. coli strains. The results were compared to the expression data obtained in conventionally reared (CR) mice. In addition, we analyzed gene expression in colon organoids derived from CR, GF, and monoassociated animals. The analysis revealed that the complete absence of intestinal microbiota mainly affected the mucosal immune system, which was not restored upon monoassociation. The most important expression changes observed in the colon mucosa indicated alterations in adipose tissue and lipid metabolism. In the comparison of differentially expressed genes in the mucosa or organoids obtained from GF and CR mice, only six genes were common for both types of samples. The results show that the increased expression of the angiopoietin-like 4 ( Angptl4 ) gene encoding a secreted regulator of lipid metabolism indicates the GF status.

Psoriasis is a chronic inflammatory skin disease, whose pathogenesis involves dysregulated interplay among immune cells, keratinocytes and environmental triggers, including microbiota. Bacterial and fungal dysbiosis has been recently associated with several chronic immune-mediated diseases including psoriasis. In this comprehensive study, we investigated how different sampling sites and methods reflect the uncovered skin microbiota composition. After establishing the most suitable approach, we further examined correlations between bacteria and fungi on the psoriatic skin. We compared microbiota composition determined in the same sample by sequencing two distinct hypervariable regions of the 16S rRNA gene. We showed that using the V3V4 region led to higher species richness and evenness than using the V1V2 region. In particular, genera, such as Staphylococcus and Micrococcus were more abundant when using the V3V4 region, while Planococcaceae , on the other hand, were detected only by the V1V2 region. We performed a detailed analysis of skin microbiota composition of psoriatic lesions, unaffected psoriatic skin, and healthy control skin from the back and elbow. Only a few discriminative features were uncovered, mostly specific for the sampling site or method (swab, scraping, or biopsy). Swabs from psoriatic lesions on the back and the elbow were associated with increased abundance of Brevibacterium and Kocuria palustris and Gordonia , respectively. In the same samples from psoriatic lesions, we found a significantly higher abundance of the fungus Malassezia restricta on the back, while Malassezia sympodialis dominated the elbow mycobiota. In psoriatic elbow skin, we found significant correlation between occurrence of Kocuria , Lactobacillus , and Streptococcus with Saccharomyces , which was not observed in healthy skin. For the first time, we showed here a psoriasis-specific correlation between fungal and bacterial species, suggesting a link between competition for niche occupancy and psoriasis. However, it still remains to be elucidated whether observed microbial shift and specific inter-kingdom relationship pattern are of primary etiological significance or secondary to the disease.

Psoriatic patients have altered microbiota, both in the intestine and on the skin. It is not clear, however, whether this is a cause or consequence of the disease. In this study, using an experimental mouse model of psoriasis induced by imiquimod (IMQ), we show that oral treatment with a broad spectrum of antibiotics (MIX) or metronidazole (MET) alone mitigates the severity of skin inflammation through downregulation of Th17 immune response in conventional mice. Since some antibiotics, including MET, can influence immune system reactivity, we also evaluated the effect of MIX in the same model under germ-free (GF) conditions. GF mice treated with MET did not show milder signs of imiquimod-induced skin inflammation (IISI) which supports the conclusion that the therapeutic effect is mediated by changes in microbiota composition. Moreover, compared to controls, mice treated with MIX had a significantly higher abundance of the genus Lactobacillus in the intestine and on the skin. Mice treated with MET had a significantly higher abundance of the genera Bifidobacterium and Enterococcus both on the skin and in the intestine and of Parabacteroides distasonis in the intestine. Additionally, GF mice and mice monocolonized with either Lactobacillus plantarum or segmented filamentous bacteria (SFB) were more resistant to IISI than conventional mice. Interestingly, compared to GF mice, IMQ induced a higher degree of systemic Th17 activation in mice monocolonized with SFB but not with L. plantarum . The present findings provide evidence that intestinal and skin microbiota directly regulates IISI and emphasizes the importance of microbiota in the pathogenesis of psoriasis.

Host's physiology is significantly influenced by microbiota colonizing the epithelial surfaces. Complex microbial communities contribute to proper mucosal barrier function, immune response, and prevention of pathogen invasion and have many other crucial functions. The oral cavity and large intestine are distant parts of the digestive tract, both heavily colonized by commensal microbiota. Nevertheless, they feature different proportions of major bacterial and fungal phyla, mostly due to distinct epithelial layers organization and different oxygen levels. A few obligate anaerobic strains inhabiting the oral cavity are involved in the pathogenesis of oral diseases. Interestingly, these microbiota components are also enriched in gut inflammatory and tumor tissue. An altered microbiota composition - dysbiosis - and formation of polymicrobial biofilms seem to play important roles in the development of oral diseases and colorectal cancer. In this review, we describe the differences in composition of commensal microbiota in the oral cavity and large intestine and the mechanisms by which microbiota affect the inflammatory and carcinogenic response of the host.

Background and Objectives: The probiotic bacterium Escherichia coli strain Nissle 1917 has previously been shown to alter the pharmacokinetics of amiodarone. The aim of this study was to determine whether the probiotic bacterium Lactobacillus casei produces similar alterations in amiodarone disposition., Methods: A suspension of live probiotic bacteria L. casei strain DN-114 001 (1.5 × 10 9 CFU/dose; probiotic pre-treated group) or a saline solution (control group) was administered directly into the stomach of male Wistar rats (N = 30 in each group) by oral gavage daily for 7 consecutive days. On the eighth day, all rats (N = 60) were given a single oral dose of an amiodarone hydrochloride suspension (model drug; 50 mg/kg). The concentrations of amiodarone and of its main metabolite N-desethylamiodarone were determined in rat plasma by high-performance liquid chromatography., Results: Comparison of the pharmacokinetics of amiodarone in the control group and probiotic pre-treated group revealed that the peak plasma concentration of amiodarone was delayed by >2 h in the probiotic pre-treated group. The plasma level of N-desethylamiodarone was unchanged in the probiotic pre-medicated group and its pharmacokinetic parameters were not altered., Conclusions: The slower absorption of amiodarone in the probiotic pre-treated rats compared to the control ones and the unchanged pharmacokinetics of its main metabolite suggest that the probiotic strain of L. casei DN-114 001 has probably no clinical consequences as the difference was not statistically significant.

Increasing numbers of clinical trials and animal experiments have shown that probiotic bacteria are promising tools for allergy prevention. Here, we analyzed the immunomodulatory properties of three selected lactobacillus strains and the impact of their mixture on allergic sensitization to Bet v 1 using a gnotobiotic mouse model. We showed that Lactobacillus (L.) rhamnosus LOCK0900, L. rhamnosus LOCK0908 and L. casei LOCK0919 are recognized via Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptors and stimulate bone marrow-derived dendritic cells to produce cytokines in species- and strain-dependent manners. Colonization of germ-free (GF) mice with a mixture of all three strains (Lmix) improved the intestinal barrier by strengthening the apical junctional complexes of enterocytes and restoring the structures of microfilaments extending into the terminal web. Mice colonized with Lmix and sensitized to the Bet v 1 allergen showed significantly lower levels of allergen-specific IgE, IgG1 and IgG2a and an elevated total IgA level in the sera and intestinal lavages as well as an increased transforming growth factor (TGF)-β level compared with the sensitized GF mice. Splenocytes and mesenteric lymph node cells from the Lmix-colonized mice showed the significant upregulation of TGF-β after in vitro stimulation with Bet v 1. Our results show that Lmix colonization improved the gut epithelial barrier and reduced allergic sensitization to Bet v 1. Furthermore, these findings were accompanied by the increased production of circulating and secretory IgA and the regulatory cytokine TGF-β. Thus, this mixture of three lactobacillus strains shows potential for use in the prevention of increased gut permeability and the onset of allergies in humans.

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.

Necrotizing enterocolitis (NEC) is severe disease of gastrointestinal tract, yet its early symptoms are nonspecific, easily interchangeable with sepsis. Therefore, reliable biomarkers for early diagnostics are needed in clinical practice. Here, we analyzed if markers of gut mucosa damage, caspase cleaved cytokeratin 18 (ccCK18) and intestinal fatty acid-binding protein (I-FABP), could be used for differential diagnostics of NEC at early stage of disease. We collected paired serum (at enrollment and week later) and urine (collected for two days in 6 h intervals) samples from 42 patients with suspected NEC. These patients were later divided into NEC (n = 24), including 13 after gastrointestinal surgery, and sepsis (n = 18) groups using standard criteria. Healthy infants (n = 12), without any previous gut surgery, served as controls. Both biomarkers were measured by a commercial ELISA assay. There were no statistically significant differences in serum ccCK18 between NEC and sepsis but NEC patients had significantly higher levels of serum and urinary I-FABP than either sepsis patients or healthy infants. Urinary I-FABP has high sensitivity (81%) and specificity (100%) and can even distinguish NEC from sepsis in patients after surgery. Urinary I-FABP can be used to distinguish NEC from neonatal sepsis, including postoperative one, better than abdominal X-ray.

Background: Disturbances in the intestinal microbial community (i.e. dysbiosis) or presence of the microbes with deleterious effects on colonic mucosa has been linked to the pathogenesis of inflammatory bowel diseases. However the role of microbiota in induction and progression of ulcerative colitis (UC) has not yet been fully elucidated., Methods: Three lines of human microbiota-associated (HMA) mice were established by gavage of colon biopsy from three patients with active UC. The shift in microbial community during its transferring from humans to mice was analyzed by next-generation sequencing using Illumina MiSeq sequencer. Spontaneous or dextran sulfate sodium (DSS)-induced colitis and microbiota composition profiling in germ-free mice and HMA mice over 3-4 generations were assessed to decipher the features of the distinctive and crucial events occurring during microbial colonization and animal reproduction., Results: None of the HMA mice developed colitis spontaneously. When treated with DSS, mice in F4 generation of one line of colonized mice (aHMA) developed colitis. Compared to the DSS-resistant earlier generations of aHMA mice, the F4 generation have increased abundance of Clostridium difficile and decrease abundance of C. symbiosum in their cecum contents measured by denaturing gradient gel electrophoresis and DNA sequencing., Conclusion: In our study, mucosa-associated microbes of UC patients were not able to induce spontaneous colitis in gnotobiotic BALB/c mice but they were able to increase the susceptibility to DSS-induced colitis, once the potentially deleterious microbes found a suitable niche.

Background. Food protein-induced proctitis/proctocolitis (FPIP) is the most common noninfectious colitis in children in the first year of life. Along with the overall clinical symptoms, diarrhoea and rectal bleeding are the main manifestations of the disease. There is no routine noninvasive test that would be specific for this type of colitis. The aim of our study was to find a noninvasive laboratory test or tests that may be helpful in differential diagnosis of food protein-induced proctitis/proctocolitis. Methods. ANA, ANCA, ASCA, a-EMA, a-tTg, specific IgE, total IgE, IgG, IgA, IgM, and concentration of serum calprotectin were measured in a group of 25 patients with colitis and 18 children with other diagnoses. Results. Atypical-pANCA antibodies of IgG isotype were detected in the sera of 24 patients by the method of indirect immunofluorescence, and 5 patients showed also the positivity of IgA isotype. In control samples these autoantibodies were not detected. Other autoantibodies were not demonstrated in either patient or control group. Conclusions. Of the parameters tested in noninfectious colitis, atypical-pANCA on ethanol-fixed granulocytes appears to be a suitable serological marker of food protein-induced proctitis/proctocolitis and suggests a possible involvement of an autoimmune mechanisms in the pathogenesis of this disease.

The mammalian microbiota plays a crucial role in the pathogenesis of many diseases. Thanks to recent advances in metagenomics, proteomics, and metabolomics, microbiome composition and metabolic activity can now be studied in detail. Results obtained by such fascinating and provocative studies would be meaningless without considering the perspective of the whole organism. Our work using gnotobiology as the major tool to unravel the mechanisms of host-microbe interaction has demonstrated the crucial role of microbiota in the initiation and progression of inflammation-associated colorectal neoplasia. Carcinogenesis in the gut is driven by the presence of potentially harmful microbes or by lack of protective ones, by the production of carcinogens generated by microbes, and by the induction of inflammation and modulation of the immune system. Here, we review these mechanisms with special emphasis on those where gnotobiology has yielded important insights.

The growing interest in the composition and effects of microbiota raised the question how drug pharmacokinetics could be influenced by concomitant application of probiotics. The aim of this study was to find whether probiotic E. coli strain Nissle 1917 (EcN) influences the pharmacokinetics of concomitantly taken antiarrhythmic drug amiodarone (AMI). Live bacterial suspension of probiotic EcN (or non-probiotic E. coli strain ATCC 25922) was applied orally to male Wistar rats for seven days, while a control group of rats was treated with a saline solution. On the eighth day, the amiodarone hydrochloride was administered as one single oral dose (50 mg/kg) to all rats (N = 60). After 0, 1, 2, 3, 4, 5.5, 7, 9, 14, 22, and 30 hours, blood samples were taken from the rat abdominal aorta. The plasma level of AMI and its metabolite N-desethylamiodarone (DEA) was determined using the HPLC with UV detection. Administration of EcN led to a 43% increase of AMI AUC0-30 in comparison with control samples. However, this effect was not observed if EcN was replaced by a reference non-probiotic E. coli strain. Thus, EcN administration was most probably responsible for better drug absorption from the gastrointestinal tract. Plasma levels of DEA were also increased in plasma samples from animals treated with EcN. This change was again not found in the experiment with the reference non-probiotic strain. Higher DEA levels in samples from EcN-treated rats may be explained either by better absorption of AMI and/or by an increased activity of CYP2C forms, known to participate in metabolism of this drug, after EcN administration. In this paper, it is documented that concomitantly taken probiotic EcN may modulate pharmacokinetics of a drug; in this case, it led to an increased bioavailability of AMI.

Background: Microbial sensing by Toll-like receptors (TLR) and its negative regulation have an important role in the pathogenesis of inflammation-related cancer. In this study, we investigated the role of negative regulation of Toll-like receptors signaling and gut microbiota in the development of colitis-associated cancer in mouse model., Methods: Colitis-associated cancer was induced by azoxymethane and dextran sodium sulfate in wild-type and in interleukin-1 receptor-associated kinase M (IRAK-M)-deficient mice with or without antibiotic (ATB) treatment. Local cytokine production was analyzed by multiplex cytokine assay or enzyme-linked immunosorbent assay, and regulatory T cells were analyzed by flow cytometry. Changes in microbiota composition during tumorigenesis were analyzed by pyrosequencing, and β-glucuronidase activity was measured in intestinal content by fluorescence assay., Results: ATB treatment of wild-type mice reduced the incidence and severity of tumors. Compared with nontreated mice, ATB-treated mice had significantly lower numbers of regulatory T cells in colon, altered gut microbiota composition, and decreased β-glucuronidase activity. However, the β-glucuronidase activity was not as low as in germ-free mice. IRAK-M-deficient mice not only developed invasive tumors, but ATB-induced decrease in β-glucuronidase activity did not rescue them from severe carcinogenesis phenotype. Furthermore, IRAK-M-deficient mice had significantly increased levels of proinflammatory cytokines in the tumor tissue., Conclusions: We conclude that gut microbiota promotes tumorigenesis by increasing the exposure of gut epithelium to carcinogens and that IRAK-M-negative regulation is essential for colon cancer resistance even in conditions of altered microbiota. Therefore, gut microbiota and its metabolic activity could be potential targets for colitis-associated cancer therapy.