The Effect of Cross-Sex Fecal Microbiota Transplantation on Metabolism and Hormonal Status in Adult Rats.

Increasing evidence of sexual dimorphism in the pathophysiology of metabolic complications caused by sex steroids is under investigation. The gut microbiota represents a complex microbial ecosystem involved in energy metabolism, immune response, nutrition acquisition, and the health of host organisms. Gender-specific differences in composition are present between females and males. The purpose of this study was to use cross-sex fecal microbiota transplantation (FMT) for the detection of sex-dependent metabolic, hormonal, and gut microbiota changes in female and male recipients. Healthy non-obese female and male Wistar rats were divided into donor, same-sex, and cross-sex recipient groups. After a 30-day period of FMT administration, biochemical markers (glucose and lipid metabolism) and sex hormones were measured, and the gut microbiota was analyzed. The cross-sex male recipients displayed a significantly lower testosterone concentration compared to the males that received same-sex FMT. Sex-dependent changes caused by cross-sex FMT were detected, while several bacterial taxa correlated with plasma testosterone levels. This study represents the first to study the effect of cross-sex changes in the gut microbiome concerning metabolic and hormonal changes/status in adult non-obese Wistar rats. Herein, we present cross-sex FMT as a potential tool to modify sex-specific pathologies.

Endogenous DNase Activity in an Animal Model of Acute Liver Failure.

Deoxyribonucleases (DNases) cleave extracellular DNA (ecDNA) and are under intense research as interventions for diseases associated with high ecDNA, such as acute live injury. DNase I treatment decreases morbidity and mortality in this animal model. Endogenous DNase activity has high interindividual variability. In this study, we tested the hypothesis that high endogenous DNase activity is beneficial in an animal model of acute liver failure. DNase activity was measured in the plasma of adult male mice taken before i.p. injection of thioacetamide to induce acute liver failure. The survival of mice was monitored for 48 h. Mice were retrospectively divided into two groups based on the median DNase activity assessed using the gel-based single-radial enzyme diffusion assay. In acute liver failure, mice with a higher baseline DNase activity had lower mortality after 48 h (by 25%). Different protection of ecDNA against nucleases by vesicles or DNA-binding proteins could play a role and should be further evaluated. Similarly, the role of endogenous DNase activity should be analyzed in other disease models associated with high ecDNA.

The Human Mycobiome: Colonization, Composition and the Role in Health and Disease.

The mycobiome is the fungal component of the human microbial ecosystem that represents only a small part of this environment but plays an essential role in maintaining homeostasis. Colonization by fungi begins immediately after birth. The initial mycobiome is influenced by the gestational age of a newborn, birth weight, delivery method and feeding method. During a human's life, the composition of the mycobiome is further influenced by a large number of endogenous and exogenous factors. The most important factors are diet, body weight, age, sex and antibiotic and antifungal therapy. The human mycobiome inhabits the oral cavity, gastrointestinal tract, respiratory tract, urogenital tract and skin. Its composition can influence the gut-brain axis through immune and non-immune mediated crosstalk systems. It also interacts with other commensals of the ecosystem through synergistic and antagonistic relationships. Moreover, colonization of the gut by opportunistic fungal pathogens in immunocompromised individuals can lead to clinically relevant disease states. Thus, the mycobiome represents an essential part of the microbiome associated with a variety of physiological and pathological processes. This review summarizes the current knowledge on the composition of the mycobiome in specific sites of the human body and its role in health and disease.

Gut Dysbiosis and Fecal Microbiota Transplantation in Autoimmune Diseases.

Gut microbiota dysbiosis has recently been reported in a number of clinical states, including neurological, psychiatric, cardiovascular, metabolic and autoimmune disorders. Yet, it is not completely understood how colonizing microorganisms are implicated in their pathophysiology and molecular pathways. There are a number of suggested mechanisms of how gut microbiota dysbiosis triggers or sustains extraintestinal diseases; however, none of these have been widely accepted as part of the disease pathogenesis. Recent studies have proposed that gut microbiota and its metabolites could play a pivotal role in the modulation of immune system responses and the development of autoimmunity in diseases such as rheumatoid arthritis, multiple sclerosis or type 1 diabetes. Fecal microbiota transplantation (FMT) is a valuable tool for uncovering the role of gut microbiota in the pathological processes. This review aims to summarize the current knowledge about gut microbiota dysbiosis and the potential of FMT in studying the pathogeneses and therapies of autoimmune diseases. Herein, we discuss the extraintestinal autoimmune pathologies with at least one published or ongoing FMT study in human or animal models.

The Citrullination-Neutrophil Extracellular Trap Axis in Chronic Diseases.

Citrullination of proteins is crucial for the formation of neutrophil extracellular traps (NETs) - strands of nuclear DNA expulsed in the extracellular environment along with antimicrobial proteins in order to halt the spread of pathogens. Paradoxically, NETs may be immunogenic and contribute to inflammation. It is known that for the externalization of DNA, a group of enzymes called peptidyl arginine deiminases (PADs) is required. Current research often looks at citrullination, NET formation, PAD overexpression, and extracellular DNA (ecDNA) accumulation in chronic diseases as separate events. In contrast, we propose that citrullination can be viewed as the primary mechanism of autoimmunity, for instance by the formation of anti-citrullinated protein antibodies (ACPAs) but also as a process contributing to chronic inflammation. Therefore, citrullination could be at the center, connecting and impacting multiple inflammatory diseases in which ACPAs, NETs, or ecDNA have already been documented. In this review, we aimed to highlight the importance of citrullination in the etiopathogenesis of a number of chronic diseases and to explore the diagnostic, prognostic, and therapeutic potential of the citrullination-NET axis.

Fecal Microbiota Transplantation as a Tool for Therapeutic Modulation of Non-gastrointestinal Disorders.

Fecal microbiota transplantation has been primarily investigated as a therapeutic tool for a number of gut disorders. Optimistic results from clinical studies on Clostridium difficile infection, inflammatory bowel disease and irritable bowel syndrome have stimulated the expansion of possible indications in which FMT might represent a game changing approach. Microbial dysbiosis was shown in a number of non-gastrointestinal disorders. Moreover, FMT was proven to be effective in therapy of numerous animal models of disease. However, only a proportion of these disorders have been addressed in clinical studies using FMT. These include obesity, non-alcoholic fatty liver disease, cardiovascular inflammation and neurological disorders such as autism, depression and Parkinson's disease. Results from preclinical and clinical studies also outlined possible molecular mechanisms that contribute to alleviation of the disease. These range from increasing the circulating levels of microbial metabolites (trimethylamine N-oxide, lipopolysaccharide, short chain fatty acids) to stimulation of the enteric nervous system. Several methodological shortcomings are still to be addressed; however, positive results of the clinical studies indicate that further investigation of FMT as a therapeutic tool for non-gastrointestinal disorders can be expected in upcoming years.

Circulating extracellular DNA is in association with continuous metabolic syndrome score in healthy adolescents.

Obesity is associated with chronic low-grade inflammation that eventually leads to metabolic complications. Extracellular DNA (ecDNA) is a damage-associated molecular pattern. Extracellular mitochondrial DNA can activate innate immunity. We hypothesized that ecDNA, especially of mitochondrial origin, could be associated with components of the metabolic syndrome in young healthy probands. In a cross-sectional study, healthy adolescents (n = 1,249) provided blood samples. Anthropometric data, blood pressure, and blood counts were assessed. In addition, biochemical analysis of sera or plasma was conducted, including the quantification of advanced oxidation protein products (AOPPs) as a marker of oxidative stress induced by neutrophil or monocyte activation. Plasma ecDNA was isolated and measured by fluorometry. Nuclear and mitochondrial DNA were quantified by real-time PCR. Males had higher total plasma ecDNA [15 (11-21) vs. 11 (8-17) ng/mL; median (interquartile range)], nuclear [1,760 (956-3,273) vs. 1,153 (600-2,292) genome equivalents (GE)/mL], and mitochondrial [37,181 (14,836-90,896) vs. 30,089 (12,587-72,286) GE/mL] DNA. ecDNA correlated positively with the continuous metabolic syndrome score (r = 0.158 for males and r = 0.134 for females). Stronger correlations were found between ecDNA of mitochondrial origin and AOPP (r = 0.202 and 0.186 for males and females, respectively). Multivariate regression analysis revealed associations of nuclear DNA with leukocyte and erythrocyte counts. The results of this study of healthy adolescents show that circulating ecDNA is associated with the risk of metabolic syndrome, not with obesity per se. The association between mitochondrial ecDNA and AOPP requires further attention as it supports a potential role of mitochondria-induced sterile inflammation in the pathogenesis of the metabolic syndrome.

Metalloproteinases in Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases of the gastrointestinal tract, encompassing two main disorders: Crohn's disease (CD) and ulcerative colitis (UC). In both these pathologies, excessive and local immune response against luminal antigens promotes a pathological process leading to various degrees of gut damage. Matrix metalloproteinases (MMPs) are a family of neutral proteases with the ability to degrade all components of extracellular matrix. In physiological conditions, MMPs are produced at very low level and generally in the latent form and are involved in the normal tissue turnover. Their function is inhibited by tissue inhibitors of metalloproteinases (TIMPs). However, in inflamed tissue of IBD patients, MMPs are produced in excess and/or the activity of TIMPs is not sufficient to block MMPs, thereby making a major contribution to the IBD-related mucosal degradation. In this review, we summarize the available evidence on the expression and role of MMPs in IBD.

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

Nuclear and Mitochondrial Circulating Cell-Free DNA Is Increased in Patients With Inflammatory Bowel Disease in Clinical Remission.

Background: The role of cell-free DNA (cfDNA) in the pathogenesis of inflammatory bowel disease (IBD) has been recently suggested. The aim of this study was to analyze circulating cfDNA and deoxyribonuclease (DNase) activity in IBD patients in clinical remission. Materials and Methods: Plasma and serum were obtained from 72 patients with Crohn's disease and 28 patients with ulcerative colitis. Total cfDNA, nuclear DNA (ncDNA), mitochondrial DNA (mtDNA) and DNase activity were measured. Results: IBD patients showed higher levels of both ncDNA and mtDNA compared to healthy controls. Concentration of ncDNA was higher in males compared to females, including patients and healthy controls. However, unlike males higher amount of ncDNA was found in female IBD patients compared to healthy controls. DNase activity was significantly lower in male IBD patients compared with healthy controls. In addition, there was a negative correlation between DNase activity and ncDNA levels in male IBD patients. Conclusions: Herein we present increased amount of circulating ncDNA and mtDNA in IBD patients in clinical remission. Thus, unlike total cfDNA, circulating ncDNA and mtDNA might not represent the optimal biomarkers of disease activity. This is also the first report on sex difference in circulating ncDNA levels, possibly associated with lower DNase activity in males.

Viruses in Cancers of the Digestive System: Active Contributors or Idle Bystanders?

The human virome, which is a collection of all the viruses that are present in the human body, is increasingly being recognized as an essential part of the human microbiota. The human gastrointestinal tract and related organs (e.g., liver, pancreas, and gallbladder)-composing the gastrointestinal (or digestive) system-contain a huge number of viral particles which contribute to maintaining tissue homeostasis and keeping our body healthy. However, perturbations of the virome steady-state may, both directly and indirectly, ignite/sustain oncogenic mechanisms contributing to the initiation of a dysplastic process and/or cancer progression. In this review, we summarize and discuss the available evidence on the association and role of viruses in the development of cancers of the digestive system.

Salivary microbiome composition changes after bariatric surgery.

Recent studies show that the salivary microbiome in subjects with obesity differ from those without obesity, but the mechanism of interaction between the salivary microbiome composition and body weight is unclear. Herein we investigate this relation by analyzing saliva samples from 35 adult patients with obesity undergoing bariatric surgery. Our aim was to describe salivary microbiome changes during body weight loss on an individual-specific level, and to elucidate the effect of bariatric surgery on the salivary microbiome which has not been studied before. Analysis of samples collected before and 1 day after surgery, as well as 3 and 12 months after surgery, showed that the salivary microbiome changed in all study participants, but these changes were heterogeneous. In the majority of participants proportions of Gemella species, Granulicatella elegans, Porphyromonas pasteri, Prevotella nanceiensis and Streptococcus oralis decreased, while Veillonella species, Megasphaera micronuciformis and Prevotella saliva increased. Nevertheless, we found participants deviating from this general trend which suggests that a variety of individual-specific factors influence the salivary microbiome composition more effectively than the body weight dynamics alone. The observed microbiome alternations could be related to dietary changes. Therefore, further studies should focus on association with altered taste preferences and potential oral health consequences.

Phages and Their Role in Gastrointestinal Disease: Focus on Inflammatory Bowel Disease.

Inflammatory bowel diseases (IBDs) are a group of chronic autoinflammatory diseases including Crohn's disease and ulcerative colitis. Although the molecular mechanisms governing the pathogenesis of gastrointestinal inflammation are not completely clear, the main factors are presumed to be genetic predisposition, environmental exposure, and the intestinal microbiome. Hitherto, most of the studies focusing on the role of the microbiome studied the action and effect of bacteria. However, the intestinal microbiome comprises other members of the microbial community as well, namely, fungi, protozoa, and viruses. We believe that bacteriophages are among the main orchestrators of the effect of microbiota on the gut mucosa. Therefore, this review aims to summarize the knowledge of the role of intestinal phageome in IBD and to discuss the concept of phage therapy and its future applications.

Cell-Free Nucleic Acids and their Emerging Role in the Pathogenesis and Clinical Management of Inflammatory Bowel Disease.

Cell-free nucleic acids (cfNAs) are defined as any nucleic acids that are present outside the cell. They represent valuable biomarkers in various diagnostic protocols such as prenatal diagnostics, the detection of cancer, and cardiovascular or autoimmune diseases. However, in the current literature, little is known about their implication in inflammatory bowel disease (IBD). IBD is a group of multifactorial, autoimmune, and debilitating diseases with increasing incidence worldwide. Despite extensive research, their etiology and exact pathogenesis is still unclear. Since cfNAs were observed in other autoimmune diseases and appear to be relevant in inflammatory processes, their role in the pathogenesis of IBD has also been suggested. This review provides a summary of knowledge from the available literature about cfDNA and cfRNA and the structures involving them such as exosomes and neutrophil extracellular traps and their association with IBD. Current studies showed the promise of cfNAs in the management of IBD not only as biomarkers distinguishing patients from healthy people and differentiating active from inactive disease state, but also as a potential therapeutic target. However, the detailed biological characteristics of cfNAs need to be fully elucidated in future experimental and clinical studies.

Beneficial effect of sugar-sweetened beverages on the risk of urinary tract infections.

Urinary tract infections (UTI) are among the most common bacterial infections. Drinking more liquids increases the frequency of urination and it is recommended as part of the prevention and/or management of UTI. The intake of sugar-sweetened beverages (SSB) is associated with obesity, diabetes and metabolic syndrome. However, cola and other SSB increase liquid intake and diuresis and could, thus, affect the risk of UTI and its complications. We hypothesize that intake of cola has a protective effect on UTI and pyelonephritis. Using an animal model of UTI, we have confirmed that dehydration with minimal urine output leads to higher bacterial counts in the kidneys in comparison to control mice (p = 0.01). The intake of SSB increased liquid intake and thus also diuresis and decreased renal bacterial counts as a marker of induced pyelonephritis (p = 0.036). The preliminary results show that dehydration is a risk factor for UTI and that higher diuresis induced by drinking SSB might be protective against pyelonephritis. The underlying mechanisms could include increased voiding frequency but potentially also active compounds in cola such as caffeine. These findings might have implications for the management of individuals at high risk of UTI. Further studies should verify the hypothesis and evaluate the practical relevance of this concept.

The effects of sucrose on urine collection in metabolic cages.

Representative urine collection that respects the standards of animal welfare is still an issue in experimental nephrology. The commonly used metabolic cages induce stress in rodents. In mice, the volume of collected urine is sometimes insufficient for further analysis. The aim of this experiment was to analyse the effects of time of day, temperature and 2%, 5% or 10% sucrose solutions on diuresis, weight change and liquid intake of adult mice placed in metabolic cages for urine collection. Mice were placed in metabolic cages for 12 h during the day or night at standard ambient (22℃) and thermoneutral (28℃) temperatures. To determine the effect of acclimatisation, mice were placed in metabolic cages for five consecutive days. Diuresis increased with concentrations of sucrose. Body weight reduction was most rapid in the group given tap water and decreased with increasing sucrose concentrations. A drastic drop in body weight was observed in mice placed in metabolic cages for four consecutive days with access to tap water and food, indicating that time spent in metabolic cages should be kept to a minimum, as prolonged confinement in metabolic cages can be harmful to mice. The administration of concentrated sucrose solutions can potentially aid in mouse urine collection by reducing the time spent in metabolic cages. Sucrose supplementation increased the albumin/creatinine ratio. However, without showing estimates of glomerular filtration rate, renal haemodynamics, plasma electrolytes and urinary electrolyte excretions, the results of this study do not provide any conclusion about the effect of sucrose on renal function.

Reverse phenotype transfer via fecal microbial transplantation in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by a disbalance in the composition of intestinal microbiota. It is not clear whether such dysbiosis is a cause or a consequence of a disease state. Fecal microbiota transplantation (FMT) from a healthy donor to a patient or diseased animal is a valuable tool for targeted modification of microbiome leading to therapeutic response. Positive effect has been shown in therapy of a number of gastrointestinal as well as non-gastrointestinal diseases. In addition, FMT has been successfully used to transfer the diseased phenotype form a donor with the disease to a healthy recipient. However, targeted modification of the microbiome before the onset of colitis has not been shown previously. Based on our preliminary results, we propose the hypothesis of so called reverse phenotype transfer in IBD. This term describes the phenomenon, in which the transplantation of gut microbiota from a donor more sensitive to IBD to a healthy recipient leads to resistance of the recipient to IBD and vice versa. Mice that received FMT from donors with severe colitis have shown improved colitis score compared with mice that received FMT from donors more resistant to development of colitis. Such reverse phenotype transfer has broad implications, especially in terms of preventive medicine. However, detailed mechanisms need to be elucidated to conclude the validity of the phenomenon.

Oxidative stress in the oral cavity is driven by individual-specific bacterial communities.

The term "bacterial dysbiosis" is being used quite extensively in metagenomic studies, however, the identification of harmful bacteria often fails due to large overlap between the bacterial species found in healthy volunteers and patients. We hypothesized that the pathogenic oral bacteria are individual-specific and they correlate with oxidative stress markers in saliva which reflect the inflammatory processes in the oral cavity. Temporally direct and lagged correlations between the markers and bacterial taxa were computed individually for 26 volunteers who provided saliva samples during one month (21.2 ± 2.7 samples/volunteer, 551 samples in total). The volunteers' microbiomes differed significantly by their composition and also by their degree of microbiome temporal variability and oxidative stress markers fluctuation. The results showed that each of the marker-taxa pairs can have negative correlations in some volunteers while positive in others. Streptococcus mutans, which used to be associated with caries before the metagenomics era, had the most prominent correlations with the oxidative stress markers, however, these correlations were not confirmed in all volunteers. The importance of longitudinal samples collections in correlation studies was underlined by simulation of single sample collections in 1000 different combinations which produced contradictory results. In conclusion, the distinct intra-individual correlation patterns suggest that different bacterial consortia might be involved in the oxidative stress induction in each human subject. In the future, decreasing cost of DNA sequencing will allow to analyze multiple samples from each patient, which might help to explore potential diagnostic applications and understand pathogenesis of microbiome-associated oral diseases.

Association between oxidative status and the composition of intestinal microbiota along the gastrointestinal tract.

Studies have shown that the microbiota along the gastrointestinal tract (GIT) plays an important role when it comes to the maintenance of its proper functions. Many studies exist that have analyzed the composition of the bacterial community in the different regions of the GIT of humans and model animals. Microbial imbalance leads to several systemic disorders, including cardiovascular and renal disease. The imbalance between the production of reactive oxygen species (ROS) and their elimination by antioxidants leads to oxidative stress. Oxidative stress plays an important role in a variety of physiological processes, as well as disease. The continuous formation of ROS in the GIT is the result of the interaction between intestinal mucosa, symbiotic bacteria and dietary factors. It has also been proven that ROS play a role in the pathogenesis of several GI disorders, including IBD. We hypothesized that the levels of advanced glycation end products (AGEs) would be the highest in the ileum, caecum or colon, where the microbiota mostly consist of butyrate producing bacteria, Bacterioides, Clostridium, Ruminococcus or Bifidobacterium, which derive energy through carbohydrate fermentation. We also assumed that advanced oxidation protein products (AOPP) mostly act in the segments, where bacteria reside and which are responsible for the amino acid fermentation, such as caecum or colon. Lipid hydroxyperoxides are generated during digestion in the stomach, which contains absorbed oxygen and has a low pH. According to this we hypothesized that the highest concentration of thiobarbituric acid reacting substances (TBARS) could be in the stomach, which, however, has not been confirmed. Because Lactobacilli are able to produce catalase, an endogenous antioxidant, and are abundant in the small intestine, we hypothesized that antioxidant capacity (measured by ferric reducing ability) would be the highest here. The highest levels of AGEs were found in the caecum. The highest level of TBARS was found in the jejunum of the rats. The assessment of our hypothesis also revealed high levels of AOPP in the caecum. It has been shown that AOPP contributes to the progression of IBD. The ferric reducing ability of tissue was the lowest in the colon of the experimental animals, which is in accordance with previous studies that show that rat colon has a lower total antioxidant capacity than the small bowel. In summary, we offer some insight into the differences between the oxidative status along the GIT of rats and some advice concerning supportive antioxidant therapy of gastrointestinal diseases.

Sex Differences in the Effect of Resveratrol on DSS-Induced Colitis in Mice.

Resveratrol is a natural polyphenol studied for its possible protective properties in inflammatory bowel diseases. Moreover, it has been shown to interact with estrogen receptors. In the present study, we aimed to investigate possible diverse effects of resveratrol on female and male mice in DSS-induced colitis. Thirty-seven C57BL/6 mice (21 female and 16 male) were divided into three groups for each sex. The first group received pure water (CTRL). The other two groups received 1.5% dextran sulfate sodium (DSS) to induce colitis from which one group was treated with resveratrol (DSS + RSV). Intake of 1.5% DSS caused weight loss in all DSS groups compared to control mice. Weight loss, stool consistency, and discomfort did not show any protective effect of resveratrol in males and showed even adverse effects in females. In females, the activity of myeloperoxidase was lower compared to that in males. However, colon length and spleen weight showed no sex differences, which can indicate the induction of only mild colitis in mice. Resveratrol did not have any effect on TNF-alpha levels. Taken together, these results for the first time propose possible diverse effects of resveratrol in DSS-induced colitis model depending on the sex of the animal. However, this conclusion must be confirmed by further analyses.