Interactions between the intestinal microbiota and epigenome in individuals with autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by variable impairment of cognitive function and interpersonal relationships. Furthermore, some individuals with ASD have gastrointestinal disorders that have been correlated with impairments in intestinal microbiota. Gut microbiota are important not only for intestinal health, but also for many other functions including food digestion, energy production, immune system regulation, and, according to current data, behavior. Disruption of the indigenous microbiota, microbial dysbiosis (imbalance between microorganisms present in the gut), overgrowth of potentially pathogenic microorganisms, a less diverse microbiome, or lower levels of beneficial bacteria in children with ASD can affect behavior. Metabolome analysis in children with ASD has identified perturbations in multiple metabolic pathways that might be associated with cognitive functions. Recent studies have shown that the intestinal microbiome provides environmental signals that can modify host response to stimuli by modifying the host epigenome, which affects DNA methylation, histone modification, and non-coding RNAs. The most studied microbiota-produced epigenetic modifiers are short-chain fatty acids, although other products of intestinal microbiota might also cause epigenetic modifications in the host's DNA. Here we review evidence suggesting that epigenetic alterations caused by modification of gene expression play an important role in understanding ASD.

Analysis of the Duodenal Microbiome in Autistic Individuals: Association With Carbohydrate Digestion.

OBJECTIVES: There is evidence that symptoms of maldigestion or malabsorption in autistic individuals are related to changes in the indigenous microbiota. Analysis of colonic bacteria has revealed microbial dysbiosis in children with autism; however, characteristics of the duodenal microbiome are not well described. In the present study the microbiome of the duodenal mucosa of subjects with autism was evaluated for dysbiosis, bacteria overgrowth, and microbiota associated with carbohydrate digestion. The relationship between the duodenal microbiome and disaccharidase activity was analyzed in biopsies from 21 autistic subjects and 19 children without autism. METHODS: Microbiota composition was determined by 16S ribosomal RNA gene sequencing, and disaccharidase activity via biochemical assays. RESULTS: Although subjects with autism had a higher frequency of constipation (P < 0.005), there was no difference in disaccharidase activity between groups. In addition, no differences in microbiome diversity (species richness and evenness) were observed. Bacteria belonging to the genus Burkholderia were more abundant in subjects with autism, whereas members of the genus Neisseria were less abundant. At the species level, a relative decrease in abundance of 2 Bacteroides species and Escherichia coli was found in autistic individuals. There was a positive correlation between the abundance of Clostridium species, and disaccharidase activity, in autistic individuals. CONCLUSIONS: There are a variety of changes at the genus and species level in duodenal microbiota in children with autism that could be influenced by carbohydrate malabsorption. These observations could be affected by variations in individual diets, but also may represent a more pervasive dysbiosis that results in metabolites that affect the behavior of autistic children.

Human mannose-binding lectin inhibitor prevents Shiga toxin-induced renal injury.

Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in STEC HUS. Using novel human MBL2-expressing mice (MBL2 KI) that lack murine Mbls (MBL2(+/+)Mbl1(-/-)Mbl2(-/-)), a novel STEC HUS model consisted of an intraperitoneal injection with Shiga toxin-2 (Stx-2) with or without anti-MBL2 antibody (3F8, intraperitoneal). Stx-2 induced weight loss, anemia, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, but a significantly decreased glomerular filtration rate compared with control/sham mice. Immunohistochemical staining revealed renal C3d deposition and fibrin deposition in glomeruli in Stx-2-injected mice. Treatment with 3F8 completely inhibited serum MBL2 levels and significantly attenuated Stx-2 induced-renal injury, free serum hemoglobin levels, renal C3d, and fibrin deposition and preserved the glomerular filtration rate. Thus, MBL2 inhibition significantly protected against complement activation and renal injury induced by Stx-2. This novel mouse model can be used to study the role of complement, particularly lectin pathway-mediated complement activation, in Stx-2-induced renal injury.

Evaluation of Intestinal Function in Children With Autism and Gastrointestinal Symptoms.

OBJECTIVE: Alterations in intestinal function, often characterized as a "leaky gut," have been attributed to children who are on the autism spectrum. Disaccharidase activity, intestinal inflammation, and permeability were analyzed in 61 children with autism and 50 nonautistic individuals with gastrointestinal symptoms. METHODS: All patients had duodenal biopsies assayed for lactase, sucrase, maltase, and palatinase activity. Intestinal permeability was evaluated by rhamnose/lactulose test and measured by high-performance liquid chromatography-mass spectrometry. Intestinal inflammation was evaluated by fecal calprotectin and lactoferrin levels using enzyme-linked immunosorbent assay and histology. RESULTS: Some children with autism had mild levels of mucosal inflammation on intestinal biopsy. Disaccharidase activity was not different in autistic and nonautistic individuals. Fecal calprotectin and lactoferrin were similar in both groups. Differences between lactulose and rhamnose recovery and lactulose/rhamnose ratio in urine were not statistically different in patients with and without autism. CONCLUSIONS: The present study supports the observation that children with autism who have symptoms of gastrointestinal disorders have objective findings similar to children without autism. Neither noninvasive testing nor endoscopic findings identify gastrointestinal pathology specific to autism, but may be of benefit in identifying children with autism who have atypical symptoms.

Gut Microbiota and Gender in Autism Spectrum Disorders.

Gender dimorphism in autism spectrum disorders (ASD) is well known; however, the reasons for gender differences in autism are poorly understood. There are several hypotheses that might explain male prevalence in ASD, including increased levels of androgens, "extreme male brain," and a combination of elevated levels of prenatal testosterone in conjunction with prenatal stress. In this comprehensive review, differences in the gut microbiome and metabolome in humans and animals are described to explain gender differences in individuals with ASD, effects on behavior and social interactions and the impact of antibiotics, probiotics and fecal transplants. The bidirectional relationship between sex hormones and intestinal microbiota could also be relevant. Such interactions have been described in autoimmune diseases, but thus far, are not implicated in ASD. Since intestinal microbiota may affect behavior, it is possible that the prevalence of ASD in boys may be associated with more significant changes in the intestinal microbiome than in affected girls.

Detached endothelial cells and microparticles as sources of tissue factor activity.

INTRODUCTION: Cytokine activation of endothelial cell monolayers is associated with cell detachment, microparticle shedding from plasma membranes, and phosphatidylserine appearance in the plasma membrane outer leaflets. While tissue factor expression on activated endothelial cells and microparticles is well documented, the contribution of detached endothelial cells to tissue factor activity is less clear. We studied tissue factor expression and the role of tissue factor pathway inhibitor on adherent and detached endothelial cells and on microparticles following endothelial cell activation with TNF-alpha. MATERIALS AND METHODS: Detached endothelial cells and microparticles were obtained from cultures of human umbilical vein endothelial cells by differential centrifugation of cell culture supernatant. For microparticle capture, an antibody directed against CD146 was used. Functional tissue factor activity was measured by chromogenic assay and tissue factor antigen by ELISA. Endothelial cell and microparticle morphology was examined by light and transmission electron microscopy. RESULTS: After cell activation for 22 h, functional tissue factor activity was distributed as follows: 60%, adherent endothelial cells; 35%, detached cells; and 5%, microparticles. Tissue factor protein followed a similar distribution. Cell detachment was 47%. Electron microscopy demonstrated shedding of microparticles with a diameter of 0.1-0.6 mum. Cy3-annexin V revealed increased phosphatidylserine on activated adherent endothelial cells and microparticles. Pre-incubation of adherent and detached endothelial cells and microparticles with anti-tissue factor antibody blocked factor Xa production. Pre-incubation with anti-tissue factor pathway inhibitor antibody increased tissue factor activity of adherent endothelial cells 2.8-fold, detached cells 1.4-fold, and microparticles 45-fold. CONCLUSIONS: Detached endothelial cells as well as microparticles from activated endothelial cell monolayers express tissue factor activity, and this activity on microparticles is markedly inhibited by microparticle-associated tissue factor pathway inhibitor.

Shiga toxin downregulates tissue factor pathway inhibitor, modulating an increase in the expression of functional tissue factor on endothelium.

INTRODUCTION: Endothelial expression of tissue factor (TF) may play a major role in (Stx)-related hemolytic uremic syndrome. We examined human umbilical vein endothelial cell (HUVEC) monolayers to determine the interaction between TF and TF pathway inhibitor (TFPI), hypothesizing that changes in TFPI modulate TF expression. MATERIALS AND METHODS: We studied 1) cell surface expression of globotriasylceramide (Gb3, the receptor for Stx) with Stx-1 (10 pM), TNFα (20 Ng/ml), or Stx-1 plus TNFα compared to control, 2) gene expression of TF and TFPI, 3) total cellular and cell surface antigenic TF and TFPI, 4) TFPI secretion into supernatant, and 5) factor Xa production. RESULTS AND CONCLUSIONS: Gb3 expression, negligible with control and Stx-1 alone, increased significantly with TNFα and with Stx-1 plus TNFα. TF mRNA increased 1.25 ± 0.32- fold (N = 9; p = 0.041) with Stx-1 alone vs. 2.82 ± 0.92-fold (N = 13; p < 0.0005) with TNFα alone. However, Stx-1 plus TNFα yielded a 6.51 ± 3.48-fold increase (N = 17; p < 0.0005). TFPI mRNA decreased with TNFα (p < 0.001) and Stx-1 plus TNFα (p < 0.0005). Total cellular and cell surface TF antigen increased significantly with TNFα, but no further with Stx-1 plus TNFα. Total TFPI cellular and cell surface antigen levels, and TFPI secretion decreased significantly with Stx-1 plus TNFα. Median factor Xa production for Stx-1 plus TNFα vs TNFα alone increased (p < 0.001) 3.24-fold. Our results indicate that a subinhibitory concentration of Stx-1 plus TNFα impairs TFPI gene expression, synthesis, cell-surface association, and secretion, leading to augmented functional TF.

Intestinal disaccharidase activity in patients with autism: effect of age, gender, and intestinal inflammation.

Intestinal disaccharidase activities were measured in 199 individuals with autism to determine the frequency of enzyme deficiency. All patients had duodenal biopsies that were evaluated morphologically and assayed for lactase, sucrase, and maltase activity. Frequency of lactase deficiency was 58% in autistic children ≤ 5 years old and 65% in older patients. As would be expected, patients with autism at age 5 > years demonstrated significant decline in lactase activity (24%, p = .02) in comparison with ≤ 5 years old autistic patients. Boys ≤ 5 years old with autism had 1.7 fold lower lactase activity than girls with autism (p = .02). Only 6% of autistic patients had intestinal inflammation. Lactase deficiency not associated with intestinal inflammation or injury is common in autistic children and may contribute to abdominal discomfort, pain and observed aberrant behavior. Most autistic children with lactose intolerance are not identified by clinical history.

Role of the renin angiotensin system in TNF-alpha and Shiga-toxin-induced tissue factor expression.

Current evidence implicates a prothrombotic state in the development of Shiga-toxin (Stx)-mediated hemolytic uremic syndrome (HUS). We recently reported that Stx modulates procoagulant activity by enhancing functional tissue factor (TF) activity on cytokine-activated human glomerular endothelial cells (HGECs). Since angiotensin II (Ang II), the key effector of the renin angiotensin system (RAS), has been shown to increase TF expression in vascular tissue, we examined the possible involvement of Ang II in TF expression in HGECs. HGECs were exposed to tumor necrosis factor (TNF)-alpha +/- Stx-1 +/- Ang II. Exogenous Ang II significantly increased TF activity and TF mRNA in TNF-alpha- +/- Stx-1-activated HGECs. This increase was mediated via Ang II type I receptor (AT(1)R), as losartan, an AT(1)R inhibitor, attenuated Ang-II-induced TF activity. To study the effect of endogenous Ang II in TF expression by TNF-alpha +/- Stx-1, HGECs were incubated with losartan or an AT(2)R inhibitor (PD 123319) or an angiotensin-converting enzyme inhibitor (enalapril). Losartan but not PD 123319 decreased TF activity induced by TNF-alpha +/- Stx-1 (P < 0.05). Enalapril, also, dose dependently, downregulated TF expression in HGECs exposed to TNF-alpha +/- Stx-1 (P < 0.05). AT(1)R mRNA was upregulated in TNF-alpha- +/- Stx-1-activated HGECs (P < 0.05). These data indicate that TF expression in TNF-alpha- and Stx-1-activated HGECs is enhanced by exogenous Ang II and that endogenous Ang II production may be upregulated by TNF-alpha +/- Stx-1. Hence, local RAS activation may be important in the development of the thrombotic microangiopathy observed in HUS.

Up-regulation of tissue factor activity on human proximal tubular epithelial cells in response to Shiga toxin.

BACKGROUND: The pathophysiology of hemolytic uremic syndrome (HUS) is incompletely established. Based on clinical studies demonstrating the presence of prothrombotic plasma markers in patients with HUS, we hypothesized that Shiga toxin might cause activation of the coagulation pathway by augmenting tissue factor, the major initiator of coagulation. METHODS: Human proximal tubular epithelial cells (PTECs) [human kidney-2 (HK-2 cells)] were exposed to Shiga toxin-1, and expression of tissue factor, cell detachment, protein synthesis, caspase-3 activity, and Shiga toxin-1 binding were examined. Results. HK-2 cells expressed constitutive surface tissue factor activity and increased their tissue factor expression upon exposure to Shiga toxin-1. Shiga toxin-1 bound to HK-2 cells and inhibited protein synthesis. The up-regulation of tissue factor was dose- and time-dependent and strongly correlated with cell detachment and increase in caspase-3 activity caused by Shiga toxin-1 exposure. A general caspase inhibitor simultaneously inhibited HK-2 cell detachment and tissue factor up-regulation while mutant Shiga toxin-1 neither caused cell detachment, protein synthesis inhibition, nor increase in tissue factor activity. Tissue factor activity elicited by Shiga toxin-1 was abrogated by a monoclonal antitissue factor antibody. Calphostin C, a protein kinase C (PKC) inhibitor, partially blocked tissue factor up-regulation, indicating possible involvement of PKC-dependent mechanism. CONCLUSION: These data, taken together, suggest a strong link between Shiga toxin-induced up-regulation of tissue factor activity, cytotoxicity, and apoptosis in HK-2 cells. The proximal tubule is a target of Shiga toxin in HUS, and it seems plausible that injured proximal tubular cells trigger the activation of the coagulation system, the formation of intrarenal platelet-fibrin thrombi, and the development of acute renal failure in HUS.

Pancreatic and intestinal enzyme activities in rats in response to balanced and unbalanced plant diets.

To simulate the effects of nutritionally adequate and inadequate vegetarian diets, rats were fed, for 28 days, an isonitrogenous, isocaloric, amino acid unbalanced cereal diet (CD) deficient in lysine and tryptophan or a balanced cereal-legume diet (CLD). The impact of these diets on enzymes responsible for digestion of proteins and carbohydrates were measured. Neither experimental diet significantly affected the animal's final weight or feed consumption in comparison with controls fed a standard mixed diet from plant and animal sources. However, during the first three weeks, the weight gain of rats fed the CD was significantly lower (p < 0.01; p < 0.05) than that of the controls. CD fed rats also had a higher feed efficiency ratio (p < 0.05), demonstrating increased feed consumption per unit of body weight. They also had decreased pancreatic alpha-amylase activity (p < 0.05), serum phytolytic and zoolytic alpha-amylase activity (p < 0.05) and serum protein level (p < 0.05) than the controls. Activity of pancreatic trypsin and intestinal enzymes (sucrase, maltase, aminopeptidase N) were the same as in the controls. In rats fed CLD, growth, food consumption, and enzyme activities did not change, however serum protein and glucose levels were higher (p < 0.025; p < 0.005) than in the controls. It is hypothesized that decrease in alpha-amylase activity was mostly related to the tryptophan deficiency in the CD because this enzyme contains the highest amount of tryptophan units among all tested enzymes.