Gloria Serena

Proof of Concept of Microbiome-Metabolome Analysis and Delayed Gluten Exposure on Celiac Disease Autoimmunity in Genetically At-Risk Infants

Celiac disease (CD) is a unique autoimmune disorder in which the genetic factors (DQ2/DQ8) and the environmental trigger (gluten) are known and necessary but not sufficient for its development. Other environmental components contributing to CD are poorly understood. Studies suggest that aspects of gluten intake might influence the risk of CD occurrence and timing of its onset, i.e., the amount and quality of ingested gluten, together with the pattern of infant feeding and the age at which gluten is introduced in the diet. In this study, we hypothesize that the intestinal microbiota as a whole rather than specific infections dictates the switch from tolerance to immune response in genetically susceptible individuals. Using a sample of infants genetically at risk of CD, we characterized the longitudinal changes in the microbial communities that colonize infants from birth to 24 months and the impact of two patterns of gluten introduction (early vs. late) on the gut microbiota and metabolome, and the switch from gluten tolerance to immune response, including onset of CD autoimmunity. We show that infants genetically susceptible to CD who are exposed to gluten early mount an immune response against gluten and develop CD autoimmunity more frequently than at-risk infants in which gluten exposure is delayed until 12 months of age. The data, while derived from a relatively small number of subjects, suggest differences between the developing microbiota of infants with genetic predisposition for CD and the microbiota from infants with a non-selected genetic background, with an overall lack of bacteria of the phylum Bacteriodetes along with a high abundance of Firmicutes and microbiota that do not resemble that of adults even at 2 years of age. Furthermore, metabolomics analysis reveals potential biomarkers for the prediction of CD. This study constitutes a definite proof-of-principle that these combined genomic and metabolomic approaches will be key to deciphering the role of the gut microbiota on CD onset.

The Role of Gluten in Celiac Disease and Type 1 Diabetes

Celiac disease (CD) and type 1 diabetes (T1D) are autoimmune conditions in which dietary gluten has been proven or suggested to play a pathogenic role. In CD; gluten is established as the instigator of autoimmunity; the autoimmune process is halted by removing gluten from the diet; which allows for resolution of celiac autoimmune enteropathy and subsequent normalization of serological markers of the disease. However; an analogous causative agent has not yet been identified for T1D. Nevertheless; the role of dietary gluten in development of T1D and the potentially beneficial effect of removing gluten from the diet of patients with T1D are still debated. In this review; we discuss the comorbid occurrence of CD and T1D and explore current evidences for the specific role of gluten in both conditions; specifically focusing on current evidence on the effect of gluten on the immune system and the gut microbiota.

Genetics and celiac disease: the importance of screening

The prevalence of celiac disease (CD) is increasing. Despite an increased awareness and an improvement in diagnostic testing, the majority of individuals with CD remain undiagnosed. Currently, genetic testing in screening for CD is used only to exclude a diagnosis or reinforce a strong clinical suspicion. In this paper, we review the most current literature regarding genetic testing in CD. In response to important data revealing that an individual’s HLA haplotype is one of the strongest known predictors of CD, we propose genetic screening for at-risk infants to stratify individuals based on genetic risk to ultimately create genetic specific screening algorithms.

Proinflammatory cytokine interferon‐γ and microbiome‐derived metabolites dictate epigenetic switch between forkhead box protein 3 isoforms in coeliac disease

Coeliac disease (CD) is an autoimmune enteropathy triggered by gluten and characterized by a strong T helper type 1 (Th1)/Th17 immune response in the small intestine. Regulatory T cells (Treg) are CD4+CD25++forkhead box protein 3 (FoxP3+) cells that regulate the immune response. Conversely to its counterpart, FoxP3 full length (FL), the alternatively spliced isoform FoxP3 Δ2, cannot properly down‐regulate the Th17‐driven immune response. As the active state of CD has been associated with impairments in Treg cell function, we aimed at determining whether imbalances between FoxP3 isoforms may be associated with the disease. Intestinal biopsies from patients with active CD showed increased expression of FOXP3 Δ2 isoform over FL, while both isoforms were expressed similarly in non‐coeliac control subjects (HC). Conversely to what we saw in the intestine, peripheral blood mononuclear cells (PBMC) from HC subjects did not show the same balance between isoforms. We therefore hypothesized that the intestinal microenvironment may play a role in modulating alternative splicing. The proinflammatory intestinal microenvironment of active patients has been reported to be enriched in butyrate‐producing bacteria, while high concentrations of lactate have been shown to characterize the preclinical stage of the disease. We show that the combination of interferon (IFN)‐γ and butyrate triggers the balance between FoxP3 isoforms in HC subjects, while the same does not occur in CD patients. Furthermore, we report that lactate increases both isoforms in CD patients. Collectively, these findings highlight the importance of the ratio between FoxP3 isoforms in CD and, for the first time, associate the alternative splicing process mechanistically with microbial‐derived metabolites.

Differential immune responses and microbiota profiles in children with autism spectrum disorders and co-morbid gastrointestinal symptoms

OBJECTIVES Many studies have reported the increased presence of gastrointestinal (GI) symptoms in children with autism spectrum disorders (ASD). Altered microbiome profiles, pro-inflammatory responses and impaired intestinal permeability have been observed in children with ASD and co-morbid GI symptoms, yet few studies have compared these findings to ASD children without GI issues or similarly aged typical developing children. The aim of this study was to determine whether there are biological signatures in terms of immune dysfunction and microbiota composition in children with ASD with GI symptoms. METHODS Children were enrolled in one of four groups: ASD and GI symptoms of irregular bowel habits (ASDGI), children with ASD but without current or previous GI symptoms (ASDNoGI), typically developing children with GI symptoms (TDGI) and typically developing children without current or previous GI symptoms (TDNoGI). Peripheral blood mononuclear cells (PBMC) were isolated from the blood, stimulated and assessed for cytokine production, while stool samples were analyzed for microbial composition. RESULTS Following Toll-Like receptor (TLR)-4 stimulation, the ASDGI group produced increased levels of mucosa-relevant cytokines including IL-5, IL-15 and IL-17 compared to ASDNoGI. The production of the regulatory cytokine TGFβ1 was decreased in the ASDGI group compared with both the ASDNoGI and TDNoGI groups. Analysis of the microbiome at the family level revealed differences in microbiome composition between ASD and TD children with GI symptoms; furthermore, a predictive metagenome functional content analysis revealed that pathways were differentially represented between ASD and TD subjects, independently of the presence of GI symptoms. The ASDGI also showed an over-representation of the gene encoding zonulin, a molecule regulating gut permeability, compared to the other groups. CONCLUSIONS Overall our findings suggest that children with ASD who experience GI symptoms have an imbalance in their immune response, possibly influenced by or influencing metagenomic changes, and may have a propensity to impaired gut barrier function which may contribute to their symptoms and clinical outcome.

[Design of a genomic, environmental, microbial and metabolomic study on celiac disease: an approach to the future of personalized prevention of celiac disease].

Over recent years we have seen rising many clinical and scientific innovations about celiac disease (CE), however the most important innovation that will contribute to change the future of the research and clinic in this field is the natural history of the disease. For many years it has been though that a genetic predisposition and the exposure to gluten were necessary and sufficient to develop CE. Recent studies, however, suggest that the loss of tolerance to gluten may occur in any moment of life upon certain conditions. Furthermore, several environmental factors known to play a role in shaping the intestinal microflora have also been considered related to the development of CE. Delivery mode, the infant diet and the use of antibiotics are included among these factors. To this day no large scale studies have determined if and how the microbiome composition and its metabolomic profile may influence the loss of tolerance to gluten and the consequent development of CE. In this paper we describe a prospective, multi-centric and longitudinal study on infants at risk for CE that will use different techniques to better understand the role of the microbome during the first steps in the development of the autoimmune disease.Over recent years we have seen rising many clinical and scientific innovations about celiac disease (CE), however the most important innovation that will contribute to change the future of the research and clinic in this field is the natural history of the disease. For many years it has been though that a genetic predisposition and the exposure to gluten were necessary and sufficient to develop CE. Recent studies, however, suggest that the loss of tolerance to gluten may occur in any moment of life upon certain conditions. Furthermore, several environmental factors known to play a role in shaping the intestinal microflora have also been considered related to the development of CE. Delivery mode, the infant diet and the use of antibiotics are included among these factors. To this day no large scale studies have determined if and how the microbiome composition and its metabolomic profile may influence the loss of tolerance to gluten and the consequent development of CE. In this paper we describe a prospective, multi-centric and longitudinal study on infants at risk for CE that will use different techniques to better understand the role of the microbome during the first steps in the development of the autoimmune disease.

167 The Microbiome Signature of Autistic Children Is Characterized by Decreased Diversity and Dysbiosis

Background: Autism Spectrum Disorders (ASDs) are complex neuro-developmental disorders characterized by cognitive defects, social interaction skills impairments and communication, language and behavioral problems. A strong correlation between autism severity and GI symptoms, alterations of intestinal permeability (IP), presence of inflammation and intestinal dysbiosis has been described. It has recently been proposed that alterations of the intestinal microbiota could contribute to the development of ASDs. Recent studies demonstrated that autistic children with gastrointestinal symptoms show major fecal microbiota alterations. In this study we aimed to study the fecal candida and bacterial microbiota, their correlation to intestinal permeability, in autistic children compared to normal developing children, in order to provide rational basis to a possible specific therapeutic intervention in restoring gut microflora in ASDs. Material and Methods: We collected stool samples from 47 subjects with ASDs (40 boys and 7 girls, mean age 6.0 ± 2.8 yr), and 33 healthy children matched controls (24 boys and 9 girls, mean age 7.3 ± 3.1 yr). Search for Candida culture and biochemical identification of the colonies were performed by Sabouraud Dextrose Agar + Chloramphenicol + Gentamicin, Oxoid Products Microbiology and analized by ID 32 C (bioMerieux) system. In a subgroup of 12 ASDs subjects (8 boys and 4 girls, mean age 6.8 ± 2.9 ), and 16 healthy children matched controls (8 boys and 8 girls, mean age 7.3 ± 3.1 yr), total microbial DNA was extracted and 16S variable region V4 amplified by PCR. The results were analyzed by QIIME software. Intestinal Permeability (IP) was assessed with the lactulose/mannitol (LA/MA) test. Results: Compared to HC, ASDs samples presented an increased number of Proteobacteria combined with a reduction of Bacteroidetes and Actinobacteria. Microbial diversity was reduced in ASDs children, albeit they were more phylogenetically different. Interestingly, β-diversity analysis showed 2 distinct clouds in ASDs children, one dominated by E. Coli and the other with no clear domination. Candida spp was detected in the stools of 15 ASD children (31.9%) compared to controls (6.1%). Interestingly in ASDs, C. albicans was most frequently identified (9 out of 15 cases). Finally, IP resulted altered in 42.4% of ASDs compared to 22.7% of controls (p < 0.05). No correlation was detected between altered IP and either microbiome composition or candidosis. Conclusions: In this proof-of-concept study we establish that ASD children have a specific microbiome signature characterized by decreased diversity and quantitative representation of some specific phyla. Candida infestation, particularly C. albicans was more frequently detected in ASD children than controls.

251 CXCR3 Splice Variants in Celiac Disease and Non-Celiac Gluten Sensitivity

Intestinal villi provide an enormous surface area for nutrient absorption. Significant loss of intestinal surface area can compromise intestinal function, causing intestinal failure. Though short-term treatments for this life-threatening condition are available, all patients need lifelong monitoring for growth and nutritional status, and would benefit from treatments that can directly increase intestinal surface area. In mice, a large increase in surface area occurs with villus development, which begins at embryonic day (E)14.5, when the thick pseudostratified epithelium with a flat luminal surface is converted to a columnar epithelium covering a field of emerging villi. Though it has long been thought that epithelial remodeling occurs by formation and fusion of secondary lumina, recent work in our laboratory showed that secondary lumina do not exist (Grosse et al., Development 138:4423, 2011). Seeking an alternative mechanism for luminal expansion, we found a unique type of cell division that is triggered specifically at E14.5, which we have named an e-division (lumen extending division). We propose that in an e-division, new apical surface is deposited at the cytokinetic plane such that the two daughter cells segregate onto adjacent villi. The e-division is distinct from cell divisions occurring before E14.5, which we have named g-divisions (girth building divisions); g-divisions do not involve deposition of new apical surface between daughter cells. Our data (lineage tracing, 3D reconstruction, and SEM) suggest that in mice deficient in the apical surface protein Ezrin, e-divisions fail stochastically, resulting in fused villi. We are modeling e-divisions in vitro using MDCK and Caco2 cell lines, which form luminal surfaces during the first cell division when plated in a 3D matrix. Using RNA interference, we have found that reducing Ezrin expression compromises lumen formation in our 3D cyst assay, providing a mechanistic explanation for the presence of fused villi in vivo. Further understanding of the process of villus development will improve in vitro bioengineering of intestinal surface, potentially yielding novel therapies for those with intestinal failure.

Peripheral and Mucosal B Cells From Celiac Disease Patients Show Increased Expression of CXCR3 and IgA Switch Markers

ponding to subdivisions of the 33mer based on the ability of ALV001 to cut several times within this gluten epitope. RESULTS: Mass spectrometry detected 33mer and all five peptide fragments resulting from degradation of the 33mer by ALV001. A large linear range was observed, with detection as low as 1 ng/ml of the peptides of interest. Intraday coefficient of variability was typically less than 5%. ALV001 cut within the 33mer epitope in the context of wheat gluten, releasing all five fragments that initially rose in concentration but plateaued as ALV001-mediated degradation slows. Using a clinical dose (150 mg, 0.3 mg/ml), ALV001 degraded 98% (10 minutes) and 99.5% (30 minutes) of the 33mer epitope. ALV001 and ALV002 functioned cooperatively; ALV002 degraded the five relatively stable peptide fragments as ALV001 activity plateaued. SUMMARY: A mass spectrometry assay was established to quantify the ability of ALV003 to degrade 33mer within wheat bread gluten in the context of a complex meal. Using this methodology, it was possible to distinguish separate but complementary contributions of ALV001 and ALV002 to 33mer degradation. The data support a model for ALV003-mediated gluten degradation in which ALV001 rapidly cleaves gluten adjacent to glutamine residues, releasing small proline-rich peptide fragments that serve as substrates for the prolyl endopeptidase ALV002. Data from a simulated gastric environment suggest that ALV003 should degrade ingested gluten in patients with celiac disease.