H influenzae LPS colocalization with Toll-like receptor 4 in eosinophilic esophagitis.

Background: Patients with eosinophilic esophagitis (EoE) have a unique esophageal microbiome with increased presence of Haemophilus influenzae, but its role in the disease is unclear. Objective: Microbiome-derived bacterial LPS activation of Toll-like receptors (TLR) is a potential mechanism for inducing inflammation in other chronic inflammatory diseases, but it has not been studied in EoE. Our aim was therefore to study microbiome-derived bacterial LPS activation of TLRs in EoE. Methods: We studied 10 patients with active EoE, 9 patients with inactive EoE, and 10 control patients. Esophageal biopsy samples from the controls, patients with active EoE (>15 eosinophils/hpf), and patients with inactive EoE were immunostained for the presence of H influenzae LPS, presence of TLR4, and colocalization of LPS and TLR4. Staining intensity was measured by using confocal laser microscopy and scored on a scale from 0 to 3 as the average score assigned by 2 blinded observers. Results: H influenzae LPS was detected by positive staining in 20 of the 29 patients (69.0%), including 9 of the 10 patients with active EoE (90.0%), 8 of the 9 patients with inactive EoE (89.9%), and 3 of the 10 controls (30%); its level was greater in the patients with active EoE than in the controls (P = .063). TLR4 was detected by positive staining in 19 of the 29 patients (65.5%), including 9 of the 10 patients with active EoE (90.0%), 4 of the 9 patients with inactive EoE (44.4%), and 6 of the 10 controls (60.0%); its level was higher in the patients with active EoE than in those with inactive EoE (P = .096). The result of testing for colocalization of LPS and TLR4 was positive in 8 of 10 patients with active EoE (80.0%), 1 of 9 patients with inactive EoE (11.1%), and 1 of 10 control patients (10.0%), with greater colocalization of H influenzae LPS and TLR4 staining density in the samples from patients with active EoE than in the controls or the patients with inactive EoE (P = .009 and P = .018, respectively). Conclusion: Esophageal microbiome-rich H influenzae LPS colocalizes to TLR4 in active EoE. These data lend further support to a role for the esophageal microbiome in modulating the activity of EoE.

G9a Modulates Lipid Metabolism in CD4 T Cells to Regulate Intestinal Inflammation.

BACKGROUND & AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.

Compositional change of gut microbiome and osteocalcin expressing endothelial progenitor cells in patients with coronary artery disease.

Osteogenic endothelial progenitor cells (EPCs) contribute to impaired endothelial repair and promote coronary artery disease (CAD) and vascular calcification. Immature EPCs expressing osteocalcin (OCN) has been linked to unstable CAD; however, phenotypic regulation of OCN-expressing EPCs is not understood. We hypothesized that gut-microbiome derived pro-inflammatory substance, trimethylamine N-oxide (TMAO) might be associated with mobilization of OCN-expressing EPCs. This study aimed to investigate the association between dysbiosis, TMAO, and circulating mature and immature OCN-expressing EPCs levels in patients with and without CAD. We included 202 patients (CAD N = 88; no CAD N = 114) who underwent assessment of EPCs using flow cytometry and gut microbiome composition. Mature and immature EPCs co-staining for OCN were identified using cell surface markers as CD34+/CD133-/kinase insert domain receptor (KDR)+ and CD34-/CD133+/KDR+ cells, respectively. The number of observed operational taxonomy units (OTU), index of microbial richness, was used to identify patients with dysbiosis. The number of immature OCN-expressing EPCs were higher in patients with CAD or dysbiosis than patients without. TMAO levels were not associated with circulating levels of OCN-expressing EPCs. The relative abundance of Ruminococcus gnavus was moderately correlated with circulating levels of immature OCN-expressing EPCs, especially in diabetic patients. Gut dysbiosis was associated with increased levels of TMAO, immature OCN-expressing EPCs, and CAD. The relative abundance of Ruminococcus gnavus was correlated with immature OCN-expressing EPCs, suggesting that the harmful effects of immature OCN-expressing EPCs on CAD and potentially vascular calcification might be mediated by gut microbiome-derived systemic inflammation.

Human gut-derived commensal suppresses generation of T-cell response to gliadin in humanized mice by modulating gut microbiota.

The human intestinal tract is colonized by a large number of diverse microorganisms that play various important physiologic functions. In inflammatory gut diseases including celiac disease (CeD), a dysbiotic state of microbiome has been observed. Interestingly, this perturbed microbiome is normalized towards eubiosis in patients showing recovery after treatment. The treatment has been observed to increase the abundance of beneficial microbes in comparison to non-treated patients. In this study, we investigated the effect of Prevotella histicola or Prevotella melaninogenica, isolated from the duodenum of a treated CeD patient, on the induction and maintenance of oral tolerance to gliadin, a CeD associated subgroup of gluten proteins, in NOD.DQ8.ABo transgenic mice. Conventionally raised mice on a gluten free diet were orally gavaged with bacteria before and after injection with pepsin trypsin digested gliadin (PTD-gliadin). P. histicola suppressed the cellular response to gliadin, whereas P. melaninogenica failed to suppress an immune response against gliadin. Interestingly, tolerance to gliadin in NOD.DQ8.ABo mice may be associated with gut microbiota as mice gavaged with P melaninogenica harbored a different microbial diversity as compared to P. histicola treated mice. This study provides experimental evidence that gut microbes like P. histicola from treated patients can suppress the immune response against gliadin epitopes.

Mucosal penetration and clearance of gluten and milk antigens in eosinophilic oesophagitis.

BACKGROUND: The Th2 allergic pathway in eosinophilic oesophagitis (EoE) responds to food antigen exposure. AIM: To compare the presence and temporal pattern of food antigen penetration in oesophageal mucosa in active and inactive EoE and controls METHODS: Thirty-two patients with EoE (20 active) and 10 controls were asked to eliminate all wheat and/or dairy 12, 24, 48, 72 or 96 hours before endoscopy. Immunostaining on endoscopic biopsies was performed for gliadin, casein and whey. RESULTS: Gluten, casein and whey were detected by positive staining in 17/32 (53.1%), 21/32 (65.6%), and 30/32 (92.0%) of patients, respectively. In active vs inactive EoE, 70.0% vs 25.0% (P < 0.05), 80.0% vs 41.5%, and 90.0% vs 90.9% patients had detectable gliadin, casein and whey, respectively. Casein and whey (20.0% and 100%, respectively) but not gliadin, were present in controls. The gliadin staining density was greater in active compared to inactive disease at ≤ 24 vs >24 hours after exposure (P = 0.05) but no differences were detected when comparing active and inactive patients for casein and whey. There was greater staining density for whey than casein for all patients at ≤24 hours (mean 2.14 ± 0.91 and 1.07 ± 1.33, P = 0.02). In active EoE, IgG4 was present in 14/20 compared to one inactive patient. CONCLUSION: The oesophageal epithelium is selectively permeable and has relatively long dwell times for food antigens known to trigger EoE. The precise mechanism of antigen-specific mucosal entry and the factors that determine the induction or effector trigger of the Th2 pathway activation merit further study.

IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease.

Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.

Community-Based Study of Celiac Disease Autoimmunity Progression in Adults.

BACKGROUND & AIMS: Celiac disease can develop at any age, but outcomes of adults with positive results from serologic tests for tissue transglutaminase antibodies (tTGA) without endoscopic determination of celiac disease (called celiac autoimmunity) have not been thoroughly evaluated. We investigated the proportion of adults with celiac autoimmunity at a community medical center and their progression to celiac disease. METHODS: We analyzed waste blood samples from a community clinic from 15,551 adults for tTGA and, if titer results were above 2 U/mL, for endomysial antibody. The blood samples had been collected at 2 time points (median interval, 8.8 years) from 2006 through 2017. We collected data from the clinic on diagnoses of celiac disease based on duodenal biopsy analysis. RESULTS: Of the serum samples collected at the first time point, 15,398 had negative results for tTGA, and 153 had positive results for tTGA (>4 U/mL). Based on medical records, 6 individuals received a diagnosis of celiac disease, for a cumulative incidence of celiac disease diagnosis of 0.06% (95% confidence interval, 0.01-0.11). Forty-nine (0.32%) individuals with a negative result from the first serologic test for tTGA had a positive result from the second test. Among the 153 adults who were tTGA positive at the first time point, 31 (20%) had a subsequent diagnosis of celiac disease, 81 (53%) remained positive for tTGA without a clinical diagnosis of celiac disease, and 41 (27%) had negative test results for tTGA at the second time point. Higher initial tTGA titers, female sex, and a history of hypothyroidism and autoimmune disease were associated with increased risks of subsequent diagnosis of celiac disease. Interestingly, adults whose first blood sample had a positive test result but second blood sample had a negative result for tTGA were older, had lower-than-average initial tTGA titer results, and had a higher mean body mass index than adults whose blood samples were positive for tTGA at both time points and adults later diagnosed with celiac disease. CONCLUSIONS: In an analysis of serum samples collected from a community clinic an average of 8.8 years apart, we found that fewer than 1% of adults with negative results from an initial test for tTGA have a positive result on a second test. Of adults with positive results from the test for tTGA, only 20% are later diagnosed with celiac disease; the remaining individuals maintain persistent increases in tTGA without diagnoses of celiac disease or have negative results from second tests.

Effect of a Gluten-free Diet on Quality of Life in Patients With Nonclassical Versus Classical Presentations of Celiac Disease.

BACKGROUND: Celiac disease (CD) often presents with symptoms of diarrhea and malabsorption, termed classical CD. However, it can also present as nonclassical CD, which is commonly associated with nongastrointestinal symptoms. Studies suggest that nonclassical CD tends to have less severe symptoms than classical CD, which may affect both adherence to a gluten-free diet (GFD) and psychological stress. Therefore, we compared adherence to a GFD and psychological measures, including quality of life (QOL) and somatization, between patients with nonclassical and classical presentations of CD. METHODS: Patients at a tertiary care center with biopsy-proven CD, who completed a Talley Bowel Disease Questionnaire and the Short Form-36 at diagnosis and who had been on a GFD for at least 1 year, were included in this study. Patients were further surveyed to assess gastrointestinal symptoms, QOL, Somatization Symptom Checklist (SSC), and adherence to a GFD. Results were compared between patients with classical versus nonclassical CD presentation. RESULTS: Among 122 patients included in this study, 62 had classical CD and 60 had nonclassical CD. At diagnosis, health-related QOL was lower in the classical CD group than in the nonclassical CD group. After following a GFD, both groups had improved QOL after following a GFD, and body mass index significantly increased in both groups. Most subscales of QOL, SSC scores, and adherence to the GFD were similar between the groups, except the Short Form-36 Mental Component summary scores that were still lower in the classical CD (48.4 vs. 52.6 nonclassical CD group; P=0.03). CONCLUSIONS: Despite QOL at diagnosis being higher in those with nonclassical CD versus lower in those with classical CD, both groups had improved QOL and achieved a similar QOL after following a GFD.

Synthetic Neoepitopes of the Transglutaminase-Deamidated Gliadin Complex as Biomarkers for Diagnosing and Monitoring Celiac Disease.

BACKGROUND & AIMS: Celiac disease (CeD) has characteristics of an autoimmune disease, such as increased antibody levels to tissue transglutaminase (tTG). However, assays to measure these biomarkers in blood samples do not identify patients with sufficient accuracy for diagnosis or monitoring of CeD. We aimed to discover biomarkers of CeD derived from neoepitopes of deamidated gliadin peptides (DGP) and tTG fragments and to determine if immune reactivity against these epitopes can identify patients with CeD with mucosal healing. METHODS: We analyzed serum samples from 90 patients with biopsy-proven CeD and 79 healthy individuals (controls) for immune reactivity against the tTG-DGP complex (discovery cohort). A fluorescent peptide microarray platform was used to estimate the antibody-binding intensity of each synthesized tTG-DGP epitope. We validated our findings in 82 patients with newly diagnosed CeD and 217 controls. We tested the ability of our peptide panel to identify patients with mucosal healing (based on the histologic analysis) using serum samples from patients with treated and healed CeD (n = 85), patients with treated but unhealed CeD (n = 81; villous atrophy despite a adhering a gluten-free diet), patients with untreated CeD (n = 82) and disease controls (n = 27), villous atrophy without CeD), and healthy controls (n = 217). Data were analyzed using principal component analysis followed by machine learning and support vector machine modeling. RESULTS: We identified 172 immunogenic epitopes of the tTG-DGP complex. We found significantly increased immune reactivity against these epitopes vs controls. In the both cohort, the set of neoepitopes derived from the tTG-DGP complex identified patients with CeD with 99% sensitivity and 100% specificity. Serum samples from patients with untreated CeD had the greatest mean antibody-binding intensity against the tTG-DGP complex (32.5 ± 16.4). The average antibody-binding intensity was significantly higher in serum from patients with treated but unhealed CeD mucosa (15.1 ± 7.5) than in patients with treated and healed CeD mucosa (5.5 ± 3.4) (P < .001). The assay identified patients with mucosa healing status with 84% sensitivity and 95% specificity. CONCLUSIONS: We identified immunogenic epitopes of the tTG-DGP complex, and found that an assay to measure the immune response to epitopes accurately identified patients with CeD, as well as patients with mucosal healing. This biomarker assay might be used in detection and monitoring of patients with CeD.

Accumulation of Heavy Metals in People on a Gluten-Free Diet.

BACKGROUND & AIMS: Specific foods such as fish and rice have high concentrations of metals such as arsenic, mercury, lead, cadmium, and cobalt. Many gluten-free diets (GFDs) include these foods, so we evaluated whether a GFD was associated with increased metal bioaccumulation. METHODS: We performed a population-based, cross-sectional study using data collected from the National Health and Nutrition Examination Survey (NHANES), from 2009 through 2012, collecting information on the diagnosis of celiac disease and adherence to a GFD. We tested NHANES blood samples to identify individuals with undiagnosed celiac disease, using assays for immunoglobulin A tissue transglutaminase followed by a confirmatory test for endomysial antibody. Among a total of 11,354 NHANES participants, celiac disease was diagnosed in 55 participants, based on test results or a reported clinical diagnosis. We collected NHANES survey data on blood levels of lead, mercury, and cadmium from subjects who were on a GFD (n = 115) and participants who were not on a GFD (n = 11,239). Levels of total arsenic in urine samples were available from 3901 subjects not following a GFD and 32 individuals following a GFD. NHANES participants were asked questions about fish and shellfish consumption. We performed multivariate logistic regression analyses to associate gluten-related conditions with blood concentrations of mercury, cadmium, and lead and urine concentration of total arsenic, adjusting for demographic characteristics, as well as for rice consumption or seafood intake. Geometric means were reported for urinary concentrations of total arsenic and blood concentrations of mercury, cadmium, and lead for demographic groups and subjects with gluten-related conditions (subjects without celiac disease who avoid gluten). RESULTS: Persons following a GFD had significantly increased total blood mercury levels (1.37 mcg/L) compared with persons not on a GFD (0.93 mcg/L) (P = .008), as well as increased blood levels of lead (1.42 vs 1.13 mcg/L; P = .007) and cadmium (0.42 vs 0.34 mcg/L; P = .03). Urine samples from subjects on a GFD had higher concentrations of total arsenic (15.15 mcg/L) than urine samples from subjects not on a GFD (8.38 mcg/L) (P = .002). After controlling for demographic characteristics, levels of all heavy metals remained significantly higher in persons following a GFD, compared with those not following a GFD. After exclusion of persons with celiac disease, people without celiac disease on a GFD (n = 101) had significantly increased blood concentrations of total mercury (1.40 mcg/L) than persons without celiac disease and not on a GFD (n = 10,890) (0.93 mcg/L; P = .02) and higher blood concentrations of lead (1.44 vs 1.13 mcg/L; P = .01) and higher urine concentrations of total arsenic (14.69 mcg/L [n = 3632] vs 8.32 mcg/L [n = 28]; P = .01). Blood samples from persons without celiac disease avoiding gluten had higher levels of cadmium (0.42 mcg/L) than persons without celiac disease and not following a GFD (0.34 mcg/L), but this difference was not significant (P = .06). CONCLUSIONS: In an analysis of data collected from NHANES, persons on a GFD had significantly higher urine levels of total arsenic and blood levels of mercury, lead, and cadmium than persons not avoiding gluten. Studies are needed to determine the long-term effects of accumulation of these elements in persons on a GFD.

Increased risk of herpes zoster in patients with coeliac disease - nationwide cohort study.

BACKGROUND AND AIMS: Clinical experience suggests that patients with coeliac disease (CD) are more prone to develop herpes zoster (HZ), but robust studies are lacking. METHODS: We identified 29,064 patients with CD 1969-2008 using biopsy report data from Sweden's 28 pathology departments. CD was equalled to villous atrophy (Marsh histopathology grade III). Each patient was matched on age, sex, calendar year and county of residence to up to five reference individuals ( n=144,342) from the general population. We then used Cox regression to estimate hazard ratios (HRs) for future HZ (defined as having a hospital-based inpatient or outpatient record of this diagnosis in the Swedish Patient Register). RESULTS: During follow-up, 154 (0.53%) individuals with CD and 499 (0.35%) reference individuals developed HZ. Among individuals aged ≥60 years, 1.06% of CD individuals and 0.85% of reference individuals had a lifetime record of HZ. Overall, CD was associated with a 1.62-fold increased risk of HZ (95% CI=1.35-1.95), and was seen also when we considered comorbidity with lymphoproliferative disease, systemic lupus erythematosus, type 1 diabetes, thyroid disease, rheumatoid disease and excluded individuals with a record of dermatitis herpetiformis. The increased risk remained significant after more than five years of follow-up (1.46; 1.16-1.84) Conclusions: CD is associated with HZ, the increased relative risk persists over time from celiac diagnosis but the absolute risk is small.

Animal models to study non-celiac gluten sensitivity.

Non-celiac gluten sensitivity (NCGS), sometimes known as non-celiac wheat sensitivity (NCWS), has recently received much attention, both scientific as well as from the alternative medical community. Over the past 5 years, there are over 200 publications on NCGS indexed on the PubMed database, the gluten-free market has been growing bigger, and it is clear that the number of consumers who are on a gluten-free diet (GFD) possibly because of a suspicion for NCGS appears to grow even faster. Nevertheless, despite these three rising events, many questions about NCGS remain unresolved. It is likely that NCGS represents a heterogeneous group of disorders linked by a common response to a GFD. It is still not fully understood how gluten, and likely other wheat proteins and components, could activate and drive the pathophysiology of NCGS. As a result, there are still no clear biomarkers, no robust clinical diagnostic criteria, nor a conclusive definition for NCGS. This heterogeneity can be approached by reducing the variables, in particular those of human behaviour and placebo effect, by studying animal models to address specific biological effects of wheat and/or gluten-related proteins. Herein we review the animal models and their potential to be used to advance our understanding of these disorders and potentially address their prevention and treatment.

Suppression of Inflammatory Arthritis by Human Gut-Derived Prevotella histicola in Humanized Mice.

OBJECTIVE: The gut microbiome regulates host immune homeostasis. Rheumatoid arthritis (RA) is associated with intestinal dysbiosis. This study was undertaken to test the ability of a human gut-derived commensal to modulate immune response and treat arthritis in a humanized mouse model. METHODS: We isolated a commensal bacterium, Prevotella histicola, that is native to the human gut and has systemic immune effects when administered enterally. Arthritis-susceptible HLA-DQ8 mice were immunized with type II collagen and treated with P histicola. Disease incidence, onset, and severity were monitored. Changes in gut epithelial proteins and immune response as well as systemic cellular and humoral immune responses were studied in treated mice. RESULTS: When treated with P histicola in prophylactic or therapeutic protocols, DQ8 mice exhibited significantly decreased incidence and severity of arthritis compared to controls. The microbial mucosal modulation of arthritis was dependent on regulation by CD103+ dendritic cells and myeloid suppressors (CD11b+Gr-1+ cells) and by generation of Treg cells (CD4+CD25+FoxP3+) in the gut, resulting in suppression of antigen-specific Th17 responses and increased transcription of interleukin-10. Treatment with P histicola led to reduced intestinal permeability by increasing expression of enzymes that produce antimicrobial peptides as well as tight junction proteins (zonula occludens 1 and occludin). However, the innate immune response via Toll-like receptor 4 (TLR-4) and TLR-9 was not affected in treated mice. CONCLUSION: Our results demonstrate that enteral exposure to P histicola suppresses arthritis via mucosal regulation. P histicola is a unique commensal that can be explored as a novel therapy for RA and may have few or no side effects.

Multiple sclerosis patients have a distinct gut microbiota compared to healthy controls.

Multiple sclerosis (MS) is an immune-mediated disease, the etiology of which involves both genetic and environmental factors. The exact nature of the environmental factors responsible for predisposition to MS remains elusive; however, it's hypothesized that gastrointestinal microbiota might play an important role in pathogenesis of MS. Therefore, this study was designed to investigate whether gut microbiota are altered in MS by comparing the fecal microbiota in relapsing remitting MS (RRMS) (n = 31) patients to that of age- and gender-matched healthy controls (n = 36). Phylotype profiles of the gut microbial populations were generated using hypervariable tag sequencing of the V3-V5 region of the 16S ribosomal RNA gene. Detailed fecal microbiome analyses revealed that MS patients had distinct microbial community profile compared to healthy controls. We observed an increased abundance of Psuedomonas, Mycoplana, Haemophilus, Blautia, and Dorea genera in MS patients, whereas control group showed increased abundance of Parabacteroides, Adlercreutzia and Prevotella genera. Thus our study is consistent with the hypothesis that MS patients have gut microbial dysbiosis and further study is needed to better understand their role in the etiopathogenesis of MS.

An expansion of rare lineage intestinal microbes characterizes rheumatoid arthritis.

BACKGROUND: The adaptive immune response in rheumatoid arthritis (RA) is influenced by an interaction between host genetics and environment, particularly the host microbiome. Association of the gut microbiota with various diseases has been reported, though the specific components of the microbiota that affect the host response leading to disease remain unknown. However, there is limited information on the role of gut microbiota in RA. In this study we aimed to define a microbial and metabolite profile that could predict disease status. In addition, we aimed to generate a humanized model of arthritis to confirm the RA-associated microbe. METHODS: To identify an RA biomarker profile, the 16S ribosomal DNA of fecal samples from RA patients, first-degree relatives (to rule out environment/background as confounding factors), and random healthy non-RA controls were sequenced. Analysis of metabolites and their association with specific taxa was performed to investigate a potential mechanistic link. The role of an RA-associated microbe was confirmed using a human epithelial cell line and a humanized mouse model of arthritis. RESULTS: Patients with RA exhibited decreased gut microbial diversity compared with controls, which correlated with disease duration and autoantibody levels. A taxon-level analysis suggested an expansion of rare taxa, Actinobacteria, with a decrease in abundant taxa in patients with RA compared with controls. Prediction models based on the random forests algorithm suggested that three genera, Collinsella, Eggerthella, and Faecalibacterium, segregated with RA. The abundance of Collinsella correlated strongly with high levels of alpha-aminoadipic acid and asparagine as well as production of the proinflammatory cytokine IL-17A. A role for Collinsella in altering gut permeability and disease severity was confirmed in experimental arthritis. CONCLUSIONS: These observations suggest dysbiosis in RA patients resulting from the abundance of certain rare bacterial lineages. A correlation between the intestinal microbiota and metabolic signatures could determine a predictive profile for disease causation and progression.

Determination of B-Cell Epitopes in Patients with Celiac Disease: Peptide Microarrays.

BACKGROUND: Most antibodies recognize conformational or discontinuous epitopes that have a specific 3-dimensional shape; however, determination of discontinuous B-cell epitopes is a major challenge in bioscience. Moreover, the current methods for identifying peptide epitopes often involve laborious, high-cost peptide screening programs. Here, we present a novel microarray method for identifying discontinuous B-cell epitopes in celiac disease (CD) by using a silicon-based peptide array and computational methods. METHODS: Using a novel silicon-based microarray platform with a multi-pillar chip, overlapping 12-mer peptide sequences of all native and deamidated gliadins, which are known to trigger CD, were synthesized in situ and used to identify peptide epitopes. RESULTS: Using a computational algorithm that considered disease specificity of peptide sequences, 2 distinct epitope sets were identified. Further, by combining the most discriminative 3-mer gliadin sequences with randomly interpolated3- or 6-mer peptide sequences, novel discontinuous epitopes were identified and further optimized to maximize disease discrimination. The final discontinuous epitope sets were tested in a confirmatory cohort of CD patients and controls, yielding 99% sensitivity and 100% specificity. CONCLUSIONS: These novel sets of epitopes derived from gliadin have a high degree of accuracy in differentiating CD from controls, compared with standard serologic tests. The method of ultra-high-density peptide microarray described here would be broadly useful to develop high-fidelity diagnostic tests and explore pathogenesis.

Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease.

Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.

Generating Transgenic Mouse Models for Studying Celiac Disease.

This chapter provides a brief overview of current animal models for studying celiac disease, with a focus on generating HLA transgenic mouse models. Human Leukocyte Antigen class II molecules have been a particular target for transgenic mice due to their tight association with celiac disease, and a number of murine models have been developed which had the endogenous MHC class II genes replaced with insertions of disease susceptible HLA class II alleles DQ2 or DQ8. Additionally, transgenic mice that overexpress interleukin-15 (IL-15), a key player in the inflammatory cascade that leads to celiac disease, have also been generated to model a state of chronic inflammation. To explore the contribution of specific bacteria in gluten-sensitive enteropathy, the nude mouse and rat models have been studied in germ-free facilities. These reductionist mouse models allow us to address single factors thought to have crucial roles in celiac disease. No single model has incorporated all of the multiple factors that make up celiac disease. Rather, these mouse models can allow the functional interrogation of specific components of the many stages of, and contributions to, the pathogenic mechanisms that will lead to gluten-dependent enteropathy. Overall, the tools for animal studies in celiac disease are many and varied, and provide ample space for further creativity as well as to characterize the complete and complex pathogenesis of celiac disease.

Isotretinoin Exposure and Risk of Celiac Disease.

BACKGROUND: Isotretinoin (13-cis retinoic acid) is a metabolite of vitamin A and has anti-inflammatory and immunoregulatory effects; however, a recent publication by DePaolo et al. demonstrated that in the presence of IL-15, retinoic acid can act as an adjuvant and promote inflammation against dietary proteins. OBJECTIVE: To evaluate the risk of overt and latent celiac disease (CD) among users of isotretinoin. MATERIAL AND METHODS: Medical records of patients from 1995 to 2011 who had a mention of isotretinoin in their records (N = 8393) were searched for CD diagnosis using ICD-09CM codes. Isotretinoin exposure was compared across overt CD patients and their age- and gender-matched controls from the same pool. To evaluate the risk of latent CD with isotretinoin exposure, patients were overlapped with a community-based list of patients with waste serum samples that were tested for CD serology, excluding those with overt CD (2006-2011). Isotretinoin exposure was defined as the use of isotretinoin prior to CD diagnosis or serology. RESULTS: Of 8393 patients, 25 had a confirmed CD diagnosis. Compared to matched controls (N = 75), isotretinoin exposure was not significantly different between overt CD patients versus controls (36% versus 39%, respectively; P = 0.712). Likewise, latent CD defined as positive serology was not statistically different between isotretinoin exposed (N = 506) versus non-exposed (N = 571) groups (1.8% versus 1.4%, respectively; P = 0.474). CONCLUSIONS: There was no association between isotretinoin use and risk of either overt or latent CD.