Inflammatory Bowel Diseases and Gut Microbiota.

Inflammatory bowel disease (IBD) is an inflammatory disease of the gastrointestinal tract, the incidence of which has rapidly increased worldwide, especially in developing and Western countries. Recent research has suggested that genetic factors, the environment, microbiota, and immune responses are involved in the pathogenesis; however, the underlying causes of IBD are unclear. Recently, gut microbiota dysbiosis, especially a decrease in the abundance and diversity of specific genera, has been suggested as a trigger for IBD-initiating events. Improving the gut microbiota and identifying the specific bacterial species in IBD are essential for understanding the pathogenesis and treatment of IBD and autoimmune diseases. Here, we review the different aspects of the role played by gut microbiota in the pathogenesis of IBD and provide a theoretical basis for modulating gut microbiota through probiotics, fecal microbiota transplantation, and microbial metabolites.

Beneficial Role of Microbial Transglutaminase in the Pathogenetic Mechanisms of Coeliac Disease.

ABSTRACT: Coeliac disease (CD) is caused by immunological intolerance to wheat gluten and related proteins of rye and barley. Consequently, gluten-free (GF) products have been developed but technological implementation is required to improve their intrinsic rheological properties. One alternative for increasing the functional properties of GF foodstuff is the incorporation of microbial transglutaminase (mTG), which allows for the cross-linking of proteins that can substitute for the gluten network in the bakery industry. mTG has been, however, suggested to mimic tissue transglutaminase and to be immunogenic in CD patients. Recently, both mTG and gliadin were found to be transported to the endoplasmic reticulum of enterocytes, suggesting cross-presentation and potential interaction with immune cells in CD. Although pathogenetic activity of mTG has not been found to date, these data naturally raise concerns among clinicians and patients about the use of mTG as a food additive. On the contrary, different studies have shown that treatment with mTG was effective in reducing the inflammatory immune response of gluten in CD. In this article, we take advantage of recent advances in gut physiology and CD pathogenesis to revise the literature data on mTG. An updated and unbiased overview of the role of mTG in this pathology allowed us to definitively highlight the beneficial use of this food additive by CD patients.

Beneficial effects of a T. monococcum wheat cultivar on diabetes incidence evaluated in non-obese diabetic mice and after in vitro simulated gastroduodenal digestion.

Wheat consumption can represent one of the nutritional factors involved in the onset of diabetes. We specifically investigated the potential diabetogenic effects of Hammurabi, a T. monococcum wheat cultivar, in non-obese diabetic (NOD) mice and analysed the levels of resistant starch in pasta manufactured with Hammurabi after in vitro gastroduodenal digestion. NOD mice were fed with Hammurabi, bread wheat or rice flour to evaluate diabetes incidence and insulitis score. An enzymatic method was applied to compare the content of resistant starch in Hammurabi pasta and durum wheat pasta (control). In NOD mice, the Hammurabi-based diet significantly delayed diabetes onset (p = 0.0042) and reduced insulitis score compared to rice or wheat-based diet. Furthermore, the resistant starch value following in vitro digestion of Hammurabi pasta was significantly higher (4.08%) than that of durum wheat pasta (2.28%). Taken together, these results highlighted the potential positive effects of the Hammurabi-based diet on diabetes incidence.

Transamidation Down-Regulates Intestinal Immunity of Recombinant α-Gliadin in HLA-DQ8 Transgenic Mice.

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.

Data-driven flood hazard zonation of Italy.

We present Flood-SHE, a data-driven, statistically-based procedure for the delineation of areas expected to be inundated by river floods. We applied Flood-SHE in the 23 River Basin Authorities (RBAs) in Italy using information on the presence or absence of inundations obtained from existing flood zonings as the dependent variable, and six hydro-morphometric variables computed from a 10 m × 10 m DEM as covariates. We trained 96 models for each RBA using 32 combinations of the hydro-morphometric covariates for the three return periods, for a total of 2208 models, which we validated using 32 model sets for each of the covariate combinations and return periods, for a total of 3072 validation models. In all the RBAs, Flood-SHE delineated accurately potentially inundated areas that matched closely the corresponding flood zonings defined by physically-based hydro-dynamic flood routing and inundation models. Flood-SHE delineated larger to much larger areas as potentially subject of being inundated than the physically-based models, depending on the quality of the flood information. Analysis of the sites with flood human consequences revealed that the new data-driven inundation zones are good predictors of flood risk to the population of Italy. Our experiment confirmed that a small number of hydro-morphometric terrain variables is sufficient to delineate accurate inundation zonings in a variety of physiographical settings, opening to the possibility of using Flood-SHE in other areas. We expect the new data-driven inundation zonings to be useful where flood zonings built on hydrological modelling are not available, and to decide where improved flood hazard zoning is needed.

Microbial transglutaminase: A biotechnological tool to manage gluten intolerance.

Celiac disease (CD) is a chronic immune-mediated disease in which gluten ingestion leads to damage of the small intestinal mucosa in genetically susceptible individuals. The enteropathy is mainly induced by the production of IFN-γ from intestinal CD4+T cells that recognise gliadin peptides following deamidation by tissue transglutaminase. The only available therapy is a strict, lifelong gluten-free diet (GFD). This diet is strongly demanding for patients, which justifies the search for alternative strategies. The enzyme approach is one promising strategy to address this issue. In particular, transamidation of wheat gliadin by microbial transglutaminase (mTG) was fully effective at inhibiting gliadin-specific IFN-γ secretion in intestinal T cells from CD patients. Furthermore, transamidated gliadin induced higher levels of the anti-inflammatory IL-10 than native gliadin in different in vitro models. These data suggest that a more balanced immune response could be induced by mTG-treated gliadin in the small intestine of celiac patients. Furthermore, the highlighted biological property of mTG-treated gliadin could be exploited to induce tolerance to native gliadin in at-risk individuals.

Thoracoabdominal Aortic Aneurysm Secondary to Aggressive Giant Cell Arteritis.

Large artery stenosis of the arm or leg arteries or the cervical arteries has been described in giant cell arteritis (GCA); aortic involvement, nevertheless, is less frequent, even if imaging tools such as positron emission tomography (PET) computed tomography have increased the frequency in the observation of aortic involvement. A 56-year-old female with a medical history of GCA presented to our emergency department with an unruptured voluminous thoracoabdominal aortic aneurysm (TAAA). The fluorodeoxyglucose PET demonstrated the presence of high inflammatory activity. The patient underwent endovascular correction using a "sandwich technique." The 3-month control CT scan shows complete aneurysm exclusion. In high risk for surgery patients with GCA, the endovascular treatment with parallel stent graft of TAAA is safe and feasible.

Contemporary Early and Long-Term Results of Open Repair for Ruptured and Symptomatic Unruptured Infrarenal AAA. Single Center Experience.

BACKGROUND: Observational studies have shown a reduced short-term mortality and increased long-term survival of EVAR over OR in the treatment of ruptured Infrarenal Abdominal Aortic Aneurysm (IAAA). Until now, none of the RCT has gained the result to demonstrate this superiority. Moreover, contemporary reports about results of OR of rIAAA are poor. METHODS: The study is observational and retrospective according to STROBE statement. The patients, unselected and strictly consecutive, were operated in urgency/emergency between 2007 and 2012. All had a preoperative CT angiography. Patients' characteristics, presentation, intraoperative and postoperative details were analyzed to identify factors that can affect the outcome. Considering the small number of events and the exploratory nature of the analyses, only univariate models were fit. RESULTS: One hundred five patients were treated in urgency/emergency setting because of symptomatic. The patients have been divided into two subgroups, based on lesion findings: 75 patients had ruptured IAAA (group A) and 30 patients had unruptured symptomatic IAAA, with CT findings of impending rupture (group B). Intraoperative, perioperative, and in-hospital mortality was respectively 0.9%, 8.6%, and 15.2%, with no difference between the groups. Unique predictors of 30-day mortality were transfusions, major and minor complications. Cumulative follow-up time was 455 person-years. Overall survival was not different between the groups. Predictors of late mortality were: age, creatinine at presentation, adjunctive procedures, length of in-hospital stay, any intraoperative complications, any perioperative complications, any major complication, and postoperative AKI. CONCLUSIONS: An experienced vascular team can achieve very good results in OR of ruptured IAAA, entirely comparable to EVAR. We are still far from a scientifically robust demonstration of the superiority of EVAR over OR in the treatment of ruptured IAAA.

Tailoring the immune response to wheat gliadin by enzymatic transamidation.

Enzymatic transamidation of wheat gliadin by microbial transglutaminase inhibits IFN-γ secretion by intestinal T cell lines from celiac disease (CD) patients. Here, we analysed its effects on intestinal biopsies from CD patients and studied the underlying mechanisms in HLA-DQ8 transgenic (tg) mice, a model of T-cell mediated gluten sensitivity. In vitro challenge with a soluble form of transamidated gliadin (spf) upregulated IL-10 transcript levels in human biopsy samples. Furthermore, the ratio of IL-10/IFN-γ transcripts was significantly increased following treatment with spf. In DQ8 tg mice, recall responses in vitro in the presence of dendritic cells pulsed with transamidated gliadin showed that gliadin-specific CD4+ T cells did not produce IFN-γ at any tested dose. On the contrary, spf-specific CD4+ T cells still secreted IFN-γ, but they also produced significant levels of IL-10 with both native and transamidated gliadin. Interestingly, this anti-inflammatory activity was restricted to a specific reverse-phase high-pressure liquid chromatography (RP-HPLC) fraction encompassing α-gliadins. These findings suggested an ability of transamidated gliadin to revert, as well as to prevent, the inflammatory phenotype triggered by native gliadin. This property was intrinsically associated with specific components of the α-gliadin fraction.

Use of Iliac Branch Endoprosthesis for Aortic Bifurcation Reconstruction.

Iliac branch devices represent a significant improvement in the treatment of common iliac artery (CIA) and aortoiliac aneurysms. Some authors have already described novel and off-label approaches of these devices. We present a case of a 75-year-old man, who underwent 3 years before to emergent aorto-aortic reconstruction, in which we used the Gore Iliac Branch Endoprosthesis as main body because of length, angulation, and narrowing of infrarenal aorta and bifurcation to treat a bilateral CIA. This off-label application of a well-recognized endovascular device appeared safe and feasible.

Conjugated linoleic acid prevents age-dependent neurodegeneration in a mouse model of neuropsychiatric lupus via the activation of an adaptive response.

Oxidative stress is a key mediator of autoimmune/neurodegenerative disorders. The antioxidant/anti-inflammatory effect of a synthetic conjugated linoleic acid (CLA) mixture in MRL/MpJ-Fas lpr mice (MRL/lpr), an animal model of neuropsychiatric lupus, was previously associated with the improvement of nuclear factor-E2-related factor 2 (Nrf2) defenses in the spleen and liver. However, little is known about the neuroprotective ability of a CLA mixture. This study investigated the age-dependent progression of oxidative stress and the hyperactivation of redox-sensitive compensatory pathways (macroautophagy, Nrf2) in old/diseased MRL/lpr mice brains and examines the effect produced by dietary CLA supplementation. Disrupted redox homeostasis was evidenced in the blood, liver, and brain of 21- to 22-week-old MRL/lpr (Old) mice compared with 8- to 10-week-old MRL/lpr (Young) animals. This alteration was associated with significant hyperactivation of compensatory mechanisms (macroautophagy, Nrf2, and astrocyte activation) in the brains of Old mice. Five-week daily supplementation with CLA (650 mg/kg-1 body weight) of 16-week-old (CLA+Old) mice diminished all the pathological hallmarks at a level comparable to Young mice or healthy controls (BALB/c). Such data demonstrated that MRL/lpr mice can serve as a valuable model for the evaluation of the effectiveness of neuroprotective drugs. Notably, the preventive effect provided by CLA supplementation against age-associated neuronal damage and hyperactivation of compensatory mechanisms suggests that the activation of an adaptive response is at least in part accountable for its neuroprotective ability.

Immune-modulating effects in mouse dendritic cells of lactobacilli and bifidobacteria isolated from individuals following omnivorous, vegetarian and vegan diets.

Lactobacilli and bifidobacteria play a primary role in modulation of gut immunity. By considering that microbiota composition depends on various factors, including diet, we asked whether functional differences could characterize faecal populations of lactobacilli and bifidobacteria isolated from individuals with different dietary habits. 155 healthy volunteers who followed omnivorous, ovo-lacto-vegetarian or vegan diets were recruited at four Italian centres (Turin, Parma, Bologna and Bari). Faecal samples were collected; lactobacilli and bifidobacteria were isolated on selective media and their immunomodulatory activity was tested in mouse dendritic cells (DCs). Pre-incubation with lactobacilli increased LPS-induced expression of the maturation markers CD80 and CD86, whereas pre-incubation with bifidobacteria decreased such expression. Analysis of the cytokine profile indicated that strains of both genera induced down-regulation of IL-12 and up-regulation of IL-10, whereas expression of TNF-α was not modulated. Notably, analysis of anti-inflammatory potential (IL-10/IL-12 ratio) showed that lactobacilli evoked a greater anti-inflammatory effect than did bifidobacteria in the omnivorous group (P<0.05). We also found significantly reduced anti-inflammatory potential in the bacterial strains isolated from Bari's volunteers in comparison with those from the cognate groups from the other centres. In conclusion, lactobacilli and bifidobacteria showed a genus-specific ability of modulating in vitro innate immunity associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the anti-inflammatory potential of some components of faecal microbiota.

Modulation of the cytokine profile in Caco-2 cells by faecal lactobacilli and bifidobacteria from individuals with distinct dietary habits.

Enterocytes are actively involved in the defense against pathogens and they limit penetration of commensal microbes into tissues. They also have an important role in gut immunity as enterocytes confer mucosal dendritic cell specialisation. On the other hand, the microbiota is directly involved in the development and modulation of the intestinal immune system. Particularly, lactobacilli and bifidobacteria play a primary role in shaping the immune response. We further explored this issue by evaluating whether functional differences in Caco-2 cells could characterise faecal populations of lactobacilli (155 samples) and bifidobacteria (110 samples) isolated from three dietary cohorts (omnivores, ovo-lacto-vegetarians and vegans) recruited at four Italian centres (Turin, Parma, Bologna and Bari). According to our findings, tested bacteria were unable to modulate expression of IL-8, IL-10, TGF-ß or thymic stromal lymphopoietin (TSLP) cytokines in unstimulated Caco-2 cells. Conversely, in phorbol 12-myristate 13-acetate and ionomycin (PMA/Io) stimulated Caco-2 cells, lactobacilli from the omnivorous group and all bifidobacteria significantly down-regulated IL-8. Notably, both genera also lowered the TSLP expression in stimulated Caco-2 cells, regardless of the diet regimen. By further examining these data on the basis of geographical origin, we found that lactobacilli from the vegetarian group recruited in Bari, significantly up-regulated this cytokine. In conclusion, we highlighted a peculiar immune-modulatory activity profile for lactobacilli on enterocytes undergoing a stimulatory signal, which was associated with a specific dietary habit. Furthermore, the geographical area had a significant impact on the inflammatory potential of members of the Lactobacillus genus.

A gut microbial metabolite of linoleic acid, 10-hydroxy-cis-12-octadecenoic acid, ameliorates intestinal epithelial barrier impairment partially via GPR40-MEK-ERK pathway.

Gut microbial metabolites of polyunsaturated fatty acids have attracted much attention because of their various physiological properties. Dysfunction of tight junction (TJ) in the intestine contributes to the pathogenesis of many disorders such as inflammatory bowel disease. We evaluated the effects of five novel gut microbial metabolites on tumor necrosis factor (TNF)-α-induced barrier impairment in Caco-2 cells and dextran sulfate sodium-induced colitis in mice. 10-Hydroxy-cis-12-octadecenoic acid (HYA), a gut microbial metabolite of linoleic acid, suppressed TNF-α and dextran sulfate sodium-induced changes in the expression of TJ-related molecules, occludin, zonula occludens-1, and myosin light chain kinase. HYA also suppressed the expression of TNF receptor 2 (TNFR2) mRNA and protein expression in Caco-2 cells and colonic tissue. In addition, HYA suppressed the protein expression of TNFR2 in murine intestinal epithelial cells. Furthermore, HYA significantly up-regulated G protein-coupled receptor (GPR) 40 expression in Caco-2 cells. It also induced [Ca(2+)]i responses in HEK293 cells expressing human GPR40 with higher sensitivity than linoleic acid, its metabolic precursor. The barrier-recovering effects of HYA were abrogated by a GPR40 antagonist and MEK inhibitor in Caco-2 cells. Conversely, 10-hydroxyoctadacanoic acid, which is a gut microbial metabolite of oleic acid and lacks a carbon-carbon double bond at Δ12 position, did not show these TJ-restoring activities and down-regulated GPR40 expression. Therefore, HYA modulates TNFR2 expression, at least partially, via the GPR40-MEK-ERK pathway and may be useful in the treatment of TJ-related disorders such as inflammatory bowel disease.

E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells.

BACKGROUND: The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. RESULTS: The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. CONCLUSIONS: The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.

Adaptive response activated by dietary cis9, trans11 conjugated linoleic acid prevents distinct signs of gliadin-induced enteropathy in mice.

PURPOSE: The beneficial effects of conjugated linoleic acid (CLA) mixture (cis9, trans11, c9; trans10, cis12, t10) against gliadin-induced toxicity in HLA-DQ8-transgenic mice (DQ8) have been associated with improved duodenal cytoprotective mechanisms [nuclear factor-E2-related factor-2, Nrf2; acylpeptide hydrolase (APEH)/proteasome]. The present study was aimed at investigating the ability of individual CLA isomers to improve the efficacy of these defensive mechanisms and to protect against duodenal injury caused by the combined administration of gliadin and indomethacin (GI). METHODS: Gluten-mediated enteropathy was induced in DQ8 mice by three intra-gastric administration of gliadin (20 mg kg(-1)/bw) and indomethacin (15 mg L(-1)) in drinking water for 10 days (GI). C9 or t10 CLA (520 mg kg(-1)/bw/day) were orally administered for 2 weeks. Pro-oxidant and toxic effects associated with GI treatment, anti-oxidant/detoxifying ability of c9 or t10-CLA and the protective effect induced by c9 pre-treatment (c9 + GI) were evaluated in DQ8 mice duodenum by combining enzymatic, immunoblotting, histological evaluation and quantitative real-time PCR assays. RESULTS: GI treatment produces the time-dependent decline of the considered detoxifying mechanisms thus leading to pro-apoptotic and pro-oxidant effects. APEH/proteasome pathway was not markedly affected by individual CLA isomers, but duodenal redox status and activity/mRNA levels of Nrf2-activated enzymes were significantly improved by c9 administration. c9 pre-treatment protects against GI-mediated accumulation of oxidative stress markers, and histological examination reveals the increase of goblet cells number in mouse duodenum but induces only a partial recovery of APEH/proteasome activity. CONCLUSIONS: The activation of and adaptive response by low doses of c9 supplementation prevents distinct signs of gliadin-induced enteropathy in DQ8 mice.

Vaccination and other antigen-specific immunomodulatory strategies in celiac disease.

BACKGROUND: Celiac disease (CD) results from an alteration in the oral tolerance to dietary gluten. The response to gluten is normally tightly regulated and involves the secretion of TGF-ß and IL-10 from different subtypes of regulatory T cells (Tregs). Interestingly, in addition to proinflammatory cytokines, the inflamed CD mucosa also contains high levels of T cell-derived IL-10 compared with treated CD patients or normal donors. Furthermore, most studies describe an increase in the number of Foxp3+ Tregs in the small intestinal mucosa in CD patients compared to controls. This paradoxical condition suggests that regulatory mechanisms might operate to counterbalance the abnormal gliadin-triggered immune activation in untreated mucosa. Indeed, addition of exogenous IL-10 to mucosal cultures from treated CD patients can suppress gliadin-induced T cell activation. Considering the central role of adaptive immunity in CD, the development of strategies to stimulate these mechanisms is a primary goal of efforts to restore gluten tolerance. Key Messages: Different immunomodulatory strategies have been explored. NexVax2, a desensitizing vaccine that uses three dominant gluten peptides administered subcutaneously to induce a tolerogenic response in CD patients, is under development. Alternatively, the potential of substituted, cyclic or dimeric peptide analogues as blockers to prevent HLA from binding to the immunodominant gliadin epitopes has been demonstrated in vitro. In line with these results, we recently found that modified (transamidated) gliadins influenced the immune response in intestinal biopsy samples from CD patients with overt disease by drastically reducing the production of IFN-γ. Notably, in a mouse model, transamidated gliadins reverted the phenotype of the gliadin-inducible immune response from an inflammatory phenotype to an anti-inflammatory phenotype. CONCLUSIONS: Various approaches are currently under investigation to recover gluten tolerance based on the use of both modified and native antigen molecules. More specific studies are now required to test the efficacy of such strategies for preventing CD.