The Combination of Gold and Silver Food Nanoparticles with Gluten Peptides Alters the Autophagic Pathway in Intestinal Crypt-like Cells.

Metallic nanoparticles (mNPs) are widely used as food additives and can interact with gliadin triggering an immune response, but evaluation of the effects on crypts, hypertrophic in celiac subjects, is still lacking. This study evaluated the effects of gold and silver mNPs in combination with gliadin on crypt-like cells (HIEC-6). Transmission electron microscopy (TEM) was used to evaluate gliadin-mNP aggregates in cells. Western blot and immunofluorescence analysis assessed autophagy-related molecule levels (p62, LC3, beclin-1, EGFR). Lysosome functionality was tested with acridine orange (AO) and Magic Red assays. TEM identified an increase in autophagic vacuoles after exposure to gliadin + mNPs, as also detected by significant increments in LC3-II and p62 expression. Immunofluorescence confirmed the presence of mature autophagosomes, showing LC3 and p62 colocalization, indicating an altered autophagic flux, further assessed with EGFR degradation, AO and Magic Red assays. The results showed a significant reduction in lysosomal enzyme activity and a modest reduction in acidity. Thus, gliadin + mNPs can block the autophagic flux inducing a lysosomal defect. The alteration of this pathway, essential for cell function, can lead to cell damage and death. The potential effects of this copresence in food should be further characterized to avoid a negative impact on celiac disease subjects.

Dietary Nanoparticles Interact with Gluten Peptides and Alter the Intestinal Homeostasis Increasing the Risk of Celiac Disease.

The introduction of metallic nanoparticles (mNPs) into the diet is a matter of concern for human health. In particular, their effect on the gastrointestinal tract may potentially lead to the increased passage of gluten peptides and the activation of the immune response. In consequence, dietary mNPs could play a role in the increasing worldwide celiac disease (CeD) incidence. We evaluated the potential synergistic effects that peptic-tryptic-digested gliadin (PT) and the most-used food mNPs may induce on the intestinal mucosa. PT interaction with mNPs and their consequent aggregation was detected by transmission electron microscopy (TEM) analyses and UV-Vis spectra. In vitro experiments on Caco-2 cells proved the synergistic cytotoxic effect of PT and mNPs, as well as alterations in the monolayer integrity and tight junction proteins. Exposure of duodenal biopsies to gliadin plus mNPs triggered cytokine production, but only in CeD biopsies. These results suggest that mNPs used in the food sector may alter intestinal homeostasis, thus representing an additional environmental risk factor for the development of CeD.

Immune-Mediated Drug-Induced Liver Injury: Immunogenetics and Experimental Models.

Drug-induced liver injury (DILI) is a challenging clinical event in medicine, particularly because of its ability to present with a variety of phenotypes including that of autoimmune hepatitis or other immune mediated liver injuries. Limited diagnostic and therapeutic tools are available, mostly because its pathogenesis has remained poorly understood for decades. The recent scientific and technological advancements in genomics and immunology are paving the way for a better understanding of the molecular aspects of DILI. This review provides an updated overview of the genetic predisposition and immunological mechanisms behind the pathogenesis of DILI and presents the state-of-the-art experimental models to study DILI at the pre-clinical level.

Accuracy of Transient Elastography in Assessing Fibrosis at Diagnosis in Naïve Patients With Primary Biliary Cholangitis: A Dual Cut-Off Approach.

BACKGROUND AND AIMS: Liver fibrosis holds a relevant prognostic meaning in primary biliary cholangitis (PBC). Noninvasive fibrosis evaluation using vibration-controlled transient elastography (VCTE) is routinely performed. However, there is limited evidence on its accuracy at diagnosis in PBC. We aimed to estimate the diagnostic accuracy of VCTE in assessing advanced fibrosis (AF) at disease presentation in PBC. APPROACH AND RESULTS: We collected data from 167 consecutive treatment-naïve PBC patients who underwent liver biopsy (LB) at diagnosis at six Italian centers. VCTE examinations were completed within 12 weeks of LB. Biopsies were scored by two blinded expert pathologists, according to the Ludwig system. Diagnostic accuracy was estimated using the area under the receiver operating characteristic curves (AUROCs) for AF (Ludwig stage ≥III). Effects of biochemical and clinical parameters on liver stiffness measurement (LSM) were appraised. The derivation cohort consisted of 126 patients with valid LSM and LB; VCTE identified patients with AF with an AUROC of 0.89. LSM cutoffs ≤6.5 and >11.0 kPa enabled to exclude and confirm, respectively, AF (negative predictive value [NPV] = 0.94; positive predictive value [PPV] = 0.89; error rate = 5.6%). These values were externally validated in an independent cohort of 91 PBC patients (NPV = 0.93; PPV = 0.89; error rate = 8.6%). Multivariable analysis found that the only parameter affecting LSM was fibrosis stage. No association was found with BMI and liver biochemistry. CONCLUSIONS: In a multicenter study of treatment-naïve PBC patients, we identified two cutoffs (LSM ≤6.5 and >11.0 kPa) able to discriminate at diagnosis the absence or presence, respectively, of AF in PBC patients, with external validation. In patients with LSM between these two cutoffs, VCTE is not reliable and liver biopsy should be evaluated for accurate disease staging. BMI and liver biochemistry did not affect LSMs.

Quality of life in patients with primary biliary cholangitis: A cross-geographical comparison.

BACKGROUND & AIMS: Several symptoms impair the quality of life (QoL) of patients with primary biliary cholangitis (PBC). They are reported to vary significantly in different countries. Aim of our study was to explore whether there is a geographical clustering that accounts for symptoms in PBC. METHODS: Data was analysed from four cohorts of PBC patients from the UK, Spain, Japan and Italy using the PBC-27 scale. RESULTS: Overall, 569 patients from four cohorts were identified, including 515 females (90.5%) with a mean age of 61 years. The analysis provided evidence for strict factorial invariance of the scale, a robust indicator of its validity for cross-cultural research. The mean of the fatigue domain of British patients was significantly greater than that of the Japanese (p â€‹< â€‹0.05), Italian (p â€‹< â€‹0.05), and Spanish patients (p â€‹< â€‹0.001). The mean of the cognitive domain after 54 years of age, was significantly greater in the British patients than in the Japanese (p â€‹< â€‹0.05) and Spanish patients (p â€‹< â€‹0.01). However, after 69 years of age, there were not significant differences between countries. The mean of the emotion domain after 54 years of age, was greater in the British that in the Spanish (p â€‹< â€‹0.01) and Italian patients (p â€‹< â€‹0.01). CONCLUSIONS: Differences in the four countries concerning fatigue, cognitive and emotional dysfunction were found. The association of latitude and symptoms might provide new insights into the role of sun exposure, genetics and/or cultural component into disease phenotype in PBC.

Gliadin, through the Activation of Innate Immunity, Triggers lncRNA NEAT1 Expression in Celiac Disease Duodenal Mucosa.

Celiac disease (CD) is an autoimmune enteropathy arising in genetically predisposed subjects exposed to gluten, which activates both innate and adaptive immunity. Although the pathogenesis is common to all patients, the clinical spectrum is quite variable, and differences could be explained by gene expression variations. Among the factors able to affect gene expression, there are lncRNAs. We evaluated the expression profile of 87 lncRNAs in CD vs. healthy control (HC) intestinal biopsies by RT-qPCR array. Nuclear enriched abundant transcript 1 (NEAT1) and taurine upregulated gene 1 (TUG1) were detected as downregulated in CD patients at diagnosis, but their expression increased in biopsies of patients on a gluten-free diet (GFD) exposed to gluten. The increase in NEAT1 expression after gluten exposure was mediated by IL-15 and STAT3 activation and binding to the NEAT1 promoter, as demonstrated by gel shift assay. NEAT1 is localized in the nucleus and can regulate gene expression by sequestering transcription factors, and it has been implicated in immune regulation and control of cell proliferation. The demonstration of its regulation by gluten thus also supports the role of lncRNAs in CD and prompts further research on these RNAs as gene expression regulators.

Immune system and cholangiocytes: A puzzling affair in primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a cholestatic liver disease characterized by the destruction of the small and medium bile ducts. Its pathogenesis is still unknown. Despite the genome wide association study findings, the therapies targeting the cytokines pathway, tested so far, have failed. The concept of the biliary epithelium as a key player of the PBC pathogenesis has emerged over the last few years. It is now well accepted that the biliary epithelial cells (BECs) actively participate to the genesis of the damage. The chronic stimulation of BECs via microbes and bile changes the cell phenotype toward an active state, which, across the production of proinflammatory mediators, can recruit, retain, and activate immune cells. The consequent immune system activation can in turn damage BECs. Thus, the crosstalk between both innate and adaptive immune cells and the biliary epithelium creates a paracrine loop responsible for the disease progression. In this review, we summarize the evidence provided in literature about the role of BECs and the immune system in the pathogenesis of PBC. We also dissect the relationship between the immune system and the BECs, focusing on the unanswered questions and the future potential directions of the translational research and the cellular therapy in this area.

New Therapeutic Targets in Autoimmune Cholangiopathies.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are autoimmune cholangiopathies characterized by limited treatment options. A more accurate understanding of the several pathways involved in these diseases has fostered the development of novel and promising targeted drugs. For PBC, the characterization of the role of farnesoid X receptor (FXR) and perixosome-proliferator activated receptor (PPAR) has paved the way to several clinical trials including different molecules with choleretic and antinflammatory action. Conversely, different pathogenetic models have been proposed in PSC such as the "leaky gut" hypothesis, a dysbiotic microbiota or a defect in mechanisms protecting against bile acid toxicity. Along these theories, new treatment approaches have been developed, ranging from drugs interfering with trafficking of lymphocytes from the gut to the liver, fecal microbiota transplantation or new biliary acids with possible immunomodulatory potential. Finally, for both diseases, antifibrotic agents are under investigation. In this review, we will illustrate current understanding of molecular mechanisms in PBC and PSC, focusing on actionable biological pathways for which novel treatments are being developed.

Autoantibodies in patients with interleukin 12 receptor beta 1 deficiency.

OBJECTIVE: Interleukin 12 receptor beta 1 (IL-12Rß1) deficiency is a primary immunodeficiency that exposes affected individuals to an augmented risk of intracellular pathogen-mediated infections. The paradoxical presence of autoimmune manifestations in immune-deficient patients has been recognized, but the basis of this phenomenon is unclear, with the role of frequent infections being a possible trigger to break tolerance. Our study aimed to analyze extensively a profile of autoantibodies in a clinically well-defined case series of patients with IL-12Rß1 deficiency. METHODS: Eight patients with IL-12Rß1 deficiency referred to Children's Medical Center in Tunis, Tunisia, during 1995-2012 were enrolled in the study. Sixteen age- and gender-matched blood donors served as controls. Serum, liver-related autoantibodies immunoglobulin (Ig)G, IgM, IgA were tested by ELISA and by standard indirect immunofluorescence on Hep-2 cells. RESULTS: We found a significant prevalence of liver autoantibodies in the study group. Regarding primary biliary cholangitis (PBC), two of eight patients were positive for MIT3 autoantibodies, both confirmed by immunofluorescence, and one patient was positive for PBC-specific antinuclear antibodies, sp100. Moreover, two patients had significantly increased gamma-glutamyltransferase levels and one had IgM levels twice the upper limit of normal. Intriguingly two patients were positive for anti-actin antibodies; a typical feature of autoimmune hepatitis type 1, along with a significant increase in IgG levels. CONCLUSIONS: This is the first report of a serological analysis in patients with an IL-12Rß1 deficiency. Despite the difficulty in interpreting the role of the IL-12, the evidence of liver-specific autoantibodies confirms the importance its signal in liver autoimmunity.

Food additives can act as triggering factors in celiac disease: Current knowledge based on a critical review of the literature.

Celiac disease (CeD) is an autoimmune disorder, mainly affecting the small intestine, triggered by the ingestion of gluten with the diet in subjects with a specific genetic status. The passage of gluten peptides through the intestinal barrier, the uptake by antigen presenting cells and their presentation to T cells represent essential steps in the pathogenesis of the disease. CeD prevalence varies in different populations, but a tendency to increase has been observed in various studies in recent years. A higher amount of gluten in modern grains could explain this increased frequency, but also food processing could play a role in this phenomenon. In particular, the common use of preservatives such as nanoparticles could intervene in the pathogenesis of CeD, due to their possible effect on the integrity of the intestinal barrier, immune response or microbiota. In fact, these alterations have been reported after exposure to metal nanoparticles, which are commonly used as preservatives or to improve food texture, consistency and color. This review will focus on the interactions between several food additives and the intestine, taking into account data obtained in vitro and in vivo, and analyzing their effect in respect to the development of CeD in genetically predisposed individuals.

miRNA-regulated gene expression differs in celiac disease patients according to the age of presentation.

Celiac disease is an intestinal disease which shows different symptoms and clinical manifestations among pediatric and adult patients. These variations could be imputable to age-related changes in gut architecture and intestinal immune system, which could be characterized by gene expression differences possibly regulated by miRNAs. We analyzed a panel of miRNAs and their target genes in duodenal biopsies of Marsh 3AB and 3C pediatric celiac patients, compared to controls. Moreover, to assess variation of expression in plasma samples, we evaluated circulating miRNA levels in controls and patients at diagnosis or on gluten-free diet. We detected a decreased miR-192-5p expression in celiac patients, but no variations in NOD2 and CXCL2, targets previously identified in adults. Conversely, we detected a significant increase in mRNA and protein levels of another target, MAD2L1, protein related to cell cycle control. miR-31-5p and miR-338-3p were down-regulated and their respective targets, FOXP3 and RUNX1, involved in Treg function, resulted up-regulated in celiac patients. Finally, we detected, in celiac patients, an increased expression of miR-21-5p, possibly caused by a regulatory loop with its putative target STAT3, which showed an increased activation in Marsh 3C patients. The analysis of plasma revealed a trend similar to that observed in biopsies, but in presence of gluten-free diet we could not detect circulating miRNAs values comparable to controls. miRNAs and their gene targets showed an altered expression in duodenal mucosa and plasma of celiac disease pediatric patients, and these alterations could be different from adult ones.