The Circulating Level of Soluble Receptor for Advanced Glycation End Products Displays Different Patterns in Ulcerative Colitis and Crohn's Disease: A Cross-Sectional Study.

BACKGROUND: RAGE is a transmembrane receptor expressed on immune and endothelial cells, whose binding with its ligands, the S100 calgranulins, leads to chronic inflammation. Conversely, its soluble form (sRAGE) plays a protective role by acting as a decoy. We carried out a cross-sectional analysis of the sRAGE and S100A12 serum levels in patients with Crohn's disease (CD) and ulcerative colitis (UC) and searched for a correlation with clinical and biological markers of activity. METHODS: We enrolled 60 CD, 67 UC patients, and 66 controls (all adults). Disease activity was scored through the clinical, endoscopic, and histologic indexes of severity, whilst disease location and behaviour were assessed according to the Montreal classification. In all cases, the levels of serum sRAGE, S100A12, C-reactive protein, and faecal calprotectin were measured. RESULTS: sRAGE levels were significantly lower in UC, both active and inactive, than in controls and CD (817.35, range 437.3-1449; 1211, range 843.7-1618; 1207.5, range 743.15-1875.75; P < 0.05 for both), and inversely correlated with clinical and endoscopic indexes of activity in both IBD groups (P < 0.05 for all) and with the histologic score in the CD group. Moreover, those CD patients with a penetrating behaviour showed a significant reduction in both sRAGE (P = 0.006) and S100A12 (P = 0.034) as compared to those with an inflammatory/stricturing pattern. Although S100A12 levels were not found up-regulated, a negative correlation appeared evident with the clinical (r = -0.38) and endoscopic (r = -0.32) indexes of activity in UC and CD, respectively. CONCLUSION: These data suggest a different role for RAGE in CD and UC, and a potential use of sRAGE as a new biomarker.

Tolerogenic effect of mesenchymal stromal cells on gliadin-specific T lymphocytes in celiac disease.

BACKGROUND AIMS: Celiac disease is caused by a dysregulated immune response toward dietary gluten, whose only treatment is a lifelong gluten-free diet. We investigated the effects of mesenchymal stromal cells (MSCs) on gliadin-specific T cells, which are known to induce intestinal lesions, in view of a possible use as new therapy. METHODS: Bone marrow-derived MSCs and gliadin-specific T-cell lines were obtained from allogeneic donors and mucosal specimens of celiac patients, respectively. The immunosuppressant effect of MSCs was evaluated in terms of proliferative response and interferon (IFN)-γ production upon gliadin stimulation of long-term T-cell lines; the immunomodulant effect was assessed in terms of apoptotic rate, immunophenotype and cytokine profile of short-term T-cell lines generated in the presence of MSCs. Different MSC:T-cell ratios were applied, and statistics were performed as appropriate. RESULTS: MSCs inhibited both proliferative response and IFN-γ production of long-term T-cell lines in a dose-dependent manner while limiting the expansion of short-term T-cell lines by increasing the apoptotic rate. Moreover, a reduction of the CD4(+) population and expansion of the regulatory FoxP3+ subset were found in T-cell lines cultured with MSCs, in which a significant decrease of interleukin (IL)-21, IFN-γ and IL-10 paralleled by an upregulation of transforming growth factor-ß1, IL-6 and IL-8 were observed. Finally, an increase of the indoleamine 2,3-dioxygenase activity was found, possibly playing a key role in mediating these effects. CONCLUSIONS: MSCs exert potent immunomodulant effects on gliadin-specific T cells, which may be exploited for future therapeutic application in celiac disease.

Insights into Energy Transfer in Light-Harvesting Complex II Through Machine-Learning Assisted Simulations.

Light-harvesting complex II (LHCII) is the major antenna of higher plants. Energy transfer processes taking place inside its aggregate of chlorophylls have been experimentally investigated with time-resolved techniques, but a complete understanding of the most relevant energy transfer pathways and relative characteristic times remains elusive. Theoretical models to disentangle experimental data in LHCII have long been challenged by the large size and complex nature of the system. Here, we show that a fully first-principles approach combining molecular dynamics and machine learning can be successfully used to reproduce transient absorption spectra and characterize the EET pathways and the involved times.

Flow cytometry for the assessment and monitoring of aberrant intraepithelial lymphocytes in non-responsive celiac disease and non-celiac enteropathies.

BACKGROUND: Few data are available on flow cytometry (FC) for monitoring intraepithelial lymphocytes (IELs) in refractory celiac disease (RCD), non-responsive celiac disease (NRCD), and non-celiac enteropathies (NCEs). AIMS: 1) To investigate the significance of monitoring IELs immunophenotype with FC in patients with NRCD, RCD and NCEs; 2) to evaluate FC concordance with immunohistochemistry (IHC) and γ-TCR clonality analysis. METHODS: Patients investigated between January-2012 and February-2023 were divided into two groups: 1)confirmed RCD or NRCD being investigated for persistent symptoms and suspected complications of celiac disease (CD); 2)NCEs lacking clinical/histological response. Clinical/molecular features and outcomes were retrospectively collected and analysed according to presence/absence of aberrant IELs on FC (cut-off≥20 % CD103+sCD3-CD8-iCD3+ IELs). RESULTS: 52 patients (18 RCD,21 NRCD,13 NCEs; 38F, 55±13 years; median follow-up 30 months, IQR 2-58) underwent 100 FC IELs determinations. 22/52 had ≥2 FC determinations and IEL phenotype remained unchanged over time in all them (κ=1.00). Aberrant IEL phenotype in CD was associated with increased mortality (HR 4.2, 95 % CI 1.5-11.9, p < 0.01). No patients with NCEs had an aberrant IEL phenotype at FC, although 3/13 developed lymphoma and 4/13 died. Concordance of FC was fair with both IHC (κ=0.40) and γ-TCR clonality analysis (κ=0.22). CONCLUSION: FC is accurate for assessing and monitoring IEL phenotype and providing important prognostic information in celiac patients. Further study is needed on its role in NCEs.

Relationship between duodenal microbiota composition, clinical features at diagnosis, and persistent symptoms in adult Coeliac disease.

BACKGROUND: Duodenal dysbiosis has been suggested to possibly influence the clinical manifestations of coeliac disease (CD), both at onset and when symptoms persist despite a gluten-free diet (GFD). AIMS: To evaluate the relationship between duodenal microbiota composition and: i) clinical phenotype of untreated CD (UCD); ii) presence and type of persistent symptoms despite a satisfactory serological and histological response to a strict GFD. METHODS: Duodenal microbiota was analyzed by 16S rRNA sequencing and compared with i) clinical features in 12 adult UCD patients; ii) presence/absence and type of persistent symptoms (diarrhea-predominant vs. non-diarrhea predominant) in 25 adult treated coeliac patients (TCD) on a strict GFD. RESULTS: UCD with iron deficiency anemia (IDA) had a pro-inflammatory shift in their duodenal microbiota (reduction of Firmicutes, p = 0.03; increase of beta-Proteobacteria, p = 0.02) than those without IDA. TCD with persistent diarrhea showed a reduction of Actinobacteria (p = 0.03) and Rothia spp (p = 0.046) compared to TCD suffering from other type of persistent symptoms. CONCLUSION: A distinctive duodenal microbiota profile is associated with IDA in UCD, and diarrhea-predominant persistent symptoms in TCD. Clinical interventions may include reconsidering patients presenting with IDA as a specific disease subtype, and dietary rebalancing if diarrhea persists despite histological response to a GFD.

Human Cytomegalovirus and Epstein-Barr virus specific immunity in patients with ulcerative colitis.

Human Cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) are endowed with the ability of establishing lifelong latency in human hosts and reactivating in immunocompromised subjects, including patients suffering from ulcerative colitis (UC). We, therefore, aimed to investigate virus-specific immunity in UC patients. A cohort of 24 UC patients (14 responders and 10 refractory to therapy) and 26 control subjects was prospectively enrolled to undergo virus-specific serology (by ELISA assay) and assessment of both CD4+ and CD8+ virus-specific T-cell response (by interferon-γ enzyme-linked immunospotanalysis). In parallel, mucosal viral load was determined by quantitative real-time PCR and the values were correlated with both clinical and endoscopic indexes of activity. For statistics, the t-test, Mann-Withney test, Fisher's exact test and Spearman rank correlation test were applied; p < 0.05 was considered significant. EBV-specific CD4+ and CD8+ T-cell responses were significantly lower in UC patients compared to controls (p < 0.0001 and p = 0.0006, respectively), whereas no difference was found for HCMV-specific T-cell response. When dividing the UC group according to response to therapy, both responders and refractory UC patients showed a deficient EBV-specific CD4+ T-cell response with respect to controls (p < 0.04 and p = 0.0003, respectively). Moreover, both EBV and HCMV mucosal loads were significantly higher in refractory UC than in responders and controls (p = 0.007 and 0.003; and p = 0.02 and 0.001, respectively), and correlated with activity indexes. Steroid therapy seemed the main risk factor for triggering EBV colitis. Finally, no cases of IgM positivity were found in the study population. An impaired EBV-specific immunity was clearly evident in UC patients, mostly in those refractory to therapy. The ELISPOT assay may serve as new tool for quantifying and monitoring virus-specific T-cell immunity in UC.

Comparative Study of Salivary, Duodenal, and Fecal Microbiota Composition Across Adult Celiac Disease.

BACKGROUND: Growing evidence suggests that an altered microbiota composition contributes to the pathogenesis and clinical features in celiac disease (CD). We performed a comparative analysis of the gut microbiota in adulthood CD to evaluate whether: (i) dysbiosis anticipates mucosal lesions, (ii) gluten-free diet restores eubiosis, (iii) refractory CD has a peculiar microbial signature, and (iv) salivary and fecal communities overlap the mucosal one. METHODS: This is a cross-sectional study where a total of 52 CD patients, including 13 active CD, 29 treated CD, 4 refractory CD, and 6 potential CD, were enrolled in a tertiary center together with 31 controls. A 16S rRNA-based amplicon metagenomics approach was applied to determine the microbiota structure and composition of salivary, duodenal mucosa, and stool samples, followed by appropriate bioinformatic analyses. RESULTS: A reduction of both α- and ß-diversity in CD, already evident in the potential form and achieving nadir in refractory CD, was evident. Taxonomically, mucosa displayed a significant abundance of Proteobacteria and an expansion of Neisseria, especially in active patients, while treated celiacs showed an intermediate profile between active disease and controls. The saliva community mirrored the mucosal one better than stool. CONCLUSION: Expansion of pathobiontic species anticipates villous atrophy and achieves the maximal divergence from controls in refractory CD. Gluten-free diet results in incomplete recovery. The overlapping results between mucosal and salivary samples indicate the use of saliva as a diagnostic fluid.

Clinical phenotype and mortality in patients with idiopathic small bowel villous atrophy: a dual-centre international study.

OBJECTIVE: Causes of small-bowel villous atrophy (VA) include coeliac disease (CD), its complications and other rare non-coeliac enteropathies. However, forms of VA of unknown aetiology may also exist. We defined them as idiopathic VA (IVA). To retrospectively classify the largest cohort of IVA patients and compare their natural history with CD. METHODS: Notes of 76 IVA patients attending two tertiary centres between January 2000 and March 2019 were retrospectively reviewed. CD, its complications and all the known causes of VA were excluded in all of them. Persistence of VA during follow-up and lymphoproliferative features were used to retrospectively classify IVA, as follows. Group 1: IVA with spontaneous histological recovery (50 patients). Group 2: persistent IVA without lymphoproliferative features (14 patients). Group 3: persistent IVA with lymphoproliferative features (12 patients). Survival was compared between IVA groups and 1114 coeliac patients. HLA was compared between IVA patients, coeliac patients and appropriate controls. RESULTS: Five-year survival was 96% in IVA group 1, 100% in IVA group 2, 27% in IVA group 3 and 97% in CD. On a multivariate analysis hypoalbuminemia (P = 0.002) and age at diagnosis (P = 0.04) predicted mortality in IVA. Group 2 showed association with HLA DQB1*0301 and DQB1*06. CONCLUSION: IVA consists of three groups of enteropathies with distinct clinical phenotypes and prognoses. Mortality in IVA is higher than in CD and mainly due to lymphoproliferative conditions necessitating more aggressive therapies.

Functional polymorphisms of the receptor for the advanced glycation end product promoter gene in inflammatory bowel disease: a case-control study.

The receptor for the advanced glycation end products (RAGE) is a multiligand transmembrane receptor involved in chronic inflammation whose specific polymorphisms of the promoter gene were found to increase its transcriptional activity. We investigated the association of both allelic and genotypic -374T/A and -429T/C polymorphisms with inflammatory bowel disease. The STREGA guidelines were applied for planning and reporting. We enrolled 133 patients with Crohn's disease (CD), 149 with ulcerative colitis (UC), and 128 blood donors. Genomic DNA was extracted from peripheral blood leukocytes collected from each patient and control. RAGE polymorphisms were analyzed by PCR-restriction fragment length polymorphism assay. The Hardy-Weinberg equilibrium was first assessed, and then, the Kruskal-Wallis test and the Fisher exact test were used for etiologic group comparisons. Distribution of patients' characteristics across genotypes was evaluated by the Fisher exact test, while that across alleles was analyzed with a probit model. A 2-sided value of p < 0.05 was considered significant. Following the evidence of the Hardy-Weinberg equilibrium, we found a higher prevalence of the allele A of the -374T/A haplotype in UC (p = 0.043), and of the allele C of the -429T/C haplotype in CD (p < 0.001) with respect to the other groups. Moreover, the homozygous AA genotype of the -374T/A polymorphism resulted associated with late onset of CD, while its TT genotype with early onset (p = 0.049). The allele C of the 429T/C haplotype was associated with early onset of UC (p = 0.03), while a higher frequency of the heterozygous TC haplotype was found in those with pancolitis (p = 0.026). The differing distribution of these polymorphisms in healthy donors and CD/UC patients suggests a role in the development and outcome of these pathological conditions.

There Is No Association between Coeliac Disease and Autoimmune Pancreatitis.

Autoimmune pancreatitis (AIP) is a rare disorder whose association with coeliac disease (CD) has never been investigated, although CD patients display a high prevalence of both endocrine and exocrine pancreatic affections. Therefore, we sought to evaluate the frequency of CD in patients with AIP and in further medical pancreatic disorders. The screening for CD was carried out through the detection of tissue transglutaminase (tTG) autoantibodies in sera of patients retrospectively enrolled and divided in four groups: AIP, chronic pancreatitis, chronic asymptomatic pancreatic hyperenzymemia (CAPH), and control subjects with functional dyspepsia. The search for anti-endomysium autoantibodies was performed in those cases with borderline or positive anti-tTG values. Duodenal biopsy was offered to all cases showing positive results. One patient out of 72 (1.4%) with AIP had already been diagnosed with CD and was following a gluten-free diet, while one case out of 71 (1.4%) with chronic pancreatitis and one out of 92 (1.1%) control subjects were diagnosed with de novo CD. No cases of CD were detected in the CAPH group. By contrast, a high prevalence of cases with ulcerative colitis was found in the AIP group (13.8%). Despite a mutual association between CD and several autoimmune disorders, our data do not support the serologic screening for CD in AIP. Further studies will clarify the usefulness of CD serologic screening in other pancreatic disorders.

Differential cellular localization of Epstein-Barr virus and human cytomegalovirus in the colonic mucosa of patients with active or quiescent inflammatory bowel disease.

The role of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in the exacerbation of inflammatory bowel disease (IBD) is still uncertain. We prospectively investigated the presence of EBV and HCMV infection in both epithelial and immune cells of colonic mucosa of IBD patients, both refractory and responders to standard therapies, in comparison with patients suffering from irritable bowel syndrome who were considered as controls, by using quantitative real-time polymerase chain reaction, immunohistochemistry and in situ hybridization, in an attempt to assess viral localization, DNA load, life cycle phase and possible correlation with disease activity indexes. We obtained clear evidence of the presence of high DNA loads of both viruses in either enterocytes or immune cells of refractory IBD patients, whereas we observed low levels in the responder group and an absence of detectable copies in all cell populations of controls. Remarkably, the values of EBV and HCMV DNA in inflamed mucosa were invariably higher than in non-inflamed areas in both IBD groups, and the EBV DNA loads in the cell populations of diseased mucosa of refractory IBD patients positively correlated with the severity of mucosal damage and clinical indexes of activity. Moreover, EBV infection resulted the most prevalent either alone or in combination with HCMV, while immunohistochemistry and in situ hybridization did not allow us to distinguish between the different phases of viral life cycle. Finally, as regards treatment, these novel findings could pave the way for the use of new antiviral molecules in the treatment of this condition.

A Refractory Celiac Patient Successfully Treated With Mesenchymal Stem Cell Infusions.

Type II refractory celiac disease (RCD), as defined according to the amount of aberrant intraepithelial lymphocytes, is a condition characterized by severe malabsorption syndrome and poor prognosis, with no effective treatment. Based on the regenerative and immunomodulatory properties of mesenchymal stem cells (MSCs), we investigated the feasibility, safety, and efficacy of serial infusions of autologous bone marrow-derived MSCs in a 51-year-old woman with type II RCD. Mesenchymal stem cells were isolated, expanded, and characterized following standard protocols. Monitoring of the patient's malabsorption indexes, mucosal architecture, and percentage of aberrant intraepithelial lymphocytes was scheduled for the time of enrollment, at each infusion, and after 6 months. Determination of mucosal expression of interleukin (IL)-15 and its receptor was also performed. Expansion of MSCs was feasible, and the patient underwent 4 systemic infusions of 2 × 10(6) MSCs/kg body weight 4 months apart, without adverse effects. During the treatment period, she experienced gradual and durable amelioration of her general condition, with normalization of stool frequency, body mass index, laboratory test results, and mucosal architecture. Remarkably, the expression of IL-15 and its receptor almost completely disappeared. Thus, treatment of RCD with serial MSC infusions seems promising, leading to recovery from the life-threatening condition while blocking the IL-15 pathogenic pathway.

The Spectrum of Differences between Childhood and Adulthood Celiac Disease.

An old saying states that ''children are not little adults" and this certainly holds true for celiac disease, as there are many peculiar aspects regarding its epidemiology, diagnosis, clinical presentations, associated diseases, and response to treatment in pediatric compared to adult populations, to such an extent that it merits a description of its own. In fact, contrary to the past when it was thought that celiac disease was a disorder predominantly affecting childhood and characterized by a malabsorption syndrome, nowadays it is well recognized that it affects also adult and elderly people with an impressive variability of clinical presentation. In general, the clinical guidelines for diagnosis recommend starting with specific serologic testing in all suspected subjects, including those suffering from extraintestinal related conditions, and performing upper endoscopy with appropriate biopsy sampling of duodenal mucosa in case of positivity. The latter may be omitted in young patients showing high titers of anti-transglutaminase antibodies. The subsequent management of a celiac patient differs substantially depending on the age at diagnosis and should be based on the important consideration that this is a lifelong condition.

Human cytomegalovirus and Epstein-Barr virus infection in inflammatory bowel disease: need for mucosal viral load measurement.

AIM: To evaluate the best diagnostic technique and risk factors of the human Cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) infection in inflammatory bowel disease (IBD). METHODS: A cohort of 40 IBD patients (17 refractory) and 40 controls underwent peripheral blood and endoscopic colonic mucosal sample harvest. Viral infection was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry, and correlations with clinical and endoscopic indexes of activity, and risk factors were investigated. RESULTS: All refractory patients carried detectable levels of HCMV and/or EBV mucosal load as compared to 13/23 (56.5%) non-refractory and 13/40 (32.5%) controls. The median DNA value was significantly higher in refractory (HCMV 286 and EBV 5.440 copies/10(5) cells) than in non-refractory (HCMV 0 and EBV 6 copies/10(5) cells; P < 0.05 and < 0.001) IBD patients and controls (HCMV and EBV 0 copies/10(5) cells; P < 0.001 for both). Refractory patients showed DNA peak values ≥ 10(3) copies/10(5) cells in diseased mucosa in comparison to non-diseased mucosa (P < 0.0121 for HCMV and < 0.0004 for EBV), while non-refractory patients and controls invariably displayed levels below this threshold, thus allowing us to differentiate viral colitis from mucosal infection. Moreover, the mucosal load positively correlated with the values found in the peripheral blood, whilst no correlation with the number of positive cells at immunohistochemistry was found. Steroid use was identified as a significant risk factor for both HCMV (P = 0.018) and EBV (P = 0.002) colitis. Finally, a course of specific antiviral therapy with ganciclovir was successful in all refractory patients with HCMV colitis, whilst refractory patients with EBV colitis did not show any improvement despite steroid tapering and discontinuation of the other medications. CONCLUSION: Viral colitis appeared to contribute to mucosal lesions in refractory IBD, and its correct diagnosis and management require quantitative real-time polymerase chain reaction assay of mucosal specimens.

Ex vivo immunosuppressive effects of mesenchymal stem cells on Crohn's disease mucosal T cells are largely dependent on indoleamine 2,3-dioxygenase activity and cell-cell contact.

INTRODUCTION: Crohn's disease (CD) is a disabling chronic enteropathy sustained by a harmful T-cell response toward antigens of the gut microbiota in genetically susceptible subjects. Growing evidence highlights the safety and possible efficacy of mesenchymal stem cells (MSCs) as a new therapeutic tool for this condition. Therefore, we aimed to investigate the effects of bone marrow-derived MSCs on pathogenic T cells with a view to clinical application. METHODS: T-cell lines from both inflamed and non-inflamed colonic mucosal specimens of CD patients and from healthy mucosa of control subjects were grown with the antigen muramyl-dipeptide in the absence or presence of donors' MSCs. The MSC effects were evaluated in terms of T-cell viability, apoptotic rate, proliferative response, immunophenotype, and cytokine profile. The role of the indoleamine 2,3-dioxygenase (IDO) was established by adding a specific inhibitor, the 1-methyl-DL-tryptophan, and by using MSCs transfected with the small interfering RNA (siRNA) targeting IDO. The relevance of cell-cell contact was evaluated by applying transwell membranes. RESULTS: A significant reduction in both cell viability and proliferative response to muramyl-dipeptide, with simultaneous increase in the apoptotic rate, was found in T cells from both inflamed and non-inflamed CD mucosa when co-cultured with MSCs and was reverted by inhibiting IDO activity and expression. A reduction of the activated CD4(+)CD25(+) subset and increase of the CD3(+)CD69(+) population were also observed when T-cell lines from CD mucosa were co-cultured with MSCs. In parallel, an inhibitory effect was evident on the expression of the pro-inflammatory cytokines tumor necrosis factor-α, interferon-γ, interleukin-17A and -21, whereas that of the transforming growth factor-ß and interleukin-6 were increased, and production of the tolerogenic molecule soluble HLA-G was high. These latter effects were almost completely eliminated by blocking the IDO, whose activity was upregulated in MSCs co-cultured with CD T cells. The use of a semipermeable membrane partially inhibited the MSC immunosuppressive effects. Finally, hardly any effects of MSCs were observed when T cells obtained from control subjects were used. CONCLUSION: MSCs exert potent immunomodulant effects on antigen-specific T cells in CD through a complex paracrine and cell-cell contact-mediated action, which may be exploited for widespread therapeutic use.

Role of the advanced glycation end products receptor in Crohn's disease inflammation.

AIM: To investigate the level of mucosal expression and the involvement of the receptor for the advanced glycation end products (RAGE) in delayed apoptosis and tumor necrosis factor (TNF)-α production in Crohn's disease (CD). METHODS: Surgical and endoscopic specimens from both inflamed and non-inflamed areas of the ileum and/or colon were collected from 20 and 14 adult CD patients, respectively, and used for the assessment of RAGE expression by means of immunohistochemistry and western blotting analysis. Normal tissues from 21 control subjects were used for comparison. The same polyclonal anti-human RAGE antibody (R and D System) was used in all experimental conditions. RAGE staining was quantized by a score including both the amount of positive cells and intensity of immunoreactivity; cellular pattern was also described. The effects of RAGE blocking on apoptotic rate and TNF-α production were investigated on immune cells freshly isolated from CD mucosa and incubated both with and without the muramyl dipeptide used as antigenic stimulus. Statistical analysis was performed via the test for trend, with regression models to account for intra-patient correlations. A 2-sided P < 0.05 was considered significant. RESULTS: In inflamed areas, RAGE expression in both the epithelial and lamina propria compartments was higher than control tissues (P = 0.001 and 0.021, respectively), and a cluster of positive cells were usually found in proximity of ulcerative lesions. Similar results were obtained in the lamina propria compartment of non-inflamed areas (P = 0.025). The pattern of staining was membranous and granular cytosolic at the epithelial level, while in the lamina propria it was diffuse cytosolic. When evaluating the amount of protein expression by immunoblotting, a significant increase of both surface area and band intensity (P < 0.0001 for both) was observed in CD inflamed areas compared to control tissue, while in non-inflamed areas a significant increase was found only for band intensity (P < 0.005). Moreover, a significantly lower expression in non-inflamed areas in comparison with inflamed areas was found for both surface area and band intensity (P < 0.0006 for both). Finally, RAGE blocking largely affects both the apoptotic rate of mucosal cells (towards an increase in both non-inflamed and inflamed areas of P < 0.001 and < 0.0001, respectively) and TNF-α secretion (towards a decrease in both non-inflamed and inflamed areas of P < 0.05 and < 0.01, respectively), mainly in the presence of antigenic stimulation. CONCLUSION: RAGE is up-regulated in CD, especially in inflamed areas, and it appears to play a role in the mechanisms involved in chronic inflammation.