Isolation and culture of fibroblasts from endoscopic duodenal biopsies of celiac patients

BACKGROUND: Fibroblasts are actually considered pivotal in inflammation and tissue remodelling process and for these reasons they are involved in the pathogenesis of autoimmune disorders such as celiac disease. Investigations to define the role of fibroblasts in celiac diseases are obstructed by the absence of specific models. Our objective is to isolate and culture primary fibroblasts from endoscopic duodenal biopsies of celiac and non-celiac subjects, to analyze their growth patterns and the morphometric characteristics. METHODS: 60 duodenal bioptic specimens from 20 celiac patients and 114 from 38 non-celiac subjects were mechanically chopped and enzymatically digested in order to obtain primary cell cultures. Growth patterns, karyotype (Q-banding analysis), expression of typing proteins (fibroblast surface protein and cytokeratin 20) and morphometric parameters (diameters and their ratio, perimeter, area and perimeter/area ratio at computerised image analysis) were investigated on cultured cells. RESULTS: Primary cells were successfully cultured in 78% of the collected duodenal biopsies. Cultured cells, expressing the fibroblast surface protein, were negative for cytokeratine 20 and maintained a normal kariotype. Cells grew slowly without differences between the celiac and the non celiac group. Morphometric analysis of celiac fibroblasts revealed significantly increased dimensions, with a preserved diameters ratio, and a reduced perimeter/area ratio. CONCLUSION: For the first time this study demonstrates the feasibility of culturing primary fibroblast cell from endoscopic duodenal biopsies in celiac and non-celiac subjects, opening a new window of opportunity in studies intended to establish the role of fibroblasts as a possible partaker in the pathogenesis of the celiac mucosal damage

A Retrospective Study on Dietary FODMAP Intake in Celiac Patients Following a Gluten-Free Diet

Our aim was to evaluate the intake of foods containing fermentable oligo/di/mono-saccharides and polyols (FODMAP) as a possible factor that induces gastrointestinal symptoms in treated celiac disease (CD) patients. We collected seven-day weighed food records for 104 CD patients and 91 healthy volunteers. All evaluated food items were from sources with high and low content of FODMAP, which were divided into cereals and sweets, sweeteners and soft drinks, fruits, dried fruits, and vegetables. Nutrient intake was calculated using the food database of the European Institute of Oncology. The symptoms reported were assessed by a Rome IV Irritable bowel syndrome (IBS) diagnostic questionnaire and by specific questions for the evaluation of functional gastrointestinal disorders (FGIDs). The 12% of CD patients met IBS symptoms criteria as opposed to 6% of controls (p = 0.09) and 27% of patients reported FGIDs symptoms vs. 22% of healthy controls (p = 0.42). The intake by CD patients was significantly higher than healthy volunteers for: sweeteners and sugars with low content of FODMAP (p = 0.0007), fruits, dried fruits, and vegetables high in FODMAP (p = 0.003) and low in FODMAP (p = 0.04) when compared to controls. CD patients had a lower intake of cereals and sweets with a high content of FODMAP (p = 0.00001). Healthy volunteers consumed significantly higher alcoholic beverages and fats high in FODMAP (both p < 0.044). The mean daily intake of other food categories did not differ between both groups. Even though CD patients had a low intake of gluten-free cereals high in FODMAP, they still consumed a significant amount of fruits and vegetables high in FODMAP. The clinical effect of a concomitant gluten-free diet and low-FODMAP diet should be prospectively evaluated as a supportive therapy in CD patients

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids In vitro

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and re- sistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutami- nase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The aris- ing protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conver- sion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the car- bohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen

Dietary gluten as a conditioning factor of the gut microbiota in celiac disease

The gut microbiota plays a relevant role in determining an individual's health status, and the diet is a major factor in modulating the composition and function of gut microbiota. Gluten constitutes an essential dietary component in Western societies and is the environmental trigger of celiac disease. The presence/absence of gluten in the diet can change the diversity and proportions of the microbial communities constituting the gut microbiota. There is an intimate relation between gluten metabolism and celiac disease pathophysiology and gut microbiota; their interrelation defines intestinal health and homeostasis. Environmental factors modify the intestinal microbiota and, in turn, its changes modulate the mucosal and immune responses. Current evidence from studies of young and adult patients with celiac disease increasingly supports that dysbiosis (i.e., compositional and functional alterations of the gut microbiome) is present in celiac disease, but to what extent this is a cause or consequence of the disease and whether the different intestinal diseases (celiac disease, ulcerative colitis, Crohn disease) have specific change patterns is not yet clear. The use of bacterial-origin enzymes that help completion of gluten digestion is of interest because of the potential application as coadjuvant in the current treatment of celiac disease. In this narrative review, we address the current knowledge on the complex interaction between gluten digestion and metabolism, celiac disease, and the intestinal microbiota

Clinical Value of Tissue Transglutaminase Antibodies in Celiac Patients over a Long Term Follow-Up

Introduction &amp; Aim: Anti-tissue transglutaminase antibody (tTGA) titer is used during the follow-up of celiac patients to evaluate gluten-free diet (GFD) responsiveness. However, no clear data are available on this issue. The aim of this study was to evaluate tTGA significance during celiac disease (CD) monitoring. Methods: From January 2017 to January 2020, consecutive CD patients on a GFD with persistent positive tTGA were enrolled. Antibody titres were evaluated on a yearly basis from CD diagnosis to the last follow-up. Urinary gluten detection tests, duodenal histology and capsule enteroscopy (CE) were performed. A tTGA-positive cohort was compared with a control group composed of 212 treated CD patients with negative tTGA. Results: 65 patients (12% males, median age at enrollment and CD diagnosis, 37 (14–86) and 31 (1–76), respectively, median follow up 4 (1–26) years) presented with positive tTGA during follow-up. Overall, the tTGA titres were 3 (1–79) fold increased (ULN). Three different tTGA trends were recognized: (I) 36 (55%) patients with a progressive titres decrease; (II) 16 (25%) patients with a fluctuating behavior; (III) 13 (20%) patients with a steady state or increased titres. tTGA+ patients did not present with different clinical and demographic parameters. Duodenal atrophy was present in 10% vs. 36% of the tTGA positive vs. negative group (p &lt; 0.005), respectively. Gluten detection results were positive in 3 (8%) cases, all in the III group. In tTGA+ patients, CE did not identify any CD-related complications. Conclusions: tTGA positivity during CD follow up did not present a relevant clinical significance without association with autoimmune comorbidities and mucosal damage

Isolation and culture of fibroblasts from endoscopic duodenal biopsies of celiac patients

Abstract Background Fibroblasts are actually considered pivotal in inflammation and tissue remodelling process and for these reasons they are involved in the pathogenesis of autoimmune disorders such as celiac disease. Investigations to define the role of fibroblasts in celiac diseases are obstructed by the absence of specific models. Our objective is to isolate and culture primary fibroblasts from endoscopic duodenal biopsies of celiac and non-celiac subjects, to analyze their growth patterns and the morphometric characteristics. Methods 60 duodenal bioptic specimens from 20 celiac patients and 114 from 38 non-celiac subjects were mechanically chopped and enzymatically digested in order to obtain primary cell cultures. Growth patterns, karyotype (Q-banding analysis), expression of typing proteins (fibroblast surface protein and cytokeratin 20) and morphometric parameters (diameters and their ratio, perimeter, area and perimeter/area ratio at computerised image analysis) were investigated on cultured cells. Results Primary cells were successfully cultured in 78% of the collected duodenal biopsies. Cultured cells, expressing the fibroblast surface protein, were negative for cytokeratine 20 and maintained a normal kariotype. Cells grew slowly without differences between the celiac and the non celiac group. Morphometric analysis of celiac fibroblasts revealed significantly increased dimensions, with a preserved diameters ratio, and a reduced perimeter/area ratio. Conclusion For the first time this study demonstrates the feasibility of culturing primary fibroblast cell from endoscopic duodenal biopsies in celiac and non-celiac subjects, opening a new window of opportunity in studies intended to establish the role of fibroblasts as a possible partaker in the pathogenesis of the celiac mucosal damage.</p

Vaccination Status and Attitudes towards Vaccines in a Cohort of Patients with Celiac Disease

(1) Background: The identification of vaccination status and attitudes towards vaccines among celiac disease (CD) patients is of great importance, but it has not yet been investigated. The aim of this study was to investigate coverage against vaccine-preventable diseases (VPDs), attitudes towards vaccinations, and its determinants among CD patients. (2) Methods: An anonymous web-based validated questionnaire was sent to a mailing list of CD adult patients. Patients were asked to self-report their previous vaccinations and attitudes towards vaccinations, which were defined as positive, negative, and partially positive/negative. The influencing factors towards vaccinations were investigated, and crude and adjusted odds ratios (AdjORs) with 95% confidence intervals (CIs) were calculated. (3) Results: The questionnaire was sent to 412 patients, with a response rate of 31.6% (130 patients, 105 women, median age 40 years, interquartile range 36–51). Patients self-reported vaccination against the following diseases: 73.8% tetanus, 42.3% flu, 20% measles, mumps and rubella, 19.2% meningitis, and 16.2% pneumococcus. Thirty-two people (24.6%) did not remember all of their previous vaccinations. In total, 104 (80%) respondents had a positive attitude towards vaccines, 25 (19.2%) a partially positive/negative one, and 1 a negative one. The determinants significantly influencing the positive attitude were being a graduate (AdjORs 7.49) and a belief in the possible return of VPDs with declining vaccination coverage rates (AdjORs 7.42), while the use of complementary and alternative medicines (AdjORs 0.11) and past negative experience (AdjORs 0.16) were associated with a negative attitude. (4) Conclusions: Despite four out of five CD patients showing a strong positive attitude towards vaccinations, one out of five had a partially negative one. Only a minority (16–20%) reported being vaccinated against some VPDs potentially harmful to their CD because of hyposplenism, such as meningitis and pneumococcus. The low vaccination rate against some VPDs, in spite of the 80% of CD patients stating a positive attitude towards vaccination, may be explained in part by patients’ vaccine hesitancy and in part by a possible role of physicians in under-prescribing vaccinations to these patients. These results may be a starting point for developing specific vaccination campaigns to increase vaccination rates against VPDs in CD patients