Targeted chitosan nanobubbles as a strategy to down-regulate microRNA-17 into B-cell lymphoma models.

Introduction: MicroRNAs represent interesting targets for new therapies because their altered expression influences tumor development and progression. miR-17 is a prototype of onco-miRNA, known to be overexpressed in B-cell non-Hodgkin lymphoma (B-NHL) with peculiar clinic-biological features. AntagomiR molecules have been largely studied to repress the regulatory functions of up-regulated onco-miRNAs, but their clinical use is mainly limited by their rapid degradation, kidney elimination and poor cellular uptake when injected as naked oligonucleotides. Methods: To overcome these problems, we exploited CD20 targeted chitosan nanobubbles (NBs) for a preferential and safe delivery of antagomiR17 to B-NHL cells. Results: Positively charged 400 nm-sized nanobubbles (NBs) represent a stable and effective nanoplatform for antagomiR encapsulation and specific release into B-NHL cells. NBs rapidly accumulated in tumor microenvironment, but only those conjugated with a targeting system (antiCD20 antibodies) were internalized into B-NHL cells, releasing antagomiR17 in the cytoplasm, both in vitro and in vivo. The result is the down-regulation of miR-17 level and the reduction in tumor burden in a human-mouse B-NHL model, without any documented side effects. Discussion: Anti-CD20 targeted NBs investigated in this study showed physico-chemical and stability properties suitable for antagomiR17 delivery in vivo and represent a useful nanoplatform to address B-cell malignancies or other cancers through the modification of their surface with specific targeting antibodies.

Rapid Immunochromatography on Fresh Intestinal Biopsy Is Highly Accurate in Detecting Intestinal Antitransglutaminase Antibodies.

INTRODUCTION: Intestinal antitransglutaminase antibodies (I-anti-TG2) are a specific marker of celiac disease (CeD). The aim of this study was to evaluate the diagnostic accuracy of a novel application of an immunochromatographic assay referred to as Rapid_AntiTG2 to detect I-anti-TG2 on intestinal biopsy lysate. METHODS: Consecutive pediatric patients referred to a single center for elective upper endoscopy were enrolled. Biopsies were taken from duodenal bulb and distal duodenum. For each sampling site, 2 biopsies were analyzed for standard histology, 1 biopsy was cultured to perform the reference standard assay for I-anti-TG2 detection (endomysium [EMA] biopsy), and 1 biopsy was mechanically lysed to perform Rapid_AntiTG2. The primary outcome was the diagnostic accuracy of Rapid_AntiTG2 on biopsy lysate compared with that of the gold standard (serology + histopathology) for CeD diagnosis. The secondary outcome was the agreement of Rapid_AntiTG2 with EMA biopsy. RESULTS: One hundred forty-eight patients were included. Of them, 79 were those with CeD (64 classical CeD, 2 seronegative CeD, and 13 potential CeD) and 69 were controls. Rapid_AntiTG2 on biopsy lysate had very high diagnostic accuracy (sensitivity 100%, specificity 97%, LR+ 34.1, LR- 0.01) in separating patients with CeD from controls. Diagnostic accuracy was unchanged in patients with potential and seronegative CeD. Rapid_AntiTG2 on biopsy lysate had almost perfect agreement with the EMA biopsy reference test (99% agreement, Cohen K 0.97). DISCUSSION: I-anti-TG2 can be detected with an immunochromatographic assay after simple mechanical lysis of fresh intestinal biopsy with very high diagnostic accuracy. The test is quick and easy to perform and can be widely available in any endoscopy unit. Its implementation would allow a better understanding of the prognostic value of I-anti-TG2 and help clinicians in cases of suspected CeD that are difficult to classify.

Celiac Disease Frequency Is Increased in IgE-Mediated Food Allergy and Could Affect Allergy Severity and Resolution.

OBJECTIVES: An increased frequency of celiac disease (CeD) has been reported in severe Immunoglobulin E (IgE) -mediated food allergy (FA). This observation requires confirmation, and whether CeD affects FA severity and resolution is unknown. The study aims to estimate the prevalence of CeD in patients with FA and to investigate whether CeD affects FA severity and oral tolerance. METHODS: Consecutive patients with FA referred for allergen reintroduction, either to evaluate allergy resolution or to start oral immunotherapy (OIT), were evaluated for CeD and for FA severity. The primary outcome was the prevalence of CeD. Secondary outcomes were the frequency of severe FA and the level of clinical tolerance at study entry and at last follow-up in patients with isolated FA versus patients with FA + CeD. RESULTS: Two hundred twenty-eight patients were included. CeD was confirmed in 15 patients (6.6%) of whom, 8 patients had a previously established diagnosis of CeD and were on a gluten-free diet. Severe FA was observed in 12 patients with FA + CeD (80%) versus 88 patients with FA (42%) ( P = 0.006). At baseline, patients with FA + CeD had significantly higher median allergen-specific IgE levels [61.8 kU/L; interquartile range (IQR) 11.6-279.0] compared to patients with FA (20.3 kU/L; IQR 2.9-72.7) ( P < 0.001). Complete clinical tolerance was observed in 1 of 15 patients (7%) with FA + CeD versus 98 of 205 patients (48%) with FA ( P = 0.002). CONCLUSIONS: CeD is highly prevalent in patients with FA and could affect FA severity and response to OIT. CeD screening should be considered in patients with severe or persistent FA.

Candidate Biomarkers for the Detection of Serious Infections in Children: A Prospective Clinical Study.

Serious bacterial infections (SBI) in children are associated with considerable morbidity and mortality, and their early identification remains challenging. The role of laboratory tests in this setting is still debated, and new biomarkers are needed. This prospective, observational, single-center study aims to evaluate the diagnostic role of blood biomarkers in detecting SBI in children presenting with signs of systemic inflammatory response syndrome (SIRS). A panel of biomarkers was performed, including C-reactive protein (CRP), procalcitonin (PCT), white blood cell count (WBC), absolute neutrophil count (ANC), interleukin (IL)-6, IL-8, IL-10, human terminal complement complex (C5b-9), Plasmalemma-Vesicle-associated protein 1 (PV-1), Intercellular Adhesion Molecule-1 (ICAM-1), and Phospholipase A2 (PLA2). Among 103 patients (median age 2.9 years, 60% males), 39 had a diagnosis of SBI (38%). Significant predictors of SBI were CRP (p = 0.001) and ICAM-1 (p = 0.043). WBC (p = 0.035), ANC (p = 0.012) and ANC/WBC ratio (p = 0.015) were also significantly associated with SBI in children without pre-existing neutropenia. ROC curves, however, revealed suboptimal performance for all variables. Nevertheless, a model that combined CRP and ANC/WBC ratio had more in-depth diagnostic accuracy than either of the two variables. Overall, this study confirms the limited usefulness of blood biomarkers for the early diagnosis of SBI. WBC, ANC, ANC/WBC ratio, CRP, and ICAM-1 showed the best, albeit moderate, diagnostic accuracy.

Spectroscopic investigation of faeces with surface-enhanced Raman scattering: a case study with coeliac patients on gluten-free diet.

Surface-enhanced Raman scattering (SERS) spectra of faecal samples can be obtained by adding AuNP to their methanol extracts according to the reported protocol, and display bands that are due to bilirubin-like species but also to xanthine and hypoxanthine, two metabolic products secreted by gut bacteria. A total of 27 faecal samples from three different groups, i.e. coeliac patients (n = 9), coeliac patients on gluten-free diet (n = 10) and a control group (n = 8), were characterized with both SERS spectroscopy and 16S rRNA sequencing analysis. Significant differences are present between SERS spectra of coeliac patients and those on gluten-free diet, with a marked increase in the relative intensity of both xanthine and hypoxanthine for the latter. Interestingly, these differences do not correlate with bacterial composition as derived from 16S rRNA sequencing.

Management of coeliac disease patients after the confirmation of diagnosis in Central Europe.

BACKGROUND: Recently published paediatric guidelines for diagnosing coeliac disease do not include recommendations on the follow-up of coeliac disease patients. GOAL: The aim of this study was to assess the management practices and experience of coeliac disease patients with their follow-up appointments in Central Europe. STUDY: Gastroenterologists and coeliac disease patients in five Central European countries were asked to complete the web-based questionnaire focusing on coeliac disease management practices. RESULTS: Answers from 147 gastroenterologists and 2041 coeliac disease patients were available for the analysis. More than half of the gastroenterologists (58.5%) schedule the first follow-up visit within 3 months after the diagnosis. At follow-up, tissue transglutaminase antibodies are checked in almost all patients (95.9%). Approximately two-thirds (60.7%) of gastroenterologists refer all of their patients to the dietitian at diagnosis. Similarly, 42.8% of coeliac disease patients reported that they had not been appointed to a dietitian. Almost one-third of coeliac disease patients (30.8%) reported that they had no follow-up appointments with gastroenterologist at all. CONCLUSIONS: Follow-up of coeliac disease patients is suboptimal in Central Europe. Many patients are not followed regularly. A lot of patients are not referred to a dietitian. The recommendations on the optimal follow-up of coeliac disease patients are needed in order to improve patient care.

20-Year Follow-up Study of Celiac Patients Identified in a Mass School Screening: Compliance to Gluten-Free Diet and Autoimmunity.

OBJECTIVES: To investigate the compliance to the gluten-free diet in a cohort of adult celiac patients 20 years after the diagnosis, received in childhood through a mass screening. METHODS: This is an observational historic cohort follow-up study. It was carried out at the Institute for Maternal and Child Health IRCCS Burlo Garofolo, Trieste, Italy. Two matched cohorts of adult celiac patients, diagnosed in childhood through a mass screening or for symptoms were enrolled. Adherence to the gluten free-diet and development of autoimmune diseases were investigated through a questionnaire administrated in the course of a phone interview.The primary study outcome was the adherence to the gluten-free diet, measured through the Biagi questionnaire, in the two cohorts of celiac patients. RESULTS: We contacted 25 patients (mean age 28 years, 19 females) diagnosed with screening and 34 patients (mean age 25 years, 26 females) diagnosed in the same period for symptoms. After 20 years, in the cohort diagnosed with screening and in the cohort diagnosed for symptoms the adherence to the gluten-free diet was optimal in 14 (56%) and 26 (81%), improvable in 5 (20%) and 3 (9%), inadequate in 6 (24%) and 3 (9%), respectively. In the two cohorts, four patients (16%) and six patients (18%) developed other autoimmune diseases. CONCLUSIONS: Twenty years after the diagnosis, near half of the patients diagnosed in a mass screening, does not have an optimal adherence to the gluten-free diet and a remarkable proportion of them have developed another autoimmune disease.

The Knowledge About Celiac Disease Among Healthcare Professionals and Patients in Central Europe.

OBJECTIVES: Celiac disease (CD) remains undiagnosed for a long time in many adult and pediatric patients. We assessed the knowledge about CD among healthcare professionals (HCPs) and CD patients in Central Europe (CE). METHODS: HCPs and CD patients from 5 CE countries were asked to complete the web-based questionnaire about CD. The questions were divided into subsections on epidemiology, clinical presentation, diagnostics, treatment, and follow-up. Achieved scores of different specialists managing patients with CD were compared and regional differences in patients' knowledge were analyzed. RESULTS: Questionnaire was completed by 1381 HCPs and 2262 CD patients or their caregivers from Croatia, Hungary, Germany, Italy, and Slovenia. Mean score achieved by HCPs was 50.9%, and by CD patients 56.4%. Pediatric gastroenterologists scored the highest (69.4%; P < 0.001). There were significant differences in knowledge of patients from different CE regions with German participants scoring the highest (58.3%). Members of CD societies scored higher compared with nonmembers (mean score 58% vs 53.2%; P < 0.001) and patients diagnosed less than 5 years ago scored higher compared with those diagnosed more than 10 years ago (mean score 57.3% vs 54.6%; P < 0.001). CONCLUSIONS: The knowledge about CD among HCPs and CD patients is not satisfactory. Further awareness-raising and learning activities are needed to improve HCPs' knowledge and to minimize the number of unrecognized patients and unnecessary diagnostic delays. Patients should be better informed about their disease to reach higher compliance with the gluten-free diet.

Clinical Presentation in Children With Coeliac Disease in Central Europe.

OBJECTIVES: During the past decades, there has been a shift in the clinical presentation of coeliac disease (CD) to nonclassical, oligosymptomatic, and asymptomatic forms. We assessed clinical presentation of CD in children and adolescents in Central Europe. METHODS: Paediatric gastroenterologists in 5 countries retrospectively reported data of their patients diagnosed with CD. Clinical presentation was analyzed and the differences among very young (<3 years) and older children and adolescents were studied. RESULTS: Data from 653 children and adolescents (median age 7 years 2 months; 63.9% girls) from Croatia, Germany, Hungary, Italy, and Slovenia were available for the analysis. One fifth (N = 134) of all children were asymptomatic. In symptomatic children, the most common leading symptom was abdominal pain (33.3%), followed by growth retardation (13.7%) and diarrhoea (13.3%). The majority of symptomatic children (47.6%; N = 247) were polysymptomatic. Abdominal pain was the most common symptom in polysymptomatic (66.4%) as well as in monosymptomatic children (29.7%). Comparing clinical presentation of CD in very young children (younger than 3 years) with older children (3 years or older), we found that symptoms and signs of malabsorption were significantly more common in younger (P < 0.001), whereas abdominal pain and asymptomatic presentation were more common in older children and adolescents (both P < 0.001). CONCLUSION: In children with CD, abdominal pain has become the most common symptom. However, in younger children, symptoms of malabsorption are still seen frequently. This raises a question about the underlying mechanism of observed change in clinical presentation in favour of nonclassical presentation and asymptomatic disease at certain age.

Circulating PV-1 as a marker of celiac disease-associated liver injury.

Aim: To investigate the role of endothelial PV-1 in patients with untreated celiac disease (CD)-associated liver injury. Materials & methods: PV-1 and PV-1 mRNA were measured in intestinal biopsies from untreated CD patients with elevated or normal alanine transaminase levels, controls, patients with inflammatory bowel disease and patients with toxic liver injury. Circulating PV-1 levels were also evaluated. Results: Circulating PV-1 levels were significantly increased in the serum of patients with CD-associated liver injury and reverted to normal following a gluten-free diet. Mucosal PV-1 and PV-1 mRNA were no different in patients with CD-associated liver injury. Conclusion: Serum but not mucosal PV-1 represents a marker of gluten-dependent liver injury and response to a gluten-free diet in patients with untreated CD.

Diagnostic accuracy and applicability of intestinal auto-antibodies in the wide clinical spectrum of coeliac disease.

BACKGROUND: Intestinal coeliac auto-antibodies are the marker of coeliac disease (CD). Since the determination of these antibodies is still not widely available, we used immunoassays to identify the most suitable technology for revealing intestinal auto-antibodies in the wide clinical spectrum of CD. METHODS: Intestinal auto-antibodies have been prospectively investigated in CD suspected children using two immunoassays: intestinal-deposits of IgA anti-tissue transglutaminase antibodies (anti-tTG) and biopsy-culture IgA anti-endomysium (AEA). Intestinal IgM antibodies have been determined in IgA-deficient subjects. FINDINGS: Two-hundred and twenty-one suspected CD patients were enrolled. Intestinal antibodies were tested positive for both assays in classical CD patients (n = 178) with villous atrophy and positive serum-CD antibodies, potential CD patients (n = 16) with normal intestinal mucosa and positive serum-CD antibodies, and pre-potential CD patients (n = 14) with normal intestinal mucosa and negative serum-CD antibodies. In 13/221 with normal intestinal mucosa, negative CD-serum antibodies and negative intestinal antibodies CD has been excluded. All classical, 14/16 potential and 11/14 pre-potential CD patients on gluten-free diet (GFD) improved their symptoms. In 9/11 pre-potential patients intestinal antibodies disappeared on GFD. Both assays were negative in 69/71 control subjects. The two assays showed high diagnostic sensitivity (100%) and specificity (99%). INTERPRETATION: Intestinal CD-antibodies make prompt diagnosis in the wide clinical spectrum of CD reducing the delay in diagnosis and treatment, especially in pre-potential CD patients. The easy handling biopsy culture assay is an effective diagnostic tool which should be carried out by any gastroenterology unit to recognize all CD clinical manifestations. FUNDING: Interreg Central-Europe, IRCCS "Burlo Garofolo".

The Use of Biopsy and "No-Biopsy" Approach for Diagnosing Paediatric Coeliac Disease in the Central European Region.

OBJECTIVES: The current European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) guidelines introduced the option to diagnose coeliac disease (CD) in children and adolescents without upper endoscopy if the defined criteria are met. The aim of our study was to evaluate how frequently paediatric gastroenterologists in Central Europe used the "no-biopsy" approach and how often the duodenal biopsy could have been omitted. METHODS: Medical records of patients aged < 19 years diagnosed with CD in 2016 from five European countries were analysed, focusing on levels of transglutaminase antibodies (TGA) at the time of diagnosis and on whether the diagnosis was confirmed using duodenal biopsy or "no-biopsy" approach. Clinical presentation and delays until final diagnosis were analysed according to diagnostic approach. RESULTS: Data from 653 children (63.9% female, median age: 7 years, range: 7 months-18.5 years) from Croatia, Hungary, Germany, Italy, and Slovenia were analysed. One fifth (n = 134) of included children were asymptomatic at diagnosis. Of 519 symptomatic children, 107 (20.6%) were diagnosed by the "no-biopsy" approach. Out of the remaining 412 children who underwent duodenal biopsies, 214 (51.9%) had TGA ≥ 10 times upper level of normal (ULN) and would have been eligible for the "no-biopsy" approach. Signs and symptoms of malabsorption were more frequent in children diagnosed without duodenal biopsies. There were no differences in diagnostic delays with respect to the diagnostic approach. CONCLUSION: In this cohort, about 60% of symptomatic CD patients could have been diagnosed without duodenal biopsies. The aim of the "no-biopsy" approach was to make the diagnostic procedure less challenging without compromising its reliability. However, this option was applied only in 20%, in spite of fewer burdens to the family and reduced costs. The reasons for this discrepancy are unknown. Physicians should be made more aware about the reliability of CD diagnosis without biopsies when the ESPGHAN guidelines for CD diagnosis are followed.

Diagnostic Delays in Children With Coeliac Disease in the Central European Region.

OBJECTIVES: Coeliac disease (CD) is a systemic autoimmune disorder affecting about 1% of the population. Many patients remain undiagnosed or are diagnosed with substantial delay. We assessed diagnostic delays in symptomatic CD children in Central Europe (CE). METHODS: Paediatric gastroenterologists in 5 CE countries retrospectively reported data of their patients diagnosed in 2016. Age at first CD-related symptom(s), first visit to paediatric gastroenterologist and confirmed diagnosis were used to determine diagnostic delays. RESULTS: Data from 393 children (65% girls, median age 7 years, range 7 months to 18.5 years) from Croatia, Hungary, Germany, Italy, and Slovenia were analysed. Median duration from first symptom(s) to visit to paediatric gastroenterologist was 5 months (range 0-10 years; preschool 4 months, school-aged 5 months), and further duration until final diagnosis was 1 month (range 0-5 years) with significant regional differences (P < 0.001). Median diagnostic delay was 6 months (range 0-10 years; preschool 5 months, school-aged 7 months). Type of clinical presentation had little, however, significant effect on delays. Reduced body mass in delays longer than 3 years compared with delays shorter than 1 year was found (z score -0.93 vs -0.39, P < 0.05). CONCLUSIONS: Time from first symptoms to CD diagnosis in children in 5 CE countries is slightly shorter compared with few other small paediatric studies, and significantly shorter than reported for adults. Nevertheless, delays of more than 3 years in 6.6% of children are worrisome. Raising awareness about the variable symptoms and implementation of reliable diagnostic tools will further reduce diagnostic delays.

Electrochemical Immunosensor Based on Nanoelectrode Ensembles for the Serological Analysis of IgG-type Tissue Transglutaminase.

Celiac disease (CD) is a gluten-dependent autoimmune disorder affecting a significant percentage of the general population, with increasing incidence particularly for children. Reliable analytical methods suitable for the serological diagnosis of the disorder are urgently required for performing both the early diagnosis and the follow-up of a patient adhering to a gluten-free diet. Herein we report on the preparation and application of a novel electrochemical immunosensor based on the use of ensembles of gold nanoelectrodes (NEEs) for the detection of anti-tissue transglutaminase (anti-tTG), which is considered one reliable serological marker for CD. To this end, we take advantage of the composite nature of the nanostructured surface of membrane-templated NEEs by functionalizing the polycarbonate surface of the track-etched membrane with tissue transglutaminase. Incubation of the functionalized NEE in anti-tTG samples results in the capture of the anti-tTG antibody. Confirmation of the recognition event is achieved by incubating the NEE with a secondary antibody labelled with horseradish peroxidase (HRP): in the presence of H2O2 as substrate and hydroquinone as redox mediator, an electrocatalytic current is indeed generated whose increment is proportional to the amount of anti-tTG captured from the sample. The optimized sensor allows a detection limit of 1.8 ng mL-1, with satisfactory selectivity and reproducibility. Analysis of serum samples from 28 individuals, some healthy and some affected by CD, furnished analytical results comparable with those achieved by classical fluoroenzyme immunoassay (FEIA). We note that the NEE-based immunosensor developed here detects the IgG isotype of anti-tTG, while FEIA detects the IgA isotype, which is not a suitable diagnostic marker for IgA-deficient patients.

A Functional Idiotype/Anti-Idiotype Network Is Active in Genetically Gluten-Intolerant Individuals Negative for Both Celiac Disease-Related Intestinal Damage and Serum Autoantibodies.

An unbalance between Abs that recognize an autoantigen (idiotypes; IDs) and Igs that bind such Abs (anti-IDs) is considered a functional event in autoimmune disorders. We investigated the presence of an ID/anti-ID network in celiac disease (CD), a condition in which antitissue transglutaminase 2 (TG2) Abs are suspected to contribute to CD pathogenesis. To characterize the ID side, we reproduced by in vitro yeast display the intestine-resident Abs from CD and control patients. These TG2-specific IDs were used to identify potential anti-IDs in the serum. We observed elevated titers of anti-IDs in asymptomatic patients with predisposition to CD and demonstrated that anti-ID depletion from the serum restores a detectable humoral response against TG2. Our study provides an alternative approach to quantify CD-related autoantibodies in cases that would be defined "negative serology" with current diagnostic applications. Therefore, we suggest that developments of this technology could be designed for perspective routine tests.

Changing Epidemiology of Liver Involvement in Children With Celiac Disease.

OBJECTIVES: Available data indicate that liver involvement is present in a significant proportion of children with celiac disease (CD) at the diagnosis (elevated transaminases 15%-57%, autoimmune liver disease 1%-2%). We sought to evaluate prevalence, clinical course, and risk factors for liver involvement in a large cohort of children with CD. METHODS: Children (age 0-18 years) diagnosed with CD from March 2010 to April 2016 were enrolled. Liver involvement was considered to be present when alanine transaminase (ALT) levels were >40 U/L (hypertransaminasemia [HTS]). Patients with HTS were re-evaluated after at least 12 months of a gluten-free diet. RESULTS: CD was diagnosed in 806 patients during the study period; of these, ALT levels were available for 700 patients (86.9%), and were elevated in 27 (3.9%, HTS group); median ALT and aspartate transaminase levels in the HTS group were 57 U/L (interquartile range 49-80 U/L) and 67 U/L (interquartile range 53-85 U/L), respectively. Younger age, malabsorption symptoms, and low hemoglobin or ferritin were significantly more common in the HTS group at univariate analysis. At multivariate analysis, only age ≤4.27 years correlated with risk of liver involvement (odds ratio 3.73; 95% confidence interval: 1.61-8.66). When retested on a gluten-free diet, all but 3 patients normalized ALT levels; of these, 1 was diagnosed with sclerosing cholangitis. CONCLUSIONS: Liver involvement in celiac children is now less frequent than previously reported, possibly due to changing CD epidemiology. Younger age is the only risk factor. Associated autoimmune liver disease is rare.

Immunohistologic analysis of the duodenal bulb: a new method for celiac disease diagnosis in children.

BACKGROUND AND AIMS: Anti-tissue transglutaminase antibodies (anti-tTG) have simplified celiac disease (CD) diagnosis. However, in atypical forms of CD, intestinal biopsy sampling is still required. This prospective study investigates whether histologic analysis of the duodenal bulb combined with intestinal IgA anti-tTG deposit immunoassay makes CD diagnosis possible in at-risk children with low concentrations of serum anti-tTG. METHODS: Histologic and intestinal IgA anti-tTG deposit immunoassays were used. RESULTS: Two hundred forty-five symptomatic children positive for serum anti-tTG (>7 U/mL) were enrolled and divided into 3 groups: extensive duodenal atrophy (n = 209), with IgA anti-tTG deposits throughout the duodenum and high serum anti-tTG concentrations (157 ± 178 U/mL); bulb duodenal atrophy (n = 22), with widespread IgA anti-tTG deposits in 9 and in the bulb alone in 13 and low serum anti-tTG concentrations (13.9 ± 8.7 U/mL); and normal duodenum (n = 14), with widespread IgA anti-tTG deposits in 8 and in the bulb alone in 6 and low serum anti-tTG concentrations (10.6 ± 6.2 U/mL). All patients in the first 2 groups were diagnosed with CD and 8 from the third group. All improved after 1 year of gluten-free diet. Bulb duodenal analysis led to a 12% (30/245) increase in CD diagnosis. No CD-related lesions were observed in the 30 control subjects. CONCLUSIONS: In children at risk for CD, bulb duodenum biopsy sampling is essential to identify villous atrophy and detect IgA anti-tTG deposits even in absence of intestinal lesions. These mucosal autoantibodies could well represent a new standard for diagnosing CD.

Potential use of MCR-ALS for the identification of coeliac-related biochemical changes in hyperspectral Raman maps from pediatric intestinal biopsies.

Raman hyperspectral imaging is an emerging practice in biological and biomedical research for label free analysis of tissues and cells. Using this method, both spatial distribution and spectral information of analyzed samples can be obtained. The current study reports the first Raman microspectroscopic characterisation of colon tissues from patients with Coeliac Disease (CD). The aim was to assess if Raman imaging coupled with hyperspectral multivariate image analysis is capable of detecting the alterations in the biochemical composition of intestinal tissues associated with CD. The analytical approach was based on a multi-step methodology: duodenal biopsies from healthy and coeliac patients were measured and processed with Multivariate Curve Resolution Alternating Least Squares (MCR-ALS). Based on the distribution maps and the pure spectra of the image constituents obtained from MCR-ALS, interesting biochemical differences between healthy and coeliac patients has been derived. Noticeably, a reduced distribution of complex lipids in the pericryptic space, and a different distribution and abundance of proteins rich in beta-sheet structures was found in CD patients. The output of the MCR-ALS analysis was then used as a starting point for two clustering algorithms (k-means clustering and hierarchical clustering methods). Both methods converged with similar results providing precise segmentation over multiple Raman images of studied tissues.

Anti-transglutaminase 6 Antibody Development in Children With Celiac Disease Correlates With Duration of Gluten Exposure.

OBJECTIVES: Antibodies against transglutaminase 6 (anti-TG6) have been implicated in neurological manifestations in adult patients with genetic gluten intolerance, and it is unclear whether autoimmunity to TG6 develops following prolonged gluten exposure. We measured the anti-TG6 in children with celiac disease (CD) at the diagnosis time to establish a correlation between these autoantibodies and the duration of gluten exposure. We investigated a correlation between anti-TG6 and the presence of neurological disorders. METHODS: Anti-TG6 (IgA/IgG) were measured by ELISA in sera of children with biopsy-proven CD and of children experiencing gastrointestinal disorders. CD patients positive for anti-TG6 were retested after 2 years of gluten-free diet (GFD). RESULTS: We analyzed the sera of 274 CD children and of 121 controls. Anti-TG6 were detected in 68/274 (25%) CD patients and in 19/121 (16%) controls, with significant difference between the 2 groups (P = 0.04). None of the CD patients and of the controls testing positive for anti-TG6 were experiencing neurological disorders. Eleven of 18 (61%) CD patients with other autoimmune diseases were positive for anti-TG6. In CD patients, a significant correlation between the gluten exposure before the CD diagnosis and anti-TG6 concentration was found (P = 0.006 for IgA; P < 0.0001 for IgG). After GFD anti-TG6 concentrations were significantly reduced (P < 0.001). No significant correlation was observed between anti-TG6 and anti-TG2 serum concentrations. CONCLUSIONS: Anti-TG6 are more prevalent in children with untreated CD in the absence of overt neurological disorders. The synthesis of the anti-TG6 is related to a longer exposure to gluten before the CD diagnosis, and the autoimmunity against TG6 is gluten dependent and disappeared during GFD.

Ex vivo response to mucosal bacteria and muramyl dipeptide in inflammatory bowel disease.

AIM: To evaluate how mucosal bacteria impact on the spontaneous and muramyl dipeptide (MDP)-induced inflammation in Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Colonic mucosal biopsies were collected from children with active or remissive CD, UC and controls. Two tissue samples were taken from inflamed mucosal segments (in patients with active disease) or from non-inflamed mucosa [in patients in remission or in healthy controls (HC)]. Experiments were performed in the presence or absence of antibiotics, to assess whether the disease-associated microbiota can modulate the cytokine response ex vivo. For this purpose, each specimen was half-cut to compare spontaneous and MDP-induced inflammation in the presence of live bacteria (LB) or antibiotics. After 24 h of culture, an array of 17 cytokines was assessed in supernatants. Statistical analyses were performed to find significant differences in single cytokines or in patterns of cytokine response in the different groups. RESULTS: We demonstrated that subjects with CD display a spontaneous production of inflammatory cytokines including granulocyte-colony stimulating factor (G-CSF), interleukin (IL) 6, IL8, IL10 and IL12, that was not significantly influenced by the addition of antibiotics. UC specimens also displayed a trend of increased spontaneous secretion of several cytokines, which however was not significant due to broader variability among patients. After the addition of antibiotics, spontaneous IL8 secretion was significantly higher in UC than in controls. In HC, a trend towards the weakening of spontaneous IL8 production was observed in the presence of live mucosal bacteria with respect to the presence of antibiotics. In contrast, in the presence of LB UC showed an increasing trend of spontaneous IL8 production, while MDP stimulation resulted in lower IL8 production in the presence of antibiotics. We also showed that subjects with CD seem to have a lowered production of IL8 in response to MDP in the presence of LB. Only with the addition of antibiotics, likely reducing the contribution of LB, multivariate statistical analysis could identify the combination of measures of G-CSF, tumor necrosis factor alpha, IL4 and IL17 as a good discriminator between CD and UC. CONCLUSION: We showed that the presence of LB or antibiotics can significantly influence the inflammatory response ex vivo in inflammatory bowel diseases.