Modulazione fisiologica della permeabilità intestinale e assorbimento di molecole idrofiliche

2009/2010La permeabilità intestinale è regolata dalle tight junctions (TJ) che costituiscono la barriera gastrointestinale e che sono soggette a regolazione fisiologica. Dati di letteratura dimostrano che digiuno e malnutrizione agiscono su tale barriera alterando la morfologia dell’epitelio intestinale e aumentando il movimento degli ioni e delle grandi molecole attraverso la via paracellulare. Partendo da queste osservazioni abbiamo ipotizzato che il digiuno potesse avere un ruolo nella modulazione delle TJ e siamo andati a valutare in vivo gli effetti del digiuno sull’assorbimento orale della tobramicina e sulla permeabilità della via paracellulare. Con questo studio abbiamo dimostrato per la prima volta che la biodisponibilità della tobramicina dipende dalla condizione fisiologica. L’assorbimento orale del farmaco è, infatti, aumentato in maniera significativa quando gli animali sono tenuti a digiuno per 15 ore.XXIII Cicl

Intestinal antitransglutaminase antibodies to discover genetic gluten intolerance

Genetic gluten intolerance is a multifactorial condition characterized by the intestinal synthesis of antitransglutaminase antibodies (anti-tTG) which might represent an early stage of this intolerance in absence of both intestinal damage and serum anti-tTG. The double immunofluorescence staining (IF) is able to point out these anti-tTG antibodies directly on intestinal biopsy. Her we describe a prospective study in which patients were analysed for genetic predisposition (HLA DQ2-DQ8) and serum anti-tTG and were monitored for clinical conditions and serum anti-tTG concentration during gluten free diet (GFD) or gluten containing diet (GCD). Our results demonstrate that the measurement of intestinal anti-tTG is a useful screening procedure to identify patients with genetic predisposition not fullfilling the current diagnostic criteria

Early diagnosis of coeliac disease

At the Immunopathology Laboratory at the IRCCS Burlo Garofolo hospital the research activity is based on autoimmune diseases, above all on celiac disease in order to diagnose it in an early stage. For this reason, we are collecting many serum samples and intestinal biopsies to analyse them with molecular (phage-display) and immunofluorescent (double staining and activated beads) assays. Within the Trans2Care project we intend to apply these methods in several areas related to the problems explored by the Project partners with the aim of promote collaboration, mobility of researchers and exchange of knowledge between partners

Supplementary data for the article: Habtamu, H. B.; Sentic, M.; Silvestrini, M.; De Leo, L.; Not, T.; Arbault, S.; Manojlovic, D.; Sojic, N.; Ugo, P. A Sensitive Electrochemiluminescence Immunosensor for Celiac Disease Diagnosis Based on Nanoelectrode Ensembles. Analytical Chemistry 2015, 87 (24), 12080–12087. https://doi.org/10.1021/acs.analchem.5b02801

Supporting information for: [https://doi.org/10.1021/acs.analchem.5b02801]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2014

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\u202f=\u202f178) with villous atrophy and positive serum-CD antibodies, potential CD patients (n\u202f=\u202f16) with normal intestinal mucosa and positive serum-CD antibodies, and pre-potential CD patients (n\u202f=\u202f14) 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

Supplementary data for the article: Habtamu, H. B.; Sentic, M.; Silvestrini, M.; De Leo, L.; Not, T.; Arbault, S.; Manojlovic, D.; Sojic, N.; Ugo, P. A Sensitive Electrochemiluminescence Immunosensor for Celiac Disease Diagnosis Based on Nanoelectrode Ensembles. Analytical Chemistry 2015, 87 (24), 12080–12087. https://doi.org/10.1021/acs.analchem.5b02801

Supporting information for: [https://doi.org/10.1021/acs.analchem.5b02801]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2014

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

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

Cross-sectional study of coeliac autoimmunity in a population of Vietnamese children

Objective: The prevalence of coeliac disease (CD) inVietnam is unknown. To fill this void, we assessed the prevalence of serological markers of CD autoimmunity in a population of children in Hanoi. Setting: The outpatient blood drawing laboratory of the largest paediatric hospital in North Vietnam was used for the study, which was part of an international project of collaboration between Italy and Vietnam. Participants: Children having blood drawn for any reason were included. Exclusion criteria were age younger than 2 years, acquired or congenital immune deficiency and inadequate sample. A total of 1961 children (96%) were enrolled (838 females, 1123 males, median age 5.3 years). Outcomes: Primary outcome was the prevalence of positive autoimmunity to both IgA antitransglutaminase antibodies (anti-tTG) assessed with an ELISA test and antiendomysial antibodies (EMA). Secondary outcome was the prevalence of CD predisposing human leucocyte antigens (HLA) (HLA DQ2/8) in the positive children and in a random group of samples negative for IgA anti-tTG. Results: The IgA anti-tTG test was positive in 21/1961 (1%; 95% CI 0.61% to 1.53%); however, EMA antibodies were negative in all. HLA DQ2/8 was present in 7/21 (33%; 95% CI 14.5% to 56.9%) of the anti-tTG-positive children and in 72/275 (26%; 95% CI 21% to 32%) of those who were negative. Conclusions: Coeliac autoimmunity is rare in Vietnam, although prevalence of HLA DQ2/8 is similar to that of other countries. We hypothesise that the scarce exposure to gluten could be responsible for these findings