Sandro Drago

Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function

Background and aims: Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability. Methods: The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers. Results: Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release. Conclusions: Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.

Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines

Objective. Little is known about the interaction of gliadin with intestinal epithelial cells and the mechanism(s) through which gliadin crosses the intestinal epithelial barrier. We investigated whether gliadin has any immediate effect on zonulin release and signaling. Material and methods. Both ex vivo human small intestines and intestinal cell monolayers were exposed to gliadin, and zonulin release and changes in paracellular permeability were monitored in the presence and absence of zonulin antagonism. Zonulin binding, cytoskeletal rearrangement, and zonula occludens-1 (ZO-1) redistribution were evaluated by immunofluorescence microscopy. Tight junction occludin and ZO-1 gene expression was evaluated by real-time polymerase chain reaction (PCR). Results. When exposed to gliadin, zonulin receptor-positive IEC6 and Caco2 cells released zonulin in the cell medium with subsequent zonulin binding to the cell surface, rearrangement of the cell cytoskeleton, loss of occludin-ZO1 protein–protein interaction, and increased monolayer permeability. Pretreatment with the zonulin antagonist FZI/0 blocked these changes without affecting zonulin release. When exposed to luminal gliadin, intestinal biopsies from celiac patients in remission expressed a sustained luminal zonulin release and increase in intestinal permeability that was blocked by FZI/0 pretreatment. Conversely, biopsies from non-celiac patients demonstrated a limited, transient zonulin release which was paralleled by an increase in intestinal permeability that never reached the level of permeability seen in celiac disease (CD) tissues. Chronic gliadin exposure caused down-regulation of both ZO-1 and occludin gene expression. Conclusions. Based on our results, we concluded that gliadin activates zonulin signaling irrespective of the genetic expression of autoimmunity, leading to increased intestinal permeability to macromolecules.

Celiac Disease: Going Against the Grains

Celiac disease (CD) occurs in genetically predisposed individuals who demonstrate a permanent intolerance to gluten, found in wheat, barley, and rye. This leads to the development of an autoimmune enteropathy, resulting in the malabsorption of critical vitamins, minerals, and calories. Signs and symptoms of the disease classically include diarrhea, short stature, iron-deficient anemia, and chronic abdominal pain. However, patients may present with another autoimmune disease (such as type I diabetes) or a family history of the disease, with no gastrointestinal symptoms. Serum antibodies can be used to screen for CD. However, the keys to confirming the diagnosis remain a small intestinal biopsy and the patients clinical response to a gluten-free diet. Clinicians in the United States must maintain a high index of suspicion for CD, because it is significantly underdiagnosed in this country.

A rapid and sensitive method to detect specific human lymphocyte antigen (HLA) class II alleles associated with celiac disease.

Abstract Background: Celiac disease (CD) is a complex disorder triggered by gluten affecting genetically predisposed individuals. More than 90% of patients carry human lymphocyte antigen (HLA)-DQ2 (DQA1*05, DQB1*02) and/or HLA-DQ8 (DQA1*03, DQB1*0302). We propose the use of the DQ-CD Typing kit that allows identification of the HLA class II alleles (DQA1*0201,*03,*05, DQB1*02,*0302, DRB1*03,*04,*07) selected to be informative in the CD risk evaluation and of a second kit, namely DQ-CD Zygosis, for DQB1*02 homozygosity determination. Methods: The study was performed on a cohort of 100 individuals previously HLA typed with commercial kits. Fresh blood or previously extracted DNA was amplified in a unique PCR program using allele-specific primers and visualized on agarose gel. Results: DNA amplification yielded strong and clear products without non specific signals or ghost bands. All the samples showed the expected alleles in accordance with the previous HLA typing. Conclusions: The DQ-CD Typing and Zygosis kits are fast, simple, economical and accurate tools that can be used to determinate the HLA-DQ2/DQ8 status in laboratory practice addressed for the diagnosis of CD. Molecular HLA testing is considered a valid support in the confirmation/exclusion of CD, especially in high-risk groups, such as CD relatives, or when serological and histological data are ambiguous. Clin Chem Lab Med 2008;46:193–6.

Recent developments in the pathogenesis, diagnosis and treatment of celiac disease

Celiac disease (CD) is a syndrome characterised by damage of the small intestinal mucosa, caused by the gliadin fraction of wheat gluten and similar alcohol-soluble proteins (prolamines) of barley and rye in genetically susceptible subjects. The clinical manifestations of CD are protean in nature and vary markedly with the age of the patient, the duration and extent of disease and the presence of extra-intestinal pathology. This article reviews three patents from the last four years which claim methods and tools for the diagnosis and treatment of CD. They are related to the antigenic role of tissue transglutaminase, the immunodominant gliadin peptide recognised by the cells in the peripheral blood of individuals with CD and the pro-inflammatory role of IL-15; however, only the first invention found application worldwide. In spite of this, the current gold standard for the diagnosis of CD is still the histological confirmation of the intestinal damage and the keystone treatment of CD patients is a life-long elimination diet, in which food products containing gluten are avoided.

Human Tissue Transglutaminase ELISA and an Old Study: A Revision of the Blood Donor Screening Study for Coeliac Disease in the USA

oeliac disease (CD) is a gluten-dependent immune-related disorder in genetically predisposed subjects.Twenty years ago the diagnosis was based entirelyon clinical suspicion and on non-specific malabsorption tests.The development of diagnostic tools in the 1990s, however,opened a new perspective; screenings were performed usingantigliadin antibodies (AGA), as initial screening assay,followed by antiendomysium antibody assay (AEA), as aconfirmation step in the tested AGA-positive samples (1).Using this approach, our first blood donor screening inUSA reported a prevalence of 1:250 (2). A few years later, theauto-antigen of CD was identified in the human tissuetransglutaminase (h-tTG) (3) and, consequently, tTG-basedELISAs were developed (4, 5). We re-screened the blooddonor’s AGA-negative sera using a h-tTG IgA.ParticipantsThe 2000 serum blood donor samples obtained from theAmerican Red Cross in Baltimore, Maryland were used in theoriginal study (2). These had been stored at –80°C since1996. Because the samples were collected anonymously, onlygeneral demographic data were available. The mean age ofblood donors was 39 years; 52.4% were men, 87% Cauca-sians, 11.5% African-Americans and 1.5% Asians.MethodsThe new protocol aimed to test for h-tTG IgA 1881 negativeAGA sera. The h-tTG positive sera were tested for AEA andalso when the whole blood was available. DNA was alsoextracted for HLA DQ2/DQ8 haplotype typing. The costeffectiveness of the two approaches was compared.In order to exclude that the storage did not influence theantibody titres, we re-measured the previously 8 AEApositive sera and 294 AGA and AEA negative sera.H-tTG IgA antibodies and AEA antibodiesH-tTG IgA was performed by ELISA (Eurospital, Italy) inaccordance with the manufacturer’s instructions, while AEAswere determined by an indirect immunofluorescence methodusing monkey oesophagus sections as antigen (Scimedex,USA). The immunological tests were performed by threedifferent operators in order to avoid misinterpretation.HLA haplotypesGenomic DNA was extracted from whole blood samplesusing QIAamp DNA Mini Kit (QIAGEN , USA) and HLAtyping performed as previously described using the Eu-DQKit (Eurospital, Italy).

Gliadin induces a zonulin-dependent increased intestinal permeability and decreased expression of tight junction proteins in an ex-vivo human intestinal model of celiac disease

Gliadin induces a zonulin-dependent increased intestinal permeability and decreased expression of tight junction proteins in an ex-vivo human intestinal model of celiac disease

Gliadin peptides induce increased Caco2 monolayer permeability and tight junction protein ZO-1 redistribution

Material and methods: Human intestinal cells (Caco2) were grown on polarized filters. Gliadin or casein pepsyn-trypsin digests were added to the luminal culture medium for a 3-hour incubation. The intestinal barrier function was assessed by evaluation of: (1) trans-epithelial electrical resistance (TEER), (2) release of zonulin, a modulator of intercellular tight junctions (tj), measured by sandwich-ELISA, (3) luminal to serosal lactulose transport measured by high performance anion exchange chromatography and, (4) localization/migration of zonula occludens protein (ZO-1), by direct immunofluorescence.

HLA A*0201 allele accounts for DQ2/DQ8 negative genome screening in celiac disease patients

Background: HLA class II alleles DQA1*0501/DQB1*0201 are the antigen presenting cells surface receptors for deamidated toxic gliadin fragments. Despite that their presence is considered necessary for celiac disease (CD) pathogenesis, these two alleles account for only 90-95% of the genomic pattern of CD HLA class II DQ2/DQ8 haplotypes. It has been recently reported that HLA DQ2 haplotype can be coded by the previously undescribed A*0201 allele.

Comparative study between American and Italian families of Celiac disease patients based on HLA profiles

Abstract Objective: To identify the HLA profiles of American celiac patients and their relatives and to compare the HLA profile of Italian and American families. Celiac disease (CD) is an autoimmune disorder triggered by gluten ingestion in genetically predisposed subjects. HLA class II alleles DQA1*0501/DQB1*02 and DQB1*0302 (encoding DQ2 and DQ8 haplotype respectively) represent the genetic markers for CD. Methods: Fourteen American celiac families (14 CD patients and 38 first degree relatives, including both parents) and 14 Italian celiac families (14 CD patients and 45 first degree relatives, including both parents) were included in this study. DNA was extracted from whole blood samples. Using the Eu-DQ ® Kit (Eurospital, Trieste—Italy), we performed the HLA typing here described. This Kit is composed of two multiplex PCR reactions, one containing DQα1*0501–DQβ1*0302 primers and Beta globin primers as internal control. The second one contains DQβ1*02 and Beta globin primers as internal control. The amplicons obtained were revealed on 2% agarose gel that is stained using Ethidium Bromide. Results: The prevalence of DQ2 alleles among American celiac patients was 64.3% as compared to 85.7% found among the Italian celiac patients (Table 1) Table 1 . American Controls Italian Controls N subjects screened 52 50 59 50 CD patients 14 14 Relatives of CD pts 38 45 Celiac DQ2 9 (64.3%) 12 (85.7%) Celiac DQ8 3 (21.4%) 2 (14.3%) Celiac DQ2/DQ8 2 (14.3%) 0 (0%) . Both the DQ8 alleles and the DQ2/DQ8 heterodimer were more frequent among the American CD subjects than the Italian patients. Interestingly, a similar difference between American and Italian subjects was also observed among the first degree relatives screened (Table 1) . Conclusions: American CD patients and their first degree relatives have a higher prevalence of DQ8 alleles, either alone or in combination with DQ2, as compared to Italian CD families.