Alberto Tommasini

Zonulin, a newly discovered modulator of intestinal permeability, and its expression in coeliac disease

We identified zonulin, a novel human protein analogue to the Vibrio cholerae derived Zonula occludens toxin, which induces tight junction disassembly and a subsequent increase in intestinal permeability in non-human primate intestinal epithelia. Zonulin expression was raised in intestinal tissues during the acute phase of coeliac disease, a clinical condition in which tight junctions are opened and permeability is increased.

Human recombinant tissue transglutaminase ELISA: an innovative diagnostic assay for celiac disease

OBJECTIVE:Tissue transglutaminase is the autoantigen recognized by the sera of celiac patients. An enzyme-linked immunosorbent assay (ELISA) based on guinea-pig tissue transglutaminase was recently used to measure serum tissue transglutaminase antibodies for the diagnosis of celiac disease. We determine the sensitivity and specificity of an ELISA test based on the use of human recombinant transglutaminase, compared with the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies.METHODS:Serum samples were tested from 65 patients with intestinal biopsy proven celiac disease, from 10 patients with Crohns disease, and from 150 healthy blood donors.RESULTS:Human transglutaminase ELISA identified 64 of 65 celiac patients, whereas the guinea pig transglutaminase ELISA and IgA antiendomysium antibodies identified 58 of 65 and 60 of 65 subjects, respectively. The three tests showed comparable specificity.CONCLUSIONS:These results proved that the human tissue transglutaminase-based ELISA represents a cost-effective strategy for identifying both symptomatic and atypical forms of celiac disease and could mean that intestinal biopsy need no longer be the gold standard for diagnosing this clinical condition. Furthermore, early identification and treatment of patients with celiac disease in an outpatient setting could have significant implications for reducing long-term morbidity and can produce major savings in future health care costs.

Mass screening for coeliac disease using antihuman transglutaminase antibody assay

Aims: To determine coeliac disease prevalence by an anti-transglutaminase antibody assay in a large paediatric population; to evaluate acceptance of the screening programme, dietary compliance, and long term health effects. Methods: Cross-sectional survey of 3188 schoolchildren (aged 6–12) and prospective follow up of diagnosed cases. Main outcome measures were: prevalence of coeliac disease defined by intestinal biopsy or positivity to both human tissue transglutaminase and anti-endomysium antibodies in HLA DQ2-8 positive subjects; percentage of children whose families accepted screening; dietary compliance as defined by negativity for anti-transglutaminase antibodies; and presence of clinical or laboratory abnormalities at 24 month follow up. Results: The families of 3188/3665 children gave their consent (87%). Thirty biopsy proven coeliacs were identified (prevalence 1:106). Three other children testing positive for both coeliac related autoantibodies and HLA DQ2-8 but refusing biopsy were considered as having coeliac disease (prevalence 1:96). Of 33 cases, 12 had coeliac related symptoms. The 30 biopsy proven coeliacs followed a gluten-free diet. Of 28 subjects completing 18–24 months follow up, 20 (71.4%) were negative for anti-transglutaminase antibodies, while eight were slightly positive; symptoms resolved in all 12 symptomatic children. Conclusions: Prevalence of coeliac disease is high in Italian schoolchildren. Two thirds of cases were asymptomatic. Acceptance of the programme was good, as was dietary compliance. Given the high prevalence and possible complications of untreated coeliac disease, the availability of a valid screening method, and evidence of willingness to comply with dietary treatment population mass screening deserves careful consideration.

Molecular Dissection of the Tissue Transglutaminase Autoantibody Response in Celiac Disease

Celiac disease (CD) is an intestinal malabsorption characterized by intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and tissue transglutaminase, an autoantigen located in the endomysium. Tissue transglutaminase belongs to the family of enzymes that catalyze protein cross-linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. The role of gliadins in eliciting the immune response in CD and how transglutaminase is linked to the primary reaction are still unclear. In this work, we report the production and analysis of six phage Ab libraries from the peripheral and intestinal lymphocytes of three CD patients. We were able to isolate Abs to transglutaminase from all intestinal lymphocytes libraries but not from those obtained from peripheral lymphocytes. This is in contrast to Abs against gliadin, which could be obtained from all libraries, indicating that the humoral response against transglutaminase occurs at the local level, whereas that against gliadin occurs both peripherally and centrally. Abs from all three patients recognized the same transglutaminase epitopes with a bias toward the use of the VH5 Ab variable region family. The possible role of these anti-transglutaminase Abs in the onset of CD and associated autoimmune pathologies is discussed.

A Diagnostic Score for Molecular Analysis of Hereditary Autoinflammatory Syndromes With Periodic Fever in Children

OBJECTIVE To identify a set of clinical parameters that can predict the probability of carrying mutations in one of the genes associated with hereditary autoinflammatory syndromes. METHODS A total of 228 consecutive patients with a clinical history of periodic fever were screened for mutations in the MVK, TNFRSF1A, and MEFV genes, and detailed clinical information was collected. A diagnostic score was formulated based on univariate and multivariate analyses in genetically positive and negative patients (training set). The diagnostic score was validated in an independent set of 77 patients (validation set). RESULTS Young age at onset (odds ratio [OR] 0.94, P = 0.003), positive family history of periodic fever (OR 4.1, P = 0.039), thoracic pain (OR 4.6, P = 0.05), abdominal pain (OR 33.1, P < 0.001), diarrhea (OR 3.3, P = 0.028), and oral aphthosis (OR 0.2, P = 0.007) were found to be independently correlated with a positive genetic test result. These variables were combined in a linear score whose ability to predict a positive result on genetic testing was validated in an independent data set. In this latter set, the diagnostic score revealed high sensitivity (82%) and specificity (72%) for discriminating patients who were genetically positive from those who were negative. In patients with a high probability of having a positive result on genetic testing, a regression tree analysis provided the most reasonable order in which the genes should be screened. CONCLUSION The proposed approach in patients with periodic fever will increase the probability of obtaining positive results on genetic testing, with good specificity and sensitivity. Our results further help to optimize the molecular analysis by suggesting the order in which the genes should be screened.

Clinical and molecular profile of a new series of patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome: Inconsistent correlation between forkhead box protein 3 expression and disease severity

BACKGROUND Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is an autoimmune genetic disorder caused by mutation of the forkhead box protein 3 gene (FOXP3), a key regulator of immune tolerance. OBJECTIVE We sought to provide clinical and molecular indicators that facilitate the understanding and diagnosis of IPEX syndrome. METHODS In 14 unrelated affected male subjects who were given diagnoses of IPEX syndrome based on FOXP3 gene sequencing, we determined whether particular FOXP3 mutations affected FOXP3 protein expression and correlated the molecular and clinical data. RESULTS Molecular analysis of FOXP3 in the 14 subjects revealed 13 missense and splice-site mutations, including 7 novel mutations. Enteropathy, generally associated with endocrinopathy and eczema, was reported in all patients, particularly in those carrying mutations within FOXP3 functional domains or mutations that altered protein expression. However, similar genotypes did not always result in similar phenotypes in terms of disease presentation and severity. In addition, FOXP3 protein expression did not correlate with disease severity. CONCLUSION Severe autoimmune enteropathy, which is often associated with increased IgE levels and eosinophilia, is the most prominent early manifestation of IPEX syndrome. Nevertheless, the disease course is variable and somewhat unpredictable. Therefore genetic analysis of FOXP3 should always be performed to ensure an accurate diagnosis, and FOXP3 protein expression analysis should not be the only diagnostic tool for IPEX syndrome.

Undiagnosed coeliac disease and risk of autoimmune disorders in subjects with Type I diabetes mellitus

Aims/hypothesis. We tested the hypothesis that silent coeliac disease is more frequent than expected in both patients with Type I (insulin-dependent) diabetes mellitus and their first-degree relatives. We evaluated how the presence of other autoimmune disorders in diabetic patients and their first-degree relatives is related to silent, unrecognized coeliac disease. Methods. Sera from 491 subjects with Type I diabetes, 824 relatives and 4000 healthy control subjects were screened for anti-endomysial antibodies and all those subjects who tested positive for anti-endomysial antibodies underwent intestinal biopsy. Results. We found that the prevalence of coeliac disease was 5.7 % among the diabetic patients and 1.9 % among the relatives, values significantly higher than those found among the control subjects (p < 0.0001; p < 0.001). The prevalence of autoimmune disorders in diabetic patients with coeliac disease was significantly higher than in subjects with Type I diabetes alone (p < 0.0001). The prevalence of autoimmune disorders in the relatives with coeliac disease was significantly higher than in those who tested negative for anti-endomysial antibodies (p = 0.01). Conclusion/interpretation. This report provides further confirmation of the high prevalence of undiagnosed coeliac disease among diabetic patients and their relatives. This interesting new finding is the increased presence of other autoimmune diseases in these patients, as well as in their relatives with a delayed diagnosis for coeliac disease. Patients newly diagnosed with coeliac disease showed excellent compliance with the gluten-free diet. This should encourage policymakers to consider introducing an easy-to-use screening programme for diabetic patients and their relatives into everyday clinical practice, in order to prevent coeliac-associated symptoms and the onset of additional, more serious auto-immune disorders. [Diabetologia (2001) 44: 151–155]

The efficacy of anakinra in an adolescent with colchicine-resistant familial Mediterranean fever

Colchicine is the treatment of choice in familial Mediterranean fever (FMF) for the prevention of both attacks and secondary amyloidosis. The overall nonresponder rate is about 5–10%. Anakinra is known to have good effectiveness in a severe autoinflammatory syndrome [chronic infantile neurological cutaneous and articular (CINCA) syndrome] and other recurrent hereditary periodic fevers. Pyrin—the protein involved in FMF—has a role in activating the proinflammatory cytokine interleukin (IL)-1β. We report the effectiveness of the addition of an IL-1-receptor inhibitor (anakinra) to colchicine in controlling the febrile attacks and acute phase response in an adolescent with FMF resistant to colchicine.

Differentiating PFAPA syndrome from monogenic periodic fevers

OBJECTIVES: To analyze whether there were clinical differences between genetically positive and negative patients fulfilling periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome criteria and to test the accuracy of the Gaslini diagnostic score for identifying patients with PFAPA syndrome with higher probabilities of carrying relevant mutations in genes associated with periodic fevers. METHODS: Complete clinical and genetic information was available for 393 children with periodic fever; 82 had positive genetic test results, 75 had incomplete genetic test results, and 236 had negative results for MVK, TNFRSF1A, and MEFV mutations. Current diagnostic criteria for PFAPA syndrome were applied. RESULTS: Of 393 children, 210 satisfied PFAPA syndrome criteria; 43 carried diagnostic mutations (mevalonate kinase deficiency: n = 33; tumor necrosis factor receptor-associated periodic syndrome: n = 3; familial Mediterranean fever: n = 7), 37 displayed low-penetrance mutations or incomplete genotypes, and 130 demonstrated negative genetic testing results. Genetically positive patients had higher frequencies of abdominal pain and diarrhea (P < .001), vomiting (P = .006), and cutaneous rash and arthralgia (P = .01). Genetically negative patients had a higher frequency of exudative pharyngitis (P = .010). Genetically undetermined patients showed the same pattern of symptom frequency as genetically negative patients. The Gaslini diagnostic score was able to identify 91% of genetically positive patients correctly, with a global accuracy of 66%. CONCLUSION: The Gaslini diagnostic score represents a useful tool to identify patients meeting PFAPA syndrome criteria and at low risk of carrying relevant mutations in genes associated with periodic fevers.

Development of a novel rapid non-invasive screening test for coeliac disease

BACKGROUND Coeliac disease is one of the commonest underdiagnosed diseases in general practice. The autoantigen recognised by the sera of patients with coeliac disease has recently been identified as tissue transglutaminase. AIMS We evaluated a simple non-invasive immunological dot blot assay for coeliac disease, suitable for use by the general physician in the ambulatory setting. The sensitivity and specificity of this dot blot assay based on recognition of recombinant human transglutaminase were compared with those of antiendomysial antibodies and an enzyme linked immunosorbent assay. METHODS Serum samples were analysed from 64 healthy controls, 58 first degree relatives of coeliacs, 74 diseased controls, and 70 biopsy confirmed untreated patients with coeliac disease. Dot blot assay and enzyme linked immunosorbent assay were performed using recombinant human transglutaminase as antigen. RESULTS The dot blot assay, which can be performed in 20 minutes, was positive in all 70 untreated coeliacs (sensitivity 100%). Among the three control groups, there were three false positive tests by dot blot (specificity 98%), all belonging to the group of healthy subjects. The antiendomysial antibodies test missed five untreated coeliac patients (sensitivity 93%) and was negative in all three control groups (specificity 100%). The specificity of the immunosorbent assay was 99% for IgA and 98% for IgG, while sensitivity was 93% for IgA, 47% for IgG, and 100% for IgA and IgG combined. CONCLUSIONS The dot blot assay is highly accurate in detecting untreated subjects with coeliac disease and can be performed in the general physicians medical office during the course of a routine examination. This innovative test is a practical, reliable alternative to both the immunofluorescent based antiendomysial test and immunosorbent assay for detection of transglutaminase antibodies for the diagnosis of coeliac disease.