Lack of evidence of rotavirus-dependent molecular mimicry as a trigger of coeliac disease.

New data suggest the involvement of rotavirus (RV) in triggering autoimmunity in coeliac disease (CD) by molecular mimicry between the human-transglutaminase protein and the dodecapeptide (260-271 aa) of the RV protein VP7 (pVP7). To assess the role of RV in the onset of CD, we measured anti-pVP7 antibodies in the sera of children with CD and of control groups. We analysed serum samples of 118 biopsy-proven CD patients and 46 patients with potential CD; 32 children with other gastrointestinal diseases; 107 no-CD children and 107 blood donors. Using enzyme-linked immunosorbent assay (ELISA) assay, we measured immunoglobulin (Ig)A-IgG antibodies against the synthetic peptides pVP7, the human transglutaminase-derived peptide (476-487 aa) which shows a homology with VP7 protein and a control peptide. The triple-layered RV particles (TLPs) containing the VP7 protein and the double-layered RV-particles (DLPs) lacking the VP7 protein were also used as antigens in ELISA assay. Antibody reactivity to the RV-TLPs was positive in 22 of 118 (18%) CD patients and in both paediatric (17 of 107, 16%) and adult (29 of 107, 27%) control groups, without showing a statistically significant difference among them (P = 0·6, P = 0·1). Biopsy-proven CD patients as well as the adult control group demonstrated a high positive antibody reactivity against both pVP7 (34 of 118, 29% CD patients; 66 of 107, 62% adult controls) and control synthetic peptides (35 of 118, 30% CD patients; 56 of 107, 52% adult controls), suggesting a non-specific response against RV pVP7. We show that children with CD do not have higher immune reactivity to RV, thus questioning the molecular mimicry mechanism as a triggering factor of CD.

Prevalence of celiac disease in patients with severe food allergy.

The association between food allergy and celiac disease (CD) is still to be clarified. We screened for CD 319 patients with severe food allergy (IgE > 85 kU/l against food proteins and a history of severe allergic reactions) who underwent specific food oral immunotherapy (OIT), together with 128 children with mild allergy who recovered without OIT, and compared the prevalence data with our historical data regarding healthy schoolchildren. Sixteen patients (5%) with severe allergy and one (0.8%) with mild allergy tested positive for both genetic and serological CD markers, while the prevalence among the schoolchildren was 1%. Intestinal biopsies were obtained in 13/16 patients with severe allergy and in the one with mild allergy, confirming the diagnosis of CD. Sufferers from severe food allergy seem to be at a fivefold increased risk of CD. Our findings suggest that routine screening for CD should be recommended in patients with severe food allergy.

Development of an enzyme-linked immunosorbent assay for Bartonella henselae infection detection.

UNLABELLED: Several serological diagnostics rely on enzyme-linked immunosorbent assay (ELISA) to detect bacterial infections. However, for some pathogens, including Bartonella henselae, diagnosis still depends on manually intensive, time-consuming assays including micro-immunofluorescence, Western blotting or indirect immunofluorescence. For such pathogens, there is obviously still a need to identify antigens to establish a reliable, fast and high-throughput assay (Dupon et al. ). We evaluated two B. henselae proteins to develop a novel serological ELISA: a well-known antigen, the 17-kDa protein, and GroEL, identified during this study by a proteomic approach. When serum IgG were tested, the specificity and sensitivity were 76 and 65·7% for 17-kDa, respectively, and 82 and 42·9% for GroEL, respectively. IgM were found to be more sensitive and specific for both proteins: 17-kDa protein, specificity 86·2% and sensitivity 75%; GroEL, specificity 97·7% and sensitivity 45·3%. IgM antibodies were also measured in lymphoma patients and patients with Mycobacterium tuberculosis infection to assess the usefulness of our ELISA to distinguish them from B. henselae infected patients. The resulting specificities were 89·1 and 93·5% for 17-kDa protein and GroEL, respectively. Combining the results from the two tests, we obtained a sensitivity of 82·8% and a specificity of 83·9%. Our work described and validated a proteomic approach suitable to identify immunogenic proteins useful for developing a serological test of B. henselae infection. SIGNIFICANCE AND IMPACT OF THE STUDY: A reliable serological assay for the diagnosis of Cat Scratch Disease (CSD) - a pathological condition caused by Bartonella henselae infection - has not yet been developed. Such an assay would be extremely useful to discriminate between CSD and other pathologies with similar symptoms but different aetiologies, for example lymphoma or tuberculosis. We investigate the use of two B. henselae proteins - GroEL and 17-kDa - to develop a serological-based ELISA, showing promising results with the potential for further development as an effective tool for the differential diagnosing of B. henselae infection.

Anti-transglutaminase antibodies in non-coeliac children suffering from infectious diseases.

Anti-transglutaminase antibodies are the diagnostic markers of coeliac disease. A role is suggested for infectious agents in the production of anti-transglutaminase antibodies. The aim was to measure positive anti-transglutaminase antibody levels in children with infectious diseases and to compare immunological and biological characteristics of the anti-transglutaminase antibodies derived from these children with that from coeliac patients. Two hundred and twenty-two children suffering from infectious diseases were enrolled prospectively along with seven biopsy-proven coeliacs. Serum samples were tested for anti-transglutaminase antibodies and anti-endomysium antibodies; positive samples were tested for coeliac-related human leucocyte antigen (HLA)-DQ2/8 and anti-viral antibodies. Purified anti-transglutaminase antibodies from the two study groups were tested for urea-dependent avidity, and their ability to induce cytoskeletal rearrangement and to modulate cell-cycle in Caco-2 cells, using phalloidin staining and bromodeoxyuridine incorporation assays, respectively. Nine of 222 children (4%) tested positive to anti-transglutaminase, one of whom also tested positive for anti-endomysium antibodies. This patient was positive for HLA-DQ2 and was diagnosed as coeliac following intestinal biopsy. Of the eight remaining children, two were positive for HLA-DQ8. Levels of anti-transglutaminase returned to normal in all subjects, despite a gluten-containing diet. Purified anti-transglutaminase of the two study groups induced actin rearrangements and cell-cycle progression. During an infectious disease, anti-transglutaminase antibodies can be produced temporarily and independently of gluten. The infection-triggered anti-transglutaminase antibodies have the same biological properties as that of the coeliacs, with the same in-vivo potential for damage.

The shared CTLA4-ICOS risk locus in celiac disease, IgA deficiency and common variable immunodeficiency.

IgA deficiency (IgAD) and common variable immunodeficiency (CVID) often co-occur in families, associating with chronic inflammatory diseases such as celiac disease (CD). ICOS (inducible co-stimulator) and CTLA4 (cytotoxic T-lymphocyte-associated protein-4) may be important in both disorders, as ICOS is necessary for Ig class-switching and CTLA4 negatively regulates T-cell activation. Linkage and association of CD with CTLA4-ICOS is well documented, we thus aimed to further pinpoint CD susceptibility by haplotype-tagging analysis. We genotyped 663 CD families from Finland and Hungary, 575 additional CD patients from Finland, Hungary and Italy; 275 Swedish and Finnish IgAD individuals and 87 CVID individuals for 14-18 genetic markers in CTLA4-ICOS. Association was found between CTLA4-ICOS and both IgAD (P=0.0015) and CVID (P=0.0064). We confirmed linkage of CTLA4-ICOS with CD (LOD 2.38, P=0.0005) and found association of CTLA4-ICOS with CD (P=0.0009). Meta-analysis of the IgAD, CVID and CD materials revealed intergenic association (P=0.0005). Disease-associated markers were associated with lower ICOS and higher CTLA4 expression, indicating that the risk haplotypes contain functional variants. In summary, we identified a novel shared risk locus for IgAD, CVID and CD, the first report of association between CTLA4-ICOS and IgAD. Association between CD and CTLA4-ICOS was also confirmed in a large European data set.

Linkage and association study of FcgammaR polymorphisms in celiac disease.

The Fcgamma receptor cluster on chromosome 1q23 contains a number of genes that may affect susceptibility to celiac disease, but previous studies have yielded contradictory results. We studied the FcgammaRIIa*A519G (rs1801274) and FcgammaRIIIa*A559C (rs396991) single nucleotide polymorphisms in celiac disease families from Hungary and Finland and in celiac disease case-control materials from Hungary and Italy. Neither the Hungarian nor the Italian case-control material or a meta-analysis of the combined case-control material showed significant single-marker or haplotype association. In addition, neither linkage nor family-based association tests showed evidence for association in the Finnish or Hungarian family material. This study thus does not support a previous publication showing FcgammaR association with celiac disease.

Combined Analysis of Methylation and Gene Expression Profiles in Separate Compartments of Small Bowel Mucosa Identified Celiac Disease Patients' Signatures.

By GWAS studies on celiac disease, gene expression was studied at the level of the whole intestinal mucosa, composed by two different compartments: epithelium and lamina propria. Our aim is to analyse the gene-expression and DNA methylation of candidate genes in each of these compartments. Epithelium was separated from lamina propria in biopsies of CeD patients and CTRs using magnetic beads. Gene-expression was analysed by RT-PC; methylation analysis required bisulfite conversion and NGS. Reverse modulation of gene-expression and methylation in the same cellular compartment was observed for the IL21 and SH2B3 genes in CeD patients relative to CTRs. Bioinformatics analysis highlighted the regulatory elements in the genomic region of SH2B3 that altered methylation levels. The cREL and TNFAIP3 genes showed methylation patterns that were significantly different between CeD patients and CTRs. In CeD, the genes linked to inflammatory processes are up-regulated, whereas the genes involved in the cell adhesion/integrity of the intestinal barrier are down-regulated. These findings suggest a correlation between gene-expression and methylation profile for the IL21 and SH2B3 genes. We identified a "gene-expression phenotype" of CeD and showed that the abnormal response to dietary antigens in CeD might be related not to abnormalities of gene structure but to the regulation of molecular pathways.

Speeding up coeliac disease diagnosis in the developing countries.

BACKGROUND: Anti-transglutaminase antibodies are highly predictive markers of active coeliac disease. Because limited facilities are available for routine use of anti-transglutaminase antibodies assays in developing countries, a simple, economical immunological test would represent a great step forward in the screening of coeliac disease. AIM: We determined the prevalence of coeliac disease in two different populations living in an urban area and in a sub-urban impoverished area of Recife (Brazil), using two rapid tests based on detection of anti-transglutaminase antibodies in serum and in one drop of whole blood. METHODS: Whole-blood and serum samples from 1074 individuals were analysed by the two rapid tests; 580 subjects were university students and 494 subjects were coming from sub-urban impoverished areas, characterized by the endemic presence of filariasis. The positive subjects were evaluated by anti-tranglutaminase enzyme linked immunosorbent assay (ELISA) assay, the coeliac disease-related HLA DQ2/8 and intestinal biopsy. RESULTS: Both rapid assays were positive in 25/1074 subjects, but only 9/25 (4/4 in urban areas, specificity 100%; 5/21 in poor areas, specificity 76%) were confirmed positive by ELISA assay. The nine subjects testing positive for HLA DQ2 and the intestinal biopsy showed the typical coeliac disease lesions (coeliac disease-prevalence: 0.84%, 9/1074); seven coeliacs were asymptomatic and two presented recurrent abdominal pain. CONCLUSIONS: The rapid assays were accurate in finding new coeliacs at a remarkably low cost. We are convinced that this new way of testing for coeliac disease can be successfully used by non-specialized personnel in daily practice in developing countries.

Coeliac disease in primary care: evaluation of a case-finding strategy.

BACKGROUND: Coeliac disease is still under-diagnosed as a consequence of poor physician awareness of the clinical spectrum of the disease. We evaluated the feasibility and the cost-effectiveness of a case-finding approach for early identification of cases, carried out by primary care practitioners. METHODS: We developed a case-finding strategy based on testing for anti-tissue transglutaminase IgA antibodies in subjects showing predefined signs and symptoms or belonging to at-risk groups. RESULTS: Sixty-nine primary care doctors and 60 primary care paediatricians agreed to participate. One thousand forty-one adults and 447 children were selected for anti-tissue transglutaminase testing during the year of the study (2001). Thirty-one (2.08%, 19 adults and 12 children) were ultimately diagnosed as coeliac patients. While no cases of coeliac disease had been diagnosed by the participating doctors in the previous year, 29 subjects were diagnosed as coeliacs in the year after the completion of the study (2002). The prevalence of confirmed coeliac disease in the population under study increased from 1:1,506 to 1:1,073 in adults and from 1:827 to 1:687 in children from year 2000 to 2001. When cases diagnosed in 2002 are included, the prevalence is 1:832 and 1:602, respectively. We calculated a cost of 923.25 euros for each new case diagnosed. CONCLUSIONS: Case-finding is a feasible and successful strategy for detecting undiagnosed coeliac patients and has the important added value of increasing the awareness of the disease among primary care physicians; it represents a cost-effective alternative to population screening for reducing the burden of undiagnosed coeliac disease.

Anti-brain but not celiac disease antibodies in Landau-Kleffner syndrome and related epilepsies.

The Landau-Kleffner syndrome, the continuous spikes-waves during slow sleep syndrome and the benign epilepsy of childhood with rolandic spikes are rare childhood epilepsies with unknown etiology. Improvement in patients treated with immunoglobulin suggests an involvement of the immune system. We provide immunohistochemical evidence of autoantibodies against rat brain auditory cortex, brainstem and cerebellum, in children suffering with one or more of these syndromes. Only 1/14 patient was celiac.

A population based seroepidemiological survey of Chlamydia pneumoniae infections in schoolchildren.

AIM: A serosurvey was carried out in schoolchildren from a northeastern area of Italy to define the burden of Chlamydia pneumoniae infection. METHODS: A sample of 649 schoolchildren underwent a simplified version of the International Study of Asthma and Allergies in Childhood questionnaire and IgG and IgA antibodies were investigated using an enzyme immunoassay, followed by a microimmunofluorescence assay in reactive sera. RESULTS: Of the children examined, 29% and 19.7% had IgG and IgA antibodies, respectively. The IgG prevalence increased with age. No other sociodemographical variable was related to C pneumoniae infection. An association was established between IgA prevalence and previous otitis media. CONCLUSIONS: A mesoendemic (intermediate between high and low endemic level) pattern of C pneumoniae infection is present in schoolchildren from this area and the prevalence rate is related to age. Moreover, this is the first epidemiological evidence of the role of C pneumoniae in otitis.

The humoral response in the pathogenesis of gluten ataxia.

OBJECTIVE: To characterize humoral response to cerebellum in patients with gluten ataxia. BACKGROUND: Gluten ataxia is a common neurologic manifestation of gluten sensitivity. METHODS: The authors assessed the reactivity of sera from patients with gluten ataxia (13), newly diagnosed patients with celiac disease without neurologic dysfunction (24), patients with other causes of cerebellar degeneration (11), and healthy control subjects (17) using indirect immunocytochemistry on human cerebellar and rat CNS tissue. Cross-reactivity of a commercial IgG antigliadin antibody with human cerebellar tissue also was studied. RESULTS: Sera from 12 of 13 patients with gluten ataxia stained Purkinje cells strongly. Less intense staining was seen in some but not all sera from patients with newly diagnosed celiac disease without neurologic dysfunction. At high dilutions (1:800) staining was seen only with sera from patients with gluten ataxia but not in control subjects. Sera from patients with gluten ataxia also stained some brainstem and cortical neurons in rat CNS tissue. Commercial anti-gliadin antibody stained human Purkinje cells in a similar manner. Adsorption of the antigliadin antibodies using crude gliadin abolished the staining in patients with celiac disease without neurologic dysfunction, but not in those with gluten ataxia. CONCLUSIONS: Patients with gluten ataxia have antibodies against Purkinje cells. Antigliadin antibodies cross-react with epitopes on Purkinje cells.

Role of human-tissue transglutaminase IgG and anti-gliadin IgG antibodies in the diagnosis of coeliac disease in patients with selective immunoglobulin A deficiency.

BACKGROUND: Selective IgA deficiency is associated with coeliac disease, and studies have shown an increased prevalence of coeliac disease in these patients ranging from 0.71 to 30.7%, depending on the test used for screening. AIMS: To determine the sensitivity of IgG anti-gliadin-antibodies and of IgG human-tissue-transglutaminase for diagnosing coeliac disease and assessing its prevalence in subjects with IgA deficiency. SUBJECTS: We tested serum samples from 126 IgA-deficient children (66 female, median age: 10.8 years). METHODS: All samples were analysed to measure IgG anti-gliadin-antibodies and IgG anti-human-tissue-transglutaminase. Patients testing positive to either test underwent intestinal biopsy. Subjects testing positive for IgG anti-human-tissue-transglutaminase underwent genetic testing for the human leucocyte antigen heterodimer. RESULTS: Twenty-seven of 126 subjects tested positive for IgG anti-gliadin-antibodies (five of whom tested positive also for IgG anti-human-tissue-transglutaminase) and 18 (including the aforementioned five) for IgG anti-human-tissue-transglutaminase. Intestinal biopsy was performed in 37 of the 40 patients who tested positive (three subjects refused). Eleven had positive intestinal biopsies all of whom tested positive for IgG anti-human-tissue-transglutaminase, but only five of these tested positive also for IgG anti-gliadin-antibodies. All 22 patients testing positive for anti-gliadin-antibody alone had normal intestinal mucosa. All the patients who tested positive for IgG anti-human-tissue-transglutaminase and underwent genetic screening (15/18) had the coeliac-related human leucocyte antigen. Overall, coeliac disease was diagnosed in 11 of the 126 subjects with IgA deficiency (8.7%). CONCLUSIONS: The prevalence of coeliac disease in subjects with total IgA deficiency was 8.7%. Assay of IgG anti-human-tissue-transglutaminase can be recommended for screening coeliac disease in IgA-deficient subjects.

Analyzing the peripheral blood antibody repertoire of a celiac disease patient using phage antibody libraries.

Celiac disease (CD) is an autoimmune enteropathy characterized by intestinal malabsorption and immunological responses to dietary gliadins and an auto antigen located in the endomysium. The latter has recently been identified as the enzyme tissue transglutaminase (tTG). The linkage between gliadins, tTG and the autoimmune response has still to be clarified. In this work we report the production and analysis of a phage antibody library from the peripheral blood lymphocytes (PLB) of a CD patient. The library contained polyreactive and monoreactive antibodies to alpha-gliadin, to the dietary antigen beta-lactoglobulin, but not to tTG. The majority of the VH regions of the anti-alpha-gliadin antibodies belonged to the VH 4 family. The possibility of exploiting phage display antibodies as tools to study the molecular events associated with CD is discussed.

[Antibodies against milk and soy proteins in specific intolerances and celiac disease]. / Anticorpi anti-proteine del latte e della soia nelle intolleranze specifiche e nella celiachia.

It has been suggested that high serum level of food proteins antibodies (especially cow's milk protein antibodies) may have a specific meaning in the diagnosis of food allergy, especially presenting with gastrointestinal complaints. In our study we tested with enzyme-linked immunoabsorbent assay test (ELISA) the antibody serum level to cow's milk and soy proteins in 123 children. The following patients were included in the study: 30 children with cow's milk enteropathy (CME), 27 children with soy proteins intolerance (SI), 19 coeliac children on gluten containing diet (CD), 9 coeliac children on gluten free diet (GFD) and 50 healthy sex and age matched control children. All coeliac patients had assumed cow's milk and soy proteins at the moment of the test or a few days before. Higher antibody serum level was found in coeliac disease to both cow's milk and soy proteins and in cases with cow's and soy allergy, than in control cases. The highest mean value of cow's milk and soy proteins antibodies have been found in the untreated coeliac children (CD), also higher than in the two groups of specific allergy. In treated coeliac children (GFD) with normalized jejunal mucosa cow's milk and soy proteins antibodies was normal. None of the 19 CD with high cow's milk and soy proteins antibodies level showed clinical intolerance to cow's milk and soy proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

[Anti-alpha-gliadin antibodies. Sensitivity, specificity and correlation with blood xylose test in the 3 diagnostic stages of celiac disease in children]. / Anticorpi antialfagliadina. Sensibilità, specificità e correlazione con la xilosemia da carico nelle tre fasi diagnostiche della malattia celiaca nel bambino.

The specificity and sensibility of IgA and IgG alfagliadin antibody test (AaGA) for screening, diagnosis and follow-up of childhood coeliac disease has been evaluated. We have compared AaGA test to D-xylose blood test and at last we have examined the false positive and negative results given by the test. Two groups of subjects were considered: 1) 90 children with untreated coeliac disease (21 newly diagnosed (I stage), 50 in gluten withdrawal (II stage), 19 in challenge (III stage); 2) 255 disease controls including: 157 healthy controls; 31 children with gastroenterological disorders other than coeliac disease; 31 children with food allergy and atopic dermatitis; 36 children with "constitutional" short stature (without GH deficiency and with normal intestinal mucosa). The sensibility of AaGA test in the first stage of coeliac disease has been of 95.2% for the IgG class antibody and 90.4% for the IgA class; on the other hand the showed a specificity of 83.6% for IgG class antibody and 96.9% for IgA class. In only two newly diagnosed coeliac children we have found false negative results: in the first case the patient was IgA-deficient, in the second the age was above 3 years. AaGA IgA resulted positive only in the 12.9% of the group of gastroenterological and atopic controls; particularly most cases were affected by multiple food allergies and two patients by chronic autoimmune disease of small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-α-enolase Antibodies in Serum from Pediatric Patients Affected by Inflammatory Diseases: Diagnostic and Pathogenetic Insights.

Human glycolytic enzyme α-enolase was associated with human diseases and with inflammation. An ELISA test was developed to measure anti-α-enolase AAE IgG and AAE IgA in the serum from patients affected by inflammatory diseases with the purpose to evaluate it as a novel diagnostic marker. 80 healthy blood donors and 194 paediatric patients affected by Juvenile idiopathic arthritis (JIA), celiac disease (CD), Crohn's Disease (CrD), hereditary periodic fever (HPF), and PFAPA syndrome were included in the study. HPF patients showed high levels of AAE antibodies, whereas JIA, CD, and CrD presented only partial results. Benign fevers such as PFAPA were almost negative for AAE Abs. These findings suggested that the genetic dysfunction of inflammasome associated with HPF could lead to the formation of AAE Abs that could be used for an early and easy diagnosis.