Fiorella Florian

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.

Anti transglutaminase antibodies cause ataxia in mice

Background Celiac disease (CD) is an autoimmune gastrointestinal disorder characterized by the presence of anti-transglutaminase 2 (TG2) and anti-gliadin antibodies. Amongst the neurological dysfunctions associated with CD, ataxia represents the most common one. Methods We analyzed by immunohistochemistry, the anti-neural reactivity of the serum from 20 CD patients. To determine the role of anti-TG2 antibodies in ataxia, two anti-TG2 single chain variable fragments (scFv), isolated from a phage-display IgA antibody library, were characterized by immunohistochemistry and ELISA, and injected in mice to study their effects on motor coordination. We found that 75% of the CD patient population without evidence of neurological involvement, has circulating anti-neural IgA and/or IgG antibodies. Two anti-TG2 scFvs, cloned from one CD patient, stained blood vessels but only one reacted with neurons. This anti-TG2 antibody showed cross reactivity with the transglutaminase isozymes TG3 and TG6. Intraventricular injection of the anti-TG2 or the anti-TG2/3/6 cross-reactive scFv provoked transient, equally intensive ataxia in mice. Conclusion The serum from CD patients contains anti-TG2, TG3 and TG6 antibodies that may potentially cause ataxia.

Molecular analysis of hyperoxaluria type 1 in Italian patients reveals eight new mutations in the alanine : glyoxylate aminotransferase gene

Abstract Systematic screening using the SSCP technique followed by sequencing of bands with abnormal mobility derived from the AGXT exons of 15 unrelated Italian patients with primary hyperoxaluria type 1 (PH1) allowed us to characterize both the mutant alleles in each individual. Eight new mutations were identified: C155del, C156ins, G244T, C252T, GAG408ins, G468A, G588A and G1098del. This study demonstrates both the effectiveness of the screening strategy chosen to identify all the mutant alleles and the high degree of allelic heterogeneity in PH1.

Majority of Children With Type 1 Diabetes Produce and Deposit Anti-Tissue Transglutaminase Antibodies in the Small Intestine

OBJECTIVE Anti-tissue transglutaminase (TG2) antibodies are the serological marker of celiac disease. Given the close association between celiac disease and type 1 diabetes, we investigated the production and deposition of anti-TG2 antibodies in the jejunal mucosa of type 1 diabetic children. RESEARCH DESIGN AND METHODS Intestinal biopsies were performed in 33 type 1 diabetic patients with a normal mucosal architecture: 14 had high levels (potential celiac disease patients) and 19 had normal levels of serum anti-TG2 antibodies. All biopsy specimens were investigated for intestinal deposits of IgA anti-TG2 antibodies by double immunofluorescence. In addition, an antibody analysis using the phage display technique was performed on the intestinal biopsy specimens from seven type 1 diabetic patients, of whom four had elevated and three had normal levels of serum anti-TG2 antibodies. RESULTS Immunofluorescence studies showed that 11 of 14 type 1 diabetic children with elevated levels and 11 of 19 with normal serum levels of anti-TG2 antibodies presented with mucosal deposits of such autoantibodies. The phage display analysis technique confirmed the intestinal production of the anti-TG2 antibodies; however, whereas the serum-positive type 1 diabetic patients showed a preferential use of the VH5 antibody gene family, in the serum-negative patients the anti-TG2 antibodies belonged to the VH1 and VH3 families, with a preferential use of the latter. CONCLUSIONS Our findings demonstrate that there is intestinal production and deposition of anti-TG2 antibodies in the jejunal mucosa of the majority of type 1 diabetic patients. However, only those with elevated serum levels of anti-TG2 antibodies showed the VH usage that is typical of the anti-TG2 antibodies that are produced in patients with celiac disease.

Characterization of the Anti-Tissue Transglutaminase Antibody Response in Nonobese Diabetic Mice

Type 1 diabetes mellitus is an autoimmune disorder characterized by destruction of insulin-producing pancreatic β cells by T lymphocytes. In nonobese diabetic (NOD) mice, a role has been hypothesized for dietary gluten proteins in the onset of diabetes, and because gluten dependence is the major feature of celiac disease, together with production of Abs to the autoantigen tissue transglutaminase (tTG), we looked for the presence of anti-tTG Abs in the serum of NOD mice and, to establish their origin, analyzed the Ab repertoire of NOD mice using phage display Ab libraries. We found significant levels of serum anti-tTG Abs and were able to isolate single-chain Ab fragments to mouse tTG mainly from the Ab libraries made from intestinal lymphocytes and to a lesser extent from splenocytes. Data from NOD mice on a gluten-free diet suggest that the anti-tTG response is not gluten-dependent. The intestinal Ab response to tTG is a feature of NOD mice, but the underlying mechanisms remain obscure.

Cryptic gluten intolerance in type 1 diabetes: identifying suitable candidates for a gluten free diet

Long term exposure to gluten in coeliacs,1 and coeliac disease (CD) diagnosis after 16 years of age2 may induce type 1 diabetes (T1D) and other autoimmune disorders. Increased prevalence of CD among diabetics and their relatives is well documented.3 Early introduction of gluten to children at high risk for T1D produces T1D associated islet autoantibodies.4 Similarly, in the absence of overt clinical symptoms of T1D, some coeliac children produce diabetes autoantibodies in a gluten dependent manner.5 In diabetics, intestinal challenge with gluten produces mucosal recruitment of lymphocytes,6 similar to that in CD patients.7 In diabetics, however, there is no production of CD related anti-tissue transglutaminase antibodies (anti-tTG).6 We have used a phage display assay8 to show that in CD patients, production of anti-tTG is limited to the intestine. Here, we …

The gut as site of production of autoimmune antibodies.

A powerful way of dissecting the antibody immune response is offered by phage display. Phage display of human antibody fragments has proved to be an effective method to investigate in vivo antibody responses in both tumor and auto-immune diseases, with the in vitro repertoire created being considered a mimic of the antibody specificity of the lymphocyte source used to create the library. In this method, V genes derived from a patient’s lymphocytes are used to express a patient’s antibody repertoire as single-chain antibody fragment (scFv) fused to the coat protein of a filamentous phage vector that carries the encoded antibody gene. Each phage carries a single antibody specificity, and by selecting specific binding phage from the library valuable information can be acquired on V gene usage and the lymphocyte source of serum antibodies. This work has been most extensively performed with thyroid disease, with the antibodies selected from such libraries having similar specificities to those found in patients’ serum and with the high affinities characteristic of immune libraries. Similar experiments have been done with type 1 diabetes, systemic lupus erythematosis, Sjogren’s syndrome, paraneoplastic encephalomyelitis and myasthenia gravis. Interestingly, this approach has not yet been attempted with Celiac disease (CD), the commonest auto-immune disease. Celiac disease is accompanied by antibody responses to dietary gliadins and tissue transglutaminase (TG2 or tTG), an autoantigen located in the endomysium. tTG 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. In a preliminary study (1) we made and selected phage antibody libraries from lymphocytes of CD patients. We were able to isolate scFv to tTG from all intestinal lymphocyte libraries but not from those obtained from peripheral lymphocytes. This is in contrast to antibodies against gliadin, which could be obtained from all libraries, indicating that the humoral response against tTG occurs at the intestinal level, whereas that against gliadin occurs both peripherally and centrally. IgA antibodies from three different patients recognized the same tTG epitopes and by enzyme linked immunosorbent assay competition experiments we demonstrated that the number of epitopic regions recognized was restricted to two. Moreover, the scFv to tTG were analyzed for the epitope(s) recognized by using cloned deletion mutants of tTG gene (2). All the scFv showed to recognize a region of tTG we identified as being conformational and located in the core domain of the enzyme. This was indentical with the region recognized by anti-tTG antibodies in the serum of celiac patients. As a further result of these studies, we found that the VH gene use of the anti-tTG antibodies was restricted to three of the seven human antibody VH families, with many of the VH genes belonging to the VH5 family with a preferential use of the DP73 allele of VH 5-51 gene. The VH5 allele was selected from libraries originated from different patients, indicating a possible biased usage in the autoimmune response to tTG. This has led us to the construction of a phagemid vector (3) for the rapid cloning and selection of anti tTG antibodies from intestinal mucosa bioptic tissue based on the selective amplification of VH5 family members and cloning into a phagemid vector in which a matching VL to produce a functional scFv has already been cloned. This result was made possible by the identification of a restricted use of a VH/VL pairing in the phage display libraries from the intestinal lymphocytes of patients with CD. This method potentially allows the detection of anti-tTG antibody synthesis in the gut mucosa when such antibodies are either borderline or undetectable in the serum. It may also provide additional useful information on biopsies that are not diagnostic of CD, and may lead to the identification of patients at an early stage of the disease, allowing prevention of long-term complications. More recently (Sblattero D, et al., unpublished data) we analyzed the intestine and spleen antibody repertoire of nonobese diabetic (NOD) mice whose major genetic trait is the onset of type 1 diabetes mellitus (T1DM). In Address correspondence and reprint requests to: Dr. R Marzari, Department of Biology, University of Trieste, Via L. Giorgieri 10, 34127, Trieste, Italy; Fax: 39-040-5587552 (e-mail: marzari@univ.trieste.it). Journal of Pediatric Gastroenterology and Nutrition 39:S730–S731

Subclinical alteration of the cervical–vaginal microbiome in women with idiopathic infertility

Biomarkers have a wide application in research and clinic, they help to choose the correct treatment for diseases. Recent studies, addressing the vaginal microbiome using next generation sequencing (NGS), reported the involvement of bacterial species in infertility. We compared the vaginal microbiome of idiopathic infertile women with that of healthy, including bacterial vaginosis affected women and non‐idiopathic infertile women, to identify bacterial species suitable as biomarkers. Information on microorganisms was obtained from the V3‐16S rDNA sequencing of cervical–vaginal fluids of 96 women using the Ion Torrent platform. Data were processed with QIIME and classified against the Vaginal 16S rDNA Reference Database. The analysis revealed a significant beta‐diversity variation (p < 0.001) between the four groups included in the study. L. iners, L. crispatus, and L. gasseri distinguished idiopathic infertile women from the other groups. In these women, a microbial profile similar to that observed in bacterial vaginosis women has been detected. Our results suggest that the quantitative assessment and identification of specific microorganisms of the cervical–vaginal microflora could increase the accuracy of available tools for the diagnosis of infertility and improve the adoption of therapeutic protocols.

Detection of AGXT gene mutations by denaturing high-performance liquid chromatography for diagnosis of hyperoxaluria type 1

Abstract Primary hyperoxaluria type 1 is an autosomal recessive disorder of glyoxylate metabolism, caused by a deficiency of alanine:glyoxylate aminotransferase, which is encoded by a single copy gene (AGXT). The aim of this research was to standardize denaturing high-performance liquid chromatography, a new, sensitive, relatively inexpensive, and automated technique, for the detection of AGXT mutation. Denaturing high-performance liquid chromatography was used to analyze in blind the AGXT gene in 20 unrelated Italian patients with primary hyperoxaluria type 1 previously studied by other standard methods (single-strand conformation polymorphism analysis and direct sequencing) and 50 controls. Denaturing high-performance liquid chromatography allowed us to identify 13 mutations and the polymorphism at position 154 in exon I of the AGXT gene. Hence the method is more sensitive and less time consuming than single-strand conformation polymorphism analysis for the detection of AGXT mutations, thus representing a useful and reliable tool for detecting the mutations responsible for primary hyperoxaluria type 1. The new technology could also be helpful in the search for healthy carriers of AGXT mutations amongst family members and their partners, and for screening of AGXT polymorphisms in patients with nephrolithiasis and healthy populations.

Profiling a North-East Italian Population by Four Highly Polymorphic DNA Probes

After the forensic validation of the SBA (Southern Blot Analysis), the use of SLPs (single locus probes) has been used in the forensic laboratory for personal identification and paternity testing since the end of the 1980s. Even if expensive and time consuming, the SBA remains the most informative approach in some circumstances, for example in those paternity cases where either the alleged father is unavailable or the paternity index achieved by PCR-based polymorphisms is too low to prove paternity. In this work the allele frequenciy distributions at four highly polymorphic loci (D1S7, D7S21, D12S11 and D7S22) are described in a population living in the Trieste and Gorizia areas (North-East Italy).