Roberto Marzari

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.

In vivo Targeting of Human Neutralizing Antibodies against CD55 and CD59 to Lymphoma Cells Increases the Antitumor Activity of Rituximab

An in vivo model of human CD20+ B-lymphoma was established in severe combined immunodeficiency mice to test the ability of human neutralizing miniantibodies to CD55 and CD59 (MB55 and MB59) to enhance the therapeutic effect of rituximab. The miniantibodies contained single-chain fragment variables and the hinge-CH2-CH3 domains of human IgG(1). LCL2 cells were selected for the in vivo study among six B-lymphoma cell lines for their high susceptibility to rituximab-dependent complement-mediated killing enhanced by MB55 and MB59. The cells injected i.p. primarily colonized the liver and spleen, leading to the death of the animals within 30 to 40 days. Thirty percent of mice receiving biotin-labeled rituximab (25 microg) i.p. on days 4 and 11 after cell injection survived to 120 days. Administration of biotin-labeled rituximab, followed by avidin (40 microg) and biotin-labeled MB55-MB59 (100 microg) at 4-h intervals after each injection resulted in the survival of 70% of mice. Surprisingly, 40% of mice survived after the sole injection of avidin and biotin-labeled MB55-MB59, an observation consistent with the in vitro data showing that the miniantibodies induced killing of approximately 25% cells through antibody-dependent cell cytotoxicity. In conclusion, MB55 and MB59 targeted to tumor cells represent a valuable tool to enhance the therapeutic effect of rituximab and other complement-fixing antitumor antibodies.

Anti-tissue transglutaminase antibodies from coeliac patients inhibit transglutaminase activity both in vitro and in situ

Background and aims: Coeliac disease (CD) is a multifactorial disorder which has an autoimmune component characterised by the occurrence of disease specific autoreactive antibodies against the enzyme tissue transglutaminase (tTG). The aim of this study was to investigate whether binding of antibodies to the enzyme influences tTG activity. Methods: tTG activity was assayed in the presence of immunoglobulin A (IgA) and immunoglobulin G (IgG) purified from the serum of coeliac patients, CUB 7402 (an anti-tTG mouse monoclonal antibody), and human anti-tTG monoclonal antibodies derived from both intestinal lymphocytes from three patients with CD and from peripheral blood lymphocytes from healthy subjects. For our studies we used calcium treated and untreated recombinant human tTG. Furthermore, the effects of antibodies were determined by immunohistochemical detection of tTG activity in sections of human umbilical cord. Results: IgG and IgA from CD patients inhibited tTG activity in vitro in a dose dependent manner, with a different rate of inhibition among patients. The monoclonal antibody CUB 7402 and human monoclonal antibodies displayed a dose dependent inhibitory effect towards the catalytic activity of the enzyme, both in vitro and in situ. Preincubation of tTG with CaCl2 caused loss of the inhibitory effect due to CUB 7402 but not that caused by human monoclonal antibodies. Conclusions: Purified CD IgA, IgG, as well as human anti-tTG monoclonal antibodies inhibited the enzymatic activity of human tTG both in vitro and in situ.

Rapid interactome profiling by massive sequencing

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.

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.

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.

Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries

Antibodies against most antigens can be isolated from high quality phage antibody libraries. However, not all antibodies binding a particular antigen are necessarily found when standard selections are performed. Here we investigate the effect of two different selection strategies on the isolation of antibodies against a number of different antigens, and find that these different strategies tend to select different antibodies, with little overlap between them. This indicates that the full diversity of these libraries is not tapped by a single selection strategy and that each selection strategy imposes different selective criteria in addition to that of antigen binding. To fully exploit such libraries, therefore, many different selection strategies should probably be employed for each antigen. The use of alternative strategies should be considered when selection apparently fails, or when the number of different antibodies recognizing an antigen needs to be maximised. Furthermore, the microtitre selection strategy developed is likely to prove useful in the application of phage antibody libraries to the human genome project, allowing the high throughput selection of antibodies against multiple antigens simultaneously.

Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59.

Expression of the complement‐regulatory proteins (CRP) CD46, CD55 and CD59 represents a strategy used by tumor cells to evade complement‐dependent cell cytoxicity stimulated by monoclonal antibodies. We have isolated two single‐chain variable fragments (scFv) to CD55 and CD59 from a human phage‐display library and from these scFv we have produced two miniantibodies (MB), MB‐55 (against CD55) and MB‐59 (against CD59), containing the human hinge–CH2–CH3 domains of IgG1. The specificity of the two MB for the corresponding CRP was assessed by ELISA using purified CD46, CD55 and CD59. MB‐55 and MB‐59 neutralized the inhibitory activity of CD55 and CD59, respectively, restoring the complement‐mediated lysis of sheep and guinea pig erythrocytes that was otherwise inhibited by the two CRP. FACS analysis revealed binding of MB‐55 and MB‐59 to the lymphoma cell line Karpas 422. The two MB induced a two‐fold increase in the complement‐dependent killing of these cells stimulated by Rituximab, a chimeric anti‐CD20 monoclonal antibody. Transfection of HEK293T cells with vectors encoding MB‐55 or MB‐59 markedly reduced the expression of CD55 and CD59. We conclude that the human antibodies MB‐55 and MB‐59 may represent a therapeutic tool to increase the complement‐dependent killing activity of Rituximab in the treatment of non‐Hodgkins lymphoma.