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.

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 Mg) i.p. on days 4 and 11 after cell injection survived to 120 days. Administration of biotin-labeled rituximab, followed by avidin (40 Mg) and biotin-labeled MB55-MB59 (100 Mg) 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 biotinlabeled MB55-MB59, an observation consistent with the in vitro data showing that the miniantibodies induced killing of f25% 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.

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.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.

BackgroundCeliac 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.MethodsWe 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.ConclusionThe serum from CD patients contains anti-TG2, TG3 and TG6 antibodies that may potentially cause ataxia.