Profiling celiac disease antibody repertoire.

The aim of this study was to dissect the autoantibody response in celiac disease (CD) that remains largely unknown, with the goal of identifying the disease-specific autoantigenic protein pattern or the so called epitome. Sera from CD patients were used to select immunoreactive antigens from a cDNA phage-display library. Candidate genes were identified, the corresponding proteins produced and their immunoreactivity validated with sera from CD patients and controls. Thirteen CD-specific antigens were identified and further validated by protein microarray. The specificity for 6 of these antigens was confirmed by ELISA. Furthermore we showed that this antibody response was not abolished on a gluten free diet and was not shared with other autoimmune diseases. These antigens appear to be CD specific and independent of gluten induction. The utility of this panel extends beyond its diagnostic value and it may drive the attention to new targets for unbiased screens in autoimmunity research.

Treatment of experimental arthritis by targeting synovial endothelium with a neutralizing recombinant antibody to C5.

OBJECTIVE: To show that a new recombinant protein (MT07) obtained by fusing a synovial-homing peptide to a neutralizing antibody to C5 can be selectively delivered to inflamed synovium and can effectively control joint inflammation in experimental models of arthritis. METHODS: Binding of MT07 to human, rat, and mouse synovial tissue was evaluated in vitro by immunofluorescence, and selective localization in the inflamed joints of rats was documented in vivo using time-domain optical imaging. The antiinflammatory effect of MT07 was tested in a rat model of antigen-induced arthritis (AIA) and in a mouse model of collagen antibody-induced arthritis (CAIA). RESULTS: MT07 was able to bind to samples of inflamed synovium from humans, mice, and rats while failing to recognize uninflamed synovium as well as inflamed mouse lung or rat kidney. In vivo analysis of the biodistribution of MT07 confirmed its preferential homing to inflamed joints, with negligible inhibition of circulating C5 levels. MT07 prevented and resolved established inflammation in a rat model of AIA, as demonstrated by changes in joint swelling, polymorphonuclear cell counts in synovial washes, release of interleukin-6 and tumor necrosis factor α, and tissue damage. A similar therapeutic effect was obtained testing MT07 in a CAIA model. CONCLUSION: Our findings show that the novel recombinant molecule MT07 has the unique ability to selectively target inflamed joints and to exert local control of the inflammatory process by neutralizing the complement system without interfering with circulating C5 levels. We believe that this approach can be extended to other antiinflammatory drugs currently used to treat patients with rheumatoid arthritis.

In vivo anti-lymphoma activity of an agonistic human recombinant anti-TRAIL-R2 minibody.

A new single-chain fragment variable (scFv) to TRAIL-R2 receptor produced as minibody (MB2.23) was characterized for anti-lymphoma activity in vivo. For this purpose, a disseminated lymphoma model was generated by intraperitoneal inoculation of BJAB cells in severe combined immunodeficiency mice. Two weekly injections with MB2.23 (10 mg/kg) were able to significantly increase the median survival time of lymphoma-bearing animals with respect to the vehicle-treated control mice, providing a rationale for further investigating the use of MB2.23 in anticancer therapy.

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.

Cryptic genetic gluten intolerance revealed by intestinal antitransglutaminase antibodies and response to gluten-free diet.

BACKGROUND AND OBJECTIVE: Antitransglutaminase (anti-TG2) antibodies are synthesised in the intestine and their presence seems predictive of future coeliac disease (CD). This study investigates whether mucosal antibodies represent an early stage of gluten intolerance even in the absence of intestinal damage and serum anti-TG2 antibodies. METHODS: This study investigated 22 relatives of patients with CD genetically predisposed to gluten intolerance but negative for both serum anti-TG2 antibodies and intestinal abnormalities. Fifteen subjects were symptomatic and seven were asymptomatic. The presence of immunoglobulin A anti-TG2 antibodies in the intestine was studied by creating phage-antibody libraries against TG-2. The presence of intestinal anti-TG2 antibodies was compared with the serum concentration of the intestinal fatty acid-binding protein (I-FABP), a marker for early intestinal mucosal damage. The effects of a 12-month gluten-free diet on anti-TG2 antibody production and the subjects' clinical condition was monitored. Twelve subjects entered the study as controls. RESULTS: The intestinal mucosa appeared normal in 18/22; 4 had a slight increase in intraepithelial lymphocytes. Mucosal anti-TG2 antibodies were isolated in 15/22 subjects (68%); in particular symptomatic subjects were positive in 13/15 cases and asymptomatic subjects in 2/7 cases (p=0.01). No mucosal antibodies were selected from the controls' biopsies. There was significant correlation between the presence of intestinal anti-TG2 antibodies and positive concentrations of I-FABP (p=0.0008). After a gluten-free diet, 19/22 subjects underwent a second intestinal biopsy, which showed that anti-TG2 antibodies had disappeared in 12/15 (p=0.002), while I-FABP decreased significantly (p<0.0001). The diet resolved both extraintestinal and intestinal symptoms. CONCLUSIONS: A new form of genetic-dependent gluten intolerance has been described in which none of the usual diagnostic markers is present. Symptoms and intestinal anti-TG2 antibodies respond to a gluten free-diet. The detection of intestinal anti-TG2 antibodies by the phage-antibody libraries has an important diagnostic and therapeutic impact for the subjects with gluten-dependent intestinal or extraintestinal symptoms. Clinical trial number NCT00677495.

Inhibition of transglutaminase 2 enzymatic activity ameliorates the anti-angiogenic effects of coeliac disease autoantibodies.

OBJECTIVE: Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. MATERIAL AND METHODS: The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. RESULTS: Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. CONCLUSIONS: Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.

Anti-idiotypic response in mice expressing human autoantibodies.

Celiac disease is an autoimmune illness characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals. The immune response is based on both cellular and humoral components, although the former seem to be more important in the pathogenesis. The autoantibody response is directed at the enzyme tissue transglutaminase, tTG or TG2, which possibly play a role in the onset of the disease. In this study we sought to develop an animal model in which to analyze the immunological regulation and significance of anti-TG2 antibodies, by expressing specific human single-chain antibody fragments in mice using adeno-associated virus vectors. Upon vector injection in the skeletal muscles, high and persistent systemic levels of anti-TG2 antibodies were obtained. Mice injected with vectors encoding antibodies also recognizing rodent TG2, also developed a strong anti-idiotypic response. This finding raises the question of whether an anti-idiotypic response to anti-TG2 antibodies is a factor associated with celiac disease.

Construction of miniantibodies for the in vivo study of human autoimmune diseases in animal models.

BACKGROUND: Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated. RESULTS: We were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". These are produced by genetic fusion of anti-tTG scFv to Human, Mouse or Rat Fc domains, making them suitable for in vivo expression. The results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous Fc domains. Further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. Finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host. CONCLUSION: In this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of Celiac Disease. This can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases.

Gluten ataxia: passive transfer in a mouse model.

Gluten sensitivity is an autoimmune disease that usually causes intestinal atrophy resulting in a malabsorption syndrome known as celiac disease. However, gluten sensitivity may involve several organs and is often associated with extraintestinal manifestations. Typically, patients with celiac disease have circulating anti-tissue transglutaminase and anti-gliadin antibodies. When patients with gluten sensitivity are affected by other autoimmune diseases, other autoantibodies may arise like anti-epidermal transglutaminase in dermatitis herpetiformis, anti-thyroid peroxidase antibodies in thyroiditis, and anti-islet cells antibodies in type 1 diabetes. The most common neurological manifestation of gluten sensitivity is ataxia, the so-called gluten ataxia (GA). In patients with GA we have demonstrated that anti-gliadin and anti-tissue transglutaminase antibodies cross-react with neurons but that additional anti-neural antibodies are present. The aim of the present article is to review the knowledge on animal models of gluten sensitivity, as well as reviewing the role of anti-neural antibodies in GA.

Characterizing monoclonal antibody epitopes by filtered gene fragment phage display.

In the present paper, we describe a novel approach to map monoclonal antibody epitopes, using three new monoclonal antibodies that recognize h-TG2 (human transglutaminase 2) as an example. The target gene was fragmented and cloned upstream of an antibiotic-resistance gene, in the vector pPAO2, to select for in-frame polypeptides. After removal of the antibiotic-resistance gene by Cre/Lox recombination, an antigen fragment phage display library was created and selected against specific monoclonal antibodies. Using the h-TG2 fragment library, we were able to identify epitopes. This technique can also be broadly applied to the study of protein-protein interactions.

Dual sugar gut-permeability testing on blood drop in animal models.

BACKGROUND: Intestinal permeability is determined by measuring nonmetabolized sugars. In animals, intestinal permeability is determined in urine, using cumbersome and expensive metabolic cages. We developed an HPLC method for determining concentrations of lactulose (L) and L-rhamnose (R) in blood-drop of rabbits and mice, and we compared these results with the procedure based on sugars excreted in urine. We measured the intestinal permeability induced by a fragment (DeltaG) of the zonula occludens toxin which opens the paracellular pathway. METHODS: The animals received sugar solution and later received the same solution+DeltaG. Five-hour urine collection and timed blood tests were performed after ingestion of sugars. Sugars were measured with HPLC, and the percentage of recovered sugars was expressed as L/R ratio. RESULTS: At 60 min after administration of sugars, the mean L/R ratio for rabbits and mice was 0.026 and 0.052, respectively. At 60 min after administration of sugars+DeltaG, the mean L/R ratio for rabbits and mice was 0.22 and 0.53. The mean L/R ratio in the urine was 0.023 at basal condition and 0.25 after DeltaG ingestion. CONCLUSIONS: Testing small serum samples for sugar permeability is effective for monitoring changes in permeability of the gut in animals. This cheap simple method allows us to measure in vivo the biological activity of other molecules which modulate the paracellular pathway.

Antibodies in proteomics I: generating antibodies.

The explosion in genome sequencing, and in subsequent DNA array experiments, has provided extensive information on gene sequence, organization and expression. This has resulted in a desire to perform similarly broad experiments on all the proteins encoded by a genome. Panels of specific antibodies, or other binding ligands, will be essential tools in this endeavour. Because traditional immunization will be unlikely to generate antibodies in sufficient quantity, and of the required quality and reproducibility, in vitro selection methods will probably be used. This review--the first of two--examines the strategies available for in vitro antibody selection. The second review discusses the adaptation of these methods to high throughput and the uses to which antibodies, once derived, can be put.

Antibodies in proteomics II: screening, high-throughput characterization and downstream applications.

There are many ways in which the use of antibodies and antibody selection can be improved and developed for high-throughput characterization. Standard protocols, such as immunoprecipitation, western blotting and immunofluorescence, can be used with antibody fragments generated by display technologies. Together with novel approaches, such as antibody chips and intracellular immunization, these methods will yield useful proteomic data following adaptation of the protocols for increased reliability and robustness. To date, most work has focused on the use of standard, well-characterized commercial antibodies. Such protocols need to be adapted for broader use, for example, with antibody fragments or other binders generated by display technologies, because it is unlikely that traditional approaches will provide the required throughput.

Predicting antigenic peptides suitable for the selection of phage antibodies.

Selection from phage antibody libraries can be considered to be an in vitro immune system in which the antibody response is reduced to the bare minimum of antigen recognition. Using selections of antibodies on peptides from a phage antibody library, we investigated what constitutes peptide antigenicity in the context of the antibody-protein binding site. We selected polyclonal antibodies in a high throughput format against 44% of 90 overlapping peptides derived from three different proteins. Of these, 33% of peptides (epitopic peptides) were able to select antibodies that recognized the protein from which the peptides were derived. Although no algorithm was able to predict all epitopic peptides, solvent accessibility was the best predictor in this cell-free antibody selection context. We subsequently applied solvent accessibility to successfully predict epitopic peptides from p53 and Znf217, and showed that such peptide selected single-chain antibodies were able to recognize soluble p53 in ELISA and Znf217 in a western blot. This is likely to have considerable utility in functional genomics and proteomics where it should be possible to select antibodies against gene products on the basis of deduced amino acid sequence in a high throughput fashion.

Prevention of arthritis by locally synthesized recombinant antibody neutralizing complement component C5.

Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have addressed these issues by developing an alternative approach consisting of the intraarticular injection of a DNA vector encoding for the anti-C5 neutralizing recombinant miniantibody MB12/22. This method allows local production of the antibody in sufficient amount to be effective in preventing joint inflammation in a rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong signal peaking at 3 days after administration. DNA encoding for the minibody was shown for 14 days in the synovial tissue and was undetectable in the controlateral knee and in other organs. The preventive effect of this approach was evaluated in rats receiving a single injection of the vector 3 days before the induction of antigen-induced arthritis and analyzed 3 days later. The treated rats exhibited a lower increase in swelling, associated with a lower number of PMN in the articular washes and reduced deposition of C9 in synovial tissue compared to control rats. These results suggest that treating the inflamed joints with a vector that induces a local production of a neutralizing anti-C5 antibody may represent a useful strategy to inhibit in situ complement activation and to treat patients with monoarthritis. Moreover, this approach may be adopted as a novel therapeutic strategy to prevent monoarthritis as an alternative to local treatment with antibodies commonly used in this form of arthritis, with the advantages of the lower cost and the longer persistence of antibody production.

Simple scale-up of recombinant antibody production using an UCOE containing vector.

Recombinant proteins, in particular antibodies, have become fundamental in biomedical research where they are used in numerous therapeutic and diagnostic applications. For this reason there is an increasing demand for quick and economical production systems for recombinant proteins in mammalian cells.

RhoB is associated with the anti-angiogenic effects of celiac patient transglutaminase 2-targeted autoantibodies.

Celiac patient-derived anti-transglutaminase 2 (TG2) antibodies disturb several steps in angiogenesis, but the detailed molecular basis is not known. Therefore, we here analyzed by microarray technology the expression of a set of genes related to angiogenesis and endothelial cell biology in order to identify factors that could explain our previous data related to vascular biology in the context of celiac disease. To this end, in vitro models using human umbilical vein endothelial cells (HUVECs) or in vivo models of angiogenesis were used. A total of 116 genes were analyzed after treatment with celiac patient autoantibodies against TG2. Compared to treatment with control IgA celiac patient, total IgA induced a consistent expression change of 10 genes, the up-regulation of four and down-regulation of six. Of these genes the up-regulated RhoB was selected for further studies. RhoB expression was found to be up-regulated at both messenger RNA and protein level in response to celiac patient total IgA as well as anti-TG2-specific antibody derived from a celiac patient. Interestingly, down-regulation of RhoB by specific small interfering RNA treatment in endothelial cells could rescue the deranged endothelial length and tubule formation caused by celiac disease autoantibodies. RhoB function is controlled by its post-translational modification by farnesylation. This modification of RhoB required for its correct function can be prevented by the cholesterol lowering drug simvastatin, which was also able to abolish the anti-angiogenic effects of celiac anti-TG2 autoantibodies. Taken together, our results would suggest that RhoB plays a key role in the response of endothelial cells to celiac disease-specific anti-TG2 autoantibodies.

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.