Insulin-degrading enzyme higher in subjects with metabolic syndrome.

Metabolic syndrome (MS) is comprised of a cluster of abnormalities in glucose, lipid, and vascular homeostasis, which is most commonly linked to abdominal obesity. MS heralds increased risk for development of diabetes and is linked to impairment in insulin signaling. Insulin-degrading enzyme (IDE) is one of the mechanisms through which insulin blood levels are maintained. It has been previously suggested that controlling IDE levels could provide yet another potential therapeutic approach in diabetes. Here we aim to investigate whether changes in serum IDE levels correlate with the severity of MS. Using a highly sensitive ELISA assay of active IDE in human serum, we found a strong correlation between circulating IDE levels and circulating levels of triglycerides, insulin, and c-peptide and an inverse correlation with HDL cholesterol (HDLc). Serum IDE levels were higher in MS subjects than in control subjects. Hence, circulating IDE may serve as a tool to identify subjects with abnormal insulin metabolism, possibly those with MS that are at risk to develop diabetes.

Cross-reactivity between annexin A2 and Beta-2-glycoprotein I in animal models of antiphospholipid syndrome.

Antiphospholipid syndrome (APS) affects coagulation and the brain by autoimmune mechanisms. The major antigen in APS is beta-2-glycoprotein I (ß2-GPI) is known to complex with annexin A2 (ANXA2), and antibodies to ANXA2 have been described in APS. We measured these antibodies in mice with experimental APS (eAPS) induced by immunization with ß2-GPI. Sera of these mice reacted significantly with recombinant ANXA2 by enzyme-linked immunosorbent assay (ELISA) and the eAPS mice had significantly high levels of immunoglobulin G (IgG) in the brain by immunoblot assays compared to adjuvant immunized controls. Immunoprecipitation performed by mixing eAPS brain tissue with protein-G beads resulted in identification of two autoantigens unique to the eAPS group, one of which was ANXA2. In order to study more directly and methodically the specific role of anti-ANXA2 antibodies in APS, we immunized mice with ß2-GPI which contained no ANXA2 or with ANXA2 and measured antibodies to these proteins. Levels of antibodies to ANXA2 measured by ELISA were 0.72 ± 0.007 arbitrary units (a.u), 0.24 ± 0.03 and 0.02 ± 0.01 a.u for sera from ANXA2, ß2-GPI and control mice, respectively (p < 0.0001 and p = 0.037 for the comparison of the ANXA2 and ß2-GPI groups to the controls). Purified IgG from ß2-GPI sera did not show cross-binding with ANXA2. Antibodies to ß2-GPI and phospholipids were found in the ß2-GPI immunized group only. The present study suggests an immune response to the ß2-GPI-ANXA2 complex in eAPS and provides a novel ANXA2 immunization model which will serve to study the role of ANXA2 antibodies in of APS.

Annexin A2, autoimmunity, anxiety and depression.

OBJECTIVES: Antiphospholipid syndrome (APS) is associated with neurological manifestations and one of the novel autoantigens associated with this disease is Annexin A2 (ANXA2). In this work we have examined the effect of high levels of autoantibodies to ANXA2 on the brain in a mouse model. METHODS: Recombinant ANXA2 emulsified in adjuvant was used to immunize mice while mice immunized with adjuvant only served as controls. At peak antibody levels the animal underwent behavioral and cognitive tests and their brains were examined for ANXA2 immunoglobulin G (IgG) and expression of ANXA2 and the closely linked protein p11. RESULTS: Very high levels of anti-ANXA2 antibodies (Abs) were associated with reduced anxiety in the open field 13.14% ± 0.89% of the time in the center compared to 8.64% ± 0.91% observed in the control mice (p < 0.001 by t-test). A forced swim test found significantly less depression manifested by immobility in the ANXA2 group. The changes in behavior were accompanied by a significant reduction in serum corticosteroid levels of ANXA2 group compared to controls. Moreover, higher levels of total IgG and p11 expression were found in ANXA2 group brains. Lower levels of circulating anti-ANXA2 Abs were not associated with behavioral changes. CONCLUSIONS: We have established an animal model with high levels of anti-ANXA2 Abs which induced IgG accumulation in the brain and specific anxiolytic and anti-depressive effects. This model promises to further our understanding of autoimmune disease such as APS and to provide better understanding of the role of the ANXA2-p11 complex in the brain.

Highly efficient selection of phage antibodies mediated by display of antigen as Lpp-OmpA' fusions on live bacteria.

Delayed infectivity panning (DIP) is a novel approach for the in vivo isolation of interacting protein pairs. DIP combines phage display and cell surface display of polypeptides as follows: an antigen is displayed in many copies on the surface of F(+) Escherichia coli cells by fusing it to a Lpp-OmpA' hybrid. To prevent premature, non-specific infection by phage, the cells are rendered functionally F(-) by growth at 16 degrees C. The antigen-displaying cells are used to capture antibody-displaying phage by virtue of the antibody-antigen interaction. Following removal of unbound phage, infection of the cells by bound phage is initiated by raising the temperature to 37 degrees C that facilitates F pilus expression. The phage then dissociate from the antigen and infect the bacteria through the F pilus. Using specific scFv antibodies and the human ErbB2 proto-oncogene and IL2-Ralpha chain as model antibody-antigen pairs, we demonstrate enrichment of those phage that display a specific antibody over phage that display an irrelevant antibody of over 1,000,000 in a single DIP cycle. We further show the successful isolation of anti-toxin, anti-receptor, anti-enzyme and anti-peptide antibodies from several immune phage libraries, a shuffled library and a large synthetic human library. The effectiveness of DIP makes it suitable for the isolation of rare clones present in large libraries. Since DIP can be applied for most of the phage libraries already existing, it could be a powerful tool for the rapid isolation and characterization of binders in numerous protein-protein interactions.