Autoimmune thrombocytopenic purpura (AITP) is a severe disease in children with a still unknown aetiology. It is not known why AITP can either be transient and self limiting or become chronic. The beneficial use of intravenous immunoglobulins (IVIG) in certain groups of AITP patients has been proven. It is, however, not clear how IVIG functions. To analyse patient-derived monoclonal IgG platelet autoantibodies that interact with IVIG in an anti-idiotypic manner, the combinatorial antibody phage display system was applied. From three different patients a large number of clones specifically reacting with IVIG molecules were derived. Many of these IVIG binders also reacted strongly with platelets in ELISA and FACS, in contrast to IVIG binders derived from a healthy individual. The heavy and light chain variable regions were sequenced and compared with each other and with databases. In all three AITP patients clones with a striking complementarity-determining region (CDR) sequence homology to each other and to many of the known anti-platelet antibodies were observed. Selected Fab-phages representing the characteristic variable regions that occurred in the investigated patients with AITP may now be used to clone potentially regulatory anti-idiotypes from healthy donors by phage display.

The beneficial use of intravenous immunoglobulins (IVIG) in certain groups of patients with autoimmune thrombocytopenic purpura (AITP) has been proven. AITP is a severe disease in children with a still unknown etiology. It is not clear how IVIG functions in this and other autoimmune diseases. To analyze and compare patient-derived monoclonal IgG antibodies that are bound by IVIG in an anti-idiotypic manner, the combinatorial antibody phage display system was applied. From three different patients with AITP, a large number of clones specifically reacting with IVIG molecules were enriched. The heavy and light chain variable regions were sequenced and compared with each other and with databases. Many variable regions showed extensive replacement mutations within the complementarity-determining regions, while two were identical to germ-line genes. Our data show that the most frequently used germ-line gene loci of these IVIG binders are identical to those observed for many other autoantibodies. This implicates a specific interaction of IVIG particularly with autoantibodies and B cell receptors derived from germ-line genes that are often used for the generation of autoantibodies.

SUMMARYIntravenous immunoglobulin preparations (IVIG) have shown positive effects in the treatment of immune defects and autoimmune diseases. It is not clear how IVIG interacts with the components of the immune system. To investigate this, we cloned previously a large number of phage displayed IgG Fab fragments derived from three patients with autoimmune thrombocytopenia (AITP) that were specifically bound by IVIG molecules. Many of these Fabs reacted with platelets. Sequencing revealed that the most frequently used germ-line gene segments of all IVIG-bound Fabs were identical to those observed for many other autoantibodies. Particularly, the loci 3±30 or 3±30/3±30.5, 3±23 and 3r, 3l, and 2a2 represented the most abundant genes used for the heavy (V H ) and light chain V region (V L ), respectively. This suggested a specific interaction of IVIG molecules with B cells that present B cell receptors derived from these germ-line genes. In the current study we determined the genetic origin of IVIG-reactive IgG and IgM cloned from a healthy person. A favoured selection of antibodies derived from the same germline origins as in AITP was observed. Because 3±30 and 3±23 are the most frequently rearranged V H germ-line gene segments among human B cells, our results suggest that this favoured anti-idiotypic interaction may have an important role for the development and control of the normal B cell repertoire.

Cerebral ischemia induces a complex transcriptional response with global changes in gene expression. It is essentially regulated by transcription factors as well as epigenetic players. While it is well known that the inhibition of transcriptionally repressive histone deacetylases leads to neuroprotection, the role of histone methyltransferases in the postischemic transcriptional response remains elusive. We investigated the effects of inhibition of the repressive H3K9 histone methyltransferases SUV39H1 and G9a on neuronal survival, H3K9 promoter signatures and gene expression. Their inhibition either with the specific blocker chaetocin or by use of RNA interference promoted neuronal survival in oxygen glucose deprivation (OGD). Brain-derived neurotrophic factor (BDNF) was upregulated and BDNF promoter regions showed an increase in histone marks characteristic for active transcription. The BDNF blockade with K252a abrogated the protective effect of chaetocin treatment. In conclusion, inhibition of histone methyltransferases SUV39H1 and G9a confers neuroprotection in a model of hypoxic metabolic stress, which is at least in part mediated by BDNF.

Objective T o perform a comparative analysis of 1) intravenous Ig (IVIG)–bound Fab fragments from a patient with autoimmune thrombocytopenia that had progressed to systemic lupus erythematosus (SLE) and 2) IVIG‐selected Fabs from an SLE patient without thrombocytopenia. Methods IVIG preparations have been successfully used to treat certain cases of autoimmune thrombocytopenia and SLE. Specific interactions of IVIG with the components of the immune system are not well characterized. To investigate these, we had previously cloned a large number of phage‐displayed IgG Fab fragments, derived from 3 patients with autoimmune thrombocytopenia, that were specifically bound by IVIG molecules during panning. Many of these Fabs reacted with platelets. Sequencing revealed that the most frequently used VH germline gene segments of all IVIG‐bound Fabs were 3‐23 and 3‐30/3‐30.5. One patient's autoimmune thrombocytopenia had progressed to SLE. Using the same cloning and panning procedures, we performed a comparative analysis of this patient's IVIG‐bound Fab fragments and the IVIG‐selected Fabs from an SLE patient without thrombocytopenia. Results We observed an exclusive selection of antibodies derived from 3‐23 and 3‐30/3‐30.5 germline segments. In contrast to the Fab fragments from the autoimmune thrombocytopenia patient who developed SLE, none of the IVIG‐selected Fabs from the SLE patient without thrombocytopenia bound to thrombocytes. Conclusion Our results suggest a preferential interaction of a subfraction of IVIG—representative of normal Ig repertoires—with antibodies and B cell receptors derived from these 2 gene segments. Importantly, these are the most frequently rearranged VH germline genes among human B cells. This kind of interaction is characteristic of a B cell superantigen, since light chains, antigen specificity, and the high variation in the third complementarity‐determining region 3 showed little influence on the selection of 3‐23– or 3‐30/3‐30.5–derived Fabs by IVIG. However, at least some of the contact residues on Fabs for IVIG appear to be different from those for staphylococcal protein A and human immunodeficiency virus gp 120. The IVIG‐selected Fabs may now be used to clone antibodies representative of this IVIG subfraction to study their possible regulatory influence on the B cell repertoire during normal development and disease.

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