Naturally Occurring Anti-Idiotypic Antibodies Portray a Largely Private Repertoire in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Rare immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a life-threatening disease resulting from a severe autoantibody-mediated ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs, member 13) deficiency. Acute iTTP episodes are medical emergencies, but when treated appropriately >95% of patients survive. However, at least half of survivors will eventually experience a relapse. How remission of an initial episode is achieved and factors contributing to reemergence of anti-ADAMTS13 Abs and a relapsing course are poorly understood. In acquired hemophilia and systemic lupus erythematosus, anti-idiotypic Abs counteracting and neutralizing pathogenic autoantibodies contribute to remission. We selected and amplified the splenic anti-idiotypic IgG1 Fab κ/λ repertoire of two relapsing iTTP patients on previously generated monoclonal inhibitory anti-ADAMTS13 Fabs by phage display to explore whether anti-idiotypic Abs have a role in iTTP. We obtained 27 single anti-idiotypic Fab clones, half of which had unique sequences, although both patients shared four H chain V region genes (VH1-69*01, VH3-15*01, VH3-23*01, and VH3-49*03). Anti-idiotypic Fab pools of both patients fully neutralized the inhibitor capacity of the monoclonal anti-ADAMTS13 Abs used for their selection. Preincubation of plasma samples of 22 unrelated iTTP patients stratified according to functional ADAMTS13 inhibitor titers (>2 Bethesda units/ml, or 1-2 Bethesda units/ml), with anti-idiotypic Fab pools neutralized functional ADAMTS13 inhibitors and restored ADAMTS13 activity in 18-45% of those cases. Taken together, we present evidence for the presence of an anti-idiotypic immune response in iTTP patients. The interindividual generalizability of this response is limited despite relatively uniform pathogenic anti-ADAMTS13 Abs recognizing a dominant epitope in the ADAMTS13 spacer domain

Genetically engineered silk and genipin-enhanced fibrin hydrogel for annulus fibrosus repair

INTRODUCTION: Around 80% of people are affected by low back pain at least once in their life, often caused by trauma provoking intervertebral disc (IVD) herniation and/or IVD degeneration. Apart from some promising approaches for nucleus pulposus repair, so far no treatment or repair is available for the outer fibrous tissue, annulus fibrosus (AF). We aimed for sealing and repairing an AF injury in a bovine IVD organ culture model in vitro over 14 days under different loading conditions. For this purpose, a silk fleece composite from Bombyx mori silk was combined with genipin-enhanced fibrin hydrogel [1]. METHODS: Bovine IVDs of 12-17 months old animals were isolated by first removing all surrounding tissue, followed by cutting out the IVDs [2]. Culturing of discs occurred in high glucose Dulbecco's Modified Eagle Medium (HG-DMEM) supplemented with 5% serum as previously described. On the next day, injury was induced using a 2mm biopsy punch (Polymed, Switzerland). The formed cavity was filled with (0.4%) genipin-enhanced human based fibrin hydrogel (35- 55mg/mL human fibrinogen, Baxter, Austria) and sealed with a silk fleece-membrane composite (Spintec Engineering, Germany). Different culture conditions were applied: free swelling, static diurnal load of 0.2MPa for 8h/d and complex loading at 0.2MPa compression combined with ± 2° torsion at 0.2Hz for 8h/d. Complex loading was applied by a custom built 2 degree of freedom bioreactor [3]. After 14 days of culture cell activity was determined with resazurin assay. Additionally, glycosaminoglycan (dimethyl-methylene blue), DNA (Hoechst) and collagen content (hydroxy-proline) were determined. Finally, real-time qPCR of major IVD marker genes was performed. RESULTS: The silk seal closing the injury site could successfully withstand the forces of all three loading conditions with no misplacement over the two weeks’ culture. Nevertheless, disc height of the repaired discs did not significantly differ from the injured group. The disc phenotype could be maintained as demonstrated by biochemical analysis of gene expression, cell activity, DNA-, collagen- and GAG content. The silk itself was evaluated to be highly biocompatible for hMSC, as revealed by cytotoxicity assays. DISCUSSION & CONCLUSIONS: The silk can be considered a highly-elastic and biocompatible material for AF closure and the genipin-enhanced fibrin hydrogel has also good biomechanical properties. However, the cyto-compatibility of genipin seems rather poor and other hydrogels and/or cross-linkers should be looked into. REFERENCES: 1 C.C. Guterl et al. (2014) Characterization of Mechanics and Cytocompatibility of Fibrin Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules, Tissue Eng Part A 2 S.C. Chan, B. Gantenbein-Ritter (2012) Preparation of intact bovine tail intervertebral discs for organ culture, J Vis Exp 3 B Gantenbein et al. (2015) Organ Culture Bioreactors - Platforms to Study Human Intervertebral Disc Degeneration and Regenerative Therapy, Curr Stem Cell Res Ther [epub ahead of print] ACKNOWLEDGEMENTS: This project is supported by the Gebert Rüf Stiftung project # GRS-028/13

Differentiation of MSC and annulus fibrosus cells on genetically-engineered silk fleece-membrane-composites enriched for GDF-6 or TGF-β3.

Intervertebral disc (IVD) repair is a high-priority topic in our active and increasingly ageing society. Since a high number of people are affected by low back pain treatment options that are able to restore the biological function of the IVD are highly warranted. Here, we investigated whether the feasibility of genetically-engineered (GE)-silk from Bombyx mori containing specific growth factors to precondition human bone-marrow derived mesenchymal stem cells (hMSC) or to activate differentiated human annulus fibrosus cells (hAFC) prior transplantation or for direct repair on the IVD. Here, we tested the hypothesis that GE-silk fleece can thrive human hMSC towards an IVD-like phenotype. We aimed to demonstrate a possible translational application of good manufacturing practice (GMP)-compliant GE-silk scaffolds in IVD repair and regeneration. GE-silk with growth and differentiation factor 6 (GDF-6-silk) or transforming growth factor β3 (TGF-β3, TGF-β3-silk) and untreated silk (cSilk) were investigated by DNA content, cell activity assay and glycosaminoglycan (GAG) content and their differentiation potential by qPCR analysis. We found that all silk types demonstrated a very high biocompatibility for both cell types, i.e., hMSC and hAFC, as revealed by cell activity, and DNA proliferation assay. Further, analyzing qPCR of marker genes revealed a trend to differentiation towards an NP-like phenotype looking at the Aggrecan/Collagen 2 ratio which was around 10:1. Our results support the conclusion that our GE-silk scaffold treatment approach can thrive hMSC towards a more IVD-like phenotype or can maintain the phenotype of native hAFC. This article is protected by copyright. All rights reserved