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Congenital disorders of glycosylation (CDG) include 150 genetically and clinically heterogeneous diseases, showing significant glycoprotein hypoglycosylation that leads to pathological consequences in multiple organs and systems whose underlying mechanisms are not yet understood. A few cellular and animal models have been used to study specific CDG characteristics, although they have given limited information due to the few CDG mutations tested and the still missing comprehensive molecular and cellular basic research. Here, we provide specific gene expression profiles, based on ribonucleic acid (RNA) microarray analysis, together with some biochemical and cellular characteristics of a total of nine control Epstein-Barr virus-transformed lymphoblastoid B cell lines (B-LCL) and 13 CDG B-LCL from patients carrying severe mutations in the phosphomannomutase 2 (PMM2) gene, strong serum protein hypoglycosylation and neurological symptoms. Significantly dysregulated genes in PMM2-CDG cells included those regulating stress responses, transcription factors, glycosylation, motility, cell junction and, importantly, those related to development and neuronal differentiation and synapse, such as carbonic anhydrase 2 (CA2) and ADAM23. PMM2-CDG-associated biological consequences involved the unfolded protein response, RNA metabolism and the endoplasmic reticulum, Golgi apparatus and mitochondria components. Changes in the transcriptional and CA2 protein levels are consistent with the CDG physiopathology. These results demonstrate the global transcriptional impact in phosphomannomutase 2-deficient cells, reveal CA2 as a potential cellular biomarker and confirm B-LCL as an advantageous model for CDG studies.

©. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Accepted version of a Published Work that appeared in final form in [Glycobiology]. To access the final edited and published work see[https://doi.org/10.1093/glycob/cwab087] Congenital disorders of glycosylation (CDG) include 150 disorders constituting in genetically and clinically heterogeneous diseases, showing significant glycoprotein hypoglycosylation that leads to pathological consequences on multiple organs and systems whose underlying mechanisms are not yet understood. A few cellular and animal models have been used to study specific CDG characteristics, although they have given limited information due to the few CDG mutations tested and the still missing comprehensive molecular and cellular basic research. Here, we provide specific gene expression profiles, based on ribonucleic acid (RNA) microarray analysis, together with some biochemical and cellular characteristics of a total of nine control Epstein– Barr virus-transformed lymphoblastoid B cell lines (B-LCL) and 13 CDG B-LCL from patients carrying severe mutations in the phosphomannomutase 2 (PMM2) gene, strong serum protein hypoglycosylation and neurological symptoms. Significantly dysregulated genes in PMM2-CDG cells included those regulating stress responses, transcription factors, glycosylation, motility, cell junction and, importantly, those related to development and neuronal differentiation and synapse, such as carbonic anhydrase 2 (CA2) and ADAM23. PMM2-CDG-associated biological consequences involved the unfolded protein response, RNA metabolism and the endoplasmic reticulum, Golgi apparatus and mitochondria components. Changes in the transcriptional and CA2 protein levels are consistent with the CDG physiopathology. These results demonstrate the global transcriptional impact in phosphomannomutase 2-deficient cells, reveal CA2 as a potential cellular biomarker and confirm B-LCL as an advantageous model for CDG studies.

Introduction: Remodeling bone tissue by insertion of resorbable bone substitution material (BSM) is a therapeutic strategy for bone defects caused by malignant diseases like multiple myeloma (MM). The migration and tube formation of vascular endothelial cells (EC) in the material is a prerequisite for successful angiogenesis as part of bone regeneration1. For the application of BSM in cancer patients it has to be considered that tumor cells may have angiogenic capacity on their own and that they are able to propagate osteolytic activity at the same time. To promote angiogenesis, growth factors (GF) like VEGF are often bound to the replacement material via heparin to induce cell migration and vessel ingrowth2. As heparin can enhance osteoclastic bone resorption by sequestering osteoprotegerin (OPG)3 we screened for alternative poly-saccharides (PS) to promote non malignant cell proliferation, GF binding and bone formation. Materials and methods: Besides heparin as an established PS we screened other PS for their angiogenic potential (tube formation assay), growth factor binding (microscale thermophoresis) and influence on proliferation of EC, fibroblasts andMMcells. All these are abilities important for angiogenesis and bone-turnover. Finally we applied co-cultures of fibroblasts and EC on collagen-based, PS coated, cylindrically BSM (3 x 5 mm) and analyzed for cellmigration and tube formation. Results: The PS investigated showed divergent results on the proliferation of selected cell types, promotion of angiogenesis and OPG binding. A promising candidate was a cellulose sulfate with special sulfatation pattern (TACS). Unlike heparin TACS did not reduce the proliferation of endothelial cells while still showing a similar angiogenic capacity. In addition TACS binds OPG to a lower extent than heparin. In 3D cultures of BSM coated with TACS vessels tend to form earlier than in control scaffolds. After 28 days in cell culture BSM were colonized to almost 90% by cells while lumen containing vessels formed in the upper parts of the scaffold (~50%) (Fig. 1). Other cellulose sulfates improved the proliferation of EC or hampered the proliferation of MM so that a combined application of these together with TACSswill be the next step. Discussion and conclusions: Co-culture of fibroblasts and HUVECs on collagen matrix leads to a vessel network in the BSM making this a promising combination of cells for preseeding. We showed that PS can improve the ability of early vessels formation, proliferation and growth factor binding. As no PS so far possesses all good attributes alone, we plan to modify the scaffolds with a combination of different PS. This way we hope to find a reliable tool to improve bone healing in multiple myeloma patients. Acknowledgments: The support by the SFB TRR79 and the DKFZ Light Microscopy Facility is gratefully acknowledged. Disclosures: The authors have nothing to disclose.

Extrafollicular (EF) B lymphocytes differ in size and morphology depending on the lymphatic organ involved and the kind of inflammatory reaction. On reevaluating EF B cells in various sites and conditions we discriminated three forms: a small (lymphoid) and intermediate (centrocytoid), and a large (monocytoid) variant. Immunohistochemically, these variants could be discriminated by their differential expression of adhesion molecules CD62L (L-selectin) and CD11c: small EF B cells were strongly L-selectin+ and CD11c−; intermediate cells were moderately CD62L+ and CD11c−; large cells were faintly CD62L+ or − but expressed CD11c. In 72 h cultures of normal peripheral and tonsillar B cells, cross-linking surface immunoglobulin in the presence of interleukin-2 or interleukin-4 led to formation of clusters in vitro together with an increase in cell size and a slight up-regulation of CD11c, as determined by flow cytometry. Stimulation with phorbol 12-myristate 13-acetate (PMA), however, gave rise to large, plastic adherent cells which also showed strong homotypic adhesion, expressed CD62L at minimal levels and CD11c at comparably highest levels and altogether mimicked the large cell variant of EF B cells. We conclude that EF B cells are subjected to cytokine-induced metamorphosis and that differences in cell size and morphology reflect their state of activation and activation-associated adhesion properties. Our data suggest that EF B cells in all anatomical sites are functionally closely related cells which — possibly mediated by CD11c/CD18 — may become sessile and proliferate locally once activated by appropriate signals.

APO-1/Fas(CD95), a member of the tumour necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily transduces apoptotic signals into apoptosis sensitive cells. In metabolic labelling experiments using the highly APO-1 positive cell lines HUT78 (adultT cell leukemia) and SKW6.4 (Blymphoblastoid cell line) APO-1 was characterised as a long living protein with a complex glycosylation pattern involving terminal sialic acid groups which account for 8-kDa of its apparent molecular weight on SDS-PAGE. APO-1 expression and the degree of sialylation were determined in additionalT and B cell lines. On the group I Burkitt's lymphoma cell line BL60 transfected with human APO-1 (K50) low sialylated species were detected only on the cell surface, suggesting that sialylation might be functionally important. Removal of terminal sialic acid groups by treatment of B and T cell lines with Vibrio cholerae neuraminidase (VCN) augmented sensitivity towards anti-APO-1 and human APO-1 ligand induced apoptosis. Similarly, VCN-treated U937 cells were rendered more sensitive to TNFalpha-induced cell death. Thus, sialylation may be one mechanism to regulate sensitivity towards ligand-mediated cell death in this receptor family.