Contributions
Abstract: P987
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - 15 Immunology
Background: Experimental Autoimmune Encephalomyelitis (EAE), animal model of Multiple Sclerosis, combines T- and B-cell production. Autoantibodies are able to reach the Central Nervous System through the disrupted blood brain barrier and target various cells.
Goals: We examined the cross reactivity and identification of the EAE autoantibodies in naive mouse brain of three developmental stages.
Methods: EAE was induced in C57BL/6 mice immunized with MOG35-55. On day 17(Acute Phase) blood-sampling was performed in EAE and NAIVE groups and corresponding antisera (EAE-AS, NAIVE-AS) were collected. The antisera were tested for the presence of autoantibodies by Western Blotting on normal spinal cord and Neural Precursor Cells (NPCs) lysates. Double and triple immunofluorescence (dIF/tIF) was performed on normal mouse brain sections from neonates, postnates and adults (P3, P17 and 3months,respectively) stained with antisera, anti-BrdU and various markers for NPC characterization. Additionally, in vitro comparative immunoreactivity assessment was perfomed, where NPCs were challenged with antisera and stained for Caspase 3.
Results: Western blot on NPCs substrate indicated specific bands (60,40-46KDa), other than MOG (26-28KDa), when using EAE-AS. dIF of Nestin+/BrdU+, EAE-AS+/BrdU+ and Musashi-1+/BrdU+ revealed abundance of positive cells in all groups: P3; Nestin: 82.30±7.464%; Musashi-1:78.86±5.851%, P17; Nestin: 77.20±9.476%; Musashi-1:75.82±4.822% and in 3 months; Nestin: 62.13±7.94%; Musashi-1:61.26±6.208%. Moreover, tIF of EAE-AS+/BrdU+/SOX-2+ in neonates showed increased colocalization while in EAE-AS+/BrdU+/DCX+ colocalization was remarkably less (78.71±8.325% vs 13.073±3.840%). Additionally, in postnates, SOX-2 diminishes while DCX escalates in adults (Postnates:SOX-2; 45.61±11.78%, DCX; 90.83±3.493%, Adults: SOX-2; 77.45±9.227%, DCX; 100.00±0.0%). Immunocytochemistry of NPCs with Caspase 3 showed increased expression when stimulated with EAE-AS versus NAIVE-AS (Caspase 3; 16.07±1.196% vs 9.554±0.5684% p< 0.001).
Conclusions: Autoantibodies produced after MOG35-55 immunization exhibit lineage-specific cross reactivity with NPC surface, with variable cell type specificity. Furthermore, autoantibodies have the potential to stimulate apoptotic pathways. These findings indicate that antibody repertoire targets epitopes other than MOG and may lead to functional alteration of NPCs.
Disclosure:
Evangelia Kesidou: Nothing to disclose
Olga Touloumi: Nothing to disclose
Roza Lagoudaki: Nothing to disclose
Kyriaki-Nepheli Poulatsidou: Nothing to disclose
Paschalis Theotokis: Nothing to disclose
Evangelia Nousiopoulou: Nothing to disclose
Evangelia Kofidou: Nothing to disclose
Nikoleta Delivanoglou: Nothing to disclose
Marina Boziki: Nothing to disclose
Hadjigeorgiou G: Nothing to disclose
Constantina Simeonidou: Nothing to disclose
Nikolaos Grigoriadis: Nothing to disclose
Abstract: P987
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - 15 Immunology
Background: Experimental Autoimmune Encephalomyelitis (EAE), animal model of Multiple Sclerosis, combines T- and B-cell production. Autoantibodies are able to reach the Central Nervous System through the disrupted blood brain barrier and target various cells.
Goals: We examined the cross reactivity and identification of the EAE autoantibodies in naive mouse brain of three developmental stages.
Methods: EAE was induced in C57BL/6 mice immunized with MOG35-55. On day 17(Acute Phase) blood-sampling was performed in EAE and NAIVE groups and corresponding antisera (EAE-AS, NAIVE-AS) were collected. The antisera were tested for the presence of autoantibodies by Western Blotting on normal spinal cord and Neural Precursor Cells (NPCs) lysates. Double and triple immunofluorescence (dIF/tIF) was performed on normal mouse brain sections from neonates, postnates and adults (P3, P17 and 3months,respectively) stained with antisera, anti-BrdU and various markers for NPC characterization. Additionally, in vitro comparative immunoreactivity assessment was perfomed, where NPCs were challenged with antisera and stained for Caspase 3.
Results: Western blot on NPCs substrate indicated specific bands (60,40-46KDa), other than MOG (26-28KDa), when using EAE-AS. dIF of Nestin+/BrdU+, EAE-AS+/BrdU+ and Musashi-1+/BrdU+ revealed abundance of positive cells in all groups: P3; Nestin: 82.30±7.464%; Musashi-1:78.86±5.851%, P17; Nestin: 77.20±9.476%; Musashi-1:75.82±4.822% and in 3 months; Nestin: 62.13±7.94%; Musashi-1:61.26±6.208%. Moreover, tIF of EAE-AS+/BrdU+/SOX-2+ in neonates showed increased colocalization while in EAE-AS+/BrdU+/DCX+ colocalization was remarkably less (78.71±8.325% vs 13.073±3.840%). Additionally, in postnates, SOX-2 diminishes while DCX escalates in adults (Postnates:SOX-2; 45.61±11.78%, DCX; 90.83±3.493%, Adults: SOX-2; 77.45±9.227%, DCX; 100.00±0.0%). Immunocytochemistry of NPCs with Caspase 3 showed increased expression when stimulated with EAE-AS versus NAIVE-AS (Caspase 3; 16.07±1.196% vs 9.554±0.5684% p< 0.001).
Conclusions: Autoantibodies produced after MOG35-55 immunization exhibit lineage-specific cross reactivity with NPC surface, with variable cell type specificity. Furthermore, autoantibodies have the potential to stimulate apoptotic pathways. These findings indicate that antibody repertoire targets epitopes other than MOG and may lead to functional alteration of NPCs.
Disclosure:
Evangelia Kesidou: Nothing to disclose
Olga Touloumi: Nothing to disclose
Roza Lagoudaki: Nothing to disclose
Kyriaki-Nepheli Poulatsidou: Nothing to disclose
Paschalis Theotokis: Nothing to disclose
Evangelia Nousiopoulou: Nothing to disclose
Evangelia Kofidou: Nothing to disclose
Nikoleta Delivanoglou: Nothing to disclose
Marina Boziki: Nothing to disclose
Hadjigeorgiou G: Nothing to disclose
Constantina Simeonidou: Nothing to disclose
Nikolaos Grigoriadis: Nothing to disclose