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Migration of neutrophils in to lymphatics structures during inflammation in vivo: a role for CCR7?
Annals of Medicine and Surgery 3 (2014) 18–19
Contents lists available at ScienceDirect
Annals of Medicine and Surgery journal homepage: www.annalsjournal.com
Abstracts from the Barts and the London Academic Medicine and Surgery Society 2013 National Research Conference on the 5th October 2013 Winning undergraduate oral presentation
GLYCOGEN SYNTHASE KINASE 3b HAS A KINASE-INDEPENDENT FUNCTION IN NEURONAL MIGRATION DURING CORTICOGENESIS Georgina Phillips, Tetsushi Kagawa, Tetsuya Taga.
Background: Disruption of neuronal migration during corticogenesis is implicated in the development of many neurological and psychiatric disorders. Overexpression of Glycogen synthase kinase 3b (GSK3b) during corticogenesis inhibits neuronal migration. GSK3b is considered a promising target in the development of new psychiatric therapies, although whether it affects neuronal migration by kinase-dependent or independent mechanisms is unknown. This study aims to determine if GSK3b inhibits neuronal migration through kinase-dependent mechanisms, using a mouse model. Methods: Plasmids co-expressing green fluorescence protein with either GSK3b with no kinase activity “kinase dead” (GSK3bKD), constitutively active GSK3b (GSK3bS9A), or no GSK3b (control) were transfected into neural stem cells lining the lateral ventricles of embryonic day 13.5 C57BL6 mice by in utero electroporation. Following two days of corticogenesis, the migratory patterns of developing neurons within the cortical plate (CP) were examined using fluorescence microscopy. Results: GSK3bKD transfected embryos had fewer neurons within the CP compared to controls (p¼0.04) reflecting significantly reduced neuronal migration. Similar inhibition of neuronal migration occurred with GSK3bS9A overexpression in the majority of mutants. Furthermore, a significant reduction (p¼0.04) in unipolar neurons at the Intermediate Zone-CP border in GSK3bKD mutants demonstrated cells’ failure to achieve the polarity crucial for migration. Conclusions: These results provide the first evidence of a kinase-independent action of GSK3b in the inhibition of neuronal polarity and subsequent migration during corticogenesis, challenging the dogma of a kinase-centric function of GSK3b. Further investigation, to determine potential kinase-independent mechanisms, could aid rational drug design for psychiatric disorders. Acknowledgments: All Researchers at the Department of Stem Cell Regulation, Tokyo Medical and Dental University, Japan.
Winning postgraduate oral presentation NG2/CSPG4 PROMOTES PROLIFERATION AND RESISTANCE TO THERAPY IN GLIOBLASTOMA MULTIFORME Richard M. Heywood, Colin Watts. Introduction: The NG2/CSPG4 proteoglycan has been shown to be a marker of an aggressive proliferative phenotype, and as a marker of treatment resistant cells in glioblastoma (GBM). However, it is unknown whether CSPG4 plays a functional role in these processes, and whether these in vitro findings have any clinical relevance Methods: Freshly derived glioblastoma cells were used for all experiments. CSPG4 shRNA knockdown cells were assessed for proliferation, clonogenic potential and in vivo tumorigenicity. Immunocytochemical staining for EGFR and pEGFR, and pharmacological inhibition of EGFR,
were used to investigate interactions between CSPG4 and EGFR. Cells were exposed to radiation, temozolomide and carmustine, and the proportion of CSPG4+ cells was assessed by flow cytometry. Cell lines were FACS sorted, and the survival of sorted cells after treatment was asessed by growth curve analysis. These experiments were repeated in CSPG4 knockdown cell lines. Results: CSPG4 knockdown cells were less proliferative and clonogenic than wild type cells. pEGFR staining was higher in wild type than in knockdown cells; wild type cells were more sensitive to pharmacological inhibition of EGFR. CSPG4 was enriched after exposure to all therapeutic modalities. FACS sorting with functional assays confirmed that CSPG4+ cells were more resistant to treatment. CSPG4 shRNA knockdown cells were more sensitive to therapy. Conclusions: CSPG4 is a marker of cells that are more proliferative and more resistant to the therapeutic modalities used clinically in the treatment of GBM. CSPG4 appears to have a functional role in these processes, by epigenetic stabilisation. Acknowledgments: Talal Al-Mayhani, Sara Piccirillo, Matt Rowland, Silvia Sonzini and Anna Kolb.
Winning posters MIGRATION OF NEUTROPHILS IN TO LYMPHATICS STRUCTURES DURING INFLAMMATION IN VIVO: A ROLE FOR CCR7? Jessica Ann Dilliway, Sussan Nourshargh, Mathieu-Benoit Voisin. Background: Blood neutrophils are the first leukocytes to arrive at sites of infection and a new interest in neutrophil regulation of the adaptive immune responses has emerged over the past decade. However, very little is known about the route of neutrophil migration into the lymphatic system and this study aimed to evaluate the migration response of neutrophils from the inflamed tissue, into the tissue-associated lymphatic vessels and finally into the draining lymph nodes (LNs). Furthermore, the expression of the lymphatic chemokine receptor CCR7 on neutrophils was investigated in vivo. Materials and Methods: WT mice received an intrascrotal injection of TNFa or chick collagen II with Complete Freund's adjuvant. Cremaster muscles, proximal LNs, spleen and blood samples were harvested and the presence of neutrophils in the different organs and expression of CCR7 were quantified by confocal microscopy and flow cytometry, respectively. Results: Both TNFa and Collagen II+CFA induced migration of neutrophils into cremaster tissues but also into the cremasteric lymphatic vessels and proximal LNs. Extracellular staining of cells revealed very low levels of surface expression of CCR7. However, significant levels of intracellular stores of CCR7 could be detected. Conclusions: Our data indicates that both cytokine-induced and immunisation-based inflammation induces the migration of neutrophils into the tissue lymphatic vessels and proximal draining LNs. Furthermore, CCR7 is expressed in mouse neutrophils mainly in intracellular stores. Collectively, these results indicate that CCR7 could play a role in the recruitment of neutrophils into lymphatic structures upon models of cremasteric inflammation in vivo.
Abstracts / Annals of Medicine and Surgery 3 (2014) 18–19
Acknowledgments: I would like to thank my tutor, Mathieu-Benoit Voisin, for all his invaluable help throughout the project which has been very much appreciated. I would also like to thank Professor Sussan Nourshargh and her colleaugues who made me feel part of the team. My thanks also extend to the Drapers Company and Arthritis Research UK who sponsored the research. THE EFFECTS OF DOCOSAHEXAENOIC ACID IN A MOUSE MODEL OF TRAUMATIC BRAIN INJURY Yasser Al Omran *, Ruth Angus *, Ping Yip, Meirion Davies, Gavin Giovanonni, Adina Michael-Titus. Introduction: Traumatic brain injury (TBI) is the leading cause of death and disability for young adults, and neuroprotective treatments that aim to address this issue have largely been disappointing. Recent studies have shown that dietary supplementation with the omega-3 fatty acid docosahexaenoic acid (DHA) exhibits neuroprotective properties after TBI. However, to date, no study has assessed the neuroprotective properties of an intravenous injection of DHA in TBI.
*
Joint first co-authors
19
Methods: Adult CD1 male mice received either a controlled cortical impact (CCI) injury or a sham surgery. Thirty minutes post-injury, CCI-injured mice were treated with either a single intravenous injection of vehicle (saline with 0.2 % ethanol), or DHA (500 nmol/kg). Functional outcome was evaluated using the Morris water maze (MWM). Twenty-eight days after injury, the animals were sacrificed and brains were sectioned and stained using a marker for astrocytes (GFAP). Results: There was no difference in the spatial learning task in the MWM between vehicle- and DHA-treated mice, but DHA-treated mice performed better in the spatial memory task in the MWM (P<0.05). From the preliminary histological analysis, a higher level of GFAP expression was seen in DHA-treated compared to vehicle-treated mice. Conclusion: This study has characterised the effects of a moderate TBI on cognitive performance and astrocytes, and has provided the first behavioural evidence that DHA may have beneficial effects when administered acutely after TBI. Acknowledgments: All experiments were performed in accordance with the Animal Care Committee of Queen Mary, University of London, and with the United Kingdom Animals (Scientific Procedures) Act 1986. Ruth Angus is supported by a Medical Research Council (MRC) doctoral studentship.
Contents lists available at ScienceDirect
Annals of Medicine and Surgery journal homepage: www.annalsjournal.com
Abstracts from the Barts and the London Academic Medicine and Surgery Society 2013 National Research Conference on the 5th October 2013 Winning undergraduate oral presentation
GLYCOGEN SYNTHASE KINASE 3b HAS A KINASE-INDEPENDENT FUNCTION IN NEURONAL MIGRATION DURING CORTICOGENESIS Georgina Phillips, Tetsushi Kagawa, Tetsuya Taga.
Background: Disruption of neuronal migration during corticogenesis is implicated in the development of many neurological and psychiatric disorders. Overexpression of Glycogen synthase kinase 3b (GSK3b) during corticogenesis inhibits neuronal migration. GSK3b is considered a promising target in the development of new psychiatric therapies, although whether it affects neuronal migration by kinase-dependent or independent mechanisms is unknown. This study aims to determine if GSK3b inhibits neuronal migration through kinase-dependent mechanisms, using a mouse model. Methods: Plasmids co-expressing green fluorescence protein with either GSK3b with no kinase activity “kinase dead” (GSK3bKD), constitutively active GSK3b (GSK3bS9A), or no GSK3b (control) were transfected into neural stem cells lining the lateral ventricles of embryonic day 13.5 C57BL6 mice by in utero electroporation. Following two days of corticogenesis, the migratory patterns of developing neurons within the cortical plate (CP) were examined using fluorescence microscopy. Results: GSK3bKD transfected embryos had fewer neurons within the CP compared to controls (p¼0.04) reflecting significantly reduced neuronal migration. Similar inhibition of neuronal migration occurred with GSK3bS9A overexpression in the majority of mutants. Furthermore, a significant reduction (p¼0.04) in unipolar neurons at the Intermediate Zone-CP border in GSK3bKD mutants demonstrated cells’ failure to achieve the polarity crucial for migration. Conclusions: These results provide the first evidence of a kinase-independent action of GSK3b in the inhibition of neuronal polarity and subsequent migration during corticogenesis, challenging the dogma of a kinase-centric function of GSK3b. Further investigation, to determine potential kinase-independent mechanisms, could aid rational drug design for psychiatric disorders. Acknowledgments: All Researchers at the Department of Stem Cell Regulation, Tokyo Medical and Dental University, Japan.
Winning postgraduate oral presentation NG2/CSPG4 PROMOTES PROLIFERATION AND RESISTANCE TO THERAPY IN GLIOBLASTOMA MULTIFORME Richard M. Heywood, Colin Watts. Introduction: The NG2/CSPG4 proteoglycan has been shown to be a marker of an aggressive proliferative phenotype, and as a marker of treatment resistant cells in glioblastoma (GBM). However, it is unknown whether CSPG4 plays a functional role in these processes, and whether these in vitro findings have any clinical relevance Methods: Freshly derived glioblastoma cells were used for all experiments. CSPG4 shRNA knockdown cells were assessed for proliferation, clonogenic potential and in vivo tumorigenicity. Immunocytochemical staining for EGFR and pEGFR, and pharmacological inhibition of EGFR,
were used to investigate interactions between CSPG4 and EGFR. Cells were exposed to radiation, temozolomide and carmustine, and the proportion of CSPG4+ cells was assessed by flow cytometry. Cell lines were FACS sorted, and the survival of sorted cells after treatment was asessed by growth curve analysis. These experiments were repeated in CSPG4 knockdown cell lines. Results: CSPG4 knockdown cells were less proliferative and clonogenic than wild type cells. pEGFR staining was higher in wild type than in knockdown cells; wild type cells were more sensitive to pharmacological inhibition of EGFR. CSPG4 was enriched after exposure to all therapeutic modalities. FACS sorting with functional assays confirmed that CSPG4+ cells were more resistant to treatment. CSPG4 shRNA knockdown cells were more sensitive to therapy. Conclusions: CSPG4 is a marker of cells that are more proliferative and more resistant to the therapeutic modalities used clinically in the treatment of GBM. CSPG4 appears to have a functional role in these processes, by epigenetic stabilisation. Acknowledgments: Talal Al-Mayhani, Sara Piccirillo, Matt Rowland, Silvia Sonzini and Anna Kolb.
Winning posters MIGRATION OF NEUTROPHILS IN TO LYMPHATICS STRUCTURES DURING INFLAMMATION IN VIVO: A ROLE FOR CCR7? Jessica Ann Dilliway, Sussan Nourshargh, Mathieu-Benoit Voisin. Background: Blood neutrophils are the first leukocytes to arrive at sites of infection and a new interest in neutrophil regulation of the adaptive immune responses has emerged over the past decade. However, very little is known about the route of neutrophil migration into the lymphatic system and this study aimed to evaluate the migration response of neutrophils from the inflamed tissue, into the tissue-associated lymphatic vessels and finally into the draining lymph nodes (LNs). Furthermore, the expression of the lymphatic chemokine receptor CCR7 on neutrophils was investigated in vivo. Materials and Methods: WT mice received an intrascrotal injection of TNFa or chick collagen II with Complete Freund's adjuvant. Cremaster muscles, proximal LNs, spleen and blood samples were harvested and the presence of neutrophils in the different organs and expression of CCR7 were quantified by confocal microscopy and flow cytometry, respectively. Results: Both TNFa and Collagen II+CFA induced migration of neutrophils into cremaster tissues but also into the cremasteric lymphatic vessels and proximal LNs. Extracellular staining of cells revealed very low levels of surface expression of CCR7. However, significant levels of intracellular stores of CCR7 could be detected. Conclusions: Our data indicates that both cytokine-induced and immunisation-based inflammation induces the migration of neutrophils into the tissue lymphatic vessels and proximal draining LNs. Furthermore, CCR7 is expressed in mouse neutrophils mainly in intracellular stores. Collectively, these results indicate that CCR7 could play a role in the recruitment of neutrophils into lymphatic structures upon models of cremasteric inflammation in vivo.
Abstracts / Annals of Medicine and Surgery 3 (2014) 18–19
Acknowledgments: I would like to thank my tutor, Mathieu-Benoit Voisin, for all his invaluable help throughout the project which has been very much appreciated. I would also like to thank Professor Sussan Nourshargh and her colleaugues who made me feel part of the team. My thanks also extend to the Drapers Company and Arthritis Research UK who sponsored the research. THE EFFECTS OF DOCOSAHEXAENOIC ACID IN A MOUSE MODEL OF TRAUMATIC BRAIN INJURY Yasser Al Omran *, Ruth Angus *, Ping Yip, Meirion Davies, Gavin Giovanonni, Adina Michael-Titus. Introduction: Traumatic brain injury (TBI) is the leading cause of death and disability for young adults, and neuroprotective treatments that aim to address this issue have largely been disappointing. Recent studies have shown that dietary supplementation with the omega-3 fatty acid docosahexaenoic acid (DHA) exhibits neuroprotective properties after TBI. However, to date, no study has assessed the neuroprotective properties of an intravenous injection of DHA in TBI.
*
Joint first co-authors
19
Methods: Adult CD1 male mice received either a controlled cortical impact (CCI) injury or a sham surgery. Thirty minutes post-injury, CCI-injured mice were treated with either a single intravenous injection of vehicle (saline with 0.2 % ethanol), or DHA (500 nmol/kg). Functional outcome was evaluated using the Morris water maze (MWM). Twenty-eight days after injury, the animals were sacrificed and brains were sectioned and stained using a marker for astrocytes (GFAP). Results: There was no difference in the spatial learning task in the MWM between vehicle- and DHA-treated mice, but DHA-treated mice performed better in the spatial memory task in the MWM (P<0.05). From the preliminary histological analysis, a higher level of GFAP expression was seen in DHA-treated compared to vehicle-treated mice. Conclusion: This study has characterised the effects of a moderate TBI on cognitive performance and astrocytes, and has provided the first behavioural evidence that DHA may have beneficial effects when administered acutely after TBI. Acknowledgments: All experiments were performed in accordance with the Animal Care Committee of Queen Mary, University of London, and with the United Kingdom Animals (Scientific Procedures) Act 1986. Ruth Angus is supported by a Medical Research Council (MRC) doctoral studentship.