38 results on '"Oszmiana A"'
Search Results
2. Nanometre-scale organization of the Natural Killer cell receptors KIR2DL1 and KIR2DS1 and its implications for signalling
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Oszmiana, Anna, Davis, Daniel, and Hussell, Tracy
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616.07 ,Natural Killer cells ,Super-resolution microscopy - Abstract
Human Natural Killer (NK) cells are regulated by a variety of germ-line encoded activating and inhibitory receptors. Broadly, activating receptors detect ligands that are expressed or up-regulated on cancerous or infected cells, while inhibitory receptors bind self-molecules to induce tolerance against healthy cells. Highly homologous pairs of activating and inhibitory receptors are also expressed on NK cells, including Killer Ig-like Receptors KIR2DL1 and KIR2DS1, which bind the same ligands, class I MHC proteins from the C2 group. Here, two super-resolution microscopy techniques, stimulated emission depletion (STED) and ground state depletion microscopy followed by individual molecule return (GSDIM) were used to examine the nanometre-scale organization of KIR2DL1 and KIR2DS1, as well as molecules engaged in their signalling. Both receptors were observed to constitutively assemble in nanometre-scale clusters at the surface of NK cells but displayed differential patterns of clustering - the activating receptor KIR2DS1 formed nanoclusters 2.3-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters was controlled by transmembrane amino-acid 233, a lysine in KIR2DS1. Mutated variant of KIR2DS1 in which lysine 233 was substituted with alanine formed significantly smaller clusters than the wild-type KIR2DS1. Reciprocally, substitution of isoleucine found at position 233 in KIR2DL1 sequence with lysine resulted in the receptor assembling into larger clusters. Super-resolution microscopy also revealed two ways in which KIR nanoclusters impact signalling. First, KIR2DS1 and DAP12 nanoclusters were juxtaposed in the resting-cell state but coalesced upon receptor ligation. Second, quantitative super-resolution microscopy revealed that membrane-proximal clusters of the kinase ZAP-70 or phosphatase SHP-1, as well as their phosphorylated active forms, were more often found in contact with larger KIR nanoclusters. Together, this work has established that size of KIR nanoclusters depends on the transmembrane sequence and impacts downstream signalling.
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- 2016
3. A systematic investigation of the effect of the fluid shear stress on Caco-2 cells towards the optimization of epithelial organ-on-chip models
- Author
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Delon, Ludivine C., Guo, Zhaobin, Oszmiana, Anna, Chien, Chia-Chi, Gibson, Rachel, Prestidge, Clive, and Thierry, Benjamin
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- 2019
- Full Text
- View/download PDF
4. Atypical cadherin FAT4 orchestrates lymphatic endothelial cell polarity in response to flow
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Betterman, Kelly L., Sutton, Drew L., Secker, Genevieve A., Kazenwadel, Jan, Oszmiana, Anna, Lim, Lillian, Miura, Naoyuki, Sorokin, Lydia, Hogan, Benjamin M., Kahn, Mark L., McNeill, Helen, and Harvey, Natasha L.
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Thermo Fisher Scientific Inc. ,R and D Systems ,Lymphatic diseases -- Genetic aspects ,Protein binding -- Genetic aspects ,Collagen -- Genetic aspects ,Transcription (Genetics) -- Genetic aspects ,Endothelium -- Genetic aspects ,Vascular endothelial growth factor -- Genetic aspects ,Software industry ,Epidermal growth factors -- Genetic aspects ,Scientific equipment industry -- Genetic aspects ,Health care industry - Abstract
The atypical cadherin FAT4 has established roles in the regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in Hennekam syndrome, features of which include lymphedema, lymphangiectasia, and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in collagen and calcium binding EGF domains 1 (CCBE1) and ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3), encoding a matrix protein and protease, respectively, that regulate activity of the key prolymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1, and ADAMTS3 mutations underlie Hennekam syndrome suggested that all 3 genes might function in a common pathway. We identified FAT4 as a target gene of GATA-binding protein 2 (GATA2), a key transcriptional regulator of lymphatic vascular development and, in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell-autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development. Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4 mutations underlie a human lymphedema syndrome., Introduction Hennekam syndrome (OMIM #235510 and #616006) is an autosomal-recessive disorder characterized by congenital lymphedema and lymphangiectasia, unusual facial morphology, attributed at least in part to intrauterine facial lymphedema, and [...]
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- 2020
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- View/download PDF
5. A Prox1 enhancer represses haematopoiesis in the lymphatic vasculature
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Jan Kazenwadel, Parvathy Venugopal, Anna Oszmiana, John Toubia, Luis Arriola-Martinez, Virginia Panara, Sandra G. Piltz, Chris Brown, Wanshu Ma, Andreas W. Schreiber, Katarzyna Koltowska, Samir Taoudi, Paul Q. Thomas, Hamish S. Scott, Natasha L. Harvey, Kazenwadel, Jan, Venugopal, Parvathy, Oszmiana, Anna, Toubia, John, Panara, Virginia, Piltz, Sandra G, Brown, Chris, Ma, Wanshu, Schreiber, Andreas W, Koltowska, Katarzyna, Taoudi, Samir, Thomas, Paul Q, and Scott, Hamish S
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Multidisciplinary - Abstract
Refereed/Peer-reviewed Transcriptional enhancer elements are responsible for orchestrating the temporal and spatial control over gene expression that is crucial for programming cell identity during development. Here we describe a novel enhancer element that is important for regulating the expression of Prox1 in lymphatic endothelial cells. This evolutionarily conserved enhancer is bound by key lymphatic transcriptional regulators including GATA2, FOXC2, NFATC1 and PROX1. Genome editing of the enhancer to remove five nucleotides encompassing the GATA2-binding site resulted in perinatal death of homozygous mutant mice due to profound lymphatic vascular defects. Lymphatic endothelial cells in enhancer mutant mice exhibited reduced expression of genes characteristic of lymphatic endothelial cell identity and increased expression of genes characteristic of haemogenic endothelium, and acquired the capacity to generate haematopoietic cells. These data not only reveal a transcriptional enhancer element important for regulating Prox1 expression and lymphatic endothelial cell identity but also demonstrate that the lymphatic endothelium has haemogenic capacity, ordinarily repressed by Prox1.
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- 2023
6. The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling
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Anna Oszmiana, David J. Williamson, Shaun-Paul Cordoba, David J. Morgan, Philippa R. Kennedy, Kevin Stacey, and Daniel M. Davis
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Biology (General) ,QH301-705.5 - Abstract
Super-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR), KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling.
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- 2016
- Full Text
- View/download PDF
7. Cytomegalovirus establishes a latent reservoir and triggers long-lasting inflammation in the eye.
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Valentina Voigt, Christopher E Andoniou, Iona S Schuster, Anna Oszmiana, Monique L Ong, Peter Fleming, John V Forrester, and Mariapia A Degli-Esposti
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Recent outbreaks of Ebola and Zika have highlighted the possibility that viruses may cause enduring infections in tissues like the eye, including the neural retina, which have been considered immune privileged. Whether this is a peculiarity of exotic viruses remains unclear, since the impact of more common viral infections on neural compartments has not been examined, especially in immunocompetent hosts. Cytomegalovirus is a common, universally distributed pathogen, generally innocuous in healthy individuals. Whether in immunocompetent hosts cytomegalovirus can access the eye, and reside there indefinitely, was unknown. Using the well-established murine cytomegalovirus infection model, we show that systemic infection of immunocompetent hosts results in broad ocular infection, chronic inflammation and establishment of a latent viral pool in the eye. Infection leads to infiltration and accumulation of anti-viral CD8+ T cells in the eye, and to the development of tissue resident memory T cells that localize to the eye, including the retina. These findings identify the eye as an unexpected reservoir for cytomegalovirus, and suggest that common viruses may target this organ more frequently than appreciated. Notably, they also highlight that infection triggers sustained inflammatory responses in the eye, including the neural retina.
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- 2018
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8. Pathogenic variants in MDFIC cause recessive central conducting lymphatic anomaly with lymphedema
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Alicia B. Byrne, Pascal Brouillard, Drew L. Sutton, Jan Kazenwadel, Saba Montazaribarforoushi, Genevieve A. Secker, Anna Oszmiana, Milena Babic, Kelly L. Betterman, Peter J. Brautigan, Melissa White, Sandra G. Piltz, Paul Q. Thomas, Christopher N. Hahn, Matthias Rath, Ute Felbor, G. Christoph Korenke, Christopher L. Smith, Kathleen H. Wood, Sarah E. Sheppard, Denise M. Adams, Ariana Kariminejad, Raphael Helaers, Laurence M. Boon, Nicole Revencu, Lynette Moore, Christopher Barnett, Eric Haan, Peer Arts, Miikka Vikkula, Hamish S. Scott, Natasha L. Harvey, UCL - SSS/DDUV/GEHU - Génétique, UCL - SSS/IREC/SLUC - Pôle St.-Luc, UCL - (SLuc) Centre de génétique médicale UCL, UCL - (SLuc) Service de chirurgie plastique, Byrne, Alicia B, Brouillard, Pascal, Sutton, Drew L, Kazenwadel, Jan, Montazaribarforoush, Saba, Secker, Genevieve A, Oszmiana, Anna, Babic, Milena, Betterman, Kelly L, Brautigan, Peter J, Hahn, Christopher N, Arts, Peer, Scott, Hamish S, and Harvey, Natasha L
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General Medicine - Abstract
Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in MDFIC , encoding the MyoD family inhibitor domain containing protein, in seven individuals with CCLA from six independent families. Clinical manifestations of affected fetuses and children included nonimmune hydrops fetalis (NIHF), pleural and pericardial effusions, and lymphedema. Generation of a mouse model of human MDFIC truncation variants revealed that homozygous mutant mice died perinatally exhibiting chylothorax. The lymphatic vasculature of homozygous Mdfic mutant mice was profoundly mispatterned and exhibited major defects in lymphatic vessel valve development. Mechanistically, we determined that MDFIC controls collective cell migration, an important early event during the formation of lymphatic vessel valves, by regulating integrin β 1 activation and the interaction between lymphatic endothelial cells and their surrounding extracellular matrix. Our work identifies MDFIC variants underlying human lymphatic disease and reveals a crucial, previously unrecognized role for MDFIC in the lymphatic vasculature. Ultimately, understanding the genetic and mechanistic basis of CCLA will facilitate the development and implementation of new therapeutic approaches to effectively treat this complex disease.
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- 2022
9. Rituximab causes a polarization of B cells that augments its therapeutic function in NK-cell–mediated antibody-dependent cellular cytotoxicity
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Rudnicka, Dominika, Oszmiana, Anna, Finch, Donna K., Strickland, Ian, Schofield, Darren J., Lowe, David C., Sleeman, Matthew A., and Davis, Daniel M.
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- 2013
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10. Pathogenic variants in
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Alicia B, Byrne, Pascal, Brouillard, Drew L, Sutton, Jan, Kazenwadel, Saba, Montazaribarforoushi, Genevieve A, Secker, Anna, Oszmiana, Milena, Babic, Kelly L, Betterman, Peter J, Brautigan, Melissa, White, Sandra G, Piltz, Paul Q, Thomas, Christopher N, Hahn, Matthias, Rath, Ute, Felbor, G Christoph, Korenke, Christopher L, Smith, Kathleen H, Wood, Sarah E, Sheppard, Denise M, Adams, Ariana, Kariminejad, Raphael, Helaers, Laurence M, Boon, Nicole, Revencu, Lynette, Moore, Christopher, Barnett, Eric, Haan, Peer, Arts, Miikka, Vikkula, Hamish S, Scott, and Natasha L, Harvey
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Mice ,Myogenic Regulatory Factors ,Pregnancy ,Hydrops Fetalis ,Animals ,Endothelial Cells ,Humans ,Female ,Lymphedema ,Chylothorax ,Lymphatic Vessels - Abstract
Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in
- Published
- 2022
11. Pathogenic variants in MDFIC cause recessive central conducting lymphatic anomaly with lymphedema
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Byrne, Alicia B., primary, Brouillard, Pascal, additional, Sutton, Drew L., additional, Kazenwadel, Jan, additional, Montazaribarforoushi, Saba, additional, Secker, Genevieve A., additional, Oszmiana, Anna, additional, Babic, Milena, additional, Betterman, Kelly L., additional, Brautigan, Peter J., additional, White, Melissa, additional, Piltz, Sandra G., additional, Thomas, Paul Q., additional, Hahn, Christopher N., additional, Rath, Matthias, additional, Felbor, Ute, additional, Korenke, G. Christoph, additional, Smith, Christopher L., additional, Wood, Kathleen H., additional, Sheppard, Sarah E., additional, Adams, Denise M., additional, Kariminejad, Ariana, additional, Helaers, Raphael, additional, Boon, Laurence M., additional, Revencu, Nicole, additional, Moore, Lynette, additional, Barnett, Christopher, additional, Haan, Eric, additional, Arts, Peer, additional, Vikkula, Miikka, additional, Scott, Hamish S., additional, and Harvey, Natasha L., additional
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- 2022
- Full Text
- View/download PDF
12. When form meets function: the cells and signals that shape the lymphatic vasculature during development
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Mathias Francois, Anna Oszmiana, Natasha L. Harvey, Francois, Mathias, Oszmiana, Anna, and Harvey, Natasha L
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lymphoedema ,Cell ,Biology ,Cardiovascular System ,valve development ,vascular development ,03 medical and health sciences ,lymphatic ,0302 clinical medicine ,Immune system ,medicine ,Animals ,Homeostasis ,Humans ,Lymphangiogenesis ,Progenitor cell ,vascular malformations ,Molecular Biology ,Lymphatic Vessels ,030304 developmental biology ,0303 health sciences ,Endothelial Cells ,Cell biology ,Endothelial stem cell ,lymphangiogenesis ,medicine.anatomical_structure ,Lymphatic system ,030217 neurology & neurosurgery ,Function (biology) ,endothelial cell heterogeneity ,Signal Transduction ,Developmental Biology - Abstract
The lymphatic vasculature is an integral component of the cardiovascular system. It is essential to maintain tissue fluid homeostasis, direct immune cell trafficking and absorb dietary lipids from the digestive tract. Major advances in our understanding of the genetic and cellular events important for constructing the lymphatic vasculature during development have recently been made. These include the identification of novel sources of lymphatic endothelial progenitor cells, the recognition of lymphatic endothelial cell specialisation and heterogeneity, and discovery of novel genes and signalling pathways underpinning developmental lymphangiogenesis. Here, we review these advances and discuss how they inform our understanding of lymphatic network formation, function and dysfunction.
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- 2021
13. Atypical cadherin FAT4 orchestrates lymphatic endothelial cell polarity in response to flow
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Genevieve A. Secker, Kelly L. Betterman, Helen McNeill, Benjamin M. Hogan, Lydia Sorokin, Drew L. Sutton, Lillian Lim, Naoyuki Miura, Mark L. Kahn, Natasha L. Harvey, Anna Oszmiana, Jan Kazenwadel, Betterman, Kelly L, Sutton, Drew L, Secker, Genevieve A, Kazenwadel, Jan, Oszmiana, Anna, Lim, Lillian, Miura, Naoyuki, Sorokin, Lydia, Hogan, Benjamin M, Kahn, Mark L, McNeill, Helen, and Harvey, Natasha L
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0301 basic medicine ,Mice, Transgenic ,Biology ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Cell polarity ,FAT4 ,medicine ,Lymphatic vessel ,Animals ,Humans ,Lymphedema ,Lymphangiogenesis ,genes ,Lymphatic Vessels ,Hippo signaling pathway ,Cadherin ,Cell Polarity ,Endothelial Cells ,General Medicine ,Syndrome ,medicine.disease ,Cadherins ,human lymphedema syndrome ,Cell biology ,Endothelial stem cell ,GATA2 Transcription Factor ,Hennekam syndrome ,030104 developmental biology ,Lymphatic system ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Female ,calcium binding EGF domains 1 (CCBE1) ,Research Article - Abstract
The atypical cadherin FAT4 has established roles in the regulation of planar cell polarity and Hippo pathway signaling that are cell context dependent. The recent identification of FAT4 mutations in Hennekam syndrome, features of which include lymphedema, lymphangiectasia, and mental retardation, uncovered an important role for FAT4 in the lymphatic vasculature. Hennekam syndrome is also caused by mutations in collagen and calcium binding EGF domains 1 (CCBE1)and ADAM metallopeptidase with thrombospondin type 1 motif 3 (ADAMTS3), encoding a matrix protein and protease,respectively, that regulate activity of the key prolymphangiogenic VEGF-C/VEGFR3 signaling axis by facilitating the proteolytic cleavage and activation of VEGF-C. The fact that FAT4, CCBE1, and ADAMTS3 mutations underlie Hennekam syndrome suggested that all 3 genes might function in a common pathway. We identified FAT4 as a target gene of GATA binding protein 2 (GATA2), a key transcriptional regulator of lymphatic vascular development and, in particular, lymphatic vessel valve development. Here, we demonstrate that FAT4 functions in a lymphatic endothelial cell–autonomous manner to control cell polarity in response to flow and is required for lymphatic vessel morphogenesis throughout development.Our data reveal a crucial role for FAT4 in lymphangiogenesis and shed light on the mechanistic basis by which FAT4mutations underlie a human lymphedema syndrome usc Refereed/Peer-reviewed
- Published
- 2020
14. A Prox1enhancer represses haematopoiesis in the lymphatic vasculature
- Author
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Kazenwadel, Jan, Venugopal, Parvathy, Oszmiana, Anna, Toubia, John, Arriola-Martinez, Luis, Panara, Virginia, Piltz, Sandra G., Brown, Chris, Ma, Wanshu, Schreiber, Andreas W., Koltowska, Katarzyna, Taoudi, Samir, Thomas, Paul Q., Scott, Hamish S., and Harvey, Natasha L.
- Abstract
Transcriptional enhancer elements are responsible for orchestrating the temporal and spatial control over gene expression that is crucial for programming cell identity during development1–3. Here we describe a novel enhancer element that is important for regulating the expression of Prox1in lymphatic endothelial cells. This evolutionarily conserved enhancer is bound by key lymphatic transcriptional regulators including GATA2, FOXC2, NFATC1 and PROX1. Genome editing of the enhancer to remove five nucleotides encompassing the GATA2-binding site resulted in perinatal death of homozygous mutant mice due to profound lymphatic vascular defects. Lymphatic endothelial cells in enhancer mutant mice exhibited reduced expression of genes characteristic of lymphatic endothelial cell identity and increased expression of genes characteristic of haemogenic endothelium, and acquired the capacity to generate haematopoietic cells. These data not only reveal a transcriptional enhancer element important for regulating Prox1expression and lymphatic endothelial cell identity but also demonstrate that the lymphatic endothelium has haemogenic capacity, ordinarily repressed by Prox1.
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- 2023
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- View/download PDF
15. When form meets function: the cells and signals that shape the lymphatic vasculature during development
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Francois, Mathias, primary, Oszmiana, Anna, additional, and Harvey, Natasha L., additional
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- 2021
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16. A 'prime-pull' vaccine strategy has a modest effect on local and systemic antibody responses to HIV gp140 in mice.
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John S Tregoning, Viviana Buffa, Anna Oszmiana, Katja Klein, Adam A Walters, and Robin J Shattock
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Medicine ,Science - Abstract
One potential strategy for the prevention of HIV infection is to induce virus specific mucosal antibody that can act as an immune barrier to prevent transmission. The mucosal application of chemokines after immunisation, termed "prime-pull", has been shown to recruit T cells to mucosal sites. We wished to determine whether this strategy could be used to increase B cells and antibody in the vaginal mucosa following immunisation with an HIV antigen. BALB/c mice were immunised intranasally with trimeric gp140 prior to vaginal application of the chemokine CCL28 or the synthetic TLR4 ligand MPLA, without antigen six days later. There was no increase in vaginal IgA, IgG or B cells following the application of CCL28, however vaginal application of MPLA led to a significant boost in antigen specific vaginal IgA. Follow up studies to investigate the effect of the timing of the "pull" stimulation demonstrated that when given 14 days after the initial immunisation MPLA significantly increased systemic antibody responses. We speculate that this may be due to residual inflammation prior to re-immunisation. Overall we conclude that in contrast to the previously observed effect on T cells, the use of "prime-pull" has only a modest effect on B cells and antibody.
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- 2013
- Full Text
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17. The central role of the cytoskeleton in mechanisms and functions of the NK cell immune synapse
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Lagrue, Kathryn, Carisey, Alex, Oszmiana, Anna, Kennedy, Philippa R., Williamson, David J., Cartwright, Adam, Barthen, Charlotte, and Davis, Daniel M.
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- 2013
- Full Text
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18. A systematic investigation of the effect of the fluid shear stress on Caco-2 cells towards the optimization of epithelial organ-on-chip models
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Ludivine C. Delon, Zhaobin Guo, Benjamin Thierry, Anna Oszmiana, Rachel J. Gibson, Clive A. Prestidge, Chia-Chi Chien, Delon, Ludivine C, Guo, Zhaobin, Oszmiana, Anna, Chien, Chia Chi, Gibson, Rachel, Prestidge, Clive, and Thierry, Benjamin
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Cell type ,fluid shear stress ,Cell Respiration ,Microfluidics ,microfluidics ,Biophysics ,Bioengineering ,Context (language use) ,02 engineering and technology ,Tight Junctions ,Biomaterials ,intestinal models ,03 medical and health sciences ,Mechanobiology ,Lab-On-A-Chip Devices ,Cytochrome P-450 CYP3A ,Humans ,Cytoskeleton ,Cell Shape ,030304 developmental biology ,0303 health sciences ,Microvilli ,Tight junction ,Chemistry ,Microfilament Proteins ,Epithelial Cells ,021001 nanoscience & nanotechnology ,Intestinal epithelium ,Actins ,Mitochondria ,Mucus ,Vacuolization ,Mechanics of Materials ,Vacuoles ,Ceramics and Composites ,organ-on-chip ,Stress, Mechanical ,Caco-2 Cells ,Energy Metabolism ,Rheology ,0210 nano-technology - Abstract
Epithelial cells experience constant mechanical forces, including fluid shear stress (FSS) on their apical surface.These forces alter both structure and function. While precise recapitulation of the complex mechanobiology of organs remains challenging, better understanding of the effect of mechanical stimuli is necessary towards the development of biorelevant in vitro models. This is especially relevant to organs-on-chip models which allow for fine control of the culture environment. In this study, the effects of the FSS on Caco-2 cell monolayers were systematically determined using a microfluidic device based on Hele-Shaw geometry. This approach allowed fora physiologically relevant range of FSS (from ∼0 to 0.03 dyn/cm2) to be applied to the cells within a single device. Exposure to microfluidic FSS induced significant phenotypical and functional changes in Caco-2 cell monolayers as compared to cells grown in static conditions. The application of FSS significantly altered the production of mucus, expression of tight junctions, vacuolization, organization of cytoskeleton, formation of microvilli, mitochondrial activity and expression of cytochrome P450. In the context of the intestinal epithelium,this detailed understanding of the effects of the FSS will enable the realization of in vitro organs-on-chip models with well-defined and tailored characteristics to a specific purpose, including for drug and nanoparticle absorption studies. The Hele-Shaw approach used in this study could be readily applied to other cell types and adapted for a wide range of physiologically relevant FSS. Refereed/Peer-reviewed
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- 2019
19. The size of activating and inhibitory killer Ig-like receptor nanoclusters is controlled by the transmembrane sequence and affects signaling
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David J. Morgan, Kevin B. Stacey, Shaun-Paul Cordoba, Daniel M. Davis, Philippa R. Kennedy, David Williamson, Anna Oszmiana, Oszmiana, Anna, Williamson, David J, Cordoba, Shaun-Paul, Morgan, David J, Kennedy, Philippa R, Stacey, Kevin, Davis, Daniel M, and Medical Research Council (MRC)
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0301 basic medicine ,chemical and pharmacologic phenomena ,Plasma protein binding ,NK cells ,Biology ,plasma membrane ,General Biochemistry, Genetics and Molecular Biology ,Article ,Nanoclusters ,Cell Line ,Cell membrane ,03 medical and health sciences ,0302 clinical medicine ,KIR2DL1 ,Receptors, KIR ,medicine ,Humans ,Phosphorylation ,Receptor ,lcsh:QH301-705.5 ,Adaptor Proteins, Signal Transducing ,ZAP-70 Protein-Tyrosine Kinase ,immunoglobulin-like receptors ,Protein Tyrosine Phosphatase, Non-Receptor Type 6 ,Cell Membrane ,tyrosine phosphorylation ,Membrane Proteins ,KIR2DS1 ,Cell Biology ,Transmembrane protein ,Cell biology ,Clone Cells ,Killer Cells, Natural ,030104 developmental biology ,medicine.anatomical_structure ,cell immune synapse ,lcsh:Biology (General) ,Amino Acid Substitution ,Receptors, KIR2DL1 ,Nanoparticles ,Signal transduction ,030215 immunology ,Protein Binding ,Signal Transduction - Abstract
Summary Super-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR), KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling., Graphical Abstract, Highlights • Activating and inhibitory NK cell receptors have a distinct nanoscale organization • The transmembrane sequence of KIR controls their nanoscale organization • Nanoclusters of KIR2DS1 and its adaptor are juxtaposed but mix upon activation • Phosphorylation of ZAP-70 or SHP-1 is favored in larger receptor nanoclusters, Oszmiana et al. use different super-resolution microscopy techniques to compare the nanoscale organization of paired activating and inhibitory receptors at the human NK cell surface. They show that the size of receptor nanoclusters is controlled by the transmembrane sequence and establish that nanocluster size affects the strength of receptor signaling.
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- 2016
20. Cytomegalovirus establishes a latent reservoir and triggers long-lasting inflammation in the eye
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Anna Oszmiana, Mariapia A. Degli-Esposti, Christopher E. Andoniou, John V. Forrester, Monique L. Ong, Valentina Voigt, Peter Fleming, Iona S. Schuster, Voigt, Valentina, Andoniou, Christopher E, Schuster, Iona S, Oszmiana, Anna, Ong, Monique L, Fleming, Peter, Forrester, John V, and Degli-Esposti, Mariapia A
- Subjects
0301 basic medicine ,Muromegalovirus ,Eye Diseases ,genetic structures ,T-Lymphocytes ,Cytomegalovirus ,Iris ,CD8-Positive T-Lymphocytes ,Eye ,Pathology and Laboratory Medicine ,Mice ,White Blood Cells ,0302 clinical medicine ,Animal Cells ,Medicine and Health Sciences ,Biology (General) ,Immune Response ,Pathogen ,Mice, Inbred BALB C ,T Cells ,Animal Models ,3. Good health ,medicine.anatomical_structure ,Experimental Organism Systems ,Virus Diseases ,Cytomegalovirus Infections ,Female ,Anatomy ,Cellular Types ,medicine.symptom ,Research Article ,QH301-705.5 ,Ocular Anatomy ,Immune Cells ,Immunology ,Congenital cytomegalovirus infection ,Mouse Models ,Inflammation ,Biology ,Research and Analysis Methods ,Microbiology ,Retina ,03 medical and health sciences ,Signs and Symptoms ,Model Organisms ,Immune system ,Ocular System ,Diagnostic Medicine ,Virology ,Genetics ,medicine ,Animals ,Iris (anatomy) ,Molecular Biology ,Disease Reservoirs ,Blood Cells ,Biology and Life Sciences ,eye diseases ,Cell Biology ,RC581-607 ,medicine.disease ,Viral Replication ,Disease Models, Animal ,Ophthalmology ,030104 developmental biology ,Viral replication ,inflammation ,030221 ophthalmology & optometry ,Eyes ,Parasitology ,sense organs ,Immunologic diseases. Allergy ,Immunologic Memory ,Head ,CD8 - Abstract
Recent outbreaks of Ebola and Zika have highlighted the possibility that viruses may cause enduring infections in tissues like the eye, including the neural retina, which have been considered immune privileged. Whether this is a peculiarity of exotic viruses remains unclear, since the impact of more common viral infections on neural compartments has not been examined, especially in immunocompetent hosts. Cytomegalovirus is a common, universally distributed pathogen, generally innocuous in healthy individuals. Whether in immunocompetent hosts cytomegalovirus can access the eye, and reside there indefinitely, was unknown. Using the well-established murine cytomegalovirus infection model, we show that systemic infection of immunocompetent hosts results in broad ocular infection, chronic inflammation and establishment of a latent viral pool in the eye. Infection leads to infiltration and accumulation of anti-viral CD8+ T cells in the eye, and to the development of tissue resident memory T cells that localize to the eye, including the retina. These findings identify the eye as an unexpected reservoir for cytomegalovirus, and suggest that common viruses may target this organ more frequently than appreciated. Notably, they also highlight that infection triggers sustained inflammatory responses in the eye, including the neural retina., Author summary Cytomegalovirus (CMV) is a common viral pathogen which is highly prevalent, but does not cause clinical disease in hosts with a fully competent immune system. After infection the virus remains with the host life-long in a chronic and then latent state. Latency is thought to establish primarily in the lung and in the salivary glands, and immune privileged tissues, such as the eye and the brain are considered inaccessible to CMV unless the host is severely immunocompromised. Here Voigt et al show that following a systemic infection of immunocompetent hosts CMV accesses the eye and establishes a reservoir of latent virus in this tissue. Ongoing inflammation in the eye, including the neural retina, is then sustained long-term in the absence of viral replication. This study reveals that virally induced inflammation in immune privileged tissues may be a general phenomenon and can occur despite a fully competent immune system.
- Published
- 2018
21. An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome
- Author
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Aleksandra, Gil-Krzewska, Mezida B, Saeed, Anna, Oszmiana, Elizabeth R, Fischer, Kathryn, Lagrue, William A, Gahl, Wendy J, Introne, John E, Coligan, Daniel M, Davis, and Konrad, Krzewski
- Subjects
actin cytoskeleton ,LAMP, Lysosome-associated membrane protein ,ICAM-1, Intercellular adhesion molecule 1 ,Vesicular Transport Proteins ,cytotoxic lymphocyte ,CHS, Chediak-Higashi syndrome ,lysosomal trafficking regulator ,Cytoplasmic Granules ,CI-MPR, Cation-independent mannose 6-phosphate receptor ,Article ,CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats ,Cell Line ,lysosomes ,NK, Natural killer ,EV, Empty vector ,Humans ,EEA1, Early Endosome Antigen 1 ,Cytoskeleton ,ARM, Armadillo repeats ,HEAT, Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A), and the lipid kinase TOR repeats ,CTL, Cytotoxic T cell ,natural killer cell ,immune deficiency ,Actins ,HLH, Hemophagocytic lymphohistiocytosis ,Killer Cells, Natural ,MTOC, Microtubule organizing center ,Chediak-Higashi syndrome ,CMFDA, 5-chloromethylfluorescein diacetate ,cytotoxicity ,lytic granules ,PSF, Point spread function ,exocytosis ,Rab, Ras-associated binding protein ,LYST, Lysosomal trafficking regulator - Abstract
Background Chediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells. Objective We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. Methods We generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells. Results Loss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells. Conclusion The cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel target for pharmaceutical intervention.
- Published
- 2017
22. Rituximab capping triggers intracellular reorganization of B cells
- Author
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Anna Oszmiana, Thomas Koller, Erdinc Sezgin, Daniel M. Davis, Christian Eggeling, Ana Filipa L.O.M. Santos, and Siebe Blok
- Subjects
Cell membrane ,medicine.anatomical_structure ,Chemistry ,medicine ,Rituximab ,Mitochondrion ,Actin ,Intracellular ,Cell biology ,medicine.drug - Published
- 2017
23. An actin cytoskeletal barrier inhibits lytic granule release from Natural Killer cells in Chediak-Higashi syndrome
- Author
-
Gil-Krzewska, Aleksandra, Saeed, Mezida B, Oszmiana, Anna, Fischer, E, Lagrue, Kathryn, Gahl, William A, Introne, Wendy J, Coligan, John E, Davis, Daniel M, and Krzewski, Konrad
- Abstract
BackgroundChediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the LYST gene, resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis due to impaired function of cytotoxic lymphocytes, mainly Natural Killer (NK) cells.ObjectiveWe sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in CHS.MethodsWe generated a human cell model of CHS, using CRISPR technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system, and super-resolution microscopy to visualize filamentous (F-)actin and lytic granules in normal and LYST-deficient NK cells.ResultsLoss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis, and impaired integrity of endo-lysosomal compartments. The large granules had an acidic pH, normal activity of lysosomal enzymes, and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunological synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunological synapse, or decreasing the lytic granule size, restored the ability of LYST-deficient NK cells to degranulate and kill target cells.ConclusionThe cortical actin and granule size play significant roles in NK cell cytotoxic function. The periodicity of sub-synaptic actin is an important factor limiting the release of large lytic granules from CHS NK cells, and could be a novel target for pharmaceutical intervention.
- Published
- 2017
24. Rituximab capping triggers intracellular reorganisation of B cells
- Author
-
Koller, T, Blok, S, Santos, A, Oszmiana, A, Davis, D, Sezgin, E, and Eggeling, C
- Subjects
immune system diseases ,hemic and lymphatic diseases - Abstract
The antibody rituximab, which binds to the protein CD20 on the surface of B-cells, has been used to treat B-cell malignancies for several years. However, the molecular mechanisms underlying this treatment are not yet fully understood. One well-established rituximab-induced mechanism, natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), has recently been described to involve the polarisation of bound rituximab and CD20 to one side of the B-cell. B-cells polarised this way were cleared more efficiently by NK-cells, which led us to further investigate the cellular events involved in the polarisation process. Using optical microscopy on rituximab-treated cells, we have found that the rituximab/CD20-rich, polarised side accumulated mitochondria and actin, whereas the nucleus was reorganised to the opposite side of the cell. Depleting actin via different methods correlated with a decrease in rituximab, mitochondria, and nucleus polarisation, suggesting polarisation to be actin-dependent, active process that triggers intracellular rearrangement. The influence of these intracellular rearrangements on the efficiency of NK-cell-mediated clearance of B-cell malignancies remains open for future investigation.
- Published
- 2017
25. An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome
- Author
-
Gil-Krzewska, Aleksandra, primary, Saeed, Mezida B., additional, Oszmiana, Anna, additional, Fischer, Elizabeth R., additional, Lagrue, Kathryn, additional, Gahl, William A., additional, Introne, Wendy J., additional, Coligan, John E., additional, Davis, Daniel M., additional, and Krzewski, Konrad, additional
- Published
- 2018
- Full Text
- View/download PDF
26. Cytomegalovirus establishes a latent reservoir and triggers long-lasting inflammation in the eye
- Author
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Voigt, Valentina, primary, Andoniou, Christopher E., additional, Schuster, Iona S., additional, Oszmiana, Anna, additional, Ong, Monique L., additional, Fleming, Peter, additional, Forrester, John V., additional, and Degli-Esposti, Mariapia A., additional
- Published
- 2018
- Full Text
- View/download PDF
27. Rituximab capping triggers intracellular reorganization of B cells
- Author
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Koller, Thomas, primary, Blok, Siebe, additional, Santos, Ana Mafalda, additional, Oszmiana, Anna, additional, Davis, Daniel, additional, Sezgin, Erdinc, additional, and Eggeling, Christian, additional
- Published
- 2017
- Full Text
- View/download PDF
28. The central role of the cytoskeleton in mechanisms and functions of the NK cell immune synapse
- Author
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David Williamson, Adam N.R. Cartwright, Alex Carisey, Charlotte Barthen, Daniel M. Davis, Philippa R. Kennedy, Anna Oszmiana, Kathryn Lagrue, Lagrue, Kathryn, Carisey, Alex, Oszmiana, Anna, Kennedy, Philippa R, Williamson, David J, Cartwright, Adam, Barthen, Charlotte, and Davis, Daniel M
- Subjects
Integrins ,Immunological Synapses ,Cellular polarity ,Immunology ,Arp2/3 complex ,Receptors, Cell Surface ,Immune receptor ,Lymphocyte Activation ,Filamentous actin ,microtubeles ,Immunological synapse ,Natural killer cell ,Cell Movement ,medicine ,Cell Adhesion ,Immunology and Allergy ,Animals ,Humans ,Cytoskeleton ,Actin ,biology ,immune synapse ,Cell Membrane ,cytoskeleton ,natural killer cell ,Actins ,Cell biology ,Killer Cells, Natural ,medicine.anatomical_structure ,biology.protein ,cellular activation ,actin ,Microtubule-Organizing Center ,Protein Binding ,Signal Transduction - Abstract
Natural killer (NK) cells discriminate between healthy and unhealthy target cells through a balance of activating and inhibitory signals at direct intercellular contacts called immune synapses. Rearrangements in the cellular cytoskeleton have long been known to be critical in assembly of immune synapses. Here, through bringing together the vast literature on this subject, the number of different ways in which the cytoskeleton is important becomes evident. The dynamics of filamentous actin are critical in (i) creating the nanometer-scale organization of NK cell receptors, (ii) establishing cellular polarity, (iii) coordinating immune receptor and integrin-mediated signaling, and (iv) directing secretion of lytic granules and cytokines. The microtubule network also is important in the delivery of lytic granules and vesicles containing cytokines to the immune synapse. Together, these data establish that the cytoskeleton acts as a central regulator of this complex and dynamic process - and an enormous amount of NK cell biology is controlled through the cytoskeleton. Refereed/Peer-reviewed
- Published
- 2013
29. Superresolution Microscopy Reveals Nanometer-Scale Reorganization of Inhibitory Natural Killer Cell Receptors upon Activation of NKG2D
- Author
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Daniel M. Davis, Anna Oszmiana, Dylan M. Owen, Stephen Rothery, Sophie V. Pageon, Shaun Paul Cordoba, Pageon, Sophie V, Cordoba, Shaun-Paul, Owen, Dylan M, Rothery, Stephen M, Oszmiana, Anna, and Davis, Daniel M
- Subjects
immune synapses ,Biochemistry & Molecular Biology ,HLA-C ,Immune receptor ,Biology ,Biochemistry ,Immunological synapse ,Natural killer cell ,CD28 Antigens ,KIR2DL1 ,HLA Antigens ,medicine ,Humans ,Receptor ,Molecular Biology ,Cytoskeleton ,Microscopy, Confocal ,natural killer cells ,optical tweezers ,Cell Membrane ,Antibodies, Monoclonal ,Cell Biology ,NKG2D ,Actin cytoskeleton ,Actins ,Cell biology ,Killer Cells, Natural ,Crosstalk (biology) ,medicine.anatomical_structure ,Microscopy, Fluorescence ,leukocyte antigen-C ,NK Cell Lectin-Like Receptor Subfamily K ,Immune System ,cytolytic interactions ,Receptors, KIR2DL1 ,superresolution ,Plasmids ,Signal Transduction - Abstract
Natural killer (NK) cell responses are regulated by a dynamic equilibrium between activating and inhibitory receptor signals at the immune synapse (or interface) with target cells. Although the organization of receptors at the immune synapse is important for appropriate integration of these signals, there is little understanding of this in detail, because research has been hampered by the limited resolution of light microscopy. Through the use of superresolution single-molecule fluorescence microscopy to reveal the organization of the NK cell surface at the single-protein level, we report that the inhibitory receptor KIR2DL1 is organized in nanometer-scale clusters at the surface of human resting NK cells. Nanoclusters of KIR2DL1 became smaller and denser upon engagement of the activating receptor NKG2D, establishing an unexpected crosstalk between activating receptor signals and the positioning of inhibitory receptors. These rearrangements in the nanoscale organization of surface NK cell receptors were dependent on the actin cytoskeleton. Together, these data establish that NK cell activation involves a nanometer-scale reorganization of surface receptors, which in turn affects models for signal integration and thresholds that control NK cell effector functions and NK cell development. Refereed/Peer-reviewed
- Published
- 2013
30. Rituximab causes a polarization of B cells that augments its therapeutic function in NK-cell-mediated antibody-dependent cellular cytotoxicity
- Author
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Darren Schofield, Matthew A. Sleeman, Daniel M. Davis, Donna K. Finch, David C. Lowe, Ian Strickland, Anna Oszmiana, Dominika Rudnicka, Rudnicka, Dominika, Oszmiana, Anna, Finch, Donna K, Strickland, Ian, Schofield, Darren J, Lowe, David C, Sleeman, Matthew A, and Davis, Daniel M
- Subjects
Immunology ,Intercellular Adhesion Molecule-1 ,chemical and pharmacologic phenomena ,Antineoplastic Agents ,myosin ,Myosins ,Lymphocyte Activation ,Biochemistry ,Immunoenzyme Techniques ,Antibodies, Monoclonal, Murine-Derived ,Immune system ,Antigen ,immune system diseases ,hemic and lymphatic diseases ,Neoplasms ,medicine ,Tumor Cells, Cultured ,Humans ,antineoplastic agent ,CD20 antigen ,CD20 ,Antibody-dependent cell-mediated cytotoxicity ,B-Lymphocytes ,natural killer cells ,biology ,B-cell lymphoma ,Chemistry ,Antibody-Dependent Cell Cytotoxicity ,Cell Biology ,Hematology ,Antigens, CD20 ,Killer Cells, Natural ,monoclonal antibody ,Monoclonal ,Cancer research ,biology.protein ,Rituximab ,Antibody ,Microtubule-Organizing Center ,medicine.drug - Abstract
Rituximab, which binds CD20 on B cells, is one of the best-characterized antibodies used in the treatment of B-cell malignancies and autoimmune diseases. Rituximab triggers natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC or what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B-cell surface independent of antibody crosslinking or intercellular contact. Unexpectedly, other proteins, including intercellular adhesion molecule 1 and moesin, were selectively recruited to the cap of CD20 and the microtubule organizing center became polarized toward the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarized compared with when they were unpolarized. Polarized B cells were lysed more frequently still when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarization of B cells and this augments its therapeutic function in triggering NK-cell-mediated ADCC. Refereed/Peer-reviewed
- Published
- 2013
31. NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus
- Author
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Mohamed O. Elasri, Halie K. Miller, Anna Oszmiana, Frances E. Rivera, Joanna Koziel, David S. Barber, Lindsey N. Shaw, James T. Riordan, Jessica E. Davenport, Vijayaraj Nagarajan, Stacey L. Kolar, Jan Potempa, Kolar, Stacey L, Nagarajan, Vijayaraj, Oszmiana, Anna, Rivera, Frances E, Miller, Halie K, Davenport, Jessica E, Riordan, James T, Potempa, Jan, Barber, David S, Koziel, Joanna, Elasri, Mohamed O, and Shaw, Lindsey N
- Subjects
Staphylococcus aureus ,Histidine Kinase ,Mutant ,antimicrobial agents ,Biology ,Microbiology ,Cell membrane ,anaerobic gene-expression ,bacillus subtilis ,Bacterial Proteins ,Genes, Reporter ,Stress, Physiological ,medicine ,Transcriptional regulation ,Escherichia coli ,methicillin resistant ,Regulation of gene expression ,Gene Expression Profiling ,Histidine kinase ,Cell Membrane ,transduction system ,Membrane Proteins ,Gene Expression Regulation, Bacterial ,beta-Galactosidase ,Two-component regulatory system ,Anti-Bacterial Agents ,Artificial Gene Fusion ,medicine.anatomical_structure ,Biochemistry ,Cell and Molecular Biology of Microbes ,biofilm formation ,Cell envelope ,Protein Kinases ,Intracellular ,Signal Transduction ,Transcription Factors - Abstract
Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter–gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.
- Published
- 2011
32. The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling
- Author
-
Oszmiana, Anna, primary, Williamson, David J., additional, Cordoba, Shaun-Paul, additional, Morgan, David J., additional, Kennedy, Philippa R., additional, Stacey, Kevin, additional, and Davis, Daniel M., additional
- Published
- 2016
- Full Text
- View/download PDF
33. A “Prime-Pull” Vaccine Strategy Has a Modest Effect on Local and Systemic Antibody Responses to HIV gp140 in Mice
- Author
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Tregoning, John S., primary, Buffa, Viviana, additional, Oszmiana, Anna, additional, Klein, Katja, additional, Walters, Adam A., additional, and Shattock, Robin J., additional
- Published
- 2013
- Full Text
- View/download PDF
34. Superresolution Microscopy Reveals Nanometer-Scale Reorganization of Inhibitory Natural Killer Cell Receptors upon Activation of NKG2D
- Author
-
Pageon, Sophie V., primary, Cordoba, Shaun-Paul, additional, Owen, Dylan M., additional, Rothery, Stephen M., additional, Oszmiana, Anna, additional, and Davis, Daniel M., additional
- Published
- 2013
- Full Text
- View/download PDF
35. NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus
- Author
-
Kolar, Stacey L., primary, Nagarajan, Vijayaraj, additional, Oszmiana, Anna, additional, Rivera, Frances E., additional, Miller, Halie K., additional, Davenport, Jessica E., additional, Riordan, James T., additional, Potempa, Jan, additional, Barber, David S., additional, Koziel, Joanna, additional, Elasri, Mohamed O., additional, and Shaw, Lindsey N., additional
- Published
- 2011
- Full Text
- View/download PDF
36. Pathogenic variants in MDFICcause recessive central conducting lymphatic anomaly with lymphedema
- Author
-
Byrne, Alicia B., Brouillard, Pascal, Sutton, Drew L., Kazenwadel, Jan, Montazaribarforoushi, Saba, Secker, Genevieve A., Oszmiana, Anna, Babic, Milena, Betterman, Kelly L., Brautigan, Peter J., White, Melissa, Piltz, Sandra G., Thomas, Paul Q., Hahn, Christopher N., Rath, Matthias, Felbor, Ute, Korenke, G. Christoph, Smith, Christopher L., Wood, Kathleen H., Sheppard, Sarah E., Adams, Denise M., Kariminejad, Ariana, Helaers, Raphael, Boon, Laurence M., Revencu, Nicole, Moore, Lynette, Barnett, Christopher, Haan, Eric, Arts, Peer, Vikkula, Miikka, Scott, Hamish S., and Harvey, Natasha L.
- Abstract
Central conducting lymphatic anomaly (CCLA), characterized by the dysfunction of core collecting lymphatic vessels including the thoracic duct and cisterna chyli, and presenting as chylothorax, pleural effusions, chylous ascites, and lymphedema, is a severe disorder often resulting in fetal or perinatal demise. Although pathogenic variants in RAS/mitogen activated protein kinase (MAPK) signaling pathway components have been documented in some patients with CCLA, the genetic etiology of the disorder remains uncharacterized in most cases. Here, we identified biallelic pathogenic variants in MDFIC, encoding the MyoD family inhibitor domain containing protein, in seven individuals with CCLA from six independent families. Clinical manifestations of affected fetuses and children included nonimmune hydrops fetalis (NIHF), pleural and pericardial effusions, and lymphedema. Generation of a mouse model of human MDFICtruncation variants revealed that homozygous mutant mice died perinatally exhibiting chylothorax. The lymphatic vasculature of homozygous Mdficmutant mice was profoundly mispatterned and exhibited major defects in lymphatic vessel valve development. Mechanistically, we determined that MDFIC controls collective cell migration, an important early event during the formation of lymphatic vessel valves, by regulating integrin β1activation and the interaction between lymphatic endothelial cells and their surrounding extracellular matrix. Our work identifies MDFICvariants underlying human lymphatic disease and reveals a crucial, previously unrecognized role for MDFIC in the lymphatic vasculature. Ultimately, understanding the genetic and mechanistic basis of CCLA will facilitate the development and implementation of new therapeutic approaches to effectively treat this complex disease.
- Published
- 2022
- Full Text
- View/download PDF
37. An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome
- Author
-
Gil-Krzewska, Aleksandra, Saeed, Mezida B, Oszmiana, Anna, Fischer, Elizabeth R, Lagrue, Kathryn, Gahl, William A, Introne, Wendy J, Coligan, John E, Davis, Daniel M, and Krzewski, Konrad
- Subjects
lysosomes ,actin cytoskeleton ,Chediak-Higashi syndrome ,cytotoxic lymphocyte ,lysosomal trafficking regulator ,cytotoxicity ,lytic granules ,natural killer cell ,immune deficiency ,exocytosis - Abstract
Background: Chediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells. Objective: We sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS. Methods: We generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells. Results: Loss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells. Conclusion: The cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel target for pharmaceutical intervention. Refereed/Peer-reviewed
- Published
- 2018
38. Rituximab capping triggers intracellular reorganisation of B cells
- Author
-
Koller, Thomas, Blok, Siebe, Santos, Ana Mafalda, Oszmiana, Anna, Davis, Daniel M, Sezgin, Erdinc, and Eggeling, Christian
- Subjects
mitochondria ,rituximab ,actins ,immune system diseases ,hemic and lymphatic diseases ,B-lymphocytes ,cell membrane - Abstract
The antibody rituximab, which binds to the protein CD20 on the surface of B-cells, has been used to treat B-cell malignancies for several years. However, the molecular mechanisms underlying this treatment are not yet fully understood. One well-established rituximab-induced mechanism, natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), has recently been described to involve the polarisation of bound rituximab and CD20 to one side of the B-cell. B-cells polarised this way were cleared more efficiently by NK-cells, which led us to further investigate the cellular events involved in the polarisation process. Using optical microscopy on rituximab-treated cells, we have found that the rituximab/CD20-rich, polarised side accumulated mitochondria and actin, whereas the nucleus was reorganised to the opposite side of the cell. Depleting actin via different methods correlated with a decrease in rituximab, mitochondria, and nucleus polarisation, suggesting polarisation to be actin-dependent, active process that triggers intracellular rearrangement. The influence of these intracellular rearrangements on the efficiency of NK-cell-mediated clearance of B-cell malignancies remains open for future investigation. B-cells polarised this way were cleared more efficiently by NK-cells, which led us to further investigate the cellular events involved in the polarisation process. Using optical microscopy on rituximab-treated cells, we have found that the rituximab/CD20-rich, polarised side accumulated mitochondria and actin, whereas the nucleus was reorganised to the opposite side of the cell. Depleting actin via different methods correlated with a decrease in rituximab, mitochondria, and nucleus polarisation, suggesting polarisation to be actin-dependent, active process that triggers intracellular rearrangement. The influence of these intracellular rearrangements on the efficiency of NK-cell-mediated clearance of B-cell malignancies remains open for future investigation. Refereed/Peer-reviewed
- Published
- 2017
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