Article
Oncology
Liye Zhou, Zexian Zeng, Ann Marie Egloff, Fan Zhang, Fei Guo, Katie M. Campbell, Peter Du, Jingxin Fu, Paul Zolkind, Xiaojing Ma, Zhe Zhang, Yi Zhang, Xiaoqing Wang, Shengqing Gu, Rachel Riley, Yasutaka Nakahori, Joshua Keegan, Robert Haddad, Jonathan D. Schoenfeld, Obi Griffith, Robert T. Manguso, James A. Lederer, X. Shirley Liu, Ravindra Uppaluri
Summary: This study reveals critical aspects of CD8+TIL heterogeneity and differentiation, and suggests the facilitation of CD8+TIL differentiation as a strategy to improve HNSCC ICB response.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2022)
Article
Oncology
Maximilian Kiessler, Ioana Plesca, Ulrich Sommer, Rebekka Wehner, Friederike Wilczkowski, Luise Mueller, Antje Tunger, Xixi Lai, Anke Rentsch, Kenneth Peuker, Sebastian Zeissig, Adrian M. Seifert, Lena Seifert, Juergen Weitz, Michael Bachmann, Martin Bornhaeuser, Daniela Aust, Gustavo Baretton, Marc Schmitz
Summary: The study found that higher densities of tumor-infiltrating pDCs are associated with lower UICC staging, increased progression-free and overall survival in colon cancer patients. A lower number of colon cancer-infiltrating pDCs was independently linked to worse prognosis, while the colocalization of activated pDCs and T cells in tumor stroma and within TLS may contribute to the correlation between higher pDC densities and better prognosis.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2021)
Article
Biochemistry & Molecular Biology
Kosuke Imamura, Yusuke Tomita, Ryo Sato, Tokunori Ikeda, Shinji Iyama, Takayuki Jodai, Misako Takahashi, Akira Takaki, Kimitaka Akaike, Shohei Hamada, Shinya Sakata, Koichi Saruwatari, Sho Saeki, Koei Ikeda, Makoto Suzuki, Takuro Sakagami
Summary: This study found that the infiltration of drebrin-expressing T cells within the tumor cell nest in resectable squamous cell lung cancer patients is associated with short relapse-free survival and overall survival. The in vitro induced drebrin(+) T cells co-express multiple exhaustion-associated molecules. Single-cell RNA-sequencing analysis confirmed that exhausted tumor-infiltrating CD8(+) T cells specifically expressed drebrin.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Review
Oncology
Karolina Frankowska, Michal Zarobkiewicz, Izabela Dabrowska, Agnieszka Bojarska-Junak
Summary: Tumor microenvironment (TME) is a complex entity that includes not only tumor cells but also various populations of immune cells. Tumor infiltrating lymphocytes (TILs) are highly reactive against the tumor component and play a key role in mediating responses to therapy. Recent studies have explored the potential utility of various imaging methods in assessing the level of TILs, particularly in breast and lung cancers.
Article
Immunology
Zexi He, Jun Gu, Ting Luan, Haihao Li, Charles Li, Zhenjie Chen, Enxiu Luo, Jiansong Wang, Yinglong Huang, Mingxia Ding
Summary: The study demonstrates the importance of the TIL-related model in predicting prognosis, immune status, and immunotherapy response in bladder cancer patients, which can help in screening patients who respond to immunotherapy.
FRONTIERS IN IMMUNOLOGY
(2022)
Article
Oncology
Mikako Nishida, Nahoko Yamashita, Taisaku Ogawa, Keita Koseki, Eiji Warabi, Tomoyuki Ohue, Masaaki Komatsu, Hirokazu Matsushita, Kazuhiro Kakimi, Eiryo Kawakami, Katsuyuki Shiroguchi, Heiichiro Udono
Summary: Metformin stimulates oxidative stress in CD8TILs, promoting antitumor immunity, and combined treatment with anti-PD-1 antibody effectively inhibits tumor growth by altering the tumor microenvironment.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2021)
Article
Oncology
Jacob Gadwa, Thomas E. Bickett, Laurel B. Darragh, Michael William Knitz, Shilpa Bhatia, Miles Piper, Benjamin Van Court, Shiv Bhuvane, Diemmy Nguyen, Varuna Nangia, Emily K. Kleczko, Raphael A. Nemenoff, Sana D. Karam
Summary: Inhibition of complement C3a and C5a signaling in HNSCC accelerates tumor growth and increases regulatory T cell populations. Local C3a and C5a signaling is important for CD4 T cell homeostasis and development into effector phenotypes. Depletion of Tregs reverses tumor growth and combining Treg depletion with C3a and C5a receptor inhibition reduces tumor growth further.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2021)
Article
Oncology
Dan P. Zandberg, Ashley V. Menk, Maria Velez, Daniel Normolle, Kristin DePeaux, Angen Liu, Robert L. Ferris, Greg M. Delgoffe
Summary: The study analyzed the effect of anti-PD-1 therapy in patients with recurrent/metastatic squamous cell carcinoma of the head and neck (HNSCC), finding that anti-PD-1 resistance is associated with changes in tumor cell metabolism, increased tumor hypoxia, and decreased CD8+ T cell numbers. Lower tumor hypoxia was associated with increased efficacy of anti-PD-1 therapy in patients.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2021)
Article
Pharmacology & Pharmacy
Liping Wang, Gui Yang, Guohong Liu, Yunbao Pan
Summary: In this study, we systematically analyzed HNSCC-infiltrating T lymphocytes lncRNAs to evaluate their predictive value for patients' survival outcome, immunotherapy response, and chemotherapeutic agents sensitivity. The findings suggested that low-risk HNSCC patients had a better prognosis, significant immune cell infiltration, and differential expression of RNA-binding proteins and immune checkpoint proteins like PD-1 and PD-L1.
FRONTIERS IN PHARMACOLOGY
(2022)
Article
Oncology
Kun Li, Yihang Gong, Dongbo Qiu, Hui Tang, Jian Zhang, Zenan Yuan, Yingqi Huang, Yunfei Qin, Linsen Ye, Yang Yang
Summary: The study found that the chemotherapy agent teniposide effectively activates the cGAS-STING signaling in HCC patients. Combining hyperbaric oxygen therapy can enhance the therapeutic effect of teniposide on HCC and improve the response to PD-1 immunotherapy.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2022)
Article
Oncology
Lauryn R. Werner, Katelin A. Gibson, Merit L. Goodman, Dominika E. Helm, Katherine R. Walter, Sean M. Holloran, Gloria M. Trinca, Richard C. Hastings, Howard H. Yang, Ying Hu, Junping Wei, Gangjun Lei, Xiao-Yi Yang, Rashna Madan, Alfredo A. Molinolo, Mary A. Markiewicz, Prabhakar Chalise, Margaret L. Axelrod, Justin M. Balko, Kent W. Hunter, Zachary C. Hartman, Carol A. Lange, Christy R. Hagan
Summary: Clinical studies have linked the use of progestins, synthetic progesterone, to an increased risk of breast cancer. However, the specific role of native progesterone signaling through the progesterone receptor in breast tumor formation remains poorly understood. Research suggests that progesterone can repress certain immune signaling pathways, potentially promoting tumor development in the mammary gland. This study investigated the effects of progesterone on immune cell populations in mouse mammary glands, as well as the impact of progesterone receptor overexpression on tumor formation and immune cell populations. The findings indicate that progesterone treatment and progesterone receptor overexpression are associated with inhibited immune responses, decreased immune cell populations, and an increased risk of mammary gland tumor development in mice.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2021)
Article
Biochemistry & Molecular Biology
Claire Y. Li, Hyeung Ju Park, Jinyeon Shin, Jung Eun Baik, Babak J. Mehrara, Raghu P. Kataru
Summary: This study suggests that lymphatic endothelial cells (LECs) can act as immunosuppressive cells in the tumor microenvironment (TME) in an MHC-II dependent manner. LECs in the TME increase MHC-II expression and co-inhibitory signals, suppressing the immune response. This finding highlights the importance of understanding the role of LECs in tumor immunity and opens up new avenues for therapeutic interventions.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Oncology
Veronica Finisguerra, Tereza Dvorakova, Matteo Formenti, Pierre Van Meerbeeck, Lionel Mignion, Bernard Gallez, Benoit J. van den Eynde
Summary: Despite limitations in certain tumor types and patients, immunotherapies have achieved revolutionary success in cancer treatment. However, the efficacy of immunotherapies is dependent on the viability and functionality of tumor antigen-specific CD8 T cells within the immunosuppressive tumor microenvironment, where oxygen levels are often low. This study reveals that the antidiabetic drug metformin can improve CD8 T cell fitness in hypoxia, increasing their proliferation and cytokine production, and enhancing their infiltration and survival in hypoxic tumor areas. This presents a promising strategy to overcome resistance to immunotherapy in hypoxic and immunosuppressive tumors.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2023)
Article
Oncology
Dan You, Stephen Hillerman, Gregory Locke, Charu Chaudhry, Caitlyn Stromko, Anwar Murtaza, Yi Fan, Jennifer Koenitzer, Yali Chen, Stephanie Briceno, Rajarshi Bhadra, Elizabeth Duperret, Johnni Gullo-Brown, Chan Gao, Dandan Zhao, John Feder, Joshua Curtin, Andrew P. Degnan, Godwin Kumi, Mark Wittman, Benjamin M. Johnson, Karen E. Parrish, Giridharan Gokulrangan, John Morrison, Michael Quigley, John T. Hunt, Luisa Salter-Cid, Emma Lees, Miguel A. Sanjuan, Jinqi Liu
Summary: This study identified a novel, potent, and selective HPK1 small molecule kinase inhibitor called Compound K (CompK), which significantly improved human T-cell immune responses in the tumor microenvironment and showed significant synergy with anti-PD-1 therapy. Animal model studies demonstrated improved immune responses and antitumor efficacy with CompK in combination with anti-PD-1, suggesting its potential as a novel pharmacological agent for cancer treatment.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2021)
Article
Oncology
Christine McInnis, Shilpa Bhatia, Brinda Vijaykumar, Qiaomu Tian, Yanbo Sun, Del Leistritz-Edwards, Charles T. Quinn, Ravi Uppaluri, Ann Marie Egloff, Lakshmi Srinivasan, Daniel C. Pregibon, Anthony J. Coyle, Glenn J. Hanna
Summary: This study conducted a comprehensive single-cell analysis of the immune landscape in HPV-related oropharyngeal squamous cell carcinomas. It identified strong cytotoxic T-cell responses to HPV16 E1 and E2 proteins, suggesting these antigens as potential targets for therapy.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2023)
Article
Oncology
Koen A. Marijt, Lisa Griffioen, Laura Blijleven, Sjoerd. H. van der Burg, Thorbald van Hall
Summary: TEIPP is a novel category of cancer antigens that emerge on cancers with functional loss of peptide pump TAP, evoking CD8 T cell immune response. By studying LRPAP1(21-30)-specific CD8 T cells, it was found that replacing the serine anchor with valine in the signal sequence enhances HLA-A2 binding affinity and T cell stimulation.
CANCER IMMUNOLOGY IMMUNOTHERAPY
(2022)
Article
Gastroenterology & Hepatology
Sheena Suthen, Chun Jye Lim, Phuong H. D. Nguyen, Charles-Antoine Dutertre, Hannah L. H. Lai, Martin Wasser, Camillus Chua, Tony K. H. Lim, Wei Qiang Leow, Tracy Jiezhen Loh, Wei Keat Wan, Yin Huei Pang, Gwyneth Soon, Peng Chung Cheow, Juinn Huar Kam, Shridhar Iyer, Alfred Kow, Wai Leong Tam, Timothy W. H. Shuen, Han Chong Toh, Yock Young Dan, Glenn K. Bonney, Chung Yip Chan, Alexander Chung, Brian K. P. Goh, Weiwei Zhai, Florent Ginhoux, Pierce K. H. Chow, Salvatore Albani, Valerie Chew
Summary: This study analyzed the immune landscapes of hypoxic regions in hepatocellular carcinoma (HCC) and identified enriched immune subsets, including regulatory T cells, M2 macrophages, and HLA-DRlo cDC2, in hypoxia-high tumor regions. In contrast, hypoxia-low tumor regions showed an abundance of active granzyme B-hi PD-1(lo) CD8(+) T cells, indicating a relatively active immune landscape. The up-regulation of cancer-associated genes in tumor tissues and immunosuppressive genes in tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC.
Article
Multidisciplinary Sciences
Florian Ingelfinger, Lisa Ann Gerdes, Vladyslav Kavaka, Sinduya Krishnarajah, Ekaterina Friebel, Edoardo Galli, Pascale Zwicky, Reinhard Furrer, Christian Peukert, Charles-Antoine Dutertre, Klara Magdalena Eglseer, Florent Ginhoux, Andrea Flierl-Hecht, Tania Kumpfel, Donatella De Feo, Bettina Schreiner, Sarah Mundt, Martin Kerschensteiner, Reinhard Hohlfeld, Eduardo Beltran, Burkhard Becher
Summary: This study investigated the influence of genetic predisposition and environmental factors on the peripheral immune signatures in monozygotic twins discordant for multiple sclerosis (MS). Through data-driven computational tools and high-throughput single-cell technologies, the researchers identified an inflammatory shift in a specific group of immune cells in twins with MS, as well as the emergence of certain hyper-responsive immune cells. They found that the variance in CD25 expression by certain helper T cells with a naive phenotype was largely driven by genetic and shared early environmental influences. However, the expansion of helper T cells in twins with MS, which were also elevated in non-twin patients with MS, appeared to be independent of individual genetic makeup and correlated with disease severity.
Article
Biochemistry & Molecular Biology
Rodrigo Nalio Ramos, Yoann Missolo-Koussou, Yohan Gerber-Ferder, Christian P. Bromley, Mattia Bugatti, Nicolas Gonzalo Nunez, Jimena Tosello Boari, Wilfrid Richer, Laurie Menger, Jordan Denizeau, Christine Sedlik, Pamela Caudana, Fiorella Kotsias, Leticia L. Niborski, Sophie Viel, Mylene Bohec, Sonia Lameiras, Sylvain Baulande, Laetitia Lesage, Andre Nicolas, Didier Meseure, Anne Vincent-Salomon, Fabien Reyal, Charles-Antoine Dutertre, Florent Ginhoux, Lene Vimeux, Emmanuel Donnadieu, Benedicte Buttard, Jerome Galon, Santiago Zelenay, William Vermi, Pierre Guermonprez, Eliane Piaggio, Julie Helft
Summary: This study identifies a specific population of FOLR2(+) tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors, which interact with CD8(+) T cells and positively correlate with better patient survival.
Article
Oncology
Nadia de Gruil, Hanno Pijl, Sjoerd H. van der Burg, Judith R. Kroep
Summary: Stimulating our body's immune response through short-term fasting diets can enhance the efficacy of cancer therapy, particularly chemotherapy. This review summarizes the preclinical and clinical evidence supporting the synergistic effects of fasting diets with cancer therapy by boosting antitumor immunity. The potential mechanisms underlying these effects include enhanced tumor immunity, decreased growth signaling, and alleviation of immunosuppression. Further studies are needed to fully evaluate the benefits of combining short-term fasting with not only chemotherapy, but also immunotherapy in cancer treatment.
Article
Biochemistry & Molecular Biology
Marit J. van Elsas, Johan M. S. van der Schoot, Alexander Bartels, Kas Steuten, Duco van Dalen, Zacharias Wijfjes, Carl G. Figdor, Thorbald van Hall, Sjoerd H. van der Burg, Martijn Verdoes, Ferenc A. Scheeren
Summary: Regulatory T cells play a crucial role in immune suppression and pose challenges in cancer therapy. By optimizing the Fc domain of an antibody, efficient depletion of tumor-resident regulatory T cells can be achieved. Using a genome engineering strategy, a stable cell line producing optimized antibodies was generated, leading to effective depletion of tumor-resident regulatory T cells and enhanced tumor eradication when combined with other antibodies.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Oncology
Christianne Groeneveldt, Priscilla Kinderman, Jordi J. C. van Stigt Thans, Camilla Labrie, Lisa Griffioen, Marjolein Sluijter, Diana J. M. van den Wollenberg, Rob C. Hoeben, Joke M. M. den Haan, Sjoerd H. van Der Burg, Thorbald van Hall, Nadine van Montfoort
Summary: The study shows that primed reovirus-specific T cells can be used as potent effector cells for anticancer treatment. By designing a synthetic long peptide vaccination strategy, the intratumoral frequency of these specific T cells can be enhanced.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2022)
Article
Biochemistry & Molecular Biology
Faezzah Baharom, Ramiro A. Ramirez-Valdez, Ahad Khalilnezhad, Shabnam Khalilnezhad, Marlon Dillon, Dalton Hermans, Sloane Fussell, Kennedy K. S. Tobin, Charles-Antoine Dutertre, Geoffrey M. Lynn, Soren Muller, Florent Ginhoux, Andrew S. Ishizuka, Robert A. Seder
Summary: Therapeutic cancer vaccines aim to enhance tumor-specific T cell immunity, but suppressive mechanisms within the tumor microenvironment can limit T cell function. This study investigated how different routes of vaccination impact intratumoral myeloid cells and found that intravenous administration of a nanoparticle vaccine induced tumor regression mediated by systemic type I interferon, leading to a reduction in intratumoral monocytes expressing immune-regulatory genes. In humans, these gene signatures are associated with worse outcomes. These results suggest that combining the generation of tumor-specific CD8+ T cells with remodeling of the tumor microenvironment holds promise for tumor immunotherapy.
Article
Biochemistry & Molecular Biology
Shidi Wu, Marion Rietveld, Marieke Hogervorst, Frank de Gruijl, Sjoerd van der Burg, Maarten Vermeer, Remco van Doorn, Marij Welters, Abdoelwaheb El Ghalbzouri
Summary: The study demonstrated that papillary and reticular fibroblasts in the dermis have distinct effects on epithelial/non-epithelial tumors, particularly in promoting EMT and invasion processes.
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
(2022)
Article
Multidisciplinary Sciences
C. Stingl, S. P. Lau, S. H. van der Burg, J. G. Aerts, C. H. J. van Eijck, T. M. Luider
Summary: This study used proteomics analysis to investigate the potential of tumor-reactive T-cell response through vaccination of pancreatic cancer patients with an allogenic tumor cell lysate vaccine. The results identified 61 tumor antigens and quantified them using TMT labels.
Article
Immunology
Bjoern E. Clausen, Lukas Amon, Ronald A. Backer, Luciana Berod, Tobias Bopp, Anna Brand, Sven Burgdorf, Luxia Chen, Meihong Da, Ute Distler, Regine J. Dress, Diana Dudziak, Charles-Antoine Dutertre, Christina Eich, Anna Gabele, Melanie Geiger, Florent Ginhoux, Lucila Giusiano, Gloria J. Godoy, Ahmed E. Hamouda, Lukas Hatscher, Lukas Heger, Gordon F. Heidkamp, Lola C. Hernandez, Lukas Jacobi, Tomasz Kaszubowski, Wan Ting Kong, Christian H. K. Lehmann, Tamara Lopez-Lopez, Karsten Mahnke, Dominik Nitsche, Jorg Renkawitz, Rifat A. Reza, Pablo J. Saez, Laura Schlautmann, Madeleine T. Schmitt, Anna Seichter, Malte Sielaff, Tim Sparwasser, Patrizia Stoitzner, Giorgi Tchitashvili, Stefan Tenzer, Nounagnon R. Tochoedo, Damir Vurnek, Fabian Zink, Thomas Hieronymus
Summary: This article provides a collection of protocols for the functional characterization of mouse and human dendritic cells (DC), including endocytosis and metabolism analysis, transcriptomic and proteomic characterization, migration characterization, and measurement of inflammasome and antigen (cross)-presentation activity. These protocols, written by experienced scientists and peer-reviewed by leading experts, are essential resources for basic and clinical DC immunologists.
EUROPEAN JOURNAL OF IMMUNOLOGY
(2022)
Article
Immunology
Akshamal M. Gamage, Wharton O. Y. Chan, Feng Zhu, Yan Ting Lim, Sandy Long, Matae Ahn, Chee Wah Tan, Randy Jee Hiang Foo, Wan Rong Sia, Xiao Fang Lim, Haopeng He, Weiwei Zhai, Danielle E. Anderson, Radoslaw Mikolaj Sobota, Charles-Antoine Dutertre, Lin-Fa Wang
Summary: Bats are reservoir hosts of zoonotic viruses with pandemic potential. This study utilized single-cell transcriptome sequencing to analyze the immune response in bat lungs upon infection with a double-stranded RNA virus. The study revealed the presence of neutrophils, NK cells, and T cells as the main immune cells in the lung tissue. Different subpopulations of CD8+ effector T cells and NK and T cells expressing genes involved in T cell activation and effector function were identified.
Article
Immunology
Egle Kvedaraite, Paul Milne, Ahad Khalilnezhad, Marion Chevrier, Raman Sethi, Hong Kai Lee, Daniel W. Hagey, Tatiana von Bahr Greenwood, Natalia Mouratidou, Martin Jadersten, Nicole Yee Shin Lee, Lara Minnerup, Yingrou Tan, Charles-Antoine Dutertre, Nathan Benac, You Yi Hwang, Josephine Lum, Amos Hong Pheng Loh, Jessica Jansson, Karen Wei Weng Teng, Shabnam Khalilnezhad, Weili Xu, Anastasia Resteu, Hong Liang Tey, Ng Lai Guan, Anis Larbi, Shanshan Wu Howland, Henrik Arnell, Samir E. L. Andaloussi, Jorge Braier, Georgios Rassidakis, Laura Galluzzo, Andrzej Dzionek, Jan-Inge Henter, Jinmiao Chen, Matthew Collin, Florent Ginhoux
Summary: This study used single-cell RNA-seq and protein analysis to dissect the heterogeneity of LCH cells and compare them with normal mononuclear phagocytes. The study found discriminatory signatures in LCH cells, indicating senescence and escape from immune surveillance. Additionally, the study uncovered two major lineages of LCH cells, DC2 and DC3/monocyte-like phenotypes.
SCIENCE IMMUNOLOGY
(2022)
Article
Oncology
Marit J. van Elsas, Camilla Labrie, Anders Etzerodt, Pornpimol Charoentong, Jordi J. C. van Stigt Thans, Thorbald Van Hall, Sjoerd H. van der Burg
Summary: A small population of CD163(hi) tissue-resident macrophages is identified to be responsible for primary and secondary resistance against T-cell-based immunotherapies. While these CD163(hi) M2 macrophages are resistant to Csf1r-targeted therapies, in-depth characterization and identification of the underlying mechanisms driving immunotherapy resistance allows the specific targeting of this subset of macrophages, thereby creating new opportunities for therapeutic intervention with the aim to overcome immunotherapy resistance.
JOURNAL FOR IMMUNOTHERAPY OF CANCER
(2023)
Article
Biochemical Research Methods
Quentin Blampey, Nadege Bercovici, Charles-Antoine Dutertre, Isabelle Pic, Joana Mourato Ribeiro, Fabrice Andre, Paul-Henry Cournede
Summary: Cytometry technology allows for precise single-cell phenotyping within heterogeneous populations. However, traditional manual gating for cell type annotation lacks reproducibility and sensitivity to batch effect. In this study, we introduce Scyan, a novel Single-cell Cytometry Annotation Network that automatically annotates cell types using prior expert knowledge. We demonstrate that Scyan outperforms current state-of-the-art models on multiple public datasets and provides faster and interpretable results. Scyan also addresses complementary tasks such as batch-effect correction, debarcoding, and population discovery.
BRIEFINGS IN BIOINFORMATICS
(2023)