Immunity & Ageing最新文献

{"title":"Repeated COVID-19 mRNA vaccination results in IgG4 class switching and decreased NK cell activation by S1-specific antibodies in older adults","authors":"Anne T. Gelderloos, Marije K. Verheul, Irene Middelhof, Mary-Lène de Zeeuw-Brouwer, Robert S. van Binnendijk, Anne-Marie Buisman, Puck B. van Kasteren","doi":"10.1186/s12979-024-00466-9","DOIUrl":"https://doi.org/10.1186/s12979-024-00466-9","url":null,"abstract":"Previous research has shown that repeated COVID-19 mRNA vaccination leads to a marked increase of SARS-CoV-2 spike-specific serum antibodies of the IgG4 subclass, indicating far-reaching immunoglobulin class switching after booster immunization. Considering that repeated vaccination has been recommended especially for older adults, the aim of this study was to investigate IgG subclass responses in the ageing population and assess their relation with Fc-mediated antibody effector functionality. Spike S1-specific IgG subclass concentrations (expressed in arbitrary units per mL), antibody-dependent NK cell activation, complement deposition and monocyte phagocytosis were quantified in serum from older adults (n = 38–50, 65–83 years) at one month post-second, -third and -fifth vaccination. Subclass distribution in serum was compared to that in younger adults (n = 64, 18–47 years) at one month post-second and -third vaccination. Compared to younger individuals, older adults showed increased levels of IgG2 and IgG4 at one month post-third vaccination (possibly related to factors other than age) and a further increase following a fifth dose. The capacity of specific serum antibodies to mediate NK cell activation and complement deposition relative to S1-specific total IgG concentrations decreased upon repeated vaccination. This decrease associated with an increased IgG4/IgG1 ratio. In conclusion, these findings show that, like younger individuals, older adults produce antibodies with reduced functional capacity upon repeated COVID-19 mRNA vaccination. Additional research is needed to better understand the mechanisms underlying these responses and their potential implications for vaccine effectiveness. Such knowledge is vital for the future design of optimal vaccination strategies in the ageing population.","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":7.9,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Ageing results in an exacerbated inflammatory response to LPS by resident lung cells","authors":"Celia Diaz-Nicieza, Laura Sahyoun, Christina Michalaki, Cecilia Johansson, Fiona J. Culley","doi":"10.1186/s12979-024-00467-8","DOIUrl":"https://doi.org/10.1186/s12979-024-00467-8","url":null,"abstract":"Ageing is associated with an increased risk of lung infection and chronic inflammatory lung disease. Innate immune responses are the first line of defence in the respiratory tract, however, age-related changes to innate immunity in the lung are not fully described. Both resident haematopoietic cells, such as alveolar macrophages, and non-haematopoeitic cells, such as epithelial and endothelial cells can contribute to inflammatory and immune responses in the lung. In this study we aimed to determine the impact of ageing on early innate responses of resident cells in the lung. Aged and young mice were inoculated intranasally with lipopolysaccharide (LPS). After 4 h, aged mice recruited higher numbers of neutrophils to the airways and lung. This exacerbated inflammatory response was associated with higher concentrations of chemokines CXCL1, CXCL2 and CCL2 in the airways. Next, precision cut lung slices (PCLS) were stimulated ex vivo with LPS for 16 h. Gene expression of Cxcl2, Tnf and Il1b were all higher in PCLS from aged than young mice and higher levels of secretion of CXCL2 and TNF were detected. To determine which lung cells were altered by age, LPS was intranasally administered to aged and young mice and individual populations of cells isolated by FACS. RT-PCR on sorted cell populations demonstrated higher expression of inflammatory cytokines Cxcl2, Ccl2 and Tnf in epithelial cells and alveolar macrophages and higher expression of Cxcl2 by endothelial cells of aged mice compared to young. These differences in expression of pro-inflammatory cytokines did not correspond to higher levels of Tlr4 expression. Ageing leads to a heightened neutrophilic inflammatory response in the lung after LPS exposure, and higher expression and production of pro-inflammatory cytokines by resident lung cells, including alveolar macrophages, epithelial cells and endothelial cells. The responses of multiple resident lung cell populations are altered by aging and contribute to the exacerbated inflammation in the lung following LPS challenge. This has implications for our understanding of respiratory infections and inflammation in older people.","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":7.9,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176292","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Warmer environmental temperature accelerates aging in mosquitoes, decreasing longevity and worsening infection outcomes","authors":"Jordyn S. Barr, Lindsay E. Martin, Ann T. Tate, Julián F. Hillyer","doi":"10.1186/s12979-024-00465-w","DOIUrl":"https://doi.org/10.1186/s12979-024-00465-w","url":null,"abstract":"Most insects are poikilotherms and ectotherms, so their body temperature is predicated by environmental temperature. With climate change, insect body temperature is rising, which affects how insects develop, survive, and respond to infection. Aging also affects insect physiology by deteriorating body condition and weakening immune proficiency via senescence. Aging is usually considered in terms of time, or chronological age, but it can also be conceptualized in terms of body function, or physiological age. We hypothesized that warmer temperature decouples chronological and physiological age in insects by accelerating senescence. To investigate this, we reared the African malaria mosquito, Anopheles gambiae, at 27 °C, 30 °C and 32 °C, and measured survival starting at 1-, 5-, 10- and 15-days of adulthood after no manipulation, injury, or a hemocoelic infection with Escherichia coli or Micrococcus luteus. Then, we measured the intensity of an E. coli infection to determine how the interaction between environmental temperature and aging shapes a mosquito’s response to infection. We demonstrate that longevity declines when a mosquito is infected with bacteria, mosquitoes have shorter lifespans when the temperature is warmer, older mosquitoes are more likely to die, and warmer temperature marginally accelerates the aging-dependent decline in survival. Furthermore, we discovered that E. coli infection intensity increases when the temperature is warmer and with aging, and that warmer temperature accelerates the aging-dependent increase in infection intensity. Finally, we uncovered that warmer temperature affects both bacterial and mosquito physiology. Warmer environmental temperature accelerates aging in mosquitoes, negatively affecting both longevity and infection outcomes. These findings have implications for how insects will serve as pollinators, agricultural pests, and disease vectors in our warming world.","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":7.9,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176293","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Characterization of age-associated inflammasome activation reveals tissue specific differences in transcriptional and post-translational inflammatory responses","authors":"Sarah Talley, Tyler Nguyen, Lily Van Ye, Rasa Valiauga, Jake DeCarlo, Jabra Mustafa, Benjamin Cook, Fletcher A. White, Edward M. Campbell","doi":"10.1186/s12979-024-00462-z","DOIUrl":"https://doi.org/10.1186/s12979-024-00462-z","url":null,"abstract":"Aging is associated with systemic chronic, low-grade inflammation, termed ‘inflammaging’. This pattern of inflammation is multifactorial and is driven by numerous inflammatory pathways, including the inflammasome. However, most studies to date have examined changes in the transcriptomes that are associated with aging and inflammaging, despite the fact that inflammasome activation is driven by a series of post-translational activation steps, culminating in the cleavage and activation of caspase-1. Here, we utilized transgenic mice expressing a caspase-1 biosensor to examine age-associated inflammasome activation in various organs and tissues to define these post-translational manifestations of inflammaging. Consistent with other studies, we observe increased inflammation, including inflammasome activation, in aged mice and specific tissues. However, we note that the degree of inflammasome activation is not uniformly associated with transcriptional changes commonly used as a surrogate for inflammasome activation in tissues. Furthermore, we used a skull thinning technique to monitor central nervous system inflammasome activation in vivo in aged mice and found that neuroinflammation is significantly amplified in aged mice in response to endotoxin challenge. Together, these data reveal that inflammaging is associated with both transcriptional and post-translational inflammatory pathways that are not uniform between tissues and establish new methodologies for measuring age-associated inflammasome activation in vivo and ex vivo.","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":7.9,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142176344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Melatonin enhances NK cell function in aged mice by increasing T-bet expression via the JAK3-STAT5 signaling pathway.","authors":"Caiying Liang, Rongrong Song, Jieyu Zhang, Jie Yao, Ziyuan Guan, Xiaokang Zeng","doi":"10.1186/s12979-024-00459-8","DOIUrl":"10.1186/s12979-024-00459-8","url":null,"abstract":"<p><p>Natural killer (NK) cells are crucial innate immune cells that provide defense against viruses and tumors. However, aging is associated with alterations in NK cell composition and compromised cell functions. Melatonin, known for its anti-tumor effects, has been reported to improve NK cell function. However, the molecular mechanism underlying melatonin's effect on senescent NK cells remains unclear. In this study, we aimed to elucidate the mechanism by which melatonin enhances the function of senescent NK cells. Our findings revealed that melatonin significantly increased the number and function of NK cells in aging mice. The results suggest that melatonin enhances NK cell proliferation, degranulation, and IFN-γ secretion. Further investigations demonstrated that melatonin promotes NK cell maturation and activation, mainly via the JAK3/STAT5 signaling pathway, leading to increased expression of T-bet. These discoveries provide a theoretical basis for potential immunotherapy strategies based on melatonin-mediated modulation of NK cell function in aging individuals.</p>","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11375890/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142141708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Heat‑killed probiotic Levilactobacillus brevis MKAK9 and its exopolysaccharide promote longevity by modulating aging hallmarks and enhancing immune responses in Caenorhabditis elegans.","authors":"Arun Kumar, Manti Kumar Saha, Vipin Kumar, Anupam Bhattacharya, Sagar Barge, Ashis K Mukherjee, Mohan C Kalita, Mojibur R Khan","doi":"10.1186/s12979-024-00464-x","DOIUrl":"10.1186/s12979-024-00464-x","url":null,"abstract":"","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11365118/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142114566","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role for BLT1 in regulating inflammation within adipose tissue immune cells of aged mice.","authors":"Wei-Ching Shih, In Hwa Jang, Victor Kruglov, Deborah Dickey, Stephanie Cholensky, David A Bernlohr, Christina D Camell","doi":"10.1186/s12979-024-00461-0","DOIUrl":"10.1186/s12979-024-00461-0","url":null,"abstract":"<p><strong>Background: </strong>Aging is a complex biological process characterized by obesity and immunosenescence throughout the organism. Immunosenescence involves a decline in immune function and the increase in chronic-low grade inflammation, called inflammaging. Adipose tissue expansion, particularly that of visceral adipose tissue (VAT), is associated with an increase in pro-inflammatory macrophages that play an important role in modulating immune responses and producing inflammatory cytokines. The leukotriene B4 receptor 1 (BLT1) is a regulator of obesity-induced inflammation. Its ligand, LTB4, acts as a chemoattractant for immune cells and induces inflammation. Studies have shown that BLT1 is crucial for cytokine production during lipopolysaccharide (LPS) endotoxemia challenge in younger organisms. However, the expression patterns and function of BLT1 in older organisms remains unknown.</p><p><strong>Results: </strong>In this study, we investigated BLT1 expression in immune cell subsets within the VAT of aged male and female mice. Moreover, we examined how antagonizing BLT1 signaling could alter the inflammatory response to LPS in aged mice. Our results demonstrate that aged mice exhibit increased adiposity and inflammation, characterized by elevated frequencies of B and T cells, along with pro-inflammatory macrophages in VAT. BLT1 expression is the highest in VAT macrophages. LPS and LTB4 treatment result in increased BLT1 in young and aged bone marrow-derived macrophages (BMDMs). However, LTB4 treatment resulted in amplified Il6 from aged, but not young BMDMs. Treatment of aged mice with the BLT1 antagonist, U75302, followed by LPS-induced endotoxemia resulted in an increase in anti-inflammatory macrophages, reduced phosphorylated NFκB and reduced Il6.</p><p><strong>Conclusions: </strong>This study provides valuable insights into the age- and sex- specific changes in BLT1 expression on immune cell subsets within VAT. This study offers support for the potential of BLT1 in modulating inflammation in aging.</p>","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11346001/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142074525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Mouse brain contains age-dependent extraparenchymal granular structures and astrocytes, both reactive to natural IgM antibodies, linked to the fissura magna.","authors":"Clara Romera, Marta Riba, Raquel Alsina, Marina Sartorio, Jordi Vilaplana, Carme Pelegrí, Jaume Del Valle","doi":"10.1186/s12979-024-00460-1","DOIUrl":"10.1186/s12979-024-00460-1","url":null,"abstract":"<p><strong>Background: </strong>Mouse brains can contain specific polyglucosan aggregates known as Periodic Acid-Schiff (PAS)-granules. Generated in astrocytes, these granules increase with age and exhibit neo-epitopes of carbohydrate nature that are recognized by natural IgM antibodies (IgMs). The existence of neoepitopes on PAS granules suggests the presence of neoepitopes in other brain structures, and this is investigated here. To this end, brain sections from SAMP8 and ICR-CD1 mice were examined at different ages.</p><p><strong>Results: </strong>We have identified two novel structures that, apart from PAS granules, are recognized by natural IgMs. On one side, IgM reactive (IgM⁺) granular structures which are placed in the longitudinal fissure, the quadrigeminal cistern, and a region that extends from the quadrigeminal cistern to the interpeduncular cistern. This last region, located between the telencephalon and both the mesencephalon and diencephalon, is designated henceforth as the fissura magna, as it is indeed a fissure and the largest in the brain. As all these regions are extraparenchymal (EP), the IgM⁺ granules found in these zones have been named EP granules. These EP granules are mainly associated with fibroblasts and are not stained with PAS. On the other side, some IgM⁺ astrocytes have been found in the glia limitans, near the above-mentioned fissures. Remarkably, EP granules are more prevalent at younger ages, while the number of IgM⁺ astrocytes increases with age, similarly to the already described evolution of PAS granules.</p><p><strong>Conclusions: </strong>The present work reports the presence of two brain-related structures that, apart from PAS granules, contain neo-epitopes of carbohydrate nature, namely EP granules and IgM⁺ astrocytes. We suggest that EP granules, associated to fibroblasts, may be part of a physiological function in brain clearance or brain-CSF immune surveillance, while both PAS granules and IgM⁺ astrocytes may be related to the increasing accumulation of harmful materials that occurs with age and linked to brain protective mechanisms. Moreover, the specific localisation of these EP granules and IgM⁺ astrocytes suggest the importance of the fissura magna in these brain-related cleaning and immune functions. The overall results reinforce the possible link between the fissura magna and the functioning of the glymphatic system.</p>","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11337560/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142019489","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Harnessing immunomodulation to combat sarcopenia: current insights and possible approaches.","authors":"Ning Zhang, Liting Zhai, Ronald Man Yeung Wong, Can Cui, Sheung-Wai Law, Simon Kwoon-Ho Chow, Stuart B Goodman, Wing-Hoi Cheung","doi":"10.1186/s12979-024-00458-9","DOIUrl":"10.1186/s12979-024-00458-9","url":null,"abstract":"<p><p>Sarcopenia is a complex age-associated syndrome of progressive loss of muscle mass and strength. Although this condition is influenced by many factors, age-related changes in immune function including immune cell dynamics, and chronic inflammation contribute to its progression. The complex interplay between the immune system, gut-muscle axis, and autophagy further underscores their important roles in sarcopenia pathogenesis. Immunomodulation has emerged as a promising strategy to counteract sarcopenia. Traditional management approaches to treat sarcopenia including physical exercise and nutritional supplementation, and the emerging technologies of biophysical stimulation demonstrated the importance of immunomodulation and regulation of macrophages and T cells and reduction of chronic inflammation. Treatments to alleviate low-grade inflammation in older adults by modulating gut microbial composition and diversity further combat sarcopenia. Furthermore, some pharmacological interventions, nano-medicine, and cell therapies targeting muscle, gut microbiota, or autophagy present additional avenues for immunomodulation in sarcopenia. This narrative review explores the immunological underpinnings of sarcopenia, elucidating the relationship between the immune system and muscle during ageing. Additionally, the review discusses new areas such as the gut-muscle axis and autophagy, which bridge immune system function and muscle health. Insights into current and potential approaches for sarcopenia management through modulation of the immune system are provided, along with suggestions for future research directions and therapeutic strategies. We aim to guide further investigation into clinical immunological biomarkers and identify indicators for sarcopenia diagnosis and potential treatment targets to combat this condition. We also aim to draw attention to the importance of considering immunomodulation in the clinical management of sarcopenia.</p>","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11299351/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141894838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The immunology of B-1 cells: from development to aging.","authors":"Matheus Silvério Mattos, Sofie Vandendriessche, Ari Waisman, Pedro Elias Marques","doi":"10.1186/s12979-024-00455-y","DOIUrl":"10.1186/s12979-024-00455-y","url":null,"abstract":"<p><p>B-1 cells have intricate biology, with distinct function, phenotype and developmental origin from conventional B cells. They generate a B cell receptor with conserved germline characteristics and biased V(D)J recombination, allowing this innate-like lymphocyte to spontaneously produce self-reactive natural antibodies (NAbs) and become activated by immune stimuli in a T cell-independent manner. NAbs were suggested as \"rheostats\" for the chronic diseases in advanced age. In fact, age-dependent loss of function of NAbs has been associated with clinically-relevant diseases in the elderly, such as atherosclerosis and neurodegenerative disorders. Here, we analyzed comprehensively the ontogeny, phenotypic characteristics, functional properties and emerging roles of B-1 cells and NAbs in health and disease. Additionally, after navigating through the complexities of B-1 cell biology from development to aging, therapeutic opportunities in the field are discussed.</p>","PeriodicalId":51289,"journal":{"name":"Immunity & Ageing","volume":null,"pages":null},"PeriodicalIF":5.2,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11295433/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141879749","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}