Annals of periodontology最新文献

{"title":"Campylobacter Surface-Layers (S-Layers) and Immune Evasion","authors":"Stuart A. Thompson Dr.","doi":"10.1902/annals.2002.7.1.43","DOIUrl":"10.1902/annals.2002.7.1.43","url":null,"abstract":"<p>Many pathogenic bacteria have evolved mechanisms for evading host immune systems. One evasion mechanism is manifest by the surface layer (S-layer), a paracrystalline protein structure composed of S-layer proteins (SLPs). The S-layer, possessed by 2 <i>Campylobacter</i> species (<i>C. fetus</i> and <i>C. rectus</i>), is external to the bacterial outer membrane and can have multiple functions in immune avoidance. <i>C. fetus</i> is a pathogen of ungulates and immunocompromised humans, in whom it causes disseminated bloodstream disease. In <i>C. fetus</i>, the S-layer is required for dissemination and is involved in 2 mechanisms of evasion. First, the S-layer confers resistance to complementmediated killing in non-immune serum by preventing the binding of complement factor C3b to the <i>C. fetus</i> cell surface. S-layer expressing <i>C. fetus</i> strains remain susceptible to complementindependent killing, utilizing opsonic antibodies directed against the S-layer. However, <i>C. fetus</i> has also evolved a mechanism for avoiding antibody-mediated killing by high-frequency antigenic variation of SLPs. Antigenic variation is accomplished by complex DNA inversion events involving a family of multiple SLPencoding genes and a single SLP promoter. Inversion events result in the expression of antigenically variant S-layers, which require distinct antibody responses for killing. <i>C. rectus</i> is implicated in the pathogenesis of periodontal disease and also possesses an S-layer that appears to be involved in evading the human system. Although studied less extensively than its <i>C. fetus</i> counterpart, the <i>C. rectus</i> S-layer appears to confer resistance to complement-mediated killing and to cause the down-regulation of proinflammatory cytokines. <i>Ann Periodontol 2002;7:43-53.</i></p>","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"43-53"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.43","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25185531","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Relationships Among Clinical Measures of Periodontal Disease and Their Associations With Systemic Markers","authors":"James D. Beck Dr.,&nbsp;Steven Offenbacher","doi":"10.1902/annals.2002.7.1.79","DOIUrl":"10.1902/annals.2002.7.1.79","url":null,"abstract":"<p>B<b>ackground:</b> Recent investigations of the relationship between periodontitis and systemic disease require that periodontal disease also must be thought of as a disease process that is an exposure for a systemic disease or condition (outcome), rather than as the outcome itself. When viewing periodontal disease as an exposure, investigators must consider the clinical, microbiological, and inflammatory components of periodontitis that potentially convey risk for the systemic outcome of interest, which may or may not be the same as those associated with the assessments used to define tooth-based disease. Another important consideration is the temporal relationship between the exposure and the outcome of interest.</p><p><b>Methods:</b> To explore which definitions of periodontal disease or clustering of clinical signs are important with regards to systemic exposure to inflammatory stress, we examined the relationship between clinical periodontal disease measures and 2 systemic inflammatory markers of increased risk for cardiovascular disease: serum soluble intercellular adhesion molecule (sICAM), which is a measure of vascular stress and serum C-reactive protein (CRP), which is a measure of hepatic acute-phase response. The Dental Arteriosclerosis Risk in Communities (ARIC) study, a cross-sectional study of the relationship between periodontal disease and cardiovascular disease, forms the basis for the examples used in this investigation.</p><p><b>Results:</b> Our findings demonstrated that while attachment loss, probing depth, (PD) and bleeding on probing (BOP) are individually associated with sICAM and CRP, only BOP remains significant for sICAM when all 3 are in the model and, for CRP, only PD remains significant. Both of these clinical parameters were more robust in estimating the degree of systemic inflammation than traditional classifications of mild, moderate, and severe periodontitis or other measures of disease severity such as attachment loss.</p><p><b>Conclusions:</b> When selecting a definition of “systemic periodontitis” (periodontal disease that represents an exposure for a systemic condition), it is helpful to think of periodontal disease as a chronic oral infection with a number of clinical signs, rather than as the dento-centrically defined entity, periodontal disease. Thus, “systemic periodontitis” should be defined predicated upon those clinical signs that best represent the underlying mechanisms and temporal sequence that may affect that systemic outcome. <i>Ann Periodontol 2002;7:79-89.</i></p>","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"79-89"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.79","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25185534","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Periodontitis in Humans and Non-Human Primates: Oral-Systemic Linkage Inducing Acute Phase Proteins","authors":"Jeffrey L. Ebersole Dr.,&nbsp;David Cappelli,&nbsp;Erik C. Mathys,&nbsp;Michelle J. Steffen,&nbsp;Robert E. Singer,&nbsp;Michael Montgomery,&nbsp;Glen E. Mott,&nbsp;M. John Novak","doi":"10.1902/annals.2002.7.1.102","DOIUrl":"10.1902/annals.2002.7.1.102","url":null,"abstract":"<p><b>Background:</b> The acute phase response (APR) represents a systemic counterpart to the localized inflammatory response. This report describes patient-oriented and non-human primate model studies to determine the effect of periodontal disease on systemic acute phase proteins (APP).</p><p><b>Methods:</b> Patient-oriented studies included comparison of the levels of APP, using enzyme-linked immunosorbent assay (ELISA), with the presence and severity of periodontitis in localized chronic periodontitis (LCP), generalized aggressive periodontitis (GAP), and Sjögren's syndrome (SS) patients. The non-human primate experiments evaluated the serum level of APPs under natural conditions, following mechanical hygiene, experimental gingivitis, and during ligature-induced periodontitis.</p><p><b>Results:</b> Analysis of the LCP population showed what appeared to be a threshold of periodontal disease severity required for elevating the C-reactive protein (CRP) and haptoglobin (HG). The results demonstrated a significant elevation in CRP in the GAP versus the control groups, as well as lower levels of all mediators in healthy non-smokers (HNS) versus smokers (HS), suggesting that these systemic inflammatory markers were altered in response to challenge by noxious materials from smoking. Significantly different levels of CRP, HG, and α1-antiproteinase were noted in the SS patients suggesting that the autoimmune aspects of Sjögren's syndrome may impact upon oral health and systemic responses. Parallel evidence was also obtained from the primate studies. Providing mechanical oral hygiene, which significantly lowered clinical inflammation and bleeding of the gingiva, decreased the serum APP levels. Both CRP and fibrinogen were significantly elevated during progressing periodontitis, which also appeared to have an impact on serum lipids and lipoproteins.</p><p><b>Conclusions:</b> These findings supported results relating chronic oral infections and the inflammation of periodontitis as contributors to and/or triggers for systemic inflammatory responses. Finally, similarities in the clinical and microbiological parameters of gingival inflammation and periodontitis between humans and non-human primates was extended to identification of changes in serum APP in the non-human primates that appeared to be in direct response to the induction of progressing periodontitis. These systemic changes provide additional evidence for the biological plausibility of periodontal infections contributing to various systemic diseases. <i>Ann Periodontol 2002;7:102-111.</i></p>","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"102-111"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.102","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25185537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bioinformatics and Approaches to Identifying Polygenic Susceptibility Traits","authors":"Bruce S. Weir Dr.","doi":"10.1902/annals.2002.7.1.1","DOIUrl":"10.1902/annals.2002.7.1.1","url":null,"abstract":"<p>The role of genetic factors in periodontal disease is now well recognized, although details for the genetic mechanisms of the disease and implications for therapy can be as obscure as they are for other human traits. This paper addresses the role that the analysis of genome-wide data might play in helping to understand the molecular determinants of periodontal risk. Very few human diseases are not polygenic, in that an individual's susceptibility depends on his or her constitution at many genetic loci, each of which may have a small effect. Not only do these loci interact, but also their actions and interactions depend on nongenetic factors. Much of the statistical machinery to handle this complexity was developed in the plant and animal breeding context, where crosses between inbred lines selected for trait differences could be conducted. Human polygenic studies began with studies on large pedigrees, but have expanded to include case-control analyses of random samples of individuals who differ in disease status, and studies of marker transmissions within nuclear families. In the area of characterizing the genetic architecture of complex traits, the relatively new field of bioinformatics is distinguished from the more mature fields of statistical genetics or genetic epidemiology by its focus on genome-wide data. The very dense sets of genetic markers now available, particularly those at single nucleotide positions (SNPs), have meant that it is possible to seek linkages or associations between chromosomal position and disease from the whole genome in a single study. Apart from the obvious problems of scale, there are real issues involved with multiple testing and recognizing interactions. Current thinking tends to focus on relatively conserved “haplotype blocks” instead of single genetic markers, although there is no consensus on the utility of this emphasis. <i>Ann Periodontol 2002;7:1-7.</i></p>","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"1-7"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25186093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Periodontal Disease Increases the Risk of Preterm Delivery Among Preeclamptic Women","authors":"Estelle L. Riché,&nbsp;Kim A. Boggess,&nbsp;Susi Lieff,&nbsp;Amy P. Murtha,&nbsp;Richard L. Auten,&nbsp;James D. Beck,&nbsp;Steven Offenbacher Dr.","doi":"10.1902/annals.2002.7.1.95","DOIUrl":"10.1902/annals.2002.7.1.95","url":null,"abstract":"<p><b>Background:</b> Preterm births are a major cause of neonatal morbidity and mortality, and represent an important public health issue. About 30% of preterm births are due to medical conditions of the mother or the fetus, among which preeclampsia plays a major role. We have previously reported that maternal periodontal disease enhances the risk for preterm delivery and preeclampsia. Our current objective was to determine whether maternal periodontal disease increases the risk for preterm delivery among preeclamptic women.</p><p><b>Methods:</b> Women were enrolled prior to their twenty-sixth week of gestation. Periodontal status was assessed at baseline and defined as healthy, mild, or moderate/severe. Repeat examinations were performed at delivery to assess changes in periodontal status.</p><p><b>Results:</b> A cohort of 1,020 women was studied, 47 of whom had preeclampsia. A strong association between periodontal disease status at enrollment and rate of premature delivery was observed among preeclamptic women after adjusting for the major risk factors for preterm delivery, including maternal race; age; marital status; WIC (women, infants, children program) or food stamps; insurance; previous preterm delivery; and chorioamnionitis. Among preeclamptic women, 49.3% with mild periodontal disease and 82.6% with moderate to severe disease delivered preterm (hazard ratios [HR] 4.11 and 11.0, respectively). Periodontal disease worsening during pregnancy in preeclamptic women was also associated with an increased risk of preterm births (HR 8.44).</p><p><b>Conclusion:</b> These results suggest that mothers with preeclampsia may be at greater risk for preterm delivery if periodontal disease is present early in pregnancy or progresses during pregnancy. <i>Ann Periodontol 2002;7:95-101.</i></p>","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"95-101"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.95","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25185536","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Population Studies of Microbial Ecology in Periodontal Health and Disease","authors":"Panos N. Papapanou Dr.","doi":"10.1902/annals.2002.7.1.54","DOIUrl":"10.1902/annals.2002.7.1.54","url":null,"abstract":"<p>It has been established that the bacterial diversity in any given environment is severely underestimated when assessed by means of culture-based techniques. Yet, almost all currently available knowledge related to the periodontal microbiota in health and disease has been generated either by culture-based surveys or by methods that require prior species identification by culture. A handful of recent studies using culture-independent molecular methods providing 16S rRNA sequences for both cultivable and not yet cultivated species of human periodontal bacteria demonstrated a high bacterial diversity in the oral cavity. It has been estimated that approximately 500 species may colonize the human oral cavity, half of which have been cultivated to date.</p><p>A review of the available epidemiological data on the prevalence of certain periodontal microbiota on a population level reveals considerable variation in estimates with respect to 1) sampling strategy, 2) mode of bacterial identification, and 3) race/ethnicity of the studied population. Nevertheless, specific bacterial profiles appear to confer high odds ratios for pathological periodontal conditions and/or progressive periodontal disease. However, the currently recognized periodontal pathogens are commonly recovered from periodontally healthy children, and their carrier rate in adults is substantial. Virulent clones, such as a highly leukotoxic strain of <i>Actinobacillus actinomycetemcomitans</i>, have been found to be closely associated with aggressive forms of periodontitis.</p><p>In conclusion, while the majority of the periodontal microbiota are commensals, a subset of likely opportunistic pathogens fulfills the epidemiologic requirements needed in order to be ascribed as risk/causative factors. Given the large proportion of the periodontal microbial habitat that is currently insufficiently explored, and assuming that the hitherto uncultivated segment of the bacterial community will include similar levels of pathogenic species, the list of periodontal pathogens should be expected to expand. <i>Ann Periodontol 2002;7:54-61.</i></p>","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"54-61"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.54","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25185530","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Subtractive Cloning: New Genes for Studying Inflammatory Disorders","authors":"Denis Rebrikov,&nbsp;Sejal Desai,&nbsp;Yakov N. Kogan,&nbsp;Angela M. Thornton,&nbsp;Luda Diatchenko Dr.","doi":"10.1902/annals.2002.7.1.17","DOIUrl":"10.1902/annals.2002.7.1.17","url":null,"abstract":"<p>Understanding of the biology of interaction between pathogens and host is the central question in studying inflammatory disorders. Subtractive DNA cloning is one of the most efficient and comprehensive methods available for identifying eukaryotic genes regulated under specific physiological conditions, including inflammation and host response. Here we explore the utility of subtractive DNA cloning and describe suppression subtractive hybridization (SSH), a polymerase chain reaction (PCR)-based DNA subtraction method that has been developed and evolved in our labs over several years. The SSH method possesses a number of advantages as compared to other subtractive cloning techniques, making it one of the most adventitious methods for cloning differentially expressed genes. Besides isolation of differentially expressed eukaryotic mRNAs, subtractive DNA cloning can be used to identify genes that are differentially expressed between diverse bacterial species. These genes can be of great interest, as some may encode strain-specific traits such as drug resistance, or bacterial surface proteins involved in determining the virulence of a particular strain. Other genes may be useful as markers for epidemiological or evolutionary studies. To demonstrate the potential of the SSH technique, we describe here the comprehensive characterization of 2 SSH subtracted libraries constructed in our laboratories. One library was created using eukaryotic cDNA subtraction and is specific for mRNAs up-regulated in CD25 positive cells from mouse lymph nodes as compared to CD25 negative cells. The second subtracted library is specific for a methicillin-resistant <i>Staphylococcus aureus</i> bacterial strain, but not in a methicillin-sensitive strain. The bacterial genomes of these 2 strains have been completely sequenced and this second library provides an excellent reference for testing the ability of SSH to recover all strain-specific gene content. The analysis of these 2 subtracted libraries serves as the basis for a discussion of the strength and limitations of the SSH technique. We will also compare and contrast subtractive DNA cloning to other current technologies used to isolate differentially expressed genes. <i>Ann Periodontol 2002;7:17-28.</i></p>","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"17-28"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.17","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25186097","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Poster Session Abstracts","authors":"","doi":"10.1902/annals.2002.7.1.112","DOIUrl":"https://doi.org/10.1902/annals.2002.7.1.112","url":null,"abstract":"","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"112-126"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.112","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138023245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cellular Responses to Oral Pathogens","authors":"Howard K. Kuramitsu Dr.,&nbsp;H. Miyakawa,&nbsp;M. Qi,&nbsp;I.C. Kang","doi":"10.1902/annals.2002.7.1.90","DOIUrl":"10.1902/annals.2002.7.1.90","url":null,"abstract":"<p><b>Background:</b> Several previous epidemiological studies, along with the results of more recent animal model approaches, have suggested a role for periodontitis in atherosclerosis. Such an association could be mediated by direct interactions of periodontopathic bacteria with host vascular tissues.</p><p><b>Methods:</b>The interactions of <i>Porphyromonas gingivalis</i> with endothelial cells and macrophages in vitro were investigated relative to modification of low-density lipoproteins (LDL).</p><p><b>Results:</b> <i>P. gingivalis</i> 381, its outer membrane vesicles, and the lipopolysaccharide (LPS) derived from these organisms were all shown to induce modification of LDL in the presence of the murine macrophage J774.A.1. Such alterations led to an increase in the migration of the particles through agarose gels. In addition,u direct modification of LDL by strain 381 was demonstrated in the absence of macrophages. This latter property appears to be related to the potent protease activities of the bacterium. These properties may contribute to modification of LDL to forms which have been strongly implicated in cholesterol lipid accumulation in vascular tissues. <i>P. gingivalis</i> 381 also appears to induce cyclooxygenase-2 expression in endothelial cells as determined with human umbilical vascular endothelial cells (HUVEC).</p><p><b>Conclusions:</b> These in vitro results with vascular cells in culture suggest a molecular basis for a potential role for periodontopathic bacteria such as <i>P. gingivalis</i> in augmenting foam cell formation characteristic of atherosclerotic lesions. <i>Ann Periodontol 2002;7:90-94.</i></p>","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"90-94"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.90","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25185535","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Reflections on the 20th International Conference on Periodontal Research: Molecular Determinants of Risk With a Commentary on Post-Genomic Periodontal Research — Musings of an Experimentalist","authors":"Steven Offenbacher","doi":"10.1902/annals.2002.7.1.i","DOIUrl":"10.1902/annals.2002.7.1.i","url":null,"abstract":"","PeriodicalId":79473,"journal":{"name":"Annals of periodontology","volume":"7 1","pages":"i-iv"},"PeriodicalIF":0.0,"publicationDate":"2002-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1902/annals.2002.7.1.i","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25186091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}