Journal of immunological methods最新文献

{"title":"Nephelometry vs. Immunoturbidimetry assay: Analytical performance on IgG subclasses","authors":"","doi":"10.1016/j.jim.2024.113725","DOIUrl":"10.1016/j.jim.2024.113725","url":null,"abstract":"<div><p>Interest in measuring immunoglobulin G Subclasses (IgG Subclasses) is increasing as more information is gathered and understanding regarding conditions associated with deficiencies of each IgG Subclass grows. Different methodologies are available for the measurement of IgG Subclasses, but their specificities vary. As a result, laboratories choose the methodology that better suits their routine, but which may not necessarily align with the needs of their population. In addition, the lack of standardization for the quantification of IgG Subclasses causes diagnostic gaps when comparing results provided by different methodologies. Thus, the purpose of our research is to compare the analytical performance of The Binding Site's (TBS) Optilite® human Immunoglobulin G (IgG) and IgG Subclasses Immunoturbidimetry assay, with the Nephelometry method routinely used in our clinical laboratory, Siemens BNII®. Our results show that the Immunoturbidimetry assay appears to be the most reliable to evaluate IgG Subclasses: the sum of IgG Subclasses and Total IgG correlate better than by Nephelometry. Although these methodologies share a similar principle, the comparison of results appears to be compromised. Therefore, prior to switching methodologies, further studies should be conducted to assess which methodology could be better applied to specific populations. It is also essential to standardise IgG Subclasses assays to reduce discrepancies that arise from comparing results.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"532 ","pages":"Article 113725"},"PeriodicalIF":1.6,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141600245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Techniques for evaluating the ATP-gated ion channel P2X7 receptor function in macrophages and microglial cells","authors":"Raíssa Leite-Aguiar ,&nbsp;Victória Gabriela Bello-Santos ,&nbsp;Newton Gonçalves Castro ,&nbsp;Robson Coutinho-Silva ,&nbsp;Luiz Eduardo Baggio Savio","doi":"10.1016/j.jim.2024.113727","DOIUrl":"10.1016/j.jim.2024.113727","url":null,"abstract":"<div><p>Resident macrophages are tissue-specific innate immune cells acting as sentinels, constantly patrolling their assigned tissue to maintain homeostasis, and quickly responding to pathogenic invaders or molecular danger signals molecules when necessary. Adenosine triphosphate (ATP), when released to the extracellular medium, acts as a danger signal through specific purinergic receptors. Interaction of ATP with the purinergic receptor P2X7 activates macrophages and microglial cells in different pathological conditions, triggering inflammation. The highly expressed P2X7 receptor in these cells induces cell membrane permeabilization, inflammasome activation, cell death, and the production of inflammatory mediators, including cytokines and nitrogen and oxygen-reactive species. This review explores the techniques to evaluate the functional and molecular aspects of the P2X7 receptor, particularly in macrophages and microglial cells. Polymerase chain reaction (PCR), Western blotting, and immunocytochemistry or immunohistochemistry are essential for assessing gene and protein expression in these cell types. Evaluation of P2X7 receptor function involves the use of ATP and selective agonists and antagonists and diverse techniques, including electrophysiology, intracellular calcium measurements, ethidium bromide uptake, and propidium iodide cell viability assays. These techniques are crucial for studying the role of P2X7 receptors in immune responses, neuroinflammation, and various pathological conditions. Therefore, a comprehensive understanding of the functional and molecular aspects of the P2X7 receptor in macrophages and microglia is vital for unraveling its involvement in immune modulation and its potential as a therapeutic target. The methodologies presented and discussed herein offer valuable tools for researchers investigating the complexities of P2X7 receptor signaling in innate immune cells in health and disease.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"532 ","pages":"Article 113727"},"PeriodicalIF":1.6,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141600246","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A 38-colour high dimensional immunophenotyping panel for human peripheral blood mononuclear cells","authors":"Jeremy Anderson ,&nbsp;Leanne Quah ,&nbsp;Kiara Mangano ,&nbsp;Daniel G. Pellicci ,&nbsp;Nadia Mazarakis ,&nbsp;Paul V. Licciardi","doi":"10.1016/j.jim.2024.113726","DOIUrl":"10.1016/j.jim.2024.113726","url":null,"abstract":"<div><p>High dimensional immunophenotyping panels are invaluable resources for performing extensive phenotyping on peripheral blood mononuclear cells (PBMCs). We designed a 38-colour high dimensional phenotyping panel to measure innate (monocytes, dendritic cells, NK cells, basophils, innate like lymphoid cells), T cell (γδ T cells, MAIT cells, CD4 and CD8 memory, Th1, Th2, Th17, Tfh, Treg) and B cell (memory, plasma cells, transitional B cells, plasmablasts, IgG, IgM) subsets in addition to their activation status using the 5-laser Cytek Aurora. We optimised optimal fluorochrome combinations and titres to minimise spread and autofluorescence of rare immune cell populations and tested this panel on PBMCs from 15 healthy adults. This high dimensional panel will be invaluable for direct ex vivo studies to evaluate immune cells in the context of human health and disease, especially when samples are limited.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"532 ","pages":"Article 113726"},"PeriodicalIF":1.6,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141590458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Belimumab concentration measurements using a homologous anti-idiotype immunoassay","authors":"Floris C. Loeff ,&nbsp;Ioannis Parodis ,&nbsp;Tomas Walhelm ,&nbsp;Andreas Jönsen ,&nbsp;Dionysis Nikolopoulos ,&nbsp;Christopher Sjöwall ,&nbsp;Anders A. Bengtsson ,&nbsp;Dorien Kos ,&nbsp;Astrid van Leeuwen ,&nbsp;Bryan van den Broek ,&nbsp;Lisanne Dijk ,&nbsp;Jorn Jeremiasse ,&nbsp;Birgit S. Blomjous ,&nbsp;Alexandre E. Voskuyl ,&nbsp;Gerrit Jan Wolbink ,&nbsp;Irene E.M. Bultink ,&nbsp;Theo Rispens","doi":"10.1016/j.jim.2024.113717","DOIUrl":"10.1016/j.jim.2024.113717","url":null,"abstract":"<div><p>Monitoring belimumab concentrations in patients can be a valuable tool for assessing treatment response and for personalizing drug doses. Various assay formats may be used to measure concentrations of therapeutic monoclonal antibodies. A particularly useful format involves the use of anti-idiotype monoclonal antibodies, selected to be highly specific to the antibody of interest. Here, we describe the development of a specific, high-affinity anti-idiotype antibody to belimumab, and the application of this antibody in a homologous sandwich ELISA to measure belimumab concentrations.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"532 ","pages":"Article 113717"},"PeriodicalIF":1.6,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498239","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"1,25-dihydroxyvitamin D3 augments low-dose PMA-based monocyte-to-macrophage differentiation in THP-1 cells","authors":"Bronwyn A. Mol,&nbsp;Janet J. Wasinda,&nbsp;Yi F. Xu,&nbsp;Nikki L. Gentle,&nbsp;Vanessa Meyer","doi":"10.1016/j.jim.2024.113716","DOIUrl":"10.1016/j.jim.2024.113716","url":null,"abstract":"<div><p>The human monocytic THP-1 cell line is the most routinely employed <em>in vitro</em> model for studying monocyte-to-macrophage differentiation. Despite the wide use of this model, differentiation protocols using phorbol 12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D₃ (1,25D₃) vary drastically between studies. Given that differences in differentiation protocols have the potential to impact the characteristics of the macrophages produced, we aimed to assess the efficacy of three different THP-1 differentiation protocols by assessing changes in morphology and gene- and cell surface macrophage marker expression. THP-1 cells were differentiated with either 5 nM PMA, 10 nM 1,25D₃, or a combination thereof, followed by a rest period. The results indicated that all three protocols significantly increased the expression of the macrophage markers, CD11b (<em>p</em> &lt; 0.001) and CD14 (<em>p</em> &lt; 0.010). Despite this, THP-1 cells exposed to 1,25D₃ alone did not adopt the morphological and expression characteristics associated with macrophages. PMA was required to produce these characteristics, which were found to be more pronounced in the presence of 1,25D₃. Both PMA- and PMA with 1,25D₃₋differentiated THP-1 cells were capable of M1 and M2 macrophage polarization, though the gene expression of polarization-associated markers was most pronounced in PMA with 1,25D₃₋differentiated THP-1 cells. Moreover, the combination of PMA with 1,25D₃ appeared to support the process of commitment to a particular polarization state.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"532 ","pages":"Article 113716"},"PeriodicalIF":1.6,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141498238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A real-time antibody-dependent cellular phagocytosis assay by live cell imaging","authors":"Yongchang Shi ,&nbsp;Yonglian Sun ,&nbsp;Akiko Seki ,&nbsp;Sascha Rutz ,&nbsp;James T. Koerber ,&nbsp;Jianyong Wang","doi":"10.1016/j.jim.2024.113715","DOIUrl":"10.1016/j.jim.2024.113715","url":null,"abstract":"<div><p>Antibody-dependent cellular phagocytosis (ADCP) is a cellular process by which antibody-opsonized targets (pathogens or cells) activate the Fc receptors on the surface of phagocytes to induce phagocytosis, resulting in internalization and degradation of pathogens or target cells through phagosome acidification. Besides NK cells-mediated antibody-dependent cellular cytotoxicity (ADCC), tumor-infiltrated monocytes and macrophages can directly kill tumor cells in the presence of tumor antigen-specific antibodies through ADCP, representing another attractive strategy for cancer immunotherapy. Even though several methods have been developed to measure ADCP, an automated and high-throughput quantitative assay should offer highly desirable advantages for drug discovery. In this study we established a new ADCP assay to identify therapeutical monoclonal antibodies (mAbs) that facilitate macrophages phagocytosis of live target cells. We used Incucyte, an imaging system for live cell analysis. By labeling the live target cells with a pH sensitive dye (pHrodo), we successfully monitored the ADCP in real time. We demonstrated that our image-based assay is robust and quantitative, suitable for screening and characterization of therapeutical mAbs that directly kill target cells through ADCP. Furthermore, we found different subtypes of macrophages have distinct ADCP activities using both mouse and human primary macrophages differentiated <em>in vitro</em>. By studying various mAbs with mutations in their Fc regions using our assay, we showed that the variants with increased binding to Fc gamma receptors (FcγRs) have enhanced ADCP activities.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113715"},"PeriodicalIF":1.6,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141468604","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A universal urinary cell gene signature of acute rejection in kidney allografts","authors":"Thalia Salinas ,&nbsp;Carol Li ,&nbsp;Catherine Snopkowski ,&nbsp;Vijay K. Sharma ,&nbsp;Darshana M. Dadhania ,&nbsp;Karsten Suhre ,&nbsp;Thangamani Muthukumar ,&nbsp;Manikkam Suthanthiran","doi":"10.1016/j.jim.2024.113714","DOIUrl":"10.1016/j.jim.2024.113714","url":null,"abstract":"<div><h3>Introduction</h3><p>Acute rejection (AR) undermines the life-extending benefits of kidney transplantation and is diagnosed using the invasive biopsy procedure. T cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR), or concurrent TCMR + ABMR (Mixed Rejection [MR]) are the three major types of AR. Development of noninvasive biomarkers diagnostic of AR due to any of the three types is a useful addition to the diagnostic armamentarium.</p></div><div><h3>Methods</h3><p>We developed customized RT-qPCR assays and measured urinary cell mRNA copy numbers in 145 biopsy-matched urine samples from 126 kidney allograft recipients. We determined whether the urinary cell three-gene signature diagnostic of TCMR (<span>Suthanthiran et al., 2013</span>) discriminates patients with no rejection biopsies (NR, <em>n</em> = 50) from those with ABMR (<em>n</em> = 28) or MR (<em>n</em> = 20) biopsies.</p></div><div><h3>Results</h3><p>The urinary cell three-gene signature discriminated all three types of rejection biopsies from NR biopsies (<em>P</em> &lt; 0.0001, One-way ANOVA). Dunnett's multiple comparisons test yielded <em>P</em> &lt; 0.0001 for NR vs. TCMR; <em>P</em> &lt; 0.001 for NR vs. ABMR; and <em>P</em> &lt; 0.0001 for NR vs. MR. By bootstrap resampling, optimism-corrected area under the receiver operating characteristic curve (AUC) was 0.749 (bias-corrected 95% confidence interval [CI], 0.638 to 0.840) for NR vs. TCMR (<em>P</em> &lt; 0.0001); 0.780 (95% CI, 0.656 to 0.878) for NR vs. ABMR (<em>P</em> &lt; 0.0001); and 0.857 (95% CI, 0.727 to 0.947) for NR vs. MR (<em>P</em> &lt; 0.0001). All three rejection categories were distinguished from NR biopsies with similar accuracy (all AUC comparisons <em>P</em> &gt; 0.05).</p></div><div><h3>Conclusion</h3><p>The urinary cell three-gene signature score discriminates AR due to TCMR, ABMR or MR from NR biopsies in human kidney allograft recipients.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"532 ","pages":"Article 113714"},"PeriodicalIF":1.6,"publicationDate":"2024-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141468597","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"ERAMER: A novel in silico tool for prediction of ERAP1 enzyme trimming","authors":"Anas Al-okaily ,&nbsp;Razan Abu Khashabeh ,&nbsp;Osama Alsmadi ,&nbsp;Yazan Ahmad ,&nbsp;Iyad Sultan ,&nbsp;Abdelghani Tbakhi ,&nbsp;Pramod K Srivastava","doi":"10.1016/j.jim.2024.113713","DOIUrl":"10.1016/j.jim.2024.113713","url":null,"abstract":"<div><p>MHC class I pathway consists of four main steps: proteasomal cleavage in the cytosol in which precursor proteins are cleaved into smaller peptides, which are then transported into the endoplasmic reticulum by the transporter associated with antigen processing, TAP, for further processing (trimming) from the N-terminal region by an ER resident aminopeptidases 1 (ERAP1) enzyme, to generate optimal peptides (8–10 amino acids in length) to produce a stable MHCI-peptide complex, that get transited <em>via</em> the Golgi apparatus to the cell surface for presentation to the cellular immune system. Several studies reported specificities related to the ERAP1 trimming process, yet there is no <em>in silico</em> tool for the prediction of the trimming process of the ERAP1 enzyme. In this paper, we provide and implement a prediction model for the trimming process of the ERAP1 enzyme.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113713"},"PeriodicalIF":1.6,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141457314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Strategies to determine positive anti-SARS-CoV-2 memory T lymphocyte response during the evolution of an epidemic","authors":"Isabelle Nel ,&nbsp;Ajeeva Ithayakumar ,&nbsp;Noémie Blumenthal ,&nbsp;Charlotte Duneton ,&nbsp;Valérie Guérin-El Khourouj ,&nbsp;Jérôme Viala ,&nbsp;Catherine Dollfus ,&nbsp;Véronique Baudouin ,&nbsp;Sophie Guilmin-Crepon ,&nbsp;Ioannis Theodorou ,&nbsp;Guislaine Carcelain","doi":"10.1016/j.jim.2024.113712","DOIUrl":"10.1016/j.jim.2024.113712","url":null,"abstract":"<div><p>During SARS-CoV-2 pandemic, the assessment of immune protection of people at risk of severe infection was an important goal. The appearance of VOCs (Variant of Concern) highlighted the limits of evaluating immune protection through the humoral response. While the humoral response partly loses its neutralizing activity, the anti-SARS-CoV-2 memory T cell response strongly cross protects against VOCs becoming an indispensable tool to assess immune protection. We compared two techniques available in laboratory to evaluate anti-SARS-CoV-2 memory T cell response in a cohort of infected or vaccinated patients with different levels of risk to develop a severe disease: the ELISpot assay and the T-Cell Lymphocyte Proliferation Assay respectively exploring IFNγ production and cell proliferation. We showed that the ELISpot assay detected more anti-Spike memory T cell response than the Lymphocyte Proliferation Assay. We next observed that the use of two different suppliers as antigenic source in the ELISpot assay did not affect the detection of anti-Spike memory T cell response. Finally, we explored a new approach for defining the positivity threshold, using unsupervised mixed Gaussian modeling, challenging the traditional ROC curve used by the supplier. That will be helpful in endemic situation where it could be difficult to recruit “negative” patients.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113712"},"PeriodicalIF":1.6,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0022175924000978/pdfft?md5=9d26956e717f00130e70ab4fbc05bb8e&pid=1-s2.0-S0022175924000978-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141436974","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discrepancies between two total IgE assays and difference in reference intervals in healthy adults","authors":"Mai Elzieny ,&nbsp;Gabriel N. Maine ,&nbsp;Robin A. Carey-Ballough ,&nbsp;Qian Sun","doi":"10.1016/j.jim.2024.113711","DOIUrl":"10.1016/j.jim.2024.113711","url":null,"abstract":"<div><h3>Objective</h3><p>To compare total immunoglobulin (Ig) E assay performance characteristics between Abbott Architect and Siemens Immulite test systems. Reference intervals were also determined for both platforms in an American population of healthy adults.</p></div><div><h3>Methods</h3><p>Agreement of the two total IgE assays was evaluated in a cohort of 331 subjects with normal complete blood count (CBC) and comprehensive metabolic panel (CMP) results. Reference intervals were established in 302 subjects after exclusion of atopic individuals on the Abbott Architect and Siemens Immulite test systems.</p></div><div><h3>Results</h3><p>We demonstrated a 32% positive bias for total IgE quantitation on the Siemens Immulite platform compared to the Abbott Architect, despite both methods calibrated against the same WHO international reference material (75/502), Furthermore, the upper limit of the reference interval (95th percentile) was determined to be higher for the Siemens Immulite assay compared to the Abbott Architect (132 and 102 IU/mL, respectively).</p></div><div><h3>Conclusion</h3><p>Despite the use of a common WHO reference material for total IgE assay calibration, significant differences in quantitation was observed between two FDA-cleared test systems. Given that, it is warranted for clinical laboratories to verify vendor established reference intervals and adjust accordingly based on internal assessment of the normal range.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"531 ","pages":"Article 113711"},"PeriodicalIF":1.6,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141327525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}