Epithelial cell biology最新文献

{"title":"Gamma delta TCR-bearing intraepithelial lymphocytes regulate class II major histocompatibility complex molecule expression on the mouse small intestinal epithelium.","authors":"S Matsumoto,&nbsp;H Setoyama,&nbsp;A Imaoka,&nbsp;Y Okada,&nbsp;H Amasaki,&nbsp;K Suzuki,&nbsp;Y Umesaki","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Introduction of fecal bacteria into germ-free (GF) Balb/c mice induces class II major histocompatibility complex (MHC) molecules on the small intestinal epithelium. In this study, we elucidated the regulatory mechanisms for the class II MHC molecule induction on the mouse small intestinal epithelium during microbial colonisation of the gut in ex-GF mice. Intraperitoneal injection of interferon-gamma (IFN-gamma) into GF Balb/c mice induced class II MHC expression on the small intestinal epithelial cells. Induction of these molecules was inhibited by peritoneal injection of a monoclonal antibody (mAb) against IFN-gamma on the conventionalisation of GF mice. RNA reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the small intestinal epithelium indicated that the class II transactivator (CIITA), a regulatory factor for the class II MHC gene, and the I-E alpha chain, but not IFN-gamma receptor mRNA, increased during conventionalisation. The induction of class II MHC on the epithelial cells during the conventionalisation of GF C.B-17 scid mice was much lower than that in GF Balb/c mice. Immunocytochemical and RT-PCR analysis showed that both the number of IFN-gamma producing IEL and the level of the IFN-gamma mRNA in gamma delta TCR IEL were very low in the GF state, and gradually increased after microbial colonisation. After in vivo treatment with a mAb against gamma delta TCR, the number of gamma delta TCR-expressing IEL greatly decreased and the expression of class II MHC molecules on the small intestinal epithelium was repressed during the conventionalisation of GF mice. Taken together, these results suggested that gamma delta TCR-bearing IEL modulate class II MHC molecule expression on the small intestinal epithelium through the production of IFN-gamma during microbial colonisation in ex-GF mice.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 4","pages":"163-70"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20367481","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":"Characterization of Na,K-ATPase isoform expression and activity in MDCK and Caco-2 epithelial cells.","authors":"K K Grindstaff,&nbsp;G Blanco,&nbsp;R W Mercer","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The asymmetric distribution of the Na,K-ATPase in the plasma membrane of epithelial cells is essential to the establishment of the transepithelial Na+ gradient that supports the vectorial transport of ions and solutes. To investigate the changes that occur during the development of polarity, we have characterized Na,K-ATPase expression and activity in two epithelial cell culture lines, Madin Darby canine kidney (MDCK) and Caco-2 cells. RNA and immunoblot analysis of both cell lines demonstrate that only the alpha 1 and beta 1 isoforms are expressed in nonpolarized and polarized cultures. Interestingly, alpha 1 and beta 1 message increases in MDCK cells with the development of polarity, yet there is little change in the amount of protein for either subunit. In contrast, alpha 1 and beta 1 polypeptide expression increases in Caco-2 cells with the development of polarity, even though the amount of both transcripts decreases. The lack of correlation between the changes that occur at the level of the message and protein suggest that appropriate expression is mediated in part by a combination of transcriptional and translational events. Furthermore, while there was a slight decrease in activity in polarized MDCK cells, there was a 1.9 fold increase in Na,K-ATPase activity in the polarized Caco-2 cells as compared to nonpolarized cells. These results demonstrate that the regulation of Na,K-ATPase alpha 1 and beta 1 isoform expression is mediated by a combination of transcriptional and translational events during the development of polarity in both cell lines.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 1","pages":"17-24"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19544358","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":"Activators of protein kinase C but not of phospholipase C modulate adenylate cyclase-responses of normal pig epidermis.","authors":"H Iizuka,&nbsp;H Takahashi,&nbsp;A Ishida-Yamamoto,&nbsp;Y Hashimoto","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>It has been reported that keratinocytes possess phospholipase C (PLC)-mediated signal transduction system(s), that can be triggered by histamine, bradykinin, thrombin, platelet-activating factor (PAF), and epidermal growth factor (EGF)/transforming growth factor-alpha (TGF-alpha). Since the activation of PLC results in release of 1,2-diacylglycerol (DAG), the physiologic activator of protein kinase C (PKC) that modulates the epidermal adenylate cyclase, we investigated the effects of these PLC activating chemicals on the adenylate cyclase responses of dispase-separated normal pig epidermis. Among these chemicals and factors only histamine decreased the successive histamine-induced cyclic AMP accumulation and increased forskolin-, and cholera toxin-induced AMP accumulations. These effects were similar to those of PKC activators. However, in contrast to the PKC-activator-induced partial and receptor-non-specific desensitization, the histamine-induced desensitization was completely-inducible and specific to the histamine receptor system, and was not affected by the PKC inhibitor, H-7. Similar modulation of the epidermal adenylate cyclase was induced by other adenylate cyclase stimulators (epinephrine, adenosine and prostaglandin E2), but not by bradykinin, thrombin, PAF, or EGF. The combined addition of bradykinin, thrombin, PAF and EGF to the culture medium had no effect on the adenylate cyclase responses, either. Thus no evidence for receptor-agonist dependent PLC-induced modulation of the adenylate cyclase was obtained in the normal pig epidermis. Although keratinocytes might contain PLC-mediated signal transduction systems, that are triggered by histamine, bradykinin, thrombin, PAF, and EGF/TGF-alpha, none of the activators singly or in combination appear to activate PKC sufficiently for the modulation of adenylate cyclase responses of the normal pig epidermis.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 1","pages":"35-41"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19544365","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":"Epidermal growth factor receptor expression on human breast luminal and basal cells in vitro.","authors":"P Monaghan,&nbsp;C L Clarke,&nbsp;N P Perusinghe,&nbsp;M G Ormerod,&nbsp;M J O'Hare","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The expression of EGF receptors has been studied on luminal and basal cells of human breast in vitro. Primary cultures of normal adult human breast epithelium were prepared as single cell suspensions containing a mixture of luminal and basal cells. The cells were simultaneously immunolabelled with antibodies recognising EMA (luminal epithelial cells), CALLA/CD10 (basal cells) and the epidermal growth factor receptor (EGFR). Flow cytometric analysis of these triple labelled cells detected low levels of EGFR on both cell types, with proportionally more EGFR on basal cells compared with luminal cells. Separated populations of basal and luminal cells were prepared from single cell suspensions by flow sorting or by immunomagnetic methods and cultured with and without EGF. Increased proliferation was detected in both cell types in the presence of EGF. To determine the localisation of the EGF receptor, purified cell populations were immunolabelled with anti-EGFR antibody and an FITC-labelled second antibody for fluorescence light microscopy and colloidal gold-labelled antibody for scanning electron microscopy (SEM). Low levels of EGFR were detected by indirect immunofluorescence on both cell types with higher levels on basal cells compared with luminal cells. The detailed subcellular distribution of the receptor was examined by SEM, with gold-labelling of EGFR detected using a field emission scanning electron microscope with a YAG crystal backscattered electron detector. Both luminal and basal cells expressed EGFR over the upper surface of individual cells when these were growing in isolation, but when cells formed part of a confluent island, levels of EGFR on the upper surface of cells were obviously reduced. Observations made by SEM on cells at the edges of such confluent islands showed that cultured basal cells expressed much higher levels of EGFR on their basal, as compared with their upper surfaces.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 2","pages":"52-62"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19663123","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":"Vectorial secretion by constitutive and regulated secretory pathways in mammary epithelial cells.","authors":"D R Blatchford,&nbsp;K A Hendry,&nbsp;M D Turner,&nbsp;R D Burgoyne,&nbsp;C J Wilde","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Lactating mammary epithelial cells synthesize large quantities of milk proteins, which they secrete vectorially at the apical membrane into the alveolar lumen of the gland. Recent work suggested that mammary protein secretion is not wholly constitutive, but may also occur in part through a regulated secretory pathway. This study used mouse mammary epithelial cells cultured on Engelbreth-Holm-Swarm (EHS) matrix to compare the proportions of basally and apically-directed proteins secreted constitutively or in a regulated manner. On EHS matrix, mammary cells formed mammospheres, multicellular structures enshrouded in matrix material, within which they became polarised, formed tight intercellular junctions, and secreted milk proteins vectorially. Protein secreted basolaterally was collected in culture medium, whereas apically-secreted milk proteins accumulated in a closed lumen within the mammosphere, and were recovered by EGTA treatment of the cells in situ. Protein secretion was measured by following the release of radiolabelled protein after pulse-labelling with [35S]-methionine. Basolateral and apical secretion of [35S]-protein appeared complete within 1 h of pulse-labelling, consistent with immediate secretion through constitutive secretory pathways. However, addition of the calcium ionophore ionomycin induced a second wave of secretion in both directions. Ca(2+)-stimulated secretion occurred within 15 min of ionomycin addition, doubled the extent of basolateral and apical secretion, but did not change the populations of proteins secreted. Ionomycin treatment did not affect mammosphere morphology or mammary cell ultrastructure. The results suggest that lactating mammary epithelial cells secrete proteins apically and basolaterally by two pathways, one a Ca(2+)-independent constitutive pathway, the other a regulated pathway stimulated by elevation of intracellular Ca2+.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 1","pages":"8-16"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19544240","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":"Assessment of inflammatory events in epithelial permeability: a rapid screening method using fluorescein dextrans.","authors":"S E Sanders,&nbsp;J L Madara,&nbsp;D K McGuirk,&nbsp;D S Gelman,&nbsp;S P Colgan","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Cultured intestinal epithelial monolayers serve as models for mechanistic studies of intestinal inflammation. One crucial aspect of epithelial function modulated by inflammation is permeability. Indices of permeability typically obtained are transepithelial resistance or more formalized assays using Ussing chambers modified for cultured monolayers. Such Ussing chamber systems are inconvenient for screening assays and their geometry precludes evaluation of important inflammatory responses such as transepithelial migration of neutrophils. Here we use a novel flux assay to investigate the kinetics of fluorescein-labelled dextran (FD) molecules across intestinal epithelial cell lines grown on permeable membrane supports. Our results show that we could consistently demonstrate picomolar flux of FD in a range of sizes (3-70 kD) across T84 epithelial monolayers. Such FD flux was time- and dose-dependent and flux increased exponentially with decreasing transepithelial resistance. Exposure of intestinal epithelia to mucosal-derived lymphocytes or to soluble lymphocyte products (interferon-gamma, interleukin-4 or interleukin-13) increased FD flux in a dose-dependent fashion. Finally, studies of neutrophil transepithelial migration revealed qualitative and quantitative differences in FD flux depending on FD size. We conclude that in vitro transepithelial FD flux may be a useful tool to study aspects of intestinal permeability in health and in disease.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 1","pages":"25-34"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19544363","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":"The generation of anchoring fibrils by epidermal keratinocytes: a quantitative long-term study.","authors":"C C Compton,&nbsp;W Press,&nbsp;J M Gill,&nbsp;G Bantick,&nbsp;K B Nadire,&nbsp;G Warland,&nbsp;J T Fallon,&nbsp;E C Vamvakas","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Anchoring fibrils (AFs) are derived from basal keratinocytes, but the kinetics of their formation is unknown. In this study, de novo generation of AFs by cultured human keratinocyte autografts was assessed from 1 week to 6 years postgrafting. Within 2 weeks, AF population densities were equal to those of normal controls and remained normal thereafter. However, AF diameters were narrow compared to controls (P < 0.05) until 3 years postgrafting. The depth of extension of AF-anchoring plaque lattices into the subjacent stroma was normal by 3 weeks but, after 1 month, was typically 1.5-to 2-fold greater than normal. The findings indicate that: (1) basal keratinocytes immediately re-establish a full complement of AFs; (2) once reformed, AF populations remain normal in density over time; (3) nascent AFs are thin and require several years to reach full maturity; and (4) abnormally thick AF lattices may form over time in healed wounds.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 3","pages":"93-103"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19934138","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":"Effects of the medium HCO3-/CO2 buffer system on differentiation and intermediary metabolism properties of rabbit proximal tubule cells in primary culture.","authors":"C Monteil,&nbsp;S Marouillat,&nbsp;J P Fillastre,&nbsp;J P Morin","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>In vivo, bicarbonate can affect proximal tubule intermediary metabolism, including gluconeogenesis, ammoniagenesis and maintenance of the mitochondrial substrate supply. In vitro, rabbit proximal tubule cells (RPTC) in primary culture revert from gluconeogenesis to glycolysis and their mitochondrial metabolism remains lower than in vivo. To determine whether the bicarbonate buffer system could have an effect on these deregulations, RPTC in primary culture grown in the absence of insulin and glucose in the culture medium were developed either with the standard sodium bicarbonate buffer with 5% CO2 or with a Hepes hydrogen ion buffer in the presence of 0.5% CO2. Duration of the bicarbonate-free cultures was increased until at least day 17 after seeding, compared with day 11 in bicarbonate-buffered cultures. As could be expected, succinate dehydrogenase activity remained stable as a function of time in bicarbonate-free cultures while an early marked decrease of this activity occurred from seeding in cultures developed in the presence of bicarbonate buffer. Compared to bicarbonate-buffered cells, higher phosphoenolpyruvate carboxykinase activity concomitant with lower intracellular lactate dehydrogenase activity was observed in cultures developed in the absence of bicarbonate, which is indicative of closer carbohydrate metabolism orientation to the in vivo situation for RPTC. Immunofluorescence staining of RPTC with monoclonal antibodies directed to neutral endopeptidase (NEP), and dipeptidyl-peptidase IV (DPP II) showed similar extensive labelling with DPP and NEP in both culture conditions. Confocal microscopy analysis of NEP subcellular distribution, showed exclusive targetting of NEP to the apical plasma membranes. In both models, cAMP production was stimulated by parathyroid hormone and unaffected by arginine vasopressin. In conclusion, bicarbonate withdrawal from the culture medium (without changing the pH of the medium) allows a marked improvement of mitochondrial capacity and carbohydrate metabolism pattern without any loss of differentiated properties.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 3","pages":"131-9"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19933407","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":"The isolation and culture of adult mouse colonic epithelium.","authors":"C Booth,&nbsp;S Patel,&nbsp;G R Bennion,&nbsp;C S Potten","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>A technique is described for the reproducible primary culture of colonic epithelium from adult mice. A collagenase-dispase digestion technique (adapted form Evans et al. 1992) is used to release the epithelium, followed by differential sedimentation to produce a high purity crypt preparation with maintained structural integrity and minimal mesenchymal contamination. The crypt units attach to collagen coated plastic within 24 h and the epithelial cells quickly begin to migrate outwards producing a monolayer surrounding the attached crypts. Electron microscopy revealed that the migrating epithelial cells possessed both desmosomes and microvilli. Proliferation in the colony supports the outward migration of cells until the migratory cells of adjacent colonies connect and a confluent monolayer begins to form. Proliferation is routinely maintained for 10 days (although cultures have now been maintained without subculturing for 35 days) and is demonstrated by increased cell numbers in spite of continuous cell loss into the culture media. Culture growth is enhanced by increasing concentrations of fetal calf and mouse serum and EGF but does not appear to respond significantly to added transferrin. Growth is also stimulated by a murine small intestinal extract thought to contain a potentially novel growth factor or cocktail of factors. This culture model has considerable potential for studies on growth factor control of this carcinoma susceptible tissue and its differentiated function as well as studies into the mechanisms of carinogenesis.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 2","pages":"76-86"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19663126","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":"Transitional differentiation patterns of principal and intercalated cells during renal collecting duct development.","authors":"J Aigner,&nbsp;S Kloth,&nbsp;M L Jennings,&nbsp;W W Minuth","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The developing renal collecting duct epithelium of neonatal rabbits exhibits 3 different zones. The ampullary tip epithelium acts as an embryonic inducer and is responsible for the generation of all of the nephron anlagen. It pilots the whole microarchitecture of the kidney. In the ampullary neck epithelium multiple cell divisions cause the elongation of the embryonic collecting duct so that the organ can grow. Finally, the cells in the ampullar shaft transdifferentiate into the functional collecting duct epithelium (CD) consisting of Principal (P) and various kinds of Intercalated (IC) cells. It is unknown by which morphogenic mechanisms the ampullar cells develop into the heterogeneously composed collecting duct epithelium. Using both morphological and immunohistochemical methods, we investigated the transdifferentiation patterns leading from the ampullar epithelium to the P and IC cells in the neonatal kidney. An electron microscope analysis of the cortico-medullary course of the developing collecting duct revealed that conspicuous morphological alterations start in the neck of the ampulla. The lumen of the neck region is narrowed to a slit. While most of the cells in the ampullar tip exhibit few, short microvilli, the neck cells bear numerous, extremely long microvilli at their apical cell poles. All of the neck cells exhibit the same cytoplasmic staining pattern and the same number of mitochondria. Farther down in the shaft, clearly recognizable P and IC cells are found. Thus, differentiation into P and IC cells starts with a transitional precursor cell type in the ampullar neck. Perfusion culture experiments with the embryonic collecting duct epithelium made it possible to generate transitional and differentiated cell types for the first time under in vitro conditions. The cultured epithelial cells showed characteristics common to both P and IC cells. Immunohistochemical findings revealed that morphological differentiation starts before the functional properties of P and IC cells can be detected.</p>","PeriodicalId":77116,"journal":{"name":"Epithelial cell biology","volume":"4 3","pages":"121-30"},"PeriodicalIF":0.0,"publicationDate":"1995-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19933406","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}