RESUMO
Point-of-care hepatitis C virus (HCV) RNA testing is advantageous, enabling diagnosis of active infection in a single visit. This study evaluated the sensitivity and specificity of the Xpert HCV Viral Load Finger-Stick assay (Xpert HCV VL FS) for HCV RNA detection (finger-stick) and the Xpert HCV Viral Load assay (plasma) compared with the Abbott RealTime HCV Viral Load assay by venepuncture. Plasma and finger-stick capillary whole-blood samples were collected from participants in an observational cohort in Australia. Of 223 participants enrolled, HCV RNA was detected in 40% of participants (85 of 210) with available Xpert HCV Viral Load testing. Participants receiving HCV therapy were excluded from subsequent analyses (n = 16). Sensitivity of the Xpert HCV Viral Load assay for HCV RNA quantification in plasma collected by venepuncture was 100.0% (95% confidence interval [CI] 96.9%-100.0%) and specificity was 100.0% (95% CI, 94.4%-100.0%). Sensitivity of the Xpert HCV VL FS assay for HCV RNA quantification in samples collected by finger-stick was 100.0% (95% CI, 93.9%-100.0%) and specificity was 100.0% (95% CI, 96.6%-100.0%). The Xpert HCV VL FS test can accurately detect active infection from a finger-stick sample in 1 hour allowing single-visit HCV diagnosis.
Assuntos
Bioensaio/métodos , Hepacivirus/genética , Hepatite C/virologia , Carga Viral/métodos , Adulto , Austrália , Coleta de Amostras Sanguíneas/métodos , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , RNA Viral/genética , Sensibilidade e Especificidade , Testes Sorológicos/métodosRESUMO
We report the establishment and characterization of immortalized human fetal liver progenitor cells by expression of the Simian virus 40 large T (SV40 LT) antigen. Well-characterized cells at various passages were transplanted into nude mice with acute liver injury and tested for functional capacity. The SV40LT antigen-immortalized fetal liver cells showed a morphology similar to primary cells. Cultured cells demonstrated stable phenotypic expression in various passages, of hepatic markers such as albumin, CK 8, CK18, transcription factors HNF-4α and HNF-1α and CYP3A/7. The cells did not stain for any of the tested cancer-associated markers. Albumin, HNF-4α and CYP3A7 expression was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Flow cytometry showed expression of some progenitor cell markers. In vivo study showed that the cells expressed both fetal and differentiated hepatocytes markers. Our study suggests new approaches to expand hepatic progenitor cells, analyze their fate in animal models aiming at cell therapy of hepatic diseases.
Assuntos
Antígenos Transformantes de Poliomavirus/análise , Diferenciação Celular , Linhagem Celular , Células-Tronco Fetais/citologia , Hepatócitos/citologia , Fenótipo , Albuminas/análise , Albuminas/genética , Animais , Antígenos CD/análise , Antígenos de Neoplasias/análise , Antígenos Transformantes de Poliomavirus/genética , Hidrocarboneto de Aril Hidroxilases/análise , Hidrocarboneto de Aril Hidroxilases/genética , Biomarcadores Tumorais/análise , Moléculas de Adesão Celular/análise , Citocromo P-450 CYP3A/análise , Citocromo P-450 CYP3A/genética , Molécula de Adesão da Célula Epitelial , Células-Tronco Fetais/química , Células-Tronco Fetais/transplante , Expressão Gênica , Fator 1-alfa Nuclear de Hepatócito/análise , Fator 4 Nuclear de Hepatócito/análise , Fator 4 Nuclear de Hepatócito/genética , Hepatócitos/química , Hepatócitos/transplante , Humanos , Queratinas/análise , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , RNA Mensageiro/análise , Vírus 40 dos Símios , Transfecção , Proteína Supressora de Tumor p53/análiseRESUMO
BACKGROUND AIMS: Cultured human hepatocytes have extensive diagnostic and clinical applications. However, the setting-up of new in vitro culture techniques allowing the long-term survival and functional maintenance of adult human hepatocytes represents a formidable challenge. Fetal liver cells (FLC) are attractive candidate donor cells because of their high proliferative capacity. METHODS: Using cell culture and molecular techniques, we studied the in vitro and in vivo characteristics of FLC grown long-term in serum-free conditions. RESULTS: Serum-free FLC obtained from 6-10-week-old human fetal livers grew as multiple clusters in suspension and could be subcultured for at least six passages. These cells maintained stable hepatocyte phenotypes and gene expression patterns in culture for up to 6 months. When a cluster of these cells in various passages was placed on collagen-coated plates, they formed a monolayer and morphologically resembled hepatocytes. The cells expressed alpha -fetoprotein, cytokeratin (CK) 8, CK18 and CK19 and albumin (ALB). Hepatocyte nuclear factor 4alpha and 1beta and cytochrome P450 (CYP) 3A4 and CYP3A7 mRNA expression was demonstrated by reverse transcriptase-polymerase chain reaction (RT-PCR). Cells at different passages, when transplanted into nude mice with liver injury, engrafted successfully, as detected by in situ hybridization using a human-specific DNA probe. Colonies of human-specific CK8, CK18, c-Met nuclear antigen (Ag), mitochondrial Ag, hepatocyte-specific Ag and ALB-expressing cells were present in the livers of recipient animals. CONCLUSIONS: Primary human FLC can be kept in culture consistently over a long time period and are potential candidates for cell therapy and in vitro diagnostics.
Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura Livres de Soro/farmacologia , Feto/citologia , Hepatócitos/citologia , Hepatócitos/transplante , Fígado/citologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Vidro , Hepatócitos/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Especificidade de Órgãos/efeitos dos fármacos , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Fatores de TempoRESUMO
To avoid the time consuming, labor intensive seed-train expansion and to improve production reliability and consistency, portions of bulk cryopreserved cells from the same cultivation can be utilized as inocula or alternatively may be used to undertake transient transfections for large-scale bioreactor production. In this study, the conditions for large-scale freezing in cryobags were optimized utilizing a design of experiment approach. We showed that relatively high density of 30-40 x 10(6) cells/mL and relatively low Me(2)SO concentrations of 5-6% in the freezing media are optimal to freeze HEK293-EBNA and CHO-S cells in a controlled manner in order to achieve high viable cell recovery and growth post-thawing. The immediate transfer of freshly thawed cells into culture medium resulted in better cell growth compared to cells that were centrifuged in order to remove Me(2)SO. This was the case as long as the residual Me(2)SO did not exceed 0.2-0.3%. The best time to perform transient 25 kDa polyethylenimine-mediated transfection of pCEP4-EGFP plasmid into freshly thawed, one-step inoculated cells is after 72-96 h in culture. At this time point, the numbers of EGFP-positive cells in the freshly thawed culture mimic perfectly that of cells grown continuously. Finally, our data showed that it is possible to freeze transiently polyethyleneimine-transfected HEK293-EBNA cells and maintain growth rate and expression of recombinant protein following thawing. The optimal time point for freezing cells was 4 h after transfection.
Assuntos
Técnicas de Cultura de Células/métodos , Criopreservação/métodos , Dimetil Sulfóxido/farmacologia , Rim/citologia , Rim/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Células CHO , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Cricetinae , Cricetulus , Crioprotetores/farmacologia , Congelamento , Humanos , Rim/efeitos dos fármacos , Polietilenoimina/química , Engenharia de Proteínas/métodos , Transfecção/métodosRESUMO
BACKGROUND: Point-of-care hepatitis C virus (HCV) RNA testing offers an advantage over antibody testing (which only indicates previous exposure), enabling diagnosis of active infection in a single visit. In this study, we evaluated the performance of the Xpert HCV Viral Load assay with venepuncture and finger-stick capillary whole-blood samples. METHODS: Plasma and finger-stick capillary whole-blood samples were collected from participants in an observational cohort enrolled at five sites in Australia (three drug and alcohol clinics, one homelessness service, and one needle and syringe programme). We compared the sensitivity and specificity of the Xpert HCV Viral Load test for HCV RNA detection by venepuncture and finger-stick collection with the Abbott RealTime HCV Viral Load assay (gold standard). FINDINGS: Of 210 participants enrolled between Feb 8, 2016, and July 27, 2016, 150 participants had viral load testing results for the three assays tested. HCV RNA was detected in 45 (30% [95% CI 23-38]) of 150 participants based on Abbott RealTime. Sensitivity of the Xpert HCV Viral Load assay for HCV RNA detection in plasma collected by venepuncture was 100·0% (95% CI 92·0-100·0) and specificity was 99·1% (95% CI 94·9-100·0). Sensitivity of the Xpert HCV Viral Load assay for HCV RNA detection in samples collected by finger-stick was 95·5% (95% CI 84·5-99·4) and specificity was 98·1% (95% CI 93·4-99·8). No adverse events caused by the index test or the reference standard were observed. IMPLICATIONS: The Xpert HCV Viral Load test can detect active infection from a finger-stick sample, which represents an advance over antibody-based tests that only indicate past or previous exposure. FUNDING: National Health and Medical Research Council (Australia), Cepheid, South Eastern Sydney Local Health District (Australia), and Merck Sharp & Dohme (Australia).
Assuntos
Hepacivirus/genética , Hepatite C/diagnóstico , Testes Imediatos , RNA Viral/sangue , Carga Viral/métodos , Adulto , Capilares , Feminino , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Flebotomia , Sensibilidade e EspecificidadeRESUMO
Effective repopulation of the liver is essential for successful clinical hepatocyte transplantation. The objective was to improve repopulation of the liver with human hepatocytes using chemokines. We used flow cytometry and immunohistochemistry assays to identify commonly expressed chemokine receptors on human fetal and adult hepatocytes. The migratory capacity of the cells to various chemokines was tested. For in vivo studies, we used a nude mouse model of partial hepatectomy followed by intraparenchymal injections of chemokine ligands at various concentrations. Human fetal liver cells transformed with human telomerase reverse transcriptase were used for intrasplenic cell transplantation. Repopulation and functionality were assessed 4 weeks after transplantation. The receptor CXCR3 was commonly expressed on both fetal and adult hepatocytes. Both cell types migrated efficiently toward corresponding CXC chemokine ligands 9, 10, and 11. In vivo, animals injected with recombinant chemokines showed the highest cell engraftment compared with controls (p<.05). The engrafted cells expressed several human hepatic markers such as cytokeratin 8 and 18 and albumin as well as transferrin, UGT1A1, hepatocyte nuclear factor (1α, 1ß, and 4α), cytochrome CYP3A1, CCAAT/enhancer binding protein (α and ß), and human albumin compared with controls. No inflammatory cells were detected in the livers at 4 weeks after transplantation. The improved repopulation of transplanted cells is likely a function of the chemokines to mediate cell homing and retention in the injured liver and might be an attractive strategy to augment repopulation of transplanted hepatocytes in vivo.
Assuntos
Quimiocinas/metabolismo , Hepatócitos/transplante , Hepatopatias/cirurgia , Animais , Western Blotting , Movimento Celular , Modelos Animais de Doenças , Citometria de Fluxo , Xenoenxertos , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: The recently developed Xpert® Ebola Assay is a novel nucleic acid amplification test for simplified detection of Ebola virus (EBOV) in whole blood and buccal swab samples. The assay targets sequences in two EBOV genes, lowering the risk for new variants to escape detection in the test. The objective of this report is to present analytical characteristics of the Xpert® Ebola Assay on whole blood samples. METHODS AND FINDINGS: This study evaluated the assay's analytical sensitivity, analytical specificity, inclusivity and exclusivity performance in whole blood specimens. EBOV RNA, inactivated EBOV, and infectious EBOV were used as targets. The dynamic range of the assay, the inactivation of virus, and specimen stability were also evaluated. The lower limit of detection (LoD) for the assay using inactivated virus was estimated to be 73 copies/mL (95% CI: 51-97 copies/mL). The LoD for infectious virus was estimated to be 1 plaque-forming unit/mL, and for RNA to be 232 copies/mL (95% CI 163-302 copies/mL). The assay correctly identified five different Ebola viruses, Yambuku-Mayinga, Makona-C07, Yambuku-Ecran, Gabon-Ilembe, and Kikwit-956210, and correctly excluded all non-EBOV isolates tested. The conditions used by Xpert® Ebola for inactivation of infectious virus reduced EBOV titer by ≥6 logs. CONCLUSION: In summary, we found the Xpert® Ebola Assay to have high analytical sensitivity and specificity for the detection of EBOV in whole blood. It offers ease of use, fast turnaround time, and remote monitoring. The test has an efficient viral inactivation protocol, fulfills inclusivity and exclusivity criteria, and has specimen stability characteristics consistent with the need for decentralized testing. The simplicity of the assay should enable testing in a wide variety of laboratory settings, including remote laboratories that are not capable of performing highly complex nucleic acid amplification tests, and during outbreaks where time to detection is critical.
Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/diagnóstico , Programas de Rastreamento/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Chlorocebus aethiops , Ebolavirus/fisiologia , Genes Virais/genética , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Interações Hospedeiro-Patógeno , Humanos , RNA Viral/sangue , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Células Vero , Inativação de VírusRESUMO
The PTCH1 tumor suppressor gene encodes a receptor for secreted hedgehog (HH) ligands and is important for proper proliferation, differentiation and patterning in almost every tissue and organ during embryogenesis. The PTCH1 protein works as a negative regulator of the HH-signaling pathway by repressing downstream signaling by the coreceptor smoothened (SMOH). Mutations in PTCH1 lead to constitutive expression of HH target genes and a relationship between mutated PTCH1 and the most common tumor form in the Western world, Basal Cell Carcinoma (BCC) has been clearly established. We here show that PTCH1 is transcriptionally regulated by three independent promoters generating transcripts with alternative first exons. We demonstrate that only one of two putative Gli-binding sites that were identified in the promoter region of PTCH1 is functional, and that the transactivating Gli proteins, GLI1, Gli2 and GLI3, bind and enhance transcription through this site. Moreover, a strong repression of both basal and induced PTCH1 transcription was observed following expression of a truncated version of GLI3. Most interestingly, the upstream components in the HH-signaling cascade, Sonic HH (SHH) and SMOH, solely operate through the functional Gli-binding site because mutation of the Gli-binding site resulted in the disappearance of the enhanced transcription induced by the Gli proteins, as well as by SHH or SMOH. This finding suggests that transcriptional activation of the PTCH1 gene mediated via the HH-signaling pathway is dependent on the single functional Gli-binding site.
Assuntos
Regulação da Expressão Gênica , Proteínas de Membrana/genética , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismo , Células 3T3 , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Genes Supressores de Tumor , Proteínas de Fluorescência Verde , Proteínas Hedgehog , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas Oncogênicas/genética , Receptores Patched , Receptor Patched-1 , Ligação Proteica , Receptores de Superfície Celular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Transfecção , Proteína GLI1 em Dedos de ZincoRESUMO
The Hedgehog signaling pathway regulates the development and function of numerous tissues and when mis-regulated causes tumorigenesis. To assess the role of a deregulated Hedgehog signaling pathway in the mammary gland we targeted the expression of the Hedgehog effector protein, GLI1, to mammary epithelial cells using a bigenic inducible system. A constitutively active Hedgehog signaling pathway resulted with 100% penetrance in an undifferentiated mammary lobuloalveolar network during pregnancy. GLI1-expressing transgenic females were unable to lactate and milk protein gene expression was essentially absent. The inability to lactate was permanent and independent of continued GLI1 transgene expression. An increased expression of the GLI1 response gene Snail coupled to reduced expression of E-cadherin and STAT5 in the transgenic mammary gland provides a likely molecular explanation, underlying the observed phenotypic changes. In addition, remodeling of the mammary gland after parturition was impaired and expression of GLI1 was associated with accumulation of cellular debris in the mammary ducts during involution, indicating a defect in the clearance of dead cells. Areas with highly proliferative epithelial cells were observed in mammary glands with induced expression of GLI1. Within such areas an increased frequency of cells expressing nuclear Cyclin D1 was observed. Taken together the data support the notion that correct regulation of Hedgehog signaling within the epithelial cell compartment is critical for pregnancy-induced mammary gland development and remodeling.