scholarly journals Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells

2021 ◽  
Author(s):  
Darrell R. Kapczynski ◽  
Ryan Sweeney ◽  
David L. Suarez ◽  
Erica Spackman ◽  
Mary Pantin-Jackwood
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Golden Hamster ◽  
In Vitro Model ◽  
Animal Species ◽  
Mdck Cells ◽  
Myotis Lucifugus ◽  
Host Susceptibility ◽  
Vitro Model

ABSTRACTThe SARS-CoV-2 (SC2) virus has caused a worldwide pandemic because of the virus’s ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SC2, there is no practical way to assess every species susceptibility to SC2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SC2, we expressed the ACE2 and/or transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that expression of both human ACE2 and TMPRSS2 genes is necessary to support SC2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SC2 in these cell lines were comparable to those observed in control Vero cells. To further test the model, we developed seven additional transgenic cell lines expressing the ACE2 and TMPRSS2 derived from Felis (cat), Equus (horse), Sus (pig), Capra (goat), Mesocricetus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SC2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SC2 and emerging variant viruses.IMPORTANCESARS-CoV-2 (SC2) is believed to have originated in animal species and jumped into humans where it has produced the greatest viral pandemic of our time. Identification of animal species susceptible to SC2 infection would provide information on potential zoonotic reservoirs, and transmission potential at the human-animal interface. Our work provides a model system to test the ability of the virus to replicate in an otherwise non-permissive cell line by transgenic insertion of the ACE2 and TMPRSS2 genes from human and other animal species. The results from our in vitro model positively correlate with animal infection studies enhancing the predicative capability of the model. Importantly, we demonstrate that both proteins are required for successful virus replication. These findings establish a framework to test other animal species for susceptibility to infection that may be critical zoonotic reservoirs for transmission, as well as to test variant viruses that arise over time.

The EOL-1 cell line as an in vitro model for the study of FIP1L1-PDGFRA–positive chronic eosinophilic leukemia

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2802-2805 ◽  
Author(s):  
Jan Cools ◽  
Hilmar Quentmeier ◽  
Brian J. P. Huntly ◽  
Peter Marynen ◽  
James D. Griffin ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
In Vitro Model ◽  
Kinase Inhibitors ◽  
Hypereosinophilic Syndrome ◽  
Leukemia Cell ◽  
Chronic Eosinophilic Leukemia ◽  
Leukemia Cell Lines ◽  
Vitro Model

Abstract We recently identified the chimeric kinase FIP1L1-platelet-derived growth factor receptor α (PDGFRα) as a cause of the hypereosinophilic syndrome and of chronic eosinophilic leukemia. To investigate the role of FIP1L1-PDGFRA in the pathogenesis of acute leukemia, we screened 87 leukemia cell lines for the presence of FIP1L1-PDGFRA. One cell line, EOL-1, expressed the FIP1L1-PDGFRA fusion. Three structurally divergent kinase inhibitors—imatinib (STI-571), PKC412, and SU5614—inhibited the growth of EOL-1 cells. These results indicate that the fusion of FIP1L1 to PDGFRA occurs rarely in leukemia cell lines, but they identify EOL-1 as an in vitro model for the study of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia and for the analysis of small molecule inhibitors of FIP1L1-PDGFRα. (Blood. 2004;103:2802-2805)

scholarly journals Development and Characterisation of an in Vitro Model of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4505-4505
Author(s):  
Thomas Lewis ◽  
Elisabeth Jane Walsby ◽  
Stephen Man ◽  
Christopher Fegan ◽  
Chris Pepper
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
In Vitro Model ◽  
Plastic Substrate ◽  
Osteosarcoma Cell ◽  
U937 Cells ◽  
Myeloma Cells ◽  
Vitro Model

Abstract Multiple myeloma is an incurable malignancy of terminally differentiated B-cells - also known as plasma cells. These malignant cells are extremely reliant on the bone marrow microenvironment for their growth and survival, as well as their acquired ability to resist therapeutic intervention. Consequently, maintaining primary myeloma cells in vitro remains a challenge. Patients suffering from this incurable disease often develop osteolytic lesions, due to an imbalance between osteoblasts and osteoclasts, which cause bone pain and a high frequency of fractures. This project aims to create a physiologically relevant in vitro model of myeloma that incorporates an osteoclast microenvironment. Osteoclasts normally work in concert with osteoblasts during bone tissue remodelling. In myeloma their activity predominates and is intrinsic to disease progression. It is now clear that osteoclasts also contribute to the survival of myeloma cells but the precise mechanism(s) for this remain unresolved. As a first step, we developed a model that can support and measure osteoclast function. We showed that the osteosarcoma cell line SAOS-2 was able to secrete a calcified matrix in a fashion similar to human osteoblasts. This was deposited on the plastic substrate following treatment with 300mM ascorbic acid and 10mM ß-glycerol phosphate for 28 days. Subsequently, the cells were removed from the mineralized plates and they were used to provide a base material to measure the resorption capacity of osteoclast-like cells and investigate how myeloma cells influence that activity. We next went on to develop and characterise an in vitro osteoclastic model using the myelo-monocytic U937 cell line. Treatment with 100nM PMA and 10nM calcitriol causes these cells to merge and form multi-nucleated (Figure 1A), TRAP positive (Figure 1B) and RANK positive cells. Culturing two different myeloma cell lines, H929 and RPMI-8226, in co-culture with the osteoclast-like cells for a period of 48 hours revealed two unique sub-populations of CD138bright and CD138dim myeloma cells. Phenotypic analysis of these distinct populations showed that they expressed similar levels of CD38 but the CD138dim cells showed a significant upregulation of CD69 (p≤0.05) in both cell lines as a result of co-culture with differentiated U937 osteoclast-like cells. This data indicates that osteoclast-like U937 cells can activate a subset of myeloma cells and may provide a means of sustaining primary myeloma cells in vitro. We are currently performing RNA-seq experiments to try to understand why only a subset of cells respond to this stimulus. We will then go on to establish whether primary myeloma cells derived from patients show similar responses when co-cultured under the same conditions. Figure 1: U937 cells have the capacity to form large multinucleated osteoclast-like cells that express tartrate resistant alkaline phosphatase (TRAP). A. Representative brightfield images coupled with images of DAPI staining following treatment with PMA and calcitriol reveals that U937 cells can become adherent and merge with one another to form large, multinucleated cells. B. Representative images of TRAP (purple) staining illustrate that these osteoclast-like U937 cells are also able to express TRAP following treatment. Disclosures Fegan: Acerta Pharma: Research Funding. Pepper:Cardiff University: Patents & Royalties: Telomere measurement patents.

The HepaRG cell line, a superior in vitro model to L-02, HepG2 and hiHeps cell lines for assessing drug-induced liver injury

2016 ◽  
Vol 32 (1) ◽  
pp. 37-59 ◽  
Author(s):  
Yu Wu ◽  
Xing-chao Geng ◽  
Ju-feng Wang ◽  
Yu-fa Miao ◽  
Yan-li Lu ◽  
...  
Keyword(s):  
Liver Injury ◽  
Cell Line ◽  
Cell Lines ◽  
In Vitro Model ◽  
Drug Induced ◽  
Heparg Cell ◽  
Drug Induced Liver Injury ◽  
Heparg Cell Line ◽  
Vitro Model

The Human Leukemic HL-60 Cell Line: An in Vitro Model for Cell Death Endpoint Identification

1996 ◽  
Vol 24 (4) ◽  
pp. 581-587
Author(s):  
Cristiana Zanetti ◽  
Arrnalaura Stammati ◽  
Orazio Sapora ◽  
Flavia Zucco
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
In Vitro Model ◽  
Leukemia Cell ◽  
Promyelocytic Leukemia ◽  
Leukemia Cell Line ◽  
Cell Type ◽  
Vitro Model ◽  
Promyelocytic Leukemia Cell Line

The aim of this study was to investigate the endpoints related to cell death, either necrosis or apoptosis, induced by four chemicals in the promyelocytic leukemia cell line, HL-60. Cell morphology, DNA fragmentation, cytofluorimetric analysis and oxygen consumption were used to classify the type of cell death observed. In our analysis, we found that not all the selected parameters reproduced the differences observed in the cell death caused by the four chemicals tested. As cell death is a very complex phenomenon, several factors should be taken into account (cell type, exposure time and chemical concentration), if chemicals are to be classified according to differences in the mechanisms more directly involved in cell death.

scholarly journals Adrenomedullin expression in a rat model of acute lung injury induced by hypoxia and LPS

2005 ◽  
Vol 288 (3) ◽  
pp. L536-L545 ◽  
Author(s):  
Jackeline Agorreta ◽  
Javier J. Zulueta ◽  
Luis M. Montuenga ◽  
Mercedes Garayoa
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Cell Lines ◽  
Rat Model ◽  
In Vitro Model ◽  
Rat Lung ◽  
Vitro Model

Adrenomedullin (ADM) is upregulated independently by hypoxia and LPS, two key factors in the pathogenesis of acute lung injury (ALI). This study evaluates the expression of ADM in ALI using experimental models combining both stimuli: an in vivo model of rats treated with LPS and acute normobaric hypoxia (9% O2) and an in vitro model of rat lung cell lines cultured with LPS and exposed to hypoxia (1% O2). ADM expression was analyzed by in situ hybridization, Northern blot, Western blot, and RIA analyses. In the rat lung, combination of hypoxia and LPS treatments overcomes ADM induction occurring after each treatment alone. With in situ techniques, the synergistic effect of both stimuli mainly correlates with ADM expression in inflammatory cells within blood vessels and, to a lesser extent, to cells in the lung parenchyma and bronchiolar epithelial cells. In the in vitro model, hypoxia and hypoxia + LPS treatments caused a similar strong induction of ADM expression and secretion in epithelial and endothelial cell lines. In alveolar macrophages, however, LPS-induced ADM expression and secretion were further increased by the concomitant exposure to hypoxia, thus paralleling the in vivo response. In conclusion, ADM expression is highly induced in a variety of key lung cell types in this rat model of ALI by combination of hypoxia and LPS, suggesting an essential role for this mediator in this syndrome.

scholarly journals Stable Transfection of the BovineNRAMP1 Gene into Murine RAW264.7 Cells: Effect onBrucella abortus Survival

2001 ◽  
Vol 69 (5) ◽  
pp. 3110-3119 ◽  
Author(s):  
Robert Barthel ◽  
Jianwei Feng ◽  
Jorge A. Piedrahita ◽  
David N. McMurray ◽  
Joe W. Templeton ◽  
...  
Keyword(s):  
Cell Lines ◽  
In Vitro Model ◽  
Regulatory Control ◽  
Natural Resistance ◽  
Ifn Γ ◽  
Flanking Region ◽  
Vitro Model ◽  
Raw264.7 Macrophage

ABSTRACT Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. BovineNRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovine NRAMP1 as stable transgenes under the regulatory control of the bovineNRAMP1 promoter in the murine RAW264.7 macrophage cell line (Bcg s ) to analyze the regulation of the NRAMP1 gene and its role in macrophage function. We demonstrated that the 5′-flanking region of bovineNRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3′ untranslated region critically affects the expression of bovineNRAMP1 and the control of in vitro replication ofBrucella abortus but not Salmonella enterica serovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-γ)- and IFN-γ–lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h.

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