scholarly journals Low Dose BCG Infection as a Model for Macrophage Activation Maintaining Cell Viability

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Leslie Chávez-Galán ◽  
Dominique Vesin ◽  
Denis Martinvalet ◽  
Irene Garcia
Keyword(s):  
Cell Activation ◽  
In Vitro Model ◽  
Low Dose ◽  
Mycobacterial Infection ◽  
Dependent Manner ◽  
Research Fields ◽  
Current Vaccine ◽  
Vitro Model

Mycobacterium bovisBCG, the current vaccine against tuberculosis, is ingested by macrophages promoting the development of effector functions including cell death and microbicidal mechanisms. Despite accumulating reports onM. tuberculosis, mechanisms of BCG/macrophage interaction remain relatively undefined. In vivo, few bacilli are sufficient to establish a mycobacterial infection; however, in vitro studies systematically use high mycobacterium doses. In this study, we analyze macrophage/BCG interactions and microenvironment upon infection with low BCG doses and propose an in vitro model to study cell activation without affecting viability. We show that RAW macrophages infected with BCG at MOI 1 activated higher and sustained levels of proinflammatory cytokines and transcription factors while MOI 0.1 was more efficient for early stimulation of IL-1β, MCP-1, and KC. Both BCG infection doses induced iNOS and NO in a dose-dependent manner and maintained nuclear and mitochondrial structures. Microenvironment generated by MOI 1 induced macrophage proliferation but not MOI 0.1 infection. In conclusion, BCG infection at low dose is an efficient in vitro model to study macrophage/BCG interactions that maintains macrophage viability and mitochondrial structures. This represents a novel model that can be applied to BCG research fields including mycobacterial infections, cancer immunotherapy, and prevention of autoimmunity and allergies.

scholarly journals Generation of a Novel In Vitro Model to Study Endothelial Dysfunction from Atherothrombotic Specimens

2021 ◽  
Author(s):  
Susan Gallogly ◽  
Takeshi Fujisawa ◽  
John D. Hung ◽  
Mairi Brittan ◽  
Elizabeth M. Skinner ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
In Vitro Model ◽  
Umbilical Vein ◽  
Coronary Syndrome ◽  
Coronary Endothelial Cells ◽  
Vitro Model ◽  
Umbilical Vein Endothelial Cells

Abstract Purpose Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction. Methods In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo. Results CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo. Conclusions CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.

scholarly journals 3D Environment Is Required In Vitro to Demonstrate Altered Bone Metabolism Characteristic for Type 2 Diabetics

2021 ◽  
Vol 22 (6) ◽  
pp. 2925
Author(s):  
Victor Häussling ◽  
Romina H Aspera-Werz ◽  
Helen Rinderknecht ◽  
Fabian Springer ◽  
Christian Arnscheidt ◽  
...  
Keyword(s):  
Bone Metabolism ◽  
In Vitro Model ◽  
Bone Diseases ◽  
3D Environment ◽  
Type 2 Diabetics ◽  
Mineralized Matrix ◽  
Vitro Model

A large British study, with almost 3000 patients, identified diabetes as main risk factor for delayed and nonunion fracture healing, the treatment of which causes large costs for the health system. In the past years, much progress has been made to treat common complications in diabetics. However, there is still a lack of advanced strategies to treat diabetic bone diseases. To develop such therapeutic strategies, mechanisms leading to massive bone alterations in diabetics have to be well understood. We herein describe an in vitro model displaying bone metabolism frequently observed in diabetics. The model is based on osteoblastic SaOS-2 cells, which in direct coculture, stimulate THP-1 cells to form osteoclasts. While in conventional 2D cocultures formation of mineralized matrix is decreased under pre-/diabetic conditions, formation of mineralized matrix is increased in 3D cocultures. Furthermore, we demonstrate a matrix stability of the 3D carrier that is decreased under pre-/diabetic conditions, resembling the in vivo situation in type 2 diabetics. In summary, our results show that a 3D environment is required in this in vitro model to mimic alterations in bone metabolism characteristic for pre-/diabetes. The ability to measure both osteoblast and osteoclast function, and their effect on mineralization and stability of the 3D carrier offers the possibility to use this model also for other purposes, e.g., drug screenings.

scholarly journals Exposure of ichnovirus particles to digitonin leads to enhanced infectivity and induces fusion from without in an in vitro model system

2007 ◽  
Vol 88 (11) ◽  
pp. 2977-2984 ◽  
Author(s):  
Don Stoltz ◽  
Renée Lapointe ◽  
Andrea Makkay ◽  
Michel Cusson
Keyword(s):  
Outer Membrane ◽  
In Vitro Model ◽  
Model System ◽  
Host Cells ◽  
Fusion Event ◽  
Susceptible Host ◽  
In Vitro Model System ◽  
Vitro Model

Unlike most viruses, the mature ichnovirus particle possesses two unit membrane envelopes. Following loss of the outer membrane in vivo, nucleocapsids are believed to gain entry into the cytosol via a membrane fusion event involving the inner membrane and the plasma membrane of susceptible host cells; accordingly, experimentally induced damage to the outer membrane might be expected to increase infectivity. Here, in an attempt to develop an in vitro model system for studying ichnovirus infection, we show that digitonin-induced disruption of the virion outer membrane not only increases infectivity, but also uncovers an activity not previously associated with any polydnavirus: fusion from without.

Uptake and Retention of Hematoporphyrin Derivative in an in Vivo/in Vitro Model of Cerebral Glioma

Neurosurgery ◽  
1985 ◽  
Vol 17 (6) ◽  
pp. 883-890 ◽  
Author(s):  
Andrew H. Kaye ◽  
George Morstyn ◽  
Robert G. Ashcroft
Keyword(s):  
In Vitro Model ◽  
Hematoporphyrin Derivative ◽  
Cerebral Glioma ◽  
Vitro Model

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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