Corticosteroids alter alveolar macrophage control of Lichtheimia corymbifera spores in an ex vivo mouse model

2020 ◽  
Author(s):  
Kévin Brunet ◽  
François Arrivé ◽  
Jean-Philippe Martellosio ◽  
Isabelle Lamarche ◽  
Sandrine Marchand ◽  
...  
Keyword(s):  
Mouse Model ◽  
Alveolar Macrophage ◽  
Oxidative Burst ◽  
Ex Vivo ◽  
Fungal Growth ◽  
Invasive Infection ◽  
Ex Vivo Model ◽  
Lichtheimia Corymbifera ◽  
The Impact

Abstract Alveolar macrophages (AM) are the first-line lung defense against Mucorales in pulmonary mucormycosis. Since corticosteroid use is a known risk factor for mucormycosis, the aim of this study was to describe the role of corticosteroids on AM capacities to control Lichtheimia corymbifera spore growth using a new ex vivo model. An in vivo mouse model was developed to determine the acetate cortisone dose able to trigger pulmonary invasive infection. Then, in the ex vivo model, male BALB/c mice were pretreated with the corticosteroid regimen triggering invasive infection, before AM collection through bronchoalveolar lavage. AMs from corticosteroid-treated mice and untreated control AMs were then exposed to L. corymbifera spores in vitro (ratio 1:5). AM control of fungal growth, adherence/phagocytosis, and oxidative burst were assessed using optical densities by spectrophotometer, flow cytometry, and 2', 7'-dichlorofluoresceine diacetate fluorescence, respectively. Cortisone acetate at 500 mg/kg, at D-3 and at D0, led to pulmonary invasive infection at D3. Co-incubated spores and AMs from corticosteroid-treated mice had significantly higher absorbance (fungal growth) than co-incubated spores and control AMs, at 24 h (P = .025), 36 h (P = .004), and 48 h (P = .001). Colocalization of spores with AMs from corticosteroid-treated mice was significantly lower than for control AMs (7.6 ± 1.9% vs 22.3 ± 5.8%; P = .003), reflecting spore adherence and phagocytosis inhibition. Finally, oxidative burst was significantly increased when control AMs were incubated with spores (P = 0.029), while corticosteroids hampered oxidative burst from treated AMs (P = 0.321). Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease in our ex vivo model. Lay Summary The aim of this study was to describe the impact of corticosteroids on alveolar macrophage (AM) capacities to control Mucorales growth in a new murine ex vivo model. Corticosteroids enhanced fungal growth of L. corymbifera through AM phagocytosis inhibition and burst oxidative decrease.

Effects of Two Different Doses of Hirudin on APTT, Determined with Eight Different Reagents

1995 ◽  
Vol 73 (02) ◽  
pp. 219-222 ◽  
Author(s):  
Manuel Monreal ◽  
Luis Monreal ◽  
Rafael Ruiz de Gopegui ◽  
Yvonne Espada ◽  
Ana Maria Angles ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Anticoagulant Activity ◽  
Subcutaneous Administration ◽  
In Vitro Study ◽  
Ex Vivo Model ◽  
Suitable Candidate ◽  
Significant Prolongation ◽  
High Doses ◽  
Different Doses

SummaryThe APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study.Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica- based reagents.In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica- containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.

Anti-Inflammatory Effects of Novel Standardized Platelet Rich Plasma Releasates on Knee Osteoarthritic Chondrocytes and Cartilage in vitro

2019 ◽  
Vol 20 (11) ◽  
pp. 920-933 ◽  
Author(s):  
Lucía Gato-Calvo ◽  
Tamara Hermida-Gómez ◽  
Cristina R. Romero ◽  
Elena F. Burguera ◽  
Francisco J. Blanco
Keyword(s):  
Gene Expression ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Cartilage Explants ◽  
Ex Vivo Model ◽  
Chondrocyte Viability ◽  
Platelet Concentration ◽  
The Absolute ◽  
Anti Inflammatory

Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

scholarly journals Cell–cell coupling and DNA methylation abnormal phenotypes in the after-hours mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Federico Tinarelli ◽  
Elena Ivanova ◽  
Ilaria Colombi ◽  
Erica Barini ◽  
Edoardo Balzani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Neuronal Networks ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Neuronal Cultures ◽  
Functional Impairments ◽  
After Hours ◽  
The Impact

Abstract Background DNA methylation has emerged as an important epigenetic regulator of brain processes, including circadian rhythms. However, how DNA methylation intervenes between environmental signals, such as light entrainment, and the transcriptional and translational molecular mechanisms of the cellular clock is currently unknown. Here, we studied the after-hours mice, which have a point mutation in the Fbxl3 gene and a lengthened circadian period. Methods In this study, we used a combination of in vivo, ex vivo and in vitro approaches. We measured retinal responses in Afh animals and we have run reduced representation bisulphite sequencing (RRBS), pyrosequencing and gene expression analysis in a variety of brain tissues ex vivo. In vitro, we used primary neuronal cultures combined to micro electrode array (MEA) technology and gene expression. Results We observed functional impairments in mutant neuronal networks, and a reduction in the retinal responses to light-dependent stimuli. We detected abnormalities in the expression of photoreceptive melanopsin (OPN4). Furthermore, we identified alterations in the DNA methylation pathways throughout the retinohypothalamic tract terminals and links between the transcription factor Rev-Erbα and Fbxl3. Conclusions The results of this study, primarily represent a contribution towards an understanding of electrophysiological and molecular phenotypic responses to external stimuli in the Afh model. Moreover, as DNA methylation has recently emerged as a new regulator of neuronal networks with important consequences for circadian behaviour, we discuss the impact of the Afh mutation on the epigenetic landscape of circadian biology.

scholarly journals Indirect cholinergic activation slows down pancreatic cancer growth and tumor-associated inflammation

2020 ◽  
Vol 39 (1) ◽  
Author(s):  
Paulo L. Pfitzinger ◽  
Laura Fangmann ◽  
Kun Wang ◽  
Elke Demir ◽  
Engin Gürlevik ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Mouse Model ◽  
Tumor Stage ◽  
Ache Inhibitors ◽  
Impact On Survival ◽  
The Impact ◽  
Ache Expression ◽  
Cholinergic Activation

Abstract Background Nerve-cancer interactions are increasingly recognized to be of paramount importance for the emergence and progression of pancreatic cancer (PCa). Here, we investigated the role of indirect cholinergic activation on PCa progression through inhibition of acetylcholinesterase (AChE) via clinically available AChE-inhibitors, i.e. physostigmine and pyridostigmine. Methods We applied immunohistochemistry, immunoblotting, MTT-viability, invasion, flow-cytometric-cell-cycle-assays, phospho-kinase arrays, multiplex ELISA and xenografted mice to assess the impact of AChE inhibition on PCa cell growth and invasiveness, and tumor-associated inflammation. Survival analyses were performed in a novel genetically-induced, surgically-resectable mouse model of PCa under adjuvant treatment with gemcitabine+/−physostigmine/pyridostigmine (n = 30 mice). Human PCa specimens (n = 39) were analyzed for the impact of cancer AChE expression on tumor stage and survival. Results We discovered a strong expression of AChE in cancer cells of human PCa specimens. Inhibition of this cancer-cell-intrinsic AChE via pyridostigmine and physostigmine, or administration of acetylcholine (ACh), diminished PCa cell viability and invasion in vitro and in vivo via suppression of pERK signaling, and reduced tumor-associated macrophage (TAM) infiltration and serum pro-inflammatory cytokine levels. In the novel genetically-induced, surgically-resectable PCa mouse model, adjuvant co-therapy with AChE blockers had no impact on survival. Accordingly, survival of resected PCa patients did not differ based on tumor AChE expression levels. Patients with higher-stage PCa also exhibited loss of the ACh-synthesizing enzyme, choline-acetyltransferase (ChAT), in their nerves. Conclusion For future clinical trials of PCa, direct cholinergic stimulation of the muscarinic signaling, rather than indirect activation via AChE blockade, may be a more effective strategy.

Precision cut lung slices: an ex vivo model for assessing the impact of immunomodulatory therapeutics on lung immune responses

2021 ◽  
Author(s):  
Guanghui Liu ◽  
Linnea Särén ◽  
Helena Douglasson ◽  
Xiao-Hong Zhou ◽  
Per M. Åberg ◽  
...  
Keyword(s):  
Immune Responses ◽  
Ex Vivo ◽  
Ex Vivo Model ◽  
Precision Cut Lung Slices ◽  
The Impact

scholarly journals Experimental Models to Study COVID-19 Effect in Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 91
Author(s):  
Rishi Man Chugh ◽  
Payel Bhanja ◽  
Andrew Norris ◽  
Subhrajit Saha
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Drug Efficacy ◽  
Cell Loss ◽  
Experimental Models ◽  
Model Systems ◽  
Virus Infectivity ◽  
Ex Vivo Model ◽  
Global Pandemic ◽  
The Impact

The new strain of coronavirus (severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)) emerged in 2019 and hence is often referred to as coronavirus disease 2019 (COVID-19). This disease causes hypoxic respiratory failure and acute respiratory distress syndrome (ARDS), and is considered as the cause of a global pandemic. Very limited reports in addition to ex vivo model systems are available to understand the mechanism of action of this virus, which can be used for testing of any drug efficacy against virus infectivity. COVID-19 induces tissue stem cell loss, resulting inhibition of epithelial repair followed by inflammatory fibrotic consequences. Development of clinically relevant models is important to examine the impact of the COVID-19 virus in tissue stem cells among different organs. In this review, we discuss ex vivo experimental models available to study the effect of COVID-19 on tissue stem cells.

scholarly journals Considerations to Model Heart Disease in Women with Preeclampsia and Cardiovascular Disease

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 899
Author(s):  
Clara Liu Chung Ming ◽  
Kimberly Sesperez ◽  
Eitan Ben-Sefer ◽  
David Arpon ◽  
Kristine McGrath ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiovascular Disease ◽  
Ex Vivo ◽  
Myocardial Damage ◽  
Cardiovascular Disorder ◽  
Model Systems ◽  
Ex Vivo Model ◽  
Disease Manifestation

Preeclampsia is a multifactorial cardiovascular disorder diagnosed after 20 weeks of gestation, and is the leading cause of death for both mothers and babies in pregnancy. The pathophysiology remains poorly understood due to the variability and unpredictability of disease manifestation when studied in animal models. After preeclampsia, both mothers and offspring have a higher risk of cardiovascular disease (CVD), including myocardial infarction or heart attack and heart failure (HF). Myocardial infarction is an acute myocardial damage that can be treated through reperfusion; however, this therapeutic approach leads to ischemic/reperfusion injury (IRI), often leading to HF. In this review, we compared the current in vivo, in vitro and ex vivo model systems used to study preeclampsia, IRI and HF. Future studies aiming at evaluating CVD in preeclampsia patients could benefit from novel models that better mimic the complex scenario described in this article.

Abstract 16485: The Nitroxyl Donor 1-Nitrosocyclohexyl Acetate Limits Cardiac Superoxide Upregulation and Diastolic Dysfunction in a Mouse Model of Diabetic Cardiomyopathy

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Rebecca H Ritchie ◽  
Nga Cao ◽  
Yung George Wong ◽  
Sarah Rosli ◽  
Helen Kiriazis ◽  
...  
Keyword(s):  
Heart Failure ◽  
Mouse Model ◽  
Diastolic Dysfunction ◽  
Diabetic Cardiomyopathy ◽  
Ex Vivo ◽  
Systolic Function ◽  
Left Ventricular ◽  
Cardiomyocyte Hypertrophy ◽  
Lv Diastolic Dysfunction ◽  
The Impact

Nitroxyl (HNO), a redox congener of NO•, is a novel regulator of cardiovascular function combining vasodilator and positive inotropic properties. Our previous studies have demonstrated these properties occur concomitantly in the intact heart; HNO moreover also exhibits antihypertrophic and superoxide-suppressing actions. HNO donors may thus offer favorable actions in heart failure. The impact of chronic HNO donor administration has however yet to be reported in this context. We tested the hypothesis that the HNO donor 1-nitrosocyclohexyl acetate (1-NCA) limits cardiomyocyte hypertrophy and left ventricular (LV) diastolic dysfunction in a mouse model of diabetic cardiomyopathy in vivo. Male 6 week-old FVB/N mice received either streptozotocin (55 mg/kg/day i.p. for 5 days, n=17), to induce type 1 diabetes, or citrate vehicle (n=16). After 4 weeks of hyperglycemia, mice were allocated to 1-NCA therapy (83mg/kg/day i.p.) or vehicle, and followed for a further 4 weeks. As shown in the table, blood glucose was unaffected by 1-NCA. LV diastolic dysfunction was evident in diabetic mice, measured as echocardiography-derived A wave velocity, deceleration time and E:A ratio; LV systolic function was preserved. Diabetes-induced diastolic dysfunction was accompanied by increased LV cardiomyocyte size, hypertrophic and pro-fibrotic gene expression, and upregulation of LV superoxide. These characteristics of diabetic cardiomyopathy were largely prevented by 1-NCA treatment. Selectivity of 1-NCA as a donor of HNO versus NO• was demonstrated by the sensitivity of the coronary vasodilation response of 1-NCA to the HNO scavenger L-cysteine (4mM), but not to the NO• scavenger hydroxocobalamin (50μM), in the normal rat heart ex vivo (n=3-7). Collectively, our studies provide the first evidence that HNO donors may represent a promising new strategy for the treatment of diabetic cardiomyopathy, and implies their therapeutic efficacy in settings of chronic heart failure.

scholarly journals Ex vivo model for studying polymicrobial biofilm formation in root canals

2017 ◽  
Vol 22 (1) ◽  
pp. 31 ◽  
Author(s):  
Hugo Díez Ortega ◽  
Francisco Correa Toral ◽  
Leylin Delgado Hernández ◽  
Carolina Echavarría González ◽  
Fátima Serna Varona ◽  
...  
Keyword(s):  
Root Canal ◽  
Ex Vivo ◽  
Ex Vivo Model ◽  
Root Canals ◽  
Gram Staining ◽  
Natural Teeth ◽  
Formation Conditions ◽  
Root Canal System ◽  
Co2 Atmosphere

Endodontic disease has mainly a microbial origin. It is caused by biofilms capable of attaching and surviving in the root canal. Therefore, it is important to study the conditions in which those biofilms grow, develop and colonize the root canal system. However, few studies have used natural teeth as models, which would take into account the root canal anatomical complexity and simulate the clinical reality. In this study, we used human premolar root canals to standardize <em>in vitro</em> biofilm optimal formation conditions for microorganisms such as <em>Enterococcus faecalis</em>, <em>Staphylococcus aureus</em> and <em>Candida albicans</em>. 128 lower premolars underwent canal preparation using K-type files, and were treated with 5.25% sodium hypochlorite and EDTA. Samples were inoculated with microorganisms and incubated for 15, 30, 45, and 60 days under anaerobiosis (CO2 atmosphere) and aerobiosis. Microorganism presence was confirmed by Gram staining, cell culture, and electron microscopy. Exopolysaccharide matrix and microorganism aggregation were observed following 15 days of incubation. Bacterial growth towards the apical third of the root canal and biofilm maturation was detected after 30 days. CO2 atmosphere favored microbial growth the most. <em>In vitro</em> biofilm maturation was confirmed after 30 days of incubation under a CO2 atmosphere for both bacteria and yeast.

scholarly journals A New Ex Vivo Model to Evaluate the Hair Protective Effect of a Biomimetic Exopolysaccharide against Water Pollution

Cosmetics ◽  
2020 ◽  
Vol 7 (4) ◽  
pp. 78
Author(s):  
Claire Tubia ◽  
Alfonso Fernández-Botello ◽  
Jan Dupont ◽  
Eni Gómez ◽  
Jérôme Desroches ◽  
...  
Keyword(s):  
Water Pollution ◽  
Heavy Metals ◽  
Heavy Metal ◽  
Adverse Effects ◽  
Inductively Coupled Plasma ◽  
Ex Vivo ◽  
Polluted Water ◽  
Ex Vivo Model ◽  
Fiber Damage ◽  
The Impact

As an external appendage, hair is exposed to multiple stresses of different origins such as particles and gases in air, or heavy metals and chemicals in water. So far, little research has addressed the impact of water pollution on hair. The present study describes a new ex vivo model that allowed us to document the adverse effects of water pollutants on the structure of hair proteins, as well as the protective potential of active cosmetic ingredients derived from a biomimetic exopolysaccharide (EPS). The impact of water pollution was evaluated on hair from a Caucasian donor repeatedly immersed in heavy metal-containing water. Heavy metal retention in and on hair was then quantified using Inductively Coupled Plasma Spectrometry (ICP/MS). The adverse effects of heavy metals on the internal structure of hair and its prevention by the EPS were assessed through measurement of keratin birefringence. Notably, the method allows the monitoring of the organization of keratin fibers and therefore the initial change on it in order to modulate the global damage in the hair. Results revealed an increasing amount of lead, cadmium and copper, following multiple exposures to polluted water. In parallel, the structure of keratin was also altered with exposures. However, heavy metal-induced keratin fiber damage could be prevented in the presence of the tested EPS, avoiding more drastic hair problems, such as lack of shine, or decrease in strength, due to damage accumulation.

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