Thymol disrupts the membrane integrity of Salmonella ser. typhimurium in vitro and recovers infected macrophages from oxidative stress in an ex vivo model

Studies from animal models suggest that myocardial mitochondrial damage contributes to cardiac dysfunction after burn injury. In this report, we used an ex vivo model of primary cardiomyocyte culture to investigate the mechanisms of burn-induced mitochondrial impairment. Briefly, blood serum was collected from Sprague-Dawley (SD) rats subjected to 40% total body surface area burn and added (10% vol/vol) to primary cardiomyocytes prepared from SD rats. The effect of the burn serum on mitochondrial function and membrane integrity in the myocytes was analyzed. Exposure of myocytes to burn serum doubled the mitochondrial membrane damage measured by two independent assays. This treatment also significantly elevated mitochondrial oxidative stress, indicated by a more than 30% increase in lipid oxidation. Downregulation of mitochondrial antioxidant defense was also evident since the activities of the antioxidant enzymes superoxide dismutase and glutathione peroxidase were reduced by about 30% and 50%, respectively. Burn serum also induced deficiency of mitochondrial metabolism, indicated by a 30% decrease in the activity of cytochrome c oxidase. These mitochondrial dysfunctions appear to be generated by oxidative stress because burn serum induced a significant increase of mitochondrial oxygen species (mtROS) in cardiomyocytes, and pretreatment of cardiomyocytes with the antioxidant N-acetyl-cysteine prevented the mitochondrial damages induced by burn serum. Remarkably, the increase in mtROS was abolished by an antibody-mediated blockade of CD14. Furthermore, burn injury-induced mitochondrial damage in cardiomyocytes was prevented in CD14 knockout mice. Taken together, these data suggested that burn injury produces CD14-dependent mitochondrial damage via oxidative stress in myocardium.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653754 ◽

1995 ◽

Vol 73 (02) ◽

pp. 219-222 ◽

Cited By ~ 4

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.

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

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666190619111118 ◽

2019 ◽

Vol 20 (11) ◽

pp. 920-933 ◽

Cited By ~ 3

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.

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

Cells ◽

10.3390/cells10040899 ◽

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.

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

Medical Mycology ◽

10.1093/mmy/myaa104 ◽

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.

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Ex vivo model for studying polymicrobial biofilm formation in root canals

Universitas Scientiarum ◽

10.11144/javeriana.sc22-1.evmf ◽

2017 ◽

Vol 22 (1) ◽

pp. 31 ◽

Cited By ~ 1

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 in vitro biofilm optimal formation conditions for microorganisms such as Enterococcus faecalis, Staphylococcus aureus and Candida albicans. 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. In vitro biofilm maturation was confirmed after 30 days of incubation under a CO2 atmosphere for both bacteria and yeast.

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Heme oxygenase-1 induction by hemin prevents oxidative stress-induced acute cholestasis in the rat

Clinical Science ◽

10.1042/cs20180675 ◽

2019 ◽

Vol 133 (1) ◽

pp. 117-134 ◽

Cited By ~ 7

Author(s):

Pamela L. Martín ◽

Paula Ceccatto ◽

María V. Razori ◽

Daniel E.A. Francés ◽

Sandra M.M. Arriaga ◽

...

Keyword(s):

Oxidative Stress ◽

Heme Oxygenase ◽

Bile Flow ◽

Driving Forces ◽

Membrane Lipids ◽

Ex Vivo ◽

Heme Oxygenase 1 ◽

Canalicular Transporters

Abstract We previously demonstrated in in vitro and ex vivo models that physiological concentrations of unconjugated bilirubin (BR) prevent oxidative stress (OS)-induced hepatocanalicular dysfunction and cholestasis. Here, we aimed to ascertain, in the whole rat, whether a similar cholestatic OS injury can be counteracted by heme oxygenase-1 (HO-1) induction that consequently elevates endogenous BR levels. This was achieved through the administration of hemin, an inducer of HO-1, the rate-limiting step in BR generation. We found that BR peaked between 6 and 8 h after hemin administration. During this time period, HO-1 induction fully prevented the pro-oxidant tert-butylhydroperoxide (tBuOOH)-induced drop in bile flow, and in the biliary excretion of bile salts and glutathione, the two main driving forces of bile flow; this was associated with preservation of the membrane localization of their respective canalicular transporters, bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2), which are otherwise endocytosed by OS. HO-1 induction counteracted the oxidation of intracellular proteins and membrane lipids induced by tBuOOH, and fully prevented the increase in the oxidized-to-total glutathione (GSHt) ratio, a sensitive parameter of hepatocellular OS. Compensatory elevations of the activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) were also prevented. We conclude that in vivo HO-1 induction protects the liver from acute oxidative injury, thus preventing consequent cholestasis. This reveals an important role for the induction of HO-1 and the consequently elevated levels of BR in preserving biliary secretory function under OS conditions, thus representing a novel therapeutic tool to limit the cholestatic injury that bears an oxidative background.

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

10.20944/preprints202104.0240.v1 ◽

2021 ◽

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 that is the leading cause of death for both mothers and babies in pregnancy. The pathophysiology remains poorly understood due to 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, that 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.

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The nanocomposite fullerol reduces oxidative stress, pulmonary injury, and mortality in a rat model of acute lung injury

10.22541/au.163252259.97677391/v1 ◽

2021 ◽

Author(s):

Rosária Aires ◽

Ildernandes Vieira-Alves ◽

Leda Maria Coimbra-Campos ◽

Marina Ladeira ◽

Teresa Socarras ◽

...

Keyword(s):

Oxidative Stress ◽

Acute Lung Injury ◽

Lung Injury ◽

High Mortality ◽

Therapeutic Potential ◽

Ex Vivo ◽

Mortality Rates ◽

Kaplan Meier ◽

Pulmonary Damage

BACKGROUND AND PURPOSE Acute lung injury (ALI) is a critical disorder that has high mortality rates, and pharmacological therapies are so far ineffective. The pathophysiology of ALI involves pulmonary oxidative stress and inflammatory response. Fullerol is a carbon nanocomposite that possesses antioxidant and anti-inflammatory properties. Here, we evaluated the therapeutic potential of fullerol and its mechanisms in a model of paraquat-induced ALI. EXPERIMENTAL APPROACH Rats were divided into ALI (paraquat alone), fullerol (paraquat plus fullerol), and control groups. Survival curves were estimated using the Kaplan-Meier method. The myeloperoxidase assay, ELISA, and hematoxylin and eosin staining were used to determine neutrophils infiltration, cytokines production, and histopathological parameters in lung samples, respectively. The antioxidant effect of fullerol was evaluated in vitro and ex vivo. KEY RESULTS Fullerol (0.01 to 0.3 mg/kg) markedly reduced the severe lung injury and high mortality rates observed in ALI rats. Moreover, fullerol (0.03 mg/kg) inhibited the reactive oxygen species formation and lipid peroxidation seen in lungs from ALI rats, and exhibited a potent concentration-dependent (10 to 10 mg/ml) in vitro antioxidant activity. Importantly, fullerol (0.03 mg/kg) inhibited neutrophils accumulation in bronchoalveolar lavage and lungs, and the increase in pulmonary levels of TNF-α, IL-1β, IL-6, and CINC-1 in ALI rats. CONCLUSIONS AND IMPLICATIONS Fullerol treatment was effective in reducing pulmonary damage and ALI-induced mortality, highlighting its therapeutic potential in an ALI condition. Searching for new pharmacological therapies to treat ALI may be desirable especially in view of the new coronavirus disease 2019 that currently plagues the world.

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Newly developed dual topoisomerase inhibitor P8-D6 is highly active in ovarian cancer

Therapeutic Advances in Medical Oncology ◽

10.1177/17588359211059896 ◽

2021 ◽

Vol 13 ◽

pp. 175883592110598

Author(s):

Inken Flörkemeier ◽

Tamara N. Steinhauer ◽

Nina Hedemann ◽

Magnus Ölander ◽

Per Artursson ◽

...

Keyword(s):

Ovarian Cancer ◽

Topoisomerase I ◽

Ex Vivo ◽

Chemotherapy Resistance ◽

Membrane Integrity ◽

Three Dimensional ◽

New Drugs ◽

Highly Active ◽

Gynaecologic Neoplasms

Background: Ovarian cancer (OvCa) constitutes a rare and highly aggressive malignancy and is one of the most lethal of all gynaecologic neoplasms. Due to chemotherapy resistance and treatment limitations because of side effects, OvCa is still not sufficiently treatable. Hence, new drugs for OvCa therapy such as P8-D6 with promising antitumour properties have a high clinical need. The benzo[ c]phenanthridine P8-D6 is an effective inductor of apoptosis by acting as a dual topoisomerase I/II inhibitor. Methods: In the present study, the effectiveness of P8-D6 on OvCa was investigated in vitro. In various OvCa cell lines and ex vivo primary cells, the apoptosis induction compared with standard therapeutic agents was determined in two-dimensional monolayers. Expanded by three-dimensional and co-culture, the P8-D6 treated cells were examined for changes in cytotoxicity, apoptosis rate and membrane integrity via scanning electron microscopy (SEM). Likewise, the effects of P8-D6 on non-cancer human ovarian surface epithelial cells and primary human hepatocytes were determined. Results: This study shows a significant P8-D6-induced increase in apoptosis and cytotoxicity in OvCa cells which surpasses the efficacy of well-established drugs like cisplatin or the topoisomerase inhibitors etoposide and topotecan. Non-cancer cells were affected only slightly by P8-D6. Moreover, no hepatotoxic effect in in vitro studies was detected. Conclusion: P8-D6 is a strong and rapid inductor of apoptosis and might be a novel treatment option for OvCa therapy.

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