In vitro Modeling of Chicken Cecal Microbiota Ecology and Metabolism Using the PolyFermS Platform

Continuous in vitro fermentation models provide a useful tool for a fast, reproducible, and direct assessment of treatment-related changes in microbiota metabolism and composition independent of the host. In this study, we used the PolyFermS model to mimic the conditions of the chicken cecum and evaluated three nutritive media for in vitro modeling of the chicken cecal microbiota ecology and metabolism. We observed that our model inoculated with immobilized cecal microbiota and fed with a modified Viande Levure medium (mVL-3) reached a high bacterial cell density of up to approximately 10.5 log cells per mL and stable microbiota composition, akin to the host, during 82 days of continuous operation. Relevant bacterial functional groups containing primary fibrolytic (Bacteroides, Bifidobacteriaceae, Ruminococcaceae), glycolytic (Enterococcus), mucolytic (Bacteroides), proteolytic (Bacteroides), and secondary acetate-utilizing butyrate-producing and propionate-producing (Lachnospiraceae) taxa were preserved in vitro. Besides, conserved metabolic and functional Kyoto Encyclopedia of Genes and Genomes pathways were observed between in vitro microbiota and cecal inoculum microbiota as predicted by functional metagenomics analysis. Furthermore, we demonstrated that the continuous inoculation provided by the inoculum reactor generated reproducible metabolic profiles in second-stage reactors comparable to the chicken cecum, allowing for the simultaneous investigation and direct comparison of different treatments with a control. In conclusion, we showed that PolyFermS is a suitable model for mimicking chicken cecal microbiota fermentation allowing ethical and ex vivo screening of environmental factors, such as dietary additives, on chicken cecal fermentation. We report here for the first time a fermentation medium (mVL-3) that closely mimics the substrate conditions in the chicken cecum and supports the growth and metabolic activity of the cecal bacterial akin to the host. Our PolyFermS chicken cecum model is a useful tool to study microbiota functionality and structure ex vivo.

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Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

Circulation Research ◽

10.1161/res.109.suppl_1.ap283 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Allen M Andres ◽

Chengqun Huang ◽

Eric P Ratliff ◽

Genaro Hernandez ◽

Pamela Lee ◽

...

Keyword(s):

Ex Vivo ◽

Wild Type ◽

Simulated Ischemia ◽

Selective Targeting ◽

Cardioprotective Effects ◽

Mitochondrial Turnover ◽

First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

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Candida albicans forms biofilms on the vaginal mucosa

Microbiology ◽

10.1099/mic.0.039354-0 ◽

2010 ◽

Vol 156 (12) ◽

pp. 3635-3644 ◽

Cited By ~ 168

Author(s):

M. M. Harriott ◽

E. A. Lilly ◽

T. E. Rodriguez ◽

P. L. Fidel ◽

M. C. Noverr

Keyword(s):

Biofilm Formation ◽

Ex Vivo ◽

Microscopic Analysis ◽

Vaginal Mucosa ◽

First Time ◽

Established Role ◽

Type Strains

Current understanding of resistance and susceptibility to vulvovaginal candidiasis challenges existing paradigms of host defence against fungal infection. While abiotic biofilm formation has a clearly established role during systemic Candida infections, it is not known whether C. albicans forms biofilms on the vaginal mucosa and the possible role of biofilms in disease. In vivo and ex vivo murine vaginitis models were employed to examine biofilm formation by scanning electron and confocal microscopy. C. albicans strains included 3153A (lab strain), DAY185 (parental control strain), and mutants defective in morphogenesis and/or biofilm formation in vitro (efg1/efg1 and bcr1/bcr1). Both 3153A and DAY815 formed biofilms on the vaginal mucosa in vivo and ex vivo as indicated by high fungal burden and microscopic analysis demonstrating typical biofilm architecture and presence of extracellular matrix (ECM) co-localized with the presence of fungi. In contrast, efg1/efg1 and bcr1/bcr1 mutant strains exhibited weak or no biofilm formation/ECM production in both models compared to wild-type strains and complemented mutants despite comparable colonization levels. These data show for the first time that C. albicans forms biofilms in vivo on vaginal epithelium, and that in vivo biotic biofilm formation requires regulators of biofilm formation (BCR1) and morphogenesis (EFG1).

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Sensory Stimuli Directly Acting at the Central Nervous System Regulate Gastric Ghrelin Secretion. An ex Vivo Organ Culture Study

Endocrinology ◽

10.1210/en.2007-0226 ◽

2007 ◽

Vol 148 (8) ◽

pp. 3998-4006 ◽

Cited By ~ 44

Author(s):

Luisa M. Seoane ◽

Omar Al-Massadi ◽

J. Eduardo Caminos ◽

Sulay A. Tovar ◽

Carlos Dieguez ◽

...

Keyword(s):

Organ Culture ◽

Ex Vivo ◽

Negative Energy ◽

Gastrointestinal Hormone ◽

Suitable Model ◽

Sensory Stimuli ◽

Orexigenic Peptide ◽

The Central Nervous System ◽

Ghrelin Secretion

Ghrelin, a novel gastrointestinal hormone involved in GH regulation, has been postulated as a relevant orexigenic peptide released by splanchnic tissues. Descriptive studies have shown that plasma ghrelin levels increase in states of negative energy balance or fasting, while decreasing in obesity and after feeding. In the present study, a novel organ-culture model of gastric tissue explants obtained from rat donors has been validated for ex vivo experiments. Fasting induced gastric ghrelin release as well as ghrelin mRNA expression that were reflected in plasma. Interestingly, those changes were fully reverted by 15 min of refeeding before stomach extraction. Unexpectedly, when animals were allowed 15 min before explant extraction to see or smell, but not eat, the food (tease feeding), ghrelin secretion was suppressed just like in gastric explants from refed animals. This effect was blocked when the animals were subjected to surgical vagotomy or treated with atropine sulphate. In conclusion, gastric explants were a suitable model for testing ghrelin mechanism of secretion in vitro, and they were found to maintain memory of the previously received signals. Similar to feeding, tease feeding resulted in suppression of ghrelin discharge by explants.

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Rational humanization of the powerful antithrombotic anti-GPIbα antibody: 6B4

Thrombosis and Haemostasis ◽

10.1160/th06-06-0297 ◽

2006 ◽

Vol 96 (11) ◽

pp. 671-684 ◽

Cited By ~ 21

Author(s):

Alexandre Fontayne ◽

Karen Vanhoorelbeke ◽

Inge Pareyn ◽

Isabel Van Rompaey ◽

Muriel Meiring ◽

...

Keyword(s):

Ex Vivo ◽

Three Dimensional ◽

Vector System ◽

Dimensional Structure ◽

Fab Fragment ◽

Variable Regions ◽

Significant Prolongation ◽

Von Willebrand ◽

First Time

SummaryFab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibα (GPIbα), have a powerful antithrombotic effect in baboons by blocking the GPIbα binding site for von Willebrand factor (VWF), without significant prolongation of the skin bleeding time. In order to bring this antibody to the clinic,we here humanized for the first time an anti-human GPIbα by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6B4 were cloned and sequenced. Based on this,a three-dimensional structure of the Fv-fragment was constructed by using homology-based modeling, and with this and comparison with antibodies with known structure,”murine” putative immunogenic residues which are exposed, were changed for “human-like” residues. The humanized Fab-fragment, h6B4-Fab, was constructed in the pKaneo vector system, expressed and purified and showed in vitro an unaltered, even slightly higher binding affinity for its antigen than the murine form as determined by different ELISA set-ups and surface plasmon resonance. Finally, injection of doses of 0.1 to 1.5 mg/kg of h6B4-Fab in baboons showed that both pharmacokinetics and ex-vivo bio-activity of the molecule were to a large extent preserved.In conclusion, the method used here to humanize 6B4 by resurfacing resulted in a fully active derivative, which is now ready for further development.

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Human Mesenchymal Stem Cell Combined with a New Strontium-Enriched Bioactive Glass: An ex-vivo Model for Bone Regeneration

Materials ◽

10.3390/ma12213633 ◽

2019 ◽

Vol 12 (21) ◽

pp. 3633 ◽

Cited By ~ 11

Author(s):

Devis Bellucci ◽

Elena Veronesi ◽

Valentina Strusi ◽

Tiziana Petrachi ◽

Alba Murgia ◽

...

Keyword(s):

Bioactive Glass ◽

Ex Vivo ◽

Human Mesenchymal Stem Cell ◽

Cellular Model ◽

Ex Vivo Model ◽

Bone Differentiation ◽

Biological Potential ◽

Orthopedic Applications ◽

First Time

A 3D cellular model that mimics the potential clinical application of a biomaterial is here applied for the first time to a bioactive glass, in order to assess its biological potential. A recently developed bioactive glass (BGMS10), whose composition contained strontium and magnesium, was produced in the form of granules and fully investigated in terms of biocompatibility in vitro. Apart from standard biological characterization (Simulated Body Fluid (SBF) testing and biocompatibility as per ISO10993), human bone marrow mesenchymal stromal/stem cells (BM-MSCs) were used to investigate the performance of the bioactive glass granules in an innovative 3D cellular model. The results showed that BGMS10 supported human BM-MSCs adhesion, colonization, and bone differentiation. Thus, bioactive glass granules seem to drive osteogenic differentiation and thus look particularly promising for orthopedic applications, bone tissue engineering and regenerative medicine.

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Astrovirus-Induced Synthesis of Nitric Oxide Contributes to Virus Control during Infection

Journal of Virology ◽

10.1128/jvi.78.3.1564-1574.2004 ◽

2004 ◽

Vol 78 (3) ◽

pp. 1564-1574 ◽

Cited By ~ 26

Author(s):

Matthew D. Koci ◽

Laura A. Kelley ◽

Diane Larsen ◽

Stacey Schultz-Cherry

Keyword(s):

Nitric Oxide ◽

Ex Vivo ◽

Small Animal ◽

Innate Immune Responses ◽

Vitro Analysis ◽

Oxide Synthase ◽

First Time

ABSTRACT Astrovirus is one of the major causes of infant and childhood diarrhea worldwide. Our understanding of astrovirus pathogenesis trails behind our knowledge of its molecular and epidemiologic properties. Using a recently developed small-animal model, we investigated the mechanisms by which astrovirus induces diarrhea and the role of both the adaptive and innate immune responses to turkey astrovirus type-2 (TAstV-2) infection. Astrovirus-infected animals were analyzed for changes in total lymphocyte populations, alterations in CD4+/CD8+ ratios, production of virus-specific antibodies (Abs), and macrophage activation. There were no changes in the numbers of circulating or splenic lymphocytes or in CD4+/CD8+ ratios compared to controls. Additionally, there was only a modest production of virus-specific Abs. However, adherent spleen cells from infected animals produced more nitric oxide (NO) in response to ex vivo stimulation with lipopolysaccharide. In vitro analysis demonstrated that TAstV-2 induced macrophage production of inducible nitric oxide synthase. Studies using NO donors and inhibitors in vivo demonstrated, for the first time, that NO inhibited astrovirus replication. These studies suggest that NO is important in limiting astrovirus replication and are the first, to our knowledge, to describe the potential role of innate immunity in astrovirus infection.

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Analysis of the role of TpUB05 antigen from Theileria parva in immune responses to malaria in humans compared to its homologue in Plasmodium falciparum; UB05 antigen

10.21203/rs.2.16899/v2 ◽

2019 ◽

Author(s):

Jerome Nyhalah Dinga ◽

Stanley Dobgima Gamua ◽

Stephanie Numenyi Perimbie ◽

Francis N. G. Chuma ◽

Dieudonné Lemuh Njimoh ◽

...

Keyword(s):

Immune Responses ◽

Protective Immunity ◽

Vaccine Development ◽

Ex Vivo ◽

Growth Inhibition Assay ◽

Potential Marker ◽

As Species ◽

Potential Vaccine ◽

First Time

Abstract Background: Despite the amount of resources deployed and technological advancements in Molecular Biology, vaccinology, immunology, genetics, and biotechnology, there is still no effective vaccines against malaria. Immunity to either malaria or East Coast fever is usually seen as species- and/or strain-specific. But there is growing body of evidence suggesting the possibility of the existence of cross strain, cross species and cross genus immune responses in apicomplexans. The principle of gene conservations indicates that homologues play similar role in closely related organisms. UB05 antigen (XP_001347656.2) from P. falciparum is part of chimeric UB05-09 antigen; a potential vaccine candidate has been demonstrated to be a marker of protective immunity in malaria. The homologue of UB05 in T. parva is TpUB05 (XP_763711.1) which was also tested and shown to be a potential marker of protective immunity in ECF as well. In a bid to identify potent markers of protective immunity to aid malaria vaccine development, TpUB05 was tested in malaria caused by P. falciparum . Results: It was observed that TpUB05 provoked stronger immune responses in malaria compared to UB05 antigen as tested using ELISA, ex-vivo ELISpot assay and in vitro growth inhibition assay. Conclusion: This study suggests for the first time that TpUB05 from T. parva is a better marker of protective immunity in malaria compared to its homologue UB05 from P. falciparum .

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The human SOX18 gene: Expression analysis and characterization of its 5’ flanking region

Archives of Biological Sciences ◽

10.2298/abs0704267p ◽

2007 ◽

Vol 59 (4) ◽

pp. 267-272 ◽

Cited By ~ 4

Author(s):

Isidora Petrovic ◽

Milena Stevanovic

Keyword(s):

Transcriptional Regulation ◽

Regulatory Elements ◽

Model System ◽

Suitable Model ◽

Flanking Region ◽

Vitro Model ◽

The Given ◽

First Time

The aim of this study was to establish an adequate in vitro model system for studying transcriptional regulation of the human SOX18 gene. The paper presents an analysis of expression of this gene in cultured cell lines and characterization of its 5' flanking region. Using RT-PCR, Northern and Western blot analysis, we demonstrated SOX18 expression in HeLa cells, indicating that this cell line provides a suitable model system for studying transcriptional regulation of the given gene. We also cloned, sequenced and for the first time characterized the human SOX18 5? flanking region. It is shown that the region 892 bp in size immediately upstream from the start codone harbors regulatory elements sufficient for transcription and represents an SOX18 promoter region.

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Comparison of the In Vitro and Ex Vivo Permeation of Existing Topical Formulations Used in the Treatment of Facial Angiofibroma and Characterization of the Variations Observed

Pharmaceutics ◽

10.3390/pharmaceutics12111060 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1060

Author(s):

Guillaume Le Guyader ◽

Bernard Do ◽

Victoire Vieillard ◽

Karine Andrieux ◽

Muriel Paul

Keyword(s):

Ex Vivo ◽

Thermodynamic Activity ◽

Physico Chemical ◽

Ex Vivo Permeation ◽

Franz Cells ◽

Great Heterogeneity ◽

The Impact ◽

First Time

Rapamycin has been used topically to treat facial angiofibromas associated with tuberous sclerosis for more than a decade. In the absence of a commercial form, a large number of formulations have been clinically tested. However, given the great heterogeneity of these studies, particularly with regard to the response criteria, it was difficult to know the impact and thus to compare the relevance of the formulations used. The objective of this work was therefore to evaluate the link between the diffusion of rapamycin and the physico-chemical characteristics of these different formulations on Strat-M® membranes as well as on human skin using Franz cells. Our results underline the importance of the type of vehicle used (hydrogel > cream > lipophilic ointment), the soluble state of rapamycin and its concentration close to saturation to ensure maximum thermodynamic activity. Thus, this is the first time that a comparative study of the different rapamycin formulations identified in the literature for the management of facial angiofibromas has been carried out using a pharmaceutical and biopharmaceutical approach. It highlights the important parameters to be considered in the development and optimization of topical rapamycin formulations with regard to cutaneous absorption for clinical efficacy.

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Comparison of methods to experimentally induce opacification and elasticity change in ex vivo porcine lenses

Scientific Reports ◽

10.1038/s41598-021-02851-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Manuel Ruiss ◽

Martin Kronschläger ◽

Andreas Schlatter ◽

Thomas Dechat ◽

Oliver Findl

Keyword(s):

Sodium Chloride ◽

Microwave Heating ◽

Culture Medium ◽

Ex Vivo ◽

Treatment Strategies ◽

Suitable Model ◽

Lens Opacification ◽

Vitro Model ◽

The Moment

AbstractAt the moment, cataract, which is the opacification of the eye’s lens, can only be treated by surgery. In order to develop and test new pharmacological treatment strategies for the disease, there is a need for an appropriate in vitro model using ex vivo animal lenses. In this study, porcine lenses were incubated in either culture medium, glucose, triamcinolone acetonide, sodium chloride, hydrogen peroxide, sodium selenite, neutral buffered formalin, or were exposed to microwave heating to experimentally induce lens opacification. Changes in the lens morphology, weight, size, and elasticity were monitored 7 days after treatment. The fastest induction of dense opacification was seen in lenses exposed to sodium chloride, neutral buffered formalin, and microwave heating. No change in the size and weight of the lenses were detected, whereas loss in elasticity could be detected in lenses treated with formalin solution or microwave heating. Thus, neutral buffered formalin- and microwave-treated ex vivo porcine lenses seem to be a suitable model for mature cataracts, whereas hypertonic sodium chloride may be useful for studies on osmolarity-induced lens opacification.

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