More Realistic Face Model Surface Improves Relevance of Pediatric In-Vitro Aerosol Studies

PubMed Central

Amirav, Israel; Halamish, Asaf; Gorenberg, Miguel; Omar, Hamza; Newhouse, Michael T.

2015-01-01

Background Various hard face models are commonly used to evaluate the efficiency of aerosol face masks. Softer more realistic “face” surface materials, like skin, deform upon mask application and should provide more relevant in-vitro tests. Studies that simultaneously take into consideration many of the factors characteristic of the in vivo face are lacking. These include airways, various application forces, comparison of various devices, comparison with a hard-surface model and use of a more representative model face based on large numbers of actual faces. Aim To compare mask to “face” seal and aerosol delivery of two pediatric masks using a soft vs. a hard, appropriately representative, pediatric face model under various applied forces. Methods Two identical face models and upper airways replicas were constructed, the only difference being the suppleness and compressibility of the surface layer of the “face.” Integrity of the seal and aerosol delivery of two different masks [AeroChamber (AC) and SootherMask (SM)] were compared using a breath simulator, filter collection and realistic applied forces. Results The soft “face” significantly increased the delivery efficiency and the sealing characteristics of both masks. Aerosol delivery with the soft “face” was significantly greater for the SM compared to the AC (p< 0.01). No statistically significant difference between the two masks was observed with the hard “face.” Conclusions The material and pliability of the model “face” surface has a significant influence on both the seal and delivery efficiency of face masks. This finding should be taken into account during in-vitro aerosol studies. PMID:26090661

Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery.

PubMed

Stock, Kristin; Estrada, Marta F; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vítor E; Barbier, Michaël; Blom, Sami; Arundkar, Sharath C; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph

2016-07-01

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.

Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery

PubMed Central

Stock, Kristin; Estrada, Marta F.; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vítor E.; Barbier, Michaël; Blom, Sami; Arundkar, Sharath C.; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph

2016-01-01

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models. PMID:27364600

Translational models of tumor angiogenesis: A nexus of in silico and in vitro models.

PubMed

Soleimani, Shirin; Shamsi, Milad; Ghazani, Mehran Akbarpour; Modarres, Hassan Pezeshgi; Valente, Karolina Papera; Saghafian, Mohsen; Ashani, Mehdi Mohammadi; Akbari, Mohsen; Sanati-Nezhad, Amir

2018-03-05

Emerging evidence shows that endothelial cells are not only the building blocks of vascular networks that enable oxygen and nutrient delivery throughout a tissue but also serve as a rich resource of angiocrine factors. Endothelial cells play key roles in determining cancer progression and response to anti-cancer drugs. Furthermore, the endothelium-specific deposition of extracellular matrix is a key modulator of the availability of angiocrine factors to both stromal and cancer cells. Considering tumor vascular network as a decisive factor in cancer pathogenesis and treatment response, these networks need to be an inseparable component of cancer models. Both computational and in vitro experimental models have been extensively developed to model tumor-endothelium interactions. While informative, they have been developed in different communities and do not yet represent a comprehensive platform. In this review, we overview the necessity of incorporating vascular networks for both in vitro and in silico cancer models and discuss recent progresses and challenges of in vitro experimental microfluidic cancer vasculature-on-chip systems and their in silico counterparts. We further highlight how these two approaches can merge together with the aim of presenting a predictive combinatorial platform for studying cancer pathogenesis and testing the efficacy of single or multi-drug therapeutics for cancer treatment. Copyright © 2018. Published by Elsevier Inc.

In vitro validation of a novel mechanical model for testing the anchorage capacity of pedicle screws using physiological load application.

PubMed

Liebsch, Christian; Zimmermann, Julia; Graf, Nicolas; Schilling, Christoph; Wilke, Hans-Joachim; Kienle, Annette

2018-01-01

Biomechanical in vitro tests analysing screw loosening often include high standard deviations caused by high variabilities in bone mineral density and pedicle geometry, whereas standardized mechanical models made of PU foam often do not integrate anatomical or physiological boundary conditions. The purpose of this study was to develop a most realistic mechanical model for the standardized and reproducible testing of pedicle screws regarding the resistance against screw loosening and the holding force as well as to validate this model by in vitro experiments. The novel mechanical testing model represents all anatomical structures of a human vertebra and is consisting of PU foam to simulate cancellous bone, as well as a novel pedicle model made of short carbon fibre filled epoxy. Six monoaxial cannulated pedicle screws (Ø6.5 × 45mm) were tested using the mechanical testing model as well as human vertebra specimens by applying complex physiological cyclic loading (shear, tension, and bending; 5Hz testing frequency; sinusoidal pulsating forces) in a dynamic materials testing machine with stepwise increasing load after each 50.000 cycles (100.0N shear force + 20.0N per step, 51.0N tension force + 10.2N per step, 4.2Nm bending moment + 0.8Nm per step) until screw loosening was detected. The pedicle screw head was fixed on a firmly clamped rod while the load was applied in the vertebral body. For the in vitro experiments, six human lumbar vertebrae (L1-3, BMD 75.4 ± 4.0mg/cc HA, pedicle width 9.8 ± 0.6mm) were tested after implanting pedicle screws under X-ray control. Relative motions of pedicle screw, specimen fixture, and rod fixture were detected using an optical motion tracking system. Translational motions of the mechanical testing model experiments in the point of load introduction (0.9-2.2mm at 240N shear force) were reproducible within the variation range of the in vitro experiments (0.6-3.5mm at 240N shear force). Screw loosening occurred continuously in each case between 140N and 280N, while abrupt failures of the specimen were observed only in vitro. In the mechanical testing model, no translational motion was detected in the screw entry point, while in vitro, translational motions of up to 2.5mm in inferior direction were found, leading to a slight shift of the centre of rotation towards the screw tip. Translational motions of the screw tip of about 5mm in superior direction were observed both in vitro and in the mechanical testing model, while they were continuous in the mechanical testing model and rapidly increasing after screw loosening initiation in vitro. The overall pedicle screw loosening characteristics were qualitatively and quantitatively similar between the mechanical testing model and the human vertebral specimens as long as there was no translation of the screw at the screw entrance point. Therefore, the novel mechanical testing model represents a promising method for the standardized testing of pedicle screws regarding screw loosening for cases where the screw rotates around a point close to the screw entry point. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cadexomer iodine provides superior efficacy against bacterial wound biofilms in vitro and in vivo.

PubMed

Fitzgerald, Daniel J; Renick, Paul J; Forrest, Emma C; Tetens, Shannon P; Earnest, David N; McMillan, Jillian; Kiedaisch, Brett M; Shi, Lei; Roche, Eric D

2017-01-01

Examination of clinical samples indicates bacterial biofilms are present in the majority of chronic wounds, and substantial evidence suggests biofilms contribute significantly to delayed healing. Bacteria in biofilms are highly tolerant of antimicrobials, and little data exist to guide the choice of anti-biofilm wound therapy. Cadexomer iodine (CI) was recently reported to have superior efficacy compared to diverse wound dressings against Pseudomonas aeruginosa biofilms in an ex vivo model. In the current study, the strong performance of CI vs. P. aeruginosa biofilm was confirmed using colony and colony drip-flow in vitro wound biofilm models. Similar in vitro efficacy of CI was also demonstrated against mature Staphylococcus aureus biofilms using the same models. Additionally, the rapid kill of mature S. aureus and P. aeruginosa colony biofilms was visualized by confocal microscopy using Live/Dead fluorescent stains. Superior in vitro efficacy of CI vs. staphylococcal biofilms was further demonstrated against methicillin-resistant S. aureus (MRSA) using multiple biofilm models with log reduction, Live/Dead, and metabolic endpoints. Comparator antimicrobial dressings, including silver-based dressings used throughout and other active agents used in individual models, elucidated only limited effects against the mature biofilms. Given the promising in vitro activity, CI was tested in an established mouse model of MRSA wound biofilm. CI had significantly greater impact on MRSA biofilm in mouse wounds than silver dressings or mupirocin based on Gram-stained histology sections and quantitative microbiology from biopsy samples (>4 log reduction in CFU/g vs. 0.7-1.6, p < 0.0001). The superior efficacy for CI in these in vitro and in vivo models suggests CI topical products may represent a better choice to address established bacterial biofilm in chronic wounds. © 2016 by the Wound Healing Society.

Bayesian Models Leveraging Bioactivity and Cytotoxicity Information for Drug Discovery

PubMed Central

Ekins, Sean; Reynolds, Robert C.; Kim, Hiyun; Koo, Mi-Sun; Ekonomidis, Marilyn; Talaue, Meliza; Paget, Steve D.; Woolhiser, Lisa K.; Lenaerts, Anne J.; Bunin, Barry A.; Connell, Nancy; Freundlich, Joel S.

2013-01-01

SUMMARY Identification of unique leads represents a significant challenge in drug discovery. This hurdle is magnified in neglected diseases such as tuberculosis. We have leveraged public high-throughput screening (HTS) data, to experimentally validate virtual screening approach employing Bayesian models built with bioactivity information (single-event model) as well as bioactivity and cytotoxicity information (dual-event model). We virtually screen a commercial library and experimentally confirm actives with hit rates exceeding typical HTS results by 1-2 orders of magnitude. The first dual-event Bayesian model identified compounds with antitubercular whole-cell activity and low mammalian cell cytotoxicity from a published set of antimalarials. The most potent hit exhibits the in vitro activity and in vitro/in vivo safety profile of a drug lead. These Bayesian models offer significant economies in time and cost to drug discovery. PMID:23521795

Mg and Mg alloys: how comparable are in vitro and in vivo corrosion rates? A review.

PubMed

Martinez Sanchez, Adela Helvia; Luthringer, Bérengère J C; Feyerabend, Frank; Willumeit, Regine

2015-02-01

Due to their biodegradability, magnesium and magnesium-based alloys could represent the third generation of biomaterials. However, their mechanical properties and time of degradation have to match the needs of applications. Several approaches, such as choice of alloying elements or tailored microstructure, are employed to tailor corrosion behaviour. Due to the high electrochemical activity of Mg, numerous environmental factors (e.g. temperature and surrounding ion composition) influence its corrosion behaviour, making it unpredictable. Nevertheless, the need of reliable in vitro model(s) to predict in vivo implant degradation is increasing. In an attempt to find a correlation between in vitro and vivo corrosion rates, this review presents a systematic literature survey, as well as an attempt to correlate the different results. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Analysis of the Material Properties of Early Chondrogenic Differentiated Adipose-Derived Stromal Cells (ASC) Using an in vitro Three-dimensional Micromass Culture System

PubMed Central

Xu, Yue; Balooch, Guive; Chiou, Michael; Bekerman, Elena; Ritchie, Robert O.; Longaker, Michael T.

2009-01-01

Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitro model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation. PMID:17543281

Pediatric in vitro and in silico models of deposition via oral and nasal inhalation.

PubMed

Carrigy, Nicholas B; Ruzycki, Conor A; Golshahi, Laleh; Finlay, Warren H

2014-06-01

Respiratory tract deposition models provide a useful method for optimizing the design and administration of inhaled pharmaceutical aerosols, and can be useful for estimating exposure risks to inhaled particulate matter. As aerosol must first pass through the extrathoracic region prior to reaching the lungs, deposition in this region plays an important role in both cases. Compared to adults, much less extrathoracic deposition data are available with pediatric subjects. Recently, progress in magnetic resonance imaging and computed tomography scans to develop pediatric extrathoracic airway replicas has facilitated addressing this issue. Indeed, the use of realistic replicas for benchtop inhaler testing is now relatively common during the development and in vitro evaluation of pediatric respiratory drug delivery devices. Recently, in vitro empirical modeling studies using a moderate number of these realistic replicas have related airway geometry, particle size, fluid properties, and flow rate to extrathoracic deposition. Idealized geometries provide a standardized platform for inhaler testing and exposure risk assessment and have been designed to mimic average in vitro deposition in infants and children by replicating representative average geometrical dimensions. In silico mathematical models have used morphometric data and aerosol physics to illustrate the relative importance of different deposition mechanisms on respiratory tract deposition. Computational fluid dynamics simulations allow for the quantification of local deposition patterns and an in-depth examination of aerosol behavior in the respiratory tract. Recent studies have used both in vitro and in silico deposition measurements in realistic pediatric airway geometries to some success. This article reviews the current understanding of pediatric in vitro and in silico deposition modeling via oral and nasal inhalation.

THE ROLE OF MICROGLIA IN PARAQUAT INDUCED DOPAMINERGIC NEUROTOXICITY.

EPA Science Inventory

This manuscript relates oxidative stress conveyed by a widely used pesticide (paraquat) to selective neurodegeneration of the in vitro models of the mouse brain. As such, it represents a provocative link to the conditions of oxidative stress, environmental chemical exposures and...

Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology.

PubMed

Frega, Monica; Tedesco, Mariateresa; Massobrio, Paolo; Pesce, Mattia; Martinoia, Sergio

2014-06-30

Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These networks are usually grown onto specific rigid substrates (also with embedded electrodes) and lack of most of the constituents of the in-vivo like environment: cell morphology, cell-to-cell interaction and neuritic outgrowth in all directions. Cells in a brain region develop in a 3D space and interact with a complex multi-cellular environment and extracellular matrix. Under this perspective, 3D networks coupled to micro-transducer arrays, represent a new and powerful in-vitro model capable of better emulating in-vivo physiology. In this work, we present a new experimental paradigm constituted by 3D hippocampal networks coupled to Micro-Electrode-Arrays (MEAs) and we show how the features of the recorded network dynamics differ from the corresponding 2D network model. Further development of the proposed 3D in-vitro model by adding embedded functionalized scaffolds might open new prospects for manipulating, stimulating and recording the neuronal activity to elucidate neurophysiological mechanisms and to design bio-hybrid microsystems.

Network dynamics of 3D engineered neuronal cultures: a new experimental model for in-vitro electrophysiology

PubMed Central

Frega, Monica; Tedesco, Mariateresa; Massobrio, Paolo; Pesce, Mattia; Martinoia, Sergio

2014-01-01

Despite the extensive use of in-vitro models for neuroscientific investigations and notwithstanding the growing field of network electrophysiology, all studies on cultured cells devoted to elucidate neurophysiological mechanisms and computational properties, are based on 2D neuronal networks. These networks are usually grown onto specific rigid substrates (also with embedded electrodes) and lack of most of the constituents of the in-vivo like environment: cell morphology, cell-to-cell interaction and neuritic outgrowth in all directions. Cells in a brain region develop in a 3D space and interact with a complex multi-cellular environment and extracellular matrix. Under this perspective, 3D networks coupled to micro-transducer arrays, represent a new and powerful in-vitro model capable of better emulating in-vivo physiology. In this work, we present a new experimental paradigm constituted by 3D hippocampal networks coupled to Micro-Electrode-Arrays (MEAs) and we show how the features of the recorded network dynamics differ from the corresponding 2D network model. Further development of the proposed 3D in-vitro model by adding embedded functionalized scaffolds might open new prospects for manipulating, stimulating and recording the neuronal activity to elucidate neurophysiological mechanisms and to design bio-hybrid microsystems. PMID:24976386

RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

PubMed

Morabito, Caterina; Steimberg, Nathalie; Mazzoleni, Giovanna; Guarnieri, Simone; Fanò-Illic, Giorgio; Mariggiò, Maria A

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

RCCS Bioreactor-Based Modelled Microgravity Induces Significant Changes on In Vitro 3D Neuroglial Cell Cultures

PubMed Central

Mazzoleni, Giovanna; Fanò-Illic, Giorgio; Mariggiò, Maria A.

2015-01-01

We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions. PMID:25654124

Screening for invasion of the individual human brain tumour in an autologous confrontation system in vitro.

PubMed

de Ridder, L

1999-01-01

Invasiveness is the major cause of death in patients bearing a brain tumour. The invasiveness or infiltrative capacity of a primary brain tumour has a prognostic value for the evaluation of the process in vivo. So a model to imitate invasion might give information on the in vivo behaviour and outcome of the disease for the individual patient. The developed in vitro model represents an assay in which the patients' brain tumour-derived cells are confronted with connective tissue from the patient himself, i.e. an autologous system to evaluate the individual behaviour of the tumour, in contrast to other invasion models. The test can be applied with tumour-derived material collected by a stereotactic biopsy.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

PubMed

Gettings, S D; Lordo, R A; Hintze, K L; Bagley, D M; Casterton, P L; Chudkowski, M; Curren, R D; Demetrulias, J L; Dipasquale, L C; Earl, L K; Feder, P I; Galli, C L; Glaza, S M; Gordon, V C; Janus, J; Kurtz, P J; Marenus, K D; Moral, J; Pape, W J; Renskers, K J; Rheins, L A; Roddy, M T; Rozen, M G; Tedeschi, J P; Zyracki, J

1996-01-01

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

Permeability of PEGylated immunoarsonoliposomes through in vitro blood brain barrier-medulloblastoma co-culture models for brain tumor therapy.

PubMed

Al-Shehri, Abdulghani; Favretto, Marco E; Ioannou, Panayiotis V; Romero, Ignacio A; Couraud, Pierre-Olivier; Weksler, Babette Barbash; Parker, Terry L; Kallinteri, Paraskevi

2015-03-01

Owing to restricted access of pharmacological agents into the brain due to blood brain barrier (BBB) there is a need: 1. to develop a more representative 3-D-co-culture model of tumor-BBB interaction to investigate drug and nanoparticle transport into the brain for diagnostic and therapeutic evaluation. 2. to address the lack of new alternative methods to animal testing according to replacement-reduction-refinement principles. In this work, in vitro BBB-medulloblastoma 3-D-co-culture models were established using immortalized human primary brain endothelial cells (hCMEC/D3). hCMEC/D3 cells were cultured in presence and in absence of two human medulloblastoma cell lines on Transwell membranes. In vitro models were characterized for BBB formation, zonula occludens-1 expression and permeability to dextran. Transferrin receptors (Tfr) expressed on hCMEC/D3 were exploited to facilitate arsonoliposome (ARL) permeability through the BBB to the tumor by covalently attaching an antibody specific to human Tfr. The effect of anticancer ARLs on hCMEC/D3 was assessed. In vitro BBB and BBB-tumor co-culture models were established successfully. BBB permeability was affected by the presence of tumor aggregates as suggested by increased permeability of ARLs. There was a 6-fold and 8-fold increase in anti-Tfr-ARL uptake into VC312R and BBB-DAOY co-culture models, respectively, compared to plain ARLs. The three-dimensional models might be appropriate models to study the transport of various drugs and nanocarriers (liposomes and immunoarsonoliposomes) through the healthy and diseased BBB. The immunoarsonoliposomes can be potentially used as anticancer agents due to good tolerance of the in vitro BBB model to their toxic effect.

Selection of a Relevant In Vitro Blood-Brain Barrier Model to Investigate Pro-Metastatic Features of Human Breast Cancer Cell Lines.

PubMed

Drolez, Aurore; Vandenhaute, Elodie; Julien, Sylvain; Gosselet, Fabien; Burchell, Joy; Cecchelli, Roméo; Delannoy, Philippe; Dehouck, Marie-Pierre; Mysiorek, Caroline

2016-01-01

Around 7-17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells to reach the brain could provide a relevant and important strategy. For that purpose an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells adhesion capacities.

Brain Aggregates: An Effective In Vitro Cell Culture System Modeling Neurodegenerative Diseases.

PubMed

Ahn, Misol; Kalume, Franck; Pitstick, Rose; Oehler, Abby; Carlson, George; DeArmond, Stephen J

2016-03-01

Drug discovery for neurodegenerative diseases is particularly challenging because of the discrepancies in drug effects between in vitro and in vivo studies. These discrepancies occur in part because current cell culture systems used for drug screening have many limitations. First, few cell culture systems accurately model human aging or neurodegenerative diseases. Second, drug efficacy may differ between dividing and stationary cells, the latter resembling nondividing neurons in the CNS. Brain aggregates (BrnAggs) derived from embryonic day 15 gestation mouse embryos may represent neuropathogenic processes in prion disease and reflect in vivo drug efficacy. Here, we report a new method for the production of BrnAggs suitable for drug screening and suggest that BrnAggs can model additional neurological diseases such as tauopathies. We also report a functional assay with BrnAggs by measuring electrophysiological activities. Our data suggest that BrnAggs could serve as an effective in vitro cell culture system for drug discovery for neurodegenerative diseases. © 2016 American Association of Neuropathologists, Inc. All rights reserved.

Mobile Genetic Elements: In Silico, In Vitro, In Vivo

PubMed Central

Arkhipova, Irina R.; Rice, Phoebe A.

2016-01-01

Mobile genetic elements (MGEs), also called transposable elements (TEs), represent universal components of most genomes and are intimately involved in nearly all aspects of genome organization, function, and evolution. However, there is currently a gap between fast-paced TE discovery in silico, stimulated by exponential growth of comparative genomic studies, and a limited number of experimental models amenable to more traditional in vitro and in vivo studies of structural, mechanistic, and regulatory properties of diverse MGEs. Experimental and computational scientists came together to bridge this gap at a recent conference, “Mobile Genetic Elements: in silico, in vitro, in vivo,” held at the Marine Biological Laboratory (MBL) in Woods Hole, MA, USA. PMID:26822117

An in vitro model of infection of chicken embryos by Cryptosporidium baileyi.

PubMed

Huang, Lei; Zhu, Huili; Zhang, Sumei; Wang, Rongjun; Liu, Limin; Jian, Fuchun; Ning, Changshen; Zhang, Longxian

2014-12-01

Cryptosporidiosis is one of the most prevalent parasitic infections in domesticated, caged and wild birds. Cryptosporidium baileyi is the most common species reported in a wide range of avian hosts. Although this parasite is well investigated, there is no adequate in vitro model for its endogenous development, and therefore, knowledge of each life cycle phase is scarce. In the present study, an in vitro model for C. baileyi in chicken embryos was developed and the complete life cycle investigated by light and electron microscopy, including both the sexual and asexual reproduction stages. The complete life cycle of C. baileyi was observed during 1-96 h post inoculation (PI), and the average reproduction number of C. baileyi oocysts in allantoic fluid of each chicken embryo was greatest at 168 h PI. These results suggest that chicken embryos could adequately represent the natural host cells and support the development of all the endogenous life cycle stages of C. baileyi, and also provide a new and effective in vitro cultivation system for further studies on antigens, virulence, infectivity, metabolites, and sensitivity of drugs against parasites. Copyright © 2014 Elsevier Inc. All rights reserved.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 1: method parameterization and comparison of in vitro digestion profiles across a range of representative formulations.

PubMed

Williams, Hywel D; Sassene, Philip; Kleberg, Karen; Bakala-N'Goma, Jean-Claude; Calderone, Marilyn; Jannin, Vincent; Igonin, Annabel; Partheil, Anette; Marchaud, Delphine; Jule, Eduardo; Vertommen, Jan; Maio, Mario; Blundell, Ross; Benameur, Hassan; Carrière, Frédéric; Müllertz, Anette; Porter, Christopher J H; Pouton, Colin W

2012-09-01

The Lipid Formulation Classification System Consortium is an industry-academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation. Copyright © 2012 Wiley Periodicals, Inc.

Comparison of experimental models for predicting laser-tissue interaction from 3.8-micron lasers

NASA Astrophysics Data System (ADS)

Williams, Piper C. M.; Winston, Golda C. H.; Randolph, Don Q.; Neal, Thomas A.; Eurell, Thomas E.; Johnson, Thomas E.

2004-07-01

The purpose of this study was to evaluate the laser-tissue interactions of engineered human skin and in-vivo pig skin following exposure to a single 3.8 micron laser light pulse. The goal of the study was to determine if these tissues shared common histologic features following laser exposure that might prove useful in developing in-vitro and in-vivo experimental models to predict the bioeffects of human laser exposure. The minimum exposure required to produce gross morphologic changes following a four microsecond, pulsed skin exposure for both models was determined. Histology was used to compare the cellular responses of the experimental models following laser exposure. Eighteen engineered skin equivalents (in-vitro model), were exposed to 3.8 micron laser light and the tissue responses compared to equivalent exposures made on five Yorkshire pigs (in-vivo model). Representative biopsies of pig skin were taken for histologic evaluation from various body locations immediately, one hour, and 24 hours following exposure. The pattern of epithelial changes seen following in-vitro laser exposure of the engineered human skin and in-vivo exposure of pig skin indicated a common histologic response for this particular combination of laser parameters.

Consumer product in vitro digestion model: Bioaccessibility of contaminants and its application in risk assessment.

PubMed

Brandon, Esther F A; Oomen, Agnes G; Rompelberg, Cathy J M; Versantvoort, Carolien H M; van Engelen, Jacqueline G M; Sips, Adrienne J A M

2006-03-01

This paper describes the applicability of in vitro digestion models as a tool for consumer products in (ad hoc) risk assessment. In current risk assessment, oral bioavailability from a specific product is considered to be equal to bioavailability found in toxicity studies in which contaminants are usually ingested via liquids or food matrices. To become bioavailable, contaminants must first be released from the product during the digestion process (i.e. become bioaccessible). Contaminants in consumer products may be less bioaccessible than contaminants in liquid or food. Therefore, the actual risk after oral exposure could be overestimated. This paper describes the applicability of a simple, reliable, fast and relatively inexpensive in vitro method for determining the bioaccessibility of a contaminant from a consumer product. Different models, representing sucking and/or swallowing were developed. The experimental design of each model can be adjusted to the appropriate exposure scenarios as determined by the risk assessor. Several contaminated consumer products were tested in the various models. Although relevant in vivo data are scare, we succeeded to preliminary validate the model for one case. This case showed good correlation and never underestimated the bioavailability. However, validation check needs to be continued.

Integrating Biological and Chemical Data for Hepatotoxicity Prediction (SOT)

EPA Science Inventory

The U.S. EPA ToxCastTM program is screening thousands of environmental chemicals for bioactivity using hundreds of high-throughput in vitro assays to build predictive models of toxicity. A set of 677 chemicals were represented by 711 bioactivity descriptors (from ToxCast assays),...

Pluripotent stem cell derived hepatocyte like cells and their potential in toxicity screening.

PubMed

Greenhough, Sebastian; Medine, Claire N; Hay, David C

2010-12-30

Despite considerable progress in modelling human liver toxicity, the requirement still exists for efficient, predictive and cost effective in vitro models to reduce attrition during drug development. Thousands of compounds fail in this process, with hepatotoxicity being one of the significant causes of failure. The cost of clinical studies is substantial, therefore it is essential that toxicological screening is performed early on in the drug development process. Human hepatocytes represent the gold standard model for evaluating drug toxicity, but are a limited resource. Current alternative models are based on immortalised cell lines and animal tissue, but these are limited by poor function, exhibit species variability and show instability in culture. Pluripotent stem cells are an attractive alternative as they are capable of self-renewal and differentiation to all three germ layers, and thereby represent a potentially inexhaustible source of somatic cells. The differentiation of human embryonic stem cells and induced pluripotent stem cells to functional hepatocyte like cells has recently been reported. Further development of this technology could lead to the scalable production of hepatocyte like cells for liver toxicity screening and clinical therapies. Additionally, induced pluripotent stem cell derived hepatocyte like cells may permit in vitro modelling of gene polymorphisms and genetic diseases. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Immune Cell-Supplemented Human Skin Model for Studying Fungal Infections.

PubMed

Kühbacher, Andreas; Sohn, Kai; Burger-Kentischer, Anke; Rupp, Steffen

2017-01-01

Human skin is a niche for various fungal species which either colonize the surface of this tissue as commensals or, primarily under conditions of immunosuppression, invade the skin and cause infection. Here we present a method for generation of a human in vitro skin model supplemented with immune cells of choice. This model represents a complex yet amenable tool to study molecular mechanisms of host-fungi interactions at human skin.

Three-Dimensional In Vitro Skin and Skin Cancer Models Based on Human Fibroblast-Derived Matrix.

PubMed

Berning, Manuel; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

2015-09-01

Three-dimensional in vitro skin and skin cancer models help to dissect epidermal-dermal and tumor-stroma interactions. In the model presented here, normal human dermal fibroblasts isolated from adult skin self-assembled into dermal equivalents with their specific fibroblast-derived matrix (fdmDE) over 4 weeks. The fdmDE represented a complex human extracellular matrix that was stabilized by its own heterogeneous collagen fiber meshwork, largely resembling a human dermal in vivo architecture. Complemented with normal human epidermal keratinocytes, the skin equivalent (fdmSE) thereof favored the establishment of a well-stratified and differentiated epidermis and importantly allowed epidermal regeneration in vitro for at least 24 weeks. Moreover, the fdmDE could be used to study the features of cutaneous skin cancer. Complementing fdmDE with HaCaT cells in different stages of malignancy or tumor-derived cutaneous squamous cell carcinoma cell lines, the resulting skin cancer equivalents (fdmSCEs) recapitulated the respective degree of tumorigenicity. In addition, the fdmSCE invasion phenotypes correlated with their individual degree of tissue organization, disturbance in basement membrane organization, and presence of matrix metalloproteinases. Together, fdmDE-based models are well suited for long-term regeneration of normal human epidermis and, as they recapitulate tumor-specific growth, differentiation, and invasion profiles of cutaneous skin cancer cells, also provide an excellent human in vitro skin cancer model.

The Microphysiology Systems Database for Analyzing and Modeling Compound Interactions with Human and Animal Organ Models

PubMed Central

Vernetti, Lawrence; Bergenthal, Luke; Shun, Tong Ying; Taylor, D. Lansing

2016-01-01

Abstract Microfluidic human organ models, microphysiology systems (MPS), are currently being developed as predictive models of drug safety and efficacy in humans. To design and validate MPS as predictive of human safety liabilities requires safety data for a reference set of compounds, combined with in vitro data from the human organ models. To address this need, we have developed an internet database, the MPS database (MPS-Db), as a powerful platform for experimental design, data management, and analysis, and to combine experimental data with reference data, to enable computational modeling. The present study demonstrates the capability of the MPS-Db in early safety testing using a human liver MPS to relate the effects of tolcapone and entacapone in the in vitro model to human in vivo effects. These two compounds were chosen to be evaluated as a representative pair of marketed drugs because they are structurally similar, have the same target, and were found safe or had an acceptable risk in preclinical and clinical trials, yet tolcapone induced unacceptable levels of hepatotoxicity while entacapone was found to be safe. Results demonstrate the utility of the MPS-Db as an essential resource for relating in vitro organ model data to the multiple biochemical, preclinical, and clinical data sources on in vivo drug effects. PMID:28781990

Fibroblasts Influence Survival and Therapeutic Response in a 3D Co-Culture Model

PubMed Central

Majety, Meher; Pradel, Leon P.; Gies, Manuela; Ries, Carola H.

2015-01-01

In recent years, evidence has indicated that the tumor microenvironment (TME) plays a significant role in tumor progression. Fibroblasts represent an abundant cell population in the TME and produce several growth factors and cytokines. Fibroblasts generate a suitable niche for tumor cell survival and metastasis under the influence of interactions between fibroblasts and tumor cells. Investigating these interactions requires suitable experimental systems to understand the cross-talk involved. Most in vitro experimental systems use 2D cell culture and trans-well assays to study these interactions even though these paradigms poorly represent the tumor, in which direct cell-cell contacts in 3D spaces naturally occur. Investigating these interactions in vivo is of limited value due to problems regarding the challenges caused by the species-specificity of many molecules. Thus, it is essential to use in vitro models in which human fibroblasts are co-cultured with tumor cells to understand their interactions. Here, we developed a 3D co-culture model that enables direct cell-cell contacts between pancreatic, breast and or lung tumor cells and human fibroblasts/ or tumor-associated fibroblasts (TAFs). We found that co-culturing with fibroblasts/TAFs increases the proliferation in of several types of cancer cells. We also observed that co-culture induces differential expression of soluble factors in a cancer type-specific manner. Treatment with blocking antibodies against selected factors or their receptors resulted in the inhibition of cancer cell proliferation in the co-cultures. Using our co-culture model, we further revealed that TAFs can influence the response to therapeutic agents in vitro. We suggest that this model can be reliably used as a tool to investigate the interactions between a tumor and the TME. PMID:26053043

In-vitro and in-vivo evidence of dose-dependent decrease of uropathogenic Escherichia coli virulence after consumption of commercial Vaccinium macrocarpon (cranberry) capsules.

PubMed

Lavigne, J-P; Bourg, G; Combescure, C; Botto, H; Sotto, A

2008-04-01

This study evaluated the antibacterial efficacy of the consumption of cranberry capsules vs. placebo in the urine of healthy volunteers. A first double-blind, randomised, crossover trial involved eight volunteers who had followed three regimens, with or without cranberry, with a wash-out period of at least 6 days between each regimen. Twelve hours after consumption of cranberry or placebo hard capsules, the first urine of the morning was collected. Different Escherichia coli strains were cultured in the urine samples. Urinary antibacterial adhesion activity was measured in vitro using the human T24 epithelial cell-line, and in vivo using the Caenorhabditis elegans killing model. With the in-vitro model, 108 mg of cranberry induced a significant reduction in bacterial adherence to T24 cells as compared with placebo (p <0.001). A significant dose-dependent decrease in bacterial adherence in vitro was noted after the consumption of 108 and 36 mg of cranberry (p <0.001). The in-vivo model confirmed that E. coli strains had a reduced ability to kill C. elegans after growth in the urine of patients who consumed cranberry capsules. Overall, these in-vivo and in-vitro studies suggested that consumption of cranberry juice represents an interesting new strategy to prevent recurrent urinary tract infection.

In-vitro and in-vivo evidence of dose-dependent decrease of uropathogenic Escherichia coli virulence after consumption of commercial Vaccinium macrocarpon (cranberry) capsules

PubMed Central

Lavigne, Jean-Philippe; Bourg, Gisèle; Combescure, Christophe; Botto, Henri; Sotto, Albert

2008-01-01

This study evaluated the antibacterial efficacy of the consumption of cranberry capsules vs. placebo in the urine of healthy volunteers. A first double-blind, randomised, crossover trial involved eight volunteers who had followed three regimens, with or without cranberry, with a wash-out period of at least 6 days between each regimen. Twelve hours after consumption of cranberry or placebo hard capsules, the first urine of the morning was collected. Different Escherichia coli strains were cultured in the urine samples. Urinary antibacterial adhesion activity was measured in vitro using the human T24 epithelial cell-line, and in vivo using the Caenorhabditis elegans killing model. With the in-vitro model, 108 mg of cranberry induced a significant reduction in bacterial adherence to T24 cells as compared with placebo (p <0.001). A significant dose-dependent decrease in bacterial adherence in vitro was noted after the consumption of 108 and 36 mg of cranberry (p <0.001). The in-vivo model confirmed that E. coli strains had a reduced ability to kill C. elegans after growth in the urine of patients who consumed cranberry capsules. Overall, these in-vivo and in-vitro studies suggested that consumption of cranberry juice represents an interesting new strategy to prevent recurrent urinary tract infection. PMID:18190583

Strategies for Determining Correct Cytochrome P450 Contributions in Hepatic Clearance Predictions: In Vitro-In Vivo Extrapolation as Modelling Approach and Tramadol as Proof-of Concept Compound.

PubMed

T'jollyn, Huybrecht; Snoeys, Jan; Van Bocxlaer, Jan; De Bock, Lies; Annaert, Pieter; Van Peer, Achiel; Allegaert, Karel; Mannens, Geert; Vermeulen, An; Boussery, Koen

2017-06-01

Although the measurement of cytochrome P450 (CYP) contributions in metabolism assays is straightforward, determination of actual in vivo contributions might be challenging. How representative are in vitro for in vivo CYP contributions? This article proposes an improved strategy for the determination of in vivo CYP enzyme-specific metabolic contributions, based on in vitro data, using an in vitro-in vivo extrapolation (IVIVE) approach. Approaches are exemplified using tramadol as model compound, and CYP2D6 and CYP3A4 as involved enzymes. Metabolism data for tramadol and for the probe substrates midazolam (CYP3A4) and dextromethorphan (CYP2D6) were gathered in human liver microsomes (HLM) and recombinant human enzyme systems (rhCYP). From these probe substrates, an activity-adjustment factor (AAF) was calculated per CYP enzyme, for the determination of correct hepatic clearance contributions. As a reference, tramadol CYP contributions were scaled-back from in vivo data (retrograde approach) and were compared with the ones derived in vitro. In this view, the AAF is an enzyme-specific factor, calculated from reference probe activity measurements in vitro and in vivo, that allows appropriate scaling of a test drug's in vitro activity to the 'healthy volunteer' population level. Calculation of an AAF, thus accounts for any 'experimental' or 'batch-specific' activity difference between in vitro HLM and in vivo derived activity. In this specific HLM batch, for CYP3A4 and CYP2D6, an AAF of 0.91 and 1.97 was calculated, respectively. This implies that, in this batch, the in vitro CYP3A4 activity is 1.10-fold higher and the CYP2D6 activity 1.97-fold lower, compared to in vivo derived CYP activities. This study shows that, in cases where the HLM pool does not represent the typical mean population CYP activities, AAF correction of in vitro metabolism data, optimizes CYP contributions in the prediction of hepatic clearance. Therefore, in vitro parameters for any test compound, obtained in a particular batch, should be corrected with the AAF for the respective enzymes. In the current study, especially the CYP2D6 contribution was found, to better reflect the average in vivo situation. It is recommended that this novel approach is further evaluated using a broader range of compounds.

Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression.

PubMed

Sawant, Sharada; Dongre, Harsh; Singh, Archana Kumari; Joshi, Shriya; Costea, Daniela Elena; Mahadik, Snehal; Ahire, Chetan; Makani, Vidhi; Dange, Prerana; Sharma, Shilpi; Chaukar, Devendra; Vaidya, Milind

2016-01-01

To study multistep tumorigenesis process, there is a need of in-vitro 3D model simulating in-vivo tissue. Present study aimed to reconstitute in-vitro tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted in-vitro models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced in-vitro. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in in-vitro grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis.

Establishment of 3D Co-Culture Models from Different Stages of Human Tongue Tumorigenesis: Utility in Understanding Neoplastic Progression

PubMed Central

Sawant, Sharada; Dongre, Harsh; Singh, Archana Kumari; Joshi, Shriya; Costea, Daniela Elena; Mahadik, Snehal; Ahire, Chetan; Makani, Vidhi; Dange, Prerana; Sharma, Shilpi; Chaukar, Devendra; Vaidya, Milind

2016-01-01

To study multistep tumorigenesis process, there is a need of in-vitro 3D model simulating in-vivo tissue. Present study aimed to reconstitute in-vitro tissue models comprising various stages of neoplastic progression of tongue tumorigenesis and to evaluate the utility of these models to investigate the role of stromal fibroblasts in maintenance of desmosomal anchoring junctions using transmission electron microscopy. We reconstituted in-vitro models representing normal, dysplastic, and malignant tissues by seeding primary keratinocytes on either fibroblast embedded in collagen matrix or plain collagen matrix in growth factor-free medium. The findings of histomorphometry, immunohistochemistry, and electron microscopy analyses of the three types of 3D cultures showed that the stratified growth, cell proliferation, and differentiation were comparable between co-cultures and their respective native tissues; however, they largely differed in cultures grown without fibroblasts. The immunostaining intensity of proteins, viz., desmoplakin, desmoglein, and plakoglobin, was reduced as the disease stage increased in all co-cultures as observed in respective native tissues. Desmosome-like structures were identified using immunogold labeling in these cultures. Moreover, electron microscopic observations revealed that the desmosome number and their length were significantly reduced and intercellular spaces were increased in cultures grown without fibroblasts when compared with their co-culture counterparts. Our results showed that the major steps of tongue tumorigenesis can be reproduced in-vitro. Stromal fibroblasts play a role in regulation of epithelial thickness, cell proliferation, differentiation, and maintenance of desmosomalanchoring junctions in in-vitro grown tissues. The reconstituted co-culture models could help to answer various biological questions especially related to tongue tumorigenesis. PMID:27501241

Structural and functional identification of vasculogenic mimicry in vitro.

PubMed

Racordon, Dusan; Valdivia, Andrés; Mingo, Gabriel; Erices, Rafaela; Aravena, Raúl; Santoro, Felice; Bravo, Maria Loreto; Ramirez, Carolina; Gonzalez, Pamela; Sandoval, Alejandra; González, Alfonso; Retamal, Claudio; Kogan, Marcelo J; Kato, Sumie; Cuello, Mauricio A; Osorio, German; Nualart, Francisco; Alvares, Pedro; Gago-Arias, Araceli; Fabri, Daniella; Espinoza, Ignacio; Sanchez, Beatriz; Corvalán, Alejandro H; Pinto, Mauricio P; Owen, Gareth I

2017-08-01

Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite its strong correlation with reduced patient survival, controversy still surrounds the existence of an in vitro model of VM. Furthermore, many studies that claim to demonstrate VM fail to provide solid evidence of true hollow channels, raising concerns as to whether actual VM is actually being examined. Herein, we provide a standardized in vitro assay that recreates the formation of functional hollow channels using ovarian cancer cell lines, cancer spheres and primary cultures derived from ovarian cancer ascites. X-ray microtomography 3D-reconstruction, fluorescence confocal microscopy and dye microinjection conclusively confirm the existence of functional glycoprotein-rich lined tubular structures in vitro and demonstrate that many of structures reported in the literature may not represent VM. This assay may be useful to design and test future VM-blocking anticancer therapies.

A high-throughput in vitro ring assay for vasoactivity using magnetic 3D bioprinting

PubMed Central

Tseng, Hubert; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Shen, Tsaiwei; Hebel, Chris; Barthlow, Herbert G.; Wagoner, Matthew; Souza, Glauco R.

2016-01-01

Vasoactive liabilities are typically assayed using wire myography, which is limited by its high cost and low throughput. To meet the demand for higher throughput in vitro alternatives, this study introduces a magnetic 3D bioprinting-based vasoactivity assay. The principle behind this assay is the magnetic printing of vascular smooth muscle cells into 3D rings that functionally represent blood vessel segments, whose contraction can be altered by vasodilators and vasoconstrictors. A cost-effective imaging modality employing a mobile device is used to capture contraction with high throughput. The goal of this study was to validate ring contraction as a measure of vasoactivity, using a small panel of known vasoactive drugs. In vitro responses of the rings matched outcomes predicted by in vivo pharmacology, and were supported by immunohistochemistry. Altogether, this ring assay robustly models vasoactivity, which could meet the need for higher throughput in vitro alternatives. PMID:27477945

Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities.

PubMed

Lan, Shih-Feng; Starly, Binil

2011-10-01

Prediction of human response to potential therapeutic drugs is through conventional methods of in vitro cell culture assays and expensive in vivo animal testing. Alternatives to animal testing require sophisticated in vitro model systems that must replicate in vivo like function for reliable testing applications. Advancements in biomaterials have enabled the development of three-dimensional (3D) cell encapsulated hydrogels as in vitro drug screening tissue model systems. In this study, we have developed an in vitro platform to enable high density 3D culture of liver cells combined with a monolayer growth of target breast cancer cell line (MCF-7) in a static environment as a representative example of screening drug compounds for hepatotoxicity and drug efficacy. Alginate hydrogels encapsulated with serial cell densities of HepG2 cells (10(5)-10(8) cells/ml) are supported by a porous poly-carbonate disc platform and co-cultured with MCF-7 cells within standard cell culture plates during a 3 day study period. The clearance rates of drug transformation by HepG2 cells are measured using a coumarin based pro-drug. The platform was used to test for HepG2 cytotoxicity 50% (CT(50)) using commercially available drugs which further correlated well with published in vivo LD(50) values. The developed test platform allowed us to evaluate drug dose concentrations to predict hepatotoxicity and its effect on the target cells. The in vitro 3D co-culture platform provides a scalable and flexible approach to test multiple-cell types in a hybrid setting within standard cell culture plates which may open up novel 3D in vitro culture techniques to screen new chemical entity compounds. Copyright © 2011 Elsevier Inc. All rights reserved.

History of leukemia-lymphoma cell lines.

PubMed

Drexler, Hans G; Macleod, Roderick A F

2010-08-01

We outline the near 50-year history of leukemia-lymphoma (LL) cell lines - a key model system in biomedicine. Due to the detailed documentation of their oncogenomic and transcriptional alterations via recent advances in molecular medicine, LL cell lines may be fitted to parent tumors with a degree of precision unattainable in other cancers. We have surveyed the corpus of published LL cell lines and found 637 examples that meet minimum standards of authentication and characterization. Alarmingly, the rate of establishment of new LL cell lines has plummeted over the last decade. Although the main hematopoietic developmental cell types are represented by cell lines, some LL categories stubbornly resist establishment in vitro. The advent of engineering techniques for immortalizing primary human cells that maintain differentiation means the time is ripe for renewed search for in vitro models from un(der)represented hematologic entities. Given their manifold applications in biomedicine, there is little doubt that LL-derived cell lines will continue to play a vital part well into the next half-century as well. © 2010 The Authors. Human Cell © 2010 Japan Human Cell Society.

Droplet-based microtumor model to assess cell-ECM interactions and drug resistance of gastric cancer cells.

PubMed

Jang, Minjeong; Koh, Ilkyoo; Lee, Seok Jae; Cheong, Jae-Ho; Kim, Pilnam

2017-01-27

Gastric cancer (GC) is a common aggressive malignant tumor with high incidence and mortality worldwide. GC is classified into intestinal and diffuse types according to the histo-morphological features. Because of distinctly different clinico-pathological features, new cancer therapy strategies and in vitro preclinical models for the two pathological variants of GC is necessary. Since extracellular matrix (ECM) influence the biological behavior of tumor cells, we hypothesized that GC might be more similarly modeled in 3D with matrix rather than in 2D. Herein, we developed a microfluidic-based a three-dimensional (3D) in vitro gastric cancer model, with subsequent drug resistance assay. AGS (intestinal type) and Hs746T (diffuse type) gastric cancer cell lines were encapsulated in collagen beads with high cellular viability. AGS exhibited an aggregation pattern with expansive growth, whereas Hs746T showed single-cell-level infiltration. Importantly, in microtumor models, epithelial-mesenchymal transition (EMT) and metastatic genes were upregulated, whereas E-cadherin was downregulated. Expression of ß-catenin was decreased in drug-resistant cells, and chemosensitivity toward the anticancer drug (5-FU) was observed in microtumors. These results suggest that in vitro microtumor models may represent a biologically relevant platform for studying gastric cancer cell biology and tumorigenesis, and for accelerating the development of novel therapeutic targets.

Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

PubMed

Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

2012-05-01

The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

Characterization of In Vitro Engineered Human Adipose Tissues: Relevant Adipokine Secretion and Impact of TNF-α

PubMed Central

Aubin, Kim; Safoine, Meryem; Proulx, Maryse; Audet-Casgrain, Marie-Alice; Côté, Jean-François; Têtu, Félix-André; Roy, Alphonse; Fradette, Julie

2015-01-01

Representative modelling of human adipose tissue functions is central to metabolic research. Tridimensional models able to recreate human adipogenesis in a physiological tissue-like context in vitro are still scarce. We describe the engineering of white adipose tissues reconstructed from their cultured adipose-derived stromal precursor cells. We hypothesize that these reconstructed tissues can recapitulate key functions of AT under basal and pro-inflammatory conditions. These tissues, featuring human adipocytes surrounded by stroma, were stable and metabolically active in long-term cultures (at least 11 weeks). Secretion of major adipokines and growth factors by the reconstructed tissues was determined and compared to media conditioned by human native fat explants. Interestingly, the secretory profiles of the reconstructed adipose tissues indicated an abundant production of leptin, PAI-1 and angiopoietin-1 proteins, while higher HGF levels were detected for the human fat explants. We next demonstrated the responsiveness of the tissues to the pro-inflammatory stimulus TNF-α, as reflected by modulation of MCP-1, NGF and HGF secretion, while VEGF and leptin protein expression did not vary. TNF-α exposure induced changes in gene expression for adipocyte metabolism-associated mRNAs such as SLC2A4, FASN and LIPE, as well as for genes implicated in NF-κB activation. Finally, this model was customized to feature adipocytes representative of progressive stages of differentiation, thereby allowing investigations using newly differentiated or more mature adipocytes. In conclusion, we produced tridimensional tissues engineered in vitro that are able to recapitulate key characteristics of subcutaneous white adipose tissue. These tissues are produced from human cells and their neo-synthesized matrix elements without exogenous or synthetic biomaterials. Therefore, they represent unique tools to investigate the effects of pharmacologically active products on human stromal cells, extracellular matrix and differentiated adipocytes, in addition to compounds modulating adipogenesis from precursor cells. PMID:26367137

A three-phase in-vitro system for studying Pseudomonas aeruginosa adhesion and biofilm formation upon hydrogel contact lenses

PubMed Central

2010-01-01

Background Pseudomonas aeruginosa is commonly associated with contact lens (CL) -related eye infections, for which bacterial adhesion and biofilm formation upon hydrogel CLs is a specific risk factor. Whilst P. aeruginosa has been widely used as a model organism for initial biofilm formation on CLs, in-vitro models that closely reproduce in-vivo conditions have rarely been presented. Results In the current investigation, a novel in-vitro biofilm model for studying the adherence of P. aeruginosa to hydrogel CLs was established. Nutritional and interfacial conditions similar to those in the eye of a CL wearer were created through the involvement of a solid:liquid and a solid:air interface, shear forces and a complex artificial tear fluid. Bioburdens varied depending on the CL material and biofilm maturation occurred after 72 h incubation. Whilst a range of biofilm morphologies were visualised including dispersed and adherent bacterial cells, aggregates and colonies embedded in extracellular polymer substances (EPS), EPS fibres, mushroom-like formations, and crystalline structures, a compact and heterogeneous biofilm morphology predominated on all CL materials. Conclusions In order to better understand the process of biofilm formation on CLs and to test the efficacy of CL care solutions, representative in-vitro biofilm models are required. Here, we present a three-phase biofilm model that simulates the environment in the eye of a CL wearer and thus generates biofilms which resemble those commonly observed in-situ. PMID:21062489

Validation of an immortalized human (hBMEC) in vitro blood-brain barrier model.

PubMed

Eigenmann, Daniela Elisabeth; Jähne, Evelyn Andrea; Smieško, Martin; Hamburger, Matthias; Oufir, Mouhssin

2016-03-01

We recently established and optimized an immortalized human in vitro blood-brain barrier (BBB) model based on the hBMEC cell line. In the present work, we validated this mono-culture 24-well model with a representative series of drug substances which are known to cross or not to cross the BBB. For each individual compound, a quantitative UHPLC-MS/MS method in Ringer HEPES buffer was developed and validated according to current regulatory guidelines, with respect to selectivity, precision, and reliability. Various biological and analytical challenges were met during method validation, highlighting the importance of careful method development. The positive controls antipyrine, caffeine, diazepam, and propranolol showed mean endothelial permeability coefficients (P e) in the range of 17-70 × 10(-6) cm/s, indicating moderate to high BBB permeability when compared to the barrier integrity marker sodium fluorescein (mean P e 3-5 × 10(-6) cm/s). The negative controls atenolol, cimetidine, and vinblastine showed mean P e values < 10 × 10(-6) cm/s, suggesting low permeability. In silico calculations were in agreement with in vitro data. With the exception of quinidine (P-glycoprotein inhibitor and substrate), BBB permeability of all control compounds was correctly predicted by this new, easy, and fast to set up human in vitro BBB model. Addition of retinoic acid and puromycin did not increase transendothelial electrical resistance (TEER) values of the BBB model.

Recreating blood-brain barrier physiology and structure on chip: A novel neurovascular microfluidic bioreactor.

PubMed

Brown, Jacquelyn A; Pensabene, Virginia; Markov, Dmitry A; Allwardt, Vanessa; Neely, M Diana; Shi, Mingjian; Britt, Clayton M; Hoilett, Orlando S; Yang, Qing; Brewer, Bryson M; Samson, Philip C; McCawley, Lisa J; May, James M; Webb, Donna J; Li, Deyu; Bowman, Aaron B; Reiserer, Ronald S; Wikswo, John P

2015-09-01

The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier.

Short-term spheroid culture of primary colorectal cancer cells as an in vitro model for personalizing cancer medicine

PubMed Central

Jeppesen, Maria; Hagel, Grith; Glenthoj, Anders; Vainer, Ben; Ibsen, Per; Harling, Henrik; Thastrup, Ole; Jørgensen, Lars N.

2017-01-01

Chemotherapy treatment of cancer remains a challenge due to the molecular and functional heterogeneity displayed by tumours originating from the same cell type. The pronounced heterogeneity makes it difficult for oncologists to devise an effective therapeutic strategy for the patient. One approach for increasing treatment efficacy is to test the chemosensitivity of cancer cells obtained from the patient’s tumour. 3D culture represents a promising method for modelling patient tumours in vitro. The aim of this study was therefore to evaluate how closely short-term spheroid cultures of primary colorectal cancer cells resemble the original tumour. Colorectal cancer cells were isolated from human tumour tissue and cultured as spheroids. Spheroid cultures were established with a high success rate and remained viable for at least 10 days. The spheroids exhibited significant growth over a period of 7 days and no difference in growth rate was observed for spheroids of different sizes. Comparison of spheroids with the original tumour revealed that spheroid culture generally preserved adenocarcinoma histology and expression patterns of cytokeratin 20 and carcinoembryonic antigen. Interestingly, spheroids had a tendency to resemble tumour protein expression more closely after 10 days of culture compared to 3 days. Chemosensitivity screening using spheroids from five patients demonstrated individual response profiles. This indicates that the spheroids maintained patient-to-patient differences in sensitivity towards the drugs and combinations most commonly used for treatment of colorectal cancer. In summary, short-term spheroid culture of primary colorectal adenocarcinoma cells represents a promising in vitro model for use in personalized medicine. PMID:28877221

IN VITRO DIGESTIVE FLUID EXTRACTION AS A MEASURE OF THE BIOAVAILABILITY OF SEDIMENT-ASSOCIATED POLYCYCLIC AROMATIC HYDROCARBONS: SOURCES OF VARIATION AND IMPLICATIONS FOR PARTITIONING MODELS. (R825353)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Meeting Report: FutureTox II: Contemporary Concepts in Toxicology “Pathways to Prediction: In Vitro and In Silico Models for Predictive Toxicology”

EPA Science Inventory

The Society of Toxicology (SOT) held avery successful FutureTox II Contemporary Concepts in Toxicology (CCT) Conference in Chapel Hill, North Carolina, on January 16th and 17th, 2014. There were over 291 attendees representing industry, government and academia; the sessions were ...

Effect of Acylglycerol Composition and Fatty Acyl Chain Length on Lipid Digestion in pH-Stat Digestion Model and Simulated In Vitro Digestion Model.

PubMed

Qi, Jin F; Jia, Cai H; Shin, Jung A; Woo, Jeong M; Wang, Xiang Y; Park, Jong T; Hong, Soon T; Lee, K-T

2016-02-01

In this study, a pH-stat digestion model and a simulated in vitro digestion model were employed to evaluate the digestion degree of lipids depending on different acylglycerols and acyl chain length (that is, diacylglycerol [DAG] compared with soybean oil representing long-chain triacylglycerol compared with medium-chain triacylglycerol [MCT]). In the pH-stat digestion model, differences were observed among the digestion degrees of 3 oils using digestion rate (k), digestion half-time (t1/2 ), and digestion extent (Φmax). The results showed the digestion rate order was MCT > soybean oil > DAG. Accordingly, the order of digestion half-times was MCT < soybean oil < DAG. In simulated in vitro digestion model, digestion rates (k') and digestion half-times (t'1/2 ) were also obtained and the results showed a digestion rate order of MCT (k' = 0.068 min(-1) ) > soybean oil (k' = 0.037 min(-1) ) > DAG (k' = 0.024 min(-1) ). Consequently, the order of digestion half-times was MCT (t'1/2 = 10.20 min) < soybean oil (t'1/2 = 18.74 min) < DAG (t'1/2 = 29.08 min). The parameters obtained using the 2 models showed MCT was digested faster than soybean oil, and that soybean oil was digested faster than DAG. © 2015 Institute of Food Technologists®

In Vitro and in Silico Tools To Assess Extent of Cellular Uptake and Lysosomal Sequestration of Respiratory Drugs in Human Alveolar Macrophages.

PubMed

Ufuk, Ayşe; Assmus, Frauke; Francis, Laura; Plumb, Jonathan; Damian, Valeriu; Gertz, Michael; Houston, J Brian; Galetin, Aleksandra

2017-04-03

Accumulation of respiratory drugs in human alveolar macrophages (AMs) has not been extensively studied in vitro and in silico despite its potential impact on therapeutic efficacy and/or occurrence of phospholipidosis. The current study aims to characterize the accumulation and subcellular distribution of drugs with respiratory indication in human AMs and to develop an in silico mechanistic AM model to predict lysosomal accumulation of investigated drugs. The data set included 9 drugs previously investigated in rat AM cell line NR8383. Cell-to-unbound medium concentration ratio (K p,cell ) of all drugs (5 μM) was determined to assess the magnitude of intracellular accumulation. The extent of lysosomal sequestration in freshly isolated human AMs from multiple donors (n = 5) was investigated for clarithromycin and imipramine (positive control) using an indirect in vitro method (±20 mM ammonium chloride, NH 4 Cl). The AM cell parameters and drug physicochemical data were collated to develop an in silico mechanistic AM model. Three in silico models differing in their description of drug membrane partitioning were evaluated; model (1) relied on octanol-water partitioning of drugs, model (2) used in vitro data to account for this process, and model (3) predicted membrane partitioning by incorporating AM phospholipid fractions. In vitro K p,cell ranged >200-fold for respiratory drugs, with the highest accumulation seen for clarithromycin. A good agreement in K p,cell was observed between human AMs and NR8383 (2.45-fold bias), highlighting NR8383 as a potentially useful in vitro surrogate tool to characterize drug accumulation in AMs. The mean K p,cell of clarithromycin (81, CV = 51%) and imipramine (963, CV = 54%) were reduced in the presence of NH 4 Cl by up to 67% and 81%, respectively, suggesting substantial contribution of lysosomal sequestration and intracellular binding in the accumulation of these drugs in human AMs. The in vitro data showed variability in drug accumulation between individual human AM donors due to possible differences in lysosomal abundance, volume, and phospholipid content, which may have important clinical implications. Consideration of drug-acidic phospholipid interactions significantly improved the performance of the in silico models; use of in vitro K p,cell obtained in the presence of NH 4 Cl as a surrogate for membrane partitioning (model (2)) captured the variability in clarithromycin and imipramine K p,cell observed in vitro and showed the best ability to predict correctly positive and negative lysosomotropic properties. The developed mechanistic AM model represents a useful in silico tool to predict lysosomal and cellular drug concentrations based on drug physicochemical data and system specific properties, with potential application to other cell types.

Experimental psychiatric illness and drug abuse models: from human to animal, an overview.

PubMed

Edwards, Scott; Koob, George F

2012-01-01

Preclinical animal models have supported much of the recent rapid expansion of neuroscience research and have facilitated critical discoveries that undoubtedly benefit patients suffering from psychiatric disorders. This overview serves as an introduction for the following chapters describing both in vivo and in vitro preclinical models of psychiatric disease components and briefly describes models related to drug dependence and affective disorders. Although there are no perfect animal models of any psychiatric disorder, models do exist for many elements of each disease state or stage. In many cases, the development of certain models is essentially restricted to the human clinical laboratory domain for the purpose of maximizing validity, whereas the use of in vitro models may best represent an adjunctive, well-controlled means to model specific signaling mechanisms associated with psychiatric disease states. The data generated by preclinical models are only as valid as the model itself, and the development and refinement of animal models for human psychiatric disorders continues to be an important challenge. Collaborative relationships between basic neuroscience and clinical modeling could greatly benefit the development of new and better models, in addition to facilitating medications development.

Human Cerebrospinal Fluid Promotes Neuronal Viability and Activity of Hippocampal Neuronal Circuits In Vitro

PubMed Central

Perez-Alcazar, Marta; Culley, Georgia; Lyckenvik, Tim; Mobarrez, Kristoffer; Bjorefeldt, Andreas; Wasling, Pontus; Seth, Henrik; Asztely, Frederik; Harrer, Andrea; Iglseder, Bernhard; Aigner, Ludwig; Hanse, Eric; Illes, Sebastian

2016-01-01

For decades it has been hypothesized that molecules within the cerebrospinal fluid (CSF) diffuse into the brain parenchyma and influence the function of neurons. However, the functional consequences of CSF on neuronal circuits are largely unexplored and unknown. A major reason for this is the absence of appropriate neuronal in vitro model systems, and it is uncertain if neurons cultured in pure CSF survive and preserve electrophysiological functionality in vitro. In this article, we present an approach to address how human CSF (hCSF) influences neuronal circuits in vitro. We validate our approach by comparing the morphology, viability, and electrophysiological function of single neurons and at the network level in rat organotypic slice and primary neuronal cultures cultivated either in hCSF or in defined standard culture media. Our results demonstrate that rodent hippocampal slices and primary neurons cultured in hCSF maintain neuronal morphology and preserve synaptic transmission. Importantly, we show that hCSF increases neuronal viability and the number of electrophysiologically active neurons in comparison to the culture media. In summary, our data indicate that hCSF represents a physiological environment for neurons in vitro and a superior culture condition compared to the defined standard media. Moreover, this experimental approach paves the way to assess the functional consequences of CSF on neuronal circuits as well as suggesting a novel strategy for central nervous system (CNS) disease modeling. PMID:26973467

Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro.

PubMed

Knight, Eleanor; Przyborski, Stefan

2015-12-01

Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co-culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue-like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two-dimensional (2D) substrates and review the popular approaches currently available that enable the development of three-dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold-free or scaffold-based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science. © 2014 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.

Calcified lesion modeling for excimer laser ablation

NASA Astrophysics Data System (ADS)

Scott, Holly A.; Archuleta, Andrew; Splinter, Robert

2009-06-01

Objective: Develop a representative calcium target model to evaluate penetration of calcified plaque lesions during atherectomy procedures using 308 nm Excimer laser ablation. Materials and Methods: An in-vitro model representing human calcified plaque was analyzed using Plaster-of-Paris and cement based composite materials as well as a fibrinogen model. The materials were tested for mechanical consistency. The most likely candidate(s) resulting from initial mechanical and chemical screening was submitted for ablation testing. The penetration rate of specific multi-fiber catheter designs and a single fiber probe was obtained and compared to that in human cadaver calcified plaque. The effects of lasing parameters and catheter tip design on penetration speed in a representative calcified model were verified against the results in human cadaver specimens. Results: In Plaster of Paris, the best penetration was obtained using the single fiber tip configuration operating at 100 Fluence, 120 Hz. Calcified human lesions are twice as hard, twice as elastic as and much more complex than Plaster of Paris. Penetration of human calcified specimens was highly inconsistent and varied significantly from specimen to specimen and within individual specimens. Conclusions: Although Plaster of Paris demonstrated predictable increases in penetration with higher energy density and repetition rate, it can not be considered a totally representative laser ablation model for calcified lesions. This is in part due to the more heterogeneous nature and higher density composition of cadaver intravascular human calcified occlusions. Further testing will require a more representative model of human calcified lesions.

In Vitro Exposure Systems and Dosimetry Assessment Tools ...

EPA Pesticide Factsheets

In 2009, the passing of The Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP) and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed “modified risk”. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference titled “In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products” to bring together stakeholders representing regulatory agencies, academia, and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapor exposure systems, as well as the various approaches and challenges to quantifying the complex exposures, in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were, 1) Tobacco Smoke And E-Cigarette Aerosols, 2) Air-Liquid Interface-In Vitro Exposure Systems, 3) Dosimetry Approaches For Particles And Vapors; In Vitro Dosimetry Determinations and 4) Exposure Microenvironment/Physiology Of Cells. The two and a half day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will re

Energetics of ligand-receptor binding affinity on endothelial cells: An in vitro model.

PubMed

Fotticchia, Iolanda; Guarnieri, Daniela; Fotticchia, Teresa; Falanga, Andrea Patrizia; Vecchione, Raffaele; Giancola, Concetta; Netti, Paolo Antonio

2016-08-01

Targeted therapies represent a challenge in modern medicine. In this contest, we propose a rapid and reliable methodology based on Isothermal Titration Calorimetry (ITC) coupled with confluent cell layers cultured around biocompatible templating microparticles to quantify the number of overexpressing receptors on cell membrane and study the energetics of receptor-ligand binding in near-physiological conditions. In the in vitro model here proposed we used the bEnd3 cell line as brain endothelial cells to mimic the blood brain barrier (BBB) cultured on dextran microbeads ranging from 67μm to 80μm in size (Cytodex) and the primary human umbilical vein cells (HUVEC) for comparison. The revealed affinity between transferrin (Tf) and transferrin receptor (TfR) in both systems is very high, Kd values are in the order of nM. Conversely, the value of TfRs/cell reveals a 100-fold increase in the number of TfRs per bEnd3 cells compared to HUVEC cells. The presented methodology can represent a novel and helpful strategy to identify targets, to address drug design and selectively deliver therapeutics that can cross biological barriers such as the blood brain barrier. Copyright © 2016 Elsevier B.V. All rights reserved.

A first vascularized skin equivalent as an alternative to animal experimentation.

PubMed

Groeber, Florian; Engelhardt, Lisa; Lange, Julia; Kurdyn, Szymon; Schmid, Freia F; Rücker, Christoph; Mielke, Stephan; Walles, Heike; Hansmann, Jan

2016-01-01

Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research.

Use of in-vitro experimental results to model in-situ experiments: bio-denitrification under geological disposal conditions.

PubMed

Masuda, Kaoru; Murakami, Hiroshi; Kurimoto, Yoshitaka; Kato, Osamu; Kato, Ko; Honda, Akira

2013-01-01

Some of the low level radioactive wastes from reprocessing of spent nuclear fuels contain nitrates. Nitrates can be present in the form of soluble salts and can be reduced by various reactions. Among them, reduction by metal compounds and microorganisms seems to be important in the underground repository. Reduction by microorganism is more important in near field area than inside the repository because high pH and extremely high salt concentration would prevent microorganism activities. In the near field, pH is more moderate (pH is around 8) and salt concentration is lower. However, the electron donor may be limited there and it might be the control factor for microorganism's denitrification activities. In this study, in-vitro experiments of the nitrate reduction reaction were conducted using model organic materials purported to exist in underground conditions relevant to geological disposal. Two kinds of organic materials were selected. A super plasticizer was selected as being representative of the geological disposal system and humic acid was selected as being representative of pre-existing organic materials in the bedrock. Nitrates were reduced almost to N2 gas in the existence of super plasticizer. In the case of humic acids, although nitrates were reduced, the rate was much lower and, in this case, dead organism was used as an electron donor instead of humic acids. A reaction model was developed based on the in-vitro experiments and verified by running simulations against data obtained from in-situ experiments using actual groundwaters and microorganisms. The simulation showed a good correlation with the experimental data and contributes to the understanding of microbially mediated denitrification in geological disposal systems.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project.

PubMed

de Hoogt, Ronald; Estrada, Marta F; Vidic, Suzana; Davies, Emma J; Osswald, Annika; Barbier, Michael; Santo, Vítor E; Gjerde, Kjersti; van Zoggel, Hanneke J A A; Blom, Sami; Dong, Meng; Närhi, Katja; Boghaert, Erwin; Brito, Catarina; Chong, Yolanda; Sommergruber, Wolfgang; van der Kuip, Heiko; van Weerden, Wytske M; Verschuren, Emmy W; Hickman, John; Graeser, Ralph

2017-11-21

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project

PubMed Central

de Hoogt, Ronald; Estrada, Marta F.; Vidic, Suzana; Davies, Emma J.; Osswald, Annika; Barbier, Michael; Santo, Vítor E.; Gjerde, Kjersti; van Zoggel, Hanneke J. A. A.; Blom, Sami; Dong, Meng; Närhi, Katja; Boghaert, Erwin; Brito, Catarina; Chong, Yolanda; Sommergruber, Wolfgang; van der Kuip, Heiko; van Weerden, Wytske M.; Verschuren, Emmy W.; Hickman, John; Graeser, Ralph

2017-01-01

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented. PMID:29160867

Complete In Vitro Life Cycle of Trypanosoma congolense: Development of Genetic Tools

PubMed Central

Plazolles, Nicolas; Baltz, Théo

2010-01-01

Background Animal African trypanosomosis, a disease mainly caused by the protozoan parasite Trypanosoma congolense, is a major constraint to livestock productivity and has a significant impact in the developing countries of Africa. RNA interference (RNAi) has been used to study gene function and identify drug and vaccine targets in a variety of organisms including trypanosomes. However, trypanosome RNAi studies have mainly been conducted in T. brucei, as a model for human infection, largely ignoring livestock parasites of economical importance such as T. congolense, which displays different pathogenesis profiles. The whole T. congolense life cycle can be completed in vitro, but this attractive model displayed important limitations: (i) genetic tools were currently limited to insect forms and production of modified infectious BSF through differentiation was never achieved, (ii) in vitro differentiation techniques lasted several months, (iii) absence of long-term bloodstream forms (BSF) in vitro culture prevented genomic analyses. Methodology/Principal Findings We optimized culture conditions for each developmental stage and secured the differentiation steps. Specifically, we devised a medium adapted for the strenuous development of stable long-term BSF culture. Using Amaxa nucleofection technology, we greatly improved the transfection rate of the insect form and designed an inducible transgene expression system using the IL3000 reference strain. We tested it by expression of reporter genes and through RNAi. Subsequently, we achieved the complete in vitro life cycle with dramatically shortened time requirements for various wild type and transgenic strains. Finally, we established the use of modified strains for experimental infections and underlined a host adaptation phase requirement. Conclusions/Significance We devised an improved T. congolense model, which offers the opportunity to perform functional genomics analyses throughout the whole life cycle. It represents a very useful tool to understand pathogenesis mechanisms and to study potential therapeutic targets either in vitro or in vivo using a mouse model. PMID:20209144

Analysis of the material properties of early chondrogenic differentiated adipose-derived stromal cells (ASC) using an in vitro three-dimensional micromass culture system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yue; Balooch, Guive; Chiou, Michael

2007-07-27

Cartilage is an avascular tissue with only a limited potential to heal and chondrocytes in vitro have poor proliferative capacity. Recently, adipose-derived stromal cells (ASC) have demonstrated a great potential for application to tissue engineering due to their ability to differentiate into cartilage, bone, and fat. In this study, we have utilized a high density three-dimensional (3D) micromass model system of early chondrogenesis with ASC. The material properties of these micromasses showed a significant increase in dynamic and static elastic modulus during the early chondrogenic differentiation process. These data suggest that the 3D micromass culture system represents an in vitromore » model of early chondrogenesis with dynamic cell signaling interactions associated with the mechanical properties of chondrocyte differentiation.« less

Establishment of pseudovirus infection mouse models for in vivo pharmacodynamics evaluation of filovirus entry inhibitors.

PubMed

Chen, Qing; Tang, Ke; Zhang, Xiaoyu; Chen, Panpan; Guo, Ying

2018-03-01

Filoviruses cause severe and fatal viral hemorrhagic fever in humans. Filovirus research has been extensive since the 2014 Ebola outbreak. Due to their high pathogenicity and mortality, live filoviruses require Biosafety Level-4 (BSL-4) facilities, which have restricted the development of anti-filovirus vaccines and drugs. An HIV-based pseudovirus cell infection assay is widely used for viral entry studies in BSL-2 conditions. Here, we successfully constructed nine in vitro pseudo-filovirus models covering all filovirus genera and three in vivo pseudo-filovirus-infection mouse models using Ebola virus, Marburg virus, and Lloviu virus as representative viruses. The pseudo-filovirus-infected mice showed visualizing bioluminescence in a dose-dependent manner. A bioluminescence peak in mice was reached on day 5 post-infection for Ebola virus and Marburg virus and on day 4 post-infection for Lloviu virus. Two known filovirus entry inhibitors, clomiphene and toremiphene, were used to validate the model. Collectively, our study shows that all genera of filoviruses can be well-pseudotyped and are infectious in vitro . The pseudo-filovirus-infection mouse models can be used for in vivo activity evaluation of anti-filovirus drugs. This sequential in vitro and in vivo evaluation system of filovirus entry inhibitors provides a secure and efficient platform for screening and assessing anti-filovirus agents in BSL-2 facilities.

A novel fully-humanised 3D skin equivalent to model early melanoma invasion

PubMed Central

Hill, David S; Robinson, Neil D P; Caley, Matthew P; Chen, Mei; O’Toole, Edel A; Armstrong, Jane L; Przyborski, Stefan; Lovat, Penny E

2015-01-01

Metastatic melanoma remains incurable, emphasising the acute need for improved research models to investigate the underlying biological mechanisms mediating tumour invasion and metastasis, and to develop more effective targeted therapies to improve clinical outcome. Available animal models of melanoma do not accurately reflect human disease and current in vitro human skin equivalent models incorporating melanoma cells are not fully representative of the human skin microenvironment. We have developed a robust and reproducible, fully-humanised 3D skin equivalent comprising a stratified, terminally differentiated epidermis and a dermal compartment consisting of fibroblast-generated extracellular matrix. Melanoma cells incorporated into the epidermis were able to invade through the basement membrane and into the dermis, mirroring early tumour invasion in vivo. Comparison of our novel 3D melanoma skin equivalent with melanoma in situ and metastatic melanoma indicates this model accurately recreates features of disease pathology, making it a physiologically representative model of early radial and vertical growth phase melanoma invasion. PMID:26330548

Combination of caspofungin or anidulafungin with antimicrobial peptides results in potent synergistic killing of Candida albicans and Candida glabrata in vitro.

PubMed

Harris, Mark R; Coote, Peter J

2010-04-01

Administering synergistic combinations of antifungals could be a route to overcome problems with toxicity and the development of resistance. Combination of the echinocandins caspofungin or anidulafungin with a range of structurally diverse antimicrobial peptides resulted in potent synergistic killing of Candida spp. in vitro. Fungicidal synergy was measured by calculating fractional inhibitory concentration indices from checkerboard assays as well as loss of viability. Inhibitory combinations of the antifungals did not induce cytotoxicity in vitro. However, in a murine model of systemic candidiasis, co-administration of caspofungin with one example of the cationic peptides tested, ranalexin, did not show enhanced efficacy compared with the single treatments alone. Further study using alternative peptides will identify whether this combination approach could represent a novel treatment for fungal pathogens. (c) 2009 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Ex vitro composite plants: an inexpensive, rapid method for root biology.

PubMed

Collier, Ray; Fuchs, Beth; Walter, Nathalie; Kevin Lutke, William; Taylor, Christopher G

2005-08-01

Plant transformation technology is frequently the rate-limiting step in gene function analysis in non-model plants. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. The novel, rapid and inexpensive ex vitro method for producing composite plants described in this report represents a significant advance over existing composite plant induction protocols, which rely on expensive and time-consuming in vitro conditions. The utility of the new system is validated by expression and RNAi silencing of GFP in transgenic roots of composite plants, and is bolstered further by experimental disruption, via RNAi silencing, of endogenous plant resistance to the plant parasitic nematode Meloidogyne incognita in transgenic roots of Lycopersicon esculentum cv. Motelle composite plants. Critical parameters of the method are described and discussed herein.

Cheese rind communities provide tractable systems for in situ and in vitro studies of microbial diversity

PubMed Central

Wolfe, Benjamin E.; Button, Julie E.; Santarelli, Marcela; Dutton, Rachel J.

2014-01-01

SUMMARY Tractable microbial communities are needed to bridge the gap between observations of patterns of microbial diversity and mechanisms that can explain these patterns. We developed cheese rinds as model microbial communities by characterizing in situ patterns of diversity and by developing an in vitro system for community reconstruction. Sequencing of 137 different rind communities across 10 countries revealed 24 widely distributed and culturable genera of bacteria and fungi as dominant community members. Reproducible community types formed independent of geographic location of production. Intensive temporal sampling demonstrated that assembly of these communities is highly reproducible. Patterns of community composition and succession observed in situ can be recapitulated in a simple in vitro system. Widespread positive and negative interactions were identified between bacterial and fungal community members. Cheese rind microbial communities represent an experimentally tractable system for defining mechanisms that influence microbial community assembly and function. PMID:25036636

In vivo and in vitro toxicological effects of titanium dioxide nanoparticles on small intestine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tassinari, Roberta; La Rocca, Cinzia; Tait, Sabrina

2015-06-23

In European Union, titanium dioxide (TiO{sub 2}) as bulk material is a food additive (E171) and - as nanoparticle (NP) - is used as a white pigment in several products (e.g. food, cosmetics, drugs). E171 contains approximately 36% of particles less than 100 nm in at least one dimension and TiO{sub 2} NP exposure is estimated fairly below 2.5 mg/person/day. The gastrointestinal tract is a route of entry for NPs, thus representing a potential target of effects. In in vivo study, the effects of TiO{sub 2} NP in adult rat small intestine have been evaluated by oral administration of 0 (CTRL), 1more » and 2 mg/kg body weight per day - relevant to human dietary intake. Detailed quali/quantitative histopathological analyses were performed on CTRL and treated rat samples. Scanning electron microscopy (SEM) analysis was performed on small intestine. An in vitro study on Caco-2 cells was also used in order to evaluate the potential cytotoxic effects directly on enterocytes through the lactate dehydrogenase (LDH) assay. Suspensions of TiO{sub 2} NPs for in vitro and in vivo study were characterized by EM. Histomorphometrical data showed treatment-related changes of villus height and widths in male rats. Significantly different from CTRL decreased LDH levels in the medium were detected in vitro at 24h with 2.5, 5, 10 and 20 µg/cm{sup 2} levels of TiO{sub 2} NPs. SEM analysis showed no damaged areas. Overall the results showed that enterocytes may represent a target of TiO{sub 2} NP toxicity by direct exposure both in vivo and in vitro models.« less

In vivo and in vitro toxicological effects of titanium dioxide nanoparticles on small intestine

NASA Astrophysics Data System (ADS)

Tassinari, Roberta; La Rocca, Cinzia; Stecca, Laura; Tait, Sabrina; De Berardis, Barbara; Ammendolia, Maria Grazia; Iosi, Francesca; Di Virgilio, Antonio; Martinelli, Andrea; Maranghi, Francesca

2015-06-01

In European Union, titanium dioxide (TiO2) as bulk material is a food additive (E171) and - as nanoparticle (NP) - is used as a white pigment in several products (e.g. food, cosmetics, drugs). E171 contains approximately 36% of particles less than 100 nm in at least one dimension and TiO2 NP exposure is estimated fairly below 2.5 mg/person/day. The gastrointestinal tract is a route of entry for NPs, thus representing a potential target of effects. In in vivo study, the effects of TiO2 NP in adult rat small intestine have been evaluated by oral administration of 0 (CTRL), 1 and 2 mg/kg body weight per day - relevant to human dietary intake. Detailed quali/quantitative histopathological analyses were performed on CTRL and treated rat samples. Scanning electron microscopy (SEM) analysis was performed on small intestine. An in vitro study on Caco-2 cells was also used in order to evaluate the potential cytotoxic effects directly on enterocytes through the lactate dehydrogenase (LDH) assay. Suspensions of TiO2 NPs for in vitro and in vivo study were characterized by EM. Histomorphometrical data showed treatment-related changes of villus height and widths in male rats. Significantly different from CTRL decreased LDH levels in the medium were detected in vitro at 24h with 2.5, 5, 10 and 20 µg/cm2 levels of TiO2 NPs. SEM analysis showed no damaged areas. Overall the results showed that enterocytes may represent a target of TiO2 NP toxicity by direct exposure both in vivo and in vitro models.

Predicting hepatotoxicity using ToxCast in vitro bioactivity and ...

EPA Pesticide Factsheets

Background: The U.S. EPA ToxCastTM program is screening thousands of environmental chemicals for bioactivity using hundreds of high-throughput in vitro assays to build predictive models of toxicity. We represented chemicals based on bioactivity and chemical structure descriptors then used supervised machine learning to predict their hepatotoxic effects.Results: A set of 677 chemicals were represented by 711 in vitro bioactivity descriptors (from ToxCast assays), 4,376 chemical structure descriptors (from QikProp, OpenBabel, PADEL, and PubChem), and three hepatotoxicity categories (from animal studies). Hepatotoxicants were defined by rat liver histopathology observed after chronic chemical testing and grouped into hypertrophy (161), injury (101) and proliferative lesions (99). Classifiers were built using six machine learning algorithms: linear discriminant analysis (LDA), Naïve Bayes (NB), support vector classification (SVM), classification and regression trees (CART), k-nearest neighbors (KNN) and an ensemble of classifiers (ENSMB). Classifiers of hepatotoxicity were built using chemical structure, ToxCast bioactivity, and a hybrid representation. Predictive performance was evaluated using 10-fold cross-validation testing and in-loop, filter-based, feature subset selection. Hybrid classifiers had the best balanced accuracy for predicting hypertrophy (0.78±0.08), injury (0.73±0.10) and proliferative lesions (0.72±0.09). Though chemical and bioactivity class

The development of a tissue-engineered tracheobronchial epithelial model using a bilayered collagen-hyaluronate scaffold.

PubMed

O'Leary, Cian; Cavanagh, Brenton; Unger, Ronald E; Kirkpatrick, C James; O'Dea, Shirley; O'Brien, Fergal J; Cryan, Sally-Ann

2016-04-01

Today, chronic respiratory disease is one of the leading causes of mortality globally. Epithelial dysfunction can play a central role in its pathophysiology. The development of physiologically-representative in vitro model systems using tissue-engineered constructs might improve our understanding of epithelial tissue and disease. This study sought to engineer a bilayered collagen-hyaluronate (CHyA-B) scaffold for the development of a physiologically-representative 3D in vitro tracheobronchial epithelial co-culture model. CHyA-B scaffolds were fabricated by integrating a thin film top-layer into a porous sub-layer with lyophilisation. The film layer firmly connected to the sub-layer with delamination occurring at stresses of 12-15 kPa. Crosslinked scaffolds had a compressive modulus of 1.9 kPa and mean pore diameters of 70 μm and 80 μm, depending on the freezing temperature. Histological analysis showed that the Calu-3 bronchial epithelial cell line attached and grew on CHyA-B with adoption of an epithelial monolayer on the film layer. Immunofluorescence and qRT-PCR studies demonstrated that the CHyA-B scaffolds facilitated Calu-3 cell differentiation, with enhanced mucin expression, increased ciliation and the formation of intercellular tight junctions. Co-culture of Calu-3 cells with Wi38 lung fibroblasts was achieved on the scaffold to create a submucosal tissue analogue of the upper respiratory tract, validating CHyA-B as a platform to support co-culture and cellular organisation reminiscent of in vivo tissue architecture. In summary, this study has demonstrated that CHyA-B is a promising tool for the development of novel 3D tracheobronchial co-culture in vitro models with the potential to unravel new pathways in drug discovery and drug delivery. Copyright © 2016 Elsevier Ltd. All rights reserved.

Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro.

PubMed

Paolinelli, Roberta; Corada, Monica; Ferrarini, Luca; Devraj, Kavi; Artus, Cédric; Czupalla, Cathrin J; Rudini, Noemi; Maddaluno, Luigi; Papa, Eleanna; Engelhardt, Britta; Couraud, Pierre Olivier; Liebner, Stefan; Dejana, Elisabetta

2013-01-01

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

An Advanced In Vitro Technology Platform to Study the Mechanism of Action of Prebiotics and Probiotics in the Gastrointestinal Tract.

PubMed

Marzorati, Massimo; Van de Wiele, Tom

The gastrointestinal tract (GIT) hosts the most complex microbial community in the human body. Given the extensive metabolic potential which is present in this community, this additional organ is of key importance to maintain a healthy status and several diseases are frequently correlated with an alteration of the composition/functionality of the gut microbiota. Consequently, there is a great interest in identifying potential approaches that could modulate the microbiota and its metabolism to bring about a positive health effect. A classical approach to reach this goal is the use of prebiotics and/or probiotics. How to study the potential effect of new prebiotics/probiotics and how to localize this effect along the full GIT? Human intervention trials are the golden standard to validate functional properties of food products. Yet, most studies on gut microbiota are based on the analysis of fecal samples because they are easily collected in a non-invasive manner. A complementary option is represented by well-designed in vitro simulation technologies. Among all the available systems, the Simulator of Human Intestinal Microbial Ecosystem has already been shown to be a useful model for nutrition studies in terms of analysis of the intestinal microbial community composition and activity. The Simulator of Human Intestinal Microbial Ecosystem is a scientifically validated platform representing the physiology and microbiology of the adult human GIT. Furthermore, recent advances in in vitro modelling also allow to combine the study of bacteria-host interactions, such as mucosal adhesion and interaction with the immune system, thereby further increasing the value of the scientific output.

What experimental approaches (eg, in vivo, in vitro, tissue retrieval) are effective in investigating the biologic effects of particles?

PubMed Central

Bostrom, Mathias; O'Keefe, Regis

2009-01-01

Understanding the complex cellular and tissue mechanisms and interactions resulting in periprosthetic osteolysis requires a number of experimental approaches, each of which has its own set of advantages and limitations. In vitro models allow for the isolation of individual cell populations and have furthered our understanding of particle-cell interactions; however, they are limited because they do not mimic the complex tissue environment in which multiple cell interactions occur. In vivo animal models investigate the tissue interactions associated with periprosthetic osteolysis, but the choice of species and whether the implant system is subjected to mechanical load or to unloaded conditions are critical in assessing whether these models can be extrapolated to the clinical condition. Rigid analysis of retrieved tissue from clinical cases of osteolysis offers a different approach to studying the biologic process of osteolysis, but it is limited in that the tissue analyzed represents the end-stage of this process and, thus, may not reflect this process adequately. PMID:18612016

What experimental approaches (eg, in vivo, in vitro, tissue retrieval) are effective in investigating the biologic effects of particles?

PubMed

Bostrom, Mathias; O'Keefe, Regis

2008-01-01

Understanding the complex cellular and tissue mechanisms and interactions resulting in periprosthetic osteolysis requires a number of experimental approaches, each of which has its own set of advantages and limitations. In vitro models allow for the isolation of individual cell populations and have furthered our understanding of particle-cell interactions; however, they are limited because they do not mimic the complex tissue environment in which multiple cell interactions occur. In vivo animal models investigate the tissue interactions associated with periprosthetic osteolysis, but the choice of species and whether the implant system is subjected to mechanical load or to unloaded conditions are critical in assessing whether these models can be extrapolated to the clinical condition. Rigid analysis of retrieved tissue from clinical cases of osteolysis offers a different approach to studying the biologic process of osteolysis, but it is limited in that the tissue analyzed represents the end-stage of this process and, thus, may not reflect this process adequately.

Formulation development of physiological environment responsive periodontal drug delivery system for local delviery of metronidazole benzoate.

PubMed

Dabhi, Mahesh R; Sheth, Navin R

2013-03-01

The objective of the present investigation was to develop and evaluate physiological environment responsive periodontal drug delivery system (PERPDDS) for local delivery of metronidazole benzoate. Poly-ϵ-caprolactone an in situ precipitating polymer was used in combination with, carbopol 934P, a pH simulative polymer to develop PERPDDS. The prepared PERPDDS was evaluated for various parameters such as in vitro gelling capacity, viscosity, rheology, compatibility study, and in vitro diffusion study. A 3(2) full factorial design was used to investigate the influence of formulation variables. Drug release data from all formulations were fitted to different kinetic models and the korsemeyer-peppas model was found the best fit model. The value of diffusional exponent (n) was in between 0.3283 and 0.3979 indicating purely fickian diffusion release mechanism. Increasing the concentration of each polymeric component increases viscosity, and time for 50% and 90% drug release was observed and graphically represented by the surface response and contour plots.

Biomimetic engineered muscle with capacity for vascular integration and functional maturation in vivo

PubMed Central

Juhas, Mark; Engelmayr, George C.; Fontanella, Andrew N.; Palmer, Gregory M.; Bursac, Nenad

2014-01-01

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration. PMID:24706792

An untargeted multi-technique metabolomics approach to studying intracellular metabolites of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

PubMed

Ruiz-Aracama, Ainhoa; Peijnenburg, Ad; Kleinjans, Jos; Jennen, Danyel; van Delft, Joost; Hellfrisch, Caroline; Lommen, Arjen

2011-05-20

In vitro cell systems together with omics methods represent promising alternatives to conventional animal models for toxicity testing. Transcriptomic and proteomic approaches have been widely applied in vitro but relatively few studies have used metabolomics. Therefore, the goal of the present study was to develop an untargeted methodology for performing reproducible metabolomics on in vitro systems. The human liver cell line HepG2, and the well-known hepatotoxic and non-genotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), were used as the in vitro model system and model toxicant, respectively. The study focused on the analysis of intracellular metabolites using NMR, LC-MS and GC-MS, with emphasis on the reproducibility and repeatability of the data. State of the art pre-processing and alignment tools and multivariate statistics were used to detect significantly altered levels of metabolites after exposing HepG2 cells to TCDD. Several metabolites identified using databases, literature and LC-nanomate-Orbitrap analysis were affected by the treatment. The observed changes in metabolite levels are discussed in relation to the reported effects of TCDD. Untargeted profiling of the polar and apolar metabolites of in vitro cultured HepG2 cells is a valid approach to studying the effects of TCDD on the cell metabolome. The approach described in this research demonstrates that highly reproducible experiments and correct normalization of the datasets are essential for obtaining reliable results. The effects of TCDD on HepG2 cells reported herein are in agreement with previous studies and serve to validate the procedures used in the present work.

An in vitro biofilm model associated to dental implants: structural and quantitative analysis of in vitro biofilm formation on different dental implant surfaces.

PubMed

Sánchez, M C; Llama-Palacios, A; Fernández, E; Figuero, E; Marín, M J; León, R; Blanc, V; Herrera, D; Sanz, M

2014-10-01

The impact of implant surfaces in dental biofilm development is presently unknown. The aim of this investigation was to assess in vitro the development of a complex biofilm model on titanium and zirconium implant surfaces, and to compare it with the same biofilm formed on hydroxyapatite surface. Six standard reference strains were used to develop an in vitro biofilm over sterile titanium, zirconium and hydroxyapatite discs, coated with saliva within the wells of pre-sterilized polystyrene tissue culture plates. The selected species used represent initial (Streptococcus oralis and Actinomyces naeslundii), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late colonizers (Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans). The developed biofilms (growth time 1 to 120h) were studied with confocal laser scanning microscopy using a vital fluorescence technique and with low-temperature scanning electron microscopy. The number (colony forming units/biofilm) and kinetics of the bacteria within the biofilm were studied with quantitative PCR (qPCR). As outcome variables, the biofilm thickness, the percentage of cell vitality and the number of bacteria were compared using the analysis of variance. The bacteria adhered and matured within the biofilm over the three surfaces with similar dynamics. Different surfaces, however, demonstrated differences both in the thickness, deposition of the extracellular polysaccharide matrix as well as in the organization of the bacterial cells. While the formation and dynamics of an in vitro biofilm model was similar irrespective of the surface of inoculation (hydroxyapatite, titanium or zirconium), there were significant differences in regards to the biofilm thickness and three-dimensional structure. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Biohybrid Membrane Systems and Bioreactors as Tools for In Vitro Drug Testing.

PubMed

Salerno, Simona; Bartolo, Loredana De

2017-01-01

In drug development, in vitro human model systems are absolutely essential prior to the clinical trials, considering the increasing number of chemical compounds in need of testing, and, keeping in mind that animals cannot predict all the adverse human health effects and reactions, due to the species-specific differences in metabolic pathways. The liver plays a central role in the clearance and biotransformation of chemicals and xenobiotics. In vitro liver model systems by using highly differentiated human cells could have a great impact in preclinical trials. Membrane biohybrid systems constituted of human hepatocytes and micro- and nano-structured membranes, represent valuable tools for studying drug metabolism and toxicity. Membranes act as an extracellular matrix for the adhesion of hepatocytes, and compartmentalise them in a well-defined physical and chemical microenvironment with high selectivity. Advanced 3-D tissue cultures are furthermore achieved by using membrane bioreactors (MBR), which ensure the continuous perfusion of cells protecting them from shear stress. MBRs with different configurations allow the culturing of cells at high density and under closely monitored high perfusion, similarly to the natural liver. These devices that promote the long-term maintenance and differentiation of primary human hepatocytes with preserved liver specific functions can be employed in drug testing for prolonged exposure to chemical compounds and for assessing repeated-dose toxicity. The use of primary human hepatocytes in MBRs is the only system providing a faster and more cost-effective method of analysis for the prediction of in vitro human drug metabolism and enzyme induction alternative and/or complementary to the animal experimentation. In this paper, in vitro models for studying drug metabolism and toxicity as advanced biohybrid membrane systems and MBRs will be reviewed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

In vitro and in vivo models of colorectal cancer: antigenotoxic activity of berries.

PubMed

Brown, Emma M; Latimer, Cheryl; Allsopp, Philip; Ternan, Nigel G; McMullan, Geoffery; McDougall, Gordon J; Stewart, Derek; Crozier, Alan; Rowland, Ian; Gill, Chris I R

2014-05-07

The etiology of colorectal cancer (CRC), a common cause of cancer-related mortality globally, has strong associations with diet. There is considerable epidemiological evidence that fruits and vegetables are associated with reduced risk of CRC. This paper reviews the extensive evidence, both from in vitro studies and animal models, that components of berry fruits can modulate biomarkers of DNA damage and that these effects may be potentially chemoprotective, given the likely role that oxidative damage plays in mutation rate and cancer risk. Human intervention trials with berries are generally consistent in indicating a capacity to significantly decrease oxidative damage to DNA, but represent limited evidence for anticarcinogenicity, relying as they do on surrogate risk markers. To understand the effects of berry consumption on colorectal cancer risk, future studies will need to be well controlled, with defined berry extracts, using suitable and clinically relevant end points and considering the importance of the gut microbiota.

Machine Learning Model Analysis and Data Visualization with Small Molecules Tested in a Mouse Model of Mycobacterium tuberculosis Infection (2014–2015)

PubMed Central

2016-01-01

The renewed urgency to develop new treatments for Mycobacterium tuberculosis (Mtb) infection has resulted in large-scale phenotypic screening and thousands of new active compounds in vitro. The next challenge is to identify candidates to pursue in a mouse in vivo efficacy model as a step to predicting clinical efficacy. We previously analyzed over 70 years of this mouse in vivo efficacy data, which we used to generate and validate machine learning models. Curation of 60 additional small molecules with in vivo data published in 2014 and 2015 was undertaken to further test these models. This represents a much larger test set than for the previous models. Several computational approaches have now been applied to analyze these molecules and compare their molecular properties beyond those attempted previously. Our previous machine learning models have been updated, and a novel aspect has been added in the form of mouse liver microsomal half-life (MLM t1/2) and in vitro-based Mtb models incorporating cytotoxicity data that were used to predict in vivo activity for comparison. Our best Mtbin vivo models possess fivefold ROC values > 0.7, sensitivity > 80%, and concordance > 60%, while the best specificity value is >40%. Use of an MLM t1/2 Bayesian model affords comparable results for scoring the 60 compounds tested. Combining MLM stability and in vitroMtb models in a novel consensus workflow in the best cases has a positive predicted value (hit rate) > 77%. Our results indicate that Bayesian models constructed with literature in vivoMtb data generated by different laboratories in various mouse models can have predictive value and may be used alongside MLM t1/2 and in vitro-based Mtb models to assist in selecting antitubercular compounds with desirable in vivo efficacy. We demonstrate for the first time that consensus models of any kind can be used to predict in vivo activity for Mtb. In addition, we describe a new clustering method for data visualization and apply this to the in vivo training and test data, ultimately making the method accessible in a mobile app. PMID:27335215

An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm.

PubMed

Zhao, Ke-Qing; Wu, Yang; Yi, Yu-Xi; Feng, Si-Jia; Wei, Ruo-Yan; Ma, Ying; Zheng, Chun-Quan; Qu, Di

2018-04-16

Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.

A new mild hyperthermia device to treat vascular involvement in cancer surgery.

PubMed

Ware, Matthew J; Nguyen, Lam P; Law, Justin J; Krzykawska-Serda, Martyna; Taylor, Kimberly M; Cao, Hop S Tran; Anderson, Andrew O; Pulikkathara, Merlyn; Newton, Jared M; Ho, Jason C; Hwang, Rosa; Rajapakshe, Kimal; Coarfa, Cristian; Huang, Shixia; Edwards, Dean; Curley, Steven A; Corr, Stuart J

2017-09-12

Surgical margin status in cancer surgery represents an important oncologic parameter affecting overall prognosis. The risk of disease recurrence is minimized and survival often prolonged if margin-negative resection can be accomplished during cancer surgery. Unfortunately, negative margins are not always surgically achievable due to tumor invasion into adjacent tissues or involvement of critical vasculature. Herein, we present a novel intra-operative device created to facilitate a uniform and mild heating profile to cause hyperthermic destruction of vessel-encasing tumors while safeguarding the encased vessel. We use pancreatic ductal adenocarcinoma as an in vitro and an in vivo cancer model for these studies as it is a representative model of a tumor that commonly involves major mesenteric vessels. In vitro data suggests that mild hyperthermia (41-46 °C for ten minutes) is an optimal thermal dose to induce high levels of cancer cell death, alter cancer cell's proteomic profiles and eliminate cancer stem cells while preserving non-malignant cells. In vivo and in silico data supports the well-known phenomena of a vascular heat sink effect that causes high temperature differentials through tissues undergoing hyperthermia, however temperatures can be predicted and used as a tool for the surgeon to adjust thermal doses delivered for various tumor margins.

Cancer Tissue Engineering: A Novel 3D Polystyrene Scaffold for In Vitro Isolation and Amplification of Lymphoma Cancer Cells from Heterogeneous Cell Mixtures

PubMed Central

Caicedo-Carvajal, Carlos E.; Liu, Qing; Remache, Yvonne; Goy, Andre; Suh, K. Stephen

2011-01-01

Isolation and amplification of primary lymphoma cells in vitro setting is technically and biologically challenging task. To optimize culture environment and mimic in vivo conditions, lymphoma cell lines were used as a test case and were grown in 3-dimension (3D) using a novel 3D tissue culture polystyrene scaffold with neonatal stromal cells to represent a lymphoma microenvironment. In this model, the cell proliferation was enhanced more than 200-fold or 20,000% neoplastic surplus in 7 days when less than 1% lymphoma cells were cocultured with 100-fold excess of neonatal stroma cells, representing 3.2-fold higher proliferative rate than 2D coculture model. The lymphoma cells grew and aggregated to form clusters during 3D coculture and did not maintained the parental phenotype to grow in single-cell suspension. The cluster size was over 5-fold bigger in the 3D coculture by day 4 than 2D coculture system and contained less than 0.00001% of neonatal fibroblast trace. This preliminary data indicate that novel 3D scaffold geometry and coculturing environment can be customized to amplify primary cancer cells from blood or tissues related to hematological cancer and subsequently used for personalized drug screening procedures. PMID:22073378

Ecological Effect of Arginine on Oral Microbiota.

PubMed

Zheng, Xin; He, Jinzhi; Wang, Lin; Zhou, Shuangshuang; Peng, Xian; Huang, Shi; Zheng, Liwei; Cheng, Lei; Hao, Yuqing; Li, Jiyao; Xu, Jian; Xu, Xin; Zhou, Xuedong

2017-08-03

Dental caries is closely associated with the microbial dybiosis between acidogenic/aciduric pathogens and alkali-generating commensal bacteria colonized in the oral cavity. Our recent studies have shown that arginine may represent a promising anti-caries agent by modulating microbial composition in an in vitro consortium. However, the effect of arginine on the oral microbiota has yet to be comprehensively delineated in either clinical cohort or in vitro biofilm models that better represent the microbial diversity of oral cavity. Here, by employing a clinical cohort and a saliva-derived biofilm model, we demonstrated that arginine treatment could favorably modulate the oral microbiota of caries-active individuals. Specifically, treatment with arginine-containing dentifrice normalized the oral microbiota of caries-active individuals similar to that of caries-free controls in terms of microbial structure, abundance of typical species, enzymatic activities of glycolysis and alkali-generation related enzymes and their corresponding transcripts. Moreover, we found that combinatory use of arginine with fluoride could better enrich alkali-generating Streptococcus sanguinis and suppress acidogenic/aciduric Streptococcus mutans, and thus significantly retard the demineralizing capability of saliva-derived oral biofilm. Hence, we propose that fluoride and arginine have a potential synergistic effect in maintaining an eco-friendly oral microbial equilibrium in favor of better caries management.

Comparison of the uptake of methacrylate-based nanoparticles in static and dynamic in vitro systems as well as in vivo.

PubMed

Rinkenauer, Alexandra C; Press, Adrian T; Raasch, Martin; Pietsch, Christian; Schweizer, Simon; Schwörer, Simon; Rudolph, Karl L; Mosig, Alexander; Bauer, Michael; Traeger, Anja; Schubert, Ulrich S

2015-10-28

Polymer-based nanoparticles are promising drug delivery systems allowing the development of new drug and treatment strategies with reduced side effects. However, it remains a challenge to screen for new and effective nanoparticle-based systems in vitro. Important factors influencing the behavior of nanoparticles in vivo cannot be simulated in screening assays in vitro, which still represent the main tools in academic research and pharmaceutical industry. These systems have serious drawbacks in the development of nanoparticle-based drug delivery systems, since they do not consider the highly complex processes influencing nanoparticle clearance, distribution, and uptake in vivo. In particular, the transfer of in vitro nanoparticle performance to in vivo models often fails, demonstrating the urgent need for novel in vitro tools that can imitate aspects of the in vivo situation more accurate. Dynamic cell culture, where cells are cultured and incubated in the presence of shear stress has the potential to bridge this gap by mimicking key-features of organs and vessels. Our approach implements and compares a chip-based dynamic cell culture model to the common static cell culture and mouse model to assess its capability to predict the in vivo success more accurately, by using a well-defined poly((methyl methacrylate)-co-(methacrylic acid)) and poly((methyl methacrylate)-co-(2-dimethylamino ethylmethacrylate)) based nanoparticle library. After characterization in static and dynamic in vitro cell culture we were able to show that physiological conditions such as cell-cell communication of co-cultured endothelial cells and macrophages as well as mechanotransductive signaling through shear stress significantly alter cellular nanoparticle uptake. In addition, it could be demonstrated by using dynamic cell cultures that the in vivo situation is simulated more accurately and thereby can be applied as a novel system to investigate the performance of nanoparticle systems in vivo more reliable. Copyright © 2015. Published by Elsevier B.V.

A Computational Model of the Rainbow Trout Hypothalamus-Pituitary-Ovary-Liver Axis

PubMed Central

Gillies, Kendall; Krone, Stephen M.; Nagler, James J.; Schultz, Irvin R.

2016-01-01

Reproduction in fishes and other vertebrates represents the timely coordination of many endocrine factors that culminate in the production of mature, viable gametes. In recent years there has been rapid growth in understanding fish reproductive biology, which has been motivated in part by recognition of the potential effects that climate change, habitat destruction and contaminant exposure can have on natural and cultured fish populations. New approaches to understanding the impacts of these stressors are being developed that require a systems biology approach with more biologically accurate and detailed mathematical models. We have developed a multi-scale mathematical model of the female rainbow trout hypothalamus-pituitary-ovary-liver axis to use as a tool to help understand the functioning of the system and for extrapolation of laboratory findings of stressor impacts on specific components of the axis. The model describes the essential endocrine components of the female rainbow trout reproductive axis. The model also describes the stage specific growth of maturing oocytes within the ovary and permits the presence of sub-populations of oocytes at different stages of development. Model formulation and parametrization was largely based on previously published in vivo and in vitro data in rainbow trout and new data on the synthesis of gonadotropins in the pituitary. Model predictions were validated against several previously published data sets for annual changes in gonadotropins and estradiol in rainbow trout. Estimates of select model parameters can be obtained from in vitro assays using either quantitative (direct estimation of rate constants) or qualitative (relative change from control values) approaches. This is an important aspect of mathematical models as in vitro, cell-based assays are expected to provide the bulk of experimental data for future risk assessments and will require quantitative physiological models to extrapolate across biological scales. PMID:27096735

A Computational Model of the Rainbow Trout Hypothalamus-Pituitary-Ovary-Liver Axis.

PubMed

Gillies, Kendall; Krone, Stephen M; Nagler, James J; Schultz, Irvin R

2016-04-01

Reproduction in fishes and other vertebrates represents the timely coordination of many endocrine factors that culminate in the production of mature, viable gametes. In recent years there has been rapid growth in understanding fish reproductive biology, which has been motivated in part by recognition of the potential effects that climate change, habitat destruction and contaminant exposure can have on natural and cultured fish populations. New approaches to understanding the impacts of these stressors are being developed that require a systems biology approach with more biologically accurate and detailed mathematical models. We have developed a multi-scale mathematical model of the female rainbow trout hypothalamus-pituitary-ovary-liver axis to use as a tool to help understand the functioning of the system and for extrapolation of laboratory findings of stressor impacts on specific components of the axis. The model describes the essential endocrine components of the female rainbow trout reproductive axis. The model also describes the stage specific growth of maturing oocytes within the ovary and permits the presence of sub-populations of oocytes at different stages of development. Model formulation and parametrization was largely based on previously published in vivo and in vitro data in rainbow trout and new data on the synthesis of gonadotropins in the pituitary. Model predictions were validated against several previously published data sets for annual changes in gonadotropins and estradiol in rainbow trout. Estimates of select model parameters can be obtained from in vitro assays using either quantitative (direct estimation of rate constants) or qualitative (relative change from control values) approaches. This is an important aspect of mathematical models as in vitro, cell-based assays are expected to provide the bulk of experimental data for future risk assessments and will require quantitative physiological models to extrapolate across biological scales.

Assessment of in vitro COPD models for tobacco regulatory science: Workshop proceedings, conclusions and paths forward for in vitro model use.

PubMed

Behrsing, Holger; Raabe, Hans; Manuppello, Joseph; Bombick, Betsy; Curren, Rodger; Sullivan, Kristie; Sethi, Sanjay; Phipps, Richard; Tesfaigzi, Yohannes; Yan, Sherwin; D'Ruiz, Carl; Tarran, Robert; Constant, Samuel; Phillips, Gary; Gaça, Marianna; Hayden, Patrick; Cao, Xuefei; Mathis, Carole; Hoeng, Julia; Braun, Armin; Hill, Erin

2016-05-01

The Family Smoking Prevention and Tobacco Control Act of 2009 established the Food and Drug Administration Center for Tobacco Products (FDA-CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 8-10 December 2014, IIVS organised a workshop conference, entitled Assessment of In Vitro COPD Models for Tobacco Regulatory Science, to bring together stakeholders representing regulatory agencies, academia, industry and animal protection, to address the research priorities articulated by the FDA-CTP. Specific topics were covered to assess the status of current in vitro technologies as they are applied to understanding the adverse pulmonary events resulting from tobacco product exposure, and in particular, the progression of chronic obstructive pulmonary disease (COPD). The four topics covered were: a) Inflammation and Oxidative Stress; b) Ciliary Dysfunction and Ion Transport; c) Goblet Cell Hyperplasia and Mucus Production; and d) Parenchymal/Bronchial Tissue Destruction and Remodelling. The 2.5 day workshop included 18 expert speakers, plus poster sessions, networking and breakout sessions, which identified key findings and provided recommendations to advance the in vitro technologies and assays used to evaluate tobacco-induced disease etiologies. The workshop summary was reported at the 2015 Society of Toxicology Annual Meeting, and the recommendations led to an IIVS-organised technical workshop in June 2015, entitled Goblet Cell Hyperplasia, Mucus Production, and Ciliary Beating Assays, to assess these assays and to conduct a proof-of-principle multi-laboratory exercise to determine their suitability for standardisation. Here, we report on the proceedings, recommendations and outcomes of the December 2014 workshop, including paths forward to continue the development of non-animal methods to evaluate tissue responses that model the disease processes that may lead to COPD, a major cause of mortality worldwide. 2016 FRAME.

3D is not enough: Building up a cell instructive microenvironment for tumoral stroma microtissues.

PubMed

Brancato, Virginia; Garziano, Alessandro; Gioiella, Filomena; Urciuolo, Francesco; Imparato, Giorgia; Panzetta, Valeria; Fusco, Sabato; Netti, Paolo A

2017-01-01

We fabricated three-dimensional microtissues with the aim to replicate in vitro the composition and the functionalities of the tumor microenvironment. By arranging either normal fibroblasts (NF) or cancer-activated fibroblasts (CAF) in two different three dimensional (3D) configurations, two kinds of micromodules were produced: spheroids and microtissues. Spheroids were obtained by means of the traditional cell aggregation technique resulting in a 3D model characterized by high cell density and low amount of extracellular proteins. The microtissues were obtained by culturing cells into porous gelatin microscaffolds. In this latter configuration, cells assembled an intricate network of collagen, fibronectin and hyaluronic acid. We investigated the biophysical properties of both 3D models in terms of cell growth, metabolic activity, texture and composition of the extracellular matrix (via histological analysis and multiphoton imaging) and cell mechanical properties (via Particle Tracking Microrheology). In the spheroid models such biophysical properties remained unchanged regardless to the cell type used. In contrast, normal-microtissues and cancer-activated-microtissues displayed marked differences. CAF-microtissues possessed higher proliferation rate, superior contraction capability, different micro-rheological properties and an extracellular matrix richer in collagen fibronectin and hyaluronic acid. At last, multiphoton investigation revealed differences in the collagen network architecture. Taken together, these results suggested that despite to cell spheroids, microtissues better recapitulate the important differences existing in vivo between normal and cancer-activated stroma representing a more suitable system to mimic in vitro the stromal element of the tumor tissues. This work concerns the engineering of tumor tissue in vitro. Tumor models serve as biological equivalent to study pathologic progression and to screen or validate the drugs efficacy. Tumor tissue is composed by malignant cells surviving in a microenvironment, or stroma. Stroma plays a pivotal role in cancer progression. Current in vitro models, i.e. spheroids, can't replicate the phenomena related to the tumor stroma remodeling. For this reason, to better replicate the tumor physiology in vitro that include functional and morphological changes, a novel 3D cancer model is proposed. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

In-vitro characterization of buccal iontophoresis: the case of sumatriptan succinate.

PubMed

Telò, Isabella; Tratta, Elena; Guasconi, Barbara; Nicoli, Sara; Pescina, Silvia; Govoni, Paolo; Santi, Patrizia; Padula, Cristina

2016-06-15

Buccal administration of sumatriptan succinate might be an interesting alternative to the present administration routes, due to its non-invasiveness and rapid onset of action, but because of its low permeability, a permeation enhancement strategy is required. The aim of this work was then to study, in-vitro, buccal iontophoresis of sumatriptan succinate. Permeation experiments were performed in-vitro across pig esophageal epithelium, a recently proposed model of human buccal mucosa, using vertical diffusion cells. The iontophoretic behavior of the tissue was characterized by measuring its isoelectric point (Na(+) transport number and the electroosmotic flow of acetaminophen determination) and by evaluating tissue integrity after current application. The results obtained confirm the usefulness of pig esophageal epithelium as an in-vitro model membrane for buccal drug delivery. The application of iontophoresis increased sumatriptan transport, proportionally to the current density applied, without tissue damage: electrotransport was the predominant mechanism. Integrating the results of the present work with literature data on the transport of other molecules across the buccal mucosa and across the skin, we can draw a general conclusion: the difference in passive transport across buccal mucosa and across the skin is influenced by permeant lipophilicity and by the penetration pathway. Finally, buccal iontophoretic administration of sumatriptan allows to administer 6mg of the drug in 1h, representing a promising alternative to the current administration routes. Copyright © 2016 Elsevier B.V. All rights reserved.

Regenerated keratin membrane to match the in vitro drug diffusion through human epidermis

PubMed Central

Selmin, Francesca; Cilurzo, Francesco; Aluigi, Annalisa; Franzè, Silvia; Minghetti, Paola

2012-01-01

This work aimed to develop membranes made of regenerated keratin and ceramides (CERs) to match the barrier property of the human stratum corneum in in vitro percutaneous absorption studies. The membrane composition was optimized on the basis of the in vitro drug diffusion profiles of ibuprofen, propranolol and testosterone chosen as model drugs on the basis of their different diffusion and solubility properties. The data were compared to those obtained using human epidermis. The ATR-FTIR and SEM analyses revealed that CERs were suspended into the regenerated keratin matrix, even if a partial solubilization occurred. It resulted in the membranes being physically stable after exposure to aqueous buffer and/or mineral oil and the fluxes of ibuprofen and propranolol from these vehicles through membranes and human skin were of the same order of magnitude. The best relationship with human epidermis data was obtained with 180 μm-thick membrane containing 1% ceramide III and 1% ceramide VI. The data on the testosterone diffusion were affected by the exposure of the membrane to a water/ethanol solution over a prolonged period of time, indicating that such an organic solvent was able to modify the supermolecular organization of keratin and CERs. The keratin/CER membranes can represent a simplified model to assay the in vitro skin permeability study of small molecules. PMID:25755997

Establishing the Effect of Brushing and a Day’s Diet on Tooth Tissue Loss in Vitro

PubMed Central

Forbes-Haley, Claire; Jones, Siân Bodfel; Davies, Maria; West, Nicola X.

2016-01-01

To develop an in vitro model to mimic the effects of meals equivalent to a day’s diet on tooth tissue loss (TTL). To identify how diet effects tooth wear and to test the efficacy of dental products designed to reduce tooth wear in a more realistic environment. A typical Friday diet was devised comprising: Breakfast then brushing, lunch, dinner then brushing. Groups of enamel samples were exposed to one meal, or all three in series, a control group was exposed to water and brushed. The daily cycle was repeated to represent two days’ consumption; TTL was quantified by non-contact profilometry. This pilot study highlighted adaptions that could be made to the model such as human enamel and saliva to further replicate natural eating habits. The sum of the TTL measured after Breakfast, lunch and dinner (bovine enamel specimens exposed to single meals) was less than that exhibited by the group of samples exposed to the series of meals but this difference was not significant (p = 0.09).In the absence and presence of brushing, TTL caused by breakfast and dinner was similar, but significantly greater than that caused by lunch (p < 0.05). While brushing increased TTL, this increase was not significant. It is possible to model a daily diet in vitro, and the data obtained confirms that the combination of food and drink affects the degree of TTL. This supports the further development of an in vitro model that includes alternative foodstuffs. This would aid understanding of the effects different diets have on TTL and could test new products designed to prevent TTL. PMID:29563467

Analytical model for real time, noninvasive estimation of blood glucose level.

PubMed

Adhyapak, Anoop; Sidley, Matthew; Venkataraman, Jayanti

2014-01-01

The paper presents an analytical model to estimate blood glucose level from measurements made non-invasively and in real time by an antenna strapped to a patient's wrist. Some promising success has been shown by the RIT ETA Lab research group that an antenna's resonant frequency can track, in real time, changes in glucose concentration. Based on an in-vitro study of blood samples of diabetic patients, the paper presents a modified Cole-Cole model that incorporates a factor to represent the change in glucose level. A calibration technique using the input impedance technique is discussed and the results show a good estimation as compared to the glucose meter readings. An alternate calibration methodology has been developed that is based on the shift in the antenna resonant frequency using an equivalent circuit model containing a shunt capacitor to represent the shift in resonant frequency with changing glucose levels. Work under progress is the optimization of the technique with a larger sample of patients.

Temozolomide-induced increase of tumorigenicity can be diminished by targeting of mitochondria in in vitro models of patient individual glioblastoma

PubMed Central

Walther, Madlin; Schneider, Björn; Linnebacher, Michael; Classen, Carl Friedrich

2018-01-01

Glioblastoma multiforme (GBM) is a highly heterogeneous and aggressive brain tumor with a dismal prognosis. Development of resistance towards cytostatic drugs like the GBM standard drug temozolomide is a severe problem in GBM treatment. One potential source of GBM relapse could be so called cancer stem like cells (CSCs). These represent an undifferentiated subpopulation of cells with high potential for tumor initiation. Furthermore, it has been shown that differentiated GBM cells can regain CSC properties when exposed to continuous temozolomide treatment in vitro. In this study, treatment of several primary GBM cell lines with clinically relevant doses of temozolomide increased their tumorigenicity as determined by colony formation assays in soft agar. Increased tumorigenicity is a known property of CSCs. Hence, therapy options that specifically target CSCs are under investigation. CSCs appear to be particularly dependent on mitochondria biogenesis which may represent a useful target for CSC elimination. Toxicity towards mitochondria is a known side effect of several antibiotics. Thus, addition of antibiotics like doxycycline may represent a useful tool to inhibit CSCs in GBM. Here, we show that combining temozolomide treatment of primary GBM cells with doxycycline could counteract the increase of tumorigenicity induced by temozolomide treatment. PMID:29352318

New use of an old drug: inhibition of breast cancer stem cells by benztropine mesylate.

PubMed

Cui, Jihong; Hollmén, Maija; Li, Lina; Chen, Yong; Proulx, Steven T; Reker, Daniel; Schneider, Gisbert; Detmar, Michael

2017-01-03

Cancer stem cells (CSCs) play major roles in cancer initiation, metastasis, recurrence and therapeutic resistance. Targeting CSCs represents a promising strategy for cancer treatment. The purpose of this study was to identify selective inhibitors of breast CSCs (BCSCs). We carried out a cell-based phenotypic screening with cell viability as a primary endpoint, using a collection of 2,546 FDA-approved drugs and drug-like molecules in spheres formed by malignant human breast gland-derived cells (HMLER-shEcad cells, representing BCSCs) and control immortalized non-tumorigenic human mammary cells (HMLE cells, representing normal stem cells). 19 compounds were identified from screening. The chemically related molecules benztropine mesylate and deptropine citrate were selected for further validation and both potently inhibited sphere formation and self-renewal of BCSCs in vitro. Benztropine mesylate treatment decreased cell subpopulations with high ALDH activity and with a CD44+/CD24- phenotype. In vivo, benztropine mesylate inhibited tumor-initiating potential in a 4T1 mouse model. Functional studies indicated that benztropine mesylate inhibits functions of CSCs via the acetylcholine receptors, dopamine transporters/receptors, and/or histamine receptors. In summary, our findings identify benztropine mesylate as an inhibitor of BCSCs in vitro and in vivo. This study also provides a screening platform for identification of additional anti-CSC agents.

In vitro to In vivo extrapolation of hepatic metabolism in fish: An inter-laboratory comparison of In vitro methods

EPA Science Inventory

Chemical biotransformation represents the single largest source of uncertainty in chemical bioaccumulation assessments for fish. In vitro methods employing isolated hepatocytes and liver subcellular fractions (S9) can be used to estimate whole-body rates of chemical metabolism, ...

Genetic toxicity assessment of engineered nanoparticles using a 3D in vitro skin model (EpiDerm™).

PubMed

Wills, John W; Hondow, Nicole; Thomas, Adam D; Chapman, Katherine E; Fish, David; Maffeis, Thierry G; Penny, Mark W; Brown, Richard A; Jenkins, Gareth J S; Brown, Andy P; White, Paul A; Doak, Shareen H

2016-09-09

The rapid production and incorporation of engineered nanomaterials into consumer products alongside research suggesting nanomaterials can cause cell death and DNA damage (genotoxicity) makes in vitro assays desirable for nanosafety screening. However, conflicting outcomes are often observed when in vitro and in vivo study results are compared, suggesting more physiologically representative in vitro models are required to minimise reliance on animal testing. BASF Levasil® silica nanoparticles (16 and 85 nm) were used to adapt the 3D reconstructed skin micronucleus (RSMN) assay for nanomaterials administered topically or into the growth medium. 3D dose-responses were compared to a 2D micronucleus assay using monocultured human B cells (TK6) after standardising dose between 2D / 3D assays by total nanoparticle mass to cell number. Cryogenic vitrification, scanning electron microscopy and dynamic light scattering techniques were applied to characterise in-medium and air-liquid interface exposures. Advanced transmission electron microscopy imaging modes (high angle annular dark field) and X-ray spectrometry were used to define nanoparticle penetration / cellular uptake in the intact 3D models and 2D monocultured cells. For all 2D exposures, significant (p < 0.002) increases in genotoxicity were observed (≥100 μg/mL) alongside cell viability decreases (p < 0.015) at doses ≥200 μg/mL (16 nm-SiO2) and ≥100 μg/mL (85 nm-SiO2). In contrast, 2D-equivalent exposures to the 3D models (≤300 μg/mL) caused no significant DNA damage or impact on cell viability. Further increasing dose to the 3D models led to probable air-liquid interface suffocation. Nanoparticle penetration / cell uptake analysis revealed no exposure to the live cells of the 3D model occurred due to the protective nature of the skin model's 3D cellular microarchitecture (topical exposures) and confounding barrier effects of the collagen cell attachment layer (in-medium exposures). 2D monocultured cells meanwhile showed extensive internalisation of both silica particles causing (geno)toxicity. The results establish the importance of tissue microarchitecture in defining nanomaterial exposure, and suggest 3D in vitro models could play a role in bridging the gap between in vitro and in vivo outcomes in nanotoxicology. Robust exposure characterisation and uptake assessment methods (as demonstrated) are essential to interpret nano(geno)toxicity studies successfully.

THE ROLE OF ELECTRICAL SIGNALS IN MURINE CORNEAL WOUND RE-EPITHELIALISATION

PubMed Central

Kucerova, R.; Walczysko, P.; Reid, B.; Ou, J.; Leiper, L. J.; Rajnicek, A. M.; McCaig, C. D.; Zhao, M.; Collinson, J. M.

2011-01-01

Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for electric fields in wound re-epithelialisation, using the Pax6+/− mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type and Pax6+/− corneal epithelial cells showed increased migration speeds in response to applied electric fields in vitro. However, only Pax6+/+ cells demonstrated directional galvanotaxis towards the cathode, with activation of pSrc signalling, polarised to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6+/− corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as wild-type. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for wild-type or mutant corneas. Furthermore, during healing in vivo, no polarisation of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous electric fields do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signalling initiation. PMID:20945376

In vitro reconstruction of human junctional and sulcular epithelium

PubMed Central

Dabija-Wolter, G; Bakken, V; Cimpan, M R; Johannessen, A C; Costea, D E

2013-01-01

BACKGROUND The aim of this study was to develop and characterize standardized in vitro three-dimensional organotypic models of human junctional epithelium (JE) and sulcular epithelium (SE). METHODS Organotypic models were constructed by growing human normal gingival keratinocytes on top of collagen matrices populated with gingival fibroblasts (GF) or periodontal ligament fibroblasts (PLF). Tissues obtained were harvested at different time points and assessed for epithelial morphology, proliferation (Ki67), expression of JE-specific markers (ODAM and FDC-SP), cytokeratins (CK), transglutaminase, filaggrin, and basement membrane proteins (collagen IV and laminin1). RESULTS The epithelial component in 3- and 5-day organotypics showed limited differentiation and expressed Ki-67, ODAM, FDC-SP, CK 8, 13, 16, 19, and transglutaminase in a similar fashion to control JE samples. PLF supported better than GF expression of CK19 and suprabasal proliferation, although statistically significant only at day 5. Basement membrane proteins started to be deposited only from day 5. The rate of proliferating cells as well as the percentage of CK19-expressing cells decreased significantly in 7- and 9-day cultures. Day 7 organotypics presented higher number of epithelial cell layers, proliferating cells in suprabasal layers, and CK expression pattern similar to SE. CONCLUSION Both time in culture and fibroblast type had impact on epithelial phenotype. Five-day cultures with PLF are suggested as JE models, 7-day cultures with PLF or GF as SE models, while 9-day cultures with GF as gingival epithelium (GE) models. Such standard, reproducible models represent useful tools to study periodontal bacteria–host interactions in vitro. PMID:22947066

A novel quantitative model of cell cycle progression based on cyclin-dependent kinases activity and population balances.

PubMed

Pisu, Massimo; Concas, Alessandro; Cao, Giacomo

2015-04-01

Cell cycle regulates proliferative cell capacity under normal or pathologic conditions, and in general it governs all in vivo/in vitro cell growth and proliferation processes. Mathematical simulation by means of reliable and predictive models represents an important tool to interpret experiment results, to facilitate the definition of the optimal operating conditions for in vitro cultivation, or to predict the effect of a specific drug in normal/pathologic mammalian cells. Along these lines, a novel model of cell cycle progression is proposed in this work. Specifically, it is based on a population balance (PB) approach that allows one to quantitatively describe cell cycle progression through the different phases experienced by each cell of the entire population during its own life. The transition between two consecutive cell cycle phases is simulated by taking advantage of the biochemical kinetic model developed by Gérard and Goldbeter (2009) which involves cyclin-dependent kinases (CDKs) whose regulation is achieved through a variety of mechanisms that include association with cyclins and protein inhibitors, phosphorylation-dephosphorylation, and cyclin synthesis or degradation. This biochemical model properly describes the entire cell cycle of mammalian cells by maintaining a sufficient level of detail useful to identify check point for transition and to estimate phase duration required by PB. Specific examples are discussed to illustrate the ability of the proposed model to simulate the effect of drugs for in vitro trials of interest in oncology, regenerative medicine and tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma

PubMed Central

Miller, Tyler E.; Liau, Brian B.; Wallace, Lisa C.; Morton, Andrew R.; Xie, Qi; Dixit, Deobrat; Factor, Daniel C.; Kim, Leo J. Y.; Morrow, James J.; Wu, Qiulian; Mack, Stephen C.; Hubert, Christopher G.; Gillespie, Shawn M.; Flavahan, William A.; Hoffmann, Thomas; Thummalapalli, Rohit; Hemann, Michael T.; Paddison, Patrick J.; Horbinski, Craig M.; Zuber, Johannes; Scacheri, Peter C.; Bernstein, Bradley E.; Tesar, Paul J.; Rich, Jeremy N.

2017-01-01

Glioblastoma is a universally lethal cancer with a median survival of approximately 15 months1. Despite substantial efforts to define druggable targets, there are no therapeutic options that meaningfully extend glioblastoma patient lifespan. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology2–4 for use in orthotopic patient-derived xenograft (PDX) models, creating a high-throughput negative selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators necessary for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, supporting targeting the transcription elongation machinery as a therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of ‘cancer dependencies’ not identified by previous in vitro approaches, which could supply untapped opportunities for therapeutic intervention. PMID:28678782

Hepatoprotective and antioxidant activity of quercetin loaded chitosan/alginate particles in vitro and in vivo in a model of paracetamol-induced toxicity.

PubMed

Tzankova, Virginia; Aluani, Denitsa; Kondeva-Burdina, Magdalena; Yordanov, Yordan; Odzhakov, Feodor; Apostolov, Alexandar; Yoncheva, Krassimira

2017-08-01

The toxic liver impairment caused by free radical injury or excessive reactive oxigen species (ROS) formation could be effectivelly attenuated by natural antioxidants. The present study aimed to explore and compare the hepatoprotective and antioxidant effects of free and encapsulated quercetin in in vitro and in vivo models of hepatotoxicity. Thus, quercetin was encapsulated in chitosan/alginate nanoparticles by gelation method. Both empty and quercetin-loaded nanoparticles revealed good safety profile in vitro, determined by the lack of cytotoxicity in human hepatoma HepG2 cells. The pretreatment of HepG2 cells with encapsulated quercetin (10μg/ml) significantly attenuated the decrease in cell viability in H 2 О 2 -induced oxidative stress (0.1mM H 2 О 2 ) , thus showing an effective in vitro protection. In vivo evaluation of the antioxidant and hepatoprotective potential of free and encapsulated quercetin was performed in a model of paracetamol - induced liver injury in male Wistar rats. The oral pretreatment with encapsulated quercetin (0.18mg/kg b.w., 7days) significantly diminished the increased levels of serum transaminases ALT and AST, attenuated the lipid peroxidation and restored the levels of gluthation (a marker of cell antioxidant defence system). The protective effects of quercetin encapsulated in chitosan-based nanoformulation were superior to those of free quercetin. The results of the study suggest that the encapsulation of quercetin in chitosan/alginate nanoformulations might represent an effective therapeutic approach against oxidative stress induced liver injury. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

SRC family kinase (SFK) inhibition reduces rhabdomyosarcoma cell growth in vitro and in vivo and triggers p38 MAP kinase-mediated differentiation

PubMed Central

Casini, Nadia; Forte, Iris Maria; Mastrogiovanni, Gianmarco; Pentimalli, Francesca; Angelucci, Adriano; Festuccia, Claudio; Tomei, Valentina; Ceccherini, Elisa; Di Marzo, Domenico; Schenone, Silvia; Botta, Maurizio; Giordano, Antonio; Indovina, Paola

2015-01-01

Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS. PMID:25762618

Effects of ozone nano-bubble water on periodontopathic bacteria and oral cells - in vitro studies

NASA Astrophysics Data System (ADS)

Hayakumo, Sae; Arakawa, Shinichi; Takahashi, Masayoshi; Kondo, Keiko; Mano, Yoshihiro; Izumi, Yuichi

2014-10-01

The aims of the present study were to evaluate the bactericidal activity of a new antiseptic agent, ozone nano-bubble water (NBW3), against periodontopathogenic bacteria and to assess the cytotoxicity of NBW3 against human oral cells. The bactericidal activities of NBW3 against representative periodontopathogenic bacteria, Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) were evaluated using in vitro time-kill assays. The cytotoxicity of NBW3 was evaluated using three-dimensional human buccal and gingival tissue models. The numbers of colony forming units (CFUs)/mL of P. gingivalis and A. actinomycetemcomitans exposed to NBW3 dropped to below the lower limit of detection (<10 CFUs mL-1) after only 0.5 min of exposure. There were only minor decreases in the viability of oral tissue cells after 24 h of exposure to NBW3. These results suggest that NBW3 possesses potent bactericidal activity against representative periodontopathogenic bacteria and is not cytotoxic to cells of human oral tissues. The use of NBW3 as an adjunct to periodontal therapy would be promising.

Baby care product development: artificial urine in vitro assay is useful for cosmetic product assessment.

PubMed

Degouy, Arnaud; Gomez-Berrada, Marie-Pierre; Ferret, Pierre-Jacques

2014-02-01

As a result of infants' inability to control urination, the skin of the diaper area has special needs for protection from irritating effects of urine and prevention of diaper dermatitis such as products for cleansing and protection of the skin. Several in vitro models are currently available to assess tolerance. In vitro testing using artificial urine allows the protective effects of diaper-region cosmetics to be ascertained. Thus, a new model defined as "artificial urine in vitro assay" has been added to our traditional pre-clinical in vitro testing program. IL1-α is a highly active and pleiotropic pro-inflammatory cytokine. It plays a key role in inflammation and is the biological mirror of irritation induced by diaper dermatitis. This study determines, on human skin explants, if a cosmetic formula is (1) tolerated equally as well in the presence of artificial urine as in its absence and (2) is able to decrease IL1-α production induced by artificial urine or Sodium Dodecyl Sulfate. 31 tests including 17 in-house formulas, 10 bench-markers and 4 combinations of products were performed and data obtained are represented on a simple four-point scale (from practically non protective to very protective). It allows determination of formula-type groups that will have predictable protective properties in subsequent clinical trials and comparison with competitors' products. It is a useful aid in the formulation stage and provides readily-useable data for the cosmetic risk assessment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biological Tools to Study the Effects of Environmental Contaminants at the Feto–Maternal Interface

PubMed Central

Mannelli, Chiara; Ietta, Francesca; Avanzati, Anna Maria; Skarzynski, Dariusz

2015-01-01

The identification of reproductive toxicants is a major scientific challenge for human health. Prenatal life is the most vulnerable and important time span of human development. For obvious ethical reasons, in vivo models cannot be used in human pregnancy, and animal models do not perfectly reflect human physiology. This review describes the in vitro test models representative of the human feto–maternal interface and the effects of environmental chemicals with estrogen-like activity, mainly bisphenol A and para-nonylphenol, with a particular emphasis on the effects at low, nontoxic doses similar to concentrations commonly detected in the population. PMID:26740808

Porcine sclera as a model of human sclera for in vitro transport experiments: histology, SEM, and comparative permeability

PubMed Central

Ferrari, G.; Quarta, M.; Macaluso, C.; Govoni, P.; Dallatana, D.; Santi, P.

2009-01-01

Purpose To evaluate porcine sclera as a model of human sclera for in vitro studies of transscleral drug delivery of both low and high molecular weight compounds. Methods Human and porcine scleras were characterized for thickness and water content. The tissue surface was examined by scanning electron microscopy (SEM), and the histology was studied with hematoxylin-eosin staining. Comparative permeation experiments were performed using three model molecules, acetaminophen as the model compound for small molecules; a linear dextran with a molecular weight of 120 kDa as the model compound for high molecular weight drugs; and insulin, which was chosen as the model protein. Permeation parameters such as flux, lag time, and permeability coefficient were determined and compared. Results Human and porcine scleras have a similar histology and collagen bundle organization. The water content is approx 70% for both tissues while a statistically significant difference was found for the thickness, porcine sclera being approximately twofold thicker than human sclera. Differences in thickness produced differences in the permeability coefficient. In fact, human sclera was found to be two to threefold more permeable toward the three molecules studied than porcine sclera. Conclusions The results obtained in the present paper prove that porcine sclera can be considered a good model for human sclera for in vitro permeation experiments of both low and high molecular weight compounds. In fact, if the different tissue thickness is taken into account, comparable permeability was demonstrated. This suggests a possible use of this model in the evaluation of the transscleral permeation of new biotech compounds, which currently represent the most innovative and efficient therapeutic options for the treatment of ocular diseases. PMID:19190734

Porcine sclera as a model of human sclera for in vitro transport experiments: histology, SEM, and comparative permeability.

PubMed

Nicoli, S; Ferrari, G; Quarta, M; Macaluso, C; Govoni, P; Dallatana, D; Santi, P

2009-01-01

To evaluate porcine sclera as a model of human sclera for in vitro studies of transscleral drug delivery of both low and high molecular weight compounds. Human and porcine scleras were characterized for thickness and water content. The tissue surface was examined by scanning electron microscopy (SEM), and the histology was studied with hematoxylin-eosin staining. Comparative permeation experiments were performed using three model molecules, acetaminophen as the model compound for small molecules; a linear dextran with a molecular weight of 120 kDa as the model compound for high molecular weight drugs; and insulin, which was chosen as the model protein. Permeation parameters such as flux, lag time, and permeability coefficient were determined and compared. Human and porcine scleras have a similar histology and collagen bundle organization. The water content is approx 70% for both tissues while a statistically significant difference was found for the thickness, porcine sclera being approximately twofold thicker than human sclera. Differences in thickness produced differences in the permeability coefficient. In fact, human sclera was found to be two to threefold more permeable toward the three molecules studied than porcine sclera. The results obtained in the present paper prove that porcine sclera can be considered a good model for human sclera for in vitro permeation experiments of both low and high molecular weight compounds. In fact, if the different tissue thickness is taken into account, comparable permeability was demonstrated. This suggests a possible use of this model in the evaluation of the transscleral permeation of new biotech compounds, which currently represent the most innovative and efficient therapeutic options for the treatment of ocular diseases.

A putative biomarker signature for clinically effective AKT inhibition: correlation of in vitro, in vivo and clinical data identifies the importance of modulation of the mTORC1 pathway.

PubMed

Cheraghchi-Bashi, Azadeh; Parker, Christine A; Curry, Ed; Salazar, Jean-Frederic; Gungor, Hatice; Saleem, Azeem; Cunnea, Paula; Rama, Nona; Salinas, Cristian; Mills, Gordon B; Morris, Shannon R; Kumar, Rakesh; Gabra, Hani; Stronach, Euan A

2015-12-08

Our identification of dysregulation of the AKT pathway in ovarian cancer as a platinum resistance specific event led to a comprehensive analysis of in vitro, in vivo and clinical behaviour of the AKT inhibitor GSK2141795. Proteomic biomarker signatures correlating with effects of GSK2141795 were developed using in vitro and in vivo models, well characterised for related molecular, phenotypic and imaging endpoints. Signatures were validated in temporally paired biopsies from patients treated with GSK2141795 in a clinical study. GSK2141795 caused growth-arrest as single agent in vitro, enhanced cisplatin-induced apoptosis in vitro and reduced tumour volume in combination with platinum in vivo. GSK2141795 treatment in vitro and in vivo resulted in ~50-90% decrease in phospho-PRAS40 and 20-80% decrease in fluoro-deoxyglucose (FDG) uptake. Proteomic analysis of GSK2141795 in vitro and in vivo identified a signature of pathway inhibition including changes in AKT and p38 phosphorylation and total Bim, IGF1R, AR and YB1 levels. In patient biopsies, prior to treatment with GSK2141795 in a phase 1 clinical trial, this signature was predictive of post-treatment changes in the response marker CA125. Development of this signature represents an opportunity to demonstrate the clinical importance of AKT inhibition for re-sensitisation of platinum resistant ovarian cancer to platinum.

The immature dentate gyrus represents a shared phenotype of mouse models of epilepsy and psychiatric disease

PubMed Central

Shin, Rick; Kobayashi, Katsunori; Hagihara, Hideo; Kogan, Jeffrey H; Miyake, Shinichi; Tajinda, Katsunori; Walton, Noah M; Gross, Adam K; Heusner, Carrie L; Chen, Qian; Tamura, Kouichi; Miyakawa, Tsuyoshi; Matsumoto, Mitsuyuki

2013-01-01

Objectives There is accumulating evidence to suggest psychiatric disorders, such as bipolar disorder and schizophrenia, share common etiologies, pathophysiologies, genetics, and drug responses with many of the epilepsies. Here, we explored overlaps in cellular/molecular, electrophysiological, and behavioral phenotypes between putative mouse models of bipolar disorder/schizophrenia and epilepsy. We tested the hypothesis that an immature dentate gyrus (iDG), whose association with psychosis in patients has recently been reported, represents a common phenotype of both diseases. Methods Behaviors of calcium/calmodulin-dependent protein kinase II alpha (α-CaMKII) heterozygous knock-out (KO) mice, which are a representative bipolar disorder/schizophrenia model displaying iDG, and pilocarpine-treated mice, which are a representative epilepsy model, were tested followed by quantitative polymerase chain reaction (qPCR)/immunohistochemistry for mRNA/protein expression associated with an iDG phenotype. In vitro electrophysiology of dentate gyrus granule cells (DG GCs) was examined in pilocarpine-treated epileptic mice. Results The two disease models demonstrated similar behavioral deficits, such as hyperactivity, poor working memory performance, and social withdrawal. Significant reductions in mRNA expression and immunoreactivity of the mature neuronal marker calbindin and concomitant increases in mRNA expression and immunoreactivity of the immature neuronal marker calretinin represent iDG signatures that are present in both mice models. Electrophysiologically, we have confirmed that DG GCs from pilocarpine-treated mice represent an immature state. A significant decrease in hippocampal α-CaMKII protein levels was also found in both models. Conclusions Our data have shown iDG signatures from mouse models of both bipolar disorder/schizophrenia and epilepsy. The evidence suggests that the iDG may, in part, be responsible for the abnormal behavioral phenotype, and that the underlying pathophysiologies in epilepsy and bipolar disorder/schizophrenia are strikingly similar. PMID:23560889

The immature dentate gyrus represents a shared phenotype of mouse models of epilepsy and psychiatric disease.

PubMed

Shin, Rick; Kobayashi, Katsunori; Hagihara, Hideo; Kogan, Jeffrey H; Miyake, Shinichi; Tajinda, Katsunori; Walton, Noah M; Gross, Adam K; Heusner, Carrie L; Chen, Qian; Tamura, Kouichi; Miyakawa, Tsuyoshi; Matsumoto, Mitsuyuki

2013-06-01

There is accumulating evidence to suggest psychiatric disorders, such as bipolar disorder and schizophrenia, share common etiologies, pathophysiologies, genetics, and drug responses with many of the epilepsies. Here, we explored overlaps in cellular/molecular, electrophysiological, and behavioral phenotypes between putative mouse models of bipolar disorder/schizophrenia and epilepsy. We tested the hypothesis that an immature dentate gyrus (iDG), whose association with psychosis in patients has recently been reported, represents a common phenotype of both diseases. Behaviors of calcium/calmodulin-dependent protein kinase II alpha (α-CaMKII) heterozygous knock-out (KO) mice, which are a representative bipolar disorder/schizophrenia model displaying iDG, and pilocarpine-treated mice, which are a representative epilepsy model, were tested followed by quantitative polymerase chain reaction (qPCR)/immunohistochemistry for mRNA/protein expression associated with an iDG phenotype. In vitro electrophysiology of dentate gyrus granule cells (DG GCs) was examined in pilocarpine-treated epileptic mice. The two disease models demonstrated similar behavioral deficits, such as hyperactivity, poor working memory performance, and social withdrawal. Significant reductions in mRNA expression and immunoreactivity of the mature neuronal marker calbindin and concomitant increases in mRNA expression and immunoreactivity of the immature neuronal marker calretinin represent iDG signatures that are present in both mice models. Electrophysiologically, we have confirmed that DG GCs from pilocarpine-treated mice represent an immature state. A significant decrease in hippocampal α-CaMKII protein levels was also found in both models. Our data have shown iDG signatures from mouse models of both bipolar disorder/schizophrenia and epilepsy. The evidence suggests that the iDG may, in part, be responsible for the abnormal behavioral phenotype, and that the underlying pathophysiologies in epilepsy and bipolar disorder/schizophrenia are strikingly similar. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Delicaflavone induces autophagic cell death in lung cancer via Akt/mTOR/p70S6K signaling pathway.

PubMed

Sui, Yuxia; Yao, Hong; Li, Shaoguang; Jin, Long; Shi, Peiying; Li, Zhijun; Wang, Gang; Lin, Shilan; Wu, Youjia; Li, Yuxiang; Huang, Liying; Liu, Qicai; Lin, Xinhua

2017-03-01

Searching for potential anticancer agents from natural sources is an effective strategy for developing novel chemotherapeutic agents. In this study, data supporting the in vitro and in vivo anticancer effects of delicaflavone, a rarely occurring biflavonoid from Selaginella doederleinii, were reported. Delicaflavone exhibited favorable anticancer properties, as shown by the MTT assay and xenograft model of human non-small cell lung cancer in male BALB/c nude mice without observable adverse effect. By transmission electron microscopy with acridine orange and Cyto-ID®Autophagy detection dyes, Western blot analysis, and RT-PCR assay, we confirmed that delicaflavone induces autophagic cell death by increasing the ratio of LC3-II to LC3-I, which are autophagy-related proteins, and promoting the generation of acidic vesicular organelles and autolysosomes in the cytoplasm of human lung cancer A549 and PC-9 cells in a time- and dose-dependent manner. Delicaflavone downregulated the expression of phospho-Akt, phospho-mTOR, and phospho-p70S6K in a time- and dose-dependent manner, suggesting that it induced autophagy by inhibiting the Akt/mTOR/p70S6K pathway in A549 and PC-9 cells. Delicaflavone is a potential anticancer agent that can induce autophagic cell death in human non-small cell lung cancer via the Akt/mTOR/p70S6K signaling pathway. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer. Delicaflavone showed anti-lung cancer effects in vitro and in vivo. Delicaflavone induced autophagic cell death via Akt/mTOR/p70S6K signaling pathway. Delicaflavone did not show observable side effects in a xenograft mouse model. Delicaflavone may represent a potential therapeutic agent for lung cancer.

In vitro exposure systems and dosimetry assessment tools for inhaled tobacco products: Workshop proceedings, conclusions and paths forward for in vitro model use.

PubMed

Behrsing, Holger; Hill, Erin; Raabe, Hans; Tice, Raymond; Fitzpatrick, Suzanne; Devlin, Robert; Pinkerton, Kent; Oberdörster, Günter; Wright, Chris; Wieczorek, Roman; Aufderheide, Michaela; Steiner, Sandro; Krebs, Tobias; Asgharian, Bahman; Corley, Richard; Oldham, Michael; Adamson, Jason; Li, Xiang; Rahman, Irfan; Grego, Sonia; Chu, Pei-Hsuan; McCullough, Shaun; Curren, Rodger

2017-07-01

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4-6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols; b) Air-Liquid Interface-In Vitro Exposure Systems; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide. 2017 FRAME.

Expert consensus on an in vitro approach to assess ...

EPA Pesticide Factsheets

Report from an international workshop with the goal of reviewing the state-of-the-science and determine the technical needs to develop an in vitro system that will reduce and eventually replace the use of animals for evaluating the potential inhalation toxicity of nanomaterials (NMs) in a regulatory setting. Workshop was co-organized in February 2015 by the PETA International Science Consortium Ltd. with the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods an international workshop that was attended by representatives from industry, government, academia, and non-governmental organizations with expertise in in vivo and in vitro lung systems, respiratory toxicology, inhalation particle dosimetry, nanotoxicology, and hazard and human health risk analysis. This report provides an overview of the presentations, discussions, and recommendations of the participants on the design of an in vitro system for the prediction of pulmonary fibrosis. The workshop participants identified multi-walled carbon nanotubes (MWCNTs), which have been shown to induce fibrosis in animal experiments and represent an important commercial nanomaterial class, as representative pro-fibrogenic NMs to use for the development of an in vitro test system. Recommendations were made for designing a system using lung relevant cells co-cultured at the air-liquid interface to assess the pro-fibrogenic potential of aerosolized MWCNTs, while consider

Cell and molecular biology of the spiny dogfish Squalus acanthias and little skate Leucoraja erinacea: insights from in vitro cultured cells.

PubMed

Barnes, D W

2012-04-01

Two of the most commonly used elasmobranch experimental model species are the spiny dogfish Squalus acanthias and the little skate Leucoraja erinacea. Comparative biology and genomics with these species have provided useful information in physiology, pharmacology, toxicology, immunology, evolutionary developmental biology and genetics. A wealth of information has been obtained using in vitro approaches to study isolated cells and tissues from these organisms under circumstances in which the extracellular environment can be controlled. In addition to classical work with primary cell cultures, continuously proliferating cell lines have been derived recently, representing the first cell lines from cartilaginous fishes. These lines have proved to be valuable tools with which to explore functional genomic and biological questions and to test hypotheses at the molecular level. In genomic experiments, complementary (c)DNA libraries have been constructed, and c. 8000 unique transcripts identified, with over 3000 representing previously unknown gene sequences. A sub-set of messenger (m)RNAs has been detected for which the 3' untranslated regions show elements that are remarkably well conserved evolutionarily, representing novel, potentially regulatory gene sequences. The cell culture systems provide physiologically valid tools to study functional roles of these sequences and other aspects of elasmobranch molecular cell biology and physiology. Information derived from the use of in vitro cell cultures is valuable in revealing gene diversity and information for genomic sequence assembly, as well as for identification of new genes and molecular markers, construction of gene-array probes and acquisition of full-length cDNA sequences. © 2012 The Author. Journal of Fish Biology © 2012 The Fisheries Society of the British Isles.

A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells.

PubMed

Magistri, Marco; Khoury, Nathalie; Mazza, Emilia Maria Cristina; Velmeshev, Dmitry; Lee, Jae K; Bicciato, Silvio; Tsoulfas, Pantelis; Faghihi, Mohammad Ali

2016-11-01

Astrocytes are a morphologically and functionally heterogeneous population of cells that play critical roles in neurodevelopment and in the regulation of central nervous system homeostasis. Studies of human astrocytes have been hampered by the lack of specific molecular markers and by the difficulties associated with purifying and culturing astrocytes from adult human brains. Human neural progenitor cells (NPCs) with self-renewal and multipotent properties represent an appealing model system to gain insight into the developmental genetics and function of human astrocytes, but a comprehensive molecular characterization that confirms the validity of this cellular system is still missing. Here we used an unbiased transcriptomic analysis to characterize in vitro culture of human NPCs and to define the gene expression programs activated during the differentiation of these cells into astrocytes using FBS or the combination of CNTF and BMP4. Our results demonstrate that in vitro cultures of human NPCs isolated during the gliogenic phase of neurodevelopment mainly consist of radial glial cells (RGCs) and glia-restricted progenitor cells. In these cells the combination of CNTF and BMP4 activates the JAK/STAT and SMAD signaling cascades, leading to the inhibition of oligodendrocytes lineage commitment and activation of astrocytes differentiation. On the other hand, FBS-derived astrocytes have properties of reactive astrocytes. Our work suggests that in vitro culture of human NPCs represents a valuable cellular system to study human disorders characterized by impairment of astrocytes development and function. Our datasets represent an important resource for researchers studying human astrocytes development and might set the basis for the discovery of novel human-specific astrocyte markers. © 2016 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

In vitro chemopreventive properties of peptides released from quinoa (Chenopodium quinoa Willd.) protein under simulated gastrointestinal digestion.

PubMed

Vilcacundo, Rubén; Miralles, Beatriz; Carrillo, Wilman; Hernández-Ledesma, Blanca

2018-03-01

Because of the continuous and direct interaction between the digestive tract and foods, dietary compounds represent an interesting source of chemopreventive agents for gastrointestinal health. In this study, the influence of a standardized static in vitro gastrointestinal digestion model on the release of peptides with chemopreventive potential from quinoa protein was investigated. Gastroduodenal digests and fractions collected by ultrafiltration were evaluated for their in plate oxygen radical absorbance capacity and in vitro colon cancer cell viability inhibitory activity. Highest effects were observed in the digests obtained during the intestinal phase, with fraction containing peptides <5kDa as the main responsible for the antioxidant activity and peptides >5kDa showing the greatest anti-cancer effects. Seventeen potential bioactive peptides derived from quinoa proteins have been identified. These proteins might be utilized as new ingredients in the development of functional foods or nutraceuticals with the aim of reducing oxidative stress-associated diseases, including cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model.

PubMed

Tamai, Miho; Aoki, Mami; Nishimura, Akihito; Morishita, Koji; Tagawa, Yoh-ichi

2013-12-01

Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVL(mES), which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVL(mES) culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. L-Ornithine is an intermediate of the urea cycle, but supplemental L-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVL(mES), supplemental L-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVL(mES) displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVL(mES) will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.

Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy

PubMed Central

Farcal, Lucian; Torres Andón, Fernando; Di Cristo, Luisana; Rotoli, Bianca Maria; Bussolati, Ovidio; Bergamaschi, Enrico; Mech, Agnieszka; Hartmann, Nanna B.; Rasmussen, Kirsten; Riego-Sintes, Juan; Ponti, Jessica; Kinsner-Ovaskainen, Agnieszka; Rossi, François; Oomen, Agnes; Bos, Peter; Chen, Rui; Bai, Ru; Chen, Chunying; Rocks, Louise; Fulton, Norma; Ross, Bryony; Hutchison, Gary; Tran, Lang; Mues, Sarah; Ossig, Rainer; Schnekenburger, Jürgen; Campagnolo, Luisa; Vecchione, Lucia; Pietroiusti, Antonio; Fadeel, Bengt

2015-01-01

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO2 NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects. PMID:25996496

Human immune cells' behavior and survival under bioenergetically restricted conditions in an in vitro fracture hematoma model

PubMed Central

Hoff, Paula; Maschmeyer, Patrick; Gaber, Timo; Schütze, Tabea; Raue, Tobias; Schmidt-Bleek, Katharina; Dziurla, René; Schellmann, Saskia; Lohanatha, Ferenz Leonard; Röhner, Eric; Ode, Andrea; Burmester, Gerd-Rüdiger; Duda, Georg N; Perka, Carsten; Buttgereit, Frank

2013-01-01

The initial inflammatory phase of bone fracture healing represents a critical step for the outcome of the healing process. However, both the mechanisms initiating this inflammatory phase and the function of immune cells present at the fracture site are poorly understood. In order to study the early events within a fracture hematoma, we established an in vitro fracture hematoma model: we cultured hematomas forming during an osteotomy (artificial bone fracture) of the femur during total hip arthroplasty (THA) in vitro under bioenergetically controlled conditions. This model allowed us to monitor immune cell populations, cell survival and cytokine expression during the early phase following a fracture. Moreover, this model enabled us to change the bioenergetical conditions in order to mimic the in vivo situation, which is assumed to be characterized by hypoxia and restricted amounts of nutrients. Using this model, we found that immune cells adapt to hypoxia via the expression of angiogenic factors, chemoattractants and pro-inflammatory molecules. In addition, combined restriction of oxygen and nutrient supply enhanced the selective survival of lymphocytes in comparison with that of myeloid derived cells (i.e., neutrophils). Of note, non-restricted bioenergetical conditions did not show any similar effects regarding cytokine expression and/or different survival rates of immune cell subsets. In conclusion, we found that the bioenergetical conditions are among the crucial factors inducing the initial inflammatory phase of fracture healing and are thus a critical step for influencing survival and function of immune cells in the early fracture hematoma. PMID:23396474

Mathematical Modeling of Cellular Cross-Talk Between Endothelial and Tumor Cells Highlights Counterintuitive Effects of VEGF-Targeted Therapies.

PubMed

Jain, Harsh; Jackson, Trachette

2018-05-01

Tumor growth and progression are critically dependent on the establishment of a vascular support system. This is often accomplished via the expression of pro-angiogenic growth factors, including members of the vascular endothelial growth factor (VEGF) family of ligands. VEGF ligands are overexpressed in a wide variety of solid tumors and therefore have inspired optimism that inhibition of the different axes of the VEGF pathway-alone or in combination-would represent powerful anti-angiogenic therapies for most cancer types. When considering treatments that target VEGF and its receptors, it is difficult to tease out the differential anti-angiogenic and anti-tumor effects of all combinations experimentally because tumor cells and vascular endothelial cells are engaged in a dynamic cross-talk that impacts key aspects of tumorigenesis, independent of angiogenesis. Here we develop a mathematical model that connects intracellular signaling responsible for both endothelial and tumor cell proliferation and death to population-level cancer growth and angiogenesis. We use this model to investigate the effect of bidirectional communication between endothelial cells and tumor cells on treatments targeting VEGF and its receptors both in vitro and in vivo. Our results underscore the fact that in vitro therapeutic outcomes do not always translate to the in vivo situation. For example, our model predicts that certain therapeutic combinations result in antagonism in vivo that is not observed in vitro. Mathematical modeling in this direction can shed light on the mechanisms behind experimental observations that manipulating VEGF and its receptors is successful in some cases but disappointing in others.

Teratoma formation of human embryonic stem cells in three-dimensional perfusion culture bioreactors.

PubMed

Stachelscheid, H; Wulf-Goldenberg, A; Eckert, K; Jensen, J; Edsbagge, J; Björquist, P; Rivero, M; Strehl, R; Jozefczuk, J; Prigione, A; Adjaye, J; Urbaniak, T; Bussmann, P; Zeilinger, K; Gerlach, J C

2013-09-01

Teratoma formation in mice is today the most stringent test for pluripotency that is available for human pluripotent cells, as chimera formation and tetraploid complementation cannot be performed with human cells. The teratoma assay could also be applied for assessing the safety of human pluripotent cell-derived cell populations intended for therapeutic applications. In our study we examined the spontaneous differentiation behaviour of human embryonic stem cells (hESCs) in a perfused 3D multi-compartment bioreactor system and compared it with differentiation of hESCs and human induced pluripotent cells (hiPSCs) cultured in vitro as embryoid bodies and in vivo in an experimental mouse model of teratoma formation. Results from biochemical, histological/immunohistological and ultrastuctural analyses revealed that hESCs cultured in bioreactors formed tissue-like structures containing derivatives of all three germ layers. Comparison with embryoid bodies and the teratomas revealed a high degree of similarity of the tissues formed in the bioreactor to these in the teratomas at the histological as well as transcriptional level, as detected by comparative whole-genome RNA expression profiling. The 3D culture system represents a novel in vitro model that permits stable long-term cultivation, spontaneous multi-lineage differentiation and tissue formation of pluripotent cells that is comparable to in vivo differentiation. Such a model is of interest, e.g. for the development of novel cell differentiation strategies. In addition, the 3D in vitro model could be used for teratoma studies and pluripotency assays in a fully defined, controlled environment, alternatively to in vivo mouse models. Copyright © 2012 John Wiley & Sons, Ltd.

In vitro and in vivo approaches to study osteocyte biology.

PubMed

Kalajzic, Ivo; Matthews, Brya G; Torreggiani, Elena; Harris, Marie A; Divieti Pajevic, Paola; Harris, Stephen E

2013-06-01

Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in the regulation of bone formation and resorption. Studies of osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of osteocyte directed transgenic and gene deficiency mouse models. We will provide an overview of the current in vitro and in vivo models utilized to study osteocyte biology. We discuss generation of osteocyte-like cell lines and isolation of primary osteocytes and summarize studies that have utilized these cellular models to understand the functional role of osteocytes. Approaches that attempt to selectively identify and isolate osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in osteocytes will be discussed. In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate in understanding the multiple roles of osteocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Purcell, James W; Davis, Jefferson; Reddy, Mamatha

2009-06-10

Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein (KSP), a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have demonstrated a 9% response rate in patients with locally advanced or metastatic breast cancer, and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer we explored the activity of ispinesib alone and in combination several therapies approved for the treatment of breast cancer. We measured the ispinesib sensitivity andmore » pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo, and tested the ability of ispinesib to enhance the anti-tumor activity of approved therapies. In vitro, ispinesib displayed broad anti-proliferative activity against a panel of 53 breast cell-lines. In vivo, ispinesib produced regressions in each of five breast cancer models, and tumor free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the anti-tumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine, and exhibited activity comparable to paclitaxel and ixabepilone. These findings support further clinical exploration of KSP inhibitors for the treatment of breast cancer.« less

Purpose-driven biomaterials research in liver-tissue engineering.

PubMed

Ananthanarayanan, Abhishek; Narmada, Balakrishnan Chakrapani; Mo, Xuejun; McMillian, Michael; Yu, Hanry

2011-03-01

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications. Copyright © 2010 Elsevier Ltd. All rights reserved.

A method for evaluating nanoparticle transport through the blood-brain barrier in vitro.

PubMed

Guarnieri, Daniela; Muscetti, Ornella; Netti, Paolo A

2014-01-01

Blood-brain barrier (BBB) represents a formidable barrier for many therapeutic drugs to enter the brain tissue. The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous system (CNS) diseases. In this context, nanoparticles are an emerging class of drug delivery systems that can be easily tailored to deliver drugs to various compartments of the body, including the brain. To identify, characterize, and validate novel nanoparticles applicable to brain delivery, in vitro BBB model systems have been developed. In this work, we describe a method to screen nanoparticles with variable size and surface functionalization in order to define the physicochemical characteristics underlying the design of nanoparticles that are able to efficiently cross the BBB.

Exploration and comparison of in vitro eye irritation tests with the ISO standard in vivo rabbit test for the evaluation of the ocular irritancy of contact lenses.

PubMed

Yun, Jun-Won; Hailian, Quan; Na, Yirang; Kang, Byeong-Cheol; Yoon, Jung-Hee; Cho, Eun-Young; Lee, Miri; Kim, Da-Eun; Bae, SeungJin; Seok, Seung Hyeok; Lim, Kyung-Min

2016-12-01

In an effort to explore the use of alternative methods to animal testing for the evaluation of the ocular irritancy of medical devices, we evaluated representative contact lenses with the bovine corneal opacity and permeability test (BCOP) and an in vitro eye irritation test using the three-dimensionally-reconstructed human corneal epithelium (RhCE) models, EpiOcular™ and MCTT HCE™. In addition, we compared the obtained results with the ISO standard in vivo rabbit eye irritation test (ISO10993-10). Along with the positive controls (benzalkonium chloride, BAK, 0.02, 0.2, and 1%), the extracts of 4 representative contact lenses (soft, disposable, hard, and colored lenses) and 2 reference lenses (dye-eluting and BAK-coated lenses) were tested. All the lenses, except for the BAK-coated lens, were determined non-irritants in all test methods, while the positive controls yielded relevant results. More importantly, BCOP, EpiOcular™, and MCTT HCE™ yielded a consistent decision for all the tested samples, with the exception of 0.2% BAK in BCOP, for which no prediction could be made. Overall, all the in vitro tests correlated well with the in vivo rabbit eye irritation test, and furthermore, the combination of in vitro tests as a tiered testing strategy was able to produce results similar to those seen in vivo. These observations suggest that such methods can be used as alternative assays to replace the conventional in vivo test method in the evaluation of the ocular irritancy of ophthalmic medical devices, although further study is necessary. Copyright © 2016. Published by Elsevier Ltd.

Screening and Development of New Inhibitors of FtsZ from M. Tuberculosis

PubMed Central

Mathew, Bini; Ross, Larry; Connelly, Michele C.; Lofton, Hava; Rajagopalan, Malini; Guy, R. Kiplin; Reynolds, Robert C.

2016-01-01

A variety of commercial analogs and a newer series of Sulindac derivatives were screened for inhibition of M. tuberculosis (Mtb) in vitro and specifically as inhibitors of the essential mycobacterial tubulin homolog, FtsZ. Due to the ease of preparing diverse analogs and a favorable in vivo pharmacokinetic and toxicity profile of a representative analog, the Sulindac scaffold may be useful for further development against Mtb with respect to in vitro bacterial growth inhibition and selective activity for Mtb FtsZ versus mammalian tubulin. Further discovery efforts will require separating reported mammalian cell activity from both antibacterial activity and inhibition of Mtb FtsZ. Modeling studies suggest that these analogs bind in a specific region of the Mtb FtsZ polymer that differs from human tubulin and, in combination with a pharmacophore model presented herein, future hybrid analogs of the reported active molecules that more efficiently bind in this pocket may improve antibacterial activity while improving other drug characteristics. PMID:27768711

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

PubMed

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

The occupational physician's point of view: the model of man-made vitreous fibers.

PubMed Central

Brochard, P; Pairon, J C; Bignon, J

1994-01-01

This article gives a detailed description of the procedure the occupational physician uses in interpreting the available scientific data to provide useful information for prevention of pulmonary diseases related to man-made mineral fibers, particularly lung cancer and mesothelioma. As it is difficult to reach definite conclusions from human data on the toxicity of specific fibers, an experimental approach is needed. Concerning animal data, we emphasize that adequate inhalation studies are the "gold standard" for extrapolating to humans. However, experiments using intracavitary injection or cells in vitro may represent indicative tests for a possible carcinogenic effect. Such tests should be used to assess the intrinsic carcinogenicity of fibers, but they must be confirmed by adequate inhalation models. Despite the present uncertainties, a proposal is made that could make it possible to classify fibers according to their toxicologic potential, grading them in accordance with physicochemical parameters, in vitro testing, and animal experiments. This procedure may be applicable to nonvitreous fibers and to organic fibers. PMID:7882952

Discovery of a Novel Class of Survival Motor Neuron 2 Splicing Modifiers for the Treatment of Spinal Muscular Atrophy.

PubMed

Pinard, Emmanuel; Green, Luke; Reutlinger, Michael; Weetall, Marla; Naryshkin, Nikolai A; Baird, John; Chen, Karen S; Paushkin, Sergey V; Metzger, Friedrich; Ratni, Hasane

2017-05-25

Spinal muscular atrophy (SMA) is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene, resulting in low levels of functional SMN protein. We have reported recently the identification of small molecules (coumarins, iso-coumarins and pyrido-pyrimidinones) that modify the alternative splicing of SMN2, a paralogous gene to SMN1, restoring the survival motor neuron (SMN) protein level in mouse models of SMA. Herein, we report our efforts to identify a novel chemotype as one strategy to potentially circumvent safety concerns from earlier derivatives such as in vitro phototoxicity and in vitro mutagenicity associated with compounds 1 and 2 or the in vivo retinal findings observed in a long-term chronic tox study with 3 at high exposures only. Optimized representative compounds modify the alternative splicing of SMN2, increase the production of full length SMN2 mRNA, and therefore levels of full length SMN protein upon oral administration in two mouse models of SMA.

Interactions between human osteoblasts and prostate cancer cells in a novel 3D in vitro model

PubMed Central

Sieh, Shirly; Lubik, Amy A; Clements, Judith A; Nelson, Colleen C

2010-01-01

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening. PMID:21197221

Determination of replicate composite bone material properties using modal analysis.

PubMed

Leuridan, Steven; Goossens, Quentin; Pastrav, Leonard; Roosen, Jorg; Mulier, Michiel; Denis, Kathleen; Desmet, Wim; Sloten, Jos Vander

2017-02-01

Replicate composite bones are used extensively for in vitro testing of new orthopedic devices. Contrary to tests with cadaveric bone material, which inherently exhibits large variability, they offer a standardized alternative with limited variability. Accurate knowledge of the composite's material properties is important when interpreting in vitro test results and when using them in FE models of biomechanical constructs. The cortical bone analogue material properties of three different fourth-generation composite bone models were determined by updating FE bone models using experimental and numerical modal analyses results. The influence of the cortical bone analogue material model (isotropic or transversely isotropic) and the inter- and intra-specimen variability were assessed. Isotropic cortical bone analogue material models failed to represent the experimental behavior in a satisfactory way even after updating the elastic material constants. When transversely isotropic material models were used, the updating procedure resulted in a reduction of the longitudinal Young's modulus from 16.00GPa before updating to an average of 13.96 GPa after updating. The shear modulus was increased from 3.30GPa to an average value of 3.92GPa. The transverse Young's modulus was lowered from an initial value of 10.00GPa to 9.89GPa. Low inter- and intra-specimen variability was found. Copyright © 2016 Elsevier Ltd. All rights reserved.

A computational model of in vitro angiogenesis based on extracellular matrix fibre orientation.

PubMed

Edgar, Lowell T; Sibole, Scott C; Underwood, Clayton J; Guilkey, James E; Weiss, Jeffrey A

2013-01-01

Recent interest in the process of vascularisation within the biomedical community has motivated numerous new research efforts focusing on the process of angiogenesis. Although the role of chemical factors during angiogenesis has been well documented, the role of mechanical factors, such as the interaction between angiogenic vessels and the extracellular matrix, remains poorly understood. In vitro methods for studying angiogenesis exist; however, measurements available using such techniques often suffer from limited spatial and temporal resolutions. For this reason, computational models have been extensively employed to investigate various aspects of angiogenesis. This paper outlines the formulation and validation of a simple and robust computational model developed to accurately simulate angiogenesis based on length, branching and orientation morphometrics collected from vascularised tissue constructs. Microvessels were represented as a series of connected line segments. The morphology of the vessels was determined by a linear combination of the collagen fibre orientation, the vessel density gradient and a random walk component. Excellent agreement was observed between computational and experimental morphometric data over time. Computational predictions of microvessel orientation within an anisotropic matrix correlated well with experimental data. The accuracy of this modelling approach makes it a valuable platform for investigating the role of mechanical interactions during angiogenesis.

Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene: an oncolytic virus superior to dl1520 (ONYX-015) for human head and neck cancer.

PubMed

Tysome, James R; Wang, Pengju; Alusi, Ghassan; Briat, Arnaud; Gangeswaran, Rathi; Wang, Jiwei; Bhakta, Vipul; Fodor, Istvan; Lemoine, Nick R; Wang, Yaohe

2011-09-01

Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.

The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes.

PubMed

Xie, Yuan; Bergström, Tobias; Jiang, Yiwen; Johansson, Patrik; Marinescu, Voichita Dana; Lindberg, Nanna; Segerman, Anna; Wicher, Grzegorz; Niklasson, Mia; Baskaran, Sathishkumar; Sreedharan, Smitha; Everlien, Isabelle; Kastemar, Marianne; Hermansson, Annika; Elfineh, Lioudmila; Libard, Sylwia; Holland, Eric Charles; Hesselager, Göran; Alafuzoff, Irina; Westermark, Bengt; Nelander, Sven; Forsberg-Nilsson, Karin; Uhrbom, Lene

2015-10-01

Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research.

Moving beyond the comprehensive in vitro proarrhythmia assay: Use of human-induced pluripotent stem cell-derived cardiomyocytes to assess contractile effects associated with drug-induced structural cardiotoxicity.

PubMed

Yang, Xi; Papoian, Thomas

2018-02-27

Drug-induced cardiotoxicity is a potentially severe side effect that can adversely affect myocardial contractility through structural or electrophysiological changes in cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising human cardiac in vitro model system to assess both proarrhythmic and non-proarrhythmic cardiotoxicity of new drug candidates. The scalable differentiation of hiPSCs into cardiomyocytes provides a renewable cell source that overcomes species differences present in current animal models of drug toxicity testing. The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative represents a paradigm shift for proarrhythmic risk assessment, and hiPSC-CMs are an integral component of that paradigm. The recent advancements in hiPSC-CMs will not only impact safety decisions for possible drug-induced proarrhythmia, but should also facilitate risk assessment for non-proarrhythmic cardiotoxicity, where current non-clinical approaches are limited in detecting this risk before initiation of clinical trials. Importantly, emerging evidence strongly suggests that the use of hiPSC-CMs with cardiac physiological relevant measurements in vitro improves the detection of structural cardiotoxicity. Here we review high-throughput drug screening using the hiPSC-CM model as an experimentally feasible approach to assess potential contractile and structural cardiotoxicity in early phase drug development. We also suggest that the assessment of structural cardiotoxicity can be added to electrophysiological tests in the same platform to complement the Comprehensive in vitro Proarrhythmia Assay for regulatory use. Ideally, application of these novel tools in early drug development will allow for more reliable risk assessment and lead to more informed regulatory decisions in making safe and effective drugs available to the public. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

In Vitro Determination of Drug Transfer from Drug-Coated Balloons

PubMed Central

Seidlitz, Anne; Kotzan, Nadine; Nagel, Stefan; Reske, Thomas; Grabow, Niels; Harder, Claus; Petersen, Svea; Sternberg, Katrin; Weitschies, Werner

2013-01-01

Drug-coated balloons are medical devices designed to locally deliver drug to diseased segments of the vessel wall. For these dosage forms, drug transfer to the vessel wall needs to be examined in detail, since drug released into the blood is cleared from the site. In order to examine drug transfer, a new in vitro setup was developed combining the estimation of drug loss during advancement to the site of application in a model coronary artery pathway with a hydrogel compartment representing, as a very simplified model, the vessel wall. The transfer of fluorescent model substances as well as the drug paclitaxel from coated balloons to the simulated vessel wall was evaluated using this method. The model was suitable to quantify the fractions transferred to the hydrogel and also to qualitatively assess distribution patterns in the hydrogel film. In the case of fluorescein sodium, rhodamin b and paclitaxel, vast amounts of the coated substance were lost during the simulated passage and only very small fractions of about 1% of the total load were transferred to the gel. This must be attributed to good water solubility of the fluorescent substances and the mechanical instability of the paclitaxel coating. Transfer of the hydrophobic model substance triamterene was however nearly unaffected by the preliminary tracking procedure with transferred fractions ranging from 8% to 14%. Analysis of model substance distribution yielded inhomogeneous distributions indicating that the coating was not evenly distributed on the balloon surface and that a great fraction of the coating liquid did not penetrate the folds of the balloon. This finding is contradictory to the generally accepted assumption of a drug depot inside the folds and emphasizes the necessity to thoroughly characterize in vitro performance of drug-coated balloons to support the very promising clinical data. PMID:24391863

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model

PubMed Central

Sirenko, Oksana; Grimm, Fabian A.; Ryan, Kristen R.; Iwata, Yasuhiro; Chiu, Weihsueh A.; Parham, Frederick; Wignall, Jessica A.; Anson, Blake; Cromwell, Evan F.; Behl, Mamta; Rusyn, Ivan; Tice, Raymond R.

2017-01-01

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30 min or 24 hrs and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca2+ flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of tested chemicals exhibited effects on cardiomyocyte rhythm after 30 min of exposure. After 24 hours, the effects on cell rhythm without cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes were visualized using Toxicological Prioritization Index (ToxPi) and showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential to alter cardiomyocyte function at high exposures, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. PMID:28259702

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model.

PubMed

Sirenko, Oksana; Grimm, Fabian A; Ryan, Kristen R; Iwata, Yasuhiro; Chiu, Weihsueh A; Parham, Frederick; Wignall, Jessica A; Anson, Blake; Cromwell, Evan F; Behl, Mamta; Rusyn, Ivan; Tice, Raymond R

2017-05-01

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30min or 24h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca 2+ flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30min of exposure. In contrast, after 24h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. Copyright © 2017. Published by Elsevier Inc.

Quetiapine and its metabolite norquetiapine: translation from in vitro pharmacology to in vivo efficacy in rodent models

PubMed Central

Widzowski, D; Maciag, C; Zacco, A; Hudzik, T; Liu, J; Nyberg, S; Wood, M W

2015-01-01

Background and Purpose Quetiapine has a range of clinical activity distinct from other atypical antipsychotic drugs, demonstrating efficacy as monotherapy in bipolar depression, major depressive disorder and generalized anxiety disorder. The neuropharmacological mechanisms underlying this clinical profile are not completely understood; however, the major active metabolite, norquetiapine, has been shown to have a distinct in vitro pharmacological profile consistent with a broad therapeutic range and may contribute to the clinical profile of quetiapine. Experimental Approach We evaluated quetiapine and norquetiapine, using in vitro binding and functional assays of targets known to be associated with antidepressant and anxiolytic drug actions and compared these activities with a representative range of established antipsychotics and antidepressants. To determine how the in vitro pharmacological properties translate into in vivo activity, we used preclinical animal models with translational relevance to established antidepressant‐like and anxiolytic‐like drug action. Key Results Norquetiapine had equivalent activity to established antidepressants at the noradrenaline transporter (NET), while quetiapine was inactive. Norquetiapine was active in the mouse forced swimming and rat learned helplessness tests. In in vivo receptor occupancy studies, norquetiapine had significant occupancy at NET at behaviourally relevant doses. Both quetiapine and norquetiapine were agonists at 5‐HT1A receptors, and the anxiolytic‐like activity of norquetiapine in rat punished responding was blocked by the 5‐HT1A antagonist, WAY100635. Conclusions and Implications Quetiapine and norquetiapine have multiple in vitro pharmacological actions, and results from preclinical studies suggest that activity at NET and 5‐HT1A receptors contributes to the antidepressant and anxiolytic effects in patients treated with quetiapine. PMID:26436896

In vitro assessment of skin irritation potential of surfactant-based formulations by using a 3-D skin reconstructed tissue model and cytokine response.

PubMed

Walters, Russel M; Gandolfi, Lisa; Mack, M Catherine; Fevola, Michael; Martin, Katharine; Hamilton, Mathew T; Hilberer, Allison; Barnes, Nicole; Wilt, Nathan; Nash, Jennifer R; Raabe, Hans A; Costin, Gertrude-Emilia

2016-12-01

The personal care industry is focused on developing safe, more efficacious, and increasingly milder products, that are routinely undergoing preclinical and clinical testing before becoming available for consumer use on skin. In vitro systems based on skin reconstructed equivalents are now established for the preclinical assessment of product irritation potential and as alternative testing methods to the classic Draize rabbit skin irritation test. We have used the 3-D EpiDerm™ model system to evaluate tissue viability and primary cytokine interleukin-1α release as a way to evaluate the potential dermal irritation of 224 non-ionic, amphoteric and/or anionic surfactant-containing formulations, or individual raw materials. As part of our testing programme, two representative benchmark materials with known clinical skin irritation potential were qualified through repeated testing, for use as references for the skin irritation evaluation of formulations containing new surfactant ingredients. We have established a correlation between the in vitro screening approach and clinical testing, and are continually expanding our database to enhance this correlation. This testing programme integrates the efforts of global manufacturers of personal care products that focus on the development of increasingly milder formulations to be applied to the skin, without the use of animal testing. 2016 FRAME.

Spatially Assembled Bilayer Cell Sheets of Stem Cells and Endothelial Cells Using Thermosensitive Hydrogels for Therapeutic Angiogenesis.

PubMed

Jun, Indong; Ahmad, Taufiq; Bak, Seongwoo; Lee, Joong-Yup; Kim, Eun Mi; Lee, Jinkyu; Lee, Yu Bin; Jeong, Hongsoo; Jeon, Hojeong; Shin, Heungsoo

2017-05-01

Although the coculture of multiple cell types has been widely employed in regenerative medicine, in vivo transplantation of cocultured cells while maintaining the hierarchical structure remains challenging. Here, a spatially assembled bilayer cell sheet of human mesenchymal stem cells and human umbilical vein endothelial cells on a thermally expandable hydrogel containing fibronectin is prepared and its effect on in vitro proangiogenic functions and in vivo ischemic injury is investigated. The expansion of hydrogels in response to a temperature change from 37 to 4 °C allows rapid harvest and delivery of the bilayer cell sheet to two different targets (an in vitro model glass surface and in vivo tissue). The in vitro study confirms that the bilayer sheet significantly increases proangiogenic functions such as the release of nitric oxide and expression of vascular endothelial cell genes. In addition, transplantation of the cell sheet from the hydrogels into a hindlimb ischemia mice model demonstrates significant retardation of necrosis particularly in the group transplated with the bilayer sheet. Collectively, the bilayer cell sheet is readily transferrable from the thermally expandable hydrogel and represents an alternative approach for recovery from ischemic injury, potentially via improved cell-cell communication. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Celecoxib Derivative AR-12 Has Broad-Spectrum Antifungal Activity In Vitro and Improves the Activity of Fluconazole in a Murine Model of Cryptococcosis

PubMed Central

Koselny, Kristy; Green, Julianne; DiDone, Louis; Halterman, Justin P.; Fothergill, Annette W.; Wiederhold, Nathan P.; Patterson, Thomas F.; Cushion, Melanie T.; Rappelye, Chad; Wellington, Melanie

2016-01-01

Only one new class of antifungal drugs has been introduced into clinical practice in the last 30 years, and thus the identification of small molecules with novel mechanisms of action is an important goal of current anti-infective research. Here, we describe the characterization of the spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib derivative which has been tested in a phase I clinical trial as an anticancer agent. AR-12 inhibits fungal acetyl coenzyme A (acetyl-CoA) synthetase in vitro and is fungicidal at concentrations similar to those achieved in human plasma. AR-12 has a broad spectrum of activity, including activity against yeasts (e.g., Candida albicans, non-albicans Candida spp., Cryptococcus neoformans), molds (e.g., Fusarium, Mucor), and dimorphic fungi (Blastomyces, Histoplasma, and Coccidioides) with MICs of 2 to 4 μg/ml. AR-12 is also active against azole- and echinocandin-resistant Candida isolates, and subinhibitory AR-12 concentrations increase the susceptibility of fluconazole- and echinocandin-resistant Candida isolates. Finally, AR-12 also increases the activity of fluconazole in a murine model of cryptococcosis. Taken together, these data indicate that AR-12 represents a promising class of small molecules with broad-spectrum antifungal activity. PMID:27645246

Sensitivity to PI3K and AKT inhibitors is mediated by divergent molecular mechanisms in subtypes of DLBCL.

PubMed

Erdmann, Tabea; Klener, Pavel; Lynch, James T; Grau, Michael; Vočková, Petra; Molinsky, Jan; Tuskova, Diana; Hudson, Kevin; Polanska, Urszula M; Grondine, Michael; Mayo, Michele; Dai, Beiying; Pfeifer, Matthias; Erdmann, Kristian; Schwammbach, Daniela; Zapukhlyak, Myroslav; Staiger, Annette M; Ott, German; Berdel, Wolfgang E; Davies, Barry R; Cruzalegui, Francisco; Trneny, Marek; Lenz, Peter; Barry, Simon T; Lenz, Georg

2017-07-20

Activated B-cell-like (ABC) and germinal center B-cell-like diffuse large B-cell lymphoma (DLBCL) represent the 2 major molecular DLBCL subtypes. They are characterized by differences in clinical course and by divergent addiction to oncogenic pathways. To determine activity of novel compounds in these 2 subtypes, we conducted an unbiased pharmacologic in vitro screen. The phosphatidylinositol-3-kinase (PI3K) α/δ (PI3Kα/δ) inhibitor AZD8835 showed marked potency in ABC DLBCL models, whereas the protein kinase B (AKT) inhibitor AZD5363 induced apoptosis in PTEN-deficient DLBCLs irrespective of their molecular subtype. These in vitro results were confirmed in various cell line xenograft and patient-derived xenograft mouse models in vivo. Treatment with AZD8835 induced inhibition of nuclear factor κB signaling, prompting us to combine AZD8835 with the Bruton's tyrosine kinase inhibitor ibrutinib. This combination was synergistic and effective both in vitro and in vivo. In contrast, the AKT inhibitor AZD5363 was effective in PTEN-deficient DLBCLs through downregulation of the oncogenic transcription factor MYC. Collectively, our data suggest that patients should be stratified according to their oncogenic dependencies when treated with PI3K and AKT inhibitors. © 2017 by The American Society of Hematology.

A Nanoscale Interface Promoting Molecular and Functional Differentiation of Neural Cells

NASA Astrophysics Data System (ADS)

Posati, Tamara; Pistone, Assunta; Saracino, Emanuela; Formaggio, Francesco; Mola, Maria Grazia; Troni, Elisabetta; Sagnella, Anna; Nocchetti, Morena; Barbalinardo, Marianna; Valle, Francesco; Bonetti, Simone; Caprini, Marco; Nicchia, Grazia Paola; Zamboni, Roberto; Muccini, Michele; Benfenati, Valentina

2016-08-01

Potassium channels and aquaporins expressed by astrocytes are key players in the maintenance of cerebral homeostasis and in brain pathophysiologies. One major challenge in the study of astrocyte membrane channels in vitro, is that their expression pattern does not resemble the one observed in vivo. Nanostructured interfaces represent a significant resource to control the cellular behaviour and functionalities at micro and nanoscale as well as to generate novel and more reliable models to study astrocytes in vitro. However, the potential of nanotechnologies in the manipulation of astrocytes ion channels and aquaporins has never been previously reported. Hydrotalcite-like compounds (HTlc) are layered materials with increasing potential as biocompatible nanoscale interface. Here, we evaluate the effect of the interaction of HTlc nanoparticles films with primary rat neocortical astrocytes. We show that HTlc films are biocompatible and do not promote gliotic reaction, while favouring astrocytes differentiation by induction of F-actin fibre alignment and vinculin polarization. Western Blot, Immunofluorescence and patch-clamp revealed that differentiation was accompanied by molecular and functional up-regulation of both inward rectifying potassium channel Kir 4.1 and aquaporin 4, AQP4. The reported results pave the way to engineering novel in vitro models to study astrocytes in a in vivo like condition.

RapA, SWI/SNF subunit of Escherichia coli RNA polymerase promotes the release of nascent RNA from transcription complexes

PubMed Central

Yawn, Brandon; Zhang, Lin; Mura, Cameron; Sukhodolets, Maxim V.

2009-01-01

RapA, a prokaryotic member of the SWI/SNF protein superfamily, is an integral part of the RNA polymerase transcription complex. RapA’s function and catalytic mechanism have been linked to nucleic acid remodeling. In this work we show that mutations in the interface between RapA’s SWI/SNF and double-stranded nucleic acid-binding domains significantly alter ATP hydrolysis in purified RapA. The effects of individual mutations on ATP hydrolysis loosely correlated with RapA’s nucleic acid-remodeling activity, indicating that the interaction between these domains may be important for the RapA-mediated remodeling of nonproductive transcription complexes. In this study we introduced a model system for in vitro transcription of a full-length E. coli gene (slyD). To study the function of RapA, we fractionated and identified in vitro transcription reaction intermediates in the presence or absence of RapA. These experiments demonstrated that RapA contributes to the formation of free RNA species during in vitro transcription. This work further refines our models for RapA function in vivo and establishes a new role in RNA management for a representative of the SWI/SNF protein superfamily. PMID:19580329

New use of an old drug: inhibition of breast cancer stem cells by benztropine mesylate

PubMed Central

Cui, Jihong; Hollmén, Maija; Li, Lina; Chen, Yong; Proulx, Steven T.; Reker, Daniel; Schneider, Gisbert; Detmar, Michael

2017-01-01

Cancer stem cells (CSCs) play major roles in cancer initiation, metastasis, recurrence and therapeutic resistance. Targeting CSCs represents a promising strategy for cancer treatment. The purpose of this study was to identify selective inhibitors of breast CSCs (BCSCs). We carried out a cell-based phenotypic screening with cell viability as a primary endpoint, using a collection of 2,546 FDA-approved drugs and drug-like molecules in spheres formed by malignant human breast gland-derived cells (HMLER-shEcad cells, representing BCSCs) and control immortalized non-tumorigenic human mammary cells (HMLE cells, representing normal stem cells). 19 compounds were identified from screening. The chemically related molecules benztropine mesylate and deptropine citrate were selected for further validation and both potently inhibited sphere formation and self-renewal of BCSCs in vitro. Benztropine mesylate treatment decreased cell subpopulations with high ALDH activity and with a CD44+/CD24− phenotype. In vivo, benztropine mesylate inhibited tumor-initiating potential in a 4T1 mouse model. Functional studies indicated that benztropine mesylate inhibits functions of CSCs via the acetylcholine receptors, dopamine transporters/receptors, and/or histamine receptors. In summary, our findings identify benztropine mesylate as an inhibitor of BCSCs in vitro and in vivo. This study also provides a screening platform for identification of additional anti-CSC agents. PMID:27894093

The use of animal models in homeopathic research--a review of 2010-2014 PubMed indexed papers.

PubMed

Bonamin, Leoni Villano; Cardoso, Thayná Neves; de Carvalho, Aloísio Cunha; Amaral, Juliana Gimenez

2015-10-01

In the 1990s, a study was performed on the effects of highly diluted thyroxine on frog metamorphosis. This model represented one of the most discussed examples of the biological effects of high dilutions over the next two decades. In 2010, another critical conceptual review of the use of animal models in homeopathy and high-dilution research was published. The main contribution of these studies was the elucidation of the biological features and phenomenology of the effects of high dilutions on living systems, representing an important step forward in our understanding of the mechanisms of action of homeopathic medicines. We performed a further review of this line of investigation using the same methods. Fifty-three articles that were indexed in the PubMed database and used 12 different animal species were systematically evaluated. Only a fraction of the studies (29/53) reported herein were performed with "ultra high" dilutions. The other studies were performed with dilutions in ranges below 10(-23) (14/53 articles) or commercial complexes (10/53 articles). Only two articles reported negative results; both used in vivo protocols to test commercial complexes, one in fish and one in bees. The quality of the employed techniques improved in 2010-2014 compared with the studies that were reviewed previously in 2010, with the inclusion of more ethically refined protocols, including in vitro primary cell cultures and ex vivo studies (10/53 articles), often with three or more replicates and analyses of epigenetic mechanisms that were previously unknown in 2010. In our updated review of the past 5 years, we found further demonstrations of the biological effects of homeopathy using more refined animal models and in vitro techniques. Copyright © 2015 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

Semen molecular and cellular features: these parameters can reliably predict subsequent ART outcome in a goat model

PubMed Central

Berlinguer, Fiammetta; Madeddu, Manuela; Pasciu, Valeria; Succu, Sara; Spezzigu, Antonio; Satta, Valentina; Mereu, Paolo; Leoni, Giovanni G; Naitana, Salvatore

2009-01-01

Currently, the assessment of sperm function in a raw or processed semen sample is not able to reliably predict sperm ability to withstand freezing and thawing procedures and in vivo fertility and/or assisted reproductive biotechnologies (ART) outcome. The aim of the present study was to investigate which parameters among a battery of analyses could predict subsequent spermatozoa in vitro fertilization ability and hence blastocyst output in a goat model. Ejaculates were obtained by artificial vagina from 3 adult goats (Capra hircus) aged 2 years (A, B and C). In order to assess the predictive value of viability, computer assisted sperm analyzer (CASA) motility parameters and ATP intracellular concentration before and after thawing and of DNA integrity after thawing on subsequent embryo output after an in vitro fertility test, a logistic regression analysis was used. Individual differences in semen parameters were evident for semen viability after thawing and DNA integrity. Results of IVF test showed that spermatozoa collected from A and B lead to higher cleavage rates (0 < 0.01) and blastocysts output (p < 0.05) compared with C. Logistic regression analysis model explained a deviance of 72% (p < 0.0001), directly related with the mean percentage of rapid spermatozoa in fresh semen (p < 0.01), semen viability after thawing (p < 0.01), and with two of the three comet parameters considered, i.e tail DNA percentage and comet length (p < 0.0001). DNA integrity alone had a high predictive value on IVF outcome with frozen/thawed semen (deviance explained: 57%). The model proposed here represents one of the many possible ways to explain differences found in embryo output following IVF with different semen donors and may represent a useful tool to select the most suitable donors for semen cryopreservation. PMID:19900288

Identification of novel antitubulin agents by using a virtual screening approach based on a 7-point pharmacophore model of the tubulin colchi-site.

PubMed

Massarotti, Alberto; Theeramunkong, Sewan; Mesenzani, Ornella; Caldarelli, Antonio; Genazzani, Armando A; Tron, Gian Cesare

2011-12-01

Tubulin inhibition represents an established target in the field of anticancer research, and over the last 20 years, an intensive search for new antimicrotubule agents has occurred. Indeed, in silico models have been presented that might aid the discovery of novel agents. Among these, a 7-point pharmacophore model has been recently proposed. As a formal proof of this model, we carried out a ligand-based virtual screening on the colchicine-binding site. In vitro testing demonstrated that two compounds displayed a cytotoxic profile on neuroblastoma cancer cells (SH-SY5H) and one had an antitubulinic profile. © 2011 John Wiley & Sons A/S.

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL.

PubMed

Katoh, Masakazu; Hamajima, Fumiyasu; Ogasawara, Takahiro; Hata, Ken-ichiro

2013-12-01

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test. Copyright © 2013 Elsevier Ltd. All rights reserved.

Comparison of different models for the testing of pilocarpine eyedrops using conventional eyedrops and a novel depot formulation (nanoparticles).

PubMed

Diepold, R; Kreuter, J; Himber, J; Gurny, R; Lee, V H; Robinson, J R; Saettone, M F; Schnaudigel, O E

1989-01-01

An objective in the development of ophthalmic formulations is the use of in vitro or animal models that closely resemble the clinical situation. For this reason, experiments with conventional pilocarpine nitrate eyedrops and a depot formulation of pilocarpine nitrate sorbed to poly (butylcyanoacrylate) nanoparticles were carried out. In vitro, the diffusion of pilocarpine through bovine cornea was measured using Edelhauser cells. In vivo, the rabbit aqueous humor concentration of pilocarpine and miosis were determined after application of the above formulations. In addition, intraocular pressure was measured. Since pilocarpine has little influence on intraocular pressure in healthy rabbits, the pressure had to be increased artificially. Three models were employed that are described in the literature, namely, the betamethasone model, the alpha-chymotrypsin model, and the water-loading model. Pilocarpine could be loaded onto nanoparticles by 15% but was rapidly released from the nanoparticles based on the bovine corneal experiment. Nanoparticles only enhanced the aqueous humor concentration at 30 min; this increase, however, led to a considerably extended period of miosis as well as a reduction in intraocular pressure. The duration of the action and the intensity of the response were different among the three models tested. According to the present results, the betamethasone model seems to represent the best correlation to the clinical situation.

Carbamazepine-loaded solid lipid nanoparticles and nanostructured lipid carriers: Physicochemical characterization and in vitro/in vivo evaluation.

PubMed

Scioli Montoto, S; Sbaraglini, M L; Talevi, A; Couyoupetrou, M; Di Ianni, M; Pesce, G O; Alvarez, V A; Bruno-Blanch, L E; Castro, G R; Ruiz, M E; Islan, G A

2018-07-01

Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) represent promising alternatives for drug delivery to the central nervous system. In the present work, four different nanoformulations of the antiepileptic drug Carbamazepine (CBZ) were designed and prepared by the homogenization/ultrasonication method, with encapsulation efficiencies ranging from 82.8 to 93.8%. The formulations remained stable at 4 °C for at least 3 months. Physicochemical and microscopic characterization were performed by photon correlation spectroscopy (PCS), transmission electron microscopy (TEM), atomic force microscopy (AFM); thermal properties by differential scanning calorimetry (DSC), thermogravimetry (TGA) and X-ray diffraction analysis (XRD). The results indicated the presence of spherical shape nanoparticles with a mean particle diameter around 160 nm in a narrow size distribution; the entrapped CBZ displayed an amorphous state. The in vitro release profile of CBZ fitted into a Baker-Lonsdale model for spherical matrices and almost the 100% of the encapsulated drug was released in a controlled manner during the first 24 h. The apparent permeability of CBZ-loaded nanoparticles through a cell monolayer model was similar to that of the free drug. In vivo experiments in a mice model of seizure suggested protection by CBZ-NLC against seizures for at least 2 h after intraperitoneal administration. The developed CBZ-loaded lipid nanocarriers displayed optimal characteristics of size, shape and drug release and possibly represent a promising tool to improve the treatment of refractory epilepsy linked to efflux transporters upregulation. Copyright © 2018 Elsevier B.V. All rights reserved.

Detection of Mutant Huntingtin Aggregation Conformers and Modulation of SDS-Soluble Fibrillar Oligomers by Small Molecules

PubMed Central

Sontag, Emily Mitchell; Lotz, Gregor P.; Yang, Guocheng; Sontag, Christopher J.; Cummings, Brian J.; Glabe, Charles G.; Muchowski, Paul J.; Thompson, Leslie Michels

2012-01-01

The Huntington’s disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a β-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models. PMID:24086178

Connexin 26 facilitates gastrointestinal bacterial infection in vitro.

PubMed

Simpson, Charlotte; Kelsell, David P; Marchès, Olivier

2013-01-01

Escherichia coli, including enteropathogenic E. coli (EPEC), represents the most common cause of diarrhoea worldwide and is therefore a serious public health burden. Treatment for gastrointestinal pathogens is hindered by the emergence of multiple antibiotic resistance, leading to the requirement for the development of new therapies. A variety of mechanisms act in combination to mediate gastrointestinal-bacterial-associated diarrhoea development. For example, EPEC infection of enterocytes induces attaching and effacing lesion formation and the disruption of tight junctions. An alternative enteric pathogen, Shigella flexneri, manipulates the expression of Connexin 26 (Cx26), a gap junction protein. S. flexneri can open Cx26 hemichannels allowing the release of ATP, whereas HeLa cells expressing mutant gap-junction-associated Cx26 are less susceptible to cellular invasion by S. flexneri than cells expressing wild-type (WT) Cx26. We have investigated further the link between Cx26 expression and gastrointestinal infection by using EPEC and S. flexneri as in vitro models of infection. In this study, a significant reduction in EPEC adherence was observed in cells expressing mutant Cx26 compared with WT Cx26. Furthermore, a significant reduction in both cellular invasion by S. flexneri and adherence by EPEC was demonstrated in human intestinal cell lines following treatment with Cx26 short interfering RNA. These in vitro results suggest that the loss of functional Cx26 expression provides improved protection against gastrointestinal bacterial pathogens. Thus, Cx26 represents a potential therapeutic target for gastrointestinal bacterial infection.

The Combined Application of the Caco-2 Cell Bioassay Coupled with In Vivo (Gallus gallus) Feeding Trial Represents an Effective Approach to Predicting Fe Bioavailability in Humans

PubMed Central

Tako, Elad; Bar, Haim; Glahn, Raymond P.

2016-01-01

Research methods that predict Fe bioavailability for humans can be extremely useful in evaluating food fortification strategies, developing Fe-biofortified enhanced staple food crops and assessing the Fe bioavailability of meal plans that include such crops. In this review, research from four recent poultry (Gallus gallus) feeding trials coupled with in vitro analyses of Fe-biofortified crops will be compared to the parallel human efficacy studies which used the same varieties and harvests of the Fe-biofortified crops. Similar to the human studies, these trials were aimed to assess the potential effects of regular consumption of these enhanced staple crops on maintenance or improvement of iron status. The results demonstrate a strong agreement between the in vitro/in vivo screening approach and the parallel human studies. These observations therefore indicate that the in vitro/Caco-2 cell and Gallus gallus models can be integral tools to develop varieties of staple food crops and predict their effect on iron status in humans. The cost-effectiveness of this approach also means that it can be used to monitor the nutritional stability of the Fe-biofortified crop once a variety has released and integrated into the food system. These screening tools therefore represent a significant advancement to the field for crop development and can be applied to ensure the sustainability of the biofortification approach. PMID:27869705

Imaging cell biology in live animals: ready for prime time.

PubMed

Weigert, Roberto; Porat-Shliom, Natalie; Amornphimoltham, Panomwat

2013-06-24

Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

PubMed

Torelli, A; Soragni, E; Bolchi, A; Petrucco, S; Ottonello, S; Branca, C

1996-12-01

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

In vitro and in vivo comparisons of constant resistance AC iontophoresis and DC iontophoresis.

PubMed

Li, S Kevin; Higuchi, William I; Zhu, Honggang; Kern, Steven E; Miller, David J; Hastings, Matthew S

2003-09-04

A previous in vitro constant electrical resistance alternating current (AC) iontophoresis study with human epidermal membrane (HEM) and a model neutral permeant has shown less inter- and intra-sample variability in iontophoretic transport relative to conventional constant direct current (DC) iontophoresis. The objectives of the present study were to address the following questions. (1) Can the skin electrical resistance be maintained at a constant level by AC in humans in vivo? (2) Are the in vitro data with HEM representative of those in vivo? (3) Does constant skin resistance AC iontophoresis have less inter- and intra-sample variability than conventional constant current DC iontophoresis in vivo? (4) What are the electrical and the barrier properties of skin during iontophoresis in vivo? In the present study, in vitro HEM experiments were carried out with the constant resistance AC and the conventional constant current DC methods using mannitol and glucose as the neutral model permeants. In vivo human experiments were performed using glucose as the permeant with a constant skin resistance AC only protocol and two conventional constant current DC methods (continuous constant current DC and constant current DC with its polarity alternated every 10 min with a 3:7 on:off duty cycle). Constant current DC iontophoresis was conducted with commercial constant current DC devices, and constant resistance AC iontophoresis was carried out by reducing and maintaining the skin resistance at a constant target value with AC supplied from a function generator. This study shows that (1) skin electrical resistance can be maintained at a constant level during AC iontophoresis in vivo; (2) HEM in vitro and human skin in vivo demonstrate similar electrical and barrier properties, and these properties are consistent with our previous findings; (3) there is general qualitative and semi-quantitative agreement between the HEM data in vitro and human skin data in vivo; and (4) constant skin resistance AC iontophoresis generally provides less inter- and intra-subject variability than conventional constant current DC.

A proposal for in vitro/GFR molecular erythema action spectrum

NASA Astrophysics Data System (ADS)

de Souza, João A. V.; Lorenzini, Fabiane; Rizzatti, Mara R.

2008-08-01

We propose an erythema action spectrum based on experimental molecular measurements named molecular erythema action spectrum or in vitro/GFR, where the acronym GFR represents our research group name, Grupo de Física das Radiaçöes. The in vitro methodology was developed by using a derma tissue simulator (TSD), as a radiation protection shield, and monochromatic ultraviolet (UV) sources of 254, 310, 365, 380, and 400 nm. The irradiance from each source was monitored through spectroradiometry in order to obtain the exposure dose over a period of time. Changes in the chemical structure were monitored by Fourier transform infrared spectroscopy (FTIR) and UV and visible spectroscopy (UV-vis). The samples were analyzed by UV-vis at each 200 up to 1000 J/m2 and at each 400 up to 2000 J/m2. FTIR was performed only for samples exposed to a maximum dose of 2000 J/m2. The in vitro action parameters were obtained by considering the redshift revealed through UV-vis analysis, as being the molecular quantification of minimal erythema, and the chemical bond rupture of TSD molecules associated with erythema, revealed through FTIR. The in vitro/GFR action spectrum shows that UV-A and UV-B radiation are equally responsible for the damage observed in TSD. When this proposal was compared to the CIE erythema action spectrum from ISO [ISO17166 CIE S 007/E, Erythema Reference Action Spectrum and Standard Erythema Dose (CIE Central Bureau, Austria, 1998)], similarities could be observed in wavelengths less than 280 nm in UV-B region. However, for wavelengths higher than 300 nm, the efficiency of this radiation to induce damage, mainly in the UV-A part, was much higher than predicted in CIE model. The increasing concern on UV-A radiation, assumed to be as responsible as UV-B for inducing most of the already observed skin injuries, may be better understood when observing the experimental model presented in in vitro/GFR action spectrum.

Development of a population pharmacokinetic model for parecoxib and its active metabolite valdecoxib after parenteral parecoxib administration in children.

PubMed

Hullett, Bruce; Salman, Sam; O'Halloran, Sean J; Peirce, Deborah; Davies, Kylie; Ilett, Kenneth F

2012-05-01

Parecoxib is a cyclooxygenase-2 selective inhibitor used in management of postoperative pain in adults. This study aimed to provide pediatric pharmacokinetic information for parecoxib and its active metabolite valdecoxib. Thirty-eight children undergoing surgery received parecoxib (1 mg/kg IV to a maximum of 40 mg) at induction of anesthesia, and plasma samples were collected for drug measurement. Population pharmacokinetic parameters were estimated using nonlinear mixed effects modeling. Area under the valdecoxib concentration-time curve and time above cyclooxygenase-2 in vitro 50% inhibitory concentration for free valdecoxib were simulated. A three-compartment model best represented parecoxib disposition, whereas one compartment was adequate for valdecoxib. Age was linearly correlated with parecoxib clearance (5.0% increase/yr). There was a sigmoid relationship between age and both valdecoxib clearance and distribution volume. Time to 50% maturation was 87 weeks postmenstrual age for both. In simulations using allometric-based doses the 90% prediction interval of valdecoxib concentration-time curve in children 2-12.7 yr included the mean for adults given 40 mg parecoxib IV. Simulated free valdecoxib plasma concentration remained above the in vitro 50% inhibitory concentrations for more than 12 h. In children younger than 2 yr, a dose reduction is likely required due to ongoing metabolic maturation. The final pharmacokinetic model gave a robust representation of parecoxib and valdecoxib disposition. Area under the valdecoxib concentration-time curve was similar to that in adults (40 mg), and simulated free valdecoxib concentration was above the cyclooxygenase-2 in vitro 50% inhibitory concentration for free valdecoxib for at least 12 h.

De novo generation of HSCs from somatic and pluripotent stem cell sources

PubMed Central

Vo, Linda T.

2015-01-01

Generating human hematopoietic stem cells (HSCs) from autologous tissues, when coupled with genome editing technologies, is a promising approach for cellular transplantation therapy and for in vitro disease modeling, drug discovery, and toxicology studies. Human pluripotent stem cells (hPSCs) represent a potentially inexhaustible supply of autologous tissue; however, to date, directed differentiation from hPSCs has yielded hematopoietic cells that lack robust and sustained multilineage potential. Cellular reprogramming technologies represent an alternative platform for the de novo generation of HSCs via direct conversion from heterologous cell types. In this review, we discuss the latest advancements in HSC generation by directed differentiation from hPSCs or direct conversion from somatic cells, and highlight their applications in research and prospects for therapy. PMID:25762177

Relevant pH and lipase for in vitro models of gastric digestion.

PubMed

Sams, Laura; Paume, Julie; Giallo, Jacqueline; Carrière, Frédéric

2016-01-01

The development of in vitro digestion models relies on the availability of in vivo data such as digestive enzyme levels and pH values recorded in the course of meal digestion. The variations of these parameters along the GI tract are important for designing dynamic digestion models but also static models for which the choice of representative conditions of the gastric and intestinal conditions is critical. Simulating gastric digestion with a static model and a single set of parameters is particularly challenging because the variations in pH and enzyme concentration occurring in the stomach are much broader than those occurring in the small intestine. A review of the literature on this topic reveals that most models of gastric digestion use very low pH values that are not representative of the fed conditions. This is illustrated here by showing the variations in gastric pH as a function of meal gastric emptying instead of time. This representation highlights those pH values that are the most relevant for testing meal digestion in the stomach. Gastric lipolysis is still largely ignored or is performed with microbial lipases. In vivo data on gastric lipase and lipolysis have however been collected in humans and dogs during test meals. The biochemical characterization of gastric lipase has shown that this enzyme is rather unique among lipases: (i) stability and activity in the pH range 2 to 7 with an optimum at pH 4-5.4; (ii) high tensioactivity that allows resistance to bile salts and penetration into phospholipid layers covering TAG droplets; (iii) sn-3 stereospecificity for TAG hydrolysis; and (iv) resistance to pepsin. Most of these properties have been known for more than two decades and should provide a rational basis for the replacement of gastric lipase by other lipases when gastric lipase is not available.

Pharmacokinetic-Pharmacodynamic Modeling of the In Vitro Activities of Oxazolidinone Antimicrobial Agents against Methicillin-Resistant Staphylococcus aureus▿

PubMed Central

Schmidt, Stephan; Sabarinath, Sreedharan Nair; Barbour, April; Abbanat, Darren; Manitpisitkul, Prasarn; Sha, Sue; Derendorf, Hartmut

2009-01-01

Linezolid is the first FDA-approved oxazolidinone with activity against clinically important gram-positive pathogens, including methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). RWJ-416457 is a new oxazolidinone with an antimicrobial spectrum similar to that of linezolid. The goal of the present study was to develop a general pharmacokinetic (PK)-pharmacodynamic (PD) model that allows the characterization and comparison of the in vitro activities of oxazolidinones, determined in time-kill curve experiments, against MRSA. The in vitro activities of RWJ-416457 and the first-in-class representative, linezolid, against MRSA OC2878 were determined in static and dynamic time-kill curve experiments over a wide range of concentrations: 0.125 to 8 μg/ml (MIC, 0.5 μg/ml) and 0.25 to 16 μg/ml (MIC, 1 μg/ml), respectively. After correction for drug degradation during the time-kill curve experiments, a two-subpopulation model was simultaneously fitted to all data in the NONMEM VI program. The robustness of the model and the precision of the parameter estimates were evaluated by internal model validation by nonparametric bootstrap analysis. A two-subpopulation model, consisting of a self-replicating, oxazolidinone-susceptible and a persistent, oxazolidinone-insusceptible pool of bacteria was appropriate for the characterization of the time-kill curve data. The PK-PD model identified was capable of accounting for saturation in growth, delays in the onsets of growth and drug-induced killing, as well as naturally occurring bacterial death. The simultaneous fit of the proposed indirect-response, maximum-effect model to the data resulted in concentrations that produced a half-maximum killing effect that were significantly (P < 0.05) lower for RWJ-416457 (0.41 μg/ml) than for linezolid (1.39 μg/ml). In combination with the appropriate PK data, the susceptibility-based two-subpopulation model identified may provide valuable guidance for the selection of oxazolidinone doses or dose regimens for use in clinical studies. PMID:19786607

Human neuron-astrocyte 3D co-culture-based assay for evaluation of neuroprotective compounds.

PubMed

Terrasso, Ana Paula; Silva, Ana Carina; Filipe, Augusto; Pedroso, Pedro; Ferreira, Ana Lúcia; Alves, Paula Marques; Brito, Catarina

Central nervous system drug development has registered high attrition rates, mainly due to the lack of efficacy of drug candidates, highlighting the low reliability of the models used in early-stage drug development and the need for new in vitro human cell-based models and assays to accurately identify and validate drug candidates. 3D human cell models can include different tissue cell types and represent the spatiotemporal context of the original tissue (co-cultures), allowing the establishment of biologically-relevant cell-cell and cell-extracellular matrix interactions. Nevertheless, exploitation of these 3D models for neuroprotection assessment has been limited due to the lack of data to validate such 3D co-culture approaches. In this work we combined a 3D human neuron-astrocyte co-culture with a cell viability endpoint for the implementation of a novel in vitro neuroprotection assay, over an oxidative insult. Neuroprotection assay robustness and specificity, and the applicability of Presto Blue, MTT and CytoTox-Glo viability assays to the 3D co-culture were evaluated. Presto Blue was the adequate endpoint as it is non-destructive and is a simpler and reliable assay. Semi-automation of the cell viability endpoint was performed, indicating that the assay setup is amenable to be transferred to automated screening platforms. Finally, the neuroprotection assay setup was applied to a series of 36 test compounds and several candidates with higher neuroprotective effect than the positive control, Idebenone, were identified. The robustness and simplicity of the implemented neuroprotection assay with the cell viability endpoint enables the use of more complex and reliable 3D in vitro cell models to identify and validate drug candidates. Copyright © 2016 Elsevier Inc. All rights reserved.

Development of an artificial neural network model for risk assessment of skin sensitization using human cell line activation test, direct peptide reactivity assay, KeratinoSens™ and in silico structure alert parameter.

PubMed

Hirota, Morihiko; Ashikaga, Takao; Kouzuki, Hirokazu

2018-04-01

It is important to predict the potential of cosmetic ingredients to cause skin sensitization, and in accordance with the European Union cosmetic directive for the replacement of animal tests, several in vitro tests based on the adverse outcome pathway have been developed for hazard identification, such as the direct peptide reactivity assay, KeratinoSens™ and the human cell line activation test. Here, we describe the development of an artificial neural network (ANN) prediction model for skin sensitization risk assessment based on the integrated testing strategy concept, using direct peptide reactivity assay, KeratinoSens™, human cell line activation test and an in silico or structure alert parameter. We first investigated the relationship between published murine local lymph node assay EC3 values, which represent skin sensitization potency, and in vitro test results using a panel of about 134 chemicals for which all the required data were available. Predictions based on ANN analysis using combinations of parameters from all three in vitro tests showed a good correlation with local lymph node assay EC3 values. However, when the ANN model was applied to a testing set of 28 chemicals that had not been included in the training set, predicted EC3s were overestimated for some chemicals. Incorporation of an additional in silico or structure alert descriptor (obtained with TIMES-M or Toxtree software) in the ANN model improved the results. Our findings suggest that the ANN model based on the integrated testing strategy concept could be useful for evaluating the skin sensitization potential. Copyright © 2017 John Wiley & Sons, Ltd.

Glutaric aciduria type I and methylmalonic aciduria: simulation of cerebral import and export of accumulating neurotoxic dicarboxylic acids in in vitro models of the blood-brain barrier and the choroid plexus.

PubMed

Sauer, Sven W; Opp, Silvana; Mahringer, Anne; Kamiński, Marcin M; Thiel, Christian; Okun, Jürgen G; Fricker, Gert; Morath, Marina A; Kölker, Stefan

2010-06-01

Intracerebral accumulation of neurotoxic dicarboxylic acids (DCAs) plays an important pathophysiological role in glutaric aciduria type I and methylmalonic aciduria. Therefore, we investigated the transport characteristics of accumulating DCAs - glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) - across porcine brain capillary endothelial cells (pBCEC) and human choroid plexus epithelial cells (hCPEC) representing in vitro models of the blood-brain barrier (BBB) and the choroid plexus respectively. We identified expression of organic acid transporters 1 (OAT1) and 3 (OAT3) in pBCEC on mRNA and protein level. For DCAs tested, transport from the basolateral to the apical site (i.e. efflux) was higher than influx. Efflux transport of GA, 3-OH-GA, and MMA across pBCEC was Na(+)-dependent, ATP-independent, and was inhibited by the OAT substrates para-aminohippuric acid (PAH), estrone sulfate, and taurocholate, and the OAT inhibitor probenecid. Members of the ATP-binding cassette transporter family or the organic anion transporting polypeptide family, namely MRP2, P-gp, BCRP, and OATP1B3, did not mediate transport of GA, 3-OH-GA or MMA confirming the specificity of efflux transport via OATs. In hCPEC, cellular import of GA was dependent on Na(+)-gradient, inhibited by NaCN, and unaffected by probenecid suggesting a Na(+)-dependent DCA transporter. Specific transport of GA across hCPEC, however, was not found. In conclusion, our results indicate a low but specific efflux transport for GA, 3-OH-GA, and MMA across pBCEC, an in vitro model of the BBB, via OAT1 and OAT3 but not across hCPEC, an in vitro model of the choroid plexus. Copyright 2010 Elsevier B.V. All rights reserved.

Exposure to Engineered Nanomaterial Results in Disruption of Brush Borders in Epithelia Models in vitro

NASA Astrophysics Data System (ADS)

Faust, James J.

Engineered nanoparticles (NP; 10-9 m) have found use in a variety of consumer goods and medical devices because of the unique changes in material properties that occur when synthesized on the nanoscale. Although many definitions for nanoparticle exist, from the perspective of size, nanoparticle is defined as particles with diameters less than 100 nm in any external dimension. Examples of their use include titanium dioxide added as a pigment in products intended to be ingested by humans, silicon dioxide NPs are used in foods as an anticaking agent, and gold or iron oxide NPs can be used as vectors for drug delivery or contrast agents for specialized medical imaging. Although the intended use of these NPs is often to improve human health, it has come to the attention of investigators that NPs can have unintended or even detrimental effects on the organism. This work describes one such unintended effect of NP exposure from the perspective of exposure via the oral route. First, this Dissertation will explain an event referred to as brush border disruption that occurred after nanoparticles interacted with an in vitro model of the human intestinal epithelium. Second, this Dissertation will identify and characterize several consumer goods that were shown to contain titanium dioxide that are intended to be ingested. Third, this Dissertation shows that sedimentation due to gravity does not artifactually result in disruption of brush borders as a consequence of exposure to food grade titanium dioxide in vitro. Finally, this Dissertation will demonstrate that iron oxide nanoparticles elicited similar effects after exposure to an in vitro brush border expressing model of the human placenta. Together, these data suggest that brush border disruption is not an artifact of the material/cell culture model, but instead represents a bona fide biological response as a result of exposure to nanomaterial.

An automated process for building reliable and optimal in vitro/in vivo correlation models based on Monte Carlo simulations.

PubMed

Sutton, Steven C; Hu, Mingxiu

2006-05-05

Many mathematical models have been proposed for establishing an in vitro/in vivo correlation (IVIVC). The traditional IVIVC model building process consists of 5 steps: deconvolution, model fitting, convolution, prediction error evaluation, and cross-validation. This is a time-consuming process and typically a few models at most are tested for any given data set. The objectives of this work were to (1) propose a statistical tool to screen models for further development of an IVIVC, (2) evaluate the performance of each model under different circumstances, and (3) investigate the effectiveness of common statistical model selection criteria for choosing IVIVC models. A computer program was developed to explore which model(s) would be most likely to work well with a random variation from the original formulation. The process used Monte Carlo simulation techniques to build IVIVC models. Data-based model selection criteria (Akaike Information Criteria [AIC], R2) and the probability of passing the Food and Drug Administration "prediction error" requirement was calculated. To illustrate this approach, several real data sets representing a broad range of release profiles are used to illustrate the process and to demonstrate the advantages of this automated process over the traditional approach. The Hixson-Crowell and Weibull models were often preferred over the linear. When evaluating whether a Level A IVIVC model was possible, the model selection criteria AIC generally selected the best model. We believe that the approach we proposed may be a rapid tool to determine which IVIVC model (if any) is the most applicable.

Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via re-specification of lineage-restricted precursors

PubMed Central

Doulatov, Sergei; Vo, Linda T.; Chou, Stephanie S.; Kim, Peter G.; Arora, Natasha; Li, Hu; Hadland, Brandon K.; Bernstein, Irwin D.; Collins, James J.; Zon, Leonard I.; Daley, George Q.

2013-01-01

Summary Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor (HSPCs) has limited their characterization to in vitro assays. We report a strategy to re-specify lineage-restricted CD34+CD45+ myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engraft in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34+CD38− cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, were required for engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription factor-mediated engraftment of blood progenitors from human pluripotent cells. PMID:24094326

A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes.

PubMed

Mueller, A J; Tew, S R; Vasieva, O; Clegg, P D; Canty-Laird, E G

2016-09-27

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

PubMed

Chiba, Eriko; Villena, Julio; Hosoya, Shoichi; Takanashi, Naoya; Shimazu, Tomoyuki; Aso, Hisashi; Tohno, Masanori; Suda, Yoshihito; Kawai, Yasushi; Saito, Tadao; Miyazawa, Kenji; He, Fang; Kitazawa, Haruki

2012-10-01

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts. Copyright © 2011 Elsevier Ltd. All rights reserved.

Decreased levels of active uPA and KLK8 assessed by [111 In]MICA-401 binding correlate with the seizure burden in an animal model of temporal lobe epilepsy.

PubMed

Missault, Stephan; Peeters, Lore; Amhaoul, Halima; Thomae, David; Van Eetveldt, Annemie; Favier, Barbara; Thakur, Anagha; Van Soom, Jeroen; Pitkänen, Asla; Augustyns, Koen; Joossens, Jurgen; Staelens, Steven; Dedeurwaerdere, Stefanie

2017-09-01

Urokinase-type plasminogen activator (uPA) and kallikrein-related peptidase 8 (KLK8) are serine proteases that contribute to extracellular matrix (ECM) remodeling after brain injury. They can be labelled with the novel radiotracer [ 111 In]MICA-401. As the first step in exploring the applicability of [ 111 In]MICA-401 in tracing the mechanisms of postinjury ECM reorganization in vivo, we performed in vitro and ex vivo studies, assessing [ 111 In]MICA-401 distribution in the brain in two animal models: kainic acid-induced status epilepticus (KASE) and controlled cortical impact (CCI)-induced traumatic brain injury (TBI). In the KASE model, in vitro autoradiography with [ 111 In]MICA-401 was performed at 7 days and 12 weeks post-SE. To assess seizure burden, rats were monitored using video-electroencephalography (EEG) for 1 month before the 12-week time point. In the CCI model, in vitro autoradiography was performed at 4 days and ex vivo autoradiography at 7 days post-TBI. At 7 days post-SE, in vitro autoradiography revealed significantly decreased [ 111 In]MICA-401 binding in hippocampal CA3 subfield and extrahippocampal temporal lobe (ETL). In the chronic phase, when animals had developed spontaneous seizures, specific binding was decreased in CA3 and CA1/CA2 subfields of hippocampus, dentate gyrus, ETL, and parietal cortex. Of interest, KASE rats with the highest frequency of seizures had the lowest hippocampal [ 111 In]MICA-401 binding (r = -0.76, p ≤ 0.05). Similarly, at 4 days post-TBI, in vitro [ 111 In]MICA-401 binding was significantly decreased in medial and lateral perilesional cortex and ipsilateral dentate gyrus. Ex vivo autoradiography at 7 days post-TBI, however, revealed increased tracer uptake in perilesional cortex and hippocampus, which was likely related to tracer leakage due to blood-brain barrier (BBB) disruption. Strong association of reduced [ 111 In]MICA-401 binding with seizure burden in the KASE model suggests that analysis of reduced levels of active uPA/KLK8 represents a novel biomarker candidate to be explored as a biomarker for epilepsy severity. However, limited BBB permeability of [ 111 In]MICA-401 currently limits its application in vivo. Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.

Comparing and Validating Machine Learning Models for Mycobacterium tuberculosis Drug Discovery.

PubMed

Lane, Thomas; Russo, Daniel P; Zorn, Kimberley M; Clark, Alex M; Korotcov, Alexandru; Tkachenko, Valery; Reynolds, Robert C; Perryman, Alexander L; Freundlich, Joel S; Ekins, Sean

2018-04-26

Tuberculosis is a global health dilemma. In 2016, the WHO reported 10.4 million incidences and 1.7 million deaths. The need to develop new treatments for those infected with Mycobacterium tuberculosis ( Mtb) has led to many large-scale phenotypic screens and many thousands of new active compounds identified in vitro. However, with limited funding, efforts to discover new active molecules against Mtb needs to be more efficient. Several computational machine learning approaches have been shown to have good enrichment and hit rates. We have curated small molecule Mtb data and developed new models with a total of 18,886 molecules with activity cutoffs of 10 μM, 1 μM, and 100 nM. These data sets were used to evaluate different machine learning methods (including deep learning) and metrics and to generate predictions for additional molecules published in 2017. One Mtb model, a combined in vitro and in vivo data Bayesian model at a 100 nM activity yielded the following metrics for 5-fold cross validation: accuracy = 0.88, precision = 0.22, recall = 0.91, specificity = 0.88, kappa = 0.31, and MCC = 0.41. We have also curated an evaluation set ( n = 153 compounds) published in 2017, and when used to test our model, it showed the comparable statistics (accuracy = 0.83, precision = 0.27, recall = 1.00, specificity = 0.81, kappa = 0.36, and MCC = 0.47). We have also compared these models with additional machine learning algorithms showing Bayesian machine learning models constructed with literature Mtb data generated by different laboratories generally were equivalent to or outperformed deep neural networks with external test sets. Finally, we have also compared our training and test sets to show they were suitably diverse and different in order to represent useful evaluation sets. Such Mtb machine learning models could help prioritize compounds for testing in vitro and in vivo.

Correlation between anti-PD-L1 tumor concentrations and tumor-specific and nonspecific biomarkers in a melanoma mouse model

PubMed Central

Contreras, Ana M.; Merino, María; Vasquez, Marcos; Trocóniz, Iñaki F.

2016-01-01

Blockade of PD-L1 with specific monoclonal antibodies (anti-PD-L1) represents a therapeutic strategy to increase the capability of the immune system to modulate the tumor immune-resistance. The relationship between anti-PD-L1 tumor exposition and anti-tumor effect represents a challenge that has been addressed in this work through the identification of certain biomarkers implicated in the antibody's mechanism of action, using a syngeneic melanoma mouse model. The development of an in-vitro/in-vivo platform has allowed us to investigate the PD-L1 behavior after its blockage with anti-PD-L1 at cellular level and in animals. In-vitro studies showed that the complex PD-L1/anti-PD-L1 was retained mainly at the cell surface. The antibody concentration and time exposure affected directly the recycling or ligand turnover. In-vivo studies showed that anti-PD-L1 was therapeutically active at all stage of the disease, with a rapid onset, a low but durable efficacy and non-relevant toxic effect. This efficacy measured as tumor shrinkage correlated with tumor-specific infiltrating lymphocytes (TILs), which increased as antibody tumor concentrations increased. Both, TILS and antibody concentrations followed similar kinetic patterns, justifying the observed anti-PD-L1 rapid onset. Interestingly, peripheral lymphocytes (PBLs) behave as infiltrating lymphocytes, suggesting that these PBLs might be considered as a possible biomarker for antibody activity. PMID:27764774

Does implant coating with antibacterial-loaded hydrogel reduce bacterial colonization and biofilm formation in vitro?

PubMed

Drago, Lorenzo; Boot, Willemijn; Dimas, Kostantinos; Malizos, Kostantinos; Hänsch, Gertrud M; Stuyck, Jos; Gawlitta, Debby; Romanò, Carlo L

2014-11-01

Implant-related infections represent one of the most severe complications in orthopaedics. A fast-resorbable, antibacterial-loaded hydrogel may reduce or prevent bacterial colonization and biofilm formation of implanted biomaterials. We asked: (1) Is a fast-resorbable hydrogel able to deliver antibacterial compounds in vitro? (2) Can a hydrogel (alone or antibacterial-loaded) coating on implants reduce bacterial colonization? And (3) is intraoperative coating feasible and resistant to press-fit implant insertion? We tested the ability of Disposable Antibacterial Coating (DAC) hydrogel (Novagenit Srl, Mezzolombardo, Italy) to deliver antibacterial agents using spectrophotometry and a microbiologic assay. Antibacterial and antibiofilm activity were determined by broth microdilution and a crystal violet assay, respectively. Coating resistance to press-fit insertion was tested in rabbit tibias and human femurs. Complete release of all tested antibacterial compounds was observed in less than 96 hours. Bactericidal and antibiofilm effect of DAC hydrogel in combination with various antibacterials was shown in vitro. Approximately 80% of the hydrogel coating was retrieved on the implant after press-fit insertion. Implant coating with an antibacterial-loaded hydrogel reduces bacterial colonization and biofilm formation in vitro. A fast-resorbable, antibacterial-loaded hydrogel coating may help prevent implant-related infections in orthopaedics. However, further validation in animal models and properly controlled human studies is required.

Dendritic cells exposed in vitro to TGF-β1 ameliorate experimental autoimmune myasthenia gravis

PubMed Central

YARILIN, D; DUAN, R; HUANG, Y-M; XIAO, B-G

2002-01-01

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG), characterized by an autoaggressive T-cell-dependent antibody-mediated immune response directed against the acetylcholine receptor (AChR) of the neuromuscular junction. Dendritic cells (DC) are unique antigen-presenting cells which control T- and B-cell functions and induce immunity or tolerance. Here, we demonstrate that DC exposed to TGF-β1 in vitro mediate protection against EAMG. Freshly prepared DC from spleen of healthy rats were exposed to TGF-β1 in vitro for 48 h, and administered subcutaneously to Lewis rats (2 × 106DC/rat) on day 5 post immunization with AChR in Freund’s complete adjuvant. Control EAMG rats were injected in parallel with untreated DC (naive DC) or PBS. Lewis rats receiving TGF-β1-exposed DC developed very mild symptoms of EAMG without loss of body weight compared with control EAMG rats receiving naive DC or PBS. This effect of TGF-β1-exposed DC was associated with augmented spontaneous and AChR-induced proliferation, IFN-γ and NO production, and decreased levels of anti-AChR antibody-secreting cells. Autologous DC exposed in vitro to TGF-β1 could represent a new opportunity for DC-based immunotherapy of antibody-mediated autoimmune diseases. PMID:11876742

Inhibition of Intrahepatic Bile Duct Dilation of the Polycystic Kidney Rat with a Novel Tyrosine Kinase Inhibitor Gefitinib

PubMed Central

Sato, Yasunori; Harada, Kenichi; Furubo, Shinichi; Kizawa, Kazuo; Sanzen, Takahiro; Yasoshima, Mitsue; Ozaki, Satoru; Isse, Kumiko; Sasaki, Motoko; Nakanuma, Yasuni

2006-01-01

The polycystic kidney (PCK) rat represents a liver and kidney cyst pathology corresponding to Caroli’s disease with congenital hepatic fibrosis and autosomal recessive polycystic kidney disease. We previously reported that an epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), significantly inhibited the abnormal growth of biliary epithelial cells of PCK rats in vitro. This study investigated the effects of gefitinib on cyst pathogenesis of the PCK rat both in vitro and in vivo. A three-dimensional culture model of biliary epithelial cells in the collagen gel matrix was used for in vitro analysis. For in vivo experiments, PCK and control rats were treated with gefitinib between 3 and 10 weeks of age. In vitro, gefitinib had strong inhibitory effects on biliary cyst formation of PCK rats. In vivo, treatment with gefitinib significantly inhibited the cystic dilatation of the intrahepatic bile ducts of PCK rats, which was accompanied by improvement of liver fibrosis. By contrast, no beneficial effects were observed on renal cyst development because of the treatment. These results suggest that signaling pathways mediated by epidermal growth factor receptor are involved in biliary dysgenesis of the PCK rat, with the mechanisms of cyst progression being different between the liver and kidney. PMID:17003482

Accounting for data variability, a key factor in in vivo/in vitro relationships: application to the skin sensitization potency (in vivo LLNA versus in vitro DPRA) example.

PubMed

Dimitrov, S; Detroyer, A; Piroird, C; Gomes, C; Eilstein, J; Pauloin, T; Kuseva, C; Ivanova, H; Popova, I; Karakolev, Y; Ringeissen, S; Mekenyan, O

2016-12-01

When searching for alternative methods to animal testing, confidently rescaling an in vitro result to the corresponding in vivo classification is still a challenging problem. Although one of the most important factors affecting good correlation is sample characteristics, they are very rarely integrated into correlation studies. Usually, in these studies, it is implicitly assumed that both compared values are error-free numbers, which they are not. In this work, we propose a general methodology to analyze and integrate data variability and thus confidence estimation when rescaling from one test to another. The methodology is demonstrated through the case study of rescaling the in vitro Direct Peptide Reactivity Assay (DPRA) reactivity to the in vivo Local Lymph Node Assay (LLNA) skin sensitization potency classifications. In a first step, a comprehensive statistical analysis evaluating the reliability and variability of LLNA and DPRA as such was done. These results allowed us to link the concept of gray zones and confidence probability, which in turn represents a new perspective for a more precise knowledge of the classification of chemicals within their in vivo OR in vitro test. Next, the novelty and practical value of our methodology introducing variability into the threshold optimization between the in vitro AND in vivo test resides in the fact that it attributes a confidence probability to the predicted classification. The methodology, classification and screening approach presented in this study are not restricted to skin sensitization only. They could be helpful also for fate, toxicity and health hazard assessment where plenty of in vitro and in chemico assays and/or QSARs models are available. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Can in vitro systems capture the characteristic differences between the flexion-extension kinematics of the healthy and TKA knee?

PubMed

Varadarajan, Kartik M; Harry, Rubash E; Johnson, Todd; Li, Guoan

2009-10-01

In vitro systems provide a powerful means to evaluate the efficacy of total knee arthroplasty (TKA) in restoring normal knee kinematics. The Oxford knee rig (OKR) and the robotic knee testing system (RKTS) represent two systems that have been extensively used to study TKA biomechanics. Nonetheless, a frequently asked question is whether in vitro simulations can capture the in vivo behavior of the knee. Here, we compared the flexion-extension kinematics of intact knees and knees after TKA tested on the OKR and RKTS, to results of representative in vivo studies. The goal was to determine if the in vitro systems could capture the key kinematic features of knees in healthy subjects and TKA patients. Results showed that the RKTS and the OKR can replicate the femoral rollback and 'screw home' tibial rotation between 0 degrees and 30 degrees flexion seen in healthy subjects, and the reduced femoral rollback and absence of 'screw home' motion in TKA patients. The RKTS also replicated the overall internally rotated position of the tibia beyond 30 degrees flexion. However, ability of the OKR to replicate the internally rotated position of the knee beyond 30 degrees flexion was inconsistent. These data could aid in validation of new in vitro systems and physiologic interpretations of in vitro results.

New aspects of the pathogenesis of canine distemper leukoencephalitis.

PubMed

Lempp, Charlotte; Spitzbarth, Ingo; Puff, Christina; Cana, Armend; Kegler, Kristel; Techangamsuwan, Somporn; Baumgärtner, Wolfgang; Seehusen, Frauke

2014-07-02

Canine distemper virus (CDV) is a member of the genus morbillivirus, which is known to cause a variety of disorders in dogs including demyelinating leukoencephalitis (CDV-DL). In recent years, substantial progress in understanding the pathogenetic mechanisms of CDV-DL has been made. In vivo and in vitro investigations provided new insights into its pathogenesis with special emphasis on axon-myelin-glia interaction, potential endogenous mechanisms of regeneration, and astroglial plasticity. CDV-DL is characterized by lesions with a variable degree of demyelination and mononuclear inflammation accompanied by a dysregulated orchestration of cytokines as well as matrix metalloproteinases and their inhibitors. Despite decades of research, several new aspects of the neuropathogenesis of CDV-DL have been described only recently. Early axonal damage seems to represent an initial and progressive lesion in CDV-DL, which interestingly precedes demyelination. Axonopathy may, thus, function as a potential trigger for subsequent disturbed axon-myelin-glia interactions. In particular, the detection of early axonal damage suggests that demyelination is at least in part a secondary event in CDV-DL, thus challenging the dogma of CDV as a purely primary demyelinating disease. Another unexpected finding refers to the appearance of p75 neurotrophin (NTR)-positive bipolar cells during CDV-DL. As p75NTR is a prototype marker for immature Schwann cells, this finding suggests that Schwann cell remyelination might represent a so far underestimated endogenous mechanism of regeneration, though this hypothesis still remains to be proven. Although it is well known that astrocytes represent the major target of CDV infection in CDV-DL, the detection of infected vimentin-positive astrocytes in chronic lesions indicates a crucial role of this cell population in nervous distemper. While glial fibrillary acidic protein represents the characteristic intermediate filament of mature astrocytes, expression of vimentin is generally restricted to immature or reactive astrocytes. Thus, vimentin-positive astrocytes might constitute an important cell population for CDV persistence and spread, as well as lesion progression. In vitro models, such as dissociated glial cell cultures, as well as organotypic brain slice cultures have contributed to a better insight into mechanisms of infection and certain morphological and molecular aspects of CDV-DL. Summarized, recent in vivo and in vitro studies revealed remarkable new aspects of nervous distemper. These new perceptions substantially improved our understanding of the pathogenesis of CDV-DL and might represent new starting points to develop novel treatment strategies.

In vitro pyrogen test for toxic or immunomodulatory drugs.

PubMed

Daneshian, Mardas; Guenther, Armin; Wendel, Albrecht; Hartung, Thomas; von Aulock, Sonja

2006-06-30

Pyrogenic contaminations of some classes of injectable drugs, e.g. toxic or immunomodulatory as well as false-positive drugs, represent a major risk which cannot yet be excluded due to the limitations of current tests. Here we describe a modification of the In vitro Pyrogen Test termed AWIPT (Adsorb, Wash, In vitro Pyrogen Test), which addresses this problem by introducing a pre-incubation step in which pyrogenic contaminations in the test sample are adsorbed to albumin-coated beads. After rinsing, the beads are incubated with human whole blood and the release of the endogenous pyrogen interleukin-1beta is measured as a marker of pyrogenic activity. Intentional contaminations with lipopolysaccharide were retrieved from the chemotherapeutic agents paclitaxel, cisplatin and liposomal daunorubicin, the antibiotic gentamicin, the antifungal agent liposomal amphotericin B, and the corticosteroid prednisolone at lower dilutions than in the standard in vitro pyrogen test. This represents a promising new approach for the detection of pyrogenic contamination in drugs or in drugs containing interfering additives and should lead to improved safety levels.

BH3 mimetics inhibit growth of chondrosarcoma--a novel targeted-therapy for candidate models.

PubMed

Morii, Takeshi; Ohtsuka, Kouki; Ohnishi, Hiroaki; Mochizuki, Kazuo; Yoshiyama, Akira; Aoyagi, Takayuki; Hornicek, Francis J; Ichimura, Shoichi

2014-11-01

Chondrosarcoma is refractory to conventional chemotherapy. BH-3 mimetics ABT-737 and ABT-263 are synthetic small-molecule inhibitors of anti-apoptotic proteins B-cell lymphoma-2 (Bcl2) and Bcl-xL, which play a critical role in survival of chondrosarcoma cells. Chondrosarcoma cell lines SW-1353 and CS-1 were used as the disease model. We used immunoblotting to assess the expression of target molecules Bcl2 and Bcl-xL, and the apoptotic inducers Bcl2-associated X (Bax) and Bcl2-antagonist/killer (Bak). In vitro growth inhibition by BH-3 mimetics was confirmed by photomicroscopic cell counting and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Apoptotic induction was confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA). In vivo growth inhibition was assessed in a non-obese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. Expression of the target and effector molecules was confirmed in chondrosarcoma cell lines. BH3 mimetics significantly inhibited cell growth and induced apoptosis in vitro. Administration of ABT-263 inhibited chondrosarcoma growth and improved survival in a mouse model. BH3 mimetics represent a novel treatment modality for chondrosarcoma. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Preparation, characterization, and transport of dexamethasone-loaded polymeric nanoparticles across a human placental in vitro model

PubMed Central

Ali, Hazem; Kalashnikova, Irina; White, Mark Andrew; Sherman, Michael; Rytting, Erik

2013-01-01

The purpose of this study was to prepare dexamethasone-loaded polymeric nanoparticles and evaluate their potential for transport across human placenta. Statistical modeling and factorial design was applied to investigate the influence of process parameters on the following nanoparticle characteristics: particle size, polydispersity index, zeta potential, and drug encapsulation efficiency. Dexamethasone and nanoparticle transport was subsequently investigated using the BeWo b30 cell line, an in vitro model of human placental trophoblast cells, which represent the rate-limiting barrier for maternal-fetal transfer. Encapsulation efficiency and drug transport were determined using a validated high performance liquid chromatography method. Nanoparticle morphology and drug encapsulation were further characterized by cryo-transmission electron microscopy and X-ray diffraction, respectively. Nanoparticles prepared from poly(lactic-co-glycolic acid) were spherical, with particle sizes ranging from 140–298 nm, and encapsulation efficiency ranging from 52–89%. Nanoencapsulation enhanced the apparent permeability of dexamethasone from the maternal compartment to the fetal compartment more than 10-fold in this model. Particle size was shown to be inversely correlated with drug and nanoparticle permeability, as confirmed with fluorescently-labeled nanoparticles. These results highlight the feasibility of designing nanoparticles capable of delivering medication to the fetus, in particular, potential dexamethasone therapy for the prenatal treatment of congenital adrenal hyperplasia. PMID:23850397

Microfluidic devices for modeling cell-cell and particle-cell interactions in the microvasculature

PubMed Central

Prabhakarpandian, Balabhaskar; Shen, Ming-Che; Pant, Kapil; Kiani, Mohammad F.

2011-01-01

Cell-fluid and cell-cell interactions are critical components of many physiological and pathological conditions in the microvasculature. Similarly, particle-cell interactions play an important role in targeted delivery of therapeutics to tissue. Development of in vitro fluidic devices to mimic these microcirculatory processes has been a critical step forward in our understanding of the inflammatory process, development of nano-particulate drug carriers, and developing realistic in vitro models of the microvasculature and its surrounding tissue. However, widely used parallel plate flow based devices and assays have a number of important limitations for studying the physiological conditions in vivo. In addition, these devices are resource hungry and time consuming for performing various assays. Recently developed, more realistic, microfluidic based devices have been able to overcome many of these limitations. In this review, an overview of the fluidic devices and their use in studying the effects of shear forces on cell-cell and cell-particle interactions is presented. In addition, use of mathematical models and Computational Fluid Dynamics (CFD) based models for interpreting the complex flow patterns in the microvasculature are highlighted. Finally, the potential of 3D microfluidic devices and imaging for better representing in vivo conditions under which cell-cell and cell-particle interactions take place are discussed. PMID:21763328

In vitro activity of monoclonal and recombinant yeast killer toxin-like antibodies against antibiotic-resistant gram-positive cocci.

PubMed Central

Conti, S.; Magliani, W.; Arseni, S.; Dieci, E.; Frazzi, R.; Salati, A.; Varaldo, P. E.; Polonelli, L.

2000-01-01

BACKGROUND: Monoclonal (mAbKT) and recombinant single-chain (scFvKT) anti-idiotypic antibodies were produced to represent the internal image of a yeast killer toxin (KT) characterized by a wide spectrum of antimicrobial activity, including gram-positive cocci. Pathogenic eukaryotic and prokaryotic microorganisms, such as Candida albicans, Pneumocystis carinii, and a multidrug-resistant strain of Mycobacterium tuberculosis, presenting specific, although yet undefined, KT-cell wall receptors (KTR), have proven to be killed in vitro by mAbKT and scFvKT. mAbKT and scFvKT exert a therapeutic effect in vivo in experimental models of candidiasis and pneumocystosis by mimicking the functional activity of protective antibodies naturally produced in humans against KTR of infecting microorganisms. The swelling tide of concern over increasing bacterial resistance to antibiotic drugs gives the impetus to develop new therapeutic compounds against microbial threat. Thus, the in vitro bactericidal activity of mAbKT and scFvKT against gram-positive, drug-resistant cocci of major epidemiological interest was investigated. MATERIALS AND METHODS: mAbKT and scFvKT generated by hybridoma and DNA recombinant technology from the spleen lymphocytes of mice immunized with a KT-neutralizing monoclonal antibody (mAb KT4) were used in a conventional colony forming unit (CFU) assay to determine, from a qualitative point of view, their bactericidal activity against Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae strains. These bacterial strains are characterized by different patterns of resistance to antibiotics, including methicillin, vancomycin, and penicillin. RESULTS: According to the experimental conditions adopted, no bacterial isolate proved to be resistant to the activity of mAbKT and scFvKT. CONCLUSIONS: scFvKT exerted a microbicidal activity against multidrug resistant bacteria, which may represent the basis for the drug modeling of new antibiotics with broad antibacterial spectra to tackle the emergence of microbial resistance. PMID:10997342

Development of a 3D bone marrow adipose tissue model.

PubMed

Fairfield, Heather; Falank, Carolyne; Farrell, Mariah; Vary, Calvin; Boucher, Joshua M; Driscoll, Heather; Liaw, Lucy; Rosen, Clifford J; Reagan, Michaela R

2018-01-26

Over the past twenty years, evidence has accumulated that biochemically and spatially defined networks of extracellular matrix, cellular components, and interactions dictate cellular differentiation, proliferation, and function in a variety of tissue and diseases. Modeling in vivo systems in vitro has been undeniably necessary, but when simplified 2D conditions rather than 3D in vitro models are used, the reliability and usefulness of the data derived from these models decreases. Thus, there is a pressing need to develop and validate reliable in vitro models to reproduce specific tissue-like structures and mimic functions and responses of cells in a more realistic manner for both drug screening/disease modeling and tissue regeneration applications. In adipose biology and cancer research, these models serve as physiologically relevant 3D platforms to bridge the divide between 2D cultures and in vivo models, bringing about more reliable and translationally useful data to accelerate benchtop to bedside research. Currently, no model has been developed for bone marrow adipose tissue (BMAT), a novel adipose depot that has previously been overlooked as "filler tissue" but has more recently been recognized as endocrine-signaling and systemically relevant. Herein we describe the development of the first 3D, BMAT model derived from either human or mouse bone marrow (BM) mesenchymal stromal cells (MSCs). We found that BMAT models can be stably cultured for at least 3 months in vitro, and that myeloma cells (5TGM1, OPM2 and MM1S cells) can be cultured on these for at least 2 weeks. Upon tumor cell co-culture, delipidation occurred in BMAT adipocytes, suggesting a bidirectional relationship between these two important cell types in the malignant BM niche. Overall, our studies suggest that 3D BMAT represents a "healthier," more realistic tissue model that may be useful for elucidating the effects of MAT on tumor cells, and tumor cells on MAT, to identify novel therapeutic targets. In addition, proteomic characterization as well as microarray data (expression of >22,000 genes) coupled with KEGG pathway analysis and gene set expression analysis (GSEA) supported our development of less-inflammatory 3D BMAT compared to 2D culture. In sum, we developed the first 3D, tissue-engineered bone marrow adipose tissue model, which is a versatile, novel model that can be used to study numerous diseases and biological processes involved with the bone marrow. Copyright © 2018. Published by Elsevier Inc.

An in vitro experimental study of flow past aortic valve under varied pulsatile conditions

NASA Astrophysics Data System (ADS)

Zhang, Ruihang; Zhang, Yan

2017-11-01

Flow past aortic valve represents a complex fluid-structure interaction phenomenon that involves pulsatile, vortical, and turbulent conditions. The flow characteristics immediately downstream of the valve, such as the variation of pulsatile flow velocity, formation of vortices, distribution of shear stresses, are of particular interest to further elucidate the role of hemodynamics in various aortic diseases. However, the fluid dynamics of a realistic aortic valve is not fully understood. Particularly, it is unclear how the flow fields downstream of the aortic valve would change under varied pulsatile inlet boundary conditions. In this study, an in vitro experiment has been conducted to investigate the flow fields downstream of a silicone aortic valve model within a cardiovascular flow simulator. Phased-locked Particle Image Velocimetry measurements were performed to map the velocity fields and Reynolds normal and shear stresses at different phases in a cardiac cycle. Temporal variations of pressure across the valve model were measured using high frequency transducers. Results have been compared for different pulsatile inlet conditions, including varied frequencies (heart rates), magnitudes (stroke volumes), and cardiac contractile functions (shapes of waveforms).

Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7.

PubMed

Darabi, Radbod; Perlingeiro, Rita C R

2016-01-01

Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

Oxidative stress resulting from exposure of a human salivary gland cells to paraoxon: an in vitro model for organophosphate oral exposure.

PubMed

Prins, John M; Chao, Chih-Kai; Jacobson, Saskia M; Thompson, Charles M; George, Kathleen M

2014-08-01

Organophosphate (OP) compounds are used as insecticides, acaricides, and chemical agents and share a common neurotoxic mechanism of action. The biochemical alterations leading to many of the deleterious effects have been studied in neuronal cell lines, however, non-neuronal toxic effects of OPs are far less well characterized in vitro, and specifically in cell lines representing oral routes of exposure. To address this void, the human salivary gland (HSG) cell line, representing likely interactions in the oral cavity, was exposed to the representative OP paraoxon (PX; O,O-diethyl-p-nitrophenoxy phosphate) over a range of concentrations (0.01-100 μM) and analyzed for cytotoxicity. PX induced cytotoxicity in HSG cells at most of the exposure concentrations as revealed by MTT assay, however, the release of LDH only occurred at the highest concentration of PX tested (100 μM) at 48 h. Slight increases in cellular ATP levels were measured in PX-exposed (10 μM) HSG cells at 24 h. Exposing HSG cells to 10 μM PX also led to an increase in DNA fragmentation prior to loss of cellular membrane integrity implicating reactive oxygen species (ROS) as a trigger of toxicity. The ROS genes gss, gstm2, gstt2 and sod2 were upregulated, and the presence of superoxide following 10 μM PX exposure was determined via dihydroethidium fluorescence studies further implicating PX-induced oxidative stress in HSG cells. Published by Elsevier Ltd.

Evaluation of antiproliferative, anti-type 2 diabetes, and antihypertension potentials of ellagitannins from strawberries (Fragaria × ananassa Duch.) using in vitro models.

PubMed

Pinto, Marcia da Silva; de Carvalho, Joao Ernesto; Lajolo, Franco Maria; Genovese, Maria Inés; Shetty, Kalidas

2010-10-01

Strawberries represent the main source of ellagic acid derivatives in the Brazilian diet, corresponding to more than 50% of all phenolic compounds found in the fruit. There is a particular interest in the determination of the ellagic acid content in fruits because of possible chemopreventive benefits. In the present study, the potential health benefits of purified ellagitannins from strawberries were evaluated in relation to the antiproliferative activity and in vitro inhibition of α-amylase, α-glucosidase, and angiotensin I-converting enzyme (ACE) relevant for potential management of hyperglycemia and hypertension. Therefore, a comparison among ellagic acid, purified ellagitannins, and a strawberry extract was done to evaluate the possible synergistic effects of phenolics. In relation to the antiproliferative activity, it was observed that ellagic acid had the highest percentage inhibition of cell proliferation. The strawberry extract had lower efficacy in inhibiting the cell proliferation, indicating that in the case of this fruit there is no synergism. Purified ellagitannins had high α-amylase and ACE inhibitory activities. However, these compounds had low α-glucosidase inhibitory activity. These results suggested that the ellagitannins and ellagic acid have good potential for the management of hyperglycemia and hypertension linked to type 2 diabetes. However, further studies with animal and human models are needed to advance the in vitro assay-based biochemical rationale from this study.

Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation

PubMed Central

Cox, R F; Hernandez-Santana, A; Ramdass, S; McMahon, G; Harmey, J H; Morgan, M P

2012-01-01

Background: Mammographic microcalcifications represent one of the most reliable features of nonpalpable breast cancer yet remain largely unexplored and poorly understood. Methods: We report a novel model to investigate the in vitro mineralisation potential of a panel of mammary cell lines. Primary mammary tumours were produced by implanting tumourigenic cells into the mammary fat pads of female BALB/c mice. Results: Hydroxyapatite (HA) was deposited only by the tumourigenic cell lines, indicating mineralisation potential may be associated with cell phenotype in this in vitro model. We propose a mechanism for mammary mineralisation, which suggests that the balance between enhancers and inhibitors of physiological mineralisation are disrupted. Inhibition of alkaline phosphatase and phosphate transport prevented mineralisation, demonstrating that mineralisation is an active cell-mediated process. Hydroxyapatite was found to enhance in vitro tumour cell migration, while calcium oxalate had no effect, highlighting potential consequences of calcium deposition. In addition, HA was also deposited in primary mammary tumours produced by implanting the tumourigenic cells into the mammary fat pads of female BALB/c mice. Conclusion: This work indicates that formation of mammary HA is a cell-specific regulated process, which creates an osteomimetic niche potentially enhancing breast tumour progression. Our findings point to the cells mineralisation potential and the microenvironment regulating it, as a significant feature of breast tumour development. PMID:22233923

Live Cell Imaging of Viscosity in 3D Tumour Cell Models.

PubMed

Shirmanova, Marina V; Shimolina, Lubov' E; Lukina, Maria M; Zagaynova, Elena V; Kuimova, Marina K

2017-01-01

Abnormal levels of viscosity in tissues and cells are known to be associated with disease and malfunction. While methods to measure bulk macroscopic viscosity of bio-tissues are well developed, imaging viscosity at the microscopic scale remains a challenge, especially in vivo. Molecular rotors are small synthetic viscosity-sensitive fluorophores in which fluorescence parameters are strongly correlated to the microviscosity of their immediate environment. Hence, molecular rotors represent a promising instrument for mapping of viscosity in living cells and tissues at the microscopic level. Quantitative measurements of viscosity can be achieved by recording time-resolved fluorescence decays of molecular rotor using fluorescence lifetime imaging microscopy (FLIM), which is also suitable for dynamic viscosity mapping, both in cellulo and in vivo. Among tools of experimental oncology, 3D tumour cultures, or spheroids, are considered a more adequate in vitro model compared to a cellular monolayer, and represent a less labour-intensive and more unified approach compared to animal tumour models. This chapter describes a methodology for microviscosity imaging in tumour spheroids using BODIPY-based molecular rotors and two photon-excited FLIM.

Development, validation and implementation of an in vitro model for the study of metabolic and immune function in normal and inflamed human colonic epithelium.

PubMed

Pedersen, Gitte

2015-01-01

Ulcerative colitis (UC) and Crohn's disease (CD), collectively referred to as inflammatory bowel disease (IBD), are chronic immune disorders affecting the gastrointestinal tract. The aetiology of IBD remains an enigma, but increasing evidence suggests that the development of IBD may be triggered by a disturbance in the balance between gut commensal bacteria and host response in the intestinal mucosa. It is now known that epithelial cells have the capacity to secrete and respond to a range of immunological mediators and this suggests that these cells play a prominent role in the pathogenesis of IBD. Current knowledge about the intestinal epithelium has mainly been obtained using models based on animal cells, transformed human intestinal cell lines and isolated cells from resected colonic bowel segments. Species difference, malignant origin and confounders related to surgery, obviously make these cell models however less applicable for patophysiological studies. Consequently, there was a clear need for models of representative intestinal epithelial cells that would allow functional and dynamic studies of the differentiated human colonic epithelium in vitro. The primary purpose of this thesis was to explore and validate the optimal conditions for establishing a model based on short-term cultures of human colonic epithelial cells obtained from endoscopical biopsies. The cell cultures were accordingly used to describe the interplay between proinflammatory cytokines and colonic epithelium, with focus on alterations in viability, butyrate metabolism and secretion of a chemokine and metalloproteinases (MMP). Finally, the model was used to characterize expression and activation of receptors like toll like receptor (TLR)9 and peroxisome activated proliferators (PPAR)- known to be important players in regulation of innate and adaptive immune responses in human colonic epithelium. The results showed that it is possible to establish short-term cultures of representative, viable human colonic epithelial cells from endoscopic mucosal biopsies of patients with IBD. Short-time isolation by EGTA/EDTA from colonic biopsies allowed establishment of small scale cultures of epithelial cells which were viable and metabolic active for up to 48 hours in vitro. The cell model preserved important cellular metabolic and immunological functions of the human colonic epithelium, including the ability to oxidate butyrate, detoxificate phenolic compounds and secrete the chemokine interleukin (IL)-8 in vitro. Tumour necrosis factor (TNF)-α and interferon (IFN)-γ are pro-inflammatory cytokines, which are present in increased amounts in inflamed colonic mucosa. The precise mechanisms of cytokine-mediated mucosal injury are unknown, but one might be that TNF-α and IFN-γ directly impair epithelial cell function similar to effects seen on distinct target cells in other autoimmune diseases. Using the model, both cytokines were found directly to impair the viability of colonic epithelial cells and to induce secretion of IL-8 in vitro. Interestingly, the cells from inflamed IBD mucosa were less sensitive to cytokine-induced damage, which suggests that an intrinsic defense mechanism is triggered in these cells, perhaps as a result of exposure to toxic luminal factors or high local cytokine levels in vivo. TNF-α and IFN-γ may also be involved in regulation of intestinal inflammation through stimulation of MMP expression and proteolytic activity. We found that colonic epithelial cells express a range of MMPs and moreover that expression of distinct MMPs is increased in cells from inflamed IBD mucosa. Using a functional peptide cleavage assay it was shown that epithelial cells secreted proteolytic active enzymes and that the functional MMP activity was increased in inflamed IBD mucosa. This suggests that colonic epithelial cells, like myofibroblasts and immune cells, may contribute to local intestinal mucosal damage, through secretion of active MMPs. Disturbance of recognition and discrimination of potentially harmful pathogens from commensals in the intestinal mucosa have increasingly been implicated in the pathogenesis of IBD. Our results revealed that colonic epithelial cells express TLR9, a key pattern recognition receptor. Interestingly, the differentiated epithelial cells, which have been exposed to the luminal bacterial flora in vivo, were unresponsive to TLR9 ligand stimulation, contrasting findings in the epithelial cell line HT-29 that is cultured continuously in bacteria free environment. These findings suggest, theoretically, that colonic epithelium may regulate immune responses to microbial antigens including commensal bacterial DNA through modulation of the TLR9 pathway. Currently, the results are in line with the emerging view, that the epithelium represents an important frontline cellular component of the innate immune system in the gut. PPARγ is a nuclear receptor involved in the regulation of lipid and carbonhydrate metabolism. Recent studies in rodent colitis models suggest that PPARγ also is involved in modulation of inflammatory processes in the colon. Using the model, we characterise expression and activity of PPARs in human colonic epithelium and, additionally, evaluated the functional significance of a possible imbalanced PPARγ regulation in relation to inflammation. Our experiments showed that colonic epithelial cells express PPARγ and furthermore that PPARγ signalling was impaired in inflamed UC epithelium. It was possible to restore PPARγ signalling in the cell cultures by stimulation with rosiglitazone (a synthetic PPARγ ligand) in vitro. Hence, these experiments prompted us to design a small controlled, clinical study exploring the possible stimulatory effects of rosiglitazone (a PPAR ligand) in vivo. Interestingly, it was found that topical application of rosiglitazone in patients with active distal UC reduced clinical activity and mucosal inflammation similar to the effects measured in patients treated with mesalazine enemas. Moreover, rectal application of rosiglitazone induced PPARγ signalling in the epithelium in vivo, supporting the view that activation of PPARγ may be a new potential therapeutic target in the treatment of UC. Overall, the in vitro model of representative human colonic epithelial cells has shown to be a useful technique for detailed studies of metabolic and immunological functions that are important for homeostasis of the colonic epithelium. Currently, the findings support the view that intestinal epithelial cells actively participate in immunological processes in the colonic mucosa. Additionally, the model seems to be applicable for generating and evaluating new therapeutic approaches from laboratory bench to bed line as illustrated by the PPARγ study. It is therefore probable, that studies in models of representative colonic epithelial cells, as the one described here, could contribute with important knowledge about the pathogenesis of human inflammatory colonic diseases also in the future.

The current challenges to efficient immature oocyte cryopreservation.

PubMed

Brambillasca, Fausta; Guglielmo, Maria Cristina; Coticchio, Giovanni; Mignini Renzini, Mario; Dal Canto, Mariabeatrice; Fadini, Rubens

2013-12-01

Oocyte cryopreservation represents an important tool for assisted reproductive technology. It offers the opportunity to preserve fertility in women at risk of loss of the ovarian function for various pathologies. It also represents a treatment alternative for couples that cannot benefit from embryo cryopreservation because of moral, religious, or legal constrains. On the other hand, in vitro oocyte maturation has a range of applications. It can be applied in patients with a contraindication to ovarian stimulation to prevent ovarian hyperstimulation syndrome or to eliminate the risk of stimulation of hormone-sensitive tumours in cancer patients. However, while mature oocyte cryopreservation has found wide-spread application and oocyte in vitro maturation has a place for the treatment of specific clinical conditions, data on the efficiency of freezing of immature or in vitro matured oocytes are poorer. In this review we will focus on the combination of oocyte in vitro maturation with oocyte cryopreservation with particular emphasis on the biological implications of the cryopreservation of immature or in vitro matured oocytes. The two cryopreservation approaches, slow freezing and vitrification, will be discussed in relation to possible cryodamage occurring to subcellular structures of the oocyte and the functional interaction between oocyte and cumulus cells.

Papillary fibroblasts differentiate into reticular fibroblasts after prolonged in vitro culture.

PubMed

Janson, David; Saintigny, Gaëlle; Mahé, Christian; El Ghalbzouri, Abdoelwaheb

2013-01-01

The dermis can be divided into two morphologically different layers: the papillary and reticular dermis. Fibroblasts isolated from these layers behave differently when cultured in vitro. During skin ageing, the papillary dermis decreases in volume. Based on the functional differences in vitro, it is hypothesized that the loss of papillary fibroblasts contributes to skin ageing. In this study, we aimed to mimic certain aspects of skin ageing by using high-passage cultures of reticular and papillary fibroblasts and investigated the effect of these cells on skin morphogenesis in reconstructed human skin equivalents. Skin equivalents generated with reticular fibroblasts showed a reduced terminal differentiation and fewer proliferating basal keratinocytes. Aged in vitro papillary fibroblasts had increased expression of biomarkers specific to reticular fibroblasts. The phenotype and morphology of skin equivalents generated with high-passage papillary fibroblasts resembled that of reticular fibroblasts. This demonstrates that papillary fibroblasts can differentiate into reticular fibroblasts in vitro. Therefore, we hypothesize that papillary fibroblasts represent an undifferentiated phenotype, while reticular fibroblasts represent a more differentiated population. The differentiation process could be a new target for anti-skin-ageing strategies. © 2013 John Wiley & Sons A/S.

Bone mechanobiology, gravity and tissue engineering: effects and insights.

PubMed

Ruggiu, Alessandra; Cancedda, Ranieri

2015-12-01

Bone homeostasis strongly depends on fine tuned mechanosensitive regulation signals from environmental forces into biochemical responses. Similar to the ageing process, during spaceflights an altered mechanotransduction occurs as a result of the effects of bone unloading, eventually leading to loss of functional tissue. Although spaceflights represent the best environment to investigate near-zero gravity effects, there are major limitations for setting up experimental analysis. A more feasible approach to analyse the effects of reduced mechanostimulation on the bone is represented by the 'simulated microgravity' experiments based on: (1) in vitro studies, involving cell cultures studies and the use of bioreactors with tissue engineering approaches; (2) in vivo studies, based on animal models; and (3) direct analysis on human beings, as in the case of the bed rest tests. At present, advanced tissue engineering methods allow investigators to recreate bone microenvironment in vitro for mechanobiology studies. This group and others have generated tissue 'organoids' to mimic in vitro the in vivo bone environment and to study the alteration cells can go through when subjected to unloading. Understanding the molecular mechanisms underlying the bone tissue response to mechanostimuli will help developing new strategies to prevent loss of tissue caused by altered mechanotransduction, as well as identifying new approaches for the treatment of diseases via drug testing. This review focuses on the effects of reduced gravity on bone mechanobiology by providing the up-to-date and state of the art on the available data by drawing a parallel with the suitable tissue engineering systems. Copyright © 2014 John Wiley & Sons, Ltd.

Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures.

PubMed Central

Hernández, C; Flores, R

1992-01-01

Peach latent mosaic viroid (PLMVd), the causal agent of peach latent mosaic disease, has been sequenced and found to be a circular RNA molecule of 337 nucleotide residues, which adopts a branched conformation when it is folded in the model of lowest free energy. PLMVd exhibits limited homologies with other viroids and some satellite RNAs, but it does not have any of the central conserved sequences characteristic of the subgroups of typical viroids. However, a segment of approximately one-third of the PLMVd sequence has the elements required to form in the RNAs of both polarities the hammerhead structures proposed to act in the in vitro self-cleavage of avocado sunblotch viroid (ASBVd) and some satellite RNAs. Plus and minus partial- and full-length RNA transcripts of PLMVd containing the hammerhead structures displayed self-cleavage during transcription and after purification as predicted by these structures. These data are consistent with the high stability of the PLMVd hammerhead structures, more similar to the corresponding structures of some satellite RNAs than to those of ASBVd, and indicate that the self-cleavage reactions of PLMVd are most probably mediated by single hammerhead structures. Our results support the inclusion of PLMVd in a viroid subgroup represented by ASBVd, whose members are characterized by their ability to self-cleave in vitro, and probably in vivo, through hammerhead structures. A consensus phylogenetic tree has been obtained suggesting that PLMVd, together with ASBVd, may represent an evolutionary link between viroids and viroid-like satellite RNAs. Images PMID:1373888

In Vitro and in Vivo Wound Healing Properties of Plasma and Serum from Crocodylus siamensis Blood.

PubMed

Jangpromma, Nisachon; Preecharram, Sutthidech; Srilert, Thanawan; Maijaroen, Surachai; Mahakunakorn, Pramote; Nualkaew, Natsajee; Daduang, Sakda; Klaynongsruang, Sompong

2016-06-28

The plasma and serum of Crocodylus siamensis have previously been reported to exhibit potent antimicrobial, antioxidant, and anti-inflammatory activities. During wound healing, these biological properties play a crucial role for supporting the formation of new tissue around the injured skin in the recovery process. Thus, this study aimed to evaluate the wound healing properties of C. siamensis plasma and serum. The collected data demonstrate that crocodile plasma and serum were able to activate in vitro proliferation and migration of HaCaT, a human keratinocyte cell line, which represents an essential phase in the wound healing process. With respect to investigating cell migration, a scratch wound experiment was performed which revealed the ability of plasma and serum to decrease the gap of wounds in a dose-dependent manner. Consistent with the in vitro results, remarkably enhanced wound repair was also observed in a mouse excisional skin wound model after treatment with plasma or serum. The effects of C. siamensis plasma and serum on wound healing were further elucidated by treating wound infections by Staphylococcus aureus ATCC 25923 on mice skin coupled with a histological method. The results indicate that crocodile plasma and serum promote the prevention of wound infection and boost the re-epithelialization necessary for the formation of new skin. Therefore, this work represents the first study to demonstrate the efficiency of C. siamensis plasma and serum with respect to their wound healing properties and strongly supports the utilization of C. siamensis plasma and serum as therapeutic products for injured skin treatment.

Use of pluripotent stem cells for reproductive medicine: are we there yet?

PubMed

Duggal, Galbha; Heindryckx, Björn; Deroo, Tom; De Sutter, Petra

2014-01-01

In recent years, pluripotent stem cells have demonstrated to be exciting tools to understand embryonic development, cell lineage specification, tissue generation and repair, and various other biological processes. In addition, the identification and isolation of germ line stem cells has given more insight into germ cell biology at the molecular level and into the underlying causes of infertility which was not possible earlier. The recent derivation of in vitro derived sperm and oocytes from pluripotent stem cells in the mouse model represents a major breakthrough in the field and substantiates the critical relevance of stem cells as a potential alternative resource for treating infertility. Although the past years have yielded compelling information in understanding germ cell development via in vitro stem cell assays, extended investigative research is necessary in order to derive fully functional 'artificial gametes' in a safe way for future therapeutic applications.

Proton pump inhibitors while belonging to the same family of generic drugs show different anti-tumor effect.

PubMed

Lugini, Luana; Federici, Cristina; Borghi, Martina; Azzarito, Tommaso; Marino, Maria Lucia; Cesolini, Albino; Spugnini, Enrico Pierluigi; Fais, Stefano

2016-08-01

Tumor acidity represents a major cause of chemoresistance. Proton pump inhibitors (PPIs) can neutralize tumor acidity, sensitizing cancer cells to chemotherapy. To compare the anti-tumor efficacy of different PPIs in vitro and in vivo. In vitro experiments PPIs anti-tumor efficacy in terms of cell proliferation and cell death/apoptosis/necrosis evaluation were performed. In vivo PPIs efficacy experiments were carried out using melanoma xenograft model in SCID mice. Lansoprazole showed higher anti-tumor effect when compared to the other PPIs. The lansoprazole effect lasted even upon drug removal from the cell culture medium and it was independent from the lipophilicity of the PPIs formulation. These PPIs have shown different anti-tumoral efficacy, and the most effective at low dose was lansoprazole. The possibility to contrast tumor acidity by off-label using PPIs opens a new field of oncology investigation.

An antiviral RISC isolated from Tobacco rattle virus-infected plants

PubMed Central

Ciomperlik, Jessica J.; Omarov, Rustem T.; Scholthof, Herman B.

2011-01-01

The RNAi model predicts that during antiviral defense a RNA-induced silencing complex (RISC) is programmed with viral short-interfering RNAs (siRNAs) to target the cognate viral RNA for degradation. We show that infection of Nicotiana benthamiana with Tobacco rattle virus (TRV) activates an antiviral nuclease that specifically cleaves TRV RNA in vitro. In agreement with known RISC properties, the nuclease activity was inhibited by NaCl and EDTA and stimulated by divalent metal cations; a novel property was its preferential targeting of elongated RNA molecules. Intriguingly, the specificity of the TRV RISC could be re-programmed by exogenous addition of RNA (containing siRNAs) from plants infected with an unrelated virus, resulting in a newly acquired ability of RISC to target this heterologous genome in vitro. Evidently the virus-specific nuclease complex from N. benthamiana represents a genuine RISC that functions as a readily employable and reprogrammable antiviral defense unit. PMID:21272908

New Class of Antitrypanosomal Agents Based on Imidazopyridines.

PubMed

Silva, Daniel G; Gillespie, J Robert; Ranade, Ranae M; Herbst, Zackary M; Nguyen, Uyen T T; Buckner, Frederick S; Montanari, Carlos A; Gelb, Michael H

2017-07-13

The present work describes the synthesis of 22 new imidazopyridine analogues arising from medicinal chemistry optimization at different sites on the molecule. Seven and 12 compounds exhibited an in vitro EC 50 ≤ 1 μM against Trypanosoma cruzi ( T. cruzi ) and Trypanosoma brucei ( T. brucei ) parasites, respectively. Based on promising results of in vitro activity (EC 50 < 100 nM), cytotoxicity, metabolic stability, protein binding, and pharmacokinetics (PK) properties, compound 20 was selected as a candidate for in vivo efficacy studies. This compound was screened in an acute mouse model against T.cruzi ( Tulahuen strain). After established infection, mice were dosed twice a day for 5 days, and then monitored for 6 weeks using an in vivo imaging system (IVIS). Compound 20 demonstrated parasite inhibition comparable to the benznidazole treatment group. Compound 20 represents a potential lead for the development of drugs to treat trypanosomiasis.

S-protected thiolated chitosan: Synthesis and in vitro characterization

PubMed Central

Dünnhaupt, Sarah; Barthelmes, Jan; Thurner, Clemens C.; Waldner, Claudia; Sakloetsakun, Duangkamon; Bernkop-Schnürch, Andreas

2012-01-01

Purpose of the present study was the generation and evaluation of novel thiolated chitosans, so-named S-protected thiolated chitosans as mucosal drug delivery systems. Stability of all conjugates concerning swelling and disintegration behavior as well as drug release was examined. Mucoadhesive properties were evaluated in vitro on intestinal mucosa. Different thiolated chitosans were generated displaying increasing amounts of attached free thiol groups on the polymer, whereby more than 50% of these thiol groups were linked with 6-mercaptonicotinamide. Based on the implementation of this hydrophobic residue, the swelling behavior was 2-fold decreased, whereas stability was essentially improved. Their mucoadhesive properties were 2- and 14-fold increased compared to corresponding thiolated and unmodified chitosans, respectively. Release studies out of matrix tablets comprising the novel conjugates revealed a controlled release of a model peptide. Accordingly, S-protected thiomers represent a promising type of mucoadhesive polymers for the development of various mucosal drug delivery systems. PMID:22839999

Evaluation of methylene blue, pyrimethamine and its combination on an in vitro Neospora caninum model.

PubMed

Pereira, Luiz Miguel; Vigato-Ferreira, Isabel Cristina; DE Luca, Gabriela; Bronzon DA Costa, Cássia Mariana; Yatsuda, Ana Patrícia

2017-05-01

Neospora caninum is an apicomplexan parasite strongly related to reproductive problems in cattle. The neosporosis control is not well established and several fronts are under development, predominantly based on immune protection, immunomodulation and chemotherapy. The use of anti-malarial drugs as therapeutic sources has, in theory, considerable potential for any apicomplexan. Drugs such as methylene blue (MB) and pyrimethamine (Pyr) represent therapeutic options for malaria; thus, their use for neosporosis should be assessed. In this work, we tested the effects of MB and Pyr on N. caninum proliferation and clearance, using LacZ-tagged tachyzoites. The drugs inhibited at nanomolar dosages and its combination demonstrated an antagonistic interaction in proliferation assays, according to the Chou and Talalay method for drug combination index. However, the drug combination significantly improved the parasite in vitro clearance. The repositioning of well-established drugs opens a short-term strategy to obtain low-cost therapeutics approaches against neosporosis.

Carbohydrate-Binding Non-Peptidic Pradimicins for the Treatment of Acute Sleeping Sickness in Murine Models.

PubMed

Castillo-Acosta, Víctor M; Ruiz-Pérez, Luis M; Etxebarria, Juan; Reichardt, Niels C; Navarro, Miguel; Igarashi, Yasuhiro; Liekens, Sandra; Balzarini, Jan; González-Pacanowska, Dolores

2016-09-01

Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis in vitro. Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT.

Incubation under fluid dynamic conditions markedly improves the structural preservation in vitro of explanted skeletal muscles.

PubMed

Carton, Flavia; Calderan, Laura; Malatesta, Manuela

2017-11-28

Explanted organs and tissues represent suitable experimental systems mimicking the functional and structural complexity of the living organism, with positive ethical and economic impact on research activities. However, their preservation in culture is generally limited, thus hindering their application as experimental models for biomedical research. In the present study, we investigated the potential of an innovative fluid dynamic culture system to improve the structural preservation in vitro of explanted mouse skeletal muscles (soleus). We used light and transmission electron microscopy to compare the morphological features of muscles maintained either in multiwell plates under conventional conditions or in a bioreactor mimicking the flow of physiological fluids. Our results demonstrate that fluid dynamic conditions markedly slowed the progressive structural deterioration of the muscle tissue occurring during the permanence in the culture medium, prolonging the preservation of some organelles such as mitochondria up to 48 h.

Incubation under fluid dynamic conditions markedly improves the structural preservation in vitro of explanted skeletal muscles

PubMed Central

Carton, Flavia; Calderan, Laura; Malatesta, Manuela

2017-01-01

Explanted organs and tissues represent suitable experimental systems mimicking the functional and structural complexity of the living organism, with positive ethical and economic impact on research activities. However, their preservation in culture is generally limited, thus hindering their application as experimental models for biomedical research. In the present study, we investigated the potential of an innovative fluid dynamic culture system to improve the structural preservation in vitro of explanted mouse skeletal muscles (soleus). We used light and transmission electron microscopy to compare the morphological features of muscles maintained either in multiwell plates under conventional conditions or in a bioreactor mimicking the flow of physiological fluids. Our results demonstrate that fluid dynamic conditions markedly slowed the progressive structural deterioration of the muscle tissue occurring during the permanence in the culture medium, prolonging the preservation of some organelles such as mitochondria up to 48 h. PMID:29313601

Substrate-based inhibitors exhibiting excellent protective and therapeutic effects against Botulinum Neurotoxin A intoxication.

PubMed

Guo, Jiubiao; Wang, Jinglin; Gao, Shan; Ji, Bin; Waichi Chan, Edward; Chen, Sheng

2015-11-20

Potent inhibitors to reverse Botulinum neurotoxins (BoNTs) activity in neuronal cells are currently not available. A better understanding of the substrate recognition mechanism of BoNTs enabled us to design a novel class of peptide inhibitors which were derivatives of the BoNT/A substrate, SNAP25. Through a combination of in vitro, cellular based, and in vivo mouse assays, several potent inhibitors of approximately one nanomolar inhibitory strength both in vitro and in vivo have been identified. These compounds represent the first set of inhibitors that exhibited full protection against BoNT/A intoxication in mice model with undetectable toxicity. Our findings validated the hypothesis that a peptide inhibitor targeting the two BoNT structural regions which were responsible for substrate recognition and cleavage respectively could exhibit excellent inhibitory effect, thereby providing insight on future development of more potent inhibitors against BoNTs.

N,N'-dihydroxyamidines: a new prodrug principle to improve the oral bioavailability of amidines.

PubMed

Reeh, Christiane; Wundt, Judith; Clement, Bernd

2007-12-27

N, N'-dihydroxybenzamdine represents a model compound for a new prodrug principle to improve the oral bioavailability of drugs containing amidine functions. The activation of the prodrug could be demonstrated in vitro by porcine and human subcellular enzyme fractions, the mitochondrial benzamidoxime reducing system, and porcine hepatocytes. In vivo, the bioavailability of benzamidine after oral application of N, N'-dihydroxybenzamidine was about 91% and exceeded that of benzamidine after oral application of benzamidoxime, being about 74% (Liu, L.; Ling, Y.; Havel, C.; Bashnick, L.; Young, W.; Rai, R.; Vijaykumar, D.; Riggs, J. R.; Ton, T.; Shaghafi, M.; Graupe, D.; Mordenti, J.; Sukbuntherng, J. Species comparison of in vitro and in vivo conversion of five N-hydroxyamidine prodrugs of fVIIA inhibitors to their corresponding active amidines. Presented at the 13th North America ISSX Meeting, Maui, HI, 2005).

Chromatic dispersive confocal technology for intra-oral scanning: first in-vitro results

NASA Astrophysics Data System (ADS)

Ertl, T.; Zint, M.; Konz, A.; Brauer, E.; Hörhold, H.; Hibst, R.

2015-02-01

Various test objects, plaster models, partially equipped with extracted teeth and pig jaws representing various clinical situations of tooth preparations were used for in-vitro scanning tests with an experimental intra-oral scanning system based on chromatic-dispersive confocal technology. Scanning results were compared against data sets of the same object captured by an industrial μCT measuring system. Compared to μCT data an average error of 18 - 30 μm was achieved for a single tooth scan area and less than 40 to 60 μm error measured over the restoration + the neighbor teeth and pontic areas up to 7 units. Mean error for a full jaw is within 100 - 140 μm. The length error for a 3 - 4 unit bridge situation form contact point to contact point is below 100 μm and excellent interproximal surface coverage and prep margin clarity was achieved.

Carbohydrate-Binding Non-Peptidic Pradimicins for the Treatment of Acute Sleeping Sickness in Murine Models

PubMed Central

Castillo-Acosta, Víctor M.; Ruiz-Pérez, Luis M.; Reichardt, Niels C.; Igarashi, Yasuhiro; Liekens, Sandra; Balzarini, Jan

2016-01-01

Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis in vitro. Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT. PMID:27662652

Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs

PubMed Central

Weinstein, Edward A.; Yano, Takahiro; Li, Lin-Sheng; Avarbock, David; Avarbock, Andrew; Helm, Douglas; McColm, Andrew A.; Duncan, Ken; Lonsdale, John T.; Rubin, Harvey

2005-01-01

Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa3 system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics. PMID:15767566

GDC-0941, a novel class I selective PI3K inhibitor, enhances the efficacy of docetaxel in human breast cancer models by increasing cell death in vitro and in vivo.

PubMed

Wallin, Jeffrey J; Guan, Jane; Prior, Wei Wei; Lee, Leslie B; Berry, Leanne; Belmont, Lisa D; Koeppen, Hartmut; Belvin, Marcia; Friedman, Lori S; Sampath, Deepak

2012-07-15

Docetaxel is a front-line standard-of-care chemotherapeutic drug for the treatment of breast cancer. Phosphoinositide 3-kinases (PI3K) are lipid kinases that regulate breast tumor cell growth, migration, and survival. The current study was intended to determine whether GDC-0941, an orally bioavailable class I selective PI3K inhibitor, enhances the antitumor activity of docetaxel in human breast cancer models in vitro and in vivo. A panel of 25 breast tumor cell lines representing HER2+, luminal, and basal subtypes were treated with GDC-0941, docetaxel, or the combination of both drugs and assayed for cellular viability, modulation of PI3K pathway markers, and apoptosis induction. Drug combination effects on cellular viability were also assessed in nontransformed MCF10A human mammary epithelial cells. Human xenografts of breast cancer cell lines and patient-derived tumors were used to assess efficacy of GDC-0941 and docetaxel in vivo. Combination of GDC-0941 and docetaxel decreased the cellular viability of breast tumor cell lines in vitro but to variable degrees of drug synergy. Compared with nontransformed MCF10A cells, the addition of both drugs resulted in stronger synergistic effects in a subset of tumor cell lines that were not predicted by breast cancer subtype. In xenograft models, GDC-0941 enhanced the antitumor activity of docetaxel with maximum combination efficacy observed within 1 hour of administering both drugs. GDC-0941 increased the rate of apoptosis in cells arrested in mitosis upon cotreatment with docetaxel. GDC-0941 augments the efficacy of docetaxel by increasing drug-induced apoptosis in breast cancer models.

In vitro large-scale experimental and theoretical studies for the realization of bi-directional brain-prostheses.

PubMed

Bonifazi, Paolo; Difato, Francesco; Massobrio, Paolo; Breschi, Gian L; Pasquale, Valentina; Levi, Timothée; Goldin, Miri; Bornat, Yannick; Tedesco, Mariateresa; Bisio, Marta; Kanner, Sivan; Galron, Ronit; Tessadori, Jacopo; Taverna, Stefano; Chiappalone, Michela

2013-01-01

Brain-machine interfaces (BMI) were born to control "actions from thoughts" in order to recover motor capability of patients with impaired functional connectivity between the central and peripheral nervous system. The final goal of our studies is the development of a new proof-of-concept BMI-a neuromorphic chip for brain repair-to reproduce the functional organization of a damaged part of the central nervous system. To reach this ambitious goal, we implemented a multidisciplinary "bottom-up" approach in which in vitro networks are the paradigm for the development of an in silico model to be incorporated into a neuromorphic device. In this paper we present the overall strategy and focus on the different building blocks of our studies: (i) the experimental characterization and modeling of "finite size networks" which represent the smallest and most general self-organized circuits capable of generating spontaneous collective dynamics; (ii) the induction of lesions in neuronal networks and the whole brain preparation with special attention on the impact on the functional organization of the circuits; (iii) the first production of a neuromorphic chip able to implement a real-time model of neuronal networks. A dynamical characterization of the finite size circuits with single cell resolution is provided. A neural network model based on Izhikevich neurons was able to replicate the experimental observations. Changes in the dynamics of the neuronal circuits induced by optical and ischemic lesions are presented respectively for in vitro neuronal networks and for a whole brain preparation. Finally the implementation of a neuromorphic chip reproducing the network dynamics in quasi-real time (10 ns precision) is presented.

A 3D Real-Scale, Biomimetic, and Biohybrid Model of the Blood-Brain Barrier Fabricated through Two-Photon Lithography.

PubMed

Marino, Attilio; Tricinci, Omar; Battaglini, Matteo; Filippeschi, Carlo; Mattoli, Virgilio; Sinibaldi, Edoardo; Ciofani, Gianni

2018-02-01

The investigation of the crossing of exogenous substances through the blood-brain barrier (BBB) is object of intensive research in biomedicine, and one of the main obstacles for reliable in vitro evaluations is represented by the difficulties at the base of developing realistic models of the barrier, which could resemble as most accurately as possible the in vivo environment. Here, for the first time, a 1:1 scale, biomimetic, and biohybrid BBB model is proposed. Microtubes inspired to the brain capillaries were fabricated through two-photon lithography and used as scaffolds for the co-culturing of endothelial-like bEnd.3 and U87 glioblastoma cells. The constructs show the maturation of tight junctions, good performances in terms of hindering dextran diffusion through the barrier, and a satisfactory trans-endothelial electrical resistance. Moreover, a mathematical model is developed, which assists in both the design of the 3D microfluidic chip and its characterization. Overall, these results show the effective formation of a bioinspired cellular barrier based on microtubes reproducing brain microcapillaries to scale. This system will be exploited as a realistic in vitro model for the investigation of BBB crossing of nanomaterials and drugs, envisaging therapeutic and diagnostic applications for several brain pathologies, including brain cancer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Xenobiotic metabolism capacities of human skin in comparison with a 3D epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: activating enzymes (Phase I).

PubMed

Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Blatz, Veronika; Jäckh, Christine; Freytag, Eva-Maria; Fabian, Eric; Landsiedel, Robert; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

2012-05-01

Skin is important for the absorption and metabolism of exposed chemicals such as cosmetics or pharmaceuticals. The Seventh Amendment to the EU Cosmetics Directive prohibits the use of animals for cosmetic testing for certain endpoints, such as genotoxicity; therefore, there is an urgent need to understand the xenobiotic metabolizing capacities of human skin and to compare these activities with reconstructed 3D skin models developed to replace animal testing. We have measured Phase I enzyme activities of cytochrome P450 (CYP) and cyclooxygenase (COX) in ex vivo human skin, the 3D skin model EpiDerm™ (EPI-200), immortalized keratinocyte-based cell lines and primary normal human epidermal keratinocytes. Our data demonstrate that basal CYP enzyme activities are very low in whole human skin and EPI-200 as well as keratinocytes. In addition, activities in monolayer cells differed from organotypic tissues after induction. COX activity was similar in skin, EPI-200 and NHEK cells, but was significantly lower in immortalized keratinocytes. Hence, the 3D model EPI-200 might represent a more suitable model for dermatotoxicological studies. Altogether, these data help to better understand skin metabolism and expand the knowledge of in vitro alternatives used for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

IN VITRO METABOLISM OF THE FUNGICIDE AND ENVIRONMENTAL CONTAMINANT TRANS-BROMUCONAZOLE AND IMPLICATIONS FOR RISK ASSESSMENT

EPA Science Inventory

Trans-Bromuconazole is a chiral chemical representative of a class of triazole-derivatives known to inhibit specific fungal cytochrome P450 (CYP) reactions. Kinetic measurements and delineation of metabolic pathways for triazole chemicals within in vitro hepatic microsomes are ne...

In vitro colony interactions among species of Trichoderma with inference toward biological control.

Treesearch

Jimmy L. Reaves

1994-01-01

Colony interactions among 15 isolates representing seven species of Trichoderma were evaluated in vitro. Interactions characterized by zones of inhibition, demarcation lines, ridges of conidia, overgrowth, intermingling, anastomosis, and hyphal coiling in self-pairings and intraspecific and interspecific pairings of the seven species were recorded...

In vitro estimation of pressure drop across tracheal tubes during high-frequency percussive ventilation.

PubMed

Ajčević, M; Lucangelo, U; Ferluga, M; Zin, W A; Accardo, A

2014-02-01

Tracheal tubes (TT) are used in clinical practice to connect an artificial ventilator to the patient's airways. It is important to know the pressure used to overcome tube impedance to avoid lung injury. Although high-frequency percussive ventilation (HFPV) has been increasingly used, the mechanical behavior of TT under HFPV has not yet been described. Thus, we aimed at characterizing in vitro the pressure drop across TT (ΔPTT) by identifying the model that best fits the measured pressure-flow (P-V̇) relationships during HFPV under different working pressures (PWork), percussive frequencies and mechanical loads. Three simple models relating ΔPTT and flow (V̇) were tested. Model 1 is characterized by linear resistive [Rtube ⋅ V̇(t)] and inertial [I · V̈(t)] terms. Model 2 takes into consideration Rohrer's approach [K1· V̇(t) + K2 ⋅V̇(t)] and inertance [I ·V̈(t)]. In model 3 the pressure drop caused by friction is represented by the non-linear Blasius component [Kb· V̇(1.75)(t)] and the inertial term [I· V̈(t)]. Model 1 presented a significantly higher root mean square error of approximation than models 2 and 3, which were similar. Thus, model 1 was not as accurate as the latter, possibly due to turbulence. Model 3 presented the most robust resistance-related coefficient. Estimated inertances did not vary among the models using the same tube. In conclusion, in HFPV ΔPTT can be easily calculated by the physician using model 3.

Discovery of potent NEK2 inhibitors as potential anticancer agents using structure-based exploration of NEK2 pharmacophoric space coupled with QSAR analyses.

PubMed

Khanfar, Mohammad A; Banat, Fahmy; Alabed, Shada; Alqtaishat, Saja

2017-02-01

High expression of Nek2 has been detected in several types of cancer and it represents a novel target for human cancer. In the current study, structure-based pharmacophore modeling combined with multiple linear regression (MLR)-based QSAR analyses was applied to disclose the structural requirements for NEK2 inhibition. Generated pharmacophoric models were initially validated with receiver operating characteristic (ROC) curve, and optimum models were subsequently implemented in QSAR modeling with other physiochemical descriptors. QSAR-selected models were implied as 3D search filters to mine the National Cancer Institute (NCI) database for novel NEK2 inhibitors, whereas the associated QSAR model prioritized the bioactivities of captured hits for in vitro evaluation. Experimental validation identified several potent NEK2 inhibitors of novel structural scaffolds. The most potent captured hit exhibited an [Formula: see text] value of 237 nM.

Interactions between Sirt1 and MAPKs regulate astrocyte activation induced by brain injury in vitro and in vivo.

PubMed

Li, Dan; Liu, Nan; Zhao, Hai-Hua; Zhang, Xu; Kawano, Hitoshi; Liu, Lu; Zhao, Liang; Li, Hong-Peng

2017-03-29

Astrocyte activation is a hallmark of traumatic brain injury resulting in neurological dysfunction or death for an overproduction of inflammatory cytokines and glial scar formation. Both the silent mating type information (Sirt1) expression and mitogen-activated protein kinase (MAPK) signal pathway activation represent a promising therapeutic target for several models of neurodegenerative diseases. We investigated the potential effects of Sirt1 upregulation and MAPK pathway pharmacological inhibition on astrocyte activation in vitro and in vivo. Moreover, we attempted to confirm the underlying interactions between Sirt1 and MAPK pathways in astrocyte activation after brain injury. The present study employs an interleukin-1β (IL-1β) stimulated primary cortical astrocyte model in vitro and a nigrostriatal pathway injury model in vivo to mimic the astrocyte activation induced by traumatic brain injury. The activation of GFAP, Sirt1, and MAPK pathways were detected by Western blot; astrocyte morphological hypertrophy was assessed using immunofluorescence staining; in order to explore the neuroprotective effect of regulation Sirt1 expression and MAPK pathway activation, the motor and neurological function tests were assessed after injury. GFAP level and morphological hypertrophy of astrocytes are elevated after injury in vitro or in vivo. Furthermore, the expressions of phosphorylated extracellular regulated protein kinases (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated p38 activation (p-p38) are upregulated, but the Sirt1 expression is downregulated. Overexpression of Sirt1 significantly increases the p-ERK expression and reduces the p-JNK and p-p38 expressions. Inhibition of ERK, JNK, or p38 activation respectively with their inhibitors significantly elevated the Sirt1 expression and attenuated the astrocyte activation. Both the overproduction of Sirt1 and inhibition of ERK, JNK, or p38 activation can alleviate the astrocyte activation, thereby improving the neurobehavioral function according to the modified neurological severity scores (mNSS) and balance latency test. Thus, Sirt1 plays a protective role against astrocyte activation, which may be associated with the regulation of the MAPK pathway activation induced by brain injury in vitro and in vivo.

Establishing best practise in the application of expert review of mutagenicity under ICH M7.

PubMed

Barber, Chris; Amberg, Alexander; Custer, Laura; Dobo, Krista L; Glowienke, Susanne; Van Gompel, Jacky; Gutsell, Steve; Harvey, Jim; Honma, Masamitsu; Kenyon, Michelle O; Kruhlak, Naomi; Muster, Wolfgang; Stavitskaya, Lidiya; Teasdale, Andrew; Vessey, Jonathan; Wichard, Joerg

2015-10-01

The ICH M7 guidelines for the assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals allows for the consideration of in silico predictions in place of in vitro studies. This represents a significant advance in the acceptance of (Q)SAR models and has resulted from positive interactions between modellers, regulatory agencies and industry with a shared purpose of developing effective processes to minimise risk. This paper discusses key scientific principles that should be applied when evaluating in silico predictions with a focus on accuracy and scientific rigour that will support a consistent and practical route to regulatory submission. Copyright © 2015 Elsevier Inc. All rights reserved.

Bridging the Gap Between In Vitro Dissolution and the Time Course of Ibuprofen-Mediating Pain Relief.

PubMed

Cristofoletti, Rodrigo; Dressman, Jennifer B

2016-12-01

In vitro-in vivo extrapolation techniques combined with physiologically based pharmacokinetic models represent a feasible approach to establishing links between critical quality attributes and the time course of drug concentrations in vivo. By further integrating the results with pharmacodynamic (PD) models, scientists can also explore the time course of drug effect. The aim of this study was to assess whether differences in dissolution rates would affect the onset, magnitude, and duration of the time course of ibuprofen-mediating pain relief. An integrated in vitro-in vivo extrapolation-physiologically based pharmacokinetic/PD model was used to simulate pharmacokinetic and PD profiles for ibuprofen free acid (IBU-H) and its salts. Two elements of the pharmacokinetic profile, the peak of exposure (C max ) and the time to peak concentration (T max ), were sensitive to dissolution rate, whereas only 1 element of the pharmacodynamic profile was affected, namely the onset of drug action. The C max differences between IBU-H and its salts seem to be mitigated in the (hypothetical) effect compartment because of the concurrent distribution and elimination processes. Furthermore, the predicted maximum concentration in the effect compartment exceeded the EC 80 value, which marks the plateau phase of the PD concentration-response curve, regardless of whether IBU-H or its salts were administered. Understanding the target site distribution kinetics and the potential nonlinearities between exposure and response will assist in setting criteria that are more scientifically based for the demonstration of therapeutic equivalence. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays.

PubMed

Heidemann, Martina; Streit, Jürg; Tscherter, Anne

2015-09-23

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Type 1 fimbriae contribute to catheter-associated urinary tract infections caused by Escherichia coli.

PubMed

Reisner, Andreas; Maierl, Mario; Jörger, Michael; Krause, Robert; Berger, Daniela; Haid, Andrea; Tesic, Dijana; Zechner, Ellen L

2014-03-01

Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI), which represent the most frequent nosocomial infections. Knowledge of genetic factors for catheter colonization is limited, since their role has not been assessed using physicochemical conditions prevailing in a catheterized human bladder. The current study aimed to combine data from a dynamic catheterized bladder model in vitro with in vivo expression analysis for understanding molecular factors relevant for CAUTI caused by Escherichia coli. By application of the in vitro model that mirrors the physicochemical environment during human infection, we found that an E. coli K-12 mutant defective in type 1 fimbriae, but not isogenic mutants lacking flagella or antigen 43, was outcompeted by the wild-type strain during prolonged catheter colonization. The importance of type 1 fimbriae for catheter colonization was verified using a fimA mutant of uropathogenic E. coli strain CFT073 with human and artificial urine. Orientation of the invertible element (IE) controlling type 1 fimbrial expression in bacterial populations harvested from the colonized catheterized bladder in vitro suggested that the vast majority of catheter-colonizing cells (up to 88%) express type 1 fimbriae. Analysis of IE orientation in E. coli populations harvested from patient catheters revealed that a median level of ∼73% of cells from nine samples have switched on type 1 fimbrial expression. This study supports the utility of the dynamic catheterized bladder model for analyzing catheter colonization factors and highlights a role for type 1 fimbriae during CAUTI.

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems.

PubMed

Pamies, David; Bal-Price, Anna; Chesné, Christophe; Coecke, Sandra; Dinnyes, Andras; Eskes, Chantra; Grillari, Regina; Gstraunthaler, Gerhard; Hartung, Thomas; Jennings, Paul; Leist, Marcel; Martin, Ulrich; Passier, Robert; Schwamborn, Jens C; Stacey, Glyn N; Ellinger-Ziegelbauer, Heidrun; Daneshian, Mardas

2018-04-13

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.

Identification of novel inhibitors of non-replicating Mycobacterium tuberculosis using a carbon starvation model

PubMed Central

Grant, Sarah Schmidt; Kawate, Tomohiko; Nag, Partha P.; Silvis, Melanie R.; Gordon, Katherine; Stanley, Sarah A.; Kazyanskaya, Ed; Nietupski, Ray; Golas, Aaron; Fitzgerald, Michael; Cho, Sanghyun; Franzblau, Scott G.; Hung, Deborah T.

2013-01-01

During Mycobacterium tuberculosis infection, a population of bacteria is thought to exist in a non-replicating state, refractory to antibiotics, which may contribute to the need for prolonged antibiotic therapy. The identification of inhibitors of the non-replicating state provides tools that can be used to probe this hypothesis and the physiology of this state. The development of such inhibitors also has the potential to shorten the duration of antibiotic therapy required. Here we describe the development of a novel non-replicating assay amenable to high-throughput chemical screening coupled with secondary assays that use carbon starvation as the in vitro model. Together these assays identify compounds with activity against replicating and non-replicating M. tuberculosis as well as compounds that inhibit the transition from non-replicating to replicating stages of growth. Using these assays we successfully screened over 300,000 compounds and identified 786 inhibitors of non-replicating M. tuberculosis. In order to understand the relationship among different non-replicating models, we teste 52 of these molecules in a hypoxia model and four different chemical scaffolds in a stochastic persist model and a streptomycin dependent model. We found that compounds display varying levels of activity in different models for the non-replicating state, suggesting important differences in bacterial physiology between models. Therefore, chemical tools identified in this assay may be useful for determining the relevance of different non-replicating in vitro models to in vivo M. tuberculosis infection. Given our current limited understanding, molecules that are active across multiple models may represent more promising candidates for further development. PMID:23898841

A new in vitro lipid digestion - in vivo absorption model to evaluate the mechanisms of drug absorption from lipid-based formulations.

PubMed

Crum, Matthew F; Trevaskis, Natalie L; Williams, Hywel D; Pouton, Colin W; Porter, Christopher J H

2016-04-01

In vitro lipid digestion models are commonly used to screen lipid-based formulations (LBF), but in vitro-in vivo correlations are in some cases unsuccessful. Here we enhance the scope of the lipid digestion test by incorporating an absorption 'sink' into the experimental model. An in vitro model of lipid digestion was coupled directly to a single pass in situ intestinal perfusion experiment in an anaesthetised rat. The model allowed simultaneous real-time analysis of the digestion and absorption of LBFs of fenofibrate and was employed to evaluate the influence of formulation digestion, supersaturation and precipitation on drug absorption. Formulations containing higher quantities of co-solvent and surfactant resulted in higher supersaturation and more rapid drug precipitation in vitro when compared to those containing higher quantities of lipid. In contrast, when the same formulations were examined using the coupled in vitro lipid digestion - in vivo absorption model, drug flux into the mesenteric vein was similar regardless of in vitro formulation performance. For some drugs, simple in vitro lipid digestion models may underestimate the potential for absorption from LBFs. Consistent with recent in vivo studies, drug absorption for rapidly absorbed drugs such as fenofibrate may occur even when drug precipitation is apparent during in vitro digestion.

An in vitro method for detecting chemical sensitization using human reconstructed skin models and its applicability to cosmetic, pharmaceutical, and medical device safety testing.

PubMed

McKim, James M; Keller, Donald J; Gorski, Joel R

2012-12-01

Chemical sensitization is a serious condition caused by small reactive molecules and is characterized by a delayed type hypersensitivity known as allergic contact dermatitis (ACD). Contact with these molecules via dermal exposure represent a significant concern for chemical manufacturers. Recent legislation in the EU has created the need to develop non-animal alternative methods for many routine safety studies including sensitization. Although most of the alternative research has focused on pure chemicals that possess reasonable solubility properties, it is important for any successful in vitro method to have the ability to test compounds with low aqueous solubility. This is especially true for the medical device industry where device extracts must be prepared in both polar and non-polar vehicles in order to evaluate chemical sensitization. The aim of this research was to demonstrate the functionality and applicability of the human reconstituted skin models (MatTek Epiderm(®) and SkinEthic RHE) as a test system for the evaluation of chemical sensitization and its potential use for medical device testing. In addition, the development of the human 3D skin model should allow the in vitro sensitization assay to be used for finished product testing in the personal care, cosmetics, and pharmaceutical industries. This approach combines solubility, chemical reactivity, cytotoxicity, and activation of the Nrf2/ARE expression pathway to identify and categorize chemical sensitizers. Known chemical sensitizers representing extreme/strong-, moderate-, weak-, and non-sensitizing potency categories were first evaluated in the skin models at six exposure concentrations ranging from 0.1 to 2500 µM for 24 h. The expression of eight Nrf2/ARE, one AhR/XRE and two Nrf1/MRE controlled gene were measured by qRT-PCR. The fold-induction at each exposure concentration was combined with reactivity and cytotoxicity data to determine the sensitization potential. The results demonstrated that both the MatTek and SkinEthic models performed in a manner consistent with data previously reported with the human keratinocyte (HaCaT) cell line. The system was tested further by evaluating chemicals known to be associated with the manufacture of medical devices. In all cases, the human skin models performed as well or better than the HaCaT cell model previously evaluated. In addition, this study identifies a clear unifying trigger that controls both the Nrf2/ARE pathway and essential biochemical events required for the development of ACD. Finally, this study has demonstrated that by utilizing human reconstructed skin models, it is possible to evaluate non-polar extracts from medical devices and low solubility finished products.

Podoplanin-Fc reduces lymphatic vessel formation in vitro and in vivo and causes disseminated intravascular coagulation when transgenically expressed in the skin

PubMed Central

Cueni, Leah N.; Chen, Lu; Zhang, Hui; Marino, Daniela; Huggenberger, Reto; Alitalo, Annamari; Bianchi, Roberta

2010-01-01

Podoplanin is a small transmembrane protein required for development and function of the lymphatic vascular system. To investigate the effects of interfering with its function, we produced an Fc fusion protein of its ectodomain. We found that podoplanin-Fc inhibited several functions of cultured lymphatic endothelial cells and also specifically suppressed lymphatic vessel growth, but not blood vessel growth, in mouse embryoid bodies in vitro and in mouse corneas in vivo. Using a keratin 14 expression cassette, we created transgenic mice that overexpressed podoplanin-Fc in the skin. No obvious outward phenotype was identified in these mice, but surprisingly, podoplanin-Fc—although produced specifically in the skin—entered the blood circulation and induced disseminated intravascular coagulation, characterized by microthrombi in most organs and by thrombocytopenia, occasionally leading to fatal hemorrhage. These findings reveal an important role of podoplanin in lymphatic vessel formation and indicate the potential of podoplanin-Fc as an inhibitor of lymphangiogenesis. These results also demonstrate the ability of podoplanin to induce platelet aggregation in vivo, which likely represents a major function of lymphatic endothelium. Finally, keratin 14 podoplanin-Fc mice represent a novel genetic animal model of disseminated intravascular coagulation. PMID:20716773

Air-liquid interface exposure to aerosols of poorly soluble nanomaterials induces different biological activation levels compared to exposure to suspensions.

PubMed

Loret, Thomas; Peyret, Emmanuel; Dubreuil, Marielle; Aguerre-Chariol, Olivier; Bressot, Christophe; le Bihan, Olivier; Amodeo, Tanguy; Trouiller, Bénédicte; Braun, Anne; Egles, Christophe; Lacroix, Ghislaine

2016-11-03

Recently, much progress has been made to develop more physiologic in vitro models of the respiratory system and improve in vitro simulation of particle exposure through inhalation. Nevertheless, the field of nanotoxicology still suffers from a lack of relevant in vitro models and exposure methods to predict accurately the effects observed in vivo, especially after respiratory exposure. In this context, the aim of our study was to evaluate if exposing pulmonary cells at the air-liquid interface to aerosols of inhalable and poorly soluble nanomaterials generates different toxicity patterns and/or biological activation levels compared to classic submerged exposures to suspensions. Three nano-TiO 2 and one nano-CeO 2 were used. An exposure system was set up using VitroCell® devices to expose pulmonary cells at the air-liquid interface to aerosols. A549 alveolar cells in monocultures or in co-cultures with THP-1 macrophages were exposed to aerosols in inserts or to suspensions in inserts and in plates. Submerged exposures in inserts were performed, using similar culture conditions and exposure kinetics to the air-liquid interface, to provide accurate comparisons between the methods. Exposure in plates using classical culture and exposure conditions was performed to provide comparable results with classical submerged exposure studies. The biological activity of the cells (inflammation, cell viability, oxidative stress) was assessed at 24 h and comparisons of the nanomaterial toxicities between exposure methods were performed. Deposited doses of nanomaterials achieved using our aerosol exposure system were sufficient to observe adverse effects. Co-cultures were more sensitive than monocultures and biological responses were usually observed at lower doses at the air-liquid interface than in submerged conditions. Nevertheless, the general ranking of the nanomaterials according to their toxicity was similar across the different exposure methods used. We showed that exposure of cells at the air-liquid interface represents a valid and sensitive method to assess the toxicity of several poorly soluble nanomaterials. We underlined the importance of the cellular model used and offer the possibility to deal with low deposition doses by using more sensitive and physiologic cellular models. This brings perspectives towards the use of relevant in vitro methods of exposure to assess nanomaterial toxicity.

Sustained Radiosensitization of Hypoxic Glioma Cells after Oxygen Pretreatment in an Animal Model of Glioblastoma and In Vitro Models of Tumor Hypoxia

PubMed Central

Clarke, Ryon H.; Moosa, Shayan; Anzivino, Matthew; Wang, Yi; Floyd, Desiree Hunt; Purow, Benjamin W.; Lee, Kevin S.

2014-01-01

Glioblastoma multiforme (GBM) is the most common and lethal form of brain cancer and these tumors are highly resistant to chemo- and radiotherapy. Radioresistance is thought to result from a paucity of molecular oxygen in hypoxic tumor regions, resulting in reduced DNA damage and enhanced cellular defense mechanisms. Efforts to counteract tumor hypoxia during radiotherapy are limited by an attendant increase in the sensitivity of healthy brain tissue to radiation. However, the presence of heightened levels of molecular oxygen during radiotherapy, while conventionally deemed critical for adjuvant oxygen therapy to sensitize hypoxic tumor tissue, might not actually be necessary. We evaluated the concept that pre-treating tumor tissue by transiently elevating tissue oxygenation prior to radiation exposure could increase the efficacy of radiotherapy, even when radiotherapy is administered after the return of tumor tissue oxygen to hypoxic baseline levels. Using nude mice bearing intracranial U87-luciferase xenografts, and in vitro models of tumor hypoxia, the efficacy of oxygen pretreatment for producing radiosensitization was tested. Oxygen-induced radiosensitization of tumor tissue was observed in GBM xenografts, as seen by suppression of tumor growth and increased survival. Additionally, rodent and human glioma cells, and human glioma stem cells, exhibited prolonged enhanced vulnerability to radiation after oxygen pretreatment in vitro, even when radiation was delivered under hypoxic conditions. Over-expression of HIF-1α reduced this radiosensitization, indicating that this effect is mediated, in part, via a change in HIF-1-dependent mechanisms. Importantly, an identical duration of transient hyperoxic exposure does not sensitize normal human astrocytes to radiation in vitro. Taken together, these results indicate that briefly pre-treating tumors with elevated levels of oxygen prior to radiotherapy may represent a means for selectively targeting radiation-resistant hypoxic cancer cells, and could serve as a safe and effective adjuvant to radiation therapy for patients with GBM. PMID:25350400

Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro kcat measurements

PubMed Central

Davidi, Dan; Noor, Elad; Liebermeister, Wolfram; Bar-Even, Arren; Flamholz, Avi; Tummler, Katja; Barenholz, Uri; Goldenfeld, Miki; Shlomi, Tomer; Milo, Ron

2016-01-01

Turnover numbers, also known as kcat values, are fundamental properties of enzymes. However, kcat data are scarce and measured in vitro, thus may not faithfully represent the in vivo situation. A basic question that awaits elucidation is: how representative are kcat values for the maximal catalytic rates of enzymes in vivo? Here, we harness omics data to calculate kmaxvivo, the observed maximal catalytic rate of an enzyme inside cells. Comparison with kcat values from Escherichia coli, yields a correlation of r2= 0.62 in log scale (p < 10−10), with a root mean square difference of 0.54 (3.5-fold in linear scale), indicating that in vivo and in vitro maximal rates generally concur. By accounting for the degree of saturation of enzymes and the backward flux dictated by thermodynamics, we further refine the correspondence between kmaxvivo and kcat values. The approach we present here characterizes the quantitative relationship between enzymatic catalysis in vitro and in vivo and offers a high-throughput method for extracting enzyme kinetic constants from omics data. PMID:26951675

Practical whole-tooth restoration utilizing autologous bioengineered tooth germ transplantation in a postnatal canine model

PubMed Central

Ono, Mitsuaki; Oshima, Masamitsu; Ogawa, Miho; Sonoyama, Wataru; Hara, Emilio Satoshi; Oida, Yasutaka; Shinkawa, Shigehiko; Nakajima, Ryu; Mine, Atsushi; Hayano, Satoru; Fukumoto, Satoshi; Kasugai, Shohei; Yamaguchi, Akira; Tsuji, Takashi; Kuboki, Takuo

2017-01-01

Whole-organ regeneration has great potential for the replacement of dysfunctional organs through the reconstruction of a fully functional bioengineered organ using three-dimensional cell manipulation in vitro. Recently, many basic studies of whole-tooth replacement using three-dimensional cell manipulation have been conducted in a mouse model. Further evidence of the practical application to human medicine is required to demonstrate tooth restoration by reconstructing bioengineered tooth germ using a postnatal large-animal model. Herein, we demonstrate functional tooth restoration through the autologous transplantation of bioengineered tooth germ in a postnatal canine model. The bioengineered tooth, which was reconstructed using permanent tooth germ cells, erupted into the jawbone after autologous transplantation and achieved physiological function equivalent to that of a natural tooth. This study represents a substantial advancement in whole-organ replacement therapy through the transplantation of bioengineered organ germ as a practical model for future clinical regenerative medicine. PMID:28300208

3D tumor tissue analogs and their orthotopic implants for understanding tumor-targeting of microenvironment-responsive nanosized chemotherapy and radiation.

PubMed

Sethi, Pallavi; Jyoti, Amar; Swindell, Elden P; Chan, Ryan; Langner, Ulrich W; Feddock, Jonathan M; Nagarajan, Radhakrishnan; O'Halloran, Thomas V; Upreti, Meenakshi

2015-11-01

An appropriate representation of the tumor microenvironment in tumor models can have a pronounced impact on directing combinatorial treatment strategies and cancer nanotherapeutics. The present study develops a novel 3D co-culture spheroid model (3D TNBC) incorporating tumor cells, endothelial cells and fibroblasts as color-coded murine tumor tissue analogs (TTA) to better represent the tumor milieu of triple negative breast cancer in vitro. Implantation of TTA orthotopically in nude mice, resulted in enhanced growth and aggressive metastasis to ectopic sites. Subsequently, the utility of the model is demonstrated for preferential targeting of irradiated tumor endothelial cells via radiation-induced stromal enrichment of galectin-1 using anginex conjugated nanoparticles (nanobins) carrying arsenic trioxide and cisplatin. Demonstration of a multimodal nanotherapeutic system and inclusion of the biological response to radiation using an in vitro/in vivo tumor model incorporating characteristics of tumor microenvironment presents an advance in preclinical evaluation of existing and novel cancer nanotherapies. Existing in-vivo tumor models are established by implanting tumor cells into nude mice. Here, the authors described their approach 3D spheres containing tumor cells, enodothelial cells and fibroblasts. This would mimic tumor micro-environment more realistically. This interesting 3D model should reflect more accurately tumor response to various drugs and would enable the design of new treatment modalities. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sirenko, Oksana, E-mail: oksana.sirenko@moldev.com

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30 min or 24 h and effects on cardiomyocyte beating andmore » cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca{sup 2+} flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30 min of exposure. In contrast, after 24 h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes. - Highlights: • The cardiotoxic potential of a broad range of environmental chemicals was evaluated. • Human iPSC cardiomyocytes were used for multiparametric in vitro profiling. • Dose-response relationships were established for functional and cytotoxicity assays. • Computational data integration provided chemical-specific bioactivity profiles. • An assessment of the profiles revealed chemical class-specific similarities.« less

Bioaccessibility of PAHs in Fuel Soot Assessed by an in Vitro Digestive Model with Absorptive Sink: Effect of Food Ingestion.

PubMed

Zhang, Yanyan; Pignatello, Joseph J; Tao, Shu; Xing, Baoshan

2015-12-15

We investigated the effects of changing physiological conditions in the digestive tract expected with food ingestion on the apparent bioaccessibility (Bapp) of 11 polycyclic aromatic hydrocarbons (PAHs) in a fuel soot. A previously established in vitro digestive model was applied that included silicone sheet as a third-phase absorptive sink simulating passive transfer of PAHs to intestinal epithelium in the small intestine stage. The Bapp is defined as the fraction found in the digestive fluid plus sheet after digestion. We determined that Bapp was independent of gastric pH and addition of nonlipid milk representing dietary proteins and carbohydrates, whereas it increased with bile acids concentration (2.0-10 g/L), small intestinal pH (5.00-7.35), and addition of soybean oil representing dietary lipid (100% and 200% of the mean daily ingestion by 2-5 year olds in the U.S.). Bapp of PAHs increases with small intestinal pH due to the combined effects of mass transfer promotion from nonlabile to labile sorbed states in the soot, weaker sorption of the labile state, and increasingly favorable partitioning from the digestive fluid to the silicone sink. Under fed conditions, Bapp increases with inclusion of lipids due to the combined effects of mass transfer promotion from nonlabile to labile states, and increasingly favorable partitioning into bile acid micelles. Our results indicate significant variability in soot PAH bioaccessibility within the range of physiological conditions experienced by humans, and suggest that bioaccessibility will increase with coconsumption of food, especially food with high fat content.

Peripheral Airway Smooth Muscle, but Not the Trachealis, Is Hypercontractile in an Equine Model of Asthma.

PubMed

Matusovsky, Oleg S; Kachmar, Linda; Ijpma, Gijs; Bates, Genevieve; Zitouni, Nedjma; Benedetti, Andrea; Lavoie, Jean-Pierre; Lauzon, Anne-Marie

2016-05-01

Heaves is a naturally occurring equine disease that shares many similarities with human asthma, including reversible antigen-induced bronchoconstriction, airway inflammation, and remodeling. The purpose of this study was to determine whether the trachealis muscle is mechanically representative of the peripheral airway smooth muscle (ASM) in an equine model of asthma. Tracheal and peripheral ASM of heaves-affected horses under exacerbation, or under clinical remission of the disease, and control horses were dissected and freed of epithelium to measure unloaded shortening velocity (Vmax), stress (force/cross-sectional area), methacholine effective concentration at which 50% of the maximum response is obtained, and stiffness. Myofibrillar Mg(2+)-ATPase activity, actomyosin in vitro motility, and contractile protein expression were also measured. Horses with heaves had significantly greater Vmax and Mg(2+)-ATPase activity in peripheral airway but not in tracheal smooth muscle. In addition, a significant correlation was found between Vmax and the time elapsed since the end of the corticosteroid treatment for the peripheral airways in horses with heaves. Maximal stress and stiffness were greater in the peripheral airways of the horses under remission compared with controls and the horses under exacerbation, potentially due to remodeling. Actomyosin in vitro motility was not different between controls and horses with heaves. These data demonstrate that peripheral ASM is mechanically and biochemically altered in heaves, whereas the trachealis behaves as in control horses. It is therefore conceivable that the trachealis muscle may not be representative of the peripheral ASM in human asthma either, but this will require further investigation.

Lactoferrin and oral diseases: current status and perspective in periodontitis

PubMed Central

Berlutti, Francesca; Pilloni, Andrea; Pietropaoli, Miriam; Polimeni, Antonella; Valenti, Piera

2012-01-01

Summary Lactoferrin (Lf), an iron-binding glycoprotein able to chelate two ferric ions per molecule, is a component of human secretions synthesized by exocrine glands and neutrophils in infection/inflammation sites. Lactoferrin in saliva represents an important defence factor against bacterial injuries including those related to Streptococcus mutans and periodontopathic bacteria through its ability to decrease bacterial growth, biofilm development, iron overload, reactive oxygen formation and inflammatory processes. A growing body of research suggests that inflammatory periodontal disease involves a failure of resolution pathways to restore tissue homeostasis. There is an important distinction between anti-inflammation and resolution; anti-inflammation is pharmacologic intervention in inflammatory pathways, whereas resolution involves biologic pathways restoring inflammatory homeostasis. An appropriate regulation of pro-inflammatory cytokine synthesis might be useful in reducing periodontal tissue destruction. Recently, the multi-functional IL-6 is emerging as an important factor able to modulate bone, iron and inflammatory homeostasis. Here, we report an overview of Lf functions as well as for the first time Lf anti-inflammatory ability against periodontitis in in vitro model and observational clinical study. In in vitro model, represented by gingival fibroblasts infected with Prevotella intermedia, Lf exerted a potent anti-inflammatory activity. In the observational clinical trial performed through bovine Lf (bLf) topically administered to volunteers suffering from periodontitis, bLf decreased cytokines, including IL-6 in crevicular fluid, edema, bleeding, pocket depth, gingival and plaque index, thus improving clinical attachment levels. Even if other clinical trials are required, these results provide strong evidence for a instead of an therapeutic potential of this multifunctional natural protein. PMID:22545184

Novel Treatment of Staphylococcus aureus Device-Related Infections Using Fibrinolytic Agents.

PubMed

Hogan, S; O'Gara, J P; O'Neill, E

2018-02-01

Staphylococcal infections involving biofilms represent a significant challenge in the treatment of patients with device-related infections. Staphylococcus aureus biofilms have been shown to be SaeRS regulated and dependent on the coagulase-catalyzed conversion of fibrinogen into fibrin on surfaces coated with human plasma. Here we investigated the treatment of staphylococcal biofilm device-related infections by digesting the fibrin biofilm matrix with and without existing antimicrobials. The fibrinolytic agents plasmin, streptokinase, and nattokinase, and TrypLE, a recombinant trypsin-like protease, were used to digest and treat S. aureus biofilms grown in vitro using in vivo -like static biofilm assays with and without antimicrobials. Cytotoxicity, the potential to induce a cytokine response in whole human blood, and the risk of induction of tolerance to fibrinolytic agents were investigated. A rat model of intravascular catheter infection was established to investigate the efficacy of selected fibrinolytic agents in vivo Under biomimetic conditions, the fibrinolytic agents effectively dispersed established S. aureus biofilms and, in combination with common antistaphylococcal antimicrobials, effectively killed bacterial cells being released from the biofilm. These fibrinolytic agents were not cytotoxic and did not affect the host immune response. The rat model of infection successfully demonstrated the activity of the selected fibrinolytic agents alone and in combination with antimicrobials on established biofilms in vivo TrypLE and nattokinase most successfully removed adherent cells from plasma-coated surfaces and significantly improved the efficacy of existing antimicrobials against S. aureus biofilms in vitro and in vivo These biofilm dispersal agents represent a viable future treatment option for S. aureus device-related infections. Copyright © 2018 American Society for Microbiology.

An insight of in vitro transport of PEGylated non-ionic surfactant vesicles (NSVs) across the intestinal polarized enterocyte monolayers.

PubMed

Primavera, Rosita; Palumbo, Paola; Celia, Christian; Cinque, Benedetta; Carata, Elisabetta; Carafa, Maria; Paolino, Donatella; Cifone, Maria Grazia; Di Marzio, Luisa

2018-06-01

PEGylated non-ionic surfactant-based vesicles (NSVs) are promising drug delivery systems for the local, oral and systemic administrations of therapeutics. The aim of this study was to test the cellular biocompatibility and transport of Nile Red-loaded NSVs (NR-NSVs) across the Caco-2-cell monolayers, which represent an in vitro model of human intestinal epithelium. The NR-NSVs assumed a spherical shape with a mean size of 140 nm, and a narrow size distribution. The NR-NSVs did not modify Caco-2 cell viability, which remained unaltered in vitro up to a concentration of 1 mM. The transport studies demonstrated that the NR-NSVs moved across the Caco-2 monolayers without affecting the transepithelial electrical resistance. These results were supported by flow cytometry analysis, which demonstrated that NR-NSVs were internalized inside the Caco-2 cells. Nanoparticle tracking and Transmission Electron Microscopy (TEM) analysis showed the presence of NR-NSVs in the basolateral side of the Caco-2 monolayers. TEM images also showed that NSVs were transported intact across the Caco-2 monolayers, thus demonstrating a predominant transcytosis mechanism of transport through endocytosis. The NSVs did not affect the integrity of the membrane barrier in vitro, and can potentially be used in clinics to increase the oral bioavailability and delivery of therapeutics. Copyright © 2018 Elsevier B.V. All rights reserved.

Low pressure ultraviolet studies for inactivation of Giardia muris cysts.

PubMed

Hayes, S L; Rice, E W; Ware, M W; Schaefer, F W

2003-01-01

The research was initiated to confirm earlier ultraviolet (u.v.) light inactivation studies performed on Giardia cysts using excystation as the viability indicator. Following this, a comparison of in vitro excystation and animal infectivity was performed for assessing cyst viability after exposure to low-pressure u.v. irradiation. Cysts of Giardia muris were inactivated using a low-pressure u.v. light source. Giardia muris was employed as a surrogate for the human pathogen Giardia lamblia. Cyst viability was determined by both in vitro excystation and animal infectivity. Cyst doses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excystation as the viability indicator, fluences as high as approximately 200 mJ cm(-2) did not prevent some cysts from excysting, thus verifying earlier work. Using animal infectivity, u.v. fluences of 1.4, 1.9 and 2.3 mJ cm(-2) yielded log10 reductions ranging from 0.3 to >or= 4.4. Results indicate that in vitro excystation is not a reliable indicator of G. muris cyst viability after u.v. disinfection. Very low doses of u.v. light rendered G. muris cysts non-infective in the mouse model employed. Data presented represent the only complete u.v. inactivation curve for G. muris. This research provides evidence that u.v. can be an effective barrier against Giardia spp. in the treatment of drinking water supplies.

Prevalidation of a model for predicting acute neutropenia by colony forming unit granulocyte/macrophage (CFU-GM) assay.

PubMed

Pessina, A; Albella, B; Bueren, J; Brantom, P; Casati, S; Gribaldo, L; Croera, C; Gagliardi, G; Foti, P; Parchment, R; Parent-Massin, D; Sibiril, Y; Van Den Heuvel, R

2001-12-01

This report describes an international prevalidation study conducted to optimise the Standard Operating Procedure (SOP) for detecting myelosuppressive agents by CFU-GM assay and to study a model for predicting (by means of this in vitro hematopoietic assay) the acute xenobiotic exposure levels that cause maximum tolerated decreases in absolute neutrophil counts (ANC). In the first phase of the study (Protocol Refinement), two SOPs were assessed, by using two cell culture media (Test A, containing GM-CSF; and Test B, containing G-CSF, GM-CSF, IL-3, IL-6 and SCF), and the two tests were applied to cells from both human (bone marrow and umbilical cord blood) and mouse (bone marrow) CFU-GM. In the second phase (Protocol Transfer), the SOPs were transferred to four laboratories to verify the linearity of the assay response and its interlaboratory reproducibility. After a further phase (Protocol Performance), dedicated to a training set of six anticancer drugs (adriamycin, flavopindol, morpholino-doxorubicin, pyrazoloacridine, taxol and topotecan), a model for predicting neutropenia was verified. Results showed that the assay is linear under SOP conditions, and that the in vitro endpoints used by the clinical prediction model of neutropenia are highly reproducible within and between laboratories. Valid tests represented 95% of all tests attempted. The 90% inhibitory concentration values (IC(90)) from Test A and Test B accurately predicted the human maximum tolerated dose (MTD) for five of six and for four of six myelosuppressive anticancer drugs, respectively, that were selected as prototype xenobiotics. As expected, both tests failed to accurately predict the human MTD of a drug that is a likely protoxicant. It is concluded that Test A offers significant cost advantages compared to Test B, without any loss of performance or predictive accuracy. On the basis of these results, we proposed a formal Phase II validation study using the Test A SOP for 16-18 additional xenobiotics that represent the spectrum of haematotoxic potential.

Electrical Polarization of Titanium Surfaces for the Enhancement of Osteoblast Differentiation

PubMed Central

Gittens, Rolando A.; Olivares-Navarrete, Rene; Rettew, Robert; Butera, Robert J.; Alamgir, Faisal M.; Boyan, Barbara D.; Schwartz, Zvi

2014-01-01

Electrical stimulation has been used clinically to promote bone regeneration in cases of fractures with delayed union or nonunion, with several in vitro and in vivo reports suggesting its beneficial effects on bone formation. However, the use of electrical stimulation of titanium (Ti) implants to enhance osseointegration is less understood, in part because of the few in vitro models that attempt to represent the in vivo environment. In this article, the design of a new in vitro system that allows direct electrical stimulation of osteoblasts through their Ti substrates without the flow of exogenous currents through the media is presented, and the effect of applied electrical polarization on osteoblast differentiation and local factor production was evaluated. A custom-made polycarbonate tissue culture plate was designed to allow electrical connections directly underneath Ti disks placed inside the wells, which were supplied with electrical polarization ranging from 100 to 500 mV to stimulate MG63 osteoblasts. Our results show that electrical polarization applied directly through Ti substrates on which the cells are growing in the absence of applied electrical currents may increase osteoblast differentiation and local factor production in a voltage-dependent manner. PMID:23996899

Astrocytes from adult Wistar rats aged in vitro show changes in glial functions.

PubMed

Souza, Débora Guerini; Bellaver, Bruna; Raupp, Gustavo Santos; Souza, Diogo Onofre; Quincozes-Santos, André

2015-11-01

Astrocytes, the most versatile cells of the central nervous system, play an important role in the regulation of neurotransmitter homeostasis, energy metabolism, antioxidant defenses and the anti-inflammatory response. Recently, our group characterized cortical astrocyte cultures from adult Wistar rats. In line with that work, we studied glial function using an experimental in vitro model of aging astrocytes (30 days in vitro after reaching confluence) from newborn (NB), adult (AD) and aged (AG) Wistar rats. We evaluated metabolic parameters, such as the glucose uptake, glutamine synthetase (GS) activity, and glutathione (GSH) content, as well as the GFAP, GLUT-1 and xCT expression. AD and AG astrocytes take up less glucose than NB astrocytes and had decreased GLUT1 expression levels. Furthermore, AD and AG astrocytes exhibited decreased GS activity compared to NB cells. Simultaneously, AD and AG astrocytes showed an increase in GSH levels, along with an increase in xCT expression. NB, AD and AG astrocytes presented similar morphology; however, differences in GFAP levels were observed. Taken together, these results improve the knowledge of cerebral senescence and represent an innovative tool for brain studies of aging. Copyright © 2015 Elsevier Ltd. All rights reserved.

Research in drug development against viral diseases of military importance (biological testing). Volume 1. Final report, 15 November 1985-31 January 1991

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shannon, W.M.; Arnett, G.; Brazier, A.D.

1991-03-01

The purpose of this program is to evaluate the efficacy of candidate antiviral compounds against a spectrum of viruses of military importance. This program involves (a) primary testing of chemical compounds and natural products for antiviral efficacy in vitro using standard CPE-inhibition assays, (b) primary testing of compounds for antiviral efficacy in vivo in animal model systems, and (c) secondary evaluation of the active candidate antiviral compounds. The target viruses for in vitro testing are Vaccinia Virus (VV), Adenovirus (AD2), Vesicular Stomatitis Virus (VSV), Punta Toro Virus (PT), Sandfly Fever Virus (SF), Yellow Fever Virus (YF), Venezuelan Equine Encephalomyelitis Virusmore » (VE), Japanese Encephalitis Virus and Vaccinia Virus infections of mice. Approximately 10,000 compounds have been received for in vitro evaluation and over 66,000 assays have been performed on this contract. Compounds have been identified in nearly all virus systems that have confirmed antiviral activity equal or exceeding that of the various positive control compounds (Ribavirin, Selenazofurin, Carbocyclic-3-deaza-adenosine, Adenosine dialdehyde, Ara-A, ddC and AZT). Many of these compounds represent potent and selective new antiviral agents.« less

Keratinocyte-driven contraction of reconstructed human skin.

PubMed

Chakrabarty, K H; Heaton, M; Dalley, A J; Dawson, R A; Freedlander, E; Khaw, P T; Mac Neil, S

2001-01-01

We have previously reported that reconstructed human skin, using deepidermized acellular sterilized dermis and allogeneic keratinocytes and fibroblasts, significantly contracts in vitro. Contracture of split skin grafts in burns injuries remains a serious problem and this in vitro model provides an opportunity to study keratinocyte/mesenchymal cell interactions and cell interactions with extracted normal human dermis. The aim of this study was to investigate the nature of this in vitro contraction and explore several approaches to prevent or reduce contraction. Three different methodologies for sterilization of the dermal matrix were examined: glycerol, ethylene oxide and a combination of glycerol and ethylene oxide. While the nature of the sterilization technique influenced the extent of contraction and thinner dermal matrices contracted proportionately more than thicker matrices, in all cases contraction was driven by the keratinocytes with relatively little influence from the fibroblasts. The contraction of the underlying dermis did not represent any change in tissue mass but rather a reorganization of the dermis which was rapidly reversed (within minutes) when the epidermal layer was removed. Pharmacological approaches to block contraction showed forskolin and mannose-6-phosphate to be ineffective and ascorbic acid-2-phosphate to exacerbate contraction. However, Galardin, a matrix metalloproteinase inhibitor and keratinocyte conditioned media, both inhibited contraction.

Fabrication of a biodegradable calcium polyphosphate/polyvinyl-urethane carbonate composite for high load bearing osteosynthesis applications.

PubMed

Ramsay, Scott D; Pilliar, Robert M; Santerre, J Paul

2010-07-01

The formation of biodegradable implants for use in osteosynthesis has been a major goal of biomaterials research for the past 2-3 decades. Self-reinforced polylactide systems represent the most significant success of this research to date, however, with elastic constants up to 12-15 GPa at best, they fail to provide the initial stiffness required of devices for stabilizing fractures of major load-bearing bones. Our research has investigated the use of calcium polyphosphate (CPP), an inorganic polymer in combination with polyvinyl-urethane carbonate (PVUC) organic polymers for such applications. Initial studies indicated that composite samples formed as interpenetrating phase composites (IPC) exhibited suitable as-made strength and stiffness, however, they displayed a rapid loss of properties when exposed to in vitro aging. An investigation to determine the mechanism of this accelerated in vitro degradation for the IPCs as well as to identify possible design changes to overcome this drawback was undertaken using a model IPC system. It was found that strong interfacial strength and minimal swelling of the PVUC are very important for obtaining and maintaining appropriate mechanical properties in vitro. (c) 2010 Wiley Periodicals, Inc.

Body-on-a-chip simulation with gastrointestinal tract and liver tissues suggests that ingested nanoparticles have the potential to cause liver injury.

PubMed

Esch, Mandy B; Mahler, Gretchen J; Stokol, Tracy; Shuler, Michael L

2014-08-21

The use of nanoparticles in medical applications is highly anticipated, and at the same time little is known about how these nanoparticles affect human tissues. Here we have simulated the oral uptake of 50 nm carboxylated polystyrene nanoparticles with a microscale body-on-a-chip system (also referred to as multi-tissue microphysiological system or micro Cell Culture Analog). Using the 'GI tract-liver-other tissues' system allowed us to observe compounding effects and detect liver tissue injury at lower nanoparticle concentrations than was expected from experiments with single tissues. To construct this system, we combined in vitro models of the human intestinal epithelium, represented by a co-culture of enterocytes (Caco-2) and mucin-producing cells (TH29-MTX), and the liver, represented by HepG2/C3A cells, within one microfluidic device. The device also contained chambers that together represented the liquid portions of all other organs of the human body. Measuring the transport of 50 nm carboxylated polystyrene nanoparticles across the Caco-2/HT29-MTX co-culture, we found that this multi-cell layer presents an effective barrier to 90.5 ± 2.9% of the nanoparticles. Further, our simulation suggests that a larger fraction of the 9.5 ± 2.9% nanoparticles that travelled across the Caco-2/HT29-MTX cell layer were not large nanoparticle aggregates, but primarily single nanoparticles and small aggregates. After crossing the GI tract epithelium, nanoparticles that were administered in high doses estimated in terms of possible daily human consumption (240 and 480 × 10(11) nanoparticles mL(-1)) induced the release of aspartate aminotransferase (AST), an intracellular enzyme of the liver that indicates liver cell injury. Our results indicate that body-on-a-chip devices are highly relevant in vitro models for evaluating nanoparticle interactions with human tissues.

Interruption of Electrical Conductivity of Titanium Dental Implants Suggests a Path Towards Elimination Of Corrosion

PubMed Central

Pozhitkov, Alex E.; Daubert, Diane; Brochwicz Donimirski, Ashley; Goodgion, Douglas; Vagin, Mikhail Y.; Leroux, Brian G.; Hunter, Colby M.; Flemmig, Thomas F.; Noble, Peter A.; Bryers, James D.

2015-01-01

Peri-implantitis is an inflammatory disease that results in the destruction of soft tissue and bone around the implant. Titanium implant corrosion has been attributed to the implant failure and cytotoxic effects to the alveolar bone. We have documented the extent of titanium release into surrounding plaque in patients with and without peri-implantitis. An in vitro model was designed to represent the actual environment of an implant in a patient’s mouth. The model uses actual oral microbiota from a volunteer, allows monitoring electrochemical processes generated by biofilms growing on implants and permits control of biocorrosion electrical current. As determined by next generation DNA sequencing, microbial compositions in experiments with the in vitro model were comparable with the compositions found in patients with implants. It was determined that the electrical conductivity of titanium implants was the key factor responsible for the biocorrosion process. The interruption of the biocorrosion current resulted in a 4–5 fold reduction of corrosion. We propose a new design of dental implant that combines titanium in zero oxidation state for osseointegration and strength, interlaid with a nonconductive ceramic. In addition, we propose electrotherapy for manipulation of microbial biofilms and to induce bone healing in peri-implantitis patients. PMID:26461491

Structural Diversity in the Dandelion (Taraxacum officinale) Polyphenol Oxidase Family Results in Different Responses to Model Substrates

PubMed Central

Dirks-Hofmeister, Mareike E.; Singh, Ratna; Leufken, Christine M.; Inlow, Jennifer K.; Moerschbacher, Bruno M.

2014-01-01

Polyphenol oxidases (PPOs) are ubiquitous type-3 copper enzymes that catalyze the oxygen-dependent conversion of o-diphenols to the corresponding quinones. In most plants, PPOs are present as multiple isoenzymes that probably serve distinct functions, although the precise relationship between sequence, structure and function has not been addressed in detail. We therefore compared the characteristics and activities of recombinant dandelion PPOs to gain insight into the structure–function relationships within the plant PPO family. Phylogenetic analysis resolved the 11 isoenzymes of dandelion into two evolutionary groups. More detailed in silico and in vitro analyses of four representative PPOs covering both phylogenetic groups were performed. Molecular modeling and docking predicted differences in enzyme-substrate interactions, providing a structure-based explanation for grouping. One amino acid side chain positioned at the entrance to the active site (position HB2+1) potentially acts as a “selector” for substrate binding. In vitro activity measurements with the recombinant, purified enzymes also revealed group-specific differences in kinetic parameters when the selected PPOs were presented with five model substrates. The combination of our enzyme kinetic measurements and the in silico docking studies therefore indicate that the physiological functions of individual PPOs might be defined by their specific interactions with different natural substrates. PMID:24918587

Interruption of Electrical Conductivity of Titanium Dental Implants Suggests a Path Towards Elimination Of Corrosion.

PubMed

Pozhitkov, Alex E; Daubert, Diane; Brochwicz Donimirski, Ashley; Goodgion, Douglas; Vagin, Mikhail Y; Leroux, Brian G; Hunter, Colby M; Flemmig, Thomas F; Noble, Peter A; Bryers, James D

2015-01-01

Peri-implantitis is an inflammatory disease that results in the destruction of soft tissue and bone around the implant. Titanium implant corrosion has been attributed to the implant failure and cytotoxic effects to the alveolar bone. We have documented the extent of titanium release into surrounding plaque in patients with and without peri-implantitis. An in vitro model was designed to represent the actual environment of an implant in a patient's mouth. The model uses actual oral microbiota from a volunteer, allows monitoring electrochemical processes generated by biofilms growing on implants and permits control of biocorrosion electrical current. As determined by next generation DNA sequencing, microbial compositions in experiments with the in vitro model were comparable with the compositions found in patients with implants. It was determined that the electrical conductivity of titanium implants was the key factor responsible for the biocorrosion process. The interruption of the biocorrosion current resulted in a 4-5 fold reduction of corrosion. We propose a new design of dental implant that combines titanium in zero oxidation state for osseointegration and strength, interlaid with a nonconductive ceramic. In addition, we propose electrotherapy for manipulation of microbial biofilms and to induce bone healing in peri-implantitis patients.

Three-Dimensional Magnetic Levitation Culture System Simulating White Adipose Tissue.

PubMed

Tseng, Hubert; Daquinag, Alexes C; Souza, Glauco R; Kolonin, Mikhail G

2018-01-01

White adipose tissue (WAT) has attracted interest for tissue engineering and cell-based therapies as an abundant source of adipose stem/stromal cells (ASC). However, technical challenges in WAT cell culture have limited its applications in regenerative medicine. Traditional two-dimensional (2D) cell culture models, which are essentially monolayers of cells on glass or plastic substrates, inadequately represent tissue architecture, biochemical concentration gradients, substrate stiffness, and most importantly for WAT research, cell phenotypic heterogeneity. Physiological cell culture platforms for WAT modeling must recapitulate the native diversity of cell types and their coordination within the organ. For this purpose, we developed a three-dimensional (3D) model using magnetic levitation. Here, we describe our protocol that we successfully employed to build adipose tissue organoids (adipospheres) that preserve the heterogeneity of the constituent cell types in vitro. We demonstrate the capacity of assembling adipospheres from multiple cell types, including ASCs, endohtelial cells, and leukocytes that recreate tissue organization. These adipospheres mimicked WAT organogenesis in that they enabled the formation of vessel-like endothelial structures with lumens and differentiation of unilocular adipocytes. Altogether, magnetic levitation is a cell culture platform that recreates tissue structure, function, and heterogeneity in vitro, and serves as a foundation for high-throughput WAT tissue culture and analysis.

Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation.

PubMed

Scheidegger, F; Ellner, Y; Guye, P; Rhomberg, T A; Weber, H; Augustin, H G; Dehio, C

2009-07-01

The zoonotic pathogen Bartonella henselae (Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.

Prevalidation of a human cornea construct as an alternative to animal corneas for in vitro drug absorption studies.

PubMed

Hahne, Matthias; Zorn-Kruppa, Michaela; Guzman, Gustavo; Brandner, Johanna M; Haltner-Ukomado, Eleonore; Wätzig, Hermann; Reichl, Stephan

2012-08-01

The use of ophthalmic drugs has increased consistently over the past few decades. Currently, most research is conducted using in vivo and ex vivo animal experiments; however, they have many disadvantages, including ethical concerns, high costs, the questionable extension of animal results to humans, and poor standardization. Although several cell culture-based cornea models have been developed, none have been validated and accepted for general use. In this study, a standardized, three-dimensional model of the human cornea (Hemicornea, HC) based on immortalized human corneal cells and cultivated in serum-free conditions was developed for drug absorption studies and prevalidated using compounds with a wide range of molecular characteristics (sodium fluorescein, rhodamine B, fluorescein isothiocyanate-labeled dextran, aciclovir, bimatoprost, dexamethasone, and timolol maleate). The HC model was independently cultured in three different laboratories, and the intralaboratory and interlaboratory reproducibility was analyzed and compared with the rabbit cornea. This analysis showed that the HC has a barrier in the same range as excised animal corneas, although with a higher reproducibility and lower variability. Because of the demonstrated transferability, the HC represents a promising in vitro alternative to the use of ex vivo tissue and offers a well-defined and standardized system for drug absorption studies. Copyright © 2012 Wiley Periodicals, Inc.

Structural diversity in the dandelion (Taraxacum officinale) polyphenol oxidase family results in different responses to model substrates.

PubMed

Dirks-Hofmeister, Mareike E; Singh, Ratna; Leufken, Christine M; Inlow, Jennifer K; Moerschbacher, Bruno M

2014-01-01

Polyphenol oxidases (PPOs) are ubiquitous type-3 copper enzymes that catalyze the oxygen-dependent conversion of o-diphenols to the corresponding quinones. In most plants, PPOs are present as multiple isoenzymes that probably serve distinct functions, although the precise relationship between sequence, structure and function has not been addressed in detail. We therefore compared the characteristics and activities of recombinant dandelion PPOs to gain insight into the structure-function relationships within the plant PPO family. Phylogenetic analysis resolved the 11 isoenzymes of dandelion into two evolutionary groups. More detailed in silico and in vitro analyses of four representative PPOs covering both phylogenetic groups were performed. Molecular modeling and docking predicted differences in enzyme-substrate interactions, providing a structure-based explanation for grouping. One amino acid side chain positioned at the entrance to the active site (position HB2+1) potentially acts as a "selector" for substrate binding. In vitro activity measurements with the recombinant, purified enzymes also revealed group-specific differences in kinetic parameters when the selected PPOs were presented with five model substrates. The combination of our enzyme kinetic measurements and the in silico docking studies therefore indicate that the physiological functions of individual PPOs might be defined by their specific interactions with different natural substrates.

Brief Report: Human Acute Myeloid Leukemia Reprogramming to Pluripotency Is a Rare Event and Selects for Patient Hematopoietic Cells Devoid of Leukemic Mutations.

PubMed

Lee, Jong-Hee; Salci, Kyle R; Reid, Jennifer C; Orlando, Luca; Tanasijevic, Borko; Shapovalova, Zoya; Bhatia, Mickie

2017-09-01

Induced pluripotent stem cell reprogramming has provided critical insights into disease processes by modeling the genetics and related clinical pathophysiology. Human cancer represents highly diverse genetics, as well as inter- and intra-patient heterogeneity, where cellular model systems capable of capturing this disease complexity would be invaluable. Acute myeloid leukemia (AML) represents one of most heterogeneous cancers and has been divided into genetic subtypes correlated with unique risk stratification over the decades. Here, we report our efforts to induce pluripotency from the heterogeneous population of human patients that represents this disease in the clinic. Using robust optimized reprogramming methods, we demonstrate that reprogramming of AML cells harboring leukemic genomic aberrations is a rare event with the exception of those with de novo mixed-lineage leukemia (MLL) mutations that can be reprogrammed and model drug responses in vitro. Our findings indicate that unlike hematopoietic cells devoid of genomic aberrations, AML cells harboring driver mutations are refractory to reprogramming. Expression of MLL fusion proteins in AML cells did not contribute to induced reprogramming success, which continued to select for patient derived cells devoid of AML patient-specific aberrations. Our study reveals that unanticipated blockades to achieving pluripotency reside within the majority of transformed AML patient cells. Stem Cells 2017;35:2095-2102. © 2017 AlphaMed Press.

Predictive Modeling of Chemical Hazard by Integrating Numerical Descriptors of Chemical Structures and Short-term Toxicity Assay Data

PubMed Central

Rusyn, Ivan; Sedykh, Alexander; Guyton, Kathryn Z.; Tropsha, Alexander

2012-01-01

Quantitative structure-activity relationship (QSAR) models are widely used for in silico prediction of in vivo toxicity of drug candidates or environmental chemicals, adding value to candidate selection in drug development or in a search for less hazardous and more sustainable alternatives for chemicals in commerce. The development of traditional QSAR models is enabled by numerical descriptors representing the inherent chemical properties that can be easily defined for any number of molecules; however, traditional QSAR models often have limited predictive power due to the lack of data and complexity of in vivo endpoints. Although it has been indeed difficult to obtain experimentally derived toxicity data on a large number of chemicals in the past, the results of quantitative in vitro screening of thousands of environmental chemicals in hundreds of experimental systems are now available and continue to accumulate. In addition, publicly accessible toxicogenomics data collected on hundreds of chemicals provide another dimension of molecular information that is potentially useful for predictive toxicity modeling. These new characteristics of molecular bioactivity arising from short-term biological assays, i.e., in vitro screening and/or in vivo toxicogenomics data can now be exploited in combination with chemical structural information to generate hybrid QSAR–like quantitative models to predict human toxicity and carcinogenicity. Using several case studies, we illustrate the benefits of a hybrid modeling approach, namely improvements in the accuracy of models, enhanced interpretation of the most predictive features, and expanded applicability domain for wider chemical space coverage. PMID:22387746

PG545 enhances anti-cancer activity of chemotherapy in ovarian models and increases surrogate biomarkers such as VEGF in preclinical and clinical plasma samples.

PubMed

Winterhoff, Boris; Freyer, Luisa; Hammond, Edward; Giri, Shailendra; Mondal, Susmita; Roy, Debarshi; Teoman, Attila; Mullany, Sally A; Hoffmann, Robert; von Bismarck, Antonia; Chien, Jeremy; Block, Matthew S; Millward, Michael; Bampton, Darryn; Dredge, Keith; Shridhar, Viji

2015-05-01

Despite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer. PG545's anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models. PG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response. Our results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

A microengineered pathophysiological model of early-stage breast cancer.

PubMed

Choi, Yoonseok; Hyun, Eunjeh; Seo, Jeongyun; Blundell, Cassidy; Kim, Hee Chan; Lee, Eunhee; Lee, Su Hyun; Moon, Aree; Moon, Woo Kyung; Huh, Dongeun

2015-08-21

A mounting body of evidence in cancer research suggests that the local microenvironment of tumor cells has a profound influence on cancer progression and metastasis. In vitro studies on the tumor microenvironment and its pharmacological modulation, however, are often hampered by the technical challenges associated with creating physiological cell culture environments that integrate cancer cells with the key components of their native niche such as neighboring cells and extracellular matrix (ECM) to mimic complex microarchitecture of cancerous tissue. Using early-stage breast cancer as a model disease, here we describe a biomimetic microengineering strategy to reconstitute three-dimensional (3D) structural organization and microenvironment of breast tumors in human cell-based in vitro models. Specifically, we developed a microsystem that enabled co-culture of breast tumor spheroids with human mammary ductal epithelial cells and mammary fibroblasts in a compartmentalized 3D microfluidic device to replicate microarchitecture of breast ductal carcinoma in situ (DCIS). We also explored the potential of this breast cancer-on-a-chip system as a drug screening platform by evaluating the efficacy and toxicity of an anticancer drug (paclitaxel). Our microengineered disease model represents the first critical step towards recapitulating pathophysiological complexity of breast cancer, and may serve as an enabling tool to systematically examine the contribution of the breast cancer microenvironment to the progression of DCIS to an invasive form of the disease.

Molecular mechanism for generation of antibody memory.

PubMed

Shivarov, Velizar; Shinkura, Reiko; Doi, Tomomitsu; Begum, Nasim A; Nagaoka, Hitoshi; Okazaki, Il-Mi; Ito, Satomi; Nonaka, Taichiro; Kinoshita, Kazuo; Honjo, Tasuku

2009-03-12

Activation-induced cytidine deaminase (AID) is the essential enzyme inducing the DNA cleavage required for both somatic hypermutation and class switch recombination (CSR) of the immunoglobulin gene. We originally proposed the RNA-editing model for the mechanism of DNA cleavage by AID. We obtained evidence that fulfils three requirements for CSR by this model, namely (i) AID shuttling between nucleus and cytoplasm, (ii) de novo protein synthesis for CSR, and (iii) AID-RNA complex formation. The alternative hypothesis, designated as the DNA-deamination model, assumes that the in vitro DNA deamination activity of AID is representative of its physiological function in vivo. Furthermore, the resulting dU was removed by uracil DNA glycosylase (UNG) to generate a basic site, followed by phosphodiester bond cleavage by AP endonuclease. We critically examined each of these provisional steps. We identified a cluster of mutants (H48A, L49A, R50A and N51A) that had particularly higher CSR activities than expected from their DNA deamination activities. The most striking was the N51A mutant that had no ability to deaminate DNA in vitro but retained approximately 50 per cent of the wild-type level of CSR activity. We also provide further evidence that UNG plays a non-canonical role in CSR, namely in the repair step of the DNA breaks. Taking these results together, we favour the RNA-editing model for the function of AID in CSR.

Characterisation of the porcine eyeball as an in-vitro model for dry eye.

PubMed

Menduni, Francesco; Davies, Leon N; Madrid-Costa, D; Fratini, Antonio; Wolffsohn, James S

2018-02-01

To characterise the anatomical parameters of the porcine eye for potentially using it as a laboratory model of dry eye. Anterior chamber depth and angle, corneal curvature, shortest and longest diameter, endothelial cell density, and pachymetry were measured in sixty freshly enucleated porcine eyeballs. Corneal steepest meridian was 7.85±0.32mm, corneal flattest meridian was 8.28±0.32mm, shortest corneal diameter was 12.69±0.58mm, longest corneal diameter was 14.88±0.66mm and central corneal ultrasonic pachymetry was 1009±1μm. Anterior chamber angle was 28.83±4.16°, anterior chamber depth was 1.77±0.27mm, and central corneal thickness measured using OCT was 1248±144μm. Corneal endothelial cell density was 3250±172 cells/mm 2 . Combining different clinical techniques produced a pool of reproducible data on the porcine eye anatomy, which can be used by researchers to assess the viability of using the porcine eye as an in-vitro/ex-vivo model for dry eye. Due to the similar morphology with the human eye, porcine eyeballs may represent a useful and cost effective model to individually study important key factors in the development of dry eye, such as environmental and mechanical stresses. Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.

Performance of dry powder inhalers with single dosed capsules in preschool children and adults using improved upper airway models.

PubMed

Lindert, Sandra; Below, Antje; Breitkreutz, Joerg

2014-02-06

The pulmonary administration of pharmaceutical aerosols to patients is affected by age-dependent variations in the anatomy of the upper airways and the inhalation pattern. Considering this aspect, different upper airway models, representing the geometries of adults and preschool children, and a conventional induction port according to the European Pharmacopeia were used for in vitro testing of dry powder inhalers with single dosed capsules (Cyclohaler®, Handihaler® and Spinhaler®). Deposition measurements were performed using steady flow rates of 30 and 60 L/min for the Handihaler®/Spinhaler® and 30, 60 and 75 L/min for the Cyclohaler®. The inhalation volume was set at 1 L. For the Cyclohaler®, the in vitro testing was supplemented by a pediatric inhalation profile. Slight differences of pulmonary deposition between the idealized adult (11%-15%) and pediatric (9%-11%) upper airway model were observed for the Cyclohaler®. The applied pediatric inhalation profile resulted in a reduction of pulmonary deposition by 5% compared to steady conditions and indicated the influence of the inhalation pattern on the amount of pulmonary deposited particles. The comparison of two pediatric upper airway models showed no differences. The performance of the Handihaler® was similar to the Cyclohaler®. The Spinhaler® showed an insufficient performance and limited reproducibility in our investigations.

On the Relative Relevance of Subject-Specific Geometries and Degeneration-Specific Mechanical Properties for the Study of Cell Death in Human Intervertebral Disk Models

PubMed Central

Malandrino, Andrea; Pozo, José M.; Castro-Mateos, Isaac; Frangi, Alejandro F.; van Rijsbergen, Marc M.; Ito, Keita; Wilke, Hans-Joachim; Dao, Tien Tuan; Ho Ba Tho, Marie-Christine; Noailly, Jérôme

2015-01-01

Capturing patient- or condition-specific intervertebral disk (IVD) properties in finite element models is outmost important in order to explore how biomechanical and biophysical processes may interact in spine diseases. However, disk degenerative changes are often modeled through equations similar to those employed for healthy organs, which might not be valid. As for the simulated effects of degenerative changes, they likely depend on specific disk geometries. Accordingly, we explored the ability of continuum tissue models to simulate disk degenerative changes. We further used the results in order to assess the interplay between these simulated changes and particular IVD morphologies, in relation to disk cell nutrition, a potentially important factor in disk tissue regulation. A protocol to derive patient-specific computational models from clinical images was applied to different spine specimens. In vitro, IVD creep tests were used to optimize poro-hyperelastic input material parameters in these models, in function of the IVD degeneration grade. The use of condition-specific tissue model parameters in the specimen-specific geometrical models was validated against independent kinematic measurements in vitro. Then, models were coupled to a transport-cell viability model in order to assess the respective effects of tissue degeneration and disk geometry on cell viability. While classic disk poro-mechanical models failed in representing known degenerative changes, additional simulation of tissue damage allowed model validation and gave degeneration-dependent material properties related to osmotic pressure and water loss, and to increased fibrosis. Surprisingly, nutrition-induced cell death was independent of the grade-dependent material properties, but was favored by increased diffusion distances in large IVDs. Our results suggest that in situ geometrical screening of IVD morphology might help to anticipate particular mechanisms of disk degeneration. PMID:25717471

A device for emulating cuff recordings of action potentials propagating along peripheral nerves.

PubMed

Rieger, Robert; Schuettler, Martin; Chuang, Sheng-Chih

2014-09-01

This paper describes a device that emulates propagation of action potentials along a peripheral nerve, suitable for reproducible testing of bio-potential recording systems using nerve cuff electrodes. The system is a microcontroller-based stand-alone instrument which uses established nerve and electrode models to represent neural activity of real nerves recorded with a nerve cuff interface, taking into consideration electrode impedance, voltages picked up by the electrodes, and action potential propagation characteristics. The system emulates different scenarios including compound action potentials with selectable propagation velocities and naturally occurring nerve traffic from different velocity fiber populations. Measured results from a prototype implementation are reported and compared with in vitro recordings from Xenopus Laevis frog sciatic nerve, demonstrating that the electrophysiological setting is represented to a satisfactory degree, useful for the development, optimization and characterization of future recording systems.

Violacein, an indole-derived purple-colored natural pigment produced by Janthinobacterium lividum, inhibits the growth of head and neck carcinoma cell lines both in vitro and in vivo.

PubMed

Masuelli, Laura; Pantanella, Fabrizio; La Regina, Giuseppe; Benvenuto, Monica; Fantini, Massimo; Mattera, Rosanna; Di Stefano, Enrica; Mattei, Maurizio; Silvestri, Romano; Schippa, Serena; Manzari, Vittorio; Modesti, Andrea; Bei, Roberto

2016-03-01

Violacein (VIO; 3-[1,2-dihydro-5-(5-hydroxy-1H-indol-3-yl)-2-oxo-3H-pyrrol-3-ylidene]-1,3-dihydro-2H-indol-2-one), an indole-derived purple-colored pigment, produced by a limited number of Gram-negative bacteria species, including Chromobacterium violaceum and Janthinobacterium lividum, has been demonstrated to have anti-cancer activity, as it interferes with survival transduction signaling pathways in different cancer models. Head and neck carcinoma (HNC) represents the sixth most common and one of the most fatal cancers worldwide. We determined whether VIO was able to inhibit head and neck cancer cell growth both in vitro and in vivo. We provide evidence that VIO treatment of human and mouse head and neck cancer cell lines inhibits cell growth and induces autophagy and apoptosis. In fact, VIO treatment increased PARP-1 cleavage, the Bax/Bcl-2 ratio, the inhibition of ERK1 and ERK2 phosphorylation, and the expression of light chain 3-II (LC3-II). Moreover, VIO was able to induce p53 degradation, cytoplasmic nuclear factor kappa B (NF-κB) accumulation, and reactive oxygen species (ROS) production. VIO induced a significant increase in ROS production. VIO administration was safe in BALB/c mice and reduced the growth of transplanted salivary gland cancer cells (SALTO) in vivo and prolonged median survival. Taken together, our results indicate that the treatment of head and neck cancer cells with VIO can be useful in inhibiting in vivo and in vitro cancer cell growth. VIO may represent a suitable tool for the local treatment of HNC in combination with standard therapies.

A method to improve the efficacy of topical eflornithine hydrochloride cream.

PubMed

Kumar, Amit; Naguib, Youssef W; Shi, Yan-Chun; Cui, Zhengrong

2016-06-01

Facial hirsutism is a cosmetic concern for women and can lead to significant anxiety and lack of self-esteem. Eflornithine cream is indicated for the treatment of facial hirsutism. However, limited success rate and overall patient's satisfaction, even with a long-term and high-frequency application, leave room for improvement. The objective of this study is to test the effect of microneedle treatment on the in vitro skin permeation and the in vivo efficacy of eflornithine cream in a mouse model. In vitro permeation study of eflornithine was performed using Franz diffusion cell. In vivo efficacy study was performed in a mouse model by monitoring the re-growth of hair in the lower dorsal skin of mice after the eflornithine cream was applied onto an area pretreated with microneedles. The skin and the hair follicles in the treated area were also examined histologically. The hair growth inhibitory activity of eflornithine was significantly enhanced when the eflornithine cream was applied onto a mouse skin area pretreated with microneedles, most likely because the micropores created by microneedles allowed the permeation of eflornithine into the skin, as confirmed in an in vitro permeation study. Immunohistochemistry data revealed that cell proliferation in the skin and hair follicles was also significantly inhibited when the eflornithine cream was applied onto a skin area pretreated with microneedles. The integration of microneedle treatment into topical eflornithine therapy represents a potentially viable approach to increase eflornithine's ability to inhibit hair growth.

In vitro perturbations of targets in cancer hallmark processes predict rodent chemical carcinogenesis.

PubMed

Kleinstreuer, Nicole C; Dix, David J; Houck, Keith A; Kavlock, Robert J; Knudsen, Thomas B; Martin, Matthew T; Paul, Katie B; Reif, David M; Crofton, Kevin M; Hamilton, Kerry; Hunter, Ronald; Shah, Imran; Judson, Richard S

2013-01-01

Thousands of untested chemicals in the environment require efficient characterization of carcinogenic potential in humans. A proposed solution is rapid testing of chemicals using in vitro high-throughput screening (HTS) assays for targets in pathways linked to disease processes to build models for priority setting and further testing. We describe a model for predicting rodent carcinogenicity based on HTS data from 292 chemicals tested in 672 assays mapping to 455 genes. All data come from the EPA ToxCast project. The model was trained on a subset of 232 chemicals with in vivo rodent carcinogenicity data in the Toxicity Reference Database (ToxRefDB). Individual HTS assays strongly associated with rodent cancers in ToxRefDB were linked to genes, pathways, and hallmark processes documented to be involved in tumor biology and cancer progression. Rodent liver cancer endpoints were linked to well-documented pathways such as peroxisome proliferator-activated receptor signaling and TP53 and novel targets such as PDE5A and PLAUR. Cancer hallmark genes associated with rodent thyroid tumors were found to be linked to human thyroid tumors and autoimmune thyroid disease. A model was developed in which these genes/pathways function as hypothetical enhancers or promoters of rat thyroid tumors, acting secondary to the key initiating event of thyroid hormone disruption. A simple scoring function was generated to identify chemicals with significant in vitro evidence that was predictive of in vivo carcinogenicity in different rat tissues and organs. This scoring function was applied to an external test set of 33 compounds with carcinogenicity classifications from the EPA's Office of Pesticide Programs and successfully (p = 0.024) differentiated between chemicals classified as "possible"/"probable"/"likely" carcinogens and those designated as "not likely" or with "evidence of noncarcinogenicity." This model represents a chemical carcinogenicity prioritization tool supporting targeted testing and functional validation of cancer pathways.

New Aspects of the Pathogenesis of Canine Distemper Leukoencephalitis

PubMed Central

Lempp, Charlotte; Spitzbarth, Ingo; Puff, Christina; Cana, Armend; Kegler, Kristel; Techangamsuwan, Somporn; Baumgärtner, Wolfgang; Seehusen, Frauke

2014-01-01

Canine distemper virus (CDV) is a member of the genus morbillivirus, which is known to cause a variety of disorders in dogs including demyelinating leukoencephalitis (CDV-DL). In recent years, substantial progress in understanding the pathogenetic mechanisms of CDV-DL has been made. In vivo and in vitro investigations provided new insights into its pathogenesis with special emphasis on axon-myelin-glia interaction, potential endogenous mechanisms of regeneration, and astroglial plasticity. CDV-DL is characterized by lesions with a variable degree of demyelination and mononuclear inflammation accompanied by a dysregulated orchestration of cytokines as well as matrix metalloproteinases and their inhibitors. Despite decades of research, several new aspects of the neuropathogenesis of CDV-DL have been described only recently. Early axonal damage seems to represent an initial and progressive lesion in CDV-DL, which interestingly precedes demyelination. Axonopathy may, thus, function as a potential trigger for subsequent disturbed axon-myelin-glia interactions. In particular, the detection of early axonal damage suggests that demyelination is at least in part a secondary event in CDV-DL, thus challenging the dogma of CDV as a purely primary demyelinating disease. Another unexpected finding refers to the appearance of p75 neurotrophin (NTR)-positive bipolar cells during CDV-DL. As p75NTR is a prototype marker for immature Schwann cells, this finding suggests that Schwann cell remyelination might represent a so far underestimated endogenous mechanism of regeneration, though this hypothesis still remains to be proven. Although it is well known that astrocytes represent the major target of CDV infection in CDV-DL, the detection of infected vimentin-positive astrocytes in chronic lesions indicates a crucial role of this cell population in nervous distemper. While glial fibrillary acidic protein represents the characteristic intermediate filament of mature astrocytes, expression of vimentin is generally restricted to immature or reactive astrocytes. Thus, vimentin-positive astrocytes might constitute an important cell population for CDV persistence and spread, as well as lesion progression. In vitro models, such as dissociated glial cell cultures, as well as organotypic brain slice cultures have contributed to a better insight into mechanisms of infection and certain morphological and molecular aspects of CDV-DL. Summarized, recent in vivo and in vitro studies revealed remarkable new aspects of nervous distemper. These new perceptions substantially improved our understanding of the pathogenesis of CDV-DL and might represent new starting points to develop novel treatment strategies. PMID:24992230

Toxicokinetic and Dosimetry Modeling Tools for Exposure ...

EPA Pesticide Factsheets

New technologies and in vitro testing approaches have been valuable additions to risk assessments that have historically relied solely on in vivo test results. Compared to in vivo methods, in vitro high throughput screening (HTS) assays are less expensive, faster and can provide mechanistic insights on chemical action. However, extrapolating from in vitro chemical concentrations to target tissue or blood concentrations in vivo is fraught with uncertainties, and modeling is dependent upon pharmacokinetic variables not measured in in vitro assays. To address this need, new tools have been created for characterizing, simulating, and evaluating chemical toxicokinetics. Physiologically-based pharmacokinetic (PBPK) models provide estimates of chemical exposures that produce potentially hazardous tissue concentrations, while tissue microdosimetry PK models relate whole-body chemical exposures to cell-scale concentrations. These tools rely on high-throughput in vitro measurements, and successful methods exist for pharmaceutical compounds that determine PK from limited in vitro measurements and chemical structure-derived property predictions. These high throughput (HT) methods provide a more rapid and less resource–intensive alternative to traditional PK model development. We have augmented these in vitro data with chemical structure-based descriptors and mechanistic tissue partitioning models to construct HTPBPK models for over three hundred environmental and pharmace

FOXD3 suppresses tumor growth and angiogenesis in non-small cell lung cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Jun-Hai; Zhao, Chun-Liu; Ding, Lan-Bao

2015-10-09

The transcription factor forkhead box D3 (FOXD3), widely studied as a transcriptional repressor in embryogenesis, participates in the carcinogenesis of many cancers. However, the expression pattern and role of FOXD3 in non-small cell lung cancer (NSCLC) have not been well characterized. We report that FOXD3 is significantly downregulated in NSCLC cell lines and clinical tissues. FOXD3 overexpression significantly inhibits cell growth and results in G1 cell cycle arrest in NSCLC A549 and H1299 cells. In a xenograft tumor model, FOXD3 overexpression inhibits tumor growth and angiogenesis. Remarkably, expression of vascular endothelial growth factor (VEGF) was reduced in FOXD3 overexpression models bothmore » in vitro and in vivo. These findings suggest that FOXD3 plays a potential tumor suppressor role in NSCLC progression and represents a promising clinical prognostic marker and therapeutic target for this disease. - Highlights: • FOXD3 is downregulated in NSCLC cell lines and tissues. • FOXD3 overexpression inhibited cell proliferation in NSCLC cells. • FOXD3 overexpression led to decreased angiogenesis in NSCLC cells in vitro and in vivo.« less

Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration.

PubMed

Urciuolo, Anna; Urbani, Luca; Perin, Silvia; Maghsoudlou, Panagiotis; Scottoni, Federico; Gjinovci, Asllan; Collins-Hooper, Henry; Loukogeorgakis, Stavros; Tyraskis, Athanasios; Torelli, Silvia; Germinario, Elena; Fallas, Mario Enrique Alvarez; Julia-Vilella, Carla; Eaton, Simon; Blaauw, Bert; Patel, Ketan; De Coppi, Paolo

2018-05-30

Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis.

Development of an in vitro cell culture model to investigate the induction and quantification of oxidative stress and its inhibition by alpha-tocopherol.

PubMed

Lawlor, S M; O'Brien, N M

1994-02-01

The ability of the natural antioxidant alpha-tocopherol to protect against oxidative stress in vitro was assessed. Primary cultures of chicken embryo fibroblasts (CEF) were oxidatively stressed by exposure to paraquat (PQ). Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured as indices of oxidative stress. CEF incubated with 0.125-1.0 mm PQ for 18 hr exhibited increased SOD activity (P < 0.05). CAT activity increased with 0.25 mm PQ (P < 0.05). GSH-Px activity decreased significantly in the presence of PQ. No cytotoxicity, as indicated by lactate dehydrogenase release, was observed at PQ concentrations below 2 mm. Incorporation of added alpha-tocopherol (100 nm) into 0.25 mm PQ-treated CEF resulted in SOD activity not significantly different from that observed in control cells not treated with PQ. Lower levels of added alpha-tocopherol (16 nm) returned CAT to its control value. However, even at 1000 nm alpha-tocopherol, GSH-Px activity was not protected in PQ-treated cells. CEF represent a useful model to study both inducers and inhibitors of oxidative stress.

Antiviral effect of vitamin A on norovirus infection via modulation of the gut microbiome

PubMed Central

Lee, Heetae; Ko, GwangPyo

2016-01-01

The effect and underlying mechanism of vitamin A on norovirus infection are largely unknown. This study aimed to investigate how vitamin A administration affects the gut microbiome after norovirus infection. Here, we demonstrate that treatment with either retinol or retinoic acid (RA) inhibits murine norovirus (MNV) replication using both in vitro and in vivo models. Compositional changes in the gut microbiome associated with RA administration and/or norovirus infection were also investigated. Oral administration of RA and/or MNV significantly altered intestinal microbiome profiles. Particularly, bacterial species belonging to the Lactobacillaceae families were remarkably increased by MNV inoculation and RA administration, suggesting that the antiviral effects of RA occur via the modulation of specific microbiota. The antiviral causal effect of Lactobacillus was identified and demonstrated using in vitro models in RAW264.7 cells. The antiviral immune response to MNV was mediated by IFN-β upregulation. This study represents the first comprehensive profiling of gut microbiota in response to RA treatment against norovirus infection. Moreover, we conclude that the abundance of Lactobacillus through gut microbiota modulation by RA is at least partially responsible for norovirus inhibition. PMID:27180604

Biofilm disruption with rotating microrods enhances antimicrobial efficacy

NASA Astrophysics Data System (ADS)

Mair, Lamar O.; Nacev, Aleksandar; Hilaman, Ryan; Stepanov, Pavel Y.; Chowdhury, Sagar; Jafari, Sahar; Hausfeld, Jeffrey; Karlsson, Amy J.; Shirtliff, Mark E.; Shapiro, Benjamin; Weinberg, Irving N.

2017-04-01

Biofilms are a common and persistent cause of numerous illnesses. Compared to planktonic microbes, biofilm residing cells often demonstrate significant resistance to antimicrobial agents. Thus, methods for dislodging cells from the biofilm may increase the antimicrobial susceptibility of such cells, and serve as a mechanical means of increasing antimicrobial efficacy. Using Aspergillus fumigatus as a model microbe, we magnetically rotate microrods in and around biofilm. We show that such rods can improve the efficacy of antimicrobial Amphotericin B treatments in vitro. This work represents a first step in using kinetic magnetic particle therapy for disrupting fungal biofilms.

The Blessing and the Curse of the Multiplicative Updates

NASA Astrophysics Data System (ADS)

Warmuth, Manfred K.

Multiplicative updates multiply the parameters by nonnegative factors. These updates are motivated by a Maximum Entropy Principle and they are prevalent in evolutionary processes where the parameters are for example concentrations of species and the factors are survival rates. The simplest such update is Bayes rule and we give an in vitro selection algorithm for RNA strands that implements this rule in the test tube where each RNA strand represents a different model. In one liter of the RNA "soup" there are approximately 1020 different strands and therefore this is a rather high-dimensional implementation of Bayes rule.

Discovery of a Potent, Dual Serotonin and Norepinephrine Reuptake Inhibitor

PubMed Central

2013-01-01

The objective of the described research effort was to identify a novel serotonin and norepinephrine reuptake inhibitor (SNRI) with improved norepinephrine transporter activity and acceptable metabolic stability and exhibiting minimal drug–drug interaction. We describe herein the discovery of a series of 3-substituted pyrrolidines, exemplified by compound 1. Compound 1 is a selective SNRI in vitro and in vivo, has favorable ADME properties, and retains inhibitory activity in the formalin model of pain behavior. Compound 1 thus represents a potential new probe to explore utility of SNRIs in central nervous system disorders, including chronic pain conditions. PMID:24900709

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform.

PubMed

Katt, Moriah E; Placone, Amanda L; Wong, Andrew D; Xu, Zinnia S; Searson, Peter C

2016-01-01

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment.

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform

PubMed Central

Katt, Moriah E.; Placone, Amanda L.; Wong, Andrew D.; Xu, Zinnia S.; Searson, Peter C.

2016-01-01

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment. PMID:26904541

Protective activity of guanosine in an in vitro model of Parkinson's disease.

PubMed

Giuliani, P; Romano, S; Ballerini, P; Ciccarelli, R; Petragnani, N; Cicchitti, S; Zuccarini, M; Jiang, S; Rathbone, M P; Caciagli, F; Di Iorio, P

2012-12-01

Parkinson's disease (PD) is a pathological condition characterized by a progressive neurodegeneration of dopaminergic neurons with the consequent reduction of dopamine content in the substantia nigra. The neurotoxin 6-hydroxydopamine (6-OHDA) is widely used to mimic the neuropathology of PD in both in vivo and in vitro experimental models. We found that, as expected, in dopaminergic human SH-SY5Y neuroblastoma cells the toxin reduced cell viability causing programmed cell death as assessed by an increase in DNA fragmentation. We also examined, in these cells, the activation/inactivation of several pro and anti apoptotic signaling pathways by 6-OHDA including p-38 kinase (p-38), c-Jun N-terminal kinase (JNK), protein kinase B (also known as Akt), glycogen synthase kinase-3β (GSK3β), and Bcl-2 protein. Guanine-based purines, exert neuroprotective effects and we previously reported that guanosine activates cell survival pathways including PI3K/Akt/PKB signaling in different kinds of cells including glia and neuroblastoma cells. In the present study we found that guanosine (300 µM) protected SH-SY5Y neuroblastoma cells when they were exposed to 6-OHDA, promoting their survival. Guanosine reduced the 6-OHDA mediated activation of p-38 and JNK. Moreover the nucleoside potentiated the early increase in the phosphorylation of the anti-apoptotic kinase Akt and the increase in the expression of the anti-apoptotic Bcl-2 protein induced by 6-OHDA. In summary our results show that guanosine results to be neuroprotective in a recognized in vitro model of PD thus suggesting that it could represent a new potential pharmacological tool to be studied in the therapeutic approach to PD.

In vitro cleaning potential of three different implant debridement methods.

PubMed

Sahrmann, Philipp; Ronay, Valerie; Hofer, Deborah; Attin, Thomas; Jung, Ronald E; Schmidlin, Patrick R

2015-03-01

To assess the cleaning potential of three different instrumentation methods commonly used for implant surface decontamination in vitro, using a bone defect-simulating model. Dental implants were stained with indelible ink and mounted in resin models, which represented standardized peri-implantitis defects with different bone defect angulations (30, 60 and 90°). Cleaning procedures were performed by either an experienced dental hygienist or a 2nd-year postgraduate student. The treatment was repeated 20 times for each instrumentation, that is, with a Gracey curette, an ultrasonic device and an air powder abrasive device (PAD) with glycine powder. After each run, implants were removed and images were taken to detect color remnants in order to measure planimetrically the cumulative uncleaned surface area. SEM images were taken to assess micromorphologic surface changes (magnification 10,000 ×). Results were tested for statistical differences using two-way ANOVA and Bonferroni correction. The areas of uncleaned surfaces (%, mean ± standard deviations) for curettes, ultrasonic tips, and airflow accounted for 24.1 ± 4.8%, 18.5 ± 3.8%, and 11.3 ± 5.4%, respectively. These results were statistically significantly different (P < 0.0001). The cleaning potential of the airflow device increased with wider defects. SEM evaluation displayed distinct surface alterations after instrumentation with steel tips, whereas glycine powder instrumentation had only a minute effect on the surface topography. Within the limitations of the present in vitro model, airflow devices using glycine powders seem to constitute an efficient therapeutic option for the debridement of implants in peri-implantitis defects. Still, some uncleaned areas remained. In wide defects, differences between instruments are more accentuated. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Co-culture of stromal and erythroleukemia cells in a perfused hollow fiber bioreactor system as an in vitro bone marrow model for myeloid leukemia.

PubMed

Usuludin, Suaidah Binte Mohamed; Cao, Xue; Lim, Mayasari

2012-05-01

We have developed a hematopoietic co-culture system using the hollow fiber bioreactor (HFBR) as a potential in vitro bone marrow model for evaluating leukemia. Supporting stroma using HS-5 cells was established in HFBR system and the current bioprocess configuration yielded an average glucose consumption of 640 mg/day and an average protein concentration of 6.40 mg/mL in the extracapillary space over 28 days. Co-culture with erythroleukemia K562 cells was used as a model for myelo-leukemic cell proliferation and differentiation. Two distinct localizations of K562 cells (loosely adhered and adherent cells) were identified and characterized after 2 weeks. The HFBR co-culture resulted in greater leukemic cell expansion (3,130 fold vs. 43 fold) compared to a standard tissue culture polystyrene (TCP) culture. Majority of expanded cells (68%) in HFBR culture were the adherent population, highlighting the importance of cell-cell contact for myelo-leukemic proliferation. Differentiation tendencies in TCP favored maturation toward monocyte and erythrocyte lineages but maintained a pool of myeloid progenitors. In contrast, HFBR co-culture exhibited greater lineage diversity, stimulating monocytic and megakaryocytic differentiation while inhibiting erythroid maturation. With the extensive stromal expansion capacity on hollow fiber surfaces, the HFBR system is able to achieve high cell densities and 3D cell-cell contacts mimicking the bone marrow microenvironment. The proposed in vitro system represents a dynamic and highly scalable 3D co-culture platform for the study of cell-stroma dependent hematopoietic/leukemic cell functions and ex vivo expansion. Copyright © 2011 Wiley Periodicals, Inc.

Tadalafil modulates aromatase activity and androgen receptor expression in a human osteoblastic cell in vitro model.

PubMed

Aversa, A; Fittipaldi, S; Bimonte, V M; Wannenes, F; Papa, V; Francomano, D; Greco, E A; Lenzi, A; Migliaccio, S

2016-02-01

Phosphodiesterase type-5 inhibitor (PDE5i) tadalafil administration in men with erectile dysfunction is associated with increased testosterone/estradiol ratio, leading to hypothesize a potential increased effect of androgen action on target tissues. We aimed to characterize, in a cellular model system in vitro, the potential modulation of aromatase and sex steroid hormone receptors upon exposure to tadalafil (TAD). Human osteoblast-like cells SAOS-2 were chosen as an in vitro model system since osteoblasts are target of steroid hormones. Cells were tested for viability upon TAD exposure, which increased cell proliferation. Then, cells were treated with/without TAD for several times to evaluate potential modulation in PDE5, aromatase (ARO), androgen (AR) and estrogen (ER) receptor expression. Osteoblasts express significant levels of both PDE5 mRNA and protein. Exposure of cells to increasing concentrations of TAD (10(-8)-10(-7) M) decreased PDE5 mRNA and protein expression. Also, TAD inhibited ARO mRNA and protein expression leading to an increase in testosterone levels in the supernatants. Interestingly, TAD increased total AR mRNA and protein expression and decreased ERα, with an increased ratio of AR/ER, suggesting preferential androgenic vs estrogenic pathway activation. Our results demonstrate for the first time that TAD decreases ARO expression and increases AR protein expression in human SAOS-2, strongly suggesting a new control of steroid hormones pathway by PDE5i. These findings might represent the first evidence of translational actions of PDE5i on AR, which leads to hypothesize a growing relevance of this molecule in men with prostate cancer long-term treated with TAD for sexual rehabilitation.

Mind the Gap! A Journey towards Computational Toxicology.

PubMed

Mangiatordi, Giuseppe Felice; Alberga, Domenico; Altomare, Cosimo Damiano; Carotti, Angelo; Catto, Marco; Cellamare, Saverio; Gadaleta, Domenico; Lattanzi, Gianluca; Leonetti, Francesco; Pisani, Leonardo; Stefanachi, Angela; Trisciuzzi, Daniela; Nicolotti, Orazio

2016-09-01

Computational methods have advanced toxicology towards the development of target-specific models based on a clear cause-effect rationale. However, the predictive potential of these models presents strengths and weaknesses. On the good side, in silico models are valuable cheap alternatives to in vitro and in vivo experiments. On the other, the unconscious use of in silico methods can mislead end-users with elusive results. The focus of this review is on the basic scientific and regulatory recommendations in the derivation and application of computational models. Attention is paid to examine the interplay between computational toxicology and drug discovery and development. Avoiding the easy temptation of an overoptimistic future, we report our view on what can, or cannot, realistically be done. Indeed, studies of safety/toxicity represent a key element of chemical prioritization programs carried out by chemical industries, and primarily by pharmaceutical companies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CdSe magic-sized quantum dots incorporated in biomembrane models at the air-water interface composed of components of tumorigenic and non-tumorigenic cells.

PubMed

Goto, Thiago E; Lopes, Carla C; Nader, Helena B; Silva, Anielle C A; Dantas, Noelio O; Siqueira, José R; Caseli, Luciano

2016-07-01

Cadmium selenide (CdSe) magic-sized quantum dots (MSQDs) are semiconductor nanocrystals with stable luminescence that are feasible for biomedical applications, especially for in vivo and in vitro imaging of tumor cells. In this work, we investigated the specific interaction of CdSe MSQDs with tumorigenic and non-tumorigenic cells using Langmuir monolayers and Langmuir-Blodgett (LB) films of lipids as membrane models for diagnosis of cancerous cells. Surface pressure-area isotherms and polarization modulation reflection-absorption spectroscopy (PM-IRRAS) showed an intrinsic interaction between the quantum dots, inserted in the aqueous subphase, and Langmuir monolayers constituted either of selected lipids or of tumorigenic and non-tumorigenic cell extracts. The films were transferred to solid supports to obtain microscopic images, providing information on their morphology. Similarity between films with different compositions representing cell membranes, with or without the quantum dots, was evaluated by atomic force microscopy (AFM) and confocal microscopy. This study demonstrates that the affinity of quantum dots for models representing cancer cells permits the use of these systems as devices for cancer diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Discriminating model for diagnosis of basal cell carcinoma and melanoma in vitro based on the Raman spectra of selected biochemicals

NASA Astrophysics Data System (ADS)

Silveira, Landulfo; Silveira, Fabrício Luiz; Bodanese, Benito; Zângaro, Renato Amaro; Pacheco, Marcos Tadeu T.

2012-07-01

Raman spectroscopy has been employed to identify differences in the biochemical constitution of malignant [basal cell carcinoma (BCC) and melanoma (MEL)] cells compared to normal skin tissues, with the goal of skin cancer diagnosis. We collected Raman spectra from compounds such as proteins, lipids, and nucleic acids, which are expected to be represented in human skin spectra, and developed a linear least-squares fitting model to estimate the contributions of these compounds to the tissue spectra. We used a set of 145 spectra from biopsy fragments of normal (30 spectra), BCC (96 spectra), and MEL (19 spectra) skin tissues, collected using a near-infrared Raman spectrometer (830 nm, 50 to 200 mW, and 20 s exposure time) coupled to a Raman probe. We applied the best-fitting model to the spectra of biochemicals and tissues, hypothesizing that the relative spectral contribution of each compound to the tissue Raman spectrum changes according to the disease. We verified that actin, collagen, elastin, and triolein were the most important biochemicals representing the spectral features of skin tissues. A classification model applied to the relative contribution of collagen III, elastin, and melanin using Euclidean distance as a discriminator could differentiate normal from BCC and MEL.

FAD-induced in vitro activation of glutathione reductase in the lens of B2 deficient rats.

PubMed

Ono, S; Hirano, H

1984-04-01

We studied the FAD-induced in vitro stimulation of lenticular glutathione reductase in riboflavin-deficient rats. The stimulatory effect of FAD on lenticular glutathione reductase in rats fed a B2-deficient diet for 4 weeks was remarkably higher than in paired control rats fed a B2-supplemented basal diet and control rats had ad libitum access to a B2-supplemented basal diet. The in vitro FAD stimulation effect on rat lenticular glutathione reductase represents a sensitive indicator of the B2 deficient status.

Potential uses of terahertz pulse imaging in dentistry: caries and erosion detection

NASA Astrophysics Data System (ADS)

Longbottom, Christopher; Crawley, David A.; Cole, Bryan E.; Arnone, Donald D.; Wallace, Vincent P.; Pepper, Michael

2002-06-01

TeraHertz Pulse Imaging (TPI) is a relatively new imaging modality for medical and dental imaging. The aim of the present study was to make a preliminary assessment of the potential uses of TPI in clinical dentistry, particularly in relation to caries detection and the detection and monitoring of erosion. Images were obtained in vitro using a new TPI system developed by TeraView Ltd. We present data showing that TPI in vitro images of approximal surfaces of whole teeth demonstrate a distinctive shadowing in the presence of natural carious lesions in enamel. The thickness of this enamel shadowing appears to be related to lesion depth. The use of non-ionizing radiation to image such lesions non-destructively in vitro represents a significant step towards such measurements in vivo. In addition, data is presented which indicates that TPI may have a potential role in the detection and monitoring of enamel erosion. In vitro experiments on whole incisor teeth show that TPI is capable of detecting relatively small artificially induced changes in the buccal or palatal surface of the enamel of these teeth. Imaging of enamel thickness at such a resolution without ionizing radiation would represent a significant breakthrough if applicable in vivo.

Engineering muscle cell alignment through 3D bioprinting.

PubMed

Mozetic, Pamela; Giannitelli, Sara Maria; Gori, Manuele; Trombetta, Marcella; Rainer, Alberto

2017-09-01

Processing of hydrogels represents a main challenge for the prospective application of additive manufacturing (AM) to soft tissue engineering. Furthermore, direct manufacturing of tissue precursors with a cell density similar to native tissues has the potential to overcome the extensive in vitro culture required for conventional cell-seeded scaffolds seeking to fabricate constructs with tailored structural and functional properties. In this work, we present a simple AM methodology that exploits the thermoresponsive behavior of a block copolymer (Pluronic ® ) as a means to obtain good shape retention at physiological conditions and to induce cellular alignment. Pluronic/alginate blends have been investigated as a model system for the processing of C2C12 murine myoblast cell line. Interestingly, C2C12 cell model demonstrated cell alignment along the deposition direction, potentially representing a new avenue to tailor the resulting cell histoarchitecture during AM process. Furthermore, the fabricated constructs exhibited high cell viability, as well as a significantly improved expression of myogenic genes vs. conventional 2D cultures. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2582-2588, 2017. © 2017 Wiley Periodicals, Inc.

Lattice model for self-assembly with application to the formation of cytoskeletal-like structures

NASA Astrophysics Data System (ADS)

Stewman, Shannon F.; Dinner, Aaron R.

2007-07-01

We introduce a stochastic approach for self-assembly in systems far from equilibrium. The building blocks are represented by a lattice of discrete variables (Potts-like spins), and physically meaningful mechanisms are obtained by restricting transitions through spatially local rules based on experimental data. We use the method to study nucleation of filopodia-like bundles in a system consisting of purified actin, fascin, actin-related protein 2/3 , and beads coated with Wiskott-Aldrich syndrome protein. Consistent with previous speculation based on static experimental images, we find that bundles derive from Λ -precursor-like patterns of spins on the lattice. The ratcheting of the actin network relative to the surface that represents beads plays an important role in determining the number and orientation of bundles due to the fact that branching is the primary means for generating barbed ends pointed in directions that allow rapid filament growth. By enabling the de novo formation of coexisting morphologies without the computational cost of explicit representation of proteins, the approach introduced complements earlier models of cytoskeletal behavior in vitro and in vivo.

Applicability of new degradable hypericin-polymer-conjugates as photosensitizers: principal mode of action demonstrated by in vitro models.

PubMed

Feinweber, Daniela; Verwanger, Thomas; Brüggemann, Oliver; Teasdale, Ian; Krammer, Barbara

2014-11-01

Two series of water soluble novel conjugates of the photosensitizer hypericin were prepared and evaluated for their use as agents for photodynamic therapy, with covalently and non-covalently loaded hypericin on functionalised, hydrolytically degradable inorganic-organic hybrid polyphosphazenes. The conjugates showed excellent aqueous solubility and similar fluorescence spectra to pristine hypericin. Detailed in vitro investigations revealed that the substances were non-toxic in the dark over a wide concentration range, but displayed phototoxicity upon irradiation. Cell uptake studies showed rapid uptake with localization of hypericin observed in endoplasmic reticulum, Golgi complex and particularly in the lysosomes. Furthermore, a DNA fragmentation assay revealed that the photosensitizer conjugates are efficient inducers of apoptosis with some tumor cell selectivity caused by faster and enhanced accumulation in A431 than in HaCaT cells, and thus a moderately higher phototoxicity of A431 compared to HaCaT cells. These novel photosensitizer conjugates hence represent viable hydrolytically degradable alternatives for the advanced delivery of hypericin.

S-protected thiolated chitosan: synthesis and in vitro characterization.

PubMed

Dünnhaupt, Sarah; Barthelmes, Jan; Thurner, Clemens C; Waldner, Claudia; Sakloetsakun, Duangkamon; Bernkop-Schnürch, Andreas

2012-10-01

Purpose of the present study was the generation and evaluation of novel thiolated chitosans, so-named S-protected thiolated chitosans as mucosal drug delivery systems. Stability of all conjugates concerning swelling and disintegration behavior as well as drug release was examined. Mucoadhesive properties were evaluated in vitro on intestinal mucosa. Different thiolated chitosans were generated displaying increasing amounts of attached free thiol groups on the polymer, whereby more than 50% of these thiol groups were linked with 6-mercaptonicotinamide. Based on the implementation of this hydrophobic residue, the swelling behavior was 2-fold decreased, whereas stability was essentially improved. Their mucoadhesive properties were 2- and 14-fold increased compared to corresponding thiolated and unmodified chitosans, respectively. Release studies out of matrix tablets comprising the novel conjugates revealed a controlled release of a model peptide. Accordingly, S-protected thiomers represent a promising type of mucoadhesive polymers for the development of various mucosal drug delivery systems. Copyright © 2012 Elsevier Ltd. All rights reserved.

Infection Spread and Virus Release in Vitro in Cell Populations as a System with Percolation

NASA Astrophysics Data System (ADS)

Ochoa, Juan G. Diaz

The comprehension of the innate immune system of cell populations is not only of interest to understand systems in vivo but also in vitro, for example, in the control of the release of viral particles for the production of vaccines. In this report I introduce a model, based on dynamical networks, that simulates the cell signaling responsible for this innate immune response and its effect on the infection spread and virus production. The central motivation is to represent a cell population that is constantly mixed in a bio-reactor where there is a cell-to-cell signaling of cytokines (which are proteins responsible for the activation of the antiviral response inside the cell). Such signaling allows the definition of clusters of linked immune cells. Additionally, depending on the density of links, it is possible to identify critical threshold parameters associated to a percolation phase transition. I show that the control of this antiviral response is equivalent to a percolation process.

An antiviral RISC isolated from Tobacco rattle virus-infected plants.

PubMed

Ciomperlik, Jessica J; Omarov, Rustem T; Scholthof, Herman B

2011-03-30

The RNAi model predicts that during antiviral defense a RNA-induced silencing complex (RISC) is programmed with viral short-interfering RNAs (siRNAs) to target the cognate viral RNA for degradation. We show that infection of Nicotiana benthamiana with Tobacco rattle virus (TRV) activates an antiviral nuclease that specifically cleaves TRV RNA in vitro. In agreement with known RISC properties, the nuclease activity was inhibited by NaCl and EDTA and stimulated by divalent metal cations; a novel property was its preferential targeting of elongated RNA molecules. Intriguingly, the specificity of the TRV RISC could be reprogrammed by exogenous addition of RNA (containing siRNAs) from plants infected with an unrelated virus, resulting in a newly acquired ability of RISC to target this heterologous genome in vitro. Evidently the virus-specific nuclease complex from N. benthamiana represents a genuine RISC that functions as a readily employable and reprogrammable antiviral defense unit. Copyright © 2011 Elsevier Inc. All rights reserved.

Impact of diabetic serum on endothelial cells: An in-vitro-analysis of endothelial dysfunction in diabetes mellitus type 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muenzel, Daniela; Lehle, Karla; Haubner, Frank

2007-10-19

Diabetic endothelial dysfunction was characterized by altered levels of adhesion molecules and cytokines. Aim of our study was to evaluate the effects of diabetic serum on cell-growth and proinflammatory markers in human saphenous vein endothelial cells (HSVEC) from diabetic and non-diabetic patients. Diabetic serum showed (1) complementary proliferative activity for non-diabetic and diabetic HSVEC, (2) unchanged surface expression of adhesion molecules, and (3) elevated levels of sICAM-1 in HSVEC of all donors. The concentration of sVCAM-1 was increased only in diabetic cells. The proinflammatory state of diabetic HSVEC characterized by increased levels of cytokines was compensated. We concluded that evenmore » under normoglycemic conditions the serum itself contains critical factors leading to abnormal regulation of inflammation in diabetics. We introduced an in vitro model of diabetes representing the endothelial situation at the beginning of diabetes (non-diabetic cells/diabetic serum) as well as the diabetic chronic state (diabetic cells/diabetic serum)« less

In vitro organogenesis of gut-like structures from mouse embryonic stem cells.

PubMed

Kuwahara, M; Ogaeri, T; Matsuura, R; Kogo, H; Fujimoto, T; Torihashi, S

2004-04-01

Embryonic stem (ES) cells have pluripotency and give rise to many cell types and tissues, including representatives of all three germ layers in the embryo. We have reported previously that mouse ES cells formed contracting gut-like organs from embryoid bodies (EBs). These gut-like structures contracted spontaneously, and had large lumens surrounded by three layers, i.e. epithelium, lamina propria and muscularis. Ganglia were scattered along the periphery, and interstitial cells of Cajal (ICC) were distributed among the smooth muscle cells. In the present study, to determine whether they can be a model of gut organogenesis, we investigated the formation process of the gut-like structures in comparison with embryonic gut development. As a result, we found that the fundamental process of formation in vitro was similar to embryonic gut development in vivo. The result indicates that the gut-like structure is a useful tool not only for developmental study to determine the factors that induce gut organogenesis, but also for studies of enteric neurone and ICC development.

Caprylic and Polygalacturonic Acid Combinations for Eradication of Microbial Organisms Embedded in Biofilm

PubMed Central

Rosenblatt, Joel; Reitzel, Ruth A.; Vargas-Cruz, Nylev; Chaftari, Anne-Marie; Hachem, Ray; Raad, Issam

2017-01-01

There is a need for non-antibiotic, antimicrobial compositions with low toxicity capable of broad-spectrum eradication of pathogenic biofilms in food preparation and healthcare settings. In this study we demonstrated complete biofilm eradication within 60 min with synergistic combinations of caprylic and polygalacturonic (PG) acids in an in vitro biofilm eradication model against representative hospital and foodborne infectious pathogen biofilms (methicillin-resistant Staphylococcus aureus, multidrug-resistant Pseudomonas aeruginosa, Candida albicans, Escherichia coli, and Salmonella enteritidis). Antimicrobial synergy against biofilms was demonstrated by quantifying viable organisms remaining in biofilms exposed to caprylic acid alone, PG acid alone, or combinations of the two. The combinations also synergistically inhibited growth of planktonic organisms. Toxicity of the combination was assessed in vitro on L929 fibroblasts incubated with extracts of caprylic and PG acid combinations using the Alamar Blue metabolic activity assay and the Trypan Blue exclusion cell viability assay. The extracts did not produce cytotoxic responses relative to untreated control fibroblasts. PMID:29093703

Macrogenomic engineering via modulation of the scaling of chromatin packing density.

PubMed

Almassalha, Luay M; Bauer, Greta M; Wu, Wenli; Cherkezyan, Lusik; Zhang, Di; Kendra, Alexis; Gladstein, Scott; Chandler, John E; VanDerway, David; Seagle, Brandon-Luke L; Ugolkov, Andrey; Billadeau, Daniel D; O'Halloran, Thomas V; Mazar, Andrew P; Roy, Hemant K; Szleifer, Igal; Shahabi, Shohreh; Backman, Vadim

2017-11-01

Many human diseases result from the dysregulation of the complex interactions between tens to thousands of genes. However, approaches for the transcriptional modulation of many genes simultaneously in a predictive manner are lacking. Here, through the combination of simulations, systems modelling and in vitro experiments, we provide a physical regulatory framework based on chromatin packing-density heterogeneity for modulating the genomic information space. Because transcriptional interactions are essentially chemical reactions, they depend largely on the local physical nanoenvironment. We show that the regulation of the chromatin nanoenvironment allows for the predictable modulation of global patterns in gene expression. In particular, we show that the rational modulation of chromatin density fluctuations can lead to a decrease in global transcriptional activity and intercellular transcriptional heterogeneity in cancer cells during chemotherapeutic responses to achieve near-complete cancer cell killing in vitro. Our findings represent a 'macrogenomic engineering' approach to modulating the physical structure of chromatin for whole-scale transcriptional modulation.

[Everted intestinal sac method for quick finding absorption ingredients of Wuzhuyu decoction].

PubMed

Gong, Muxin; Wang, Yaxun; Song, Yafang; Wang, Zhimin; Zhang, Qiwei; Wang, Weihao; Zhu, Jingjing

2010-06-01

To establish a method for quick finding the absorption ingredients of Wuzhuyu decoction in order to select the index to control its quality. The absorption of three concentration of Wuzhuyu decotion was investigated with the in vitro-everted intestinal sac model. The intestinal bag fluid of jejunum and ileum were collected in different time and the eight ingredients, which were evodiamine (Ev), rutaecarpine (Ru), limonin (Li), ginsenoside-Rb1, -Rg1, -Re (Rb1, Rg1, Re), isorhamnetin-3-O-beta-D-glucosyl(6''-->1'")-alpha-L-rhamnoside (Irs)and 6-gingerol (6-Gi), were detected by HPLC as the represent constituents in samples. Eight ingredients except Ru in samples could be detected, but Ev could not be detected in high concentration samples. The ratios between absorption ingredients were different from in Wuzhuyu decotion. The in vitro-everted intestinal sac canc absorb the ingredients of Wuzhuyu decotion selectivity. Compare with the ileum, the jejunum can provide the more absorption information and faster, the best test time is 60-90 min.

Selective arylsulfonamide inhibitors of ADAM-17: hit optimization and activity in ovarian cancer cell models.

PubMed

Nuti, Elisa; Casalini, Francesca; Santamaria, Salvatore; Fabbi, Marina; Carbotti, Grazia; Ferrini, Silvano; Marinelli, Luciana; La Pietra, Valeria; Novellino, Ettore; Camodeca, Caterina; Orlandini, Elisabetta; Nencetti, Susanna; Rossello, Armando

2013-10-24

Activated leukocyte cell adhesion molecule (ALCAM) is expressed at the surface of epithelial ovarian cancer (EOC) cells and is released in a soluble form (sALCAM) by ADAM-17-mediated shedding. This process is relevant to EOC cell motility and invasiveness, which is reduced by inhibitors of ADAM-17. In addition, ADAM-17 plays a key role in EGFR signaling and thus may represent a useful target in anticancer therapy. Herein we report our hit optimization effort to identify potent and selective ADAM-17 inhibitors, starting with previously identified inhibitor 1. A new series of secondary sulfonamido-based hydroxamates was designed and synthesized. The biological activity of the newly synthesized compounds was tested in vitro on isolated enzymes and human EOC cell lines. The optimization process led to compound 21, which showed an IC50 of 1.9 nM on ADAM-17 with greatly increased selectivity. This compound maintained good inhibitory properties on sALCAM shedding in several in vitro assays.

Expression profiles of phases 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models Episkin and full thickness model from Episkin.

PubMed

Luu-The, Van; Duche, Daniel; Ferraris, Corinne; Meunier, Jean-Roch; Leclaire, Jacques; Labrie, Fernand

2009-09-01

Episkin and full thickness model from Episkin (FTM) are human skin models obtained from in vitro growth of keratinocytes into the five typical layers of the epidermis. FTM is a full thickness reconstructed skin model that also contains fibroblasts seeded in a collagen matrix. To assess whether enzymes involved in chemical detoxification are expressed in Episkin and FTM and how their levels compare with the human epidermis, dermis and total skin. Quantification of the mRNA expression levels of phases 1 and 2 metabolizing enzymes in cultured Episkin and FTM and human epidermis, dermis and total skin using Realtime PCR. The data show that the expression profiles of 61 phases 1 and 2 metabolizing enzymes in Episkin, FTM and epidermis are generally similar, with some exceptions. Cytochrome P450-dependent enzymes and flavin monooxygenases are expressed at low levels, while phase 2 metabolizing enzymes are expressed at much higher levels, especially, glutathione-S-transferase P1 (GSTP1) catechol-O-methyl transferase (COMT), steroid sulfotransferase (SULT2B1b), and N-acetyl transferase (NAT5). The present study also identifies the presence of many enzymes involved in cholesterol, arachidonic acid, leukotriene, prostaglandin, eicosatrienoic acids, and vitamin D3 metabolisms. The present data strongly suggest that Episkin and FTM represent reliable and valuable in vitro human skin models for studying the function of phases 1 and 2 metabolizing enzymes in xenobiotic metabolisms. They could be used to replace invasive methods or laboratory animals for skin experiments.

[The investigation of genomes of some species of the genus Gentiana in nature and in vitro cell culture].

PubMed

Mel'nyk, V M; Spiridonova, K V; Andrieiev, I O; Strashniuk, N M; Kunakh, V A

2002-01-01

The comparative study of the genomes of intact plants-representatives of some species of the genus Gentiana L. as well as cultured cells of G. lutea and G. punctata was performed using restriction analysis. Species specificity of restriction fragment patterns for studied representatives of this genus was revealed. The differences between electrophoretic patterns of digested DNA purified from rhizome and leaves of G. lutea and G. punctata were found. The changes in genomes of G. lutea and G. punctata cells cultured in vitro compared with the genomes of intact plants were detected. The data obtained evidence that some of them may be of nonrandom character.

Predicting the extent of metabolism using in vitro permeability rate measurements and in silico permeability rate predictions

PubMed Central

Hosey, Chelsea M; Benet, Leslie Z

2015-01-01

The Biopharmaceutics Drug Disposition Classification System (BDDCS) can be utilized to predict drug disposition, including interactions with other drugs and transporter or metabolizing enzyme effects based on the extent of metabolism and solubility of a drug. However, defining the extent of metabolism relies upon clinical data. Drugs exhibiting high passive intestinal permeability rates are extensively metabolized. Therefore, we aimed to determine if in vitro measures of permeability rate or in silico permeability rate predictions could predict the extent of metabolism, to determine a reference compound representing the permeability rate above which compounds would be expected to be extensively metabolized, and to predict the major route of elimination of compounds in a two-tier approach utilizing permeability rate and a previously published model predicting the major route of elimination of parent drug. Twenty-two in vitro permeability rate measurement data sets in Caco-2 and MDCK cell lines and PAMPA were collected from the literature, while in silico permeability rate predictions were calculated using ADMET Predictor™ or VolSurf+. The potential for permeability rate to differentiate between extensively and poorly metabolized compounds was analyzed with receiver operating characteristic curves. Compounds that yielded the highest sensitivity-specificity average were selected as permeability rate reference standards. The major route of elimination of poorly permeable drugs was predicted by our previously published model and the accuracies and predictive values were calculated. The areas under the receiver operating curves were >0.90 for in vitro measures of permeability rate and >0.80 for the VolSurf+ model of permeability rate, indicating they were able to predict the extent of metabolism of compounds. Labetalol and zidovudine predicted greater than 80% of extensively metabolized drugs correctly and greater than 80% of poorly metabolized drugs correctly in Caco-2 and MDCK, respectively, while theophylline predicted greater than 80% of extensively and poorly metabolized drugs correctly in PAMPA. A two-tier approach predicting elimination route predicts 72±9%, 49±10%, and 66±7% of extensively metabolized, biliarily eliminated, and renally eliminated parent drugs correctly when the permeability rate is predicted in silico and 74±7%, 85±2%, and 73±8% of extensively metabolized, biliarily eliminated, and renally eliminated parent drugs correctly, respectively when the permeability rate is determined in vitro. PMID:25816851

The rationale for liquid biopsy in colorectal cancer: a focus on circulating tumor cells.

PubMed

Gazzaniga, Paola; Raimondi, Cristina; Nicolazzo, Chiara; Carletti, Raffaella; di Gioia, Cira; Gradilone, Angela; Cortesi, Enrico

2015-01-01

Capturing circulating tumor cells (CTCs) and/or circulating tumor DNA from blood, which represents a precious source of biological material derived from both primary and metastatic tumors, has been named a 'liquid biopsy'. While the circulating tumor DNA might be more representative of the bulk of the metastatic tumor, CTCs are thought to reflect more of the metastases-initiating cells. Consequently, a liquid biopsy made of tumor cells and tumor DNA that is able to track cancer evolution, as a fingerprint of the patient's individual tumor, and is easy to perform at every stage of the disease course, sounds attractive. This article mainly focuses on the applications of CTCs to track tumor dynamics in real time using colorectal cancer as a model system. The analysis of viable CTCs at DNA, RNA and protein levels, as well as their expansion in vitro, may allow deep investigation of the features of metastases-initiating cells.

Effect of micro-environment modification and polymer type on the in-vitro dissolution behavior and in-vivo performance of amorphous solid dispersions.

PubMed

Sun, Weiwei; Pan, Baoliang

2017-06-15

This study investigates the effects of micro-environment modification and polymer type on the in-vitro dissolution behavior and in-vivo performance of micro-environment pH modifying solid dispersions (pH M -SD) for the poorly water-soluble model drug Toltrazuril (TOL). Various pH M -SDs were prepared using Ca(OH) 2 as a pH-modifier in hydrophilic polymers, including polyethylene glycol 6000 (PEG6000), polyvinylpyrrolidone k30 (PVPk30) and hydroxypropyl methylcellulose (HPMC). Based on the results of physicochemical characterizations and in-vitro dissolution testing, the representative ternary (Ca(OH) 2 :TOL:PEG6000/HPMC/PVPk30=1:8:24, w/w/w) and binary (TOL:PVPk30=1:3, w/w) solid dispersions were selected and optimized to perform in-vivo pharmacokinetic study. The micro-environment pH modification improved the in-vitro water-solubility and in-vivo bioavailability of parent drug TOL. Furthermore, the addition of alkalizers not only enhanced the release and absorption of prototype drug, but also promoted the generation of active metabolites, including toltrazuril sulfoxide (TOLSO) and toltrazuril sulfone (TOLSO 2 ). The in-vitro dissolution profiles and in-vivo absorption, distribution and metabolism behaviors of the pH M -SDs varied with polymer type. Moreover, in-vivo bioavailability of three active pharmaceutical ingredients increased with an increase in in-vitro dissolution rates of the drug from the pH M -SDs prepared with various polymers. Therefore, a non-sink in-vitro dissolution method can be used to predict the in-vivo performance of pH M -SDs formulated with various polymers with trend consistency. In-vitro and in-vivo screening procedures revealed that the pH M -SD composed of Ca(OH) 2 , TOL and PVPk30 at a weight ratio of 1:8:24, of which the safety was adequately proved via histopathological examination, may be a promising candidate for providing better clinical outcomes. Copyright © 2017. Published by Elsevier B.V.

Bruton's tyrosine kinase inhibitor BMS-986142 in experimental models of rheumatoid arthritis enhances efficacy of agents representing clinical standard-of-care.

PubMed

Gillooly, Kathleen M; Pulicicchio, Claudine; Pattoli, Mark A; Cheng, Lihong; Skala, Stacey; Heimrich, Elizabeth M; McIntyre, Kim W; Taylor, Tracy L; Kukral, Daniel W; Dudhgaonkar, Shailesh; Nagar, Jignesh; Banas, Dana; Watterson, Scott H; Tino, Joseph A; Fura, Aberra; Burke, James R

2017-01-01

Bruton's tyrosine kinase (BTK) regulates critical signal transduction pathways involved in the pathobiology of rheumatoid arthritis (RA) and other autoimmune disorders. BMS-986142 is a potent and highly selective reversible small molecule inhibitor of BTK currently being investigated in clinical trials for the treatment of both RA and primary Sjögren's syndrome. In the present report, we detail the in vitro and in vivo pharmacology of BMS-986142 and show this agent provides potent and selective inhibition of BTK (IC50 = 0.5 nM), blocks antigen receptor-dependent signaling and functional endpoints (cytokine production, co-stimulatory molecule expression, and proliferation) in human B cells (IC50 ≤ 5 nM), inhibits Fcγ receptor-dependent cytokine production from peripheral blood mononuclear cells, and blocks RANK-L-induced osteoclastogenesis. Through the benefits of impacting these important drivers of autoimmunity, BMS-986142 demonstrated robust efficacy in murine models of rheumatoid arthritis (RA), including collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA). In both models, robust efficacy was observed without continuous, complete inhibition of BTK. When a suboptimal dose of BMS-986142 was combined with other agents representing the current standard of care for RA (e.g., methotrexate, the TNFα antagonist etanercept, or the murine form of CTLA4-Ig) in the CIA model, improved efficacy compared to either agent alone was observed. The results suggest BMS-986142 represents a potential therapeutic for clinical investigation in RA, as monotherapy or co-administered with agents with complementary mechanisms of action.

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip.

PubMed

Dauth, Stephanie; Maoz, Ben M; Sheehy, Sean P; Hemphill, Matthew A; Murty, Tara; Macedonia, Mary Kate; Greer, Angie M; Budnik, Bogdan; Parker, Kevin Kit

2017-03-01

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features. NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections. Copyright © 2017 the American Physiological Society.

Neurons derived from different brain regions are inherently different in vitro: a novel multiregional brain-on-a-chip

PubMed Central

Dauth, Stephanie; Maoz, Ben M.; Sheehy, Sean P.; Hemphill, Matthew A.; Murty, Tara; Macedonia, Mary Kate; Greer, Angie M.; Budnik, Bogdan

2017-01-01

Brain in vitro models are critically important to developing our understanding of basic nervous system cellular physiology, potential neurotoxic effects of chemicals, and specific cellular mechanisms of many disease states. In this study, we sought to address key shortcomings of current brain in vitro models: the scarcity of comparative data for cells originating from distinct brain regions and the lack of multiregional brain in vitro models. We demonstrated that rat neurons from different brain regions exhibit unique profiles regarding their cell composition, protein expression, metabolism, and electrical activity in vitro. In vivo, the brain is unique in its structural and functional organization, and the interactions and communication between different brain areas are essential components of proper brain function. This fact and the observation that neurons from different areas of the brain exhibit unique behaviors in vitro underline the importance of establishing multiregional brain in vitro models. Therefore, we here developed a multiregional brain-on-a-chip and observed a reduction of overall firing activity, as well as altered amounts of astrocytes and specific neuronal cell types compared with separately cultured neurons. Furthermore, this multiregional model was used to study the effects of phencyclidine, a drug known to induce schizophrenia-like symptoms in vivo, on individual brain areas separately while monitoring downstream effects on interconnected regions. Overall, this work provides a comparison of cells from different brain regions in vitro and introduces a multiregional brain-on-a-chip that enables the development of unique disease models incorporating essential in vivo features. NEW & NOTEWORTHY Due to the scarcity of comparative data for cells from different brain regions in vitro, we demonstrated that neurons isolated from distinct brain areas exhibit unique behaviors in vitro. Moreover, in vivo proper brain function is dependent on the connection and communication of several brain regions, underlining the importance of developing multiregional brain in vitro models. We introduced a novel brain-on-a-chip model, implementing essential in vivo features, such as different brain areas and their functional connections. PMID:28031399

Development of an in vitro Hepatocyte Model to Investigate Chemical Mode of Action

EPA Science Inventory

There is a clear need to identify and characterize the potential of liver in vitro models that can be used to replace animals for mode of action analysis. Our goal is to use in vitro models for mode of action prediction which recapitulate critical cellular processes underlying in...

COMPARISON OF MEDIUM CONCENTRATION VS. ACTUAL TISSUE DOSE IN IN VITRO NEUROTOXICANT MODELS.

EPA Science Inventory

In vitro methods have long been used to model the effects of toxicants on the nervous system. Generally, it is assumed that concentrations of toxicant present in the medium surrounding cells in in vitro models are an adequate biomarker of cell or tissue levels. However, this assu...

Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

PubMed

Reisinger, Kerstin; Blatz, Veronika; Brinkmann, Joep; Downs, Thomas R; Fischer, Anja; Henkler, Frank; Hoffmann, Sebastian; Krul, Cyrille; Liebsch, Manfred; Luch, Andreas; Pirow, Ralph; Reus, Astrid A; Schulz, Markus; Pfuhler, Stefan

2018-03-01

Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion ® Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion ® Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will continue by testing an additional 22 chemicals. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Expression of XBP1s in fibroblasts is critical for TiAl6 V4 particle-induced RANKL expression and osteolysis.

PubMed

Wang, Zhenheng; Liu, Naicheng; Zhou, Gang; Shi, Tongguo; Wang, Zhenzhen; Gan, Jingjing; Wang, Rui; Qian, Hongbo; Bao, Nirong; Guo, Ting; Zhao, Jianning

2017-04-01

Wear particle-induced osteolysis is a major cause of aseptic loosening, which is one of the most common reasons for total hip arthroplasty (THA) failure. Previous studies have shown that the expression of Receptor activation of nuclear factor (NF)-kB (RANKL) by fibroblasts in periprosthetic membrane played a crucial role in wear particle-induced osteolysis. However, the underlying mechanism of RANKL expression remains largely unknown. In the present study, we investigated the effect of TiAl 6 V 4 particle (TiPs)-induced XBP1s (spliced form of X-box binding protein 1) on RANKL expression and osteoclastogenesis both in vitro and in vivo. The levels of XBP1s in peri-implant membrane, animal models, and TiPs-stimulated fibroblasts were determined by western blots. To assess the effect of XBP1s on RANKL expression, fibroblasts were treated with both a small interfering RNA (siRNA) and an inhibitor of XBP1 prior to exposure to TiPs. The effect of XBP1s on osteoclasts formation was determined by tartrate-resistant acid phosphatase (TRAP) staining in vitro osteoclastogenesis assay and in animal models. The resorption of bone was assessed by micro-computed tomography (micro-CT) with three-dimensional reconstruction. Our results demonstrated that XBP1s was activated in periprosthetic membrane, mouse calvaria models, and TiPs-stimulated human synovial fibroblasts. Further, inhibition of XBP1s decreased the expression of RANKL and osteoclasts formation in vitro. In mouse calvaria models, both of the osteoclastogenesis and osteolysis were inhibited XBP1s inhibitor. Our results suggested that XBP1s mediated TiPs-induced of RANKL expression in fibroblasts, and down regulating XBP1s may represent a potential therapy for wear particle-induced osteolysis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:752-759, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

The anti-inflammatory and immunomodulatory potential of braylin: Pharmacological properties and mechanisms by in silico, in vitro and in vivo approaches

PubMed Central

Espírito-Santo, Renan Fernandes; Meira, Cassio Santana; Costa, Rafael dos Santos; Souza Filho, Otávio Passos; Evangelista, Afranio Ferreira; Trossini, Gustavo Henrique Goulart; Ferreira, Glaucio Monteiro; Velozo, Eudes da Silva; Pereira Soares, Milena Botelho

2017-01-01

Braylin belongs to the group of natural coumarins, a group of compounds with a wide range of pharmacological properties. Here we characterized the pharmacological properties of braylin in vitro, in silico and in vivo in models of inflammatory/immune responses. In in vitro assays, braylin exhibited concentration-dependent suppressive activity on activated macrophages. Braylin (10–40 μM) reduced the production of nitrite, IL-1β, TNF-α and IL-6 by J774 cells or peritoneal exudate macrophages stimulated with LPS and IFN-γ. Molecular docking calculations suggested that braylin present an interaction pose to act as a glucocorticoid receptor ligand. Corroborating this idea, the inhibitory effect of braylin on macrophages was prevented by RU486, a glucocorticoid receptor antagonist. Furthermore, treatment with braylin strongly reduced the NF-κB-dependent transcriptional activity on RAW 264.7 cells. Using the complete Freund’s adjuvant (CFA)-induced paw inflammation model in mice, the pharmacological properties of braylin were demonstrated in vivo. Braylin (12.5–100 mg/kg) produced dose-related antinociceptive and antiedematogenic effects on CFA model. Braylin did not produce antinociception on the tail flick and hot plate tests in mice, suggesting that braylin-induced antinociception is not a centrally-mediated action. Braylin exhibited immunomodulatory properties on the CFA model, inhibiting the production of pro-inflammatory cytokines IL-1β, TNF-α and IL-6, while increased the anti-inflammatory cytokine TGF-β. Our results show, for the first time, anti-inflammatory, antinociceptive and immunomodulatory effects of braylin, which possibly act through the glucocorticoid receptor activation and by inhibition of the transcriptional activity of NF-κB. Because braylin is a phosphodiesterase-4 inhibitor, this coumarin could represent an ideal prototype of glucocorticoid receptor ligand, able to induce synergic immunomodulatory effects. PMID:28594906

Skin metabolism of aminophenols: Human keratinocytes as a suitable in vitro model to qualitatively predict the dermal transformation of 4-amino-2-hydroxytoluene in vivo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goebel, C.; Hewitt, N.J.; Kunze, G.

2009-02-15

4-Amino-2-hydroxytolune (AHT) is an aromatic amine ingredient in oxidative hair colouring products. As skin contact occurs during hair dyeing, characterisation of dermal metabolism is important for the safety assessment of this chemical class. We have compared the metabolism of AHT in the human keratinocyte cell line HaCaT with that observed ex-vivo in human skin and in vivo (topical application versus oral (p.o.) and intravenous (i.v.) route). Three major metabolites of AHT were excreted, i.e. N-acetyl-AHT, AHT-sulfate and AHT-glucuronide. When 12.5 mg/kg AHT was applied topically, the relative amounts of each metabolite were altered such that N-acetyl-AHT product was the majormore » metabolite (66% of the dose in comparison with 37% and 32% of the same applied dose after i.v. and p.o. administration, respectively). N-acetylated products were the only metabolites detected in HaCaT cells and ex-vivo whole human skin discs for AHT and p-aminophenol (PAP), an aromatic amine known to undergo N-acetylation in vivo. Since N-acetyltransferase 1 (NAT1) is the responsible enzyme, kinetics of AHT was further compared to the standard NAT1 substrate p-aminobenzoic acid (PABA) in the HaCaT model revealing similar values for K{sub m} and V{sub max}. In conclusion NAT1 dependent dermal N-acetylation of AHT represents a 'first-pass' metabolism effect in the skin prior to entering the systemic circulation. Since the HaCaT cell model represents a suitable in vitro assay for addressing the qualitative contribution of the skin to the metabolism of topically-applied aromatic amines it may contribute to a reduction in animal testing.« less

[Effects of electromagnetic pulse exposure on the permeability of inner blood-retinal barrier model in vitro].

PubMed

Li, Hai-juan; Yang, Long-long; Tian, Wei; Liu, Jun-ju; Xie, Xue-jun; Guo, Guo-zhen

2012-03-01

To establish the inner blood-retinal barrier (BRB) model in vitro by co-culturing RF/6A cells and C6 cells and to investigate the effects of EMP (200 kV/m, 200 pulses) exposure on the permeability of the inner BRB model in vitro. RF/6A cells and C6 cells were co-cultured on transwell, and the characteristic of the inner BRB model was assessed by detecting transendothelial electrical resistance (TEER) and the permeability of horseradish peroxidase (HRP). The co-cultured model was exposed or sham exposed to the EMP (200 kV/m 200 pulses) for 0.5, 3, 6, 12, 24 h in vitro, then TEER and the permeability of HRP were measured for studying the effects of EMP on the permeability of inner BRB model in vitro. TEER value (145 Ωcm(2)) of the co-culturing inner BRB model significantly increased, as compared to that of RF/6A cells alone model (P < 0.05) on the 6th day after inoculation. There was significant difference of permeability of HRP between the co-culturing inner BRB model and RF/6A cells alone model (P < 0.05). The ability of inhibiting large molecular materials in the co-culturing inner BRB model enhanced. The TEER value decreased and the permeability of HRP increased as compared to the sham group at 0.5, 3, 6 h after the exposure. The inner BRB model by co-culturing RF/6A cells and C6 cells in vitro is efficient and suitable to study the alterations of the restricted permeability function of the inner BRB. EMP (200 kV/m for 200 pulses) could induce the enhanced permeability of the inner BRB model in vitro.

Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations.

PubMed

Chen, Qiyu; Jia, Ai; Snyder, Shane A; Gong, Zhiyuan; Lam, Siew Hong

2016-02-01

Glucocorticoids are pharmaceutical contaminants of emerging concern due to their incomplete removal during wastewater treatment, increased presence in aquatic environment and their biological potency. The zebrafish is a popular model for aquatic toxicology and environmental risk assessment. This study aimed to determine if glucocorticoids at environmental concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and to investigate their potentials as an in vivo zebrafish assay in complementing in vitro glucocorticoid receptor bioassay. The relative expression of eleven glucocorticoid-responsive genes in zebrafish larvae and liver of adult male zebrafish exposed to three representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) was determined. The expression of pepck, baiap2 and pxr was up-regulated in zebrafish larvae and the expression of baiap2, pxr and mmp-2 was up-regulated in adult zebrafish exposed to glucocorticoids at concentrations equivalent to total glucocorticoids reported in environmental samples. The responsiveness of the specific genes were sufficiently robust in zebrafish larvae exposed to a complex environmental sample detected with in vitro glucocorticoid activity equivalent to 478 pM dexamethasone (DEX-EQ) and confirmed to contain low concentration (0.2 ng/L or less) of the targeted glucocorticoids, and possibly other glucocorticoid-active compounds. The findings provided in vivo relevance to the in vitro glucocorticoid activity and suggested that the environmental sample can perturb glucocorticoid-responsive genes in its original, or half the diluted, concentration as may be found in the environment. The study demonstrated the important complementary roles of in vivo zebrafish and in vitro bioassays coupled with analytical chemistry in monitoring environmental glucocorticoid contaminants. Copyright © 2015 Elsevier Ltd. All rights reserved.

D-BMAP18 antimicrobial peptide is active in vitro, resists to pulmonary proteases but loses its activity in a murine model of Pseudomonas aeruginosa lung infection.

NASA Astrophysics Data System (ADS)

Mardirossian, Mario; Pompilio, Arianna; Degasperi, Margherita; Runti, Giulia; Pacor, Sabrina; Di Bonaventura, Giovanni; Scocchi, Marco

2017-06-01

The spread of antibiotic resistant-pathogens is driving the search for new antimicrobial compounds. Pulmonary infections experienced by cystic fibrosis patients are a dramatic example of this health-care emergency. Antimicrobial peptides could answer the need for new antibiotics but translating them from basic research to the clinic is a challenge. We have previously evaluated the potential of the small membranolytic peptide BMAP-18 to treat CF-related infections, discovering that while this molecule had a good activity in vitro it was not active in vivo because of its rapid degradation by pulmonary proteases. In this study, we synthesized and tested the proteases-resistant all-D enantiomer. In spite of a good antimicrobial activity against Pseudomonas aeruginosa and Stenotrophomonas maltophilia clinical isolates and of a tolerable cytotoxicity in vitro, D-BMAP18 was ineffective to treat P. aeruginosa pulmonary infection in mice, in comparison to tobramycin. We observed that different factors other than peptide degradation hampered its efficacy for pulmonary application. These results indicate that D-BMAP18 needs further optimization before being suitable for clinical application and this approach may represent a guide for optimization of other anti-infective peptides eligible for the treatment of pulmonary infections.

In Vitro, In Vivo and Post Explantation Testing of Glucose-Detecting Biosensors: Current Methods and Recommendations

PubMed Central

Koschwanez, Heidi E.; Reichert, W. Monty

2007-01-01

To date, there have been a number of cases where glucose sensors have performed well over long periods of implantation; however, it remains difficult to predict whether a given sensor will perform reliably, will exhibit gradual degradation of performance, or will fail outright soon after implantation. Typically, the literature emphasizes the sensor that performed well, while only briefly (if at all) mentioning the failed devices. This leaves open the question of whether current sensor designs are adequate for the hostile in vivo environment, and whether these sensors have been assessed by the proper regimen of testing protocols. This paper reviews the current in vitro and in vivo testing procedures used to evaluate the functionality and biocompatibility of implantable glucose sensors. An overview of the standards and regulatory bodies that govern biomaterials and end-product device testing precedes a discussion of up-to-date invasive and non-invasive technologies for diabetes management. Analysis of current in vitro, in vivo, and then post implantation testing is presented. Given the underlying assumption that the success of the sensor in vivo foreshadows the long-term reliability of the sensor in the human body, the relative merits of these testing methods are evaluated with respect to how representative they are of human models. PMID:17524479

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

PubMed

Toscano, Miguel G; Navarro-Montero, Oscar; Ayllon, Veronica; Ramos-Mejia, Veronica; Guerrero-Carreno, Xiomara; Bueno, Clara; Romero, Tamara; Lamolda, Mar; Cobo, Marien; Martin, Francisco; Menendez, Pablo; Real, Pedro J

2015-01-01

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

In vitro characterisation of the fermentation profile and prebiotic capacity of gold-fleshed kiwifruit.

PubMed

Blatchford, P; Bentley-Hewitt, K L; Stoklosinski, H; McGhie, T; Gearry, R; Gibson, G; Ansell, J

2015-01-01

A new Actinidia chinensis gold-fleshed kiwifruit cultivar 'Zesy002' was tested to investigate whether it could positively modulate the composition of the human colonic microbiota. Digested Zesy002 kiwifruit was added to in vitro pH-controlled anaerobic batch fermenters that were inoculated with representative human faecal microbiota. Alterations to the gut microbial ecology were determined by 16S rRNA gene sequencing and metabolic end products were measured using gas chromatography and liquid chromatography - mass spectrometry. Results indicated a substantial shift in the composition of bacteria within the gut models caused by kiwifruit supplementation. Zesy002 supplemented microbiota had a significantly higher abundance of Bacteroides spp., Parabacteroides spp. and Bifidobacterium spp. after 48 h of fermentation compared with the start of the fermentation. Organic acids from kiwifruit were able to endure simulated gastrointestinal digestion and were detectable in the first 10 h of fermentation. The fermentable carbohydrates were converted to beneficial organic acids with a particular predilection for propionate production, corresponding with the rise in Bacteroides spp. and Parabacteroides spp. These results support the claim that Zesy002 kiwifruit non-digestible fractions can effect favourable changes to the human colonic microbial community and primary metabolites, and demonstrate a hitherto unknown effect of Zesy002 on colonic microbiota under in vitro conditions.

Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination.

PubMed

Fievez, Virginie; Plapied, Laurence; des Rieux, Anne; Pourcelle, Vincent; Freichels, Hélène; Wascotte, Valentine; Vanderhaeghen, Marie-Lyse; Jerôme, Christine; Vanderplasschen, Alain; Marchand-Brynaert, Jacqueline; Schneider, Yves-Jacques; Préat, Véronique

2009-09-01

The presence of RGD on nanoparticles allows the targeting of beta1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL-PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization.

Correlation between melanogenic and catalase activity in in vitro human melanocytes: a synergic strategy against oxidative stress.

PubMed

Maresca, Vittoria; Flori, Enrica; Briganti, Stefania; Mastrofrancesco, Arianna; Fabbri, Claudia; Mileo, Anna M; Paggi, Marco G; Picardo, Mauro

2008-04-01

UV-induced DNA damage can lead to melanoma, the most dangerous form of skin cancer. Understanding the mechanisms employed by melanocytes to protect against UV is therefore a key issue. In melanocytes, catalase is the main enzyme responsible for degrading hydrogen peroxide and we have previously shown that that low basal levels of catalase activity are associated with the light phototype in in vitro and ex vivo models. Here we investigate the possible correlation between its activity and melanogenesis in primary cultures of human melanocytes. We show that while the total melanin concentration is directly correlated to the level of pigmentation, the more the degree of pigmentation increased, the lower the proportion of pheomelanin present. Moreover, in human melanocytes in vitro, catalase-specific mRNA, protein and enzymatic activity were all directly correlated with total cellular melanin content. We also observed that immediately after a peroxidative treatment, the increase in reactive oxygen species was inversely associated with pigmentation level. Darkly pigmented melanocytes therefore possess two protective strategies represented by melanins and catalase activity that are likely to act synergistically to counteract the deleterious effects of UV radiation. By contrast, lightly pigmented melanocytes possess lower levels of melanogenic and catalase activity and are therefore more susceptible to accumulate damage after UV exposition.

Incorporating Human Interindividual Biotransformation ...

EPA Pesticide Factsheets

The protection of sensitive individuals within a population dictates that measures other than central tendencies be employed to estimate risk. The refinement of human health risk assessments for chemicals metabolized by the liver to reflect data on human variability can be accomplished through (1) the characterization of enzyme expression in large banks of human liver samples, (2) the employment of appropriate techniques for the quantification and extrapolation of metabolic rates derived in vitro, and (3) the judicious application of physiologically based pharmacokinetic (PBPK) modeling. While in vitro measurements of specific biochemical reactions from multiple human samples can yield qualitatively valuable data on human variance, such measures must be put into the perspective of the intact human to yield the most valuable predictions of metabolic differences among humans. For quantitative metabolism data to be the most valuable in risk assessment, they must be tied to human anatomy and physiology, and the impact of their variance evaluated under real exposure scenarios. For chemicals metabolized in the liver, the concentration of parent chemical in the liver represents the substrate concentration in the MichaelisMenten description of metabolism. Metabolic constants derived in vitro may be extrapolated to the intact liver, when appropriate conditions are met. Metabolic capacity Vmax; the maximal rate of the reaction) can be scaled directly to the concentration

Experimental characterization of post rigor mortis human muscle subjected to small tensile strains and application of a simple hyper-viscoelastic model.

PubMed

Gras, Laure-Lise; Laporte, Sébastien; Viot, Philippe; Mitton, David

2014-10-01

In models developed for impact biomechanics, muscles are usually represented with one-dimensional elements having active and passive properties. The passive properties of muscles are most often obtained from experiments performed on animal muscles, because limited data on human muscle are available. The aim of this study is thus to characterize the passive response of a human muscle in tension. Tensile tests at different strain rates (0.0045, 0.045, and 0.45 s⁻¹) were performed on 10 extensor carpi ulnaris muscles. A model composed of a nonlinear element defined with an exponential law in parallel with one or two Maxwell elements and considering basic geometrical features was proposed. The experimental results were used to identify the parameters of the model. The results for the first- and second-order model were similar. For the first-order model, the mean parameters of the exponential law are as follows: Young's modulus E (6.8 MPa) and curvature parameter α (31.6). The Maxwell element mean values are as follows: viscosity parameter η (1.2 MPa s) and relaxation time τ (0.25 s). Our results provide new data on a human muscle tested in vitro and a simple model with basic geometrical features that represent its behavior in tension under three different strain rates. This approach could be used to assess the behavior of other human muscles. © IMechE 2014.

Blood histamine release: A new allergy blood test

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faraj, B.A.; Gottlieb, G.R.; Camp, V.M.

1985-05-01

Allergen-mediated histamine release from human leukocytes represents an important model for in vitro studies of allergic reactions. The purpose of this study was to determine whether the measurement of histamine released in allergic patients (pts) by radioenzymatic assay following mixing of their blood with common allergens represents a reliable index for diagnosis of atopic allergy. Three categories of allergies were used: (1) housedust and mite; (2) cat and dog dander; (3) trees and grasses and ragweed mixture. The presence of allergy was established by intradermal skin testing in the study group of 82 pts. Significant atopy was defined as greatermore » than or equal to 3+ (overall range 0-4 +, negative to maximum) on skin testing. The test was carried out in tubes with 0.5 ml heparinized blood, 0.5 ml tris albumin buffer, and one of the allergens (60-100 PNU/ml). In 20 controls without allergy, there always was less than or equal to 4% histamine release (normal response). A significant allergen-mediated histamine release, ranging from 12 to 30% of the total blood histamine content, was observed in 96% of the pts with skin test sensitivity of greater than or equal to 3+. There was good agreement between skin testing and histamine release in terms of the allergen causing the response. Thus, measurement of histamine release in blood in response to allergen challenge represents a clinically useful in vitro test for the diagnosis of atopic allergy. Because data can be obtained from a single sample and are highly quantitative, this new method should have application to the longitudinal study of allergic pts and to the assessment of interventions.« less

Energy-based culture medium design for biomanufacturing optimization: A case study in monoclonal antibody production by GS-NS0 cells.

PubMed

Quiroga-Campano, Ana L; Panoskaltsis, Nicki; Mantalaris, Athanasios

2018-03-02

Demand for high-value biologics, a rapidly growing pipeline, and pressure from competition, time-to-market and regulators, necessitate novel biomanufacturing approaches, including Quality by Design (QbD) principles and Process Analytical Technologies (PAT), to facilitate accelerated, efficient and effective process development platforms that ensure consistent product quality and reduced lot-to-lot variability. Herein, QbD and PAT principles were incorporated within an innovative in vitro-in silico integrated framework for upstream process development (UPD). The central component of the UPD framework is a mathematical model that predicts dynamic nutrient uptake and average intracellular ATP content, based on biochemical reaction networks, to quantify and characterize energy metabolism and its adaptive response, metabolic shifts, to maintain ATP homeostasis. The accuracy and flexibility of the model depends on critical cell type/product/clone-specific parameters, which are experimentally estimated. The integrated in vitro-in silico platform and the model's predictive capacity reduced burden, time and expense of experimentation resulting in optimal medium design compared to commercially available culture media (80% amino acid reduction) and a fed-batch feeding strategy that increased productivity by 129%. The framework represents a flexible and efficient tool that transforms, improves and accelerates conventional process development in biomanufacturing with wide applications, including stem cell-based therapies. Copyright © 2018. Published by Elsevier Inc.

In vitro and in vivo antitumor effects of the VO-chrysin complex on a new three-dimensional osteosarcoma spheroids model and a xenograft tumor in mice.

PubMed

León, Ignacio E; Cadavid-Vargas, Juan F; Resasco, Agustina; Maschi, Fabricio; Ayala, Miguel A; Carbone, Cecilia; Etcheverry, Susana B

2016-12-01

Osteosarcoma (OS) is the most common primary tumor of bone, occurring predominantly in the second decade of life. High-dose cytotoxic chemotherapy and surgical resection have improved prognosis, with long-term survival for patients with localized disease. Vanadium is an ultra-trace element that after being absorbed accumulates in bone. Besides, vanadium compounds have been studied during recent years to be considered as representative of a new class of non-platinum antitumor agents. Moreover, flavonoids are a wide family of polyphenolic compounds that display many interesting biological effects. Since coordination of ligands to metals can improve the pharmacological properties, we report herein, for the first time, the in vitro and in vivo effects of an oxidovanadium(IV) complex with the flavonoid chrysin on the new 3D human osteosarcoma and xenograft osteosarcoma mice models. The pharmacological results show that VOchrys inhibited the cell viability affecting the shape and volume of the spheroids and VOchrys suppressed MG-63 tumor growth in the nude mice without inducing toxicity and side effects. As a whole, the results presented herein demonstrate that the antitumor action of the complex was very promissory on human osteosarcoma models, whereby suggesting that VOchrys is a potentially good candidate for future use in alternative antitumor treatments.

Influence of different abutment diameter of implants on the peri-implant stress in the crestal bone: A three-dimensional finite element analysis--In vitro study.

PubMed

Aradya, Anupama; Kumar, U Krishna; Chowdhary, Ramesh

2016-01-01

The study was designed to evaluate and compare stress distribution in transcortical section of bone with normal abutment and platform switched abutment under vertical and oblique forces in posterior mandible region. A three-dimensional finite element model was designed using ANSYS 13.0 software. The type of bone selection for the model was made of type II mandibular bone, having cortical bone thickness ranging from 0.595 mm to 1.515 mm with the crestal region measuring 1.5 mm surrounding dense trabecular bone. The implant will be modulated at 5 mm restorative platform and tapering down to 4.5 mm wide at the threads, 13 mm long with an abutment 3 mm in height. The models will be designed for two situations: (1) An implant with a 5 mm diameter abutment representing a standard platform in the posterior mandible region. (2) An implant with a 4.5 mm diameter abutment representing platform switching in the posterior mandible region. Force application was performed in both oblique and vertical conditions using 100 N as a representative masticatory force. For oblique loading, a force of 100 N was applied at 15° from the vertical axis. von Mises stress analysis was evaluated. The results of the study showed cortical stress in the conventional and platform switching model under oblique forces were 59.329 MPa and 39.952 MPa, respectively. Cortical stress in the conventional and platform switching model under vertical forces was 13.914 MPa and 12.793 MPa, respectively. Results from this study showed the platform switched abutment led to relative decrease in von Mises stress in transcortical section of bone compared to normal abutment under vertical and oblique forces in posterior mandible region.

Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints.

PubMed

Terrasso, Ana Paula; Pinto, Catarina; Serra, Margarida; Filipe, Augusto; Almeida, Susana; Ferreira, Ana Lúcia; Pedroso, Pedro; Brito, Catarina; Alves, Paula Marques

2015-07-10

There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers' expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This 3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid prototyping compliant arterial phantoms for in-vitro studies and device testing

PubMed Central

2013-01-01

Background Compliant vascular phantoms are desirable for in-vitro patient-specific experiments and device testing. TangoPlus FullCure 930® is a commercially available rubber-like material that can be used for PolyJet rapid prototyping. This work aims to gather preliminary data on the distensibility of this material, in order to assess the feasibility of its use in the context of experimental cardiovascular modelling. Methods The descending aorta anatomy of a volunteer was modelled in 3D from cardiovascular magnetic resonance (CMR) images and rapid prototyped using TangoPlus. The model was printed with a range of increasing wall thicknesses (0.6, 0.7, 0.8, 1.0 and 1.5 mm), keeping the lumen of the vessel constant. Models were also printed in both vertical and horizontal orientations, thus resulting in a total of ten specimens. Compliance tests were performed by monitoring pressure variations while gradually increasing and decreasing internal volume. Knowledge of distensibility was thus derived and then implemented with CMR data to test two applications. Firstly, a patient-specific compliant model of hypoplastic aorta suitable for connection in a mock circulatory loop for in-vitro tests was manufactured. Secondly, the right ventricular outflow tract (RVOT) of a patient necessitating pulmonary valve replacement was printed in order to physically test device insertion and assess patient’s suitability for percutaneous pulmonary valve intervention. Results The distensibility of the material was identified in a range from 6.5 × 10-3 mmHg-1 for the 0.6 mm case, to 3.0 × 10-3 mmHg-1 for the 1.5 mm case. The models printed in the vertical orientation were always more compliant than their horizontal counterpart. Rapid prototyping of a compliant hypoplastic aorta and of a RVOT anatomical model were both feasible. Device insertion in the RVOT model was successful. Conclusion Values of distensibility, compared with literature data, show that TangoPlus is suitable for manufacturing arterial phantoms, with the added benefit of being compatible with PolyJet printing, thus guaranteeing representative anatomical finishing, and quick and inexpensive fabrication. The appealing possibility of printing models of non-uniform wall thickness, resembling more closely certain physiological scenarios, can also be explored. However, this material appears to be too stiff for modelling the more compliant systemic venous system. PMID:23324211

Rapid prototyping compliant arterial phantoms for in-vitro studies and device testing.

PubMed

Biglino, Giovanni; Verschueren, Peter; Zegels, Raf; Taylor, Andrew M; Schievano, Silvia

2013-01-16

Compliant vascular phantoms are desirable for in-vitro patient-specific experiments and device testing. TangoPlus FullCure 930 is a commercially available rubber-like material that can be used for PolyJet rapid prototyping. This work aims to gather preliminary data on the distensibility of this material, in order to assess the feasibility of its use in the context of experimental cardiovascular modelling. The descending aorta anatomy of a volunteer was modelled in 3D from cardiovascular magnetic resonance (CMR) images and rapid prototyped using TangoPlus. The model was printed with a range of increasing wall thicknesses (0.6, 0.7, 0.8, 1.0 and 1.5 mm), keeping the lumen of the vessel constant. Models were also printed in both vertical and horizontal orientations, thus resulting in a total of ten specimens. Compliance tests were performed by monitoring pressure variations while gradually increasing and decreasing internal volume. Knowledge of distensibility was thus derived and then implemented with CMR data to test two applications. Firstly, a patient-specific compliant model of hypoplastic aorta suitable for connection in a mock circulatory loop for in-vitro tests was manufactured. Secondly, the right ventricular outflow tract (RVOT) of a patient necessitating pulmonary valve replacement was printed in order to physically test device insertion and assess patient's suitability for percutaneous pulmonary valve intervention. The distensibility of the material was identified in a range from 6.5 × 10(-3) mmHg(-1) for the 0.6 mm case, to 3.0 × 10(-3) mmHg(-1) for the 1.5 mm case. The models printed in the vertical orientation were always more compliant than their horizontal counterpart. Rapid prototyping of a compliant hypoplastic aorta and of a RVOT anatomical model were both feasible. Device insertion in the RVOT model was successful. Values of distensibility, compared with literature data, show that TangoPlus is suitable for manufacturing arterial phantoms, with the added benefit of being compatible with PolyJet printing, thus guaranteeing representative anatomical finishing, and quick and inexpensive fabrication. The appealing possibility of printing models of non-uniform wall thickness, resembling more closely certain physiological scenarios, can also be explored. However, this material appears to be too stiff for modelling the more compliant systemic venous system.

[Evaluation on a fast weight reduction model in vitro].

PubMed

Li, Songtao; Li, Ying; Wen, Ying; Sun, Changhao

2010-03-01

To establish a fast and effective model in vitro for screening weight-reducing drugs and taking preliminary evaluation of the model. Mature adipocytes of SD rat induced by oleic acid were used to establish a obesity model in vitro. Isoprel, genistein, caffeine were selected as positive agents and curcumine as negative agent to evaluate the obesity model. Lipolysis of adipocytes was stimulated significantly by isoprel, genistein and caffeine rather than curcumine. This model could be used efficiently for screening weight-losing drugs.

The cytokine-dependent MUTZ-3 cell line as an in vitro model for the screening of contact sensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azam, Philippe; Peiffer, Jean-Luc; Chamousset, Delphine

2006-04-01

Langerhans cells (LC) are key mediators of contact allergenicity in the skin. However, no in vitro methods exist which are based on the activation process of LC to predict the sensitization potential of chemicals. In this study, we have evaluated the performances of MUTZ-3, a cytokine-dependent human monocytic cell line, in its response to sensitizers. First, we compared undifferentiated MUTZ-3 cells with several standard human cells such as THP-1, KG-1, HL-60, K-562, and U-937 in their response to the strong sensitizer DNCB and the irritant SDS by monitoring the expression levels of HLA-DR, CD54, and CD86 by flow cytometry. Onlymore » MUTZ-3 and THP-1 cells show a strong and specific response to sensitizer, while other cell lines showed very variable responses. Then, we tested MUTZ-3 cells against a wider panel of sensitizers and irritants on a broader spectrum of cell surface markers (HLA-DR, CD40, CD54, CD80, CD86, B7-H1, B7-H2, B7-DC). Of these markers, CD86 proved to be the most reliable since it detected all sensitizers, including benzocaine, a classical false negative in local lymph node assay (LLNA) but not irritants. We confirmed the MUTZ-3 response to DNCB by real-time PCR analysis. Taken together, our data suggest that undifferentiated MUTZ-3 cells may represent a valuable in vitro model for the screening of potential sensitizers.« less

A method to improve the efficacy of topical eflornithine hydrochloride cream

PubMed Central

Kumar, Amit; Naguib, Youssef W.; Shi, Yan-chun; Cui, Zhengrong

2015-01-01

Context Facial hirsutism is a cosmetic concern for women and can lead to significant anxiety and lack of self-esteem. Eflornithine cream is indicated for the treatment of facial hirsutism. However, limited success rate and overall patient’s satisfaction, even with a long-term and high frequency application, leave room for improvement. Objective The objective of this study is to test the effect of microneedle treatment on the in vitro skin permeation and the in vivo efficacy of eflornithine cream in a mouse model. Materials and method In vitro permeation study of eflornithine was performed using Franz diffusion cell. In vivo efficacy study was performed in a mouse model by monitoring the re-growth of hair in the lower dorsal skin of mice after the eflornithine cream was applied onto an area pretreated with microneedles. The skin and the hair follicles in the treated area were also examined histologically. Results and discussion The hair growth inhibitory activity of eflornithine was significantly enhanced when the eflornithine cream was applied onto a mouse skin area pretreated with microneedles, most likely because the micropores created by microneedles allowed the permeation of eflornithine into the skin, as confirmed in an in vitro permeation study. Immunohistochemistry data revealed that cell proliferation in the skin and hair follicles was also significantly inhibited when the eflornithine cream was applied onto a skin area pretreated with microneedles. Conclusion The integration of microneedle treatment into topical eflornithine therapy represents a potentially viable approach to increase eflornithine’s ability to inhibit hair growth. PMID:25182303

Recording axonal conduction to evaluate the integration of pluripotent cell-derived neurons into a neuronal network.

PubMed

Shimba, Kenta; Sakai, Koji; Takayama, Yuzo; Kotani, Kiyoshi; Jimbo, Yasuhiko

2015-10-01

Stem cell transplantation is a promising therapy to treat neurodegenerative disorders, and a number of in vitro models have been developed for studying interactions between grafted neurons and the host neuronal network to promote drug discovery. However, methods capable of evaluating the process by which stem cells integrate into the host neuronal network are lacking. In this study, we applied an axonal conduction-based analysis to a co-culture study of primary and differentiated neurons. Mouse cortical neurons and neuronal cells differentiated from P19 embryonal carcinoma cells, a model for early neural differentiation of pluripotent stem cells, were co-cultured in a microfabricated device. The somata of these cells were separated by the co-culture device, but their axons were able to elongate through microtunnels and then form synaptic contacts. Propagating action potentials were recorded from these axons by microelectrodes embedded at the bottom of the microtunnels and sorted into clusters representing individual axons. While the number of axons of cortical neurons increased until 14 days in vitro and then decreased, those of P19 neurons increased throughout the culture period. Network burst analysis showed that P19 neurons participated in approximately 80% of the bursting activity after 14 days in vitro. Interestingly, the axonal conduction delay of P19 neurons was significantly greater than that of cortical neurons, suggesting that there are some physiological differences in their axons. These results suggest that our method is feasible to evaluate the process by which stem cell-derived neurons integrate into a host neuronal network.

Overactive and Underactive Bladder Dysfunction is Reflected by Alterations in Urothelial ATP and NO Release

PubMed Central

Munoz, Alvaro; Smith, Christopher P.; Boone, Timothy B.; Somogyi, George T.

2011-01-01

ATP and NO are released from the urothelium in the bladder. Detrusor Overactivity (DO) following spinal cord injury results in higher ATP and lower NO release from the bladder urothelium. Our aim was to study the relationship between ATP and NO release in 1) early diabetic bladders, an overactive bladder model; and 2) in “diuretic” bladders, an underactive bladder model. To induce diabetes mellitus female rats received 65 mg/kg streptozocin (i.v.). To induce chronic diuresis rats were fed with 5% sucrose. At 28 days, in vivo open cystometry was performed. Bladder wash was collected to analyze the amount of ATP and NO released into the bladder lumen. For in vitro analysis of ATP and NO release, a Ussing chamber was utilized and hypoosmotic Krebs was perfused on the urothelial side of the chamber. ATP was analyzed with luminometry or HPLC-fluorometry while NO was measured with a Sievers NO-analyzer. In vivo ATP release was increased in diabetic bladders and unchanged in diuretic bladders. In vitro release from the urothelium followed the same pattern. NO release was unchanged both in vitro and in vivo in overactive bladders whereas it was enhanced in underactive bladders. We found that the ratio of ATP/NO, representing sensory transmission in the bladder, was high in overactive and low in underactive bladder dysfunction. In summary, ATP release has a positive correlation while NO release has a negative correlation with the bladder contraction frequency. The urinary ATP/NO ratio may be a clinically relevant biomarker to characterize the extent of bladder dysfunction. PMID:21145365

Characterization of the anti-inflammatory properties of NCX 429, a dual-acting compound releasing nitric oxide and naproxen.

PubMed

Amoruso, Angela; Fresu, Luigia Grazia; Dalli, Jesmond; Miglietta, Daniela; Bardelli, Claudio; Federici Canova, Donata; Perretti, Mauro; Brunelleschi, Sandra

2015-04-01

Cyclooxygenase (COX)-inhibiting nitric oxide donors (CINODs) are a new class of drugs that structurally combine a COX inhibitor with a nitric oxide (NO) donating moiety. This combination reduces potential toxicity of the non-steroidal anti-inflammatory drugs (NSAIDs) whilst maintaining the analgesic and anti-inflammatory effects. The present study was undertaken to investigate the anti-inflammatory effects of NCX 429, a naproxen-based CINOD, and to assess the additional properties of NO donation beyond those related to naproxen. We evaluated the in vitro effects of NCX 429 on oxy-radical production, phagocytosis, cytokine release, MMP-9, PPARγ expression and NF-κB activation in human monocytes/MDM and compared to naproxen. Moreover, we compared the in vivo efficacy of NCX 429 and naproxen in a murine model of peritonitis. In all the experiments performed in vitro, NCX 429 reduced the inflammatory responses with equal or higher efficacy compared to naproxen. Moreover, in in vivo experiments, NCX 429, at the lowest dose tested, was able to significantly inhibit cell influx in response to IL-1β administration although naproxen was found to be more potent than NCX 429 at reducing PGE2 in inflammatory exudates. These results demonstrate that both in vitro and in vivo--in a murine model of peritonitis--NCX 429 elicits significant anti-inflammatory activity, beyond the simple COX inhibition or pure NO release. Therefore, NO donation along with COX inhibition may represent a strategy for investigating inflammatory diseases in which pain and function are not fully resolved by analgesics/anti-inflammatory drugs. © 2015.

Insights on in vitro models for safety and toxicity assessment of cosmetic ingredients.

PubMed

Almeida, Andreia; Sarmento, Bruno; Rodrigues, Francisca

2017-03-15

According to the current European legislation, the safety assessment of each individual cosmetic ingredient of any formulation is the basis for the safety evaluation of a cosmetic product. Also, animal testing in the European Union is prohibited for cosmetic ingredients and products since 2004 and 2009, respectively. Additionally, the commercialization of any cosmetic products containing ingredients tested on animal models was forbidden in 2009. In consequence of these boundaries, the European Centre for the Validation of Alternative Methods (ECVAM) proposes a list of validated cell-based in vitro models for predicting the safety and toxicity of cosmetic ingredients. These models have been demonstrated as valuable and effective tools to overcome the limitations of animal in vivo studies. Although the use of in vitro cell-based models for the evaluation of absorption and permeability of cosmetic ingredients is widespread, a detailed study on the properties of these platforms and the in vitro-in vivo correlation compared with human data are required. Moreover, additional efforts must be taken to develop in vitro models to predict carcinogenicity, repeat dose toxicity and reproductive toxicity, for which no alternative in vitro methods are currently available. This review paper summarizes and characterizes the most relevant in vitro models validated by ECVAM employed to predict the safety and toxicology of cosmetic ingredients. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of bacteriophage therapy to control Clostridium difficile and toxin production in an in vitro human colon model system.

PubMed

Meader, Emma; Mayer, Melinda J; Steverding, Dietmar; Carding, Simon R; Narbad, Arjan

2013-08-01

Clostridium difficile is a leading cause of hospital-acquired diarrhoea and represents a major challenge for healthcare providers. Due to the decreasing efficacy and associated problems of antibiotic therapy there is a need for synergistic and alternative treatments. In this study we investigated the use of a specific bacteriophage, ΦCD27, in a human colon model of C. difficile infection. Our findings demonstrate a significant reduction in the burden of C. difficile cells and toxin production with phage treatment relative to an untreated control, with no detrimental effect on commensal bacterial populations. The results demonstrate the potential of phage therapy, and highlight the limitations of using phages that have lysogenic capacity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of In Vitro-In Vivo Correlation/Relationship Modeling Approaches for Immediate Release Formulations Using Compartmental Dynamic Dissolution Data from “Golem”: A Novel Apparatus

PubMed Central

Tuszyński, Paweł K.; Polak, Sebastian; Jachowicz, Renata; Mendyk, Aleksander; Dohnal, Jiří

2015-01-01

Different batches of atorvastatin, represented by two immediate release formulation designs, were studied using a novel dynamic dissolution apparatus, simulating stomach and small intestine. A universal dissolution method was employed which simulated the physiology of human gastrointestinal tract, including the precise chyme transit behavior and biorelevant conditions. The multicompartmental dissolution data allowed direct observation and qualitative discrimination of the differences resulting from highly pH dependent dissolution behavior of the tested batches. Further evaluation of results was performed using IVIVC/IVIVR development. While satisfactory correlation could not be achieved using a conventional deconvolution based-model, promising results were obtained through the use of a nonconventional approach exploiting the complex compartmental dissolution data. PMID:26120580

Neurochemical measurements in the zebrafish brain

PubMed Central

Jones, Lauren J.; McCutcheon, James E.; Young, Andrew M. J.; Norton, William H. J.

2015-01-01

The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain. PMID:26441575

A Multiscale Approach to Blast Neurotrauma Modeling: Part I – Development of Novel Test Devices for in vivo and in vitro Blast Injury Models

PubMed Central

Panzer, Matthew B.; Matthews, Kyle A.; Yu, Allen W.; Morrison, Barclay; Meaney, David F.; Bass, Cameron R.

2012-01-01

The loading conditions used in some current in vivo and in vitro blast-induced neurotrauma models may not be representative of real-world blast conditions. To address these limitations, we developed a compressed-gas driven shock tube with different driven lengths that can generate Friedlander-type blasts. The shock tube can generate overpressures up to 650 kPa with durations between 0.3 and 1.1 ms using compressed helium driver gas, and peak overpressures up to 450 kPa with durations between 0.6 and 3 ms using compressed nitrogen. This device is used for short-duration blast overpressure loading for small animal in vivo injury models, and contrasts the more frequently used long duration/high impulse blast overpressures in the literature. We also developed a new apparatus that is used with the shock tube to recreate the in vivo intracranial overpressure response for loading in vitro culture preparations. The receiver device surrounds the culture with materials of similar impedance to facilitate the propagation of a single overpressure pulse through the tissue. This method prevents pressure waves reflecting off the tissue that can cause unrealistic deformation and injury. The receiver performance was characterized using the longest helium-driven shock tube, and produced in-fluid overpressures up to 1500 kPa at the location where a culture would be placed. This response was well correlated with the overpressure conditions from the shock tube (R2 = 0.97). Finite element models of the shock tube and receiver were developed and validated to better elucidate the mechanics of this methodology. A demonstration exposing a culture to the loading conditions created by this system suggest tissue strains less than 5% for all pressure levels simulated, which was well below functional deficit thresholds for strain rates less than 50 s−1. This novel system is not limited to a specific type of culture model and can be modified to reproduce more complex pressure pulses. PMID:22470367

Agent-Based Computational Modeling of Cell Culture: Understanding Dosimetry In Vitro as Part of In Vitro to In Vivo Extrapolation

EPA Science Inventory

Quantitative characterization of cellular dose in vitro is needed for alignment of doses in vitro and in vivo. We used the agent-based software, CompuCell3D (CC3D), to provide a stochastic description of cell growth in culture. The model was configured so that isolated cells assu...

Osteosarcoma Models: From Cell Lines to Zebrafish

PubMed Central

Mohseny, Alexander B.; Hogendoorn, Pancras C. W.; Cleton-Jansen, Anne-Marie

2012-01-01

High-grade osteosarcoma is an aggressive tumor most commonly affecting adolescents. The early age of onset might suggest genetic predisposition; however, the vast majority of the tumors are sporadic. Early onset, most often lack of a predisposing condition or lesion, only infrequent (<2%) prevalence of inheritance, extensive genomic instability, and a wide histological heterogeneity are just few factors to mention that make osteosarcoma difficult to study. Therefore, it is sensible to design and use models representative of the human disease. Here we summarize multiple osteosarcoma models established in vitro and in vivo, comment on their utilities, and highlight newest achievements, such as the use of zebrafish embryos. We conclude that to gain a better understanding of osteosarcoma, simplification of this extremely complex tumor is needed. Therefore, we parse the osteosarcoma problem into parts and propose adequate models to study them each separately. A better understanding of osteosarcoma provides opportunities for discovering and assaying novel effective treatment strategies. “Sometimes the model is more interesting than the original disease” PJ Hoedemaeker (1937–2007). PMID:22566751

The Volume of Three-Dimensional Cultures of Cancer Cells InVitro Influences Transcriptional Profile Differences and Similarities with Monolayer Cultures and Xenografted Tumors.

PubMed

Boghaert, Erwin R; Lu, Xin; Hessler, Paul E; McGonigal, Thomas P; Oleksijew, Anatol; Mitten, Michael J; Foster-Duke, Kelly; Hickson, Jonathan A; Santo, Vitor E; Brito, Catarina; Uziel, Tamar; Vaidya, Kedar S

2017-09-01

Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro. Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of applying these models for drug development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Fibronectin connecting segment-1 peptide inhibits pathogenic leukocyte trafficking and inflammatory demyelination in experimental models of chronic inflammatory demyelinating polyradiculoneuropathy.

PubMed

Dong, Chaoling; Greathouse, Kelsey M; Beacham, Rebecca L; Palladino, Steven P; Helton, E Scott; Ubogu, Eroboghene E

2017-06-01

The molecular determinants of pathogenic leukocyte migration across the blood-nerve barrier (BNB) in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) are unknown. Specific disease modifying therapies for CIDP are also lacking. Fibronectin connecting segment-1 (FNCS1), an alternatively spliced fibronectin variant expressed by microvascular endothelial cells at sites of inflammation in vitro and in situ, is a counterligand for leukocyte α 4 integrin (also known as CD49d) implicated in pathogenic leukocyte trafficking in multiple sclerosis and inflammatory bowel disease. We sought to determine the role of FNCS1 in CIDP patient leukocyte trafficking across the BNB in vitro and in severe chronic demyelinating neuritis in vivo using a representative spontaneous murine CIDP model. Peripheral blood mononuclear leukocytes from 7 untreated CIDP patients were independently infused into a cytokine-treated, flow-dependent in vitro BNB model system. Time-lapse digital video microscopy was performed to visualize and quantify leukocyte trafficking, comparing FNCS1 peptide blockade to relevant controls. Fifty 24-week old female B7-2 deficient non-obese diabetic mice with spontaneous autoimmune peripheral polyneuropathy (SAPP) were treated daily with 2mg/kg FNCS1 peptide for 5days via intraperitoneal injection with appropriate controls. Neurobehavioral measures of disease severity, motor nerve electrophysiology assessments and histopathological quantification of inflammation and morphometric assessment of demyelination were performed to determine in vivo efficacy. The biological relevance of FNCS1 and CD49d in CIDP was evaluated by immunohistochemical detection in affected patient sural nerve biopsies. 25μM FNCS1 peptide maximally inhibited CIDP leukocyte trafficking at the human BNB in vitro. FNCS1 peptide treatment resulted in significant improvements in disease severity, motor electrophysiological parameters of demyelination and histological measures of inflammatory demyelination. Microvessels demonstrating FNCS1 expression and CD49d+ leukocytes were seen within the endoneurium of patient nerve biopsies. Taken together, these results imply a role for FNCS1 in pathogenic leukocyte trafficking in CIDP, providing a potential target for therapeutic modulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Development of an in vitro cell culture model to study milk to plasma ratios of therapeutic drugs.

PubMed

Athavale, Maithili A; Maitra, Anurupa; Patel, Shahnaz; Bhate, Vijay R; Toddywalla, Villi S

2013-01-01

To create an in vitro cell culture model to predict the M/P (concentration of drug in milk/concentration in maternal plasma) ratios of therapeutic drugs viz. rifampicin, theophylline, paracetamol, and aspirin. An in vitro cell culture model using CIT3 cells (mouse mammary epithelial cells) was created by culturing the cells on transwells. The cells formed an integral monolayer, allowing only transcellular transport as it happens in vivo. Functionality of the cells was confirmed through scanning electron microscopy. Time wise transfer of the study drugs from plasma to milk was studied and compared with actual (in vivo) M/P ratios obtained at reported tmax for the respective drugs. The developed model mimicked two important intrinsic factors of mammary epithelial cells viz. secretory and tight-junction properties and also the passive route of drug transport. The in vitro M/P ratios at reported tmax were 0.23, 0.61, 0.87, and 0.03 respectively, for rifampicin, theophylline, paracetamol, and salicylic acid as compared to 0.29, 0.65, 0.65, and 0.22, respectively, in vitro. Our preliminary effort to develop an in vitro physiological model showed promising results. Transfer rate of the drugs using the developed model compared well with the transfer potential seen in vivo except for salicylic acid, which was transferred in far lower concentration in vitro. The model has a potential to be developed as a non-invasive alternative to the in vitro technique for determining the transfer of therapeutic drugs into breast milk.

IN VITRO SCREENING BATTERIES FOR NEUROTOXICANTS

EPA Science Inventory

The need to develop, validate and utilize in vitro models to test chemicals for neurotoxic potential is widely appreciated. his lecture discusses the major advantages and disadvantages of using cell and tissue culture, the carious in vitro models amenable for neurotoxicity studie...

In Silico and in Vitro Modeling of Hepatocyte Drug Transport Processes: Importance of ABCC2 Expression Levels in the Disposition of CarboxydichloroflurosceinS⃞

PubMed Central

Howe, Katharine; Gibson, G. Gordon; Coleman, Tanya; Plant, Nick

2009-01-01

The impact of transport proteins in the disposition of chemicals is becoming increasingly evident. Alteration in disposition can cause altered pharmacokinetic and pharmacodynamic parameters, potentially leading to reduced efficacy or overt toxicity. We have developed a quantitative in silico model, based upon literature and experimentally derived data, to model the disposition of carboxydichlorofluroscein (CDF), a substrate for the SLCO1A/B and ABCC subfamilies of transporters. Kinetic parameters generated by the in silico model closely match both literature and experimentally derived kinetic values, allowing this model to be used for the examination of transporter action in primary rat hepatocytes. In particular, we show that the in silico model is suited to the rapid, accurate determination of Ki values, using 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571) as a prototypical pan-ABCC inhibitor. In vitro-derived data are often used to predict in vivo response, and we have examined how differences in protein expression levels between these systems may affect chemical disposition. We show that ABCC2 and ABCC3 are overexpressed in sandwich culture hepatocytes by 3.5- and 2.3-fold, respectively, at the protein level. Correction for this in markedly different disposition of CDF, with the area under the concentration versus time curve and Cmax of intracellular CDF increasing by 365 and 160%, respectively. Finally, using kinetic simulations we show that ABCC2 represents a fragile node within this pathway, with alterations in ABCC2 having the most prominent effects on both the Km and Vmax through the pathway. This is the first demonstration of the utility of modeling approaches to estimate the impact of drug transport processes on chemical disposition. PMID:19022944

Elasticity-based development of functionally enhanced multicellular 3D liver encapsulated in hybrid hydrogel.

PubMed

Lee, Ho-Joon; Son, Myung Jin; Ahn, Jiwon; Oh, Soo Jin; Lee, Mihee; Kim, Ansoon; Jeung, Yun-Ji; Kim, Han-Gyeul; Won, Misun; Lim, Jung Hwa; Kim, Nam-Soon; Jung, Cho-Rock; Chung, Kyung-Sook

2017-12-01

Current in vitro liver models provide three-dimensional (3-D) microenvironments in combination with tissue engineering technology and can perform more accurate in vivo mimicry than two-dimensional models. However, a human cell-based, functionally mature liver model is still desired, which would provide an alternative to animal experiments and resolve low-prediction issues on species differences. Here, we prepared hybrid hydrogels of varying elasticity and compared them with a normal liver, to develop a more mature liver model that preserves liver properties in vitro. We encapsulated HepaRG cells, either alone or with supporting cells, in a biodegradable hybrid hydrogel. The elastic modulus of the 3D liver dynamically changed during culture due to the combined effects of prolonged degradation of hydrogel and extracellular matrix formation provided by the supporting cells. As a result, when the elastic modulus of the 3D liver model converges close to that of the in vivo liver (≅ 2.3 to 5.9 kPa), both phenotypic and functional maturation of the 3D liver were realized, while hepatic gene expression, albumin secretion, cytochrome p450-3A4 activity, and drug metabolism were enhanced. Finally, the 3D liver model was expanded to applications with embryonic stem cell-derived hepatocytes and primary human hepatocytes, and it supported prolonged hepatocyte survival and functionality in long-term culture. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. We provide a functionally improved 3D liver model that recapitulates in vivo liver stiffness. We have experimentally addressed the issues of orchestrated effects of mechanical compliance, controlled matrix formation by stromal cells in conjunction with hepatic differentiation, and functional maturation of hepatocytes in a dynamic 3D microenvironment. Our model represents critical progress in developing a biomimetic liver system to simulate liver tissue remodeling, and provides a versatile platform in drug development and disease modeling, ranging from physiology to pathology. Additionally, recent advances in the stem-cell technologies have made the development of 3D organoid possible, and thus, our study also provides further contribution to the development of physiologically relevant stem-cell-based 3D tissues that provide an elasticity-based predefined biomimetic 3D microenvironment. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Synchronous Oscillations in Microtubule Polymerization

NASA Astrophysics Data System (ADS)

Carlier, M. F.; Melki, R.; Pantaloni, D.; Hill, T. L.; Chen, Y.

1987-08-01

Under conditions where microtubule nucleation and growth are fast (i.e., high magnesium ion and tubulin concentrations and absence of glycerol), microtubule assembly in vitro exhibits an oscillatory regime preceding the establishment of steady state. The amplitude of the oscillations can represent >50% of the maximum turbidity change and oscillations persist for up to 20 periods of 80 s each. Oscillations are accompanied by extensive length redistribution of microtubules. Preliminary work suggests that the oscillatory kinetics can be simulated using a model in which many microtubules undergo synchronous transitions between growing and rapidly depolymerizing phases, complicated by the kinetically limiting rate of nucleotide exchange on free tubulin.

Biological agents for controlling excessive scarring.

PubMed

Berman, Brian

2010-01-01

The potential of various biological agents to reduce or prevent excessive scar formation has now been evaluated in numerous in-vitro studies, experimental animal models and preliminary clinical trials, in some cases with particularly promising results. Perhaps prominent among this group of biological agents, and, to some degree, possibly representing marketed compounds already being used 'off label' to manage excessive scarring, are the tumor necrosis factor alpha antagonist etanercept, and immune-response modifiers such as IFNalpha2b and imiquimod. Additional assessment of these novel agents is now justified with a view to reducing or preventing hypertrophic scars, keloid scars and the recurrence of post-excision keloid lesions.

Pyrimidine sulfonylacetanilides with improved potency against key mutant viruses of HIV-1 by specific targeting of a highly conserved residue.

PubMed

Wan, Zheng-Yong; Yao, Jin; Mao, Tian-Qi; Wang, Xin-Long; Wang, Hai-Feng; Chen, Wen-Xue; Yin, Hong; Chen, Fen-Er; De Clercq, Erik; Daelemans, Dirk; Pannecouque, Christophe

2015-09-18

Based on molecular simulation, the etravirine-VRX-480773 hybrids previously disclosed by our group were optimized to yield novel pyrimidine sulfonylacetanilides 8 with improved activity against a panel of seven clinically relevant single and double mutant strains of HIV-1. The improvement in potency in this in vitro model of HIV RNA replication partly validates the mechanism by which this class of allosteric pyrimidine derivatives inhibits the reverse transcriptase (RT), and represents a remarkable step forward in the development of anti-HIV drugs. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Varic acid analogues from fungus as PTP1B inhibitors: Biological evaluation and structure-activity relationships.

PubMed

Sun, Wenlong; Zhuang, Chunlin; Li, Xia; Zhang, Bowei; Lu, Xinhua; Zheng, Zhihui; Dong, Yuesheng

2017-08-01

Protein tyrosine phosphatase 1B (PTP1B) inhibitors as potential therapies for diabetes and obesity have attracted much attention in recent years. Six varic acid analogues were isolated from two strains of fungi and evaluated for PTP1B inhibition activities. The structure-activity relationships were also characterized and predicted by molecular modeling. Further kinetic studies indicated the reversible and competitive inhibition manner of varic acid analogues. Trivaric acid showed insulin-sensitizing effect not only in vitro but also in vivo, representing a promising lead compound for further optimization. Copyright © 2017 Elsevier Ltd. All rights reserved.

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus

PubMed Central

Rodriguez, Michelle D.; Paul, Zubin; Wood, Charles E.; Rice, Kelly C.; Triplett, Eric W.

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus. These three reporter plasmids are available through BEI Resources. PMID:29312199

Construction of Stable Fluorescent Reporter Plasmids for Use in Staphylococcus aureus.

PubMed

Rodriguez, Michelle D; Paul, Zubin; Wood, Charles E; Rice, Kelly C; Triplett, Eric W

2017-01-01

Here, the genes encoding three different fluorescent proteins were cloned into the stably maintained Staphylococcus aureus shuttle vector pKK30. The resulting plasmids were transformed into two S. aureus strains; SH1000 and RN4220. Stability assays illustrated that the three recombinant plasmids retained near 100% maintenance in vitro for 160 generations. S. aureus strain SH1000 expressing green fluorescent protein was then inoculated in an ovine model and in vivo stability for 6 days was demonstrated. In essence, these reporter plasmids represent a useful set of tools for dynamic imaging studies in S. aureus . These three reporter plasmids are available through BEI Resources.

An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates.

PubMed

Molle, Virginie; Leiba, Jade; Zanella-Cléon, Isabelle; Becchi, Michel; Kremer, Laurent

2010-11-01

Identification of the phosphorylated residues of bacterial Ser/Thr protein kinase (STPK) substrates still represents a challenging task. Herein, we present a new strategy allowing the rapid determination of phosphoacceptors in kinase substrates, essentially based on the dual expression of the kinase with its substrate in the surrogate E. coli, followed by MS analysis in a single-step procedure. The performance of this strategy is illustrated using two distinct proteins from Mycobacterium tuberculosis as model substrates, the GroEL2 and HspX chaperones. A comparative analysis with a standard method that includes mass spectrometry analysis of in vitro phosphorylated substrates is also addressed.

In vitro porcine blood-brain barrier model for permeability studies: pCEL-X software pKa(FLUX) method for aqueous boundary layer correction and detailed data analysis.

PubMed

Yusof, Siti R; Avdeef, Alex; Abbott, N Joan

2014-12-18

In vitro blood-brain barrier (BBB) models from primary brain endothelial cells can closely resemble the in vivo BBB, offering valuable models to assay BBB functions and to screen potential central nervous system drugs. We have recently developed an in vitro BBB model using primary porcine brain endothelial cells. The model shows expression of tight junction proteins and high transendothelial electrical resistance, evidence for a restrictive paracellular pathway. Validation studies using small drug-like compounds demonstrated functional uptake and efflux transporters, showing the suitability of the model to assay drug permeability. However, one limitation of in vitro model permeability measurement is the presence of the aqueous boundary layer (ABL) resulting from inefficient stirring during the permeability assay. The ABL can be a rate-limiting step in permeation, particularly for lipophilic compounds, causing underestimation of the permeability. If the ABL effect is ignored, the permeability measured in vitro will not reflect the permeability in vivo. To address the issue, we explored the combination of in vitro permeability measurement using our porcine model with the pKa(FLUX) method in pCEL-X software to correct for the ABL effect and allow a detailed analysis of in vitro (transendothelial) permeability data, Papp. Published Papp using porcine models generated by our group and other groups are also analyzed. From the Papp, intrinsic transcellular permeability (P0) is derived by simultaneous refinement using a weighted nonlinear regression, taking into account permeability through the ABL, paracellular permeability and filter restrictions on permeation. The in vitro P0 derived for 22 compounds (35 measurements) showed good correlation with P0 derived from in situ brain perfusion data (r(2)=0.61). The analysis also gave evidence for carrier-mediated uptake of naloxone, propranolol and vinblastine. The combination of the in vitro porcine model and the software analysis provides a useful tool to better predict BBB permeability in vivo and gain better mechanistic information about BBB permeation. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

A first step towards a consensus static in vitro model for simulating full-term infant digestion.

PubMed

Ménard, O; Bourlieu, C; De Oliveira, S C; Dellarosa, N; Laghi, L; Carrière, F; Capozzi, F; Dupont, D; Deglaire, A

2018-02-01

In vitro alternatives to clinical trials are used for studying human food digestion. For simulating infant digestion, only a few models, lacking physiological relevance, are available. Thanks to an extensive literature review of the in vivo infant digestive conditions, a gastrointestinal static in vitro model was developed for infants born at term and aged 28days. The model was applied to the digestion of a commercial infant formula. Kinetics of digestion, as well as the structural evolution, were compared with those obtained while submitting the same formula to the adult international consensus protocol of in vitro static digestion. The kinetics of proteolysis and lipolysis differed according to the physiological stage resulting mainly from the reduced level of enzymes and bile salts, as well as the higher gastric pH in the infant model. This in vitro static model of infant digestion is of interest for scientists, food or pharmaceutical manufacturers. Copyright © 2017 Elsevier Ltd. All rights reserved.

In vitro pharmacokinetic/pharmacodynamic models in anti-infective drug development: focus on TB

PubMed Central

Vaddady, Pavan K; Lee, Richard E; Meibohm, Bernd

2011-01-01

For rapid anti-tuberculosis (TB) drug development in vitro pharmacokinetic/pharmacodynamic (PK/PD) models are useful in evaluating the direct interaction between the drug and the bacteria, thereby guiding the selection of candidate compounds and the optimization of their dosing regimens. Utilizing in vivo drug-clearance profiles from animal and/or human studies and simulating them in an in vitro PK/PD model allows the in-depth characterization of antibiotic activity of new and existing antibacterials by generating time–kill data. These data capture the dynamic interplay between mycobacterial growth and changing drug concentration as encountered during prolonged drug therapy. This review focuses on important PK/PD parameters relevant to anti-TB drug development, provides an overview of in vitro PK/PD models used to evaluate the efficacy of agents against mycobacteria and discusses the related mathematical modeling approaches of time–kill data. Overall, it provides an introduction to in vitro PK/PD models and their application as critical tools in evaluating anti-TB drugs. PMID:21359155

Coupling of computer modeling with in vitro methodologies to reduce animal usage in toxicity testing.

PubMed

Clewell, H J

1993-05-01

The use of in vitro data to support the development of physiologically based pharmacokinetic (PBPK) models and to reduce the requirement for in vivo testing is demonstrated by three examples. In the first example, polychlorotrifluoroethylene, in vitro studies comparing metabolism and tissue response in rodents and primates made it possible to obtain definitive data for a human risk assessment without resorting to additional in vivo studies with primates. In the second example, a PBPK model for organophosphate esters was developed in which the parameters defining metabolism, tissue partitioning, and enzyme inhibition were all characterized by in vitro studies, and the rest of the model parameters were established from the literature. The resulting model was able to provide a coherent description of enzyme inhibition following both acute and chronic exposures in mice, rats, and humans. In the final example, the carcinogenic risk assessment for methylene chloride was refined by the incorporation of in vitro data on human metabolism into a PBPK model.

Alternative Reproductive Technologies: Implications for Children and Families. Hearing before the Select Committee on Children, Youth, and Families. House of Representatives, One Hundredth Congress, First Session (May 21, 1987).

ERIC Educational Resources Information Center

Congress of the U.S., Washington, DC. House Select Committee on Children, Youth, and Families.

A hearing was held for the purpose of receiving testimony about alternative reproductive technologies and their implications for children, families, and society. Testimony provided: (1) a comparison of in vitro fertilization and gamete intrafallopian transfer, and trends in in vitro fertilization; (2) a summary of definitions, statistics, and the…

Mechanical testing of bones: the positive synergy of finite-element models and in vitro experiments.

PubMed

Cristofolini, Luca; Schileo, Enrico; Juszczyk, Mateusz; Taddei, Fulvia; Martelli, Saulo; Viceconti, Marco

2010-06-13

Bone biomechanics have been extensively investigated in the past both with in vitro experiments and numerical models. In most cases either approach is chosen, without exploiting synergies. Both experiments and numerical models suffer from limitations relative to their accuracy and their respective fields of application. In vitro experiments can improve numerical models by: (i) preliminarily identifying the most relevant failure scenarios; (ii) improving the model identification with experimentally measured material properties; (iii) improving the model identification with accurately measured actual boundary conditions; and (iv) providing quantitative validation based on mechanical properties (strain, displacements) directly measured from physical specimens being tested in parallel with the modelling activity. Likewise, numerical models can improve in vitro experiments by: (i) identifying the most relevant loading configurations among a number of motor tasks that cannot be replicated in vitro; (ii) identifying acceptable simplifications for the in vitro simulation; (iii) optimizing the use of transducers to minimize errors and provide measurements at the most relevant locations; and (iv) exploring a variety of different conditions (material properties, interface, etc.) that would require enormous experimental effort. By reporting an example of successful investigation of the femur, we show how a combination of numerical modelling and controlled experiments within the same research team can be designed to create a virtuous circle where models are used to improve experiments, experiments are used to improve models and their combination synergistically provides more detailed and more reliable results than can be achieved with either approach singularly.

Mimicking brain tissue binding in an in vitro model of the blood-brain barrier illustrates differences between in vitro and in vivo methods for assessing the rate of brain penetration.

PubMed

Heymans, Marjolein; Sevin, Emmanuel; Gosselet, Fabien; Lundquist, Stefan; Culot, Maxime

2018-06-01

Assessing the rate of drug delivery to the central nervous system (CNS) in vitro has been used for decades to predict whether CNS drug candidates are likely to attain their pharmacological targets, located within the brain parenchyma, at an effective dose. The predictive value of in vitro blood-brain barrier (BBB) models is therefore frequently assessed by comparing in vitro BBB permeability, usually quoted as the endothelial permeability coefficient (P e ) or apparent permeability (P app ), to their rate of BBB permeation measured in vivo, the latter being commonly assessed in rodents. In collaboration with AstraZeneca (DMPK department, Södertälje, Sweden), the in vitro BBB permeability (P app and P e ) of 27 marketed CNS drugs has been determined using a bovine in vitro BBB model and compared to their in vivo permeability (P vivo ), obtained by rat in-situ brain perfusion. The latter was taken from published data from Summerfield et al. (2007). This comparison confirmed previous reports, showing a strong in vitro/in vivo correlation for hydrophilic compounds, characterized by low brain tissue binding and a weak correlation for lipophilic compounds, characterized by high brain tissue binding. This observation can be explained by the influence of brain tissue binding on the uptake of drugs into the CNS in vivo and the absence of possible brain tissue binding in vitro. The use of glial cells (GC) in the in vitro BBB model to mimic brain tissue binding and the introduction of a new calculation method for in vitro BBB permeability (P vitro ) resulted in a strong correlation between the in vitro and in vivo rate of BBB permeation for the whole set of compounds. These findings might facilitate further in vitro to in vivo extrapolation for CNS drug candidates. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Canine osteosarcoma cell lines contain stem-like cancer cells: biological and pharmacological characterization.

PubMed

Gatti, Monica; Wurth, Roberto; Vito, Guendalina; Pattarozzi, Alessandra; Campanella, Chiara; Thellung, Stefano; Maniscalco, Lorella; De Maria, Raffaella; Villa, Valentina; Corsaro, Alessandro; Nizzari, Mario; Bajetto, Adriana; Ratto, Alessandra; Ferrari, Angelo; Barbieri, Federica; Florio, Tullio

2016-05-01

Cancer stem cells (CSCs) represent a small subpopulation of cells responsible for tumor formation and progression, drug resistance, tumor recurrence and metastasization. CSCs have been identified in many human tumors including osteosarcoma (OSA). CSC distinctive properties are the expression of stem cell markers, sustained growth, self-renewal and tumorigenicity. Here we report the isolation of stem-like cells from two canine OSA cultures, characterized by self-renewal, evaluated by sphere formation ability, differential marker expression, and in vitro proliferation when cultured in a medium containing EGF and bFGF. Current therapies for OSA increased survival time, but prognosis remains poor, due to the development of drug resistance and metastases. Chemotherapy shrinks the tumor mass but CSCs remain unaffected, leading to tumor recurrence. Metformin, a drug for type 2 diabetes, has been shown to possess antitumor properties affecting CSC survival in different human and animal cancers. Here we show that metformin has a significant antiproliferative effect on canine OSA stem-like cells, validating this in vitro model for further pre-clinical drug evaluations. In conclusion, our results demonstrate the feasibility of obtaining CSC-enriched cultures from primary canine OSA cells as a promising model for biological and pharmacological studies of canine and human OSAs.

Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

NASA Technical Reports Server (NTRS)

Bancroft, Gregory N.; Sikavitsas, Vassilios I.; van den Dolder, Juliette; Sheffield, Tiffany L.; Ambrose, Catherine G.; Jansen, John A.; Mikos, Antonios G.; McIntire, L. V. (Principal Investigator)

2002-01-01

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

Antioxidant functionalized polymer capsules to prevent oxidative stress.

PubMed

Larrañaga, Aitor; Isa, Isma Liza Mohd; Patil, Vaibhav; Thamboo, Sagana; Lomora, Mihai; Fernández-Yague, Marc A; Sarasua, Jose-Ramon; Palivan, Cornelia G; Pandit, Abhay

2018-02-01

Polymeric capsules exhibit significant potential for therapeutic applications as microreactors, where the bio-chemical reactions of interest are efficiently performed in a spatial and time defined manner due to the encapsulation of an active biomolecule (e.g., enzyme) and control over the transfer of reagents and products through the capsular membrane. In this work, catalase loaded polymer capsules functionalized with an external layer of tannic acid (TA) are fabricated via a layer-by-layer approach using calcium carbonate as a sacrificial template. The capsules functionalised with TA exhibit a higher scavenging capacity for hydrogen peroxide and hydroxyl radicals, suggesting that the external layer of TA shows intrinsic antioxidant properties, and represents a valid strategy to increase the overall antioxidant potential of the developed capsules. Additionally, the hydrogen peroxide scavenging capacity of the capsules is enhanced in the presence of the encapsulated catalase. The capsules prevent oxidative stress in an in vitro inflammation model of degenerative disc disease. Moreover, the expression of matrix metalloproteinase-3 (MMP-3), and disintegrin and metalloproteinase with thrombospondin motif-5 (ADAMTS-5), which represents the major proteolytic enzymes in intervertebral disc, are attenuated in the presence of the polymer capsules. This platform technology exhibits potential to reduce oxidative stress, a key modulator in the pathology of a broad range of inflammatory diseases. Oxidative stress damages important cell structures leading to cellular apoptosis and senescence, for numerous disease pathologies including cancer, neurodegeneration or osteoarthritis. Thus, the development of biomaterials-based systems to control oxidative stress has gained an increasing interest. Herein, polymer capsules loaded with catalase and functionalized with an external layer of tannic acid are fabricated, which can efficiently scavenge important reactive oxygen species (i.e., hydroxyl radicals and hydrogen peroxide) and modulate extracellular matrix activity in an in vitro inflammation model of nucleus pulposus. The present work represents accordingly, an important advance in the development and application of polymer capsules with antioxidant properties for the treatment of oxidative stress, which is applicable for multiple inflammatory disease targets. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

In vivo potency revisited - Keep the target in sight.

PubMed

Gabrielsson, Johan; Peletier, Lambertus A; Hjorth, Stephan

2018-04-01

Potency is a central parameter in pharmacological and biochemical sciences, as well as in drug discovery and development endeavors. It is however typically defined in terms only of ligand to target binding affinity also in in vivo experimentation, thus in a manner analogous to in in vitro studies. As in vivo potency is in fact a conglomerate of events involving ligand, target, and target-ligand complex processes, overlooking some of the fundamental differences between in vivo and in vitro may result in serious mispredictions of in vivo efficacious dose and exposure. The analysis presented in this paper compares potency measures derived from three model situations. Model A represents the closed in vitro system, defining target binding of a ligand when total target and ligand concentrations remain static and constant. Model B describes an open in vivo system with ligand input and clearance (Cl (L) ), adding in parallel to the turnover (k syn , k deg ) of the target. Model C further adds to the open in vivo system in Model B also the elimination of the target-ligand complex (k e(RL) ) via a first-order process. We formulate corresponding equations of the equilibrium (steady-state) relationships between target and ligand, and complex and ligand for each of the three model systems and graphically illustrate the resulting simulations. These equilibrium relationships demonstrate the relative impact of target and target-ligand complex turnover, and are easier to interpret than the more commonly used ligand-, target- and complex concentration-time courses. A new potency expression, labeled L 50 , is then derived. L 50 is the ligand concentration at half-maximal target and complex concentrations and is an amalgamation of target turnover, target-ligand binding and complex elimination parameters estimated from concentration-time data. L 50 is then compared to the dissociation constant K d (target-ligand binding affinity), the conventional Black & Leff potency estimate EC 50 , and the derived Michaelis-Menten parameter K m (target-ligand binding and complex removal) across a set of literature data. It is evident from a comparison between parameters derived from in vitro vs. in vivo experiments that L 50 can be either numerically greater or smaller than the K d (or K m ) parameter, primarily depending on the ratio of k deg -to-k e(RL) . Contrasting the limit values of target R and target-ligand complex RL for ligand concentrations approaching infinity demonstrates that the outcome of the three models differs to a great extent. Based on the analysis we propose that a better understanding of in vivo pharmacological potency requires simultaneous assessment of the impact of its underlying determinants in the open system setting. We propose that L 50 will be a useful parameter guiding predictions of the effective concentration range, for translational purposes, and assessment of in vivo target occupancy/suppression by ligand, since it also encompasses target turnover - in turn also subject to influence by pathophysiology and drug treatment. Different compounds may have similar binding affinity for a target in vitro (same K d ), but vastly different potencies in vivo. L 50 points to what parameters need to be taken into account, and particularly that closed-system (in vitro) parameters should not be first choice when ranking compounds in vivo (open system). Copyright © 2017 Elsevier Inc. All rights reserved.

Tumor-Targeting Anti-CD20 Antibodies Mediate In Vitro Expansion of Memory Natural Killer Cells: Impact of CD16 Affinity Ligation Conditions and In Vivo Priming.

PubMed

Capuano, Cristina; Battella, Simone; Pighi, Chiara; Franchitti, Lavinia; Turriziani, Ombretta; Morrone, Stefania; Santoni, Angela; Galandrini, Ricciarda; Palmieri, Gabriella

2018-01-01

Natural killer (NK) cells represent a pivotal player of innate anti-tumor immune responses. The impact of environmental factors in shaping the representativity of different NK cell subsets is increasingly appreciated. Human cytomegalovirus (HCMV) infection profoundly affects NK cell compartment, as documented by the presence of a CD94/NKG2C + FcεRIγ - long-lived "memory" NK cell subset, endowed with enhanced CD16-dependent functional capabilities, in a fraction of HCMV-seropositive subjects. However, the requirements for memory NK cell pool establishment/maintenance and activation have not been fully characterized yet. Here, we describe the capability of anti-CD20 tumor-targeting therapeutic monoclonal antibodies (mAbs) to drive the selective in vitro expansion of memory NK cells and we show the impact of donor' HCMV serostatus and CD16 affinity ligation conditions on this event. In vitro expanded memory NK cells maintain the phenotypic and functional signature of their freshly isolated counterpart; furthermore, our data demonstrate that CD16 affinity ligation conditions differently affect memory NK cell proliferation and functional activation, as rituximab-mediated low-affinity ligation represents a superior proliferative stimulus, while high-affinity aggregation mediated by glycoengineered obinutuzumab results in improved multifunctional responses. Our work also expands the molecular and functional characterization of memory NK cells, and investigates the possible impact of CD16 functional allelic variants on their in vivo and in vitro expansions. These results reveal new insights in Ab-driven memory NK cell responses in a therapeutic setting and may ultimately inspire new NK cell-based intervention strategies against cancer, in which the enhanced responsiveness to mAb-bound target could significantly impact therapeutic efficacy.

Prediction of in vitro and in vivo oestrogen receptor activity using hierarchical clustering

EPA Science Inventory

In this study, hierarchical clustering classification models were developed to predict in vitro and in vivo oestrogen receptor (ER) activity. Classification models were developed for binding, agonist, and antagonist in vitro ER activity and for mouse in vivo uterotrophic ER bindi...

Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Atienzar, Franck A., E-mail: franck.atienzar@ucb.com; Novik, Eric I.; Gerets, Helga H.

Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine modelmore » along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.« less

Comparative proteome analysis of monolayer and spheroid culture of canine osteosarcoma cells.

PubMed

Gebhard, Christiane; Miller, Ingrid; Hummel, Karin; Neschi Née Ondrovics, Martina; Schlosser, Sarah; Walter, Ingrid

2018-04-15

Osteosarcoma is an aggressive bone tumor with high metastasis rate in the lungs and affects both humans and dogs in a similar way. Three-dimensional tumor cell cultures mimic the in vivo situation of micro-tumors and metastases and are therefore better experimental in vitro models than the often applied two-dimensional monolayer cultures. The aim of the present study was to perform comparative proteomics of standard monolayer cultures of canine osteosarcoma cells (D17) and three-dimensional spheroid cultures, to better characterize the 3D model before starting with experiments like migration assays. Using DIGE in combination with MALDI-TOF/TOF we found 27 unique canine proteins differently represented between these two culture systems, most of them being part of a functional network including mainly chaperones, structural proteins, stress-related proteins, proteins of the glycolysis/gluconeogenesis pathway and oxidoreductases. In monolayer cells, a noticeable shift to more acidic pI values was noticed for several proteins of medium to high abundance; two proteins (protein disulfide isomerase A3, stress-induced-phosphoprotein 1) showed an increase of phosphorylated protein species. Protein distribution within the cells, as detected by immunohistochemistry, displayed a switch of stress-induced-phosphoprotein 1 from the cytoplasm (in monolayer cultures) to the nucleus (in spheroid cultures). Additionally, Western blot testing revealed upregulated concentrations of metastasin (S100A4), triosephosphate isomerase 1 and septin 2 in spheroid cultures, in contrast to decreased concentrations of CCT2, a subunit of the T-complex. Results indicate regulation of stress proteins in the process of three-dimensional organization characterized by a hypoxic and nutrient-deficient environment comparable to tumor micro-metastases. Osteosarcoma is an aggressive bone tumor that early spreads to the lungs. Three-dimensional tumor cell cultures represent the avascular stage of micro-tumors and metastases, and should therefore represent a better experimental in vitro model compared to two-dimensional monolayer cultures. Significant differences have been reported in response to drug and radiation treatment between these two culture systems. A gel-based proteomic investigation was performed to compare protein patterns of a canine osteosarcoma cell line cultivated under those two conditions, to learn more about altered cell composition and its impact on cell behaviour. Due to the fact that the canine osteosarcoma is an accepted model for the human disease, results will be relevant for the human species as well. Copyright © 2018 Elsevier B.V. All rights reserved.

Ultrasound-guided direct delivery of 3-bromopyruvate blocks tumor progression in an orthotopic mouse model of human pancreatic cancer.

PubMed

Ota, Shinichi; Geschwind, Jean-Francois H; Buijs, Manon; Wijlemans, Joost W; Kwak, Byung Kook; Ganapathy-Kanniappan, Shanmugasundaram

2013-06-01

Studies in animal models of cancer have demonstrated that targeting tumor metabolism can be an effective anticancer strategy. Previously, we showed that inhibition of glucose metabolism by the pyruvate analog, 3-bromopyruvate (3-BrPA), induces anticancer effects both in vitro and in vivo. We have also documented that intratumoral delivery of 3-BrPA affects tumor growth in a subcutaneous tumor model of human liver cancer. However, the efficacy of such an approach in a clinically relevant orthotopic tumor model has not been reported. Here, we investigated the feasibility of ultrasound (US) image-guided delivery of 3-BrPA in an orthotopic mouse model of human pancreatic cancer and evaluated its therapeutic efficacy. In vitro, treatment of Panc-1 cells with 3-BrPA resulted in a dose-dependent decrease in cell viability. The loss of viability correlated with a dose-dependent decrease in the intracellular ATP level and lactate production confirming that disruption of energy metabolism underlies these 3-BrPA-mediated effects. In vivo, US-guided delivery of 3-BrPA was feasible and effective as demonstrated by a marked decrease in tumor size on imaging. Further, the antitumor effect was confirmed by (1) a decrease in the proliferative potential by Ki-67 immunohistochemical staining and (2) the induction of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling staining. We therefore demonstrate the technical feasibility of US-guided intratumoral injection of 3-BrPA in a mouse model of human pancreatic cancer as well as its therapeutic efficacy. Our data suggest that this new therapeutic approach consisting of a direct intratumoral injection of antiglycolytic agents may represent an exciting opportunity to treat patients with pancreas cancer.

Modeling neutralization kinetics of HIV by broadly neutralizing monoclonal antibodies in genital secretions coating the cervicovaginal mucosa.

PubMed

McKinley, Scott A; Chen, Alex; Shi, Feng; Wang, Simi; Mucha, Peter J; Forest, M Gregory; Lai, Samuel K

2014-01-01

Eliciting broadly neutralizing antibodies (bnAb) in cervicovaginal mucus (CVM) represents a promising "first line of defense" strategy to reduce vaginal HIV transmission. However, it remains unclear what levels of bnAb must be present in CVM to effectively reduce infection. We approached this complex question by modeling the dynamic tally of bnAb coverage on HIV. This analysis introduces a critical, timescale-dependent competition: to protect, bnAb must accumulate at sufficient stoichiometry to neutralize HIV faster than virions penetrate CVM and reach target cells. We developed a model that incorporates concentrations and diffusivities of HIV and bnAb in semen and CVM, kinetic rates for binding (kon) and unbinding (koff) of select bnAb, and physiologically relevant thicknesses of CVM and semen layers. Comprehensive model simulations lead to robust conclusions about neutralization kinetics in CVM. First, due to the limited time virions in semen need to penetrate CVM, substantially greater bnAb concentrations than in vitro estimates must be present in CVM to neutralize HIV. Second, the model predicts that bnAb with more rapid kon, almost independent of koff, should offer greater neutralization potency in vivo. These findings suggest the fastest arriving virions at target cells present the greatest likelihood of infection. It also implies the marked improvements in in vitro neutralization potency of many recently discovered bnAb may not translate to comparable reduction in the bnAb dose needed to confer protection against initial vaginal infections. Our modeling framework offers a valuable tool to gaining quantitative insights into the dynamics of mucosal immunity against HIV and other infectious diseases.

A comparison of quantitative methods for clinical imaging with hyperpolarized (13)C-pyruvate.

PubMed

Daniels, Charlie J; McLean, Mary A; Schulte, Rolf F; Robb, Fraser J; Gill, Andrew B; McGlashan, Nicholas; Graves, Martin J; Schwaiger, Markus; Lomas, David J; Brindle, Kevin M; Gallagher, Ferdia A

2016-04-01

Dissolution dynamic nuclear polarization (DNP) enables the metabolism of hyperpolarized (13)C-labelled molecules, such as the conversion of [1-(13)C]pyruvate to [1-(13)C]lactate, to be dynamically and non-invasively imaged in tissue. Imaging of this exchange reaction in animal models has been shown to detect early treatment response and correlate with tumour grade. The first human DNP study has recently been completed, and, for widespread clinical translation, simple and reliable methods are necessary to accurately probe the reaction in patients. However, there is currently no consensus on the most appropriate method to quantify this exchange reaction. In this study, an in vitro system was used to compare several kinetic models, as well as simple model-free methods. Experiments were performed using a clinical hyperpolarizer, a human 3 T MR system, and spectroscopic imaging sequences. The quantitative methods were compared in vivo by using subcutaneous breast tumours in rats to examine the effect of pyruvate inflow. The two-way kinetic model was the most accurate method for characterizing the exchange reaction in vitro, and the incorporation of a Heaviside step inflow profile was best able to describe the in vivo data. The lactate time-to-peak and the lactate-to-pyruvate area under the curve ratio were simple model-free approaches that accurately represented the full reaction, with the time-to-peak method performing indistinguishably from the best kinetic model. Finally, extracting data from a single pixel was a robust and reliable surrogate of the whole region of interest. This work has identified appropriate quantitative methods for future work in the analysis of human hyperpolarized (13)C data. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

A physiologically based pharmacokinetic model for ethylene oxide in mouse, rat, and human.

PubMed

Fennell, T R; Brown, C D

2001-06-15

Ethylene oxide (EO) is widely used as a gaseous sterilant and industrial intermediate and is a direct-acting mutagen and carcinogen. The objective of these studies was to develop physiologically based pharmacokinetic (PB-PK) models for EO to describe the exposure-tissue dose relationship in rodents and humans. We previously reported results describing in vitro and in vivo kinetics of EO metabolism in male and female F344 rats and B6C3F1 mice. These studies were extended by determining the kinetics of EO metabolism in human liver cytosol and microsomes. The results indicate enzymatically catalyzed GSH conjugation via cytosolic glutathione S-transferase (cGST) and hydrolysis via microsomal epoxide hydrolase (mEH) occur in both rodents and humans. The in vitro kinetic constants were scaled to account for cytosolic (cGST) and microsomal (mEH) protein content and incorporated into PB-PK descriptions for mouse, rat, and human. Flow-limited models adequately predicted blood and tissue EO levels, disposition, and elimination kinetics determined experimentally in rats and mice, with the exception of testis concentrations, which were overestimated. Incorporation of a diffusion-limited description for testis improved the ability of the model to describe testis concentrations. The model accounted for nonlinear increases in blood and tissue concentrations that occur in mice on exposure to EO concentrations greater than 200 ppm. Species differences are predicted in the metabolism and exposure-dose relationship, with a nonlinear relationship observed in the mouse as a result of GSH depletion. These models represent an essential step in developing a mechanistically based EO exposure-dose-response description for estimating human risk from exposure to EO. Copyright 2001 Academic Press.

Enteropathogenic Escherichia coli O125:H6 Triggers Attaching and Effacing Lesions on Human Intestinal Biopsy Specimens Independently of Nck and TccP/TccP2▿

PubMed Central

Bai, Li; Schüller, Stephanie; Whale, Andrew; Mousnier, Aurelie; Marches, Olivier; Wang, Lei; Ooka, Tadasuke; Heuschkel, Robert; Torrente, Franco; Kaper, James B.; Gomes, Tânia A. T.; Xu, Jianguo; Phillips, Alan D.; Frankel, Gad

2008-01-01

Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) employ either Nck, TccP/TccP2, or Nck and TccP/TccP2 pathways to activate the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and to trigger actin polymerization in cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains. In this paper we report that EPEC O125:H6, which represents a large category of strains, lacks the ability to utilize either Nck or TccP/TccP2 and hence triggers actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expression of TccP in EPEC O125:H6, which harbors an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron-dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck- and TccP-independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence. PMID:17984209

Enteropathogenic Escherichia coli O125:H6 triggers attaching and effacing lesions on human intestinal biopsy specimens independently of Nck and TccP/TccP2.

PubMed

Bai, Li; Schüller, Stephanie; Whale, Andrew; Mousnier, Aurelie; Marches, Olivier; Wang, Lei; Ooka, Tadasuke; Heuschkel, Robert; Torrente, Franco; Kaper, James B; Gomes, Tânia A T; Xu, Jianguo; Phillips, Alan D; Frankel, Gad

2008-01-01

Typical enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) employ either Nck, TccP/TccP2, or Nck and TccP/TccP2 pathways to activate the neuronal Wiskott-Aldrich syndrome protein (N-WASP) and to trigger actin polymerization in cultured cells. This phenotype is used as a marker for the pathogenic potential of EPEC and EHEC strains. In this paper we report that EPEC O125:H6, which represents a large category of strains, lacks the ability to utilize either Nck or TccP/TccP2 and hence triggers actin polymerization in vitro only inefficiently. However, we show that infection of human intestinal biopsies with EPEC O125:H6 results in formation of typical attaching and effacing lesions. Expression of TccP in EPEC O125:H6, which harbors an EHEC O157-like Tir, resulted in efficient actin polymerization in vitro and enhanced colonization of human intestinal in vitro organ cultures with detectable N-WASP and electron-dense material at the site of bacterial adhesion. These results show the existence of a natural category of EPEC that colonizes the gut mucosa using Nck- and TccP-independent mechanisms. Importantly, the results highlight yet again the fact that conclusions made on the basis of in vitro cell culture models cannot be extrapolated wholesale to infection of mucosal surfaces and that the ability to induce actin polymerization on cultured cells should not be used as a definitive marker for EPEC and EHEC virulence.

The design of an environmentally relevant mixture of persistent organic pollutants for use in in vivo and in vitro studies.

PubMed

Berntsen, Hanne Friis; Berg, Vidar; Thomsen, Cathrine; Ropstad, Erik; Zimmer, Karin Elisabeth

2017-01-01

Amongst the substances listed as persistent organic pollutants (POP) under the Stockholm Convention on Persistent Organic Pollutants (SCPOP) are chlorinated, brominated, and fluorinated compounds. Most experimental studies investigating effects of POP employ single compounds. Studies focusing on effects of POP mixtures are limited, and often conducted using extracts from collected specimens. Confounding effects of unmeasured substances in such extracts may bias the estimates of presumed causal relationships being examined. The aim of this investigation was to design a model of an environmentally relevant mixture of POP for use in experimental studies, containing 29 different chlorinated, brominated, and perfluorinated compounds. POP listed under the SCPOP and reported to occur at the highest levels in Scandinavian food, blood, or breast milk prior to 2012 were selected, and two different mixtures representing varying exposure scenarios constructed. The in vivo mixture contained POP concentrations based upon human estimated daily intakes (EDIs), whereas the in vitro mixture was based upon levels in human blood. In addition to total in vitro mixture, 6 submixtures containing the same concentration of chlorinated + brominated, chlorinated + perfluorinated, brominated + perfluorinated, or chlorinated, brominated or perfluorinated compounds only were constructed. Using submixtures enables investigating the effect of adding or removing one or more chemical groups. Concentrations of compounds included in feed and in vitro mixtures were verified by chemical analysis. It is suggested that this method may be utilized to construct realistic mixtures of environmental contaminants for toxicity studies based upon the relative levels of POP to which individuals are exposed.

Radiation-force-based Estimation of Acoustic Attenuation Using Harmonic Motion Imaging (HMI) in Phantoms and in vitro Livers Before and After HIFU Ablation

PubMed Central

Chen, Jiangang; Hou, Gary Y.; Marquet, Fabrice; Han, Yang; Camarena, Francisco

2015-01-01

Acoustic attenuation represents the energy loss of the propagating wave through biological tissues and plays a significant role in both therapeutic and diagnostic ultrasound applications. Estimation of acoustic attenuation remains challenging but critical for tissue characterization. In this study, an attenuation estimation approach was developed using the radiation-force-based method of Harmonic Motion Imaging (HMI). 2D tissue displacement maps were acquired by moving the transducer in a raster-scan format. A linear regression model was applied on the logarithm of the HMI displacements at different depths in order to estimate the acoustic attenuation. Commercially available phantoms with known attenuations (n=5) and in vitro canine livers (n=3) were tested, as well as HIFU lesions in in vitro canine livers (n=5). Results demonstrated that attenuations obtained from the phantoms showed a good correlation (R2=0.976) with the independently obtained values reported by the manufacturer with an estimation error (compared to the values independently measured) varying within the range of 15-35%. The estimated attenuation in the in vitro canine livers was equal to 0.32±0.03 dB/cm/MHz, which is in good agreement with the existing literature. The attenuation in HIFU lesions was found to be higher (0.58±0.06 dB/cm/MHz) than that in normal tissues, also in agreement with the results from previous publications. Future potential applications of the proposed method include estimation of attenuation in pathological tissues before and after thermal ablation. PMID:26371501

Inhibition of inflammatory gene expression in keratinocytes using a composition containing carnitine, thioctic Acid and saw palmetto extract.

PubMed

Chittur, Sridar; Parr, Brian; Marcovici, Geno

2011-01-01

Chronic inflammation of the hair follicle (HF) is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA). Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr) and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid) could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS) provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4) associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities.

Induced Pluripotent Stem Cells for Disease Modeling and Drug Discovery in Neurodegenerative Diseases.

PubMed

Cao, Lei; Tan, Lan; Jiang, Teng; Zhu, Xi-Chen; Yu, Jin-Tai

2015-08-01

Although most neurodegenerative diseases have been closely related to aberrant accumulation of aggregation-prone proteins in neurons, understanding their pathogenesis remains incomplete, and there is no treatment to delay the onset or slow the progression of many neurodegenerative diseases. The availability of induced pluripotent stem cells (iPSCs) in recapitulating the phenotypes of several late-onset neurodegenerative diseases marks the new era in in vitro modeling. The iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in these diseases and provides a novel human stem cell platform for screening new candidate therapeutics. Modeling human diseases using iPSCs has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. In this review, we introduce iPSC-based disease modeling in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. In addition, we discuss the implementation of iPSCs in drug discovery associated with some new techniques.

Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors

PubMed Central

Schütte, Moritz; Risch, Thomas; Abdavi-Azar, Nilofar; Boehnke, Karsten; Schumacher, Dirk; Keil, Marlen; Yildiriman, Reha; Jandrasits, Christine; Borodina, Tatiana; Amstislavskiy, Vyacheslav; Worth, Catherine L.; Schweiger, Caroline; Liebs, Sandra; Lange, Martin; Warnatz, Hans- Jörg; Butcher, Lee M.; Barrett, James E.; Sultan, Marc; Wierling, Christoph; Golob-Schwarzl, Nicole; Lax, Sigurd; Uranitsch, Stefan; Becker, Michael; Welte, Yvonne; Regan, Joseph Lewis; Silvestrov, Maxine; Kehler, Inge; Fusi, Alberto; Kessler, Thomas; Herwig, Ralf; Landegren, Ulf; Wienke, Dirk; Nilsson, Mats; Velasco, Juan A.; Garin-Chesa, Pilar; Reinhard, Christoph; Beck, Stephan; Schäfer, Reinhold; Regenbrecht, Christian R. A.; Henderson, David; Lange, Bodo; Haybaeck, Johannes; Keilholz, Ulrich; Hoffmann, Jens; Lehrach, Hans; Yaspo, Marie-Laure

2017-01-01

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab. PMID:28186126

Culture models of human mammary epithelial cell transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stampfer, Martha R.; Yaswen, Paul

2000-11-10

Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcomemore » stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.« less

Cyanidin-3-O-Glucoside Modulates the In Vitro Inflammatory Crosstalk between Intestinal Epithelial and Endothelial Cells.

PubMed

Ferrari, Daniela; Cimino, Francesco; Fratantonio, Deborah; Molonia, Maria Sofia; Bashllari, Romina; Busà, Rossana; Saija, Antonella; Speciale, Antonio

2017-01-01

Intestinal epithelium represents a protective physical barrier and actively contributes to the mucosal immune system. Polarized basolateral intestinal secretion of inflammatory mediators, followed by activation of NF- κ B signaling and inflammatory pathways in endothelial cells, efficiently triggers extravasation of neutrophils from the vasculature, therefore contributing to the development and maintenance of intestinal inflammation. Proper regulation of NF- κ B activation at the epithelial interface is crucial for the maintenance of physiological tissue homeostasis. Many papers reported that anthocyanins, a group of compounds belonging to flavonoids, possess anti-inflammatory effects and modulate NF- κ B activity. In this study, by using a coculture in vitro system, we aimed to evaluate the effects of TNF- α -stimulated intestinal cells on endothelial cells activation, as well as the protective effects of cyanidin-3-glucoside (C3G). In this model, TNF- α induced nuclear translocation of NF- κ B and TNF- α and IL-8 gene expression in Caco-2 cells, whereas C3G pretreatment dose-dependently reduced these effects. Furthermore, TNF- α -stimulated Caco-2 cells induced endothelial cells activation with increased E-selectin and VCAM-1 mRNA, leukocyte adhesion, and NF- κ B levels in HUVECs, which were inhibited by C3G. We demonstrated that selective inhibition of the NF- κ B pathway in epithelial cells represents the main mechanism by which C3G exerts these protective effects. Thus, anthocyanins could contribute to the management of chronic gut inflammatory diseases.

Proton pump inhibitors protect mice from acute systemic inflammation and induce long-term cross-tolerance

PubMed Central

Balza, E; Piccioli, P; Carta, S; Lavieri, R; Gattorno, M; Semino, C; Castellani, P; Rubartelli, A

2016-01-01

Incidence of sepsis is increasing, representing a tremendous burden for health-care systems. Death in acute sepsis is attributed to hyperinflammatory responses, but the underlying mechanisms are still unclear. We report here that proton pump inhibitors (PPIs), which block gastric acid secretion, selectively inhibited tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion by Toll-like receptor (TLR)-activated human monocytes in vitro, in the absence of toxic effects. Remarkably, the oversecretion of IL-1β that represents a hallmark of monocytes from patients affected by cryopyrin-associated periodic syndrome is also blocked. Based on these propaedeutic experiments, we tested the effects of high doses of PPIs in vivo in the mouse model of endotoxic shock. Our data show that a single administration of PPI protected mice from death (60% survival versus 5% of untreated mice) and decreased TNF-α and IL-1β systemic production. PPIs were efficacious even when administered after lipopolysaccharide (LPS) injection. PPI-treated mice that survived developed a long-term cross-tolerance, becoming resistant to LPS- and zymosan-induced sepsis. In vitro, their macrophages displayed impaired TNF-α and IL-1β to different TLR ligands. PPIs also prevented sodium thioglycollate-induced peritoneal inflammation, indicating their efficacy also in a non-infectious setting independent of TLR stimulation. Lack of toxicity and therapeutic effectiveness make PPIs promising new drugs against sepsis and other severe inflammatory conditions. PMID:27441656

Handling of computational in vitro/in vivo correlation problems by Microsoft Excel: III. Convolution and deconvolution.

PubMed

Langenbucher, Frieder

2003-11-01

Convolution and deconvolution are the classical in-vitro-in-vivo correlation tools to describe the relationship between input and weighting/response in a linear system, where input represents the drug release in vitro, weighting/response any body response in vivo. While functional treatment, e.g. in terms of polyexponential or Weibull distribution, is more appropriate for general survey or prediction, numerical algorithms are useful for treating actual experimental data. Deconvolution is not considered an algorithm by its own, but the inversion of a corresponding convolution. MS Excel is shown to be a useful tool for all these applications.

In Vitro Activities of the Everninomicin SCH 27899 and Other Newer Antimicrobial Agents against Borrelia burgdorferi

PubMed Central

Dever, Lisa L.; Torigian, Christine V.; Barbour, Alan G.

1999-01-01

The in vitro activity of the everninomicin antibiotic SCH 27899 against 17 isolates of Borrelia spp. was investigated. MICs ranged from 0.06 to 0.5 μg/ml. Time-kill studies with the B31 strain of B. burgdorferi demonstrated ≥3-log10-unit killing after 72 h with concentrations representing four times the MIC. The in vitro activity of four other newer antimicrobial agents, meropenem, cefepime, quinupristin-dalfopristin, and linezolid, was also tested against the B31 strain. Meropenem was the most potent of the latter agents, with an MIC of 0.125 μg/ml. PMID:10390242

CD71(high) population represents primitive erythroblasts derived from mouse embryonic stem cells.

PubMed

Chao, Ruihua; Gong, Xueping; Wang, Libo; Wang, Pengxiang; Wang, Yuan

2015-01-01

The CD71/Ter119 combination has been widely used to reflect dynamic maturation of erythrocytes in vivo. However, because CD71 is expressed on all proliferating cells, it is unclear whether it can be utilized as an erythrocyte-specific marker during differentiation of embryonic stem cells (ESCs). In this study, we revealed that a population expressing high level of CD71 (CD71(high)) during mouse ESC differentiation represented an in vitro counterpart of yolk sac-derived primitive erythroblasts (EryPs) isolated at 8.5days post coitum. In addition, these CD71(high) cells went through "maturational globin switching" and enucleated during terminal differentiation in vitro that were similar to the yolk sac-derived EryPs in vivo. We further demonstrated that the formation of CD71(high) population was regulated differentially by key factors including Scl, HoxB4, Eaf1, and Klf1. Taken together, our study provides a technical advance that allows efficient segregation of EryPs from differentiated ESCs in vitro for further understanding molecular regulation during primitive erythropoiesis. Copyright © 2014. Published by Elsevier B.V.

A Comprehensive Tutorial on In Vitro Characterization of New Photosensitizers for Photodynamic Antitumor Therapy and Photodynamic Inactivation of Microorganisms

PubMed Central

Maisch, Tim; Berneburg, Mark; Plaetzer, Kristjan

2013-01-01

In vitro research performed on eukaryotic or prokaryotic cell cultures usually represents the initial step for characterization of a novel photosensitizer (PS) intended for application in photodynamic therapy (PDT) of cancer or photodynamic inactivation (PDI) of microorganisms. Although many experimental steps of PS testing make use of the wide spectrum of methods readily employed in cell biology, special aspects of working with photoactive substances, such as the autofluorescence of the PS molecule or the requirement of light protection, need to be considered when performing in vitro experiments in PDT/PDI. This tutorial represents a comprehensive collection of operative instructions, by which, based on photochemical and photophysical properties of a PS, its uptake into cells, the intracellular localization and photodynamic action in both tumor cells and microorganisms novel photoactive molecules may be characterized for their suitability for PDT/PDI. Furthermore, it shall stimulate the efforts to expand the convincing benefits of photodynamic therapy and photodynamic inactivation within both established and new fields of applications and motivate scientists of all disciplines to get involved in photodynamic research. PMID:23762860

Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice.

PubMed

Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S; Pushko, Peter

2014-11-01

Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. Copyright © 2014 Elsevier Inc. All rights reserved.

A quantitative framework to evaluate modeling of cortical development by neural stem cells

PubMed Central

Stein, Jason L.; de la Torre-Ubieta, Luis; Tian, Yuan; Parikshak, Neelroop N.; Hernandez, Israel A.; Marchetto, Maria C.; Baker, Dylan K.; Lu, Daning; Hinman, Cassidy R.; Lowe, Jennifer K.; Wexler, Eric M.; Muotri, Alysson R.; Gage, Fred H.; Kosik, Kenneth S.; Geschwind, Daniel H.

2014-01-01

Summary Neural stem cells have been adopted to model a wide range of neuropsychiatric conditions in vitro. However, how well such models correspond to in vivo brain has not been evaluated in an unbiased, comprehensive manner. We used transcriptomic analyses to compare in vitro systems to developing human fetal brain and observed strong conservation of in vivo gene expression and network architecture in differentiating primary human neural progenitor cells (phNPCs). Conserved modules are enriched in genes associated with ASD, supporting the utility of phNPCs for studying neuropsychiatric disease. We also developed and validated a machine learning approach called CoNTExT that identifies the developmental maturity and regional identity of in vitro models. We observed strong differences between in vitro models, including hiPSC-derived neural progenitors from multiple laboratories. This work provides a systems biology framework for evaluating in vitro systems and supports their value in studying the molecular mechanisms of human neurodevelopmental disease. PMID:24991955

In vitro production of M. × piperita not containing pulegone and menthofuran.

PubMed

Bertoli, Alessandra; Leonardi, Michele; Krzyzanowska, Justyna; Oleszek, Wieslaw; Pistelli, Luisa

2012-01-01

The essential oils (EOs) and static headspaces (HSs) of in vitro plantlets and callus of Mentha x piperita were characterized by GC-MS analysis. Leaves were used as explants to induce in vitro plant material. The EO yields of the in vitro biomass were much lower (0.1% v/w) than those of the parent plants (2% v/w). Many typical mint volatiles were emitted by the in vitro production, but the callus and in vitro plantelet EOs were characterized by the lack of both pulegone and menthofuran. This was an important difference between in vitro and in vivo plant material as huge amounts of pulegone and menthofuran may jeopardise the safety of mint essential oil. Regarding the other characteristic volatiles, menthone was present in reduced amounts (2%) in the in vitro plantlets and was not detected in the callus, even if it represented the main constituent of the stem and leaf EOs obtained from the cultivated mint (26% leaves; 33% stems). The M. piperita callus was characterized by menthol (9%) and menthone (2%), while the in vitro plantlet EO showed lower amounts of both these compounds in favour of piperitenone oxide (45%). Therefore, the established callus and in vitro plantlets showed peculiar aromatic profiles characterized by the lack of pulegone and menthofuran which have to be monitored in the mint oil for their toxicity.

Generation of Distal Airway Epithelium from Multipotent Human Foregut Stem Cells.

PubMed

Hannan, Nicholas R F; Sampaziotis, Fotios; Segeritz, Charis-Patricia; Hanley, Neil A; Vallier, Ludovic

2015-07-15

Collectively, lung diseases are one of the largest causes of premature death worldwide and represent a major focus in the field of regenerative medicine. Despite significant progress, only few stem cell platforms are currently available for cell-based therapy, disease modeling, and drug screening in the context of pulmonary disorders. Human foregut stem cells (hFSCs) represent an advantageous progenitor cell type that can be used to amplify large quantities of cells for regenerative medicine applications and can be derived from any human pluripotent stem cell line. Here, we further demonstrate the application of hFSCs by generating a near homogeneous population of early pulmonary endoderm cells coexpressing NKX2.1 and FOXP2. These progenitors are then able to form cells that are representative of distal airway epithelium that express NKX2.1, GATA6, and cystic fibrosis transmembrane conductance regulator (CFTR) and secrete SFTPC. This culture system can be applied to hFSCs carrying the CFTR mutation Δf508, enabling the development of an in vitro model for cystic fibrosis. This platform is compatible with drug screening and functional validations of small molecules, which can reverse the phenotype associated with CFTR mutation. This is the first demonstration that multipotent endoderm stem cells can differentiate not only into both liver and pancreatic cells but also into lung endoderm. Furthermore, our study establishes a new approach for the generation of functional lung cells that can be used for disease modeling as well as for drug screening and the study of lung development.

Adaptation of the ICRP publication 66 respiratory tract model to data on plutonium biokinetics for Mayak workers.

PubMed

Khokhryakov, V F; Suslova, K G; Vostrotin, V V; Romanov, S A; Eckerman, K F; Krahenbuhl, M P; Miller, S C

2005-02-01

The biokinetics of inhaled plutonium were analyzed using compartment models representing their behavior within the respiratory tract, the gastrointestinal tract, and in systemic tissues. The processes of aerosol deposition, particle transport, absorption, and formation of a fixed deposit in the respiratory tract were formulated in the framework of the Human Respiratory Tract Model described in ICRP Publication 66. The values of parameters governing absorption and formation of the fixed deposit were established by fitting the model to the observations in 530 autopsy cases. The influence of smoking on mechanical clearance of deposited plutonium activity was considered. The dependence of absorption on the aerosol transportability, as estimated by in vitro methods (dialysis), was demonstrated. The results of this study were compared to those obtained from an earlier model of plutonium behavior in the respiratory tract, which was based on the same set of autopsy data. That model did not address the early phases of respiratory clearance and hence underestimated the committed lung dose by about 25% for plutonium oxides. Little difference in lung dose was found for nitrate forms.

Toward a computational model of hemostasis

NASA Astrophysics Data System (ADS)

Leiderman, Karin; Danes, Nicholas; Schoeman, Rogier; Neeves, Keith

2017-11-01

Hemostasis is the process by which a blood clot forms to prevent bleeding at a site of injury. The formation time, size and structure of a clot depends on the local hemodynamics and the nature of the injury. Our group has previously developed computational models to study intravascular clot formation, a process confined to the interior of a single vessel. Here we present the first stage of an experimentally-validated, computational model of extravascular clot formation (hemostasis) in which blood through a single vessel initially escapes through a hole in the vessel wall and out a separate injury channel. This stage of the model consists of a system of partial differential equations that describe platelet aggregation and hemodynamics, solved via the finite element method. We also present results from the analogous, in vitro, microfluidic model. In both models, formation of a blood clot occludes the injury channel and stops flow from escaping while blood in the main vessel retains its fluidity. We discuss the different biochemical and hemodynamic effects on clot formation using distinct geometries representing intra- and extravascular injuries.

The Pathogenic Potential of Proteus mirabilis Is Enhanced by Other Uropathogens during Polymicrobial Urinary Tract Infection.

PubMed

Armbruster, Chelsie E; Smith, Sara N; Johnson, Alexandra O; DeOrnellas, Valerie; Eaton, Kathryn A; Yep, Alejandra; Mody, Lona; Wu, Weisheng; Mobley, Harry L T

2017-02-01

Urinary catheter use is prevalent in health care settings, and polymicrobial colonization by urease-positive organisms, such as Proteus mirabilis and Providencia stuartii, commonly occurs with long-term catheterization. We previously demonstrated that coinfection with P. mirabilis and P. stuartii increased overall urease activity in vitro and disease severity in a model of urinary tract infection (UTI). In this study, we expanded these findings to a murine model of catheter-associated UTI (CAUTI), delineated the contribution of enhanced urease activity to coinfection pathogenesis, and screened for enhanced urease activity with other common CAUTI pathogens. In the UTI model, mice coinfected with the two species exhibited higher urine pH values, urolithiasis, bacteremia, and more pronounced tissue damage and inflammation compared to the findings for mice infected with a single species, despite having a similar bacterial burden within the urinary tract. The presence of P. stuartii, regardless of urease production by this organism, was sufficient to enhance P. mirabilis urease activity and increase disease severity, and enhanced urease activity was the predominant factor driving tissue damage and the dissemination of both organisms to the bloodstream during coinfection. These findings were largely recapitulated in the CAUTI model. Other uropathogens also enhanced P. mirabilis urease activity in vitro, including recent clinical isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa We therefore conclude that the underlying mechanism of enhanced urease activity may represent a widespread target for limiting the detrimental consequences of polymicrobial catheter colonization, particularly by P. mirabilis and other urease-positive bacteria. Copyright © 2017 American Society for Microbiology.

Systems Biology for Organotypic Cell Cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grego, Sonia; Dougherty, Edward R.; Alexander, Francis J.

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomicmore » data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data. This consensus report summarizes the discussions held.« less

Workshop Report: Systems Biology for Organotypic Cell Cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grego, Sonia; Dougherty, Edward R.; Alexander, Francis Joseph

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomicmore » data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.« less

Molecular dynamics modeling the synthetic and biological polymers interactions pre-studied via docking

NASA Astrophysics Data System (ADS)

Tsvetkov, Vladimir B.; Serbin, Alexander V.

2014-06-01

In previous works we reported the design, synthesis and in vitro evaluations of synthetic anionic polymers modified by alicyclic pendant groups (hydrophobic anchors), as a novel class of inhibitors of the human immunodeficiency virus type 1 ( HIV-1) entry into human cells. Recently, these synthetic polymers interactions with key mediator of HIV-1 entry-fusion, the tri-helix core of the first heptad repeat regions [ HR1]3 of viral envelope protein gp41, were pre-studied via docking in terms of newly formulated algorithm for stepwise approximation from fragments of polymeric backbone and side-group models toward real polymeric chains. In the present article the docking results were verified under molecular dynamics ( MD) modeling. In contrast with limited capabilities of the docking, the MD allowed of using much more large models of the polymeric ligands, considering flexibility of both ligand and target simultaneously. Among the synthesized polymers the dinorbornen anchors containing alternating copolymers of maleic acid were selected as the most representative ligands (possessing the top anti-HIV activity in vitro in correlation with the highest binding energy in the docking). To verify the probability of binding of the polymers with the [HR1]3 in the sites defined via docking, various starting positions of polymer chains were tried. The MD simulations confirmed the main docking-predicted priority for binding sites, and possibilities for axial and belting modes of the ligands-target interactions. Some newly MD-discovered aspects of the ligand's backbone and anchor units dynamic cooperation in binding the viral target clarify mechanisms of the synthetic polymers anti-HIV activity and drug resistance prevention.

Workshop Report: Systems Biology for Organotypic Cell Cultures

DOE PAGES

Grego, Sonia; Dougherty, Edward R.; Alexander, Francis Joseph; ...

2016-11-14

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomicmore » data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.« less

The Pathogenic Potential of Proteus mirabilis Is Enhanced by Other Uropathogens during Polymicrobial Urinary Tract Infection

PubMed Central

Smith, Sara N.; Johnson, Alexandra O.; DeOrnellas, Valerie; Eaton, Kathryn A.; Yep, Alejandra; Mody, Lona; Wu, Weisheng

2016-01-01

ABSTRACT Urinary catheter use is prevalent in health care settings, and polymicrobial colonization by urease-positive organisms, such as Proteus mirabilis and Providencia stuartii, commonly occurs with long-term catheterization. We previously demonstrated that coinfection with P. mirabilis and P. stuartii increased overall urease activity in vitro and disease severity in a model of urinary tract infection (UTI). In this study, we expanded these findings to a murine model of catheter-associated UTI (CAUTI), delineated the contribution of enhanced urease activity to coinfection pathogenesis, and screened for enhanced urease activity with other common CAUTI pathogens. In the UTI model, mice coinfected with the two species exhibited higher urine pH values, urolithiasis, bacteremia, and more pronounced tissue damage and inflammation compared to the findings for mice infected with a single species, despite having a similar bacterial burden within the urinary tract. The presence of P. stuartii, regardless of urease production by this organism, was sufficient to enhance P. mirabilis urease activity and increase disease severity, and enhanced urease activity was the predominant factor driving tissue damage and the dissemination of both organisms to the bloodstream during coinfection. These findings were largely recapitulated in the CAUTI model. Other uropathogens also enhanced P. mirabilis urease activity in vitro, including recent clinical isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We therefore conclude that the underlying mechanism of enhanced urease activity may represent a widespread target for limiting the detrimental consequences of polymicrobial catheter colonization, particularly by P. mirabilis and other urease-positive bacteria. PMID:27895127

Systems biology for organotypic cell cultures.

PubMed

Grego, Sonia; Dougherty, Edward R; Alexander, Francis J; Auerbach, Scott S; Berridge, Brian R; Bittner, Michael L; Casey, Warren; Cooley, Philip C; Dash, Ajit; Ferguson, Stephen S; Fennell, Timothy R; Hawkins, Brian T; Hickey, Anthony J; Kleensang, Andre; Liebman, Michael N J; Martin, Florian; Maull, Elizabeth A; Paragas, Jason; Qiao, Guilin Gary; Ramaiahgari, Sreenivasa; Sumner, Susan J; Yoon, Miyoung

2017-01-01

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, "organotypic" cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.

A Tick Antivirulence Protein Potentiates Antibiotics against Staphylococcus aureus

PubMed Central

Abraham, Nabil M.; Liu, Lei; Jutras, Brandon L.; Murfin, Kristen; Acar, Ali; Yarovinsky, Timur O.; Sutton, Erica; Heisig, Martin; Jacobs-Wagner, Christine

2017-01-01

ABSTRACT New strategies are needed to combat antibiotic resistance, especially against pathogens such as methicillin-resistant Staphylococcus aureus. A tick antifreeze glycoprotein, IAFGP, possesses potent antibiofilm properties against a variety of clinical pathogens, including S. aureus. Synergy between IAFGP, or a peptide (P1) representative of a repeat region of the protein, with different antibiotics was assessed in vitro. Antibiotics that synergized with either IAFPG or P1 were further evaluated in vivo using vertebrate and invertebrate infection models. IAFGP readily enhanced the efficacy of antibiotics against S. aureus. Synergy with daptomycin, an antibiotic used to treat methicillin-resistant S. aureus, was observed in vitro and in vivo using iafgp-transgenic mice and flies. Furthermore, synergy with ciprofloxacin or gentamicin, antibiotics not generally used to treat S. aureus, was also perceived. The combined effect of the antibiotic and IAFGP was associated with improved permeation of the antibiotic into the cell. Our results highlight that synergy of IAFGP with antibiotics traditionally used to treat this pathogen, and enhancement of the potency of antibiotics not commonly used against this microbe, can provide novel alternative therapeutic strategies to combat bacterial infections. PMID:28438938

Accurate and noninvasive embryos screening during in vitro fertilization (IVF) assisted by Raman analysis of embryos culture medium Accurate and noninvasive embryos screening during IVF

NASA Astrophysics Data System (ADS)

Shen, A. G.; Peng, J.; Zhao, Q. H.; Su, L.; Wang, X. H.; Hu, J. M.; Yang, J.

2012-04-01

In combination with morphological evaluation tests, we employ Raman spectroscopy to select higher potential reproductive embryos during in vitro fertilization (IVF) based on chemical composition of embryos culture medium. In this study, 57 Raman spectra are acquired from both higher and lower quality embryos culture medium (ECM) from 10 patients which have been preliminarily confirmed by clinical assay. Data are fit by using a linear combination model of least squares method in which 12 basis spectra represent the chemical features of ECM. The final fitting coefficients provide insight into the chemical compositions of culture medium samples and are subsequently used as criterion to evaluate the quality of embryos. The relative fitting coefficients ratios of sodium pyruvate/albumin and phenylalanine/albumin seem act as key roles in the embryo screening, attaining 85.7% accuracy in comparison with clinical pregnancy. The good results demonstrate that Raman spectroscopy therefore is an important candidate for an accurate and noninvasive screening of higher quality embryos, which potentially decrease the time-consuming clinical trials during IVF.

De novo epidermal regeneration using human eccrine sweat gland cells: higher competence of secretory over absorptive cells.

PubMed

Pontiggia, Luca; Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Oliveira, Carol; Braziulis, Erik; Klar, Agnieszka S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

2014-06-01

In our previous work, we showed that human sweat gland-derived epithelial cells represent an alternative source of keratinocytes to grow a near normal autologous epidermis. The role of subtypes of sweat gland cells in epidermal regeneration and maintenance remained unclear. In this study, we compare the regenerative potential of both secretory and absorptive sweat gland cell subpopulations. We demonstrate the superiority of secretory over absorptive cells in forming a new epidermis on two levels: first, the proliferative and colony-forming efficiencies in vitro are significantly higher for secretory cells (SCs), and second, SCs show a higher frequency of successful epidermis formation as well as an increase in the thickness of the formed epidermis in the in vitro and in vivo functional analyses using a 3D dermo-epidermal skin model. However, the ability of forming functional skin substitutes is not limited to SCs, which supports the hypothesis that multiple subtypes of sweat gland epithelial cells hold regenerative properties, while the existence and exact localization of a keratinocyte stem cell population in the human eccrine sweat gland remain elusive.

The use of FDTD in establishing in vitro experimentation conditions representative of lifelike cell phone radiation on the spermatozoa.

PubMed

Mouradi, Rand; Desai, Nisarg; Erdemir, Ahmet; Agarwal, Ashok

2012-01-01

Recent studies have shown that exposing human semen samples to cell phone radiation leads to a significant decline in sperm parameters. In daily living, a cell phone is usually kept in proximity to the groin, such as in a trouser pocket, separated from the testes by multiple layers of tissue. The aim of this study was to calculate the distance between cell phone and semen sample to set up an in vitro experiment that can mimic real life conditions (cell phone in trouser pocket separated by multiple tissue layers). For this reason, a computational model of scrotal tissues was designed by considering these separating layers, the results of which were used in a series of simulations using the Finite Difference Time Domain (FDTD) method. To provide an equivalent effect of multiple tissue layers, these results showed that the distance between a cell phone and semen sample should be 0.8 cm to 1.8 cm greater than the anticipated distance between a cell phone and the testes.

Effects of generation time on spray aerosol transport and deposition in models of the mouth-throat geometry.

PubMed

Worth Longest, P; Hindle, Michael; Das Choudhuri, Suparna

2009-06-01

For most newly developed spray aerosol inhalers, the generation time is a potentially important variable that can be fully controlled. The objective of this study was to determine the effects of spray aerosol generation time on transport and deposition in a standard induction port (IP) and more realistic mouth-throat (MT) geometry. Capillary aerosol generation (CAG) was selected as a representative system in which spray momentum was expected to significantly impact deposition. Sectional and total depositions in the IP and MT geometries were assessed at a constant CAG flow rate of 25 mg/sec for aerosol generation times of 1, 2, and 4 sec using both in vitro experiments and a previously developed computational fluid dynamics (CFD) model. Both the in vitro and numerical results indicated that extending the generation time of the spray aerosol, delivered at a constant mass flow rate, significantly reduced deposition in the IP and more realistic MT geometry. Specifically, increasing the generation time of the CAG system from 1 to 4 sec reduced the deposition fraction in the IP and MT geometries by approximately 60 and 33%, respectively. Furthermore, the CFD predictions of deposition fraction were found to be in good agreement with the in vitro results for all times considered in both the IP and MT geometries. The numerical results indicated that the reduction in deposition fraction over time was associated with temporal dissipation of what was termed the spray aerosol "burst effect." Based on these results, increasing the spray aerosol generation time, at a constant mass flow rate, may be an effective strategy for reducing deposition in the standard IP and in more realistic MT geometries.

Dendritic cells infected by Ad-sh-SOCS1 enhance cytokine-induced killer (CIK) cell immunotherapeutic efficacy in cervical cancer models.

PubMed

Zheng, Yi; Hu, Bicheng; Xie, Shenggao; Chen, Xiaofan; Hu, Yuqian; Chen, Wanping; Li, Shanshan; Hu, Bo

2017-05-01

Cervical cancer constitutes a major problem in women's health worldwide, but the efficacy of the standard therapy is unsatisfactory. Cytokine-induced killer (CIK) cells exhibit antitumor activity against a variety of malignancies in preclinical models and have proven safe and effective in clinical trials. However, current CIK therapy has limitations and needs to be improved to meet the clinical requirements. The aim of this study was to investigate whether suppressing the expression of cytokine signaling 1 (SOCS1) in dendritic cells (DCs) can shorten in vitro CIK culture time and improve its antitumor efficacy. DCs were pre-cultured for 3 days before infected with adenovirus-mediated-SOCS1 short hairpin RNA (Ad-sh-SOCS1) and pulsed with CTL epitope peptides E7. The DCs infected by Ad-sh-SOCS1 (gmDCs) and CIKs were then co-cultured for 5 or 9 days, and CIK proliferation and antitumor activity were evaluated both in vitro and in vivo. Our data show that gmDCs significantly stimulated the expansion of co-cultured CIKs and increased the secretion of interferon-γ and interleukin-12. Moreover, gmDCs-activated CIKs showed higher cytotoxic activity against TC-1 cells expressing HPV16E6 and E7. Our in vivo study showed that the mice infused with gmDCs-activated CIKs on day 10 had an increased survival rate and prolonged survival time compared with the controls. Taken together, these results indicate that DCs modified by adenovirus-mediated SOCS1 silencing can promote CIKs expansion and enhance the efficacy of antitumor immunotherapy both in vitro and in vivo, which represents an effective therapeutic approach for cervical cancer and other tumors. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Therapeutic potentials of naringin on polymethylmethacrylate induced osteoclastogenesis and osteolysis, in vitro and in vivo assessments

PubMed Central

Li, Nianhu; Xu, Zhanwang; Wooley, Paul H; Zhang, Jianxin; Yang, Shang-You

2014-01-01

Wear debris associated periprosthetic osteolysis represents a major pathological process associated with the aseptic loosening of joint prostheses. Naringin is a major flavonoid identified in grapefruit. Studies have shown that naringin possesses many pharmacological properties including effects on bone metabolism. The current study evaluated the influence of naringin on wear debris induced osteoclastic bone resorption both in vitro and in vivo. The osteoclast precursor cell line RAW 264.7 was cultured and stimulated with polymethylmethacrylate (PMMA) particles followed by treatment with naringin at several doses. Tartrate resistant acid phosphatase (TRAP), calcium release, and gene expression profiles of TRAP, cathepsin K, and receptor activator of nuclear factor-kappa B were sequentially evaluated. PMMA challenged murine air pouch and the load bearing tibia titanium pin-implantation mouse models were used to evaluate the effects of naringin in controlling PMMA induced bone resorption. Histological analyses and biomechanical pullout tests were performed following the animal experimentation. The in vitro data clearly demonstrated the inhibitory effects of naringin in PMMA induced osteoclastogenesis. The naringin dose of 10 μg/mL exhibited the most significant influence on the suppression of TRAP activities. Naringin treatment also markedly decreased calcium release in the stimulated cell culture medium. The short-term air pouch mouse study revealed that local injection of naringin ameliorated the PMMA induced inflammatory tissue response and subsequent bone resorption. The long-term tibia pin-implantation mouse model study suggested that daily oral gavage of naringin at 300 mg/kg dosage for 30 days significantly alleviated the periprosthetic bone resorption. A significant increase of periprosthetic bone volume and regaining of the pin stability were found in naringin treated mice. Overall, this study suggests that naringin may serve as a potential therapeutic agent to treat wear debris associated osteolysis. PMID:24376342

Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening

PubMed Central

Cheong, Wing-Lam; Tsang, Ming-San; So, Pui-Kin; Chung, Wai-Hong; Leung, Yun-Chung; Chan, Pak-Ho

2014-01-01

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening. PMID:25074398

Convection enhanced delivery of panobinostat (LBH589)-loaded pluronic nano-micelles prolongs survival in the F98 rat glioma model.

PubMed

Singleton, W G; Collins, A M; Bienemann, A S; Killick-Cole, C L; Haynes, H R; Asby, D J; Butts, C P; Wyatt, M J; Barua, N U; Gill, S S

2017-01-01

The pan-histone deacetylase inhibitor panobinostat is a potential therapy for malignant glioma, but it is water insoluble and does not cross the blood-brain barrier when administered systemically. In this article, we describe the in vitro and in vivo efficacy of a novel water-soluble nano-micellar formulation of panobinostat designed for administration by convection enhanced delivery (CED). The in vitro efficacy of panobinostat-loaded nano-micelles against rat F98, human U87-MG and M059K glioma cells and against patient-derived glioma stem cells was measured using a cell viability assay. Nano-micelle distribution in rat brain was analyzed following acute CED using rhodamine-labeled nano-micelles, and toxicity was assayed using immunofluorescent microscopy and synaptophysin enzyme-linked immunosorbent assay. We compared the survival of the bioluminescent syngenic F98/Fischer344 rat glioblastoma model treated by acute CED of panobinostat-loaded nano-micelles with that of untreated and vehicle-only-treated controls. Nano-micellar panobinostat is cytotoxic to rat and human glioma cells in vitro in a dose-dependent manner following short-time exposure to drug. Fluorescent rhodamine-labelled nano-micelles distribute with a volume of infusion/volume of distribution (Vi/Vd) ratio of four and five respectively after administration by CED. Administration was not associated with any toxicity when compared to controls. CED of panobinostat-loaded nano-micelles was associated with significantly improved survival when compared to controls (n=8 per group; log-rank test, P <0.001). One hundred percent of treated animals survived the 60-day experimental period and had tumour response on post-mortem histological examination. CED of nano-micellar panobinostat represents a potential novel therapeutic option for malignant glioma and warrants translation into the clinic.

In vitro and in vivo anti-glioma activity of a chalcone-quinoxaline hybrid.

PubMed

Loch-Neckel, Gecioni; Bicca, Maíra Assunção; Leal, Paulo César; Mascarello, Alessandra; Siqueira, Jarbas Mota; Calixto, João B

2015-01-27

Chalcones are important compounds that exhibit multiple biological activities, including anti-inflammatory, antimitotic and antibacterial properties. In the present study, we have analyzed the potential anti-cancer activity of a chalcone named N9 (a hybrid chalcone-quinoxaline compound) using in vitro and in vivo experimental glioma models. Here, we report N9-induced inhibition of cell proliferation and also N9-induced cell death in a concentration-dependent manner in U87-MG glioma cells. These effects of N9 appear to be associated with its ability to inhibit the expression of cell cycle-associated proteins, and also the augmentation in the expression of the p21 (p21/Cip1) protein, a cyclin-dependent kinase inhibitor. Additionally, N9 also potentiates the production of the pro-apoptotic markers Bax and p53 via inhibition of MDM2. Moreover, our results show that N9 also significantly enhanced apoptosis of U87-MG cells with disruption of mitochondrial membrane potential, generation of ROS and caspase-9 activation. In vivo experiments carried out in a murine xenograft model of U87-MG revealed that N9 produced a significant reduction of tumors volume when compared to vehicle treated mice. Collectively, data demonstrate that N9 possess in vitro and in vivo anti-cancer activity, an effect that seems to involve the induction of p53 and p21 proteins, as well as, the activation of mitochondrial apoptosis pathway associated with the inhibition of protein MDM2. Overall, this study suggests N9 is affecting a variety of intracellular pathways related to tumor apoptosis. Perhaps N9 or derivate molecules could represent new potential drugs for cancer therapeutics. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system.

PubMed

Cheng, Liang; Zhang, Zheng; Li, Guangming; Li, Feng; Wang, Li; Zhang, Liguo; Zurawski, Sandra M; Zurawski, Gerard; Levy, Yves; Su, Lishan

2017-10-27

TLR ligands (TLR-Ls) represent a class of novel vaccine adjuvants. However, their immunologic effects in humans remain poorly defined in vivo. Using a humanized mouse model with a functional human immune system, we investigated how different TLR-Ls stimulated human innate immune response in vivo and their applications as vaccine adjuvants for enhancing human cellular immune response. We found that splenocytes from humanized mice showed identical responses to various TLR-Ls as human PBMCs in vitro. To our surprise, various TLR-Ls stimulated human cytokines and chemokines differently in vivo compared to that in vitro. For example, CpG-A was most efficient to induce IFN-α production in vitro. In contrast, CpG-B, R848 and Poly I:C stimulated much more IFN-α than CpG-A in vivo. Importantly, the human innate immune response to specific TLR-Ls in humanized mice was different from that reported in C57BL/6 mice, but similar to that reported in nonhuman primates. Furthermore, we found that different TLR-Ls distinctively activated and mobilized human plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs) and monocytes in different organs. Finally, we showed that, as adjuvants, CpG-B, R848 and Poly I:C can all enhance antigen specific CD4 + T cell response, while only R848 and Poly I:C induced CD8 + cytotoxic T cells response to a CD40-targeting HIV vaccine in humanized mice, correlated with their ability to activate human mDCs but not pDCs. We conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

Utilization of Mytilus digestive gland cells for the in vitro screening of potential metabolic disruptors in aquatic invertebrates.

PubMed

Balbi, Teresa; Ciacci, Caterina; Grasselli, Elena; Smerilli, Arianna; Voci, Adriana; Canesi, Laura

2017-01-01

In vertebrate systems, many endocrine disruptors (EDs) can also interfere with energy and lipid metabolism, thus acting as metabolic disruptors. At the cellular level, these effects are mainly mediated by interactions with nuclear receptors/transcription factors, leading to the modulation of genes involved in lipid homeostasis, as well as by rapid, receptor-independent pathways. Several potential metabolic disruptors are found in aquatic environments. In fish, different EDs have been shown to affect hepatic lipid homeostasis both in vivo and in vitro. However, little information is available in aquatic invertebrates due to our poor knowledge of the regulatory pathways of lipid metabolism. In this work, primary cell cultures from the digestive gland of the bivalve Mytilus galloprovincialis were utilized to investigate the effects of model EDs (bisphenol A (BPA) and perfluorooctane sulphonate (PFOS)) on lipid homeostasis. Both compounds (at 24 and 3h of exposure) increased intracellular lipid and tryglyceride-TAG content, with strongest effects of PFOS at 10 -7 M. Acyl-CoA oxidase activity was unaffected, whereas some changes in the activity of glycolytic, antioxidant/biotransformation enzymes were observed; however, no clear relationship was found with lipid accumulation. Evaluation of mitochondrial membrane potential Δψm and determination of extracellular TAG content indicate that PFOS interferes with mitochondrial function and lipid secretion, whereas BPA mainly affects lipid secretion. Experiments with specific inhibitors showed that activation of PI-3 kinase and extracellularly regulated mitogen-activated protein kinase (ERK MAPK) plays a key role in mediating lipid accumulation. Mussel digestive gland cells represent a simple in vitro model for screening the metabolic effects of EDs in marine invertebrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Potential Molecular Targets of Statins in the Prevention of Hepatocarcinogenesis.

PubMed

Ridruejo, Ezequiel; Romero-Caími, Giselle; Obregón, María J; Kleiman de Pisarev, Diana; Alvarez, Laura

2018-04-09

Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-β1) and thyroid hormones (TH) regulation. The aim of our study is to establish the statins mechanism of action and the potential key molecules involved in an in vivo and in vitro HCC model. We used two models: in vivo (in rats) using diethylnitrosamine (DEN) and hexachlorobenzene (HCB) to develop HCC, we analyzed cell proliferation parameters (proliferating cell nuclear antigen, PCNA) and cholesterol metabolism (hydroxy-methylglutaryl-CoA reductase, HMGCoAR). In vitro (Hep-G2 cells) we evaluated the effects of different doses of Atorvastatin (AT) and Simvastatin (SM) on HCB induced proliferation and analyzed proliferative parameters, colesterol metabolism, TGF-β1 mRNA, c-Src and TH levels. In vivo, we observed that cell proliferation significantly increased as well as cholesterol serum levels in rats treated with HCB. In vitro, we observed the same results on PCNA as in vivo. The statins prevented the increase in HMG-CoAR mRNA levels induced by HCB, reaching levels similar to controls at máximum doses: AT (30 μM), and SM (20 μM). Increases in PCNA, TGF-β1, and pc-Src, and decreases in deiodinase I mRNA levels induced by HCB were not observed when cells were pre-treated with AT and SM at maximum doses. Statins can prevent the proliferative HCB effects on Hep-G2 cells. TGF-β1, c-Src and TH may be the statins molecular targets in hepatocarcinogenesis.

Antiviral activities of 2,6-diaminopurine-based acyclic nucleoside phosphonates against herpesviruses: In vitro study results with pseudorabies virus (PrV, SuHV-1).

PubMed

Zouharova, Darina; Lipenska, Ivana; Fojtikova, Martina; Kulich, Pavel; Neca, Jiri; Slany, Michal; Kovarcik, Kamil; Turanek-Knotigova, Pavlina; Hubatka, Frantisek; Celechovska, Hana; Masek, Josef; Koudelka, Stepan; Prochazka, Lubomir; Eyer, Ludek; Plockova, Jana; Bartheldyova, Eliska; Miller, Andrew D; Ruzek, Daniel; Raska, Milan; Janeba, Zlatko; Turanek, Jaroslav

2016-02-29

Pseudorabies virus (PrV), a causative agent of Aujeszky's disease, is deadly to most mammals with the exception of higher primates and men. This disease causes serious economic loses among farm animals, especially pigs, yet many European countries are today claimed to be Aujeszky's disease free because of the discovery of an efficient vaccination for pigs. In reality, the virus is still present in wild boar. Current vaccines are neither suitable for dogs nor are there anti-PrV drugs approved for veterinary use. Therefore, the disease still represents a high threat, particularly for expensive hunting dogs that can come into close contact with infected boars. Here we report on the anti-PrV activities of a series of synthetic diaminopurine-based acyclic nucleoside phosphonate (DAP-ANP) analogues. Initially, all synthetic DAP-ANPs under investigation are shown to exhibit minimal cytotoxicity by MTT and XTT tests (1-100μM range). Thereafter in vitro infection models are established using PrV virus SuHV-1, optimized on PK-15 and RK-13 cell lines. Out of the six DAP-ANP analogues tested, analogue VI functionalized with a cyclopropyl group on the 6-amino position of the purine ring proves the most effective antiviral DAP-ANP analogue against PrV infection, aided by sufficient hydrophobic character to enhance bioavailability to its cellular target viral DNA-polymerase. Four other DAP-ANP analogues with functional groups introduced to the C2'position are shown ineffective against PrV infection, even with favourable hydrophobic properties. Cidofovir(®), a drug approved against various herpesvirus infections, is found to exert only low activity against PrV in these same in vitro models. Copyright © 2016 Elsevier B.V. All rights reserved.

Inhibition of gamma-secretase activity impedes uterine serous carcinoma growth in a human xenograft model.

PubMed

Groeneweg, Jolijn W; Hall, Tracilyn R; Zhang, Ling; Kim, Minji; Byron, Virginia F; Tambouret, Rosemary; Sathayanrayanan, Sriram; Foster, Rosemary; Rueda, Bo R; Growdon, Whitfield B

2014-06-01

Uterine serous carcinoma (USC) represents an aggressive subtype of endometrial cancer. We sought to understand Notch pathway activity in USC and determine if pathway inhibition has anti-tumor activity. Patient USC tissue blocks were obtained and used to correlate clinical outcomes with Notch1 expression. Three established USC cell lines were treated with gamma-secretase inhibitor (GSI) in vitro. Mice harboring cell line derived or patient derived USC xenografts (PDXs) were treated with vehicle, GSI, paclitaxel and carboplatin (P/C), or combination GSI and P/C. Levels of cleaved Notch1 protein and Hes1 mRNA were determined in GSI treated samples. Statistical analysis was performed using the Wilcoxon rank sum and Kaplan-Meier methods. High nuclear Notch1 protein expression was observed in 58% of USC samples with no correlation with overall survival. GSI induced dose-dependent reductions in cell number and decreased levels of cleaved Notch1 protein and Hes1 mRNA in vitro. Treatment of mice with GSI led to decreased Hes1 mRNA expression in USC xenografts. In addition, GSI impeded tumor growth of cell line xenografts as well as UT1 USC PDXs. When GSI and P/C were combined, synergistic anti-tumor activity was observed in UT1 xenografts. Notch1 is expressed in a large subset of USC. GSI-mediated Notch pathway inhibition led to both reduced cell numbers in vitro and decreased tumor growth of USC some xenograft models. When combined with conventional chemotherapy, GSI augmented anti-tumor activity in one USC PDX line suggesting that targeting of the Notch signaling pathway is a potential therapeutic strategy for future investigation. Copyright © 2014 Elsevier Inc. All rights reserved.

Heterogeneity among Isolates Reveals that Fitness in Low Oxygen Correlates with Aspergillus fumigatus Virulence.

PubMed

Kowalski, Caitlin H; Beattie, Sarah R; Fuller, Kevin K; McGurk, Elizabeth A; Tang, Yi-Wei; Hohl, Tobias M; Obar, Joshua J; Cramer, Robert A

2016-09-20

Previous work has shown that environmental and clinical isolates of Aspergillus fumigatus represent a diverse population that occupies a variety of niches, has extensive genetic diversity, and exhibits virulence heterogeneity in a number of animal models of invasive pulmonary aspergillosis (IPA). However, mechanisms explaining differences in virulence among A. fumigatus isolates remain enigmatic. Here, we report a significant difference in virulence of two common lab strains, CEA10 and AF293, in the murine triamcinolone immunosuppression model of IPA, in which we previously identified severe low oxygen microenvironments surrounding fungal lesions. Therefore, we hypothesize that the ability to thrive within these lesions of low oxygen promotes virulence of A. fumigatus in this model. To test this hypothesis, we performed in vitro fitness and in vivo virulence analyses in the triamcinolone murine model of IPA with 14 environmental and clinical isolates of A. fumigatus Among these isolates, we observed a strong correlation between fitness in low oxygen in vitro and virulence. In further support of our hypothesis, experimental evolution of AF293, a strain that exhibits reduced fitness in low oxygen and reduced virulence in the triamcinolone model of IPA, results in a strain (EVOL20) that has increased hypoxia fitness and a corresponding increase in virulence. Thus, the ability to thrive in low oxygen correlates with virulence of A. fumigatus isolates in the context of steroid-mediated murine immunosuppression. Aspergillus fumigatus occupies multiple environmental niches, likely contributing to the genotypic and phenotypic heterogeneity among isolates. Despite reports of virulence heterogeneity, pathogenesis studies often utilize a single strain for the identification and characterization of virulence and immunity factors. Here, we describe significant variation between A. fumigatus isolates in hypoxia fitness and virulence, highlighting the advantage of including multiple strains in future studies. We also illustrate that hypoxia fitness correlates strongly with increased virulence exclusively in the nonleukopenic murine triamcinolone immunosuppression model of IPA. Through an experimental evolution experiment, we observe that chronic hypoxia exposure results in increased virulence of A. fumigatus We describe here the first observation of a model-specific virulence phenotype correlative with in vitro fitness in hypoxia and pave the way for identification of hypoxia-mediated mechanisms of virulence in the fungal pathogen A. fumigatus. Copyright © 2016 Kowalski et al.

Defined, serum/feeder-free conditions for expansion and drug screening of primary B-acute lymphoblastic leukemia.

PubMed

Jiang, Zhiwu; Wu, Di; Ye, Wei; Weng, Jianyu; Lai, Peilong; Shi, Pengcheng; Guo, Xutao; Huang, Guohua; Deng, Qiuhua; Tang, Yanlai; Zhao, Hongyu; Cui, Shuzhong; Lin, Simiao; Wang, Suna; Li, Baiheng; Wu, Qiting; Li, Yangqiu; Liu, Pentao; Pei, Duanqing; Du, Xin; Yao, Yao; Li, Peng

2017-12-05

Functional screening for compounds represents a major hurdle in the development of rational therapeutics for B-acute lymphoblastic leukemia (B-ALL). In addition, using cell lines as valid models for evaluating responses to novel drug therapies raises serious concerns, as cell lines are prone to genotypic/phenotypic drift and loss of heterogeneity in vitro . Here, we reported that OP9 cells, not OP9-derived adipocytes (OP9TA), support the growth of primary B-ALL cells in vitro . To identify the factors from OP9 cells that support the growth of primary B-ALL cells, we performed RNA-Seq to analyze the gene expression profiles of OP9 and OP9TA cells. We thus developed a defined, serum/feeder-free condition (FI76V) that can support the expansion of a range of clinically distinct primary B-ALL cells that still maintain their leukemia-initiating ability. We demonstrated the suitability of high-throughput drug screening based on our B-ALL cultured conditions. Upon screening 378 kinase inhibitors, we identified a cluster of 17 kinase inhibitors that can efficiently kill B-ALL cells in vitro . Importantly, we demonstrated the synergistic cytotoxicity of dinaciclib/BTG226 to B-ALL cells. Taken together, we developed a defined condition for the ex vivo expansion of primary B-ALL cells that is suitable for high-throughput screening of novel compounds.

Activation of Wnt Planar Cell Polarity (PCP) signaling promotes growth plate column formation in vitro.

PubMed

Randall, Rachel M; Shao, Yvonne Y; Wang, Lai; Ballock, R Tracy

2012-12-01

Disrupting the Wnt Planar Cell Polarity (PCP) signaling pathway in vivo results in loss of columnar growth plate architecture, but it is unknown whether activation of this pathway in vitro is sufficient to promote column formation. We hypothesized that activation of the Wnt PCP pathway in growth plate chondrocyte cell pellets would promote columnar organization in these cells that are normally oriented randomly in culture. Rat growth plate chondrocytes were transfected with plasmids encoding the Fzd7 cell-surface Wnt receptor, a Fzd7 deletion mutant lacking the Wnt-binding domain, or Wnt receptor-associated proteins Ror2 or Vangl2, and then cultured as three-dimensional cell pellets in the presence of recombinant Wnt5a or Wnt5b for 21 days. Cellular morphology was evaluated using histomorphometric measurements. Activation of Wnt PCP signaling components promoted the initiation of columnar morphogenesis in the chondrocyte pellet culture model, as measured by histomorphometric analysis of the column index (ANOVA p = 0.01). Activation of noncanonical Wnt signaling through overexpression of both the cell-surface Wnt receptor Fzd7 and receptor-associated protein Ror2 with addition of recombinant Wnt5a promotes the initiation of columnar architecture of growth plate chondrocytes in vitro, representing an important step toward growth plate regeneration. Copyright © 2012 Orthopaedic Research Society.

Research in drug development against viral diseases of military importance (biological testing). Volume 2. Final report, 15 November 1985-31 January 1991

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shannon, W.M.; Arnett, G.; Brazier, A.D.

1991-03-01

The purpose of this program is to evaluate the efficacy of candidate antiviral compounds against a spectrum of viruses of military importance. This program involves (a) primary testing of chemical compounds and natural products for antiviral efficacy in vitro using standard CPE-inhibition assays, (b) primary testing of compounds for antiviral efficacy in vivo in animal model systems, and (c) secondary evaluation of the active candidate antiviral compounds. The target viruses for in vitro testing are Vaccinia Virus (VV), Adenovirus (AD2), Vesicular Stomatitis Virus (VSV), Punta Toro Virus (PT), Sandfly fever Virus (SF), Yellow Fever Virus (YF), Venezuelan Equine Encephalomyelitis Virusmore » (VE), Japanese Encephalitis Virus, Pichinde Virus (PIC), Hantaan Virus (HTN), and Human Immunodeficiency Virus (HIV). The in vivo systems are Pichinde Virus infection of hamsters, Venezuelan Equine Encephalomyelitis Virus, Japanese Encephalitis Virus and Vaccinia virus infections of mice. Approximately 10,000 compounds have been received for in vitro evaluation and over 66,000 assays have been performed on this contract. Compounds have been identified in nearly all virus systems that have confirmed antiviral activity equal or exceeding that of the various positive control compounds (ribavirin, selenazofurin, carbocyclic-3-aza-adenosine, adenosine dialdehyde, Ara-A, ddC and AZT). Many of these compounds represent potent and selective new antiviral agents.« less

Expression profiling of Yersinia pestis during mouse pulmonary infection.

PubMed

Lawson, Jonathan N; Lyons, C Rick; Johnston, Stephen Albert

2006-11-01

Yersinia pestis, the causative agent of plague, can be transmitted by infected flea bite or inhaled aerosol. Both routes of infection have a high mortality rate, and pneumonic infections of Y. pestis represent a significant concern as a tool of bioterrorism. Understanding the transcriptional program of this pathogen during pulmonary infection should be valuable in understanding plague pathogenesis, as well as potentially offering insights into new vaccines and therapeutics. Toward this goal we developed a long oligonucleotide microarray to the plague bacillus and evaluated the expression profiles of Y. pestis in vitro and in the mouse pulmonary infection model in vivo. The in vitro analysis compared expression patterns at 27 versus 37 degrees C, as a surrogate of the transition from the flea to the mammalian host. The in vivo analysis used intranasal challenge to the mouse lung. By amplifying the Y. pestis RNA from individual mouse lungs we were able to map the transcriptional profile of plague at postinfection days 1 to 3. Our data present a very different transcriptional profile between in vivo and in vitro expression, suggesting Y. pestis responds to a variety of host signals during infection. Of note was the number of genes found in genomic regions with altered %GC content that are upregulated within the mouse lung environment. These data suggest these regions may provide particularly promising targets for both vaccines and therapeutics.

The effect of a combination of 0.1% octenidine dihydrochloride and 2% 2-phenoxyethanol (octenisept) on wound healing in pigs in vivo and its in vitro percutaneous permeation through intact and barrier disrupted porcine skin.

PubMed

Stahl, Jessica; Braun, Michael; Siebert, Joerg; Kietzmann, Manfred

2010-02-01

A combination of 0.1% octenidine dihydrochloride and 2% 2-phenoxyethanol (octenisept) is a commonly used disinfectant in human medicine. As porcine skin represents an adequate model for human skin, the effect of octenidine dihydrochloride and phenoxyethanol on wound healing is studied in pigs. Furthermore, the in vitro percutaneous permeation of the test substances is studied. The impact of the test formulations on wound healing is examined (A) under non occlusive conditions and (B) in comparison to another disinfectant based on povidone-iodine under occlusive conditions, while wounds are treated daily with the test substances. The percutaneous permeation of octenidine dihydrochloride and phenoxyethanol is studied in Franz-type diffusion cells with intact skin as well as barrier disrupted after tape stripping. Compared with povidone-iodine or vehicle treatment as well as untreated control wounds the treatment of wounds with the test formulation has no influence on the healing rate in pigs and does not induce retardation of wound healing. The in vitro diffusion experiment reveals that octenidine dihydrochloride is only detectable in the acceptor chamber of three-barrier disrupted skin samples. Phenoxyethanol permeates through intact porcine skin in amounts of 11.3% and through barrier disrupted skin in amounts of 43.9%

In Vitro Cultivation of 'Unculturable' Oral Bacteria, Facilitated by Community Culture and Media Supplementation with Siderophores.

PubMed

Vartoukian, Sonia R; Adamowska, Aleksandra; Lawlor, Megan; Moazzez, Rebecca; Dewhirst, Floyd E; Wade, William G

2016-01-01

Over a third of oral bacteria are as-yet-uncultivated in-vitro. Siderophores have been previously shown to enable in-vitro growth of previously uncultivated bacteria. The objective of this study was to cultivate novel oral bacteria in siderophore-supplemented culture media. Various compounds with siderophore activity, including pyoverdines-Fe-complex, desferricoprogen and salicylic acid, were found to stimulate the growth of difficult-to-culture strains Prevotella sp. HOT-376 and Fretibacterium fastidiosum. Furthermore, pyrosequencing analysis demonstrated increased proportions of the as-yet-uncultivated phylotypes Dialister sp. HOT-119 and Megasphaera sp. HOT-123 on mixed culture plates supplemented with siderophores. Therefore a culture model was developed, which incorporated 15 μg siderophore (pyoverdines-Fe-complex or desferricoprogen) or 150 μl neat subgingival-plaque suspension into a central well on agar plates that were inoculated with heavily-diluted subgingival-plaque samples from subjects with periodontitis. Colonies showing satellitism were passaged onto fresh plates in co-culture with selected helper strains. Five novel strains, representatives of three previously-uncultivated taxa (Anaerolineae bacterium HOT-439, the first oral taxon from the Chloroflexi phylum to have been cultivated; Bacteroidetes bacterium HOT-365; and Peptostreptococcaceae bacterium HOT-091) were successfully isolated. All novel isolates required helper strains for growth, implying dependence on a biofilm lifestyle. Their characterisation will further our understanding of the human oral microbiome.

Effect of mean diameter and polydispersity of PLG microspheres on drug release: experiment and theory.

PubMed

Berchane, N S; Carson, K H; Rice-Ficht, A C; Andrews, M J

2007-06-07

The need to tailor release rate profiles from polymeric microspheres is a significant problem. Microsphere size, which has a significant effect on drug release rate, can potentially be varied to design a controlled drug delivery system with desired release profile. In this work the effects of microspheres mean diameter, polydispersity, and polymer degradation on drug release rate from poly(lactide-co-glycolide) (PLG) microspheres are described. Piroxicam containing PLG microspheres were fabricated at 20% loading, and at three different impeller speeds. A portion of the microspheres was then sieved giving five different size distributions. In vitro release kinetics were determined for each preparation. Based on these experimental results, a suitable mathematical theory has been developed that incorporates the effect of microsphere size distribution and polymer degradation on drug release. We show from in vitro release experiments that microsphere size has a significant effect on drug release rate. The initial release rate decreased with an increase in microsphere size. In addition, the release profile changed from first order to concave-upward (sigmoidal) as the microsphere size was increased. The mathematical model gave a good fit to the experimental release data. For highly polydisperse populations (polydispersity parameter b<3), incorporating the microsphere size distribution into the mathematical model gave a better fit to the experimental results than using the representative mean diameter. The validated mathematical model can be used to predict small-molecule drug release from PLG microsphere populations.

Functional, thermodynamics, structural and biological studies of in silico-identified inhibitors of Mycobacterium tuberculosis enoyl-ACP(CoA) reductase enzyme

NASA Astrophysics Data System (ADS)

Martinelli, Leonardo K. B.; Rotta, Mariane; Villela, Anne D.; Rodrigues-Junior, Valnês S.; Abbadi, Bruno L.; Trindade, Rogério V.; Petersen, Guilherme O.; Danesi, Giuliano M.; Nery, Laura R.; Pauli, Ivani; Campos, Maria M.; Bonan, Carla D.; de Souza, Osmar Norberto; Basso, Luiz A.; Santos, Diogenes S.

2017-04-01

Novel chemotherapeutics agents are needed to kill Mycobacterium tuberculosis, the main causative agent of tuberculosis (TB). The M. tuberculosis 2-trans-enoyl-ACP(CoA) reductase enzyme (MtInhA) is the druggable bona fide target of isoniazid. New chemotypes were previously identified by two in silico approaches as potential ligands to MtInhA. The inhibition mode was determined by steady-state kinetics for seven compounds that inhibited MtInhA activity. Dissociation constant values at different temperatures were determined by protein fluorescence spectroscopy. van’t Hoff analyses of ligand binding to MtInhA:NADH provided the thermodynamic signatures of non-covalent interactions (ΔH°, ΔS°, ΔG°). Phenotypic screening showed that five compounds inhibited in vitro growth of M. tuberculosis H37Rv strain. Labio_16 and Labio_17 compounds also inhibited the in vitro growth of PE-003 multidrug-resistant strain. Cytotoxic effects on Hacat, Vero and RAW 264.7 cell lines were assessed for the latter two compounds. The Labio_16 was bacteriostatic and Labio_17 bactericidal in an M. tuberculosis-infected macrophage model. In Zebrafish model, Labio_16 showed no cardiotoxicity whereas Labio_17 showed dose-dependent cardiotoxicity. Accordingly, a model was built for the MtInhA:NADH:Labio_16 ternary complex. The results show that the Labio_16 compound is a direct inhibitor of MtInhA, and it may represent a hit for the development of chemotherapeutic agents to treat TB.

Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis.

PubMed

Schmelcher, Mathias; Powell, Anne M; Camp, Mary J; Pohl, Calvin S; Donovan, David M

2015-10-01

Bovine mastitis results in billion dollar losses annually in the USA alone. Streptococci are among the most relevant causative agents of this disease. Conventional antibiotic therapy is often unsuccessful and contributes to development of antibiotic resistance. Bacteriophage endolysins represent a new class of antimicrobials against these bacteria. In this work, we characterized the endolysins (lysins) of the streptococcal phages λSA2 and B30 and evaluated their potential as anti-mastitis agents. When tested in vitro against live streptococci, both enzymes exhibited near-optimum lytic activities at ionic strengths, pH, and Ca(2+) concentrations consistent with cow milk. When tested in combination in a checkerboard assay, the lysins were found to exhibit strong synergy. The λSA2 lysin displayed high activity in milk against Streptococcus dysgalactiae (reduction of CFU/ml by 3.5 log units at 100 μg/ml), Streptococcus agalactiae (2 log), and Streptococcus uberis (4 log), whereas the B30 lysin was less effective. In a mouse model of bovine mastitis, both enzymes significantly reduced intramammary concentrations of all three streptococcal species (except for B30 vs. S. dysgalactiae), and the effects on mammary gland wet weights and TNFα concentrations were consistent with these findings. Unexpectedly, the synergistic effect determined for the two enzymes in vitro was not observed in the mouse model. Overall, our results illustrate the potential of endolysins for treatment of Streptococcus-induced bovine mastitis.

Synergistic streptococcal phage λSA2 and B30 endolysins kill streptococci in cow milk and in a mouse model of mastitis

PubMed Central

Schmelcher, Mathias; Powell, Anne M.; Camp, Mary J.; Pohl, Calvin S.; Donovan, David M.

2015-01-01

Bovine mastitis results in billion dollar losses annually in the United States alone. Streptococci are among the most relevant causative agents of this disease. Conventional antibiotic therapy is often unsuccessful and contributes to development of antibiotic resistance. Bacteriophage endolysins represent a new class of antimicrobials against these bacteria. In this work, we characterized the endolysins (lysins) of the streptococcal phages λSA2 and B30 and evaluated their potential as anti-mastitis agents. When tested in vitro against live streptococci, both enzymes exhibited near-optimum lytic activities at ionic strengths, pH, and Ca2+ concentrations consistent with cow milk. When tested in combination in a checkerboard assay, the lysins were found to exhibit strong synergy. The λSA2 lysin displayed high activity in milk against Streptococcus dysgalactiae (reduction of CFU/ml by 3.5 log units at 100 μg/ml), Streptococcus agalactiae (2 log), and Streptococcus uberis (4 log), whereas the B30 lysin was less effective. In a mouse model of bovine mastitis, both enzymes significantly reduced intramammary concentrations of all three streptococcal species (except for B30 vs. S. dysgalactiae), and the effects on mammary gland wet weights and TNFα concentrations were consistent with these findings. Unexpectedly, the synergistic effect determined for the two enzymes in vitro was not observed in the mouse model. Overall, our results illustrate the potential of endolysins for treatment of Streptococcus-induced bovine mastitis. PMID:25895090

Comparative Iron Oxide Nanoparticle Cellular Dosimetry and Response in Mice by the Inhalation and Liquid Cell Culture Exposure Routes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teeguarden, Justin G.; Mikheev, Vladimir B.; Minard, Kevin R.

testing the rapidly growing number of nanomaterials requires large scale use of in vitro systems under the presumption that these systems are sufficiently predictive or descriptive of responses in in vivo systems for effective use in hazard ranking. We hypothesized that improved relationships between in vitro and in vivo models of experimental toxicology for nanomaterials would result from placing response data in vitro and in vivo on the same dose scale, the amount of material associated with cells (target cell dose). Methods: Balb/c mice were exposed nose-only to an aerosol of 12.8 nm (68.6 nm CMD, 19.9 mg/m3, 4 hours)more » super paramagnetic iron oxide particles, target cell doses were calculated and biomarkers of response anchored with histological evidence were identified by global transcriptomics. Representative murine epithelial and macrophage cell types were exposed in vitro to the same material in liquid suspension for four hours and levels nanoparticle regulated cytokine transcripts identified in vivo were quantified as a function of measured nanoparticle cellular dose. Results. Target tissue doses of 0.009-0.4 μg SPIO/cm2 lung led to an inflammatory response in the alveolar region characterized by interstitial inflammation and macrophage infiltration. In vitro, higher target tissue doses of ~1.2-4 μg SPIO/ cm2 of cells were required to induce transcriptional regulation of markers of inflammation, CXCL2 CCL3, in C10 lung epithelial cells. Estimated in vivo macrophage SPIO nanoparticle doses ranged from 1-100 pg/cell, and induction of inflammatory markers was observed in vitro in macrophages at doses of 8-35 pg/cell. Conclusions: Application of target tissue dosimetry revealed good correspondence between target cell doses triggering inflammatory processes in vitro and in vivo in the alveolar macrophage population, but not in the epithelial cells of the alveolar region. These findings demonstrate the potential for target tissue dosimetry to enable the more quantitative comparison of in vitro and in vivo systems advance their use for hazard assessment and extrapolation to humans. The mildly inflammogentic cellular doses experienced by mice were similar those calculated for humans exposed to the same at the existing permissible exposure limit of 10 mg/m3 iron oxide (as Fe).« less

Anti-HIV-1 Activity of Flavonoid Myricetin on HIV-1 Infection in a Dual-Chamber In Vitro Model

PubMed Central

Pasetto, Silvana; Pardi, Vanessa; Murata, Ramiro Mendonça

2014-01-01

HIV infection by sexual transmission remains an enormous global health concern. More than 1 million new infections among women occur annually. Microbicides represent a promising prevention strategy that women can easily control. Among emerging therapies, natural small molecules such as flavonoids are an important source of new active substances. In this study we report the in vitro cytotoxicity and anti-HIV-1 and microbicide activity of the following flavonoids: Myricetin, Quercetin and Pinocembrin. Cytotoxicity tests were conducted on TZM-bl, HeLa, PBMC, and H9 cell cultures using 0.01–100 µM concentrations. Myricetin presented the lowest toxic effect, with Quercetin and Pinocembrin relatively more toxic. The anti-HIV-1 activity was tested with TZM-bl cell plus HIV-1 BaL (R5 tropic), H9 and PBMC cells plus HIV-1 MN (X4 tropic), and the dual tropic (X4R5) HIV-1 89.6. All flavonoids showed anti-HIV activity, although Myricetin was more effective than Quercetin or Pinocembrin. In TZM-bl cells, Myricetin inhibited ≥90% of HIV-1 BaL infection. The results were confirmed by quantification of HIV-1 p24 antigen in supernatant from H9 and PBMC cells following flavonoid treatment. In H9 and PBMC cells infected by HIV-1 MN and HIV-1 89.6, Myricetin showed more than 80% anti-HIV activity. Quercetin and Pinocembrin presented modest anti-HIV activity in all experiments. Myricetin activity was tested against HIV-RT and inhibited the enzyme by 49%. Microbicide activities were evaluated using a dual-chamber female genital tract model. In the in vitro microbicide activity model, Myricetin showed promising results against different strains of HIV-1 while also showing insignificant cytotoxic effects. Further studies of Myricetin should be performed to identify its molecular targets in order to provide a solid biological foundation for translational research. PMID:25546350

Per- and polyfluoroalkyl substances impact human spermatogenesis in a stem-cell-derived model.

PubMed

Steves, Alyse N; Turry, Adam; Gill, Brittany; Clarkson-Townsend, Danielle; Bradner, Joshua M; Bachli, Ian; Caudle, W Michael; Miller, Gary W; Chan, Anthony W S; Easley, Charles A

2018-06-18

Per- and polyfluoroalkyl substances (PFASs) represent a highly ubiquitous group of synthetic chemicals used in products ranging from water and oil repellents and lubricants to firefighting foam. These substances can enter and accumulate in multiple tissue matrices in up to 100% of people assessed. Though animal models strongly identify these compounds as male reproductive toxicants, with exposed rodents experiencing declines in sperm count, alterations in hormones, and DNA damage in spermatids, among other adverse outcomes, human studies report conflicting conclusions as to the reproductive toxicity of these chemicals. Using an innovative, human stem-cell-based model of spermatogenesis, we assessed the effects of the PFASs perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and a mixture of PFOS, PFOA, and PFNA for their impacts on human spermatogenesis in vitro under conditions relevant to the general and occupationally exposed populations. Here, we show that PFOS, PFOA, PFNA, and a mixture of PFOS, PFOA, and PFNA do not decrease in vitro germ cell viability, consistent with reports from human studies. These compounds do not affect mitochondrial membrane potential or increase reactive oxygen species generation, and they do not decrease cell viability of spermatogonia, primary spermatocytes, secondary spermatocytes, or spermatids in vitro under the conditions examined. However, exposure to PFOS, PFOA, and PFNA reduces expression of markers for spermatogonia and primary spermatocytes. While not having direct effects on germ cell viability, these effects suggest the potential for long-term impacts on male fertility through the exhaustion of the spermatogonial stem cell pool and abnormalities in primary spermatocytes. CDC: Centers for Disease Control; DMSO: dimethyl sulfoxide; GHR: growth hormone receptor; hESCs: human embryonic stem cells; PFASs: per- and polyfluoroalkyl substances; PFCs: perfluorinated compounds; PFNA: perfluorononanoic acid; PFOS: perfluorooctanesulfonic acid; PFOA: perfluorooctanoic acid; PLZF: promyelocytic leukemia zinc finger; ROS: reactive oxygen species; HILI: RNA-mediated gene silencing 2; SSC: spermatogonial stem cell.

The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paes, Camila, E-mail: camilaquinetti@gmail.com; Nakagami, Gojiro, E-mail: gojiron-tky@umin.ac.jp; Minematsu, Takeo, E-mail: tminematsu-tky@umin.ac.jp

2012-10-19

Highlights: Black-Right-Pointing-Pointer An evidence of the positive effect of AHL on epithelialization process is provided. Black-Right-Pointing-Pointer AHL enhances keratinocyte's ability to migrate in an in vitro scratch wound model. Black-Right-Pointing-Pointer AHL induces the expression of Mmp13. Black-Right-Pointing-Pointer Topical application of AHL represents a possible strategy to treat chronic wounds. -- Abstract: Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalianmore » cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte's activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte's ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration process.« less

Effect of intermittent shear stress on corneal epithelial cells using an in vitro flow culture model.

PubMed

Hampel, Ulrike; Garreis, Fabian; Burgemeister, Fabian; Eßel, Nicole; Paulsen, Friedrich

2018-04-27

The aim of this study was to establish and to evaluate an in vitro model for culturing human telomerase-immortalized corneal epithelial (hTCEpi) cells under adjustable medium flow mimicking the movements of the tear film on the ocular surface. Using an IBIDI pump system, cells were cultured under unidirectional, continuous or oscillating, discontinuous medium flow. Cell surface and cytoskeletal architecture were investigated by scanning electron microscopy and immunofluorescence. Gene expression of e-cadherin, occludin, tight junction protein (TJP), desmoplakin, desmocollin and mucins was investigated by real-time PCR. Protein expression of desmoplakin, TJP, occludin and e-cadherin was analyzed by western blot and localization was detected by immunofluorescence. Rose bengal staining was used to assess mucin (MUC) barrier integrity. MUC1, -4 and -16 proteins were localized by immunofluorescence. Medium flow-induced shear stress dramatically changed cellular morphology of hTCEpi. Cells subjected to discontinuous shear stress displayed the typical flattened, polygonal cell shape of the superficial layer of stratified squamous epithelia. Cell surfaces showed less bulging under shear stress and less extracellular gaps. The mRNA expression of E-cadherin, occludin and TJP were increased under oscillatory medium flow. Desmoplakin and occludin protein were upregulated under oscillatory shear stress. Stress fiber formation was not aligned to flow direction. MUC1, -4, and -16 protein were localized under all culture conditions, a regulation on mRNA expression was not detectable. Rose Bengal uptake was diminished under unidirectional conditions. Our findings suggest that shear stress as it occurs at the ocular surface during blinking exerts marked effects on corneal epithelial cells, such as changes in cellular morphology and expression of cell junctions. The described model may be useful for in vitro investigations of ocular surface epithelia as it represents a much more physiologic reproduction of the in vivo situation than the commonly applied static culture conditions. Copyright © 2018. Published by Elsevier Inc.

Lung Toxicity of Condensed Aerosol from E-CIG Liquids: Influence of the Flavor and the In Vitro Model Used.

PubMed

Bengalli, Rossella; Ferri, Emanuele; Labra, Massimo; Mantecca, Paride

2017-10-20

The diffusion of e-cigarette (e-CIG) opens a great scientific and regulatory debate about its safety. The huge number of commercialized devices, e-liquids with almost infinite chemical formulations and the growing market demand for a rapid and efficient toxicity screen system that is able to test all of these references and related aerosols. A consensus on the best protocols for the e-CIG safety assessment is still far to be achieved, since the huge number of variables characterizing these products (e.g., flavoring type and concentration, nicotine concentration, type of the device, including the battery and the atomizer). This suggests that more experimental evidences are needed to support the regulatory frameworks. The present study aims to contribute in this field by testing the effects of condensed aerosols (CAs) from three main e-liquid categories (tobacco, mint, and cinnamon as food-related flavor), with (18 mg/mL) or without nicotine. Two in vitro models, represented by a monoculture of human epithelial alveolar cells and a three-dimensional (3D) co-culture of alveolar and lung microvascular endothelial cells were used. Cell viability, pro-inflammatory cytokines release and alveolar-blood barrier (ABB) integrity were investigated as inhalation toxicity endpoints. Results showed that nicotine itself had almost no influence on the modulation of the toxicity response, while flavor composition did have. The cell viability was significantly decreased in monoculture and ABB after exposure to the mints and cinnamon CAs. The barrier integrity was significantly affected in the ABB after exposure to cytotoxic CAs. With the exception of the significant IL-8 release in the monoculture after Cinnamon exposure, no increase of inflammatory cytokines (IL-8 and MCP-1) release was observed. These findings point out that multiple assays with different in vitro models are able to discriminate the acute inhalation toxicity of CAs from liquids with different flavors, providing the companies and regulatory bodies with useful tools for the preliminary screening of marketable products.

Propagation of uncertainty in nasal spray in vitro performance models using Monte Carlo simulation: Part II. Error propagation during product performance modeling.

PubMed

Guo, Changning; Doub, William H; Kauffman, John F

2010-08-01

Monte Carlo simulations were applied to investigate the propagation of uncertainty in both input variables and response measurements on model prediction for nasal spray product performance design of experiment (DOE) models in the first part of this study, with an initial assumption that the models perfectly represent the relationship between input variables and the measured responses. In this article, we discard the initial assumption, and extended the Monte Carlo simulation study to examine the influence of both input variable variation and product performance measurement variation on the uncertainty in DOE model coefficients. The Monte Carlo simulations presented in this article illustrate the importance of careful error propagation during product performance modeling. Our results show that the error estimates based on Monte Carlo simulation result in smaller model coefficient standard deviations than those from regression methods. This suggests that the estimated standard deviations from regression may overestimate the uncertainties in the model coefficients. Monte Carlo simulations provide a simple software solution to understand the propagation of uncertainty in complex DOE models so that design space can be specified with statistically meaningful confidence levels. (c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association

Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model.

PubMed

Conti, Stefania; Magliani, Walter; Arseni, Simona; Frazzi, Raffaele; Salati, Antonella; Ravanetti, Lara; Polonelli, Luciano

2002-06-01

Monoclonal (KTmAb) and recombinant (KTscFv) anti-idiotypic antibodies, representing the internal image of a yeast killer toxin, proved to be microbicidal in vitro against important eukaryotic and prokaryotic pathogens such as Candida albicans, Pneumocystis carinii, Mycobacterium tuberculosis, Staphylococcus aureus, S. haemolyticus, Enterococcus faecalis, E. faecium, and Streptococcus pneumoniae, including multidrug-resistant strains. KTmAb and KTscFv exerted a strong therapeutic effect in well-established animal models of candidiasis and pneumocystosis. Streptococcus mutans is the most important etiologic agent of dental caries that might result from the metabolic end products of dental plaque. Effective strategies to reduce the disease potential of dental plaque have considered the possibility of using antibiotics or antibodies against oral streptococci in general and S. mutans in particular. In this study, the activity of KTmAb and KTscFv against S. mutans and the inhibition and reduction by KTmAb of dental colonization by S. mutans and other oral streptococci in an ex vivo model of human teeth were investigated. KTscFv and KTmAb were used in a conventional colony forming unit (CFU) assay against a serotype C strain of S. mutans, and other oral streptococci (S. intermedius, S. mitis, S. oralis, S. salivarius). An ex vivo model of human teeth submerged in saliva was used to establish KTmAb potential of inhibiting or reducing the adhesion to dental surfaces by S. mutans and other oral streptococci. KTmAb and KTscFv kill in vitro S. mutans and other oral streptococci. KTmAb inhibit colonization of dental surfaces by S. mutans and oral streptococci in the ex vivo model. Killer antibodies with antibiotic activity or their engineered derivatives may have a potential in the prevention of dental caries in vivo.

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

PubMed Central

Shipley, Mackenzie M.; Mangold, Colleen A.; Szpara, Moriah L.

2016-01-01

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease. PMID:26967710

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line.

PubMed

Shipley, Mackenzie M; Mangold, Colleen A; Szpara, Moriah L

2016-02-17

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods(1-4) and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

In Vivo Predictive Dissolution (IPD) and Biopharmaceutical Modeling and Simulation: Future Use of Modern Approaches and Methodologies in a Regulatory Context.

PubMed

Lennernäs, H; Lindahl, A; Van Peer, A; Ollier, C; Flanagan, T; Lionberger, R; Nordmark, A; Yamashita, S; Yu, L; Amidon, G L; Fischer, V; Sjögren, E; Zane, P; McAllister, M; Abrahamsson, B

2017-04-03

The overall objective of OrBiTo, a project within Innovative Medicines Initiative (IMI), is to streamline and optimize the development of orally administered drug products through the creation and efficient application of biopharmaceutics tools. This toolkit will include both experimental and computational models developed on improved understanding of the highly dynamic gastrointestinal (GI) physiology relevant to the GI absorption of drug products in both fasted and fed states. A part of the annual OrBiTo meeting in 2015 was dedicated to the presentation of the most recent progress in the development of the regulatory use of PBPK in silico modeling, in vivo predictive dissolution (IPD) tests, and their application to biowaivers. There are still several areas for improvement of in vitro dissolution testing by means of generating results relevant for the intraluminal conditions in the GI tract. The major opportunity is probably in combining IPD testing and physiologically based in silico models where the in vitro data provide input to the absorption predictions. The OrBiTo project and other current research projects include definition of test media representative for the more distal parts of the GI tract, models capturing supersaturation and precipitation phenomena, and influence of motility waves on shear and other forces of hydrodynamic origin, addressing the interindividual variability in composition and characteristics of GI fluids, food effects, definition of biorelevant buffer systems, and intestinal water volumes. In conclusion, there is currently a mismatch between the extensive industrial usage of modern in vivo predictive tools and very limited inclusion of such data in regulatory files. However, there is a great interest among all stakeholders to introduce recent progresses in prediction of in vivo GI drug absorption into regulatory context.

Establishment of induced pluripotent stem cell (iPSC) line from 55-year old male patient with hemorrhagic Moyamoya disease.

PubMed

Cardano, Marina; Marsoner, Fabio; Marcatili, Matteo; Karnavas, Thodoris; Zasso, Jacopo; Lanterna, Luigi Andrea; Conti, Luciano

2016-11-01

Peripheral blood mononuclear cells (PBMCs) were collected from 55-year old male patient with a confirmed diagnosis of hemorrhagic Moyamoya disease (MMD). PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. A footprint-free hiPSC line was characterized by the expression of pluripotency markers and a normal karyotype. These cells were able to give rise to Embryoid Bodies and to a progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a suitable tool for modelling in vitro MMD disease to investigate the cellular mechanisms underlying the occurrence of this pathology. Copyright © 2016. Published by Elsevier B.V.

Endoplasmic reticulum stress is induced in the human placenta during labour

PubMed Central

Veerbeek, J.H.W.; Tissot Van Patot, M.C.; Burton, G.J.; Yung, H.W.

2015-01-01

Placental endoplasmic reticulum (ER) stress has been postulated in the pathophysiology of pre-eclampsia (PE) and intrauterine growth restriction (IUGR), but its activation remains elusive. Oxidative stress induced by ischaemia/hypoxia-reoxygenation activates ER stress in vitro. Here, we explored whether exposure to labour represents an in vivo model for the study of acute placental ER stress. ER stress markers, GRP78, P-eIF2α and XBP-1, were significantly higher in laboured placentas than in Caesarean-delivered controls localised mainly in the syncytiotrophoblast. The similarities to changes observed in PE/IUGR placentas suggest exposure to labour can be used to investigate induction of ER stress in pathological placentas. PMID:25434970

Computational modeling of the bat HKU4 coronavirus 3CLpro inhibitors as a tool for the development of antivirals against the emerging Middle East respiratory syndrome (MERS) coronavirus.

PubMed

Abuhammad, Areej; Al-Aqtash, Rua'a A; Anson, Brandon J; Mesecar, Andrew D; Taha, Mutasem O

2017-11-01

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that poses a major challenge to clinical management. The 3C-like protease (3CL pro ) is essential for viral replication and thus represents a potential target for antiviral drug development. Presently, very few data are available on MERS-CoV 3CL pro inhibition by small molecules. We conducted extensive exploration of the pharmacophoric space of a recently identified set of peptidomimetic inhibitors of the bat HKU4-CoV 3CL pro . HKU4-CoV 3CL pro shares high sequence identity (81%) with the MERS-CoV enzyme and thus represents a potential surrogate model for anti-MERS drug discovery. We used 2 well-established methods: Quantitative structure-activity relationship (QSAR)-guided modeling and docking-based comparative intermolecular contacts analysis. The established pharmacophore models highlight structural features needed for ligand recognition and revealed important binding-pocket regions involved in 3CL pro -ligand interactions. The best models were used as 3D queries to screen the National Cancer Institute database for novel nonpeptidomimetic 3CL pro inhibitors. The identified hits were tested for HKU4-CoV and MERS-CoV 3CL pro inhibition. Two hits, which share the phenylsulfonamide fragment, showed moderate inhibitory activity against the MERS-CoV 3CL pro and represent a potential starting point for the development of novel anti-MERS agents. To the best of our knowledge, this is the first pharmacophore modeling study supported by in vitro validation on the MERS-CoV 3CL pro . MERS-CoV is an emerging virus that is closely related to the bat HKU4-CoV. 3CL pro is a potential drug target for coronavirus infection. HKU4-CoV 3CL pro is a useful surrogate model for the identification of MERS-CoV 3CL pro enzyme inhibitors. dbCICA is a very robust modeling method for hit identification. The phenylsulfonamide scaffold represents a potential starting point for MERS coronavirus 3CL pro inhibitors development. Copyright © 2017 John Wiley & Sons, Ltd.

Patient-specific in vitro models for hemodynamic analysis of congenital heart disease - Additive manufacturing approach.

PubMed

Medero, Rafael; García-Rodríguez, Sylvana; François, Christopher J; Roldán-Alzate, Alejandro

2017-03-21

Non-invasive hemodynamic assessment of total cavopulmonary connection (TCPC) is challenging due to the complex anatomy. Additive manufacturing (AM) is a suitable alternative for creating patient-specific in vitro models for flow measurements using four-dimensional (4D) Flow MRI. These in vitro systems have the potential to serve as validation for computational fluid dynamics (CFD), simulating different physiological conditions. This study investigated three different AM technologies, stereolithography (SLA), selective laser sintering (SLS) and fused deposition modeling (FDM), to determine differences in hemodynamics when measuring flow using 4D Flow MRI. The models were created using patient-specific MRI data from an extracardiac TCPC. These models were connected to a perfusion pump circulating water at three different flow rates. Data was processed for visualization and quantification of velocity, flow distribution, vorticity and kinetic energy. These results were compared between each model. In addition, the flow distribution obtained in vitro was compared to in vivo. The results showed significant difference in velocities measured at the outlets of the models that required internal support material when printing. Furthermore, an ultrasound flow sensor was used to validate flow measurements at the inlets and outlets of the in vitro models. These results were highly correlated to those measured with 4D Flow MRI. This study showed that commercially available AM technologies can be used to create patient-specific vascular models for in vitro hemodynamic studies at reasonable costs. However, technologies that do not require internal supports during manufacturing allow smoother internal surfaces, which makes them better suited for flow analyses. Copyright © 2017 Elsevier Ltd. All rights reserved.

Estimating the long-term effects of in vitro fertilization in Greece: an analysis based on a lifetime-investment model

PubMed Central

Fragoulakis, Vassilis; Maniadakis, Nikolaos

2013-01-01

Objective To quantify the economic effects of a child conceived by in vitro fertilization (IVF) in terms of net tax revenue from the state’s perspective in Greece. Methods Based on previous international experience, a mathematical model was developed to assess the lifetime productivity of a single individual and his/her lifetime transactions with governmental agencies. The model distinguished among three periods in the economic life cycle of an individual: (1) early life, when the government primarily contributes resources through child tax credits, health care, and educational expenses; (2) employment, when individuals begin returning resources through taxes; and (3) retirement, when the government expends additional resources on pensions and health care. The cost of a live birth with IVF was based on the modification of a previously published model developed by the authors. All outcomes were discounted at a 3% discount rate. The data inputs – namely, the economic or demographic variables – were derived from the National Statistical Secretariat of Greece and other relevant sources. To deal with uncertainty, bias-corrected uncertainty intervals (UIs) were calculated based on 5000 Monte Carlo simulations. In addition, to examine the robustness of our results, other one-way sensitivity analyses were also employed. Results The cost of IVF per birth was estimated at €17,015 (95% UI: €13,932–€20,200). The average projected income generated by an individual throughout his/her productive life was €258,070 (95% UI: €185,376–€339,831). In addition, his/her life tax contribution was estimated at €133,947 (95% UI: €100,126–€177,375), while the discounted governmental expenses for elderly and underage individuals were €67,624 (95% UI: €55,211–€83,930). Hence, the net present value of IVF was €60,435 (95% UI: €33,651–€94,330), representing a 182% net return on investment. Results remained constant under various assumptions for the main model parameters. Conclusion State-funded IVF may represent good value for money in the Greek setting, since it has positive tax benefits for the government, notwithstanding its beneficial psychological effect on infertile couples. PMID:23818800

The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile

PubMed Central

Wiedemuth, Ralf; Binner, Aline; Navratiel, Katrin; Anastassiadis, Konstantinos; Brenner, Sebastian

2018-01-01

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC. PMID:29489861

The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile.

PubMed

Thieme, Sebastian; Holzbaur, Alexander; Wiedemuth, Ralf; Binner, Aline; Navratiel, Katrin; Anastassiadis, Konstantinos; Brenner, Sebastian; Richter, Cornelia

2018-01-01

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

Amniotic fluid stem cells: an ideal resource for therapeutic application in bone tissue engineering.

PubMed

Pantalone, A; Antonucci, I; Guelfi, M; Pantalone, P; Usuelli, F G; Stuppia, L; Salini, V

2016-07-01

Skeletal diseases, both degenerative and secondary to trauma, infections or tumors, represent an ideal target for regenerative medicine and in the last years, stem cells have been considered as good candidates for in vitro and in vivo bone regeneration. To date, several stem cell sources, such as adult mesenchymal stem cells, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have shown significant osteogenic potential. In this narrative review, we analyze the possible advantages of the use of AFSCs in the treatment of skeletal diseases, especially through the application of tissue engineering and biomaterials. Among the different sources of stem cells, great attention has been recently devoted to amniotic fluid-derived stem cells (AFSC) characterized by high renewal capacity and ability to differentiate along several different lineages. Due to these features, AFSCs represent an interesting model for regenerative medicine, also considering their low immunogenicity and the absence of tumor formation after transplantation in nude mice.

Discovery and Optimization of Indolyl-Containing 4-Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria.

PubMed

Gerasyuto, Aleksey I; Arnold, Michael A; Wang, Jiashi; Chen, Guangming; Zhang, Xiaoyan; Smith, Sean; Woll, Matthew G; Baird, John; Zhang, Nanjing; Almstead, Neil G; Narasimhan, Jana; Peddi, Srinivasa; Dumble, Melissa; Sheedy, Josephine; Weetall, Marla; Branstrom, Arthur A; Prasad, J V N; Karp, Gary M

2018-05-14

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure-activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC 90 values against Escherichia coli (0.5-1 μg/mL) and Acinetobacter baumannii (8-16 μg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.

Immortalized endothelial cell lines for in vitro blood-brain barrier models: A systematic review.

PubMed

Rahman, Nurul Adhwa; Rasil, Alifah Nur'ain Haji Mat; Meyding-Lamade, Uta; Craemer, Eva Maria; Diah, Suwarni; Tuah, Ani Afiqah; Muharram, Siti Hanna

2016-07-01

Endothelial cells play the most important role in construction of the blood-brain barrier. Many studies have opted to use commercially available, easily transfected or immortalized endothelial cell lines as in vitro blood-brain barrier models. Numerous endothelial cell lines are available, but we do not currently have strong evidence for which cell lines are optimal for establishment of such models. This review aimed to investigate the application of immortalized endothelial cell lines as in vitro blood-brain barrier models. The databases used for this review were PubMed, OVID MEDLINE, ProQuest, ScienceDirect, and SpringerLink. A narrative systematic review was conducted and identified 155 studies. As a result, 36 immortalized endothelial cell lines of human, mouse, rat, porcine and bovine origins were found for the establishment of in vitro blood-brain barrier and brain endothelium models. This review provides a summary of immortalized endothelial cell lines as a guideline for future studies and improvements in the establishment of in vitro blood-brain barrier models. It is important to establish a good and reproducible model that has the potential for multiple applications, in particular a model of such a complex compartment such as the blood-brain barrier. Copyright © 2016 Elsevier B.V. All rights reserved.

Modeling Effects of RNA on Capsid Assembly Pathways via Coarse-Grained Stochastic Simulation

PubMed Central

Smith, Gregory R.; Xie, Lu; Schwartz, Russell

2016-01-01

The environment of a living cell is vastly different from that of an in vitro reaction system, an issue that presents great challenges to the use of in vitro models, or computer simulations based on them, for understanding biochemistry in vivo. Virus capsids make an excellent model system for such questions because they typically have few distinct components, making them amenable to in vitro and modeling studies, yet their assembly can involve complex networks of possible reactions that cannot be resolved in detail by any current experimental technology. We previously fit kinetic simulation parameters to bulk in vitro assembly data to yield a close match between simulated and real data, and then used the simulations to study features of assembly that cannot be monitored experimentally. The present work seeks to project how assembly in these simulations fit to in vitro data would be altered by computationally adding features of the cellular environment to the system, specifically the presence of nucleic acid about which many capsids assemble. The major challenge of such work is computational: simulating fine-scale assembly pathways on the scale and in the parameter domains of real viruses is far too computationally costly to allow for explicit models of nucleic acid interaction. We bypass that limitation by applying analytical models of nucleic acid effects to adjust kinetic rate parameters learned from in vitro data to see how these adjustments, singly or in combination, might affect fine-scale assembly progress. The resulting simulations exhibit surprising behavioral complexity, with distinct effects often acting synergistically to drive efficient assembly and alter pathways relative to the in vitro model. The work demonstrates how computer simulations can help us understand how assembly might differ between the in vitro and in vivo environments and what features of the cellular environment account for these differences. PMID:27244559

Formulation development of retrocyclin 1 analog RC-101 as an anti-HIV vaginal microbicide product.

PubMed

Sassi, A B; Cost, M R; Cole, A L; Cole, A M; Patton, D L; Gupta, P; Rohan, L C

2011-05-01

RC-101 is a synthetic microbicide analog of retrocyclin, which has shown in vitro activity against X4 and R5 HIV-1. In an effort to develop a safe and effective RC-101 vaginal microbicide product, we assessed safety in ex vivo macaque and human models and efficacy using in vitro and ex vivo models. A polyvinyl-alcohol vaginal film containing RC-101 (100 μg/film) was developed. Formulation assessment was conducted by evaluating disintegration, drug content, mechanical properties, and stability. Efficacy was evaluated by in vitro peripheral blood mononuclear cells (PBMC) assay and ex vivo human ectocervical tissue explant model. Ex vivo safety studies were conducted by exposing RC-101 to an excised monkey reproductive tract and excised human ectocervical tissue. RC-101 100 μg films were shown to be safe to human and monkey tissue and effective against HIV-1 in vitro and ex vivo in human ectocervical tissue. The 90% inhibitory concentration (IC90) for RC-101 films at 2,000 μg (IC90=57.5 μM) using an ex vivo model was 10-fold higher than the IC90 observed using an in vitro model (IC90=5.0 μM). RC-101 films were stable for 1 month at 25°C, with in vitro bioactivity maintained for up to 6 months. RC-101 was developed in a quick-dissolve film formulation that was shown to be safe in an ex vivo model and effective in in vitro and ex vivo models. RC-101 film formulations were shown to maintain bioactivity for a period of 6 months. Findings from the present study contribute to the development of a safe and effective topical microbicide product.

3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments

PubMed Central

Albritton, Jacob L.

2017-01-01

ABSTRACT Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo. Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies. PMID:28067628

3D bioprinting: improving in vitro models of metastasis with heterogeneous tumor microenvironments.

PubMed

Albritton, Jacob L; Miller, Jordan S

2017-01-01

Even with many advances in treatment over the past decades, cancer still remains a leading cause of death worldwide. Despite the recognized relationship between metastasis and increased mortality rate, surprisingly little is known about the exact mechanism of metastatic progression. Currently available in vitro models cannot replicate the three-dimensionality and heterogeneity of the tumor microenvironment sufficiently to recapitulate many of the known characteristics of tumors in vivo Our understanding of metastatic progression would thus be boosted by the development of in vitro models that could more completely capture the salient features of cancer biology. Bioengineering groups have been working for over two decades to create in vitro microenvironments for application in regenerative medicine and tissue engineering. Over this time, advances in 3D printing technology and biomaterials research have jointly led to the creation of 3D bioprinting, which has improved our ability to develop in vitro models with complexity approaching that of the in vivo tumor microenvironment. In this Review, we give an overview of 3D bioprinting methods developed for tissue engineering, which can be directly applied to constructing in vitro models of heterogeneous tumor microenvironments. We discuss considerations and limitations associated with 3D printing and highlight how these advances could be harnessed to better model metastasis and potentially guide the development of anti-cancer strategies. © 2017. Published by The Company of Biologists Ltd.

Anticipatory Life Cycle Analysis of In Vitro Biomass Cultivation for Cultured Meat Production in the United States.

PubMed

Mattick, Carolyn S; Landis, Amy E; Allenby, Braden R; Genovese, Nicholas J

2015-10-06

Cultured, or in vitro, meat consists of edible biomass grown from animal stem cells in a factory, or carnery. In the coming decades, in vitro biomass cultivation could enable the production of meat without the need to raise livestock. Using an anticipatory life cycle analysis framework, the study described herein examines the environmental implications of this emerging technology and compares the results with published impacts of beef, pork, poultry, and another speculative analysis of cultured biomass. While uncertainty ranges are large, the findings suggest that in vitro biomass cultivation could require smaller quantities of agricultural inputs and land than livestock; however, those benefits could come at the expense of more intensive energy use as biological functions such as digestion and nutrient circulation are replaced by industrial equivalents. From this perspective, large-scale cultivation of in vitro meat and other bioengineered products could represent a new phase of industrialization with inherently complex and challenging trade-offs.

Improving in vitro to in vivo extrapolation by incorporating toxicokinetic measurements: A case study of lindane-induced neurotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Croom, Edward L.; Shafer, Timothy J.; Evans, Marina V.

Approaches for extrapolating in vitro toxicity testing results for prediction of human in vivo outcomes are needed. The purpose of this case study was to employ in vitro toxicokinetics and PBPK modeling to perform in vitro to in vivo extrapolation (IVIVE) of lindane neurotoxicity. Lindane cell and media concentrations in vitro, together with in vitro concentration-response data for lindane effects on neuronal network firing rates, were compared to in vivo data and model simulations as an exercise in extrapolation for chemical-induced neurotoxicity in rodents and humans. Time- and concentration-dependent lindane dosimetry was determined in primary cultures of rat cortical neuronsmore » in vitro using “faux” (without electrodes) microelectrode arrays (MEAs). In vivo data were derived from literature values, and physiologically based pharmacokinetic (PBPK) modeling was used to extrapolate from rat to human. The previously determined EC{sub 50} for increased firing rates in primary cultures of cortical neurons was 0.6 μg/ml. Media and cell lindane concentrations at the EC{sub 50} were 0.4 μg/ml and 7.1 μg/ml, respectively, and cellular lindane accumulation was time- and concentration-dependent. Rat blood and brain lindane levels during seizures were 1.7–1.9 μg/ml and 5–11 μg/ml, respectively. Brain lindane levels associated with seizures in rats and those predicted for humans (average = 7 μg/ml) by PBPK modeling were very similar to in vitro concentrations detected in cortical cells at the EC{sub 50} dose. PBPK model predictions matched literature data and timing. These findings indicate that in vitro MEA results are predictive of in vivo responses to lindane and demonstrate a successful modeling approach for IVIVE of rat and human neurotoxicity. - Highlights: • In vitro to in vivo extrapolation for lindane neurotoxicity was performed. • Dosimetry of lindane in a micro-electrode array (MEA) test system was assessed. • Cell concentrations at the MEA EC{sub 50} equaled rat brain levels associated with seizure. • PBPK-predicted human brain levels at seizure also equaled EC{sub 50} cell concentrations. • In vitro MEA results are predictive of lindane in vivo dose–response in rats/humans.« less

Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

PubMed

Gao, Bin; Wang, Lin; Han, Shuang; Pingguan-Murphy, Belinda; Zhang, Xiaohui; Xu, Feng

2016-08-01

Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.

In-vitro bacterial identification using fluorescence spectroscopy with an optical fiber system

NASA Astrophysics Data System (ADS)

Spector, Brian C.; Werkhaven, Jay A.; Smith, Dana; Reinisch, Lou

2000-05-01

Acute otitis media (AOM) remains a source of significant morbidity in children. With the emergence of antibiotic resistant strains of bacteria, tympanocentesis has become an important method of bacterial identification in the setting of treatment failures. Previous studies described a prototype system for the non-invasive fluorescence identification of bacteria in vitro. We demonstrate the addition of an optical fiber to allow for the identification of a specimen distant to the spectrofluorometer. Emission spectra from three bacteria, Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus were successfully obtained in vitro. This represents a necessary step prior to the study of in vivo identification of bacteria in AOM using fluorescence spectroscopy.

A Validated Multiscale In-Silico Model for Mechano-sensitive Tumour Angiogenesis and Growth

PubMed Central

Loizidou, Marilena; Stylianopoulos, Triantafyllos; Hawkes, David J.

2017-01-01

Vascularisation is a key feature of cancer growth, invasion and metastasis. To better understand the governing biophysical processes and their relative importance, it is instructive to develop physiologically representative mathematical models with which to compare to experimental data. Previous studies have successfully applied this approach to test the effect of various biochemical factors on tumour growth and angiogenesis. However, these models do not account for the experimentally observed dependency of angiogenic network evolution on growth-induced solid stresses. This work introduces two novel features: the effects of hapto- and mechanotaxis on vessel sprouting, and mechano-sensitive dynamic vascular remodelling. The proposed three-dimensional, multiscale, in-silico model of dynamically coupled angiogenic tumour growth is specified to in-vivo and in-vitro data, chosen, where possible, to provide a physiologically consistent description. The model is then validated against in-vivo data from murine mammary carcinomas, with particular focus placed on identifying the influence of mechanical factors. Crucially, we find that it is necessary to include hapto- and mechanotaxis to recapitulate observed time-varying spatial distributions of angiogenic vasculature. PMID:28125582

Vitamin D regulates tyrosine hydroxylase expression: N-cadherin a possible mediator.

PubMed

Cui, X; Pertile, R; Liu, P; Eyles, D W

2015-09-24

Vitamin D is a neuroactive steroid. Its genomic actions are mediated via the active form of vitamin D, 1,25(OH)2D3, binding to the vitamin D receptor (VDR). The VDR emerges in the rat mesencephalon at embryonic day 12, representing the peak period of dopaminergic cell birth. Our prior studies reveal that developmental vitamin D (DVD)-deficiency alters the ontogeny of dopaminergic neurons in the developing mesencephalon. There is also consistent evidence from others that 1,25(OH)2D3 promotes the survival of dopaminergic neurons in models of dopaminergic toxicity. In both developmental and toxicological studies it has been proposed that 1,25(OH)2D3 may modulate the differentiation and maturation of dopaminergic neurons; however, to date there is lack of direct evidence. The aim of the current study is to investigate this both in vitro using a human SH-SY5Y cell line transfected with rodent VDR and in vivo using a DVD-deficient model. Here we show that in VDR-expressing SH-SY5Y cells, 1,25(OH)2D3 significantly increased production of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. This effect was dose- and time-dependent, but was not due to an increase in TH-positive cell number, nor was it due to the production of trophic survival factors for dopamine neurons such as glial-derived neurotrophic factor (GDNF). In accordance with 1,25(OH)2D3's anti-proliferative actions in the brain, 1,25(OH)2D3 reduced the percentage of dividing cells from approximately 15-10%. Given the recently reported role of N-cadherin in the direct differentiation of dopaminergic neurons, we examined here whether it may be elevated by 1,25(OH)2D3. We confirmed this in vitro and more importantly, we showed DVD-deficiency decreases N-cadherin expression in the embryonic mesencephalon. In summary, in our in vitro model we have shown 1,25(OH)2D3 increases TH expression, decreases proliferation and elevates N-cadherin, a potential factor that mediates these processes. Accordingly all of these findings are reversed in the developing brain in our DVD-deficiency model. Remarkably our findings in the DVD-deficiency model phenocopy those found in a recent model where N-cadherin was regionally ablated from the mesencephalon. This study has, for the first time, shown that vitamin D directly modulates TH expression and strongly suggests N-cadherin may be a plausible mediator of this process both in vitro and in vivo. Our findings may help to explain epidemiological data linking DVD deficiency with schizophrenia. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

Stress Distribution in Single Dental Implant System: Three-Dimensional Finite Element Analysis Based on an In Vitro Experimental Model.

PubMed

Rezende, Carlos Eduardo Edwards; Chase-Diaz, Melody; Costa, Max Doria; Albarracin, Max Laurent; Paschoeto, Gabriela; Sousa, Edson Antonio Capello; Rubo, José Henrique; Borges, Ana Flávia Sanches

2015-10-01

This study aimed to analyze the stress distribution in single implant system and to evaluate the compatibility of an in vitro model with finite element (FE) model. The in vitro model consisted of Brånemark implant; multiunit set abutment of 5 mm height; metal-ceramic screw-retained crown, and polyurethane simulating the bone. Deformations were recorded in the peri-implant region in the mesial and distal aspects, after an axial 300 N load application at the center of the occlusal aspect of the crown, using strain gauges. This in vitro model was scanned with micro CT to design a three-dimensional FE model and the strains in the peri-implant bone region were registered to check the compatibility between both models. The FE model was used to evaluate stress distribution in different parts of the system. The values obtained from the in vitro model (20-587 με) and the finite element analysis (81-588 με) showed agreement among them. The highest stresses because of axial and oblique load, respectively were 5.83 and 40 MPa for the cortical bone, 55 and 1200 MPa for the implant, and 80 and 470 MPa for the abutment screw. The FE method proved to be effective for evaluating the deformation around single implant. Oblique loads lead to higher stress concentrations.

Application of in vitro bioaccessibility and bioavailability methods for calcium, carotenoids, folate, iron, magnesium, polyphenols, zinc and vitamins B6, B12, D, and E

USDA-ARS?s Scientific Manuscript database

A review of in vitro bioaccessibility and bioavailability methods for polyphenols and selected nutrients is presented. The review focuses on in vitro solubility, dialyzability, the dynamic gastrointestinal model (TIM), and Caco-2 cell models, the latter primarily for uptake and transport, and a disc...

Rat aorta as a pharmacological tool for in vitro and in vivo studies.

PubMed

Rameshrad, Maryam; Babaei, Hossein; Azarmi, Yadollah; Fouladi, Daniel Fadaei

2016-01-15

Rat aorta assay provides a low cost and rapid platform, especially for preclinical in vivo models. The signaling pathways of the analog on the vessels could be evaluated separately on the endothelium or smooth muscle cells by rings of the rat aorta in vitro. The rat aorta is used for angiogenesis modeling to integrate the benefits of the both in vivo and in vitro models. These explain the importance and usage of rat aorta in researches. Furthermore, about 4503 articles have been published with the key word "rat aorta" in title or abstract from 1955 until the end of 2013 in Medline. In this review, these articles were organized into two main categories: in vivo and in vitro studies. The in vitro section focused on the rat aorta model, as a tool for evaluate the mechanism of vasodilation, vasoconstriction and angiogenesis. In the in vivo section, the most important usage of this tissue was evaluated. Also, the vasotonic signaling pathways in the vessel are explained briefly and some rat aorta applications in vitro and in vivo have been discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

In vitro oral drug permeation models: the importance of taking physiological and physico-chemical factors into consideration.

PubMed

Joubert, Ruan; Steyn, Johan Dewald; Heystek, Hendrik Jacobus; Steenekamp, Jan Harm; Du Preez, Jan Lourens; Hamman, Josias Hendrik

2017-02-01

The assessment of intestinal membrane permeability properties of new chemical entities is a crucial step in the drug discovery and development process and a variety of in vitro models, methods and techniques are available to estimate the extent of oral drug absorption in humans. However, variations in certain physiological and physico-chemical factors are often not reflected in the results and the complex dynamic interplay between these factors is sometimes oversimplified with in vitro models. Areas covered: In vitro models to evaluate drug pharmacokinetics are briefly outlined, while both physiological and physico-chemical factors that may have an influence on these techniques are critically reviewed. The shortcomings identified for some of the in vitro techniques are discussed in conjunction with novel ways to improve and thereby overcome some challenges. Expert opinion: Although conventional in vitro methods and theories are used as basic guidelines to predict drug absorption, critical evaluations have identified some shortcomings. Advancements in technology have made it possible to investigate and understand the role of physiological and physico-chemical factors in drug delivery more clearly, which can be used to improve and refine the techniques to more closely mimic the in vivo environment.

In-Vitro Approaches for Studying Blast-Induced Traumatic Brain Injury

PubMed Central

Chen, Yung Chia; Smith, Douglas H.

2009-01-01

Abstract Traumatic brain injury caused by explosive or blast events is currently divided into four phases: primary, secondary, tertiary, and quaternary blast injury. These phases of blast-induced traumatic brain injury (bTBI) are biomechanically distinct, and can be modeled in both in-vivo and in-vitro systems. The purpose of this review is to consider the mechanical phases of bTBI, how these phases are reproduced with in-vitro models, and to review findings from these models to assess how each phase of bTBI can be examined in more detail. Highlighted are some important gaps in the literature that may be addressed in the future to better identify the exact contributing mechanisms for bTBI. These in-vitro models, viewed in combination with in-vivo models and clinical studies, can be used to assess both the mechanisms and possible treatments for this type of trauma. PMID:19397424

Addition of Epidermal Growth Factor Improves the Rate of Sulfur Mustard Wound Healing in an In Vitro Model

DTIC Science & Technology

2008-03-26

the wound site.1 Inflammation begins within minutes after the injury. Neutrophils clear out contami- nating bacteria and release cytokines to activate...of an in vitro wound healing model for periodontal cells. J Periodontol. 2000;71:226–37. 27. Oates TW, Mumford JH, Carnes DL, Cochran DL...Characterization of proliferation and cellular wound fill in periodontal cells using an in vitro wound model. J Periodontol. 2001;72:324–30. 28. Chuang AH

In vitro-in vivo correlation study for the dermatopharmacokinetics of terbinafine hydrochloride topical cream.

PubMed

Saeheng, Suwadee; Nosoongnoen, Wichit; Varothai, Supenya; Sathirakul, Korbtham

2013-09-01

To investigate the relationship between dermatopharmacokinetic (DPK) tape stripping from in vitro and in vivo using 1% terbinafine hydrochloride topical cream as the model formulation. In vitro and in vivo tape strippings were conducted on separated pig ear skin used as a biological membrane for franz diffusion cell testing and the non-hairy skin area at the ventral forearms of healthy volunteers, respectively. Terbinafine (1%) topical cream was applied to the skin for 0.5, 2, and 4 h. The drug profiles of terbinafine across the stratum corneum were determined immediately (time 0 h), and at 0.5, 1, 2, and 4 h after removing the formulation. The amounts of terbinafine were analyzed by a validated high-performance liquid chromatography-ultraviolet method. The area under the curve (AUC) and the maximum amounts of terbinafine absorption (Q(max)) were obtained from pharmacokinetic software. Partition coefficient (K(SC/veh)) and diffusion parameter (D/L²) were derived from the Fick's second law equation. During the schedule time of 8 h, the deviations of in vitro and in vivo data were 6.61 and 30.46% for AUC and Q(max), respectively. There was insignificant difference of the K(SC/veh) and the D/L² between excised pig ear and human skin. In addition, K(SC/veh) and D/L² at T(max) of 2 h were used to predict the AUC presented the value of 4.7481 %h whereas the true value calculated from pharmacokinetic software provided the value of 5.9311 %h differing from each other in approximate of 20%. In vitro tape stripping using the separated pig ear skin as a viable membrane of the franz diffusion cell testing demonstrates the potential to represent in vivo tape stripping in human for topical bioavailability/bioequivalence study of terbinafine hydrochloride 1% topical cream.

Biotransformation of aesculin by human gut bacteria and identification of its metabolites in rat urine.

PubMed

Ding, Wei-Jun; Deng, Yun; Feng, Hao; Liu, Wei-Wei; Hu, Rong; Li, Xiang; Gu, Zhe-Ming; Dong, Xiao-Ping

2009-03-28

To observe the biotransformation process of a Chinese compound, aesculin, by human gut bacteria, and to identify its metabolites in rat urine. Representative human gut bacteria were collected from 20 healthy volunteers, and then utilized in vitro to biotransform aesculin under anaerobic conditions. At 0, 2, 4, 8, 12, 16, 24, 48 and 72 h post-incubation, 10 mL of culture medium was collected. Metabolites of aesculin were extracted 3 x from rat urine with methanol and analyzed by HPLC. For in vivo metabolite analysis, aesculetin (100 mg/kg) was administered to rats via stomach gavage, rat urine was collected from 6 to 48 h post-administration, and metabolite analysis was performed by LC/ESI-MS and MS/MS in the positive and negative modes. Human gut bacteria could completely convert aesculin into aesculetin in vitro. The biotransformation process occurred from 8 to 24 h post-incubation, with its highest activity was seen from 8 to 12 h. The in vitro process was much slower than the in vivo process. In contrast to the in vitro model, six aesculetin metabolites were identified in rat urine, including 6-hydroxy-7-gluco-coumarin (M1), 6-hydroxy-7-sulf-coumarin (M2), 6, 7-di-gluco-coumarin (M3), 6-glc-7-gluco-coumarin (M4), 6-O-methyl-7-gluco-coumarin (M5) and 6-O-methyl-7-sulf-coumarin (M6). Of which, M2 and M6 were novel metabolites. Aesculin can be transferred into aesculetin by human gut bacteria and is further modified by the host in vivo. The diverse metabolites of aesculin may explain its pleiotropic pharmaceutical effects.

In-vitro transdentinal diffusion of monomers from adhesives.

PubMed

Putzeys, Eveline; Duca, Radu Corneliu; Coppens, Lieve; Vanoirbeek, Jeroen; Godderis, Lode; Van Meerbeek, Bart; Van Landuyt, Kirsten L

2018-06-01

Biocompatibility of adhesives is important since adhesives may be applied on dentin near the pulp. Accurate knowledge of the quantity of monomers reaching the pulp is important to determine potential side effects. The aim of this study was to assess the transdentinal diffusion of residual monomers from dental adhesive systems using an in-vitro pulp chamber model. Dentin disks with a thickness of 300 µm were produced from human third molars. These disks were fixed between two open-ended glass tubes, representing an in-vitro pulp chamber. The etch-and-rinse adhesive OptiBond FL and the self-etch adhesive Clearfil SE Bond were applied to the dentin side of the disks, while on in the pulpal side, the glass tube was filled with 600 µL water. The transdentinal diffusion of different monomers was quantified using ultra-performance liquid chromatography-tandem mass spectrometry. The monomers HEMA, CQ, BisGMA, GPDM, 10-MDP and UDMA eluted from the dental materials and were able to diffuse through the dentin disks to a certain extent. Compounds with a lower molecular weight (uncured group: HEMA 7850 nmol and CQ 78.2 nmol) were more likely to elute and diffuse compared to monomers with a higher molecular weight (uncured group: BisGMA 0.42 nmol). When the adhesives were left uncured, diffusion was up to 10 times higher compared to the cured conditions. This in-vitro research resulted in the quantification of various monomers able to diffuse through dentin and therefore contributes to a more detailed understanding about the potential exposure of the dental pulp to monomers from dental adhesives. Biocompatibility of adhesives is important since adhesives may be applied on dentin near the pulp, where tubular density and diameter are greatest. Copyright © 2018. Published by Elsevier Ltd.

In vitro toxicity testing with microplate cell cultures: Impact of cell binding.

PubMed

Gülden, Michael; Schreiner, Jeannine; Seibert, Hasso

2015-06-05

In vitro generated data on toxic potencies are generally based on nominal concentrations. However, cellular and extracellular binding and elimination processes may reduce the available free fraction of a compound. Then, nominal effective concentrations do not represent appropriate measures of toxic exposure in vitro and underestimate toxic potencies. In this study it was investigated whether cell binding can affect the availability of chemicals in microplate based toxicity assays. To this end the cytotoxicity of compounds like mercury chloride, digitonin and alcohol ethoxylates, accumulated by cells via different modes, was investigated in 96-well microplate cultures with varying concentrations of Balb/c 3T3 cells. The median effective nominal concentrations of all but one of the tested compounds depended linearly from the cell concentration. Applying a previously developed equilibrium distribution model cell concentration-independent median effective extracellular concentrations and cell burdens, respectively, could be calculated. The compounds were accumulated by the cells with bioconcentration factors, BCF, between 480 and ≥ 25,000. Cell binding of the alcohol ethoxylates was correlated with their lipophilicity. The results show that significant cell binding can occur even at the small cell volume fractions (∼ 1 × 10(-5) to 3 × 10(-3) L/L) encountered in microplate assays. To what extent cell binding affects the bioavailability depends on the BCF and the cell volume fraction. EC50 measurements in the presence of at least two different cell concentrations allow for excluding or detecting significant cell binding and for determining more appropriate measures of toxic exposure in vitro like median effective extracellular (free) concentrations or cell burdens. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Radiation-force-based estimation of acoustic attenuation using harmonic motion imaging (HMI) in phantoms and in vitro livers before and after HIFU ablation

NASA Astrophysics Data System (ADS)

Chen, Jiangang; Hou, Gary Y.; Marquet, Fabrice; Han, Yang; Camarena, Francisco; Konofagou, Elisa

2015-10-01

Acoustic attenuation represents the energy loss of the propagating wave through biological tissues and plays a significant role in both therapeutic and diagnostic ultrasound applications. Estimation of acoustic attenuation remains challenging but critical for tissue characterization. In this study, an attenuation estimation approach was developed using the radiation-force-based method of harmonic motion imaging (HMI). 2D tissue displacement maps were acquired by moving the transducer in a raster-scan format. A linear regression model was applied on the logarithm of the HMI displacements at different depths in order to estimate the acoustic attenuation. Commercially available phantoms with known attenuations (n=5 ) and in vitro canine livers (n=3 ) were tested, as well as HIFU lesions in in vitro canine livers (n=5 ). Results demonstrated that attenuations obtained from the phantoms showed a good correlation ({{R}2}=0.976 ) with the independently obtained values reported by the manufacturer with an estimation error (compared to the values independently measured) varying within the range of 15-35%. The estimated attenuation in the in vitro canine livers was equal to 0.32   ±   0.03 dB cm-1 MHz-1, which is in good agreement with the existing literature. The attenuation in HIFU lesions was found to be higher (0.58   ±   0.06 dB cm-1 MHz-1) than that in normal tissues, also in agreement with the results from previous publications. Future potential applications of the proposed method include estimation of attenuation in pathological tissues before and after thermal ablation.

Precision-cut liver slices of Atlantic cod (Gadus morhua): an in vitro system for studying the effects of environmental contaminants.

PubMed

Eide, M; Karlsen, O A; Kryvi, H; Olsvik, P A; Goksøyr, A

2014-08-01

The Atlantic cod (Gadus morhua) is an economically important species commonly consumed by humans. The widespread distribution of cod in the North Atlantic Ocean makes it vulnerable to effluents from human activities, such as coastal industries and offshore petroleum exploration. It has been demonstrated that many effluents have adverse effects on cod reproduction and health, e.g. by disrupting endocrine signaling pathways. The liver, expressing important components of the biotransformation and the endocrine system, is one of the main target organs. Thus, reliable and reproducible in vitro systems of the liver are important for studying effects of environmental contaminants. The aim of this study was to investigate precision-cut liver slices (PCLS) as an alternative in vitro system for toxicological studies of the Atlantic cod liver. Slices of 8 mm in diameter and 250 μm thickness were prepared and cultivated from immature cod. Several analyses to measure the liver slice viability were performed: enzyme assays, histology, and morphometric analysis, all confirming cell viability for up to 72 h in culture. The liver slices were also exposed to two well-known model environmental contaminants, β-naphthoflavone (BNF) and 17α-ethynylestradiol (EE2), representing established agonists for the aryl hydrocarbon receptor (AHR) and the estrogen receptor (ER), respectively. The results showed increased transcription of the target genes cytochrome P450 1A (CYP1A) and vitellogenin (VTG), both well-established biomarkers for exposure of fish to the selected compounds. In conclusion, PCLS is a promising in vitro system for toxicological studies of cod liver cells. The liver slices are viable in culture for several days and respond to environmental contaminants in a dose- and time-specific manner. Copyright © 2013 Elsevier B.V. All rights reserved.

Comparative assessment of three in vitro exposure methods for combustion toxicity.

PubMed

Lestari, Fatma; Markovic, Boban; Green, Anthony R; Chattopadhyay, Gautam; Hayes, Amanda J

2006-01-01

A comparative assessment of three approaches for the use of human cells in vitro to investigate combustion toxicity was conducted. These included one indirect and two direct (passive and dynamic) exposure methods. The indirect method used an impinger system in which culture medium was used to trap the toxicants, whilst the direct exposure involved the use of a Horizontal Harvard Navicyte Chamber at the air/liquid interface. The cytotoxic effects of thermal decomposition products were assessed using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay (Promega) on a selection of human cells including: HepG2, A549 and skin fibroblasts. A small scale laboratory fire test using a vertical tube furnace was designed for the generation of combustion products. Polymethyl methacrylate (PMMA) was selected as a model polymer to study the cytotoxic effects of combustion products. NOAEC (no observable adverse effect concentration), IC10 (10% inhibitory concentration), IC50 (50% inhibitory concentration) and TLC (total lethal concentration) values were determined from dose response curves. Assessment using the NRU (neutral red uptake) and ATP (adenosine triphosphate) assays on human lung derived cells (A549) was also undertaken. Comparison between in vitro cytotoxicity results against published toxicity data for PMMA combustion and predicted LC50 (50% lethal concentration) values calculated from identified compounds using GCMS (gas chromatography mass spectrometry) was determined. The results suggested that the indirect exposure method did not appear to simulate closely exposure via inhalation, whilst exposure at the air/liquid interface by using the dynamic method proved to be a more representative method of human inhalation. This exposure method may be a potential system for in vitro cytotoxicity testing in combustion toxicity. Copyright 2005 John Wiley & Sons, Ltd.

Gold(I)-Triphenylphosphine Complexes with Hypoxanthine-Derived Ligands: In Vitro Evaluations of Anticancer and Anti-Inflammatory Activities

PubMed Central

Křikavová, Radka; Hošek, Jan; Vančo, Ján; Hutyra, Jakub; Dvořák, Zdeněk; Trávníček, Zdeněk

2014-01-01

A series of gold(I) complexes involving triphenylphosphine (PPh3) and one N-donor ligand derived from deprotonated mono- or disubstituted hypoxanthine (HLn) of the general composition [Au(Ln)(PPh3)] (1–9) is reported. The complexes were thoroughly characterized, including multinuclear high resolution NMR spectroscopy as well as single crystal X-ray analysis (for complexes 1 and 3). The complexes were screened for their in vitro cytotoxicity against human cancer cell lines MCF7 (breast carcinoma), HOS (osteosarcoma) and THP-1 (monocytic leukaemia), which identified the complexes 4–6 as the most promising representatives, who antiproliferative activity was further tested against A549 (lung adenocarcinoma), G-361 (melanoma), HeLa (cervical cancer), A2780 (ovarian carcinoma), A2780R (ovarian carcinoma resistant to cisplatin), 22Rv1 (prostate cancer) cell lines. Complexes 4–6 showed a significantly higher in vitro anticancer effect against the employed cancer cells, except for G-361, as compared with the commercially used anticancer drug cisplatin, with IC50 ≈ 1–30 µM. Anti-inflammatory activity was evaluated in vitro by the assessment of the ability of the complexes to modulate secretion of the pro-inflammatory cytokines, i.e. tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in the lipopolysaccharide-activated macrophage-like THP-1 cell model. The results of this study identified the complexes as auspicious anti-inflammatory agents with similar or better activity as compared with the clinically applied gold-based antiarthritic drug Auranofin. In an effort to explore the possible mechanisms responsible for the biological effect, the products of interactions of selected complexes with sulfur-containing biomolecules (L-cysteine and reduced glutathione) were studied by means of the mass-spectrometry study. PMID:25226034

The iron chelator Dp44mT suppresses osteosarcoma’s proliferation, invasion and migration: in vitro and in vivo

PubMed Central

Li, Pengcheng; Zheng, Xun; Shou, Kangquan; Niu, Yahui; Jian, Chao; Zhao, Yong; Yi, Wanrong; Hu, Xiang; Yu, Aixi

2016-01-01

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells, including neuroblastoma, neuroepithelioma and prostate cancer. However, whether Dp44mT has anticancer effects in osteosarcoma is still unknown. Here, we investigated the antitumor action of Dp44mT in osteosarcoma and its underlying mechanisms. A human osteosarcoma 143B cell line in vitro and 143B xenograft in nude mice in vivo were utilized, the anticancer effects of Dp44mT were examined through methods of MTT assay, transwell, wound healing assay, flow cytometry, western blot, immunohistochemistry and H&E staining. We showed that Dp44mT inhibits cell proliferation, invasion and migration in vitro. In addition, flow cytometry further illustrated that Dp44mT suppression of 143B cell proliferation, invasion and migration were partially due to induction of cell apoptosis, cell cycle arrest in S phase and ROS production. Also in vitro and in vivo, the expression levels of Bcl2, Bax, Caspase3, Caspase9, LC3-II, β-catenin and its downstream targets such as C-myc and Cyclin D1 demonstrated that cell apoptosis and autophagy, as well as Wnt/β-catenin pathway were involved in Dp44mT induced osteosarcoma suppression. The Dp44mT inhibition of osteosarcoma was further verified via animal models. The findings indicated that in vivo Dp44mT showed a significant reduction in the 143B xenograft tumor growth and metastasis. In conclusion, our data demonstrated that Dp44mT has effective anticancer capability in osteosarcoma and that may represent a promising treatment strategy for osteosarcoma. PMID:28078009